register interest

Professor Kevin Marsh

Research Area: Global Health
Scientific Themes: Tropical Medicine & Global Health
Keywords: malaria, immunology and epidemiology
Web Links:

Professor Kevin Marsh has a broad research interest in child health in the tropics, with a particular focus in the immune epidemiology of malaria. From 1989 to 2014 he directed the KEMRI Wellcome Programme in Kenya. Kevin's research interests  focus on three main areas:

  • Identifying the protective immune response to malaria
  • Regulation of immune responses
  • Determinants of virulence

Kevin has a particular interest is in the development of  science in Africa. He is currently seconded for 50% of his time as senior adviser to the African Academy of Sciences. Kevin is chair of the WHO Malaria policy advisory committee (MPAC) and  a member of many global health advisory groups.

Name Department Institution Country
Prof Chris I Newbold (RDM) Investigative Medicine Division Oxford University, Weatherall Institute of Molecular Medicine United Kingdom
Professor Adrian VS Hill Jenner Institute Oxford University, Old Road Campus Research Building United Kingdom
Prof David Roberts (RDM) Nuffield Division of Clinical Laboratory Sciences Oxford University, John Radcliffe Hospital United Kingdom
David Conway MRC Laboratories Gambia
S Polley London School of Hygiene and Tropical Medicine United Kingdom
Paul Milligan London School of Hygiene and Tropical Medicine United Kingdom
Tony Holder National Institute of Medical Research Mill Hill United Kingdom
Jean Langhorne National Institute of Medical Research Mill Hill United Kingdom
Dr Alex Rowe Centers for Disease Control United States
James Beeson Walter and Eliza Hall Institute Melbourne Australia
Anna Farnert Karolinska Institute Stockholm Sweden
Ibrahim Ei Hassan Institute of Endemic Diseases Khartoum Sudan
Gilbert Kokwaro University of Nairobi Kenya
Murungi LM, Sondén K, Odera D, Oduor LB, Guleid F, Nkumama IN, Otiende M, Kangoye DT, Fegan G, Färnert A et al. 2017. Cord blood IgG and the risk of severe Plasmodium falciparum malaria in the first year of life. Int J Parasitol, 47 (2-3), pp. 153-162. | Show Abstract | Read more

Young infants are less susceptible to severe episodes of malaria but the targets and mechanisms of protection are not clear. Cord blood antibodies may play an important role in mediating protection but many studies have examined their association with the outcome of infection or non-severe malaria. Here, we investigated whether cord blood IgG to Plasmodium falciparum merozoite antigens and antibody-mediated effector functions were associated with reduced odds of developing severe malaria at different time points during the first year of life. We conducted a case-control study of well-defined severe falciparum malaria nested within a longitudinal birth cohort of Kenyan children. We measured cord blood total IgG levels against five recombinant merozoite antigens and antibody function in the growth inhibition activity and neutrophil antibody-dependent respiratory burst assays. We also assessed the decay of maternal antibodies during the first 6months of life. The mean antibody half-life range was 2.51months (95% confidence interval (CI): 2.19-2.92) to 4.91months (95% CI: 4.47-6.07). The rate of decline of maternal antibodies was inversely proportional to the starting concentration. The functional assay of antibody-dependent respiratory burst activity predicted significantly reduced odds of developing severe malaria during the first 6months of life (Odds ratio (OR) 0.07, 95% CI: 0.007-0.74, P=0.007). Identification of the targets of antibodies mediating antibody-dependent respiratory burst activity could contribute to the development of malaria vaccines that protect against severe episodes of malaria in early infancy.

Clarke GM, Rockett K, Kivinen K, Hubbart C, Jeffreys AE, Rowlands K, Jallow M, Conway DJ, Bojang KA, Pinder M et al. 2017. Characterisation of the opposing effects of G6PD deficiency on cerebral malaria and severe malarial anaemia. Elife, 6 | Show Abstract | Read more

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is believed to confer protection against Plasmodium falciparum malaria, but the precise nature of the protective effecthas proved difficult to define as G6PD deficiency has multiple allelic variants with different effects in males and females, and it has heterogeneous effects on the clinical outcome of P. falciparum infection. Here we report an analysis of multiple allelic forms of G6PD deficiency in a large multi-centre case-control study of severe malaria, using the WHO classification of G6PD mutations to estimate each individual's level of enzyme activity from their genotype. Aggregated across all genotypes, we find that increasing levels of G6PD deficiency are associated with decreasing risk of cerebral malaria, but with increased risk of severe malarial anaemia. Models of balancing selection based on these findings indicate that an evolutionary trade-off between different clinical outcomes of P. falciparum infection could have been a major cause of the high levels of G6PD polymorphism seen in human populations.

Offeddu V, Olotu A, Osier F, Marsh K, Matuschewski K, Thathy V. 2017. High Sporozoite Antibody Titers in Conjunction with Microscopically Detectable Blood Infection Display Signatures of Protection from Clinical Malaria. Front Immunol, 8 (MAY), pp. 488. | Show Abstract | Read more

Immunoepidemiological studies typically reveal slow, age-dependent acquisition of immune responses against Plasmodium falciparum sporozoites. Naturally acquired immunity against preerythrocytic stages is considered inadequate to confer protection against clinical malaria. To explore previously unrecognized antisporozoite responses, we measured serum levels of naturally acquired antibodies to whole Plasmodium falciparum sporozoites (Pfspz) and the immunodominant (NANP)5 repeats of the major sporozoite surface protein, circumsporozoite protein, in a well-characterized Kenyan cohort. Sera were sampled at the start of the malaria transmission season, and all subjects were prospectively monitored for uncomplicated clinical malaria in the ensuing 6 months. We used Kaplan-Meier analysis and multivariable regression to investigate the association of antisporozoite immunity with incidence of clinical malaria. Although naturally acquired humoral responses against Pfspz and (NANP)5 were strongly correlated (p < 0.0001), 37% of Pfspz responders did not recognize (NANP)5. The prevalence and magnitude of antisporozoite responses increased with age, although some high Pfspz responders were identified among children. Survival analysis revealed a reduced risk of and increased time to first or only episode of clinical malaria among Pfspz or (NANP)5 responders carrying microscopically detectable Plasmodium falciparum (Pf) parasitemia at the start of the transmission season (p < 0.03). Our Cox regression interaction models indicated a potentially protective interaction between high anti-Pfspz (p = 0.002) or anti-(NANP)5 (p = 0.001) antibody levels and microscopically detectable Pf parasitemia on the risk of subsequent clinical malaria. Our findings indicate that robust antisporozoite immune responses can be naturally acquired already at an early age. A potentially protective role of high levels of anti-Pfspz antibodies against clinical episodes of uncomplicated malaria was detected, suggesting that antibody-mediated preerythrocytic immunity might indeed contribute to protection in nature.

McCallum FJ, Persson KE, Fowkes FJ, Reiling L, Mugyenyi CK, Richards JS, Simpson JA, Williams TN, Gilson PR, Hodder AN et al. 2017. Differing rates of antibody acquisition to merozoite antigens in malaria: implications for immunity and surveillance. J Leukoc Biol, 101 (4), pp. 913-925. | Show Abstract | Read more

Antibodies play a key role in acquired human immunity to Plasmodium falciparum (Pf) malaria and target merozoites to reduce or prevent blood-stage replication and the development of disease. Merozoites present a complex array of antigens to the immune system, and currently, there is only a partial understanding of the targets of protective antibodies and how responses to different antigens are acquired and boosted. We hypothesized that there would be differences in the rate of acquisition of antibodies to different antigens and how well they are boosted by infection, which impacts the acquisition of immunity. We examined responses to a range of merozoite antigens in 2 different cohorts of children and adults with different age structures and levels of malaria exposure. Overall, antibodies were associated with age, exposure, and active infection, and the repertoire of responses increased with age and active infection. However, rates of antibody acquisition varied between antigens and different regions within an antigen following exposure to malaria, supporting our hypothesis. Antigen-specific responses could be broadly classified into early response types in which antibodies were acquired early in childhood exposure and late response types that appear to require substantially more exposure for the development of substantial levels. We identified antigen-specific responses that were effectively boosted after recent infection, whereas other responses were not. These findings advance our understanding of the acquisition of human immunity to malaria and are relevant to the development of malaria vaccines targeting merozoite antigens and the selection of antigens for use in malaria surveillance.

Cowman AF, Healer J, Marapana D, Marsh K. 2016. Malaria: Biology and Disease. Cell, 167 (3), pp. 610-624. | Show Abstract | Read more

Malaria has been a major global health problem of humans through history and is a leading cause of death and disease across many tropical and subtropical countries. Over the last fifteen years renewed efforts at control have reduced the prevalence of malaria by over half, raising the prospect that elimination and perhaps eradication may be a long-term possibility. Achievement of this goal requires the development of new tools including novel antimalarial drugs and more efficacious vaccines as well as an increased understanding of the disease and biology of the parasite. This has catalyzed a major effort resulting in development and regulatory approval of the first vaccine against malaria (RTS,S/AS01) as well as identification of novel drug targets and antimalarial compounds, some of which are in human clinical trials.

Hodgson SH, Llewellyn D, Silk SE, Milne KH, Elias SC, Miura K, Kamuyu G, Juma EA, Magiri C, Muia A et al. 2016. Changes in Serological Immunology Measures in UK and Kenyan Adults Post-controlled Human Malaria Infection. Front Microbiol, 7 (OCT), pp. 1604. | Show Abstract | Read more

Background: The timing of infection is closely determined in controlled human malaria infection (CHMI) studies, and as such they provide a unique opportunity to dissect changes in immunological responses before and after a single infection. The first Kenyan Challenge Study (KCS) (Pan African Clinical Trial Registry: PACTR20121100033272) was performed in 2013 with the aim of establishing the CHMI model in Kenya. This study used aseptic, cryopreserved, attenuated Plasmodium falciparum sporozoites administered by needle and syringe (PfSPZ Challenge) and was the first to evaluate parasite dynamics post-CHMI in individuals with varying degrees of prior exposure to malaria. Methods: We describe detailed serological and functional immunological responses pre- and post-CHMI for participants in the KCS and compare these with those from malaria-naïve UK volunteers who also underwent CHMI (VAC049) (ClinicalTrials.gov NCT01465048) using PfSPZ Challenge. We assessed antibody responses to three key blood-stage merozoite antigens [merozoite surface protein 1 (MSP1), apical membrane protein 1 (AMA1), and reticulocyte-binding protein homolog 5 (RH5)] and functional activity using two candidate measures of anti-merozoite immunity; the growth inhibition activity (GIA) assay and the antibody-dependent respiratory burst activity (ADRB) assay. Results:Clear serological differences were observed pre- and post-CHMI by ELISA between malaria-naïve UK volunteers in VAC049, and Kenyan volunteers who had prior malaria exposure. Antibodies to AMA1 and schizont extract correlated with parasite multiplication rate (PMR) post-CHMI in KCS. Serum from volunteer 110 in KCS, who demonstrated a dramatically reduced PMR in vivo, had no in vitro GIA prior to CHMI but the highest level of ADRB activity. A significant difference in ADRB activity was seen between KCS volunteers with minimal and definite prior exposure to malaria and significant increases were seen in ADRB activity post-CHMI in Kenyan volunteers. Quinine and atovaquone/proguanil, previously assumed to be removed by IgG purification, were identified as likely giving rise to aberrantly high in vitro GIA results. Conclusions: The ADRB activity assay is a promising functional assay that warrants further investigation as a measure of prior exposure to malaria and predictor of control of parasite growth. The CHMI model can be used to evaluate potential measures of naturally-acquired immunity to malaria.

Bediako Y, Ngoi JM, Nyangweso G, Wambua J, Opiyo M, Nduati EW, Bejon P, Marsh K, Ndungu FM. 2016. The effect of declining exposure on T cell-mediated immunity to Plasmodium falciparum - an epidemiological "natural experiment". BMC Med, 14 (1), pp. 143. | Show Abstract | Read more

BACKGROUND: Naturally acquired immunity to malaria may be lost with lack of exposure. Recent heterogeneous reductions in transmission in parts of Africa mean that large populations of previously protected people may lose their immunity while remaining at risk of infection. METHODS: Using two ethnically similar long-term cohorts of children with historically similar levels of exposure to Plasmodium falciparum who now experience very different levels of exposure, we assessed the effect of decreased parasite exposure on antimalarial immunity. Peripheral blood mononuclear cells (PBMCs) from children in each cohort were stimulated with P. falciparum and their P. falciparum-specific proliferative and cytokine responses were compared. RESULTS: We demonstrate that, while P. falciparum-specific CD4(+) T cells are maintained in the absence of exposure, the proliferative capacity of these cells is altered considerably. P. falciparum-specific CD4(+) T cells isolated from children previously exposed, but now living in an area of minimal exposure ("historically exposed") proliferate significantly more upon stimulation than cells isolated from children continually exposed to the parasite. Similarly, PBMCs from historically exposed children expressed higher levels of pro-inflammatory cytokines and lower levels of anti-inflammatory cytokines after stimulation with P. falciparum. Notably, we found a significant positive association between duration since last febrile episode and P. falciparum-specific CD4(+) T cell proliferation, with more recent febrile episodes associated with lower proliferation. CONCLUSION: Considered in the context of existing knowledge, these data suggest a model explaining how immunity is lost in absence of continuing exposure to P. falciparum.

Olotu A, Fegan G, Wambua J, Nyangweso G, Leach A, Lievens M, Kaslow DC, Njuguna P, Marsh K, Bejon P. 2016. Seven-Year Efficacy of RTS,S/AS01 Malaria Vaccine among Young African Children. N Engl J Med, 374 (26), pp. 2519-2529. | Show Abstract | Read more

BACKGROUND: The candidate malaria vaccine RTS,S/AS01 is being evaluated in order to inform a decision regarding its inclusion in routine vaccination schedules. METHODS: We conducted 7 years of follow-up in children who had been randomly assigned, at 5 to 17 months of age, to receive three doses of either the RTS,S/AS01 vaccine or a rabies (control) vaccine. The end point was clinical malaria (temperature of ≥37.5°C and infection with Plasmodium falciparum of >2500 parasites per cubic millimeter). In an analysis that was not prespecified, the malaria exposure of each child was estimated with the use of information on the prevalence of malaria among residents within a 1-km radius of the child's home. Vaccine efficacy was defined as 1 minus the hazard ratio or the incidence-rate ratio, multiplied by 100, in the RTS,S/AS01 group versus the control group. RESULTS: Over 7 years of follow-up, we identified 1002 episodes of clinical malaria among 223 children randomly assigned to the RTS,S/AS01 group and 992 episodes among 224 children randomly assigned to the control group. The vaccine efficacy, as assessed by negative binomial regression, was 4.4% (95% confidence interval [CI], -17.0 to 21.9; P=0.66) in the intention-to-treat analysis and 7.0% (95% CI, -14.5 to 24.6; P=0.52) in the per-protocol analysis. Vaccine efficacy waned over time (P=0.006 for the interaction between vaccination and time), including negative efficacy during the fifth year among children with higher-than-average exposure to malaria parasites (intention-to-treat analysis: -43.5%; 95% CI, -100.3 to -2.8 [P=0.03]; per-protocol analysis: -56.8%; 95% CI, -118.7 to -12.3 [P=0.008]). CONCLUSIONS: A three-dose vaccination with RTS,S/AS01 was initially protective against clinical malaria, but this result was offset by rebound in later years in areas with higher-than-average exposure to malaria parasites. (Funded by the PATH Malaria Vaccine Initiative and others; ClinicalTrials.gov number, NCT00872963.).

Mogeni P, Williams TN, Fegan G, Nyundo C, Bauni E, Mwai K, Omedo I, Njuguna P, Newton CR, Osier F et al. 2016. Age, Spatial, and Temporal Variations in Hospital Admissions with Malaria in Kilifi County, Kenya: A 25-Year Longitudinal Observational Study. PLoS Med, 13 (6), pp. e1002047. | Show Abstract | Read more

BACKGROUND: Encouraging progress has been seen with reductions in Plasmodium falciparum malaria transmission in some parts of Africa. Reduced transmission might lead to increasing susceptibility to malaria among older children due to lower acquired immunity, and this has implications for ongoing control strategies. METHODS AND FINDINGS: We conducted a longitudinal observational study of children admitted to Kilifi County Hospital in Kenya and linked it to data on residence and insecticide-treated net (ITN) use. This included data from 69,104 children aged from 3 mo to 13 y admitted to Kilifi County Hospital between 1 January 1990 and 31 December 2014. The variation in malaria slide positivity among admissions was examined in logistic regression models using the following predictors: location of the residence, calendar time, the child's age, ITN use, and the enhanced vegetation index (a proxy for soil moisture). The proportion of malaria slide-positive admissions declined from 0.56 (95% confidence interval [CI] 0.54-0.58) in 1998 to 0.07 (95% CI 0.06-0.08) in 2009 but then increased again through to 0.24 (95% CI 0.22-0.25) in 2014. Older children accounted for most of the increase after 2009 (0.035 [95% CI 0.030-0.040] among young children compared to 0.22 [95% CI 0.21-0.23] in older children). There was a nonlinear relationship between malaria risk and prevalence of ITN use within a 2 km radius of an admitted child's residence such that the predicted malaria positive fraction varied from ~0.4 to <0.1 as the prevalence of ITN use varied from 20% to 80%. In this observational analysis, we were unable to determine the cause of the decline in malaria between 1998 and 2009, which pre-dated the dramatic scale-up in ITN distribution and use. CONCLUSION: Following a period of reduced transmission, a cohort of older children emerged who have increased susceptibility to malaria. Further reductions in malaria transmission are needed to mitigate the increasing burden among older children, and universal ITN coverage is a promising strategy to achieve this goal.

Abdulla S, Achan J, Yeka A, D'Alessandro U, Adam I, Alemayehu BH, Allan R, Temu EA, Allen EN, Barnes KI et al. 2016. Gametocyte carriage in uncomplicated Plasmodium falciparum malaria following treatment with artemisinin combination therapy: a systematic review and meta-analysis of individual patient data BMC Medicine, 14 (1), | Show Abstract | Read more

© 2016 WWARN Gametocyte Study Group.Background: Gametocytes are responsible for transmission of malaria from human to mosquito. Artemisinin combination therapy (ACT) reduces post-treatment gametocyte carriage, dependent upon host, parasite and pharmacodynamic factors. The gametocytocidal properties of antimalarial drugs are important for malaria elimination efforts. An individual patient clinical data meta-analysis was undertaken to identify the determinants of gametocyte carriage and the comparative effects of four ACTs: artemether-lumefantrine (AL), artesunate/amodiaquine (AS-AQ), artesunate/mefloquine (AS-MQ), and dihydroartemisinin-piperaquine (DP). Methods: Factors associated with gametocytaemia prior to, and following, ACT treatment were identified in multivariable logistic or Cox regression analysis with random effects. All relevant studies were identified through a systematic review of PubMed. Risk of bias was evaluated based on study design, methodology, and missing data. Results: The systematic review identified 169 published and 9 unpublished studies, 126 of which were shared with the WorldWide Antimalarial Resistance Network (WWARN) and 121 trials including 48,840 patients were included in the analysis. Prevalence of gametocytaemia by microscopy at enrolment was 12.1 % (5887/48,589), and increased with decreasing age, decreasing asexual parasite density and decreasing haemoglobin concentration, and was higher in patients without fever at presentation. After ACT treatment, gametocytaemia appeared in 1.9 % (95 % CI, 1.7-2.1) of patients. The appearance of gametocytaemia was lowest after AS-MQ and AL and significantly higher after DP (adjusted hazard ratio (AHR), 2.03; 95 % CI, 1.24-3.12; P = 0.005 compared to AL) and AS-AQ fixed dose combination (FDC) (AHR, 4.01; 95 % CI, 2.40-6.72; P < 0.001 compared to AL). Among individuals who had gametocytaemia before treatment, gametocytaemia clearance was significantly faster with AS-MQ (AHR, 1.26; 95 % CI, 1.00-1.60; P = 0.054) and slower with DP (AHR, 0.74; 95 % CI, 0.63-0.88; P = 0.001) compared to AL. Both recrudescent (adjusted odds ratio (AOR), 9.05; 95 % CI, 3.74-21.90; P < 0.001) and new (AOR, 3.03; 95 % CI, 1.66-5.54; P < 0.001) infections with asexual-stage parasites were strongly associated with development of gametocytaemia after day 7. Conclusions: AS-MQ and AL are more effective than DP and AS-AQ FDC in preventing gametocytaemia shortly after treatment, suggesting that the non-artemisinin partner drug or the timing of artemisinin dosing are important determinants of post-treatment gametocyte dynamics.

Chan JA, Howell KB, Langer C, Maier AG, Hasang W, Rogerson SJ, Petter M, Chesson J, Stanisic DI, Duffy MF et al. 2016. A single point in protein trafficking by Plasmodium falciparum determines the expression of major antigens on the surface of infected erythrocytes targeted by human antibodies. Cell Mol Life Sci, 73 (21), pp. 4141-4158. | Show Abstract | Read more

Antibodies to blood-stage antigens of Plasmodium falciparum play a pivotal role in human immunity to malaria. During parasite development, multiple proteins are trafficked from the intracellular parasite to the surface of P. falciparum-infected erythrocytes (IEs). However, the relative importance of different proteins as targets of acquired antibodies, and key pathways involved in trafficking major antigens remain to be clearly defined. We quantified antibodies to surface antigens among children, adults, and pregnant women from different malaria-exposed regions. We quantified the importance of antigens as antibody targets using genetically engineered P. falciparum with modified surface antigen expression. Genetic deletion of the trafficking protein skeleton-binding protein-1 (SBP1), which is involved in trafficking the surface antigen PfEMP1, led to a dramatic reduction in antibody recognition of IEs and the ability of human antibodies to promote opsonic phagocytosis of IEs, a key mechanism of parasite clearance. The great majority of antibody epitopes on the IE surface were SBP1-dependent. This was demonstrated using parasite isolates with different genetic or phenotypic backgrounds, and among antibodies from children, adults, and pregnant women in different populations. Comparisons of antibody reactivity to parasite isolates with SBP1 deletion or inhibited PfEMP1 expression suggest that PfEMP1 is the dominant target of acquired human antibodies, and that other P. falciparum IE surface proteins are minor targets. These results establish SBP1 as part of a critical pathway for the trafficking of major surface antigens targeted by human immunity, and have key implications for vaccine development, and quantifying immunity in populations.

Ochola-Oyier LI, Okombo J, Wagatua N, Ochieng J, Tetteh KK, Fegan G, Bejon P, Marsh K. 2016. Comparison of allele frequencies of Plasmodium falciparum merozoite antigens in malaria infections sampled in different years in a Kenyan population. Malar J, 15 (1), pp. 261. | Show Abstract | Read more

BACKGROUND: Plasmodium falciparum merozoite antigens elicit antibody responses in malaria-endemic populations, some of which are clinically protective, which is one of the reasons why merozoite antigens are the focus of malaria vaccine development efforts. Polymorphisms in several merozoite antigen-encoding genes are thought to arise as a result of selection by the human immune system. METHODS: The allele frequency distribution of 15 merozoite antigens over a two-year period, 2007 and 2008, was examined in parasites obtained from children with uncomplicated malaria. In the same population, allele frequency changes pre- and post-anti-malarial treatment were also examined. Any gene which showed a significant shift in allele frequencies was also assessed longitudinally in asymptomatic and complicated malaria infections. RESULTS: Fluctuating allele frequencies were identified in codons 147 and 148 of reticulocyte-binding homologue (Rh) 5, with a shift from HD to YH haplotypes over the two-year period in uncomplicated malaria infections. However, in both the asymptomatic and complicated malaria infections YH was the dominant and stable haplotype over the two-year and ten-year periods, respectively. A logistic regression analysis of all three malaria infection populations between 2007 and 2009 revealed, that the chance of being infected with the HD haplotype decreased with time from 2007 to 2009 and increased in the uncomplicated and asymptomatic infections. CONCLUSION: Rh5 codons 147 and 148 showed heterogeneity at both an individual and population level and may be under some degree of immune selection.

Marsh K. 2016. Africa in transition: the case of malaria. Int Health, 8 (3), pp. 155-156. | Read more

Warimwe GM, Abdi AI, Muthui M, Fegan G, Musyoki JN, Marsh K, Bull PC. 2016. Serological Conservation of Parasite-Infected Erythrocytes Predicts Plasmodium falciparum Erythrocyte Membrane Protein 1 Gene Expression but Not Severity of Childhood Malaria. Infect Immun, 84 (5), pp. 1331-1335. | Show Abstract | Read more

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), expressed on P. falciparum-infected erythrocytes, is a major family of clonally variant targets of naturally acquired immunity to malaria. Previous studies have demonstrated that in areas where malaria is endemic, antibodies to infected erythrocytes from children with severe malaria tend to be more seroprevalent than antibodies to infected erythrocytes from children with nonsevere malaria. These data have led to a working hypothesis that PfEMP1 variants associated with parasite virulence are relatively conserved in structure. However, the longevity of such serologically conserved variants in the parasite population is unknown. Here, using infected erythrocytes from recently sampled clinical P. falciparum samples, we measured serological conservation using pools of antibodies in sera that had been sampled 10 to 12 years earlier. The serological conservation of infected erythrocytes strongly correlated with the expression of specific PfEMP1 subsets previously found to be associated with severe malaria. However, we found no association between serological conservation per se and disease severity within these data. This contrasts with the simple hypothesis that P. falciparum isolates with a serologically conserved group of PfEMP1 variants cause severe malaria. The data are instead consistent with periodic turnover of the immunodominant epitopes of PfEMP1 associated with severe malaria.

Murungi LM, Sondén K, Llewellyn D, Rono J, Guleid F, Williams AR, Ogada E, Thairu A, Färnert A, Marsh K et al. 2016. Targets and Mechanisms Associated with Protection from Severe Plasmodium falciparum Malaria in Kenyan Children. Infect Immun, 84 (4), pp. 950-963. | Show Abstract | Read more

Severe malaria (SM) is a life-threatening complication of infection with Plasmodium falciparum Epidemiological observations have long indicated that immunity against SM is acquired relatively rapidly, but prospective studies to investigate its immunological basis are logistically challenging and have rarely been undertaken. We investigated the merozoite targets and antibody-mediated mechanisms associated with protection against SM in Kenyan children aged 0 to 2 years. We designed a unique prospective matched case-control study of well-characterized SM clinical phenotypes nested within a longitudinal birth cohort of children (n= 5,949) monitored over the first 2 years of life. We quantified immunological parameters in sera collected before the SM event in cases and their individually matched controls to evaluate the prospective odds of developing SM in the first 2 years of life. Anti-AMA1 antibodies were associated with a significant reduction in the odds of developing SM (odds ratio [OR] = 0.37; 95% confidence interval [CI] = 0.15 to 0.90; P= 0.029) after adjustment for responses to all other merozoite antigens tested, while those against MSP-2, MSP-3, Plasmodium falciparum Rh2 [PfRh2], MSP-119, and the infected red blood cell surface antigens were not. The combined ability of total IgG to inhibit parasite growth and mediate the release of reactive oxygen species from neutrophils was associated with a marked reduction in the odds of developing SM (OR = 0.07; 95% CI = 0.006 to 0.82;P= 0.03). Assays of these two functional mechanisms were poorly correlated (Spearman rank correlation coefficient [rs] = 0.12;P= 0.07). Our data provide epidemiological evidence that multiple antibody-dependent mechanisms contribute to protective immunity via distinct targets whose identification could accelerate the development of vaccines to protect against SM.

Tan J, Pieper K, Piccoli L, Abdi A, Foglierini M, Geiger R, Tully CM, Jarrossay D, Ndungu FM, Wambua J et al. 2016. A LAIR1 insertion generates broadly reactive antibodies against malaria variant antigens. Nature, 529 (7584), pp. 105-109. | Show Abstract | Read more

Plasmodium falciparum antigens expressed on the surface of infected erythrocytes are important targets of naturally acquired immunity against malaria, but their high number and variability provide the pathogen with a powerful means of escape from host antibodies. Although broadly reactive antibodies against these antigens could be useful as therapeutics and in vaccine design, their identification has proven elusive. Here we report the isolation of human monoclonal antibodies that recognize erythrocytes infected by different P. falciparum isolates and opsonize these cells by binding to members of the RIFIN family. These antibodies acquired broad reactivity through a novel mechanism of insertion of a large DNA fragment between the V and DJ segments. The insert, which is both necessary and sufficient for binding to RIFINs, encodes the entire 98 amino acid collagen-binding domain of LAIR1, an immunoglobulin superfamily inhibitory receptor encoded on chromosome 19. In each of the two donors studied, the antibodies are produced by a single expanded B-cell clone and carry distinct somatic mutations in the LAIR1 domain that abolish binding to collagen and increase binding to infected erythrocytes. These findings illustrate, with a biologically relevant example, a novel mechanism of antibody diversification by interchromosomal DNA transposition and demonstrate the existence of conserved epitopes that may be suitable candidates for the development of a malaria vaccine.

Kangoye DT, Mensah VA, Murungi LM, Nkumama I, Nebie I, Marsh K, Cisse B, Bejon P, Osier FH, Sirima SB, MVVC Infant Immunology Study Group. 2016. Dynamics and role of antibodies to Plasmodium falciparum merozoite antigens in children living in two settings with differing malaria transmission intensity. Vaccine, 34 (1), pp. 160-166. | Show Abstract | Read more

BACKGROUND: Young infants have reduced susceptibility to febrile malaria compared with older children, but the mechanism for this remains unclear. There are conflicting data on the role of passively acquired antibodies. Here, we examine antibody titres to merozoite surface antigens in the protection of children in their first two years of life in two settings with differing malaria transmission intensity and compare these titres to previously established protective thresholds. METHODS: Two cohorts of children aged four to six weeks were recruited in Banfora, Burkina and Keur Soce, Senegal and followed up for two years. Malaria infections were detected by light microscopic examination of blood smears collected at active and passive case detection visits. The titres of antibodies to the Plasmodium falciparum recombinant merozoite proteins (AMA1-3D7, MSP1-19, MSP2-Dd2, and MSP3-3D7) were measured by enzyme-linked immunosorbent assay at 1-6, 9, 12, 15 and 18 months of age and compared with the protective thresholds established in Kenyan children. RESULTS: Antibody titres were below the protective thresholds throughout the study period and we did not find any association with protection against febrile malaria. Antibodies to AMA1 and MSP1-19 appeared to be markers of exposure in the univariate analysis (and so associated with increasing risk) and adjusting for exposure reduced the strength and significance of this association. CONCLUSION: The antibody levels we measured are unlikely to be responsible for the apparent protection against febrile malaria seen in young infants. Further work to identify protective antibody responses might include functional assays and a wider range of antigens.

MalariaGEN Plasmodium falciparum Community Project. 2016. Genomic epidemiology of artemisinin resistant malaria. Elife, 5 (MARCH2016), | Show Abstract | Read more

The current epidemic of artemisinin resistant Plasmodium falciparum in Southeast Asia is the result of a soft selective sweep involving at least 20 independent kelch13 mutations. In a large global survey, we find that kelch13 mutations which cause resistance in Southeast Asia are present at low frequency in Africa. We show that African kelch13 mutations have originated locally, and that kelch13 shows a normal variation pattern relative to other genes in Africa, whereas in Southeast Asia there is a great excess of non-synonymous mutations, many of which cause radical amino-acid changes. Thus, kelch13 is not currently undergoing strong selection in Africa, despite a deep reservoir of variations that could potentially allow resistance to emerge rapidly. The practical implications are that public health surveillance for artemisinin resistance should not rely on kelch13 data alone, and interventions to prevent resistance must account for local evolutionary conditions, shown by genomic epidemiology to differ greatly between geographical regions.

Kangoye DT, Noor A, Midega J, Mwongeli J, Mkabili D, Mogeni P, Kerubo C, Akoo P, Mwangangi J, Drakeley C et al. 2016. Malaria hotspots defined by clinical malaria, asymptomatic carriage, PCR and vector numbers in a low transmission area on the Kenyan Coast. Malar J, 15 (1), pp. 213. | Show Abstract | Read more

BACKGROUND: Targeted malaria control interventions are expected to be cost-effective. Clinical, parasitological and serological markers of malaria transmission have been used to detect malaria transmission hotspots, but few studies have examined the relationship between the different potential markers in low transmission areas. The present study reports on the relationships between clinical, parasitological, serological and entomological markers of malaria transmission in an area of low transmission intensity in Coastal Kenya. METHODS: Longitudinal data collected from 831 children aged 5-17 months, cross-sectional survey data from 800 older children and adults, and entomological survey data collected in Ganze on the Kenyan Coast were used in the present study. The spatial scan statistic test used to detect malaria transmission hotspots was based on incidence of clinical malaria episodes, prevalence of asymptomatic asexual parasites carriage detected by microscopy and polymerase chain reaction (PCR), seroprevalence of antibodies to two Plasmodium falciparum merozoite antigens (AMA1 and MSP1-19) and densities of Anopheles mosquitoes in CDC light-trap catches. RESULTS: There was considerable overlapping of hotspots by these different markers, but only weak to moderate correlation between parasitological and serological markers. PCR prevalence and seroprevalence of antibodies to AMA1 or MSP1-19 appeared to be more sensitive markers of hotspots at very low transmission intensity. CONCLUSION: These findings may support the choice of either serology or PCR as markers in the detection of malaria transmission hotspots for targeted interventions.

Neafsey DE, Juraska M, Bedford T, Benkeser D, Valim C, Griggs A, Lievens M, Abdulla S, Adjei S, Agbenyega T et al. 2015. Genetic Diversity and Protective Efficacy of the RTS,S/AS01 Malaria Vaccine. N Engl J Med, 373 (21), pp. 2025-2037. | Show Abstract | Read more

BACKGROUND: The RTS,S/AS01 vaccine targets the circumsporozoite protein of Plasmodium falciparum and has partial protective efficacy against clinical and severe malaria disease in infants and children. We investigated whether the vaccine efficacy was specific to certain parasite genotypes at the circumsporozoite protein locus. METHODS: We used polymerase chain reaction-based next-generation sequencing of DNA extracted from samples from 4985 participants to survey circumsporozoite protein polymorphisms. We evaluated the effect that polymorphic positions and haplotypic regions within the circumsporozoite protein had on vaccine efficacy against first episodes of clinical malaria within 1 year after vaccination. RESULTS: In the per-protocol group of 4577 RTS,S/AS01-vaccinated participants and 2335 control-vaccinated participants who were 5 to 17 months of age, the 1-year cumulative vaccine efficacy was 50.3% (95% confidence interval [CI], 34.6 to 62.3) against clinical malaria in which parasites matched the vaccine in the entire circumsporozoite protein C-terminal (139 infections), as compared with 33.4% (95% CI, 29.3 to 37.2) against mismatched malaria (1951 infections) (P=0.04 for differential vaccine efficacy). The vaccine efficacy based on the hazard ratio was 62.7% (95% CI, 51.6 to 71.3) against matched infections versus 54.2% (95% CI, 49.9 to 58.1) against mismatched infections (P=0.06). In the group of infants 6 to 12 weeks of age, there was no evidence of differential allele-specific vaccine efficacy. CONCLUSIONS: These results suggest that among children 5 to 17 months of age, the RTS,S vaccine has greater activity against malaria parasites with the matched circumsporozoite protein allele than against mismatched malaria. The overall vaccine efficacy in this age category will depend on the proportion of matched alleles in the local parasite population; in this trial, less than 10% of parasites had matched alleles. (Funded by the National Institutes of Health and others.).

Malaria Genomic Epidemiology Network, Band G, Rockett KA, Spencer CC, Kwiatkowski DP. 2015. A novel locus of resistance to severe malaria in a region of ancient balancing selection. Nature, 526 (7572), pp. 253-257. | Show Abstract | Read more

The high prevalence of sickle haemoglobin in Africa shows that malaria has been a major force for human evolutionary selection, but surprisingly few other polymorphisms have been proven to confer resistance to malaria in large epidemiological studies. To address this problem, we conducted a multi-centre genome-wide association study (GWAS) of life-threatening Plasmodium falciparum infection (severe malaria) in over 11,000 African children, with replication data in a further 14,000 individuals. Here we report a novel malaria resistance locus close to a cluster of genes encoding glycophorins that are receptors for erythrocyte invasion by P. falciparum. We identify a haplotype at this locus that provides 33% protection against severe malaria (odds ratio = 0.67, 95% confidence interval = 0.60-0.76, P value = 9.5 × 10(-11)) and is linked to polymorphisms that have previously been shown to have features of ancient balancing selection, on the basis of haplotype sharing between humans and chimpanzees. Taken together with previous observations on the malaria-protective role of blood group O, these data reveal that two of the strongest GWAS signals for severe malaria lie in or close to genes encoding the glycosylated surface coat of the erythrocyte cell membrane, both within regions of the genome where it appears that evolution has maintained diversity for millions of years. These findings provide new insights into the host-parasite interactions that are critical in determining the outcome of malaria infection.

Ashley EA, Aweeka F, Barnes KI, Bassat Q, Borrmann S, Dahal P, Davis TME, Deloron P, Denis MB, Djimde AA et al. 2015. Artemether-lumefantrine treatment of uncomplicated Plasmodium falciparum malaria: a systematic review and meta-analysis of day 7 lumefantrine concentrations and therapeutic response using individual patient data BMC Medicine, 13 (1), | Show Abstract | Read more

© 2015 WorldWide Antimalarial Resistance Network (WWARN) Lumefantrine PK/PD Study Group.Background: Achieving adequate antimalarial drug exposure is essential for curing malaria. Day 7 blood or plasma lumefantrine concentrations provide a simple measure of drug exposure that correlates well with artemether-lumefantrine efficacy. However, the 'therapeutic' day 7 lumefantrine concentration threshold needs to be defined better, particularly for important patient and parasite sub-populations. Methods: The WorldWide Antimalarial Resistance Network (WWARN) conducted a large pooled analysis of individual pharmacokinetic-pharmacodynamic data from patients treated with artemether-lumefantrine for uncomplicated Plasmodium falciparum malaria, to define therapeutic day 7 lumefantrine concentrations and identify patient factors that substantially alter these concentrations. A systematic review of PubMed, Embase, Google Scholar, ClinicalTrials.gov and conference proceedings identified all relevant studies. Risk of bias in individual studies was evaluated based on study design, methodology and missing data. Results: Of 31 studies identified through a systematic review, 26 studies were shared with WWARN and 21 studies with 2,787 patients were included. Recrudescence was associated with low day 7 lumefantrine concentrations (HR 1.59 (95 % CI 1.36 to 1.85) per halving of day 7 concentrations) and high baseline parasitemia (HR 1.87 (95 % CI 1.22 to 2.87) per 10-fold increase). Adjusted for mg/kg dose, day 7 concentrations were lowest in very young children (<3 years), among whom underweight-for-age children had 23 % (95 % CI -1 to 41 %) lower concentrations than adequately nourished children of the same age and 53 % (95 % CI 37 to 65 %) lower concentrations than adults. Day 7 lumefantrine concentrations were 44 % (95 % CI 38 to 49 %) lower following unsupervised treatment. The highest risk of recrudescence was observed in areas of emerging artemisinin resistance and very low transmission intensity. For all other populations studied, day 7 concentrations ≥200 ng/ml were associated with >98 % cure rates (if parasitemia <135,000/μL). Conclusions: Current artemether-lumefantrine dosing recommendations achieve day 7 lumefantrine concentrations ≥200 ng/ml and high cure rates in most uncomplicated malaria patients. Three groups are at increased risk of treatment failure: very young children (particularly those underweight-for-age); patients with high parasitemias; and patients in very low transmission intensity areas with emerging parasite resistance. In these groups, adherence and treatment response should be monitored closely. Higher, more frequent, or prolonged dosage regimens should now be evaluated in very young children, particularly if malnourished, and in patients with hyperparasitemia.

WWARN Artemisinin based Combination Therapy (ACT) Africa Baseline Study Group, Dahal P, d'Alessandro U, Dorsey G, Guerin PJ, Nsanzabana C, Price RN, Sibley CH, Stepniewska K, Talisuna AO. 2015. Clinical determinants of early parasitological response to ACTs in African patients with uncomplicated falciparum malaria: a literature review and meta-analysis of individual patient data. BMC Med, 13 (1), pp. 212. | Show Abstract | Read more

BACKGROUND: Artemisinin-resistant Plasmodium falciparum has emerged in the Greater Mekong sub-region and poses a major global public health threat. Slow parasite clearance is a key clinical manifestation of reduced susceptibility to artemisinin. This study was designed to establish the baseline values for clearance in patients from Sub-Saharan African countries with uncomplicated malaria treated with artemisinin-based combination therapies (ACTs). METHODS: A literature review in PubMed was conducted in March 2013 to identify all prospective clinical trials (uncontrolled trials, controlled trials and randomized controlled trials), including ACTs conducted in Sub-Saharan Africa, between 1960 and 2012. Individual patient data from these studies were shared with the WorldWide Antimalarial Resistance Network (WWARN) and pooled using an a priori statistical analytical plan. Factors affecting early parasitological response were investigated using logistic regression with study sites fitted as a random effect. The risk of bias in included studies was evaluated based on study design, methodology and missing data. RESULTS: In total, 29,493 patients from 84 clinical trials were included in the analysis, treated with artemether-lumefantrine (n = 13,664), artesunate-amodiaquine (n = 11,337) and dihydroartemisinin-piperaquine (n = 4,492). The overall parasite clearance rate was rapid. The parasite positivity rate (PPR) decreased from 59.7 % (95 % CI: 54.5-64.9) on day 1 to 6.7 % (95 % CI: 4.8-8.7) on day 2 and 0.9 % (95 % CI: 0.5-1.2) on day 3. The 95th percentile of observed day 3 PPR was 5.3 %. Independent risk factors predictive of day 3 positivity were: high baseline parasitaemia (adjusted odds ratio (AOR) = 1.16 (95 % CI: 1.08-1.25); per 2-fold increase in parasite density, P <0.001); fever (>37.5 °C) (AOR = 1.50 (95 % CI: 1.06-2.13), P = 0.022); severe anaemia (AOR = 2.04 (95 % CI: 1.21-3.44), P = 0.008); areas of low/moderate transmission setting (AOR = 2.71 (95 % CI: 1.38-5.36), P = 0.004); and treatment with the loose formulation of artesunate-amodiaquine (AOR = 2.27 (95 % CI: 1.14-4.51), P = 0.020, compared to dihydroartemisinin-piperaquine). CONCLUSIONS: The three ACTs assessed in this analysis continue to achieve rapid early parasitological clearance across the sites assessed in Sub-Saharan Africa. A threshold of 5 % day 3 parasite positivity from a minimum sample size of 50 patients provides a more sensitive benchmark in Sub-Saharan Africa compared to the current recommended threshold of 10 % to trigger further investigation of artemisinin susceptibility.

Ndungu FM, Marsh K, Fegan G, Wambua J, Nyangweso G, Ogada E, Mwangi T, Nyundo C, Macharia A, Uyoga S et al. 2015. Identifying children with excess malaria episodes after adjusting for variation in exposure: identification from a longitudinal study using statistical count models. BMC Med, 13 (1), pp. 183. | Show Abstract | Read more

BACKGROUND: The distribution of Plasmodium falciparum clinical malaria episodes is over-dispersed among children in endemic areas, with more children experiencing multiple clinical episodes than would be expected based on a Poisson distribution. There is consistent evidence for micro-epidemiological variation in exposure to P. falciparum. The aim of the current study was to identify children with excess malaria episodes after controlling for malaria exposure. METHODS: We selected the model that best fit the data out of the models examined and included the following covariates: age, a weighted local prevalence of infection as an index of exposure, and calendar time to predict episodes of malaria on active surveillance malaria data from 2,463 children of under 15 years of age followed for between 5 and 15 years each. Using parameters from the zero-inflated negative binomial model which best fitted our data, we ran 100 simulations of the model based on our population to determine the variation that might be seen due to chance. RESULTS: We identified 212 out of 2,463 children who had a number of clinical episodes above the 95(th) percentile of the simulations run from the model, hereafter referred to as "excess malaria (EM)". We then identified exposure-matched controls with "average numbers of malaria" episodes, and found that the EM group had higher parasite densities when asymptomatically infected or during clinical malaria, and were less likely to be of haemoglobin AS genotype. CONCLUSIONS: Of the models tested, the negative zero-inflated negative binomial distribution with exposure, calendar year, and age acting as independent predictors, fitted the distribution of clinical malaria the best. Despite accounting for these factors, a group of children suffer excess malaria episodes beyond those predicted by the model. An epidemiological framework for identifying these children will allow us to study factors that may explain excess malaria episodes.

Tanner M, Greenwood B, Whitty CJ, Ansah EK, Price RN, Dondorp AM, von Seidlein L, Baird JK, Beeson JG, Fowkes FJ et al. 2015. Malaria eradication and elimination: views on how to translate a vision into reality. BMC Med, 13 (1), pp. 167. | Show Abstract | Read more

Although global efforts in the past decade have halved the number of deaths due to malaria, there are still an estimated 219 million cases of malaria a year, causing more than half a million deaths. In this forum article, we asked experts working in malaria research and control to discuss the ways in which malaria might eventually be eradicated. Their collective views highlight the challenges and opportunities, and explain how multi-factorial and integrated processes could eventually make malaria eradication a reality.

Atkinson SH, Uyoga SM, Armitage AE, Khandwala S, Mugyenyi CK, Bejon P, Marsh K, Beeson JG, Prentice AM, Drakesmith H, Williams TN. 2015. Malaria and Age Variably but Critically Control Hepcidin Throughout Childhood in Kenya. EBioMedicine, 2 (10), pp. 1478-1486. | Show Abstract | Read more

Both iron deficiency (ID) and malaria are common among African children. Studies show that the iron-regulatory hormone hepcidin is induced by malaria, but few studies have investigated this relationship longitudinally. We measured hepcidin concentrations, markers of iron status, and antibodies to malaria antigens during two cross-sectional surveys within a cohort of 324 Kenyan children ≤ 8 years old who were under intensive surveillance for malaria and other febrile illnesses. Hepcidin concentrations were the highest in the youngest, and female infants, declined rapidly in infancy and more gradually thereafter. Asymptomatic malaria and malaria antibody titres were positively associated with hepcidin concentrations. Recent episodes of febrile malaria were associated with high hepcidin concentrations that fell over time. Hepcidin concentrations were not associated with the subsequent risk of either malaria or other febrile illnesses. Given that iron absorption is impaired by hepcidin, our data suggest that asymptomatic and febrile malaria contribute to the high burden of ID seen in African children. Further, the effectiveness of iron supplementation may be sub-optimal in the presence of asymptomatic malaria. Thus, strategies to prevent and eliminate malaria may have the added benefit of addressing an important cause of ID for African children.

Hodgson SH, Juma E, Salim A, Magiri C, Njenga D, Molyneux S, Njuguna P, Awuondo K, Lowe B, Billingsley PF et al. 2015. Lessons learnt from the first controlled human malaria infection study conducted in Nairobi, Kenya. Malar J, 14 (1), pp. 182. | Show Abstract | Read more

BACKGROUND: Controlled human malaria infection (CHMI) studies, in which healthy volunteers are infected with Plasmodium falciparum to assess the efficacy of novel malaria vaccines and drugs, have become a vital tool to accelerate vaccine and drug development. CHMI studies provide a cost-effective and expeditious way to circumvent the use of large-scale field efficacy studies to deselect intervention candidates. However, to date few modern CHMI studies have been performed in malaria-endemic countries. METHODS: An open-label, randomized pilot CHMI study was conducted using aseptic, purified, cryopreserved, infectious P. falciparum sporozoites (SPZ) (Sanaria® PfSPZ Challenge) administered intramuscularly (IM) to healthy Kenyan adults (n = 28) with varying degrees of prior exposure to P. falciparum. The purpose of the study was to establish the PfSPZ Challenge CHMI model in a Kenyan setting with the aim of increasing the international capacity for efficacy testing of malaria vaccines and drugs, and allowing earlier assessment of efficacy in a population for which interventions are being developed. This was part of the EDCTP-funded capacity development of the CHMI platform in Africa. DISCUSSION: This paper discusses in detail lessons learnt from conducting the first CHMI study in Kenya. Issues pertinent to the African setting, including community sensitization, consent and recruitment are considered. Detailed reasoning regarding the study design (for example, dose and route of administration of PfSPZ Challenge, criteria for grouping volunteers according to prior exposure to malaria and duration of follow-up post CHMI) are given and changes other centres may want to consider for future studies are suggested. CONCLUSIONS: Performing CHMI studies in an African setting presents unique but surmountable challenges and offers great opportunity for acceleration of malaria vaccine and drug development. The reflections in this paper aim to aid other centres and partners intending to use the CHMI model in Africa.

Worldwide Antimalarial Resistance Network (WWARN) AL Dose Impact Study Group. 2015. The effect of dose on the antimalarial efficacy of artemether-lumefantrine: a systematic review and pooled analysis of individual patient data. Lancet Infect Dis, 15 (6), pp. 692-702. | Show Abstract | Read more

BACKGROUND: Artemether-lumefantrine is the most widely used artemisinin-based combination therapy for malaria, although treatment failures occur in some regions. We investigated the effect of dosing strategy on efficacy in a pooled analysis from trials done in a wide range of malaria-endemic settings. METHODS: We searched PubMed for clinical trials that enrolled and treated patients with artemether-lumefantrine and were published from 1960 to December, 2012. We merged individual patient data from these trials by use of standardised methods. The primary endpoint was the PCR-adjusted risk of Plasmodium falciparum recrudescence by day 28. Secondary endpoints consisted of the PCR-adjusted risk of P falciparum recurrence by day 42, PCR-unadjusted risk of P falciparum recurrence by day 42, early parasite clearance, and gametocyte carriage. Risk factors for PCR-adjusted recrudescence were identified using Cox's regression model with frailty shared across the study sites. FINDINGS: We included 61 studies done between January, 1998, and December, 2012, and included 14,327 patients in our analyses. The PCR-adjusted therapeutic efficacy was 97·6% (95% CI 97·4-97·9) at day 28 and 96·0% (95·6-96·5) at day 42. After controlling for age and parasitaemia, patients prescribed a higher dose of artemether had a lower risk of having parasitaemia on day 1 (adjusted odds ratio [OR] 0·92, 95% CI 0·86-0·99 for every 1 mg/kg increase in daily artemether dose; p=0·024), but not on day 2 (p=0·69) or day 3 (0·087). In Asia, children weighing 10-15 kg who received a total lumefantrine dose less than 60 mg/kg had the lowest PCR-adjusted efficacy (91·7%, 95% CI 86·5-96·9). In Africa, the risk of treatment failure was greatest in malnourished children aged 1-3 years (PCR-adjusted efficacy 94·3%, 95% CI 92·3-96·3). A higher artemether dose was associated with a lower gametocyte presence within 14 days of treatment (adjusted OR 0·92, 95% CI 0·85-0·99; p=0·037 for every 1 mg/kg increase in total artemether dose). INTERPRETATION: The recommended dose of artemether-lumefantrine provides reliable efficacy in most patients with uncomplicated malaria. However, therapeutic efficacy was lowest in young children from Asia and young underweight children from Africa; a higher dose regimen should be assessed in these groups. FUNDING: Bill & Melinda Gates Foundation.

Rono J, Färnert A, Murungi L, Ojal J, Kamuyu G, Guleid F, Nyangweso G, Wambua J, Kitsao B, Olotu A et al. 2015. Multiple clinical episodes of Plasmodium falciparum malaria in a low transmission intensity setting: exposure versus immunity. BMC Med, 13 (1), pp. 114. | Show Abstract | Read more

BACKGROUND: Epidemiological studies indicate that some children experience many more episodes of clinical malaria than their age mates in a given location. Whether this is as a result of the micro-heterogeneity of malaria transmission with some children effectively getting more exposure to infectious mosquitoes than others, or reflects a failure in the acquisition of immunity needs to be elucidated. Here, we investigated the determinants of increased susceptibility to clinical malaria by comparing the intensity of exposure to Plasmodium falciparum and the acquisition of immunity in children at the extreme ends of the over-dispersed distribution of the incidence of clinical malaria. METHODS: The study was nested within a larger cohort in an area where the intensity of malaria transmission was low. We identified children who over a five-year period experienced 5 to 16 clinical malaria episodes (children at the tail-end of the over-dispersed distribution, n = 35), remained malaria-free (n = 12) or had a single episode (n = 26). We quantified antibodies against seven Plasmodium falciparum merozoite antigens in plasma obtained at six cross-sectional surveys spanning these five years. We analyzed the antibody responses to identify temporal dynamics that associate with disease susceptibility. RESULTS: Children experiencing multiple episodes of malaria were more likely to be parasite positive by microscopy at cross-sectional surveys (X (2) test for trend 14.72 P = 0.001) and had a significantly higher malaria exposure index, than those in the malaria-free or single episode groups (Kruskal-Wallis test P = 0.009). In contrast, the five-year temporal dynamics of anti-merozoite antibodies were similar in the three groups. Importantly in all groups, antibody levels were below the threshold concentrations previously observed to be correlated with protective immunity. CONCLUSIONS: We conclude that in the context of a low malaria transmission setting, susceptibility to clinical malaria is not accounted for by anti-merozoite antibodies but appears to be a consequence of increased parasite exposure. We hypothesize that intensive exposure is a prerequisite for protective antibody concentrations, while little to modest exposure may manifest as multiple clinical infections with low levels of antibodies. These findings have implications for interventions that effectively lower malaria transmission intensity.

Ogwang C, Kimani D, Edwards NJ, Roberts R, Mwacharo J, Bowyer G, Bliss C, Hodgson SH, Njuguna P, Viebig NK et al. 2015. Prime-boost vaccination with chimpanzee adenovirus and modified vaccinia Ankara encoding TRAP provides partial protection against Plasmodium falciparum infection in Kenyan adults. Sci Transl Med, 7 (286), pp. 286re5. | Show Abstract | Read more

Protective immunity to the liver stage of the malaria parasite can be conferred by vaccine-induced T cells, but no subunit vaccination approach based on cellular immunity has shown efficacy in field studies. We randomly allocated 121 healthy adult male volunteers in Kilifi, Kenya, to vaccination with the recombinant viral vectors chimpanzee adenovirus 63 (ChAd63) and modified vaccinia Ankara (MVA), both encoding the malaria peptide sequence ME-TRAP (the multiple epitope string and thrombospondin-related adhesion protein), or to vaccination with rabies vaccine as a control. We gave antimalarials to clear parasitemia and conducted PCR (polymerase chain reaction) analysis on blood samples three times a week to identify infection with the malaria parasite Plasmodium falciparum. On Cox regression, vaccination reduced the risk of infection by 67% [95% confidence interval (CI), 33 to 83%; P = 0.002] during 8 weeks of monitoring. T cell responses to TRAP peptides 21 to 30 were significantly associated with protection (hazard ratio, 0.24; 95% CI, 0.08 to 0.75; P = 0.016).

Boyle MJ, Reiling L, Feng G, Langer C, Osier FH, Aspeling-Jones H, Cheng YS, Stubbs J, Tetteh KK, Conway DJ et al. 2015. Human antibodies fix complement to inhibit Plasmodium falciparum invasion of erythrocytes and are associated with protection against malaria. Immunity, 42 (3), pp. 580-590. | Show Abstract | Read more

Antibodies play major roles in immunity to malaria; however, a limited understanding of mechanisms mediating protection is a major barrier to vaccine development. We have demonstrated that acquired human anti-malarial antibodies promote complement deposition on the merozoite to mediate inhibition of erythrocyte invasion through C1q fixation and activation of the classical complement pathway. Antibody-mediated complement-dependent (Ab-C') inhibition was the predominant invasion-inhibitory activity of human antibodies; most antibodies were non-inhibitory without complement. Inhibitory activity was mediated predominately via C1q fixation, and merozoite surface proteins 1 and 2 were identified as major targets. Complement fixation by antibodies was very strongly associated with protection from both clinical malaria and high-density parasitemia in a prospective longitudinal study of children. Ab-C' inhibitory activity could be induced by human immunization with a candidate merozoite surface-protein vaccine. Our findings demonstrate that human anti-malarial antibodies have evolved to function by fixing complement for potent invasion-inhibitory activity and protective immunity.

Hodgson SH, Douglas AD, Edwards NJ, Kimani D, Elias SC, Chang M, Daza G, Seilie AM, Magiri C, Muia A et al. 2015. Increased sample volume and use of quantitative reverse-transcription PCR can improve prediction of liver-to-blood inoculum size in controlled human malaria infection studies. Malar J, 14 (1), pp. 33. | Show Abstract | Read more

BACKGROUND: Controlled human malaria infection (CHMI) studies increasingly rely on nucleic acid test (NAT) methods to detect and quantify parasites in the blood of infected participants. The lower limits of detection and quantification vary amongst the assays used throughout the world, which may affect the ability of mathematical models to accurately estimate the liver-to-blood inoculum (LBI) values that are used to judge the efficacy of pre-erythrocytic vaccine and drug candidates. METHODS: Samples were collected around the time of onset of pre-patent parasitaemia from subjects who enrolled in two different CHMI clinical trials. Blood samples were tested for Plasmodium falciparum 18S rRNA and/or rDNA targets by different NAT methods and results were compared. Methods included an ultrasensitive, large volume modification of an established quantitative reverse transcription PCR (qRT-PCR) assay that achieves detection of as little as one parasite/mL of whole blood. RESULTS: Large volume qRT-PCR at the University of Washington was the most sensitive test and generated quantifiable data more often than any other NAT methodology. Standard quantitative PCR (qPCR) performed at the University of Oxford and standard volume qRT-PCR performed at the University of Washington were less sensitive than the large volume qRT-PCR, especially at 6.5 days after CHMI. In these trials, the proportion of participants for whom LBI could be accurately quantified using parasite density value greater than or equal to the lower limit of quantification was increased. A greater improvement would be expected in trials in which numerous subjects receive a lower LBI or low dose challenge. CONCLUSIONS: Standard qPCR and qRT-PCR methods with analytical sensitivities of ~20 parasites/mL probably suffice for most CHMI purposes, but the newly developed large volume qRT-PCR may be able to answer specific questions when more analytical sensitivity is required.

White MT, Verity R, Griffin JT, Asante KP, Owusu-Agyei S, Greenwood B, Drakeley C, Gesase S, Lusingu J, Ansong D et al. 2015. Immunogenicity of the RTS,S/AS01 malaria vaccine and implications for duration of vaccine efficacy: secondary analysis of data from a phase 3 randomised controlled trial. Lancet Infect Dis, 15 (12), pp. 1450-1458. | Show Abstract | Read more

BACKGROUND: The RTS,S/AS01 malaria vaccine targets the circumsporozoite protein, inducing antibodies associated with the prevention of Plasmodium falciparum infection. We assessed the association between anti-circumsporozoite antibody titres and the magnitude and duration of vaccine efficacy using data from a phase 3 trial done between 2009 and 2014. METHODS: Using data from 8922 African children aged 5-17 months and 6537 African infants aged 6-12 weeks at first vaccination, we analysed the determinants of immunogenicity after RTS,S/AS01 vaccination with or without a booster dose. We assessed the association between the incidence of clinical malaria and anti-circumsporozoite antibody titres using a model of anti-circumsporozoite antibody dynamics and the natural acquisition of protective immunity over time. FINDINGS: RTS,S/AS01-induced anti-circumsporozoite antibody titres were greater in children aged 5-17 months than in those aged 6-12 weeks. Pre-vaccination anti-circumsporozoite titres were associated with lower immunogenicity in children aged 6-12 weeks and higher immunogenicity in those aged 5-17 months. The immunogenicity of the booster dose was strongly associated with immunogenicity after primary vaccination. Anti-circumsporozoite titres wane according to a biphasic exponential distribution. In participants aged 5-17 months, the half-life of the short-lived component of the antibody response was 45 days (95% credible interval 42-48) and that of the long-lived component was 591 days (557-632). After primary vaccination 12% (11-13) of the response was estimated to be long-lived, rising to 30% (28-32%) after a booster dose. An anti-circumsporozoite antibody titre of 121 EU/mL (98-153) was estimated to prevent 50% of infections. Waning anti-circumsporozoite antibody titres predict the duration of efficacy against clinical malaria across different age categories and transmission intensities, and efficacy wanes more rapidly at higher transmission intensity. INTERPRETATION: Anti-circumsporozoite antibody titres are a surrogate of protection for the magnitude and duration of RTS,S/AS01 efficacy, with or without a booster dose, providing a valuable surrogate of effectiveness for new RTS,S formulations in the age groups considered. FUNDING: UK Medical Research Council.

Shelton JM, Corran P, Risley P, Silva N, Hubbart C, Jeffreys A, Rowlands K, Craik R, Cornelius V, Hensmann M et al. 2015. Genetic determinants of anti-malarial acquired immunity in a large multi-centre study. Malar J, 14 (1), pp. 333. | Show Abstract | Read more

BACKGROUND: Many studies report associations between human genetic factors and immunity to malaria but few have been reliably replicated. These studies are usually country-specific, use small sample sizes and are not directly comparable due to differences in methodologies. This study brings together samples and data collected from multiple sites across Africa and Asia to use standardized methods to look for consistent genetic effects on anti-malarial antibody levels. METHODS: Sera, DNA samples and clinical data were collected from 13,299 individuals from ten sites in Senegal, Mali, Burkina Faso, Sudan, Kenya, Tanzania, and Sri Lanka using standardized methods. DNA was extracted and typed for 202 Single Nucleotide Polymorphisms with known associations to malaria or antibody production, and antibody levels to four clinical grade malarial antigens [AMA1, MSP1, MSP2, and (NANP)4] plus total IgE were measured by ELISA techniques. Regression models were used to investigate the associations of clinical and genetic factors with antibody levels. RESULTS: Malaria infection increased levels of antibodies to malaria antigens and, as expected, stable predictors of anti-malarial antibody levels included age, seasonality, location, and ethnicity. Correlations between antibodies to blood-stage antigens AMA1, MSP1 and MSP2 were higher between themselves than with antibodies to the (NANP)4 epitope of the pre-erythrocytic circumsporozoite protein, while there was little or no correlation with total IgE levels. Individuals with sickle cell trait had significantly lower antibody levels to all blood-stage antigens, and recessive homozygotes for CD36 (rs321198) had significantly lower anti-malarial antibody levels to MSP2. CONCLUSION: Although the most significant finding with a consistent effect across sites was for sickle cell trait, its effect is likely to be via reducing a microscopically positive parasitaemia rather than directly on antibody levels. However, this study does demonstrate a framework for the feasibility of combining data from sites with heterogeneous malaria transmission levels across Africa and Asia with which to explore genetic effects on anti-malarial immunity.

Ochola-Oyier LI, Okombo J, Mwai L, Kiara SM, Pole L, Tetteh KK, Nzila A, Marsh K. 2015. The MSPDBL2 codon 591 polymorphism is associated with lumefantrine in vitro drug responses in Plasmodium falciparum isolates from Kilifi, Kenya. Antimicrob Agents Chemother, 59 (3), pp. 1770-1775. | Show Abstract | Read more

The mechanisms of drug resistance development in the Plasmodium falciparum parasite to lumefantrine (LUM), commonly used in combination with artemisinin, are still unclear. We assessed the polymorphisms of Pfmspdbl2 for associations with LUM activity in a Kenyan population. MSPDBL2 codon 591S was associated with reduced susceptibility to LUM (P = 0.04). The high frequency of Pfmspdbl2 codon 591S in Kenya may be driven by the widespread use of lumefantrine in artemisinin combination therapy (Coartem).

Olotu A, Clement F, Jongert E, Vekemans J, Njuguna P, Ndungu FM, Marsh K, Leroux-Roels G, Bejon P. 2014. Avidity of anti-circumsporozoite antibodies following vaccination with RTS,S/AS01E in young children. PLoS One, 9 (12), pp. e115126. | Show Abstract | Read more

BACKGROUND: The nature of protective immune responses elicited by immunization with the candidate malaria vaccine RTS,S is still incompletely understood. Antibody levels correlate with protection against malaria infection, but considerable variation in outcome is unexplained (e.g., children may experience malaria despite high anti-circumsporozoite [CS] titers). METHODS AND FINDINGS: We measured the avidity index (AI) of the anti-CS antibodies raised in subgroup of 5-17 month old children in Kenya who were vaccinated with three doses of RTS,S/AS01E between March and August 2007. We evaluated the association between the AI and the subsequent risk of clinical malaria. We selected 19 cases (i.e., with clinical malaria) and 42 controls (i.e., without clinical malaria), matching for anti-CS antibody levels and malaria exposure. We assessed their sera collected 1 month after the third dose of the vaccine, in March 2008 (range 4-10 months after the third vaccine), and at 12 months after the third vaccine dose. The mean AI was 45.2 (95% CI: 42.4 to 48.1), 45.3 (95% CI: 41.4 to 49.1) and 46.2 (95% CI; 43.2 to 49.3) at 1 month, in March 2008 (4-10 months), and at 12 months after the third vaccination, respectively (p = 0.9 by ANOVA test for variation over time). The AI was not associated with protection from clinical malaria (OR = 0.90; 95% CI: 0.49 to 1.66; p = 0.74). The AI was higher in children with high malaria exposure, as measured using the weighted local prevalence of malaria, compared to those with low malaria exposure at 1 month post dose 3 (p = 0.035). CONCLUSION: Our data suggest that in RTS,S/AS01E-vaccinated children residing in malaria endemic countries, the avidity of anti-circumsporozoite antibodies, as measured using an elution ELISA method, was not associated with protection from clinical malaria. Prior natural malaria exposure might have primed the response to RTS,S/AS01E vaccination.

Okombo J, Kamau AW, Marsh K, Sutherland CJ, Ochola-Oyier LI. 2014. Temporal trends in prevalence of Plasmodium falciparum drug resistance alleles over two decades of changing antimalarial policy in coastal Kenya. Int J Parasitol Drugs Drug Resist, 4 (3), pp. 152-163. | Show Abstract | Read more

Molecular surveillance of drug resistance markers through time provides crucial information on genomic adaptations, especially in parasite populations exposed to changing drug pressures. To assess temporal trends of established genotypes associated with tolerance to clinically important antimalarials used in Kenya over the last two decades, we sequenced a region of the pfcrt locus encompassing codons 72-76 of the Plasmodium falciparum chloroquine resistance transporter, full-length pfmdr1 - encoding multi-drug resistance protein, P-glycoprotein homolog (Pgh1) and pfdhfr encoding dihydrofolate reductase, in 485 archived Plasmodium falciparum positive blood samples collected in coastal Kenya at four different time points between 1995 and 2013. Microsatellite loci were also analyzed to compare the genetic backgrounds of parasite populations circulating before and after the withdrawal of chloroquine and sulfadoxine/pyrimethamine. Our results reveal a significant increase in the prevalence of the pfcrt K76 wild-type allele between 1995 and 2013 from 38% to 81.7% (p < 0.0001). In contrast, we noted a significant decline in wild-type pfdhfr S108 allele (p < 0.0001) culminating in complete absence of this allele in 2013. We also observed a significant increase in the prevalence of the wild-type pfmdr1 N86/Y184/D1246 haplotype from 14.6% in 1995 to 66.0% in 2013 (p < 0.0001) and a corresponding decline of the mutant pfmdr1 86Y/184Y/1246Y allele from 36.4% to 0% in 19 years (p < 0.0001). We also show extensive genetic heterogeneity among the chloroquine-sensitive parasites before and after the withdrawal of the drug in contrast to a selective sweep around the triple mutant pfdhfr allele, leading to a mono-allelic population at this locus. These findings highlight the importance of continual surveillance and characterization of parasite genotypes as indicators of the therapeutic efficacy of antimalarials, particularly in the context of changes in malaria treatment policy.

Malaria Genomic Epidemiology Network, Malaria Genomic Epidemiology Network. 2014. Reappraisal of known malaria resistance loci in a large multicenter study. Nat Genet, 46 (11), pp. 1197-1204. | Show Abstract | Read more

Many human genetic associations with resistance to malaria have been reported, but few have been reliably replicated. We collected data on 11,890 cases of severe malaria due to Plasmodium falciparum and 17,441 controls from 12 locations in Africa, Asia and Oceania. We tested 55 SNPs in 27 loci previously reported to associate with severe malaria. There was evidence of association at P < 1 × 10(-4) with the HBB, ABO, ATP2B4, G6PD and CD40LG loci, but previously reported associations at 22 other loci did not replicate in the multicenter analysis. The large sample size made it possible to identify authentic genetic effects that are heterogeneous across populations or phenotypes, with a striking example being the main African form of G6PD deficiency, which reduced the risk of cerebral malaria but increased the risk of severe malarial anemia. The finding that G6PD deficiency has opposing effects on different fatal complications of P. falciparum infection indicates that the evolutionary origins of this common human genetic disorder are more complex than previously supposed.

Terheggen U, Drew DR, Hodder AN, Cross NJ, Mugyenyi CK, Barry AE, Anders RF, Dutta S, Osier FH, Elliott SR et al. 2014. Limited antigenic diversity of Plasmodium falciparum apical membrane antigen 1 supports the development of effective multi-allele vaccines. BMC Med, 12 (1), pp. 183. | Show Abstract | Read more

BACKGROUND: Polymorphism in antigens is a common mechanism for immune evasion used by many important pathogens, and presents major challenges in vaccine development. In malaria, many key immune targets and vaccine candidates show substantial polymorphism. However, knowledge on antigenic diversity of key antigens, the impact of polymorphism on potential vaccine escape, and how sequence polymorphism relates to antigenic differences is very limited, yet crucial for vaccine development. Plasmodium falciparum apical membrane antigen 1 (AMA1) is an important target of naturally-acquired antibodies in malaria immunity and a leading vaccine candidate. However, AMA1 has extensive allelic diversity with more than 60 polymorphic amino acid residues and more than 200 haplotypes in a single population. Therefore, AMA1 serves as an excellent model to assess antigenic diversity in malaria vaccine antigens and the feasibility of multi-allele vaccine approaches. While most previous research has focused on sequence diversity and antibody responses in laboratory animals, little has been done on the cross-reactivity of human antibodies. METHODS: We aimed to determine the extent of antigenic diversity of AMA1, defined by reactivity with human antibodies, and to aid the identification of specific alleles for potential inclusion in a multi-allele vaccine. We developed an approach using a multiple-antigen-competition enzyme-linked immunosorbent assay (ELISA) to examine cross-reactivity of naturally-acquired antibodies in Papua New Guinea and Kenya, and related this to differences in AMA1 sequence. RESULTS: We found that adults had greater cross-reactivity of antibodies than children, although the patterns of cross-reactivity to alleles were the same. Patterns of antibody cross-reactivity were very similar between populations (Papua New Guinea and Kenya), and over time. Further, our results show that antigenic diversity of AMA1 alleles is surprisingly restricted, despite extensive sequence polymorphism. Our findings suggest that a combination of three different alleles, if selected appropriately, may be sufficient to cover the majority of antigenic diversity in polymorphic AMA1 antigens. Antigenic properties were not strongly related to existing haplotype groupings based on sequence analysis. CONCLUSIONS: Antigenic diversity of AMA1 is limited and a vaccine including a small number of alleles might be sufficient for coverage against naturally-circulating strains, supporting a multi-allele approach for developing polymorphic antigens as malaria vaccines.

Warimwe GM, Fegan G, Kiragu EW, Musyoki JN, Macharia AW, Marsh K, Williams TN, Bull PC. 2014. An assessment of the impact of host polymorphisms on Plasmodium falciparum var gene expression patterns among Kenyan children. BMC Infect Dis, 14 (1), pp. 524. | Show Abstract | Read more

BACKGROUND: Host genotype accounts for a component of the variability in susceptibility to childhood Plasmodium falciparum malaria. However, despite numerous examples of host polymorphisms associated with tolerance or resistance to infection, direct evidence for an impact of host genetic polymorphisms on the in vivo parasite population is difficult to obtain. Parasite molecules whose expression is most likely to be associated with such adaptation are those that are directly involved in the host-parasite interaction. A prime candidate is the family of parasite var gene-encoded molecules on P. falciparum-infected erythrocytes, PfEMP1, which binds various host molecules and facilitates parasite sequestration in host tissues to avoid clearance by the spleen. METHODS: To assess the impact of host genotype on the infecting parasite population we used a published parasite var gene sequence dataset to compare var gene expression patterns between parasites from children with polymorphisms in molecules thought to interact with or modulate display of PfEMP1 on the infected erythrocyte surface: ABO blood group, haemoglobin S, alpha-thalassaemia, the T188G polymorphism of CD36 and the K29M polymorphism of ICAM1. RESULTS: Expression levels of 'group A-like' var genes, which encode a specific group of PfEMP1 variants previously associated with low host immunity and severe malaria, showed signs of elevation among children of blood group AB. No other host factor tested showed evidence for an association with var expression. CONCLUSIONS: Our preliminary findings suggest that host ABO blood group may have a measurable impact on the infecting parasite population. This needs to be verified in larger studies.

Osier FH, Mackinnon MJ, Crosnier C, Fegan G, Kamuyu G, Wanaguru M, Ogada E, McDade B, Rayner JC, Wright GJ, Marsh K. 2014. New antigens for a multicomponent blood-stage malaria vaccine. Sci Transl Med, 6 (247), pp. 247ra102. | Show Abstract | Read more

An effective blood-stage vaccine against Plasmodium falciparum remains a research priority, but the number of antigens that have been translated into multicomponent vaccines for testing in clinical trials remains limited. Investigating the large number of potential targets found in the parasite proteome has been constrained by an inability to produce natively folded recombinant antigens for immunological studies. We overcame these constraints by generating a large library of biochemically active merozoite surface and secreted full-length ectodomain proteins. We then systematically examined the antibody reactivity against these proteins in a cohort of Kenyan children (n = 286) who were sampled at the start of a malaria transmission season and prospectively monitored for clinical episodes of malaria over the ensuing 6 months. We found that antibodies to previously untested or little-studied proteins had superior or equivalent potential protective efficacy to the handful of current leading malaria vaccine candidates. Moreover, cumulative responses to combinations comprising 5 of the 10 top-ranked antigens, including PF3D7_1136200, MSP2, RhopH3, P41, MSP11, MSP3, PF3D7_0606800, AMA1, Pf113, and MSRP1, were associated with 100% protection against clinical episodes of malaria. These data suggest not only that there are many more potential antigen candidates for the malaria vaccine development pipeline but also that effective vaccination may be achieved by combining a selection of these antigens.

Osier FH, Feng G, Boyle MJ, Langer C, Zhou J, Richards JS, McCallum FJ, Reiling L, Jaworowski A, Anders RF et al. 2014. Opsonic phagocytosis of Plasmodium falciparum merozoites: mechanism in human immunity and a correlate of protection against malaria. BMC Med, 12 (1), pp. 108. | Show Abstract | Read more

BACKGROUND: An understanding of the mechanisms mediating protective immunity against malaria in humans is currently lacking, but critically important to advance the development of highly efficacious vaccines. Antibodies play a key role in acquired immunity, but the functional basis for their protective effect remains unclear. Furthermore, there is a strong need for immune correlates of protection against malaria to guide vaccine development. METHODS: Using a validated assay to measure opsonic phagocytosis of Plasmodium falciparum merozoites, we investigated the potential role of this functional activity in human immunity against clinical episodes of malaria in two independent cohorts (n = 109 and n = 287) experiencing differing levels of malaria transmission and evaluated its potential as a correlate of protection. RESULTS: Antibodies promoting opsonic phagocytosis of merozoites were cytophilic immunoglobulins (IgG1 and IgG3), induced monocyte activation and production of pro-inflammatory cytokines, and were directed against major merozoite surface proteins (MSPs). Consistent with protective immunity in humans, opsonizing antibodies were acquired with increasing age and malaria exposure, were boosted on re-infection, and levels were related to malaria transmission intensity. Opsonic phagocytosis was strongly associated with a reduced risk of clinical malaria in longitudinal studies in children with current or recent infections. In contrast, antibodies to the merozoite surface in standard immunoassays, or growth-inhibitory antibodies, were not significantly associated with protection. In multivariate analyses including several antibody responses, opsonic phagocytosis remained significantly associated with protection against malaria, highlighting its potential as a correlate of immunity. Furthermore, we demonstrate that human antibodies against MSP2 and MSP3 that are strongly associated with protection in this population are effective in opsonic phagocytosis of merozoites, providing a functional link between these antigen-specific responses and protection for the first time. CONCLUSIONS: Opsonic phagocytosis of merozoites appears to be an important mechanism contributing to protective immunity in humans. The opsonic phagocytosis assay appears to be a strong correlate of protection against malaria, a valuable biomarker of immunity, and provides a much-needed new tool for assessing responses to blood-stage malaria vaccines and measuring immunity in populations.

Ocholla H, Preston MD, Mipando M, Jensen AT, Campino S, MacInnis B, Alcock D, Terlouw A, Zongo I, Oudraogo JB et al. 2014. Whole-genome scans provide evidence of adaptive evolution in Malawian Plasmodium falciparum isolates. J Infect Dis, 210 (12), pp. 1991-2000. | Show Abstract | Read more

BACKGROUND: Selection by host immunity and antimalarial drugs has driven extensive adaptive evolution in Plasmodium falciparum and continues to produce ever-changing landscapes of genetic variation. METHODS: We performed whole-genome sequencing of 69 P. falciparum isolates from Malawi and used population genetics approaches to investigate genetic diversity and population structure and identify loci under selection. RESULTS: High genetic diversity (π = 2.4 × 10(-4)), moderately high multiplicity of infection (2.7), and low linkage disequilibrium (500-bp) were observed in Chikhwawa District, Malawi, an area of high malaria transmission. Allele frequency-based tests provided evidence of recent population growth in Malawi and detected potential targets of host immunity and candidate vaccine antigens. Comparison of the sequence variation between isolates from Malawi and those from 5 geographically dispersed countries (Kenya, Burkina Faso, Mali, Cambodia, and Thailand) detected population genetic differences between Africa and Asia, within Southeast Asia, and within Africa. Haplotype-based tests of selection to sequence data from all 6 populations identified signals of directional selection at known drug-resistance loci, including pfcrt, pfdhps, pfmdr1, and pfgch1. CONCLUSIONS: The sequence variations observed at drug-resistance loci reflect differences in each country's historical use of antimalarial drugs and may be useful in formulating local malaria treatment guidelines.

Bejon P, Williams TN, Nyundo C, Hay SI, Benz D, Gething PW, Otiende M, Peshu J, Bashraheil M, Greenhouse B et al. 2014. A micro-epidemiological analysis of febrile malaria in Coastal Kenya showing hotspots within hotspots. Elife, 3 pp. e02130. | Show Abstract | Read more

Malaria transmission is spatially heterogeneous. This reduces the efficacy of control strategies, but focusing control strategies on clusters or 'hotspots' of transmission may be highly effective. Among 1500 homesteads in coastal Kenya we calculated (a) the fraction of febrile children with positive malaria smears per homestead, and (b) the mean age of children with malaria per homestead. These two measures were inversely correlated, indicating that children in homesteads at higher transmission acquire immunity more rapidly. This inverse correlation increased gradually with increasing spatial scale of analysis, and hotspots of febrile malaria were identified at every scale. We found hotspots within hotspots, down to the level of an individual homestead. Febrile malaria hotspots were temporally unstable, but 4 km radius hotspots could be targeted for 1 month following 1 month periods of surveillance.DOI: http://dx.doi.org/10.7554/eLife.02130.001.

Abdi AI, Fegan G, Muthui M, Kiragu E, Musyoki JN, Opiyo M, Marsh K, Warimwe GM, Bull PC. 2014. Plasmodium falciparum antigenic variation: relationships between widespread endothelial activation, parasite PfEMP1 expression and severe malaria. BMC Infect Dis, 14 (1), pp. 170. | Show Abstract | Read more

BACKGROUND: Plasmodium falciparum erythrocyte membrane protein 1(PfEMP1) is a family of variant surface antigens (VSA) that mediate the adhesion of parasite infected erythrocytes to capillary endothelial cells within host tissues. Opinion is divided over the role of PfEMP1 in the widespread endothelial activation associated with severe malaria. In a previous study we found evidence for differential associations between defined VSA subsets and specific syndromes of severe malaria: group A-like PfEMP1 expression and the "rosetting" phenotype were associated with impaired consciousness and respiratory distress, respectively. This study explores the involvement of widespread endothelial activation in these associations. METHODS: We used plasma angiopoietin-2 as a marker of widespread endothelial activation. Using logistic regression analysis, we explored the relationships between plasma angiopoietin-2 levels, parasite VSA expression and the two syndromes of severe malaria, impaired consciousness and respiratory distress. RESULTS: Plasma angiopoietin-2 was associated with both syndromes. The rosetting phenotype did not show an independent association with respiratory distress when adjusted for angiopoietin-2, consistent with a single pathogenic mechanism involving widespread endothelial activation. In contrast, group A-like PfEMP1 expression and angiopoietin-2 maintained independent associations with impaired consciousness when adjusted for each other. CONCLUSION: The results are consistent with multiple pathogenic mechanisms leading to severe malaria and heterogeneity in the pathophysiology of impaired consciousness. The observed association between group A-like PfEMP1 and impaired consciousness does not appear to involve widespread endothelial activation.

Gitau EN, Tuju J, Karanja H, Stevenson L, Requena P, Kimani E, Olotu A, Kimani D, Marsh K, Bull P, Urban BC. 2014. CD4+ T cell responses to the Plasmodium falciparum erythrocyte membrane protein 1 in children with mild malaria. J Immunol, 192 (4), pp. 1753-1761. | Show Abstract | Read more

The immune response against the variant surface Ag Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a key component of clinical immunity against malaria. We have investigated the development and maintenance of CD4(+) T cell responses to a small semiconserved area of the Duffy binding-like domain (DBL)α-domain of PfEMP1, the DBLα-tag. Young children were followed up longitudinally, and parasites and PBMCs were isolated from 35 patients presenting with an acute case of uncomplicated malaria. The DBLα-tag from the PfEMP1 dominantly expressed by the homologous parasite isolate was cloned and expressed as recombinant protein. The recombinant DBLα-tag was used to activate PBMCs collected from each acute episode and from an annual cross-sectional survey performed after the acute malaria episode. In this article, we report that CD4(+) T cell responses to the homologous DBLα-tag were induced in 75% of the children at the time of the acute episode and in 62% of the children at the following cross-sectional survey on average 235 d later. Furthermore, children who had induced DBLα-tag-specific CD4(+)IL-4(+) T cells at the acute episode remained episode free for longer than children who induced other types of CD4(+) T cell responses. These results suggest that a wide range of DBLα-tag-specific CD4(+) T cell responses were induced in children with mild malaria and, in the case of CD4(+)IL-4(+) T cell responses, were associated with protection from clinical episodes.

Hodgson SH, Juma E, Salim A, Magiri C, Kimani D, Njenga D, Muia A, Cole AO, Ogwang C, Awuondo K et al. 2014. Evaluating controlled human malaria infection in Kenyan adults with varying degrees of prior exposure to Plasmodium falciparum using sporozoites administered by intramuscular injection. Front Microbiol, 5 (DEC), pp. 686. | Show Abstract | Read more

BACKGROUND: Controlled human malaria infection (CHMI) studies are a vital tool to accelerate vaccine and drug development. As CHMI trials are performed in a controlled environment, they allow unprecedented, detailed evaluation of parasite growth dynamics (PGD) and immunological responses. However, CHMI studies have not been routinely performed in malaria-endemic countries or used to investigate mechanisms of naturally-acquired immunity (NAI) to Plasmodium falciparum. METHODS: We conducted an open-label, randomized CHMI pilot-study using aseptic, cryopreserved P. falciparum sporozoites (PfSPZ Challenge) to evaluate safety, infectivity and PGD in Kenyan adults with low to moderate prior exposure to P. falciparum (Pan African Clinical Trial Registry: PACTR20121100033272). RESULTS: All participants developed blood-stage infection confirmed by quantitative polymerase chain reaction (qPCR). However one volunteer (110) remained asymptomatic and blood-film negative until day 21 post-injection of PfSPZ Challenge. This volunteer had a reduced parasite multiplication rate (PMR) (1.3) in comparison to the other 27 volunteers (median 11.1). A significant correlation was seen between PMR and screening anti-schizont Enzyme Linked Immunosorbent Assays (ELISA) OD (p = 0.044, R = -0.384) but not when volunteer 110 was excluded from the analysis (p = 0.112, R = -0.313). CONCLUSIONS: PfSPZ Challenge is safe and infectious in malaria-endemic populations and could be used to assess the efficacy of malaria vaccines and drugs in African populations. Whilst our findings are limited by sample size, our pilot study has demonstrated for the first time that NAI may impact on PMR post-CHMI in a detectable fashion, an important finding that should be evaluated in further CHMI studies.

Kangoye DT, Nebie I, Yaro JB, Debe S, Traore S, Ouedraogo O, Sanou G, Soulama I, Diarra A, Tiono A et al. 2014. Plasmodium falciparum malaria in children aged 0-2 years: the role of foetal haemoglobin and maternal antibodies to two asexual malaria vaccine candidates (MSP3 and GLURP). PLoS One, 9 (9), pp. e107965. | Show Abstract | Read more

BACKGROUND: Children below six months are reported to be less susceptible to clinical malaria. Maternally derived antibodies and foetal haemoglobin are important putative protective factors. We examined antibodies to Plasmodium falciparum merozoite surface protein 3 (MSP3) and glutamate-rich protein (GLURP), in children in their first two years of life in Burkina Faso and their risk of malaria. METHODS: A cohort of 140 infants aged between four and six weeks was recruited in a stable transmission area of south-western Burkina Faso and monitored for 24 months by active and passive surveillance. Malaria infections were detected by examining blood smears using light microscopy. Enzyme-linked immunosorbent assay was used to quantify total Immunoglobulin G to Plasmodium falciparum antigens MSP3 and two regions of GLURP (R0 and R2) on blood samples collected at baseline, three, six, nine, 12, 18 and 24 months. Foetal haemoglobin and variant haemoglobin fractions were measured at the baseline visit using high pressure liquid chromatography. RESULTS: A total of 79.6% of children experienced one or more episodes of febrile malaria during monitoring. Antibody titres to MSP3 were prospectively associated with an increased risk of malaria while antibody responses to GLURP (R0 and R2) did not alter the risk. Antibody titres to MSP3 were higher among children in areas of high malaria risk. Foetal haemoglobin was associated with delayed first episode of febrile malaria and haemoglobin CC type was associated with reduced incidence of febrile malaria. CONCLUSIONS: We did not find any evidence of association between titres of antibodies to MSP3, GLURP-R0 or GLURP-R2 as measured by enzyme-linked immunosorbent assay and early protection against malaria, although anti-MSP3 antibody titres may reflect increased exposure to malaria and therefore greater risk. Foetal haemoglobin was associated with protection against febrile malaria despite the study limitations and its role is therefore worthy further investigation.

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Agnandji ST, Lell B, Fernandes JF, Abossolo BP, Kabwende AL, Adegnika AA, Mordmueller B, Issifou S, Kremsner PG, Loembe MM et al. 2014. Efficacy and Safety of the RTS,S/AS01 Malaria Vaccine during 18 Months after Vaccination: A Phase 3 Randomized, Controlled Trial in Children and Young Infants at 11 African Sites PLOS MEDICINE, 11 (7), pp. e1001685-e1001685. | Show Abstract | Read more

Background:A malaria vaccine could be an important addition to current control strategies. We report the safety and vaccine efficacy (VE) of the RTS,S/AS01 vaccine during 18 mo following vaccination at 11 African sites with varying malaria transmission.Methods and Findings:6,537 infants aged 6-12 wk and 8,923 children aged 5-17 mo were randomized to receive three doses of RTS,S/AS01 or comparator vaccine.VE against clinical malaria in children during the 18 mo after vaccine dose 3 (per protocol) was 46% (95% CI 42% to 50%) (range 40% to 77%; VE, p<0.01 across all sites). VE during the 20 mo after vaccine dose 1 (intention to treat [ITT]) was 45% (95% CI 41% to 49%). VE against severe malaria, malaria hospitalization, and all-cause hospitalization was 34% (95% CI 15% to 48%), 41% (95% CI 30% to 50%), and 19% (95% CI 11% to 27%), respectively (ITT).VE against clinical malaria in infants was 27% (95% CI 20% to 32%, per protocol; 27% [95% CI 21% to 33%], ITT), with no significant protection against severe malaria, malaria hospitalization, or all-cause hospitalization.Post-vaccination anti-circumsporozoite antibody geometric mean titer varied from 348 to 787 EU/ml across sites in children and from 117 to 335 EU/ml in infants (per protocol).VE waned over time in both age categories (Schoenfeld residuals p<0.001). The number of clinical and severe malaria cases averted per 1,000 children vaccinated ranged across sites from 37 to 2,365 and from -1 to 49, respectively; corresponding ranges among infants were -10 to 1,402 and -13 to 37, respectively (ITT). Meningitis was reported as a serious adverse event in 16/5,949 and 1/2,974 children and in 9/4,358 and 3/2,179 infants in the RTS,S/AS01 and control groups, respectively.Conclusions:RTS,S/AS01 prevented many cases of clinical and severe malaria over the 18 mo after vaccine dose 3, with the highest impact in areas with the greatest malaria incidence. VE was higher in children than in infants, but even at modest levels of VE, the number of malaria cases averted was substantial. RTS,S/AS01 could be an important addition to current malaria control in Africa.Trial registration:http://www.ClinicalTrials.gov NCT00866619. Please see later in the article for the Editors' Summary.

Wendler JP, Okombo J, Amato R, Miotto O, Kiara SM, Mwai L, Pole L, O'Brien J, Manske M, Alcock D et al. 2014. A genome wide association study of Plasmodium falciparum susceptibility to 22 antimalarial drugs in Kenya. PLoS One, 9 (5), pp. e96486. | Show Abstract | Read more

BACKGROUND: Drug resistance remains a chief concern for malaria control. In order to determine the genetic markers of drug resistant parasites, we tested the genome-wide associations (GWA) of sequence-based genotypes from 35 Kenyan P. falciparum parasites with the activities of 22 antimalarial drugs. METHODS AND PRINCIPAL FINDINGS: Parasites isolated from children with acute febrile malaria were adapted to culture, and sensitivity was determined by in vitro growth in the presence of anti-malarial drugs. Parasites were genotyped using whole genome sequencing techniques. Associations between 6250 single nucleotide polymorphisms (SNPs) and resistance to individual anti-malarial agents were determined, with false discovery rate adjustment for multiple hypothesis testing. We identified expected associations in the pfcrt region with chloroquine (CQ) activity, and other novel loci associated with amodiaquine, quinazoline, and quinine activities. Signals for CQ and primaquine (PQ) overlap in and around pfcrt, and interestingly the phenotypes are inversely related for these two drugs. We catalog the variation in dhfr, dhps, mdr1, nhe, and crt, including novel SNPs, and confirm the presence of a dhfr-164L quadruple mutant in coastal Kenya. Mutations implicated in sulfadoxine-pyrimethamine resistance are at or near fixation in this sample set. CONCLUSIONS/SIGNIFICANCE: Sequence-based GWA studies are powerful tools for phenotypic association tests. Using this approach on falciparum parasites from coastal Kenya we identified known and previously unreported genes associated with phenotypic resistance to anti-malarial drugs, and observe in high-resolution haplotype visualizations a possible signature of an inverse selective relationship between CQ and PQ.

Abdulla S, Alonso P, Binka F, Graves P, Greenwood B, Leke R, Malik E, Marsh K, Meek S, Mendis K et al. 2013. Malaria Policy Advisory Committee to the WHO: conclusions and recommendations of September 2013 meeting MALARIA JOURNAL, 12 (1), pp. 456-456. | Read more

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Achan J, Adam I, Arinaitwe E, Ashley EA, Awab GR, Ba MS, Barnes KI, Bassat Q, Borrmann S, Bousema T et al. 2013. The Effect of Dosing Regimens on the Antimalarial Efficacy of Dihydroartemisinin-Piperaquine: A Pooled Analysis of Individual Patient Data PLOS MEDICINE, 10 (12), pp. e1001564-e1001564. | Show Abstract | Read more

Background:Dihydroartemisinin-piperaquine (DP) is increasingly recommended for antimalarial treatment in many endemic countries; however, concerns have been raised over its potential under dosing in young children. We investigated the influence of different dosing schedules on DP's clinical efficacy.Methods and Findings:A systematic search of the literature was conducted to identify all studies published between 1960 and February 2013, in which patients were enrolled and treated with DP. Principal investigators were approached and invited to share individual patient data with the WorldWide Antimalarial Resistance Network (WWARN). Data were pooled using a standardised methodology. Univariable and multivariable risk factors for parasite recrudescence were identified using a Cox's regression model with shared frailty across the study sites. Twenty-four published and two unpublished studies (n = 7,072 patients) were included in the analysis. After correcting for reinfection by parasite genotyping, Kaplan-Meier survival estimates were 97.7% (95% CI 97.3%-98.1%) at day 42 and 97.2% (95% CI 96.7%-97.7%) at day 63. Overall 28.6% (979/3,429) of children aged 1 to 5 years received a total dose of piperaquine below 48 mg/kg (the lower limit recommended by WHO); this risk was 2.3-2.9-fold greater compared to that in the other age groups and was associated with reduced efficacy at day 63 (94.4% [95% CI 92.6%-96.2%], p<0.001). After adjusting for confounding factors, the mg/kg dose of piperaquine was found to be a significant predictor for recrudescence, the risk increasing by 13% (95% CI 5.0%-21%) for every 5 mg/kg decrease in dose; p = 0.002. In a multivariable model increasing the target minimum total dose of piperaquine in children aged 1 to 5 years old from 48 mg/kg to 59 mg/kg would halve the risk of treatment failure and cure at least 95% of patients; such an increment was not associated with gastrointestinal toxicity in the ten studies in which this could be assessed.Conclusions:DP demonstrates excellent efficacy in a wide range of transmission settings; however, treatment failure is associated with a lower dose of piperaquine, particularly in young children, suggesting potential for further dose optimisation.Please see later in the article for the Editors' Summary. © 2013 Price et al.

Crosnier C, Wanaguru M, McDade B, Osier FH, Marsh K, Rayner JC, Wright GJ. 2013. A library of functional recombinant cell-surface and secreted P. falciparum merozoite proteins. Mol Cell Proteomics, 12 (12), pp. 3976-3986. | Show Abstract | Read more

Malaria, an infectious disease caused by parasites of the Plasmodium genus, is one of the world's major public health concerns causing up to a million deaths annually, mostly because of P. falciparum infections. All of the clinical symptoms are associated with the blood stage of the disease, an obligate part of the parasite life cycle, when a form of the parasite called the merozoite recognizes and invades host erythrocytes. During erythrocyte invasion, merozoites are directly exposed to the host humoral immune system making the blood stage of the parasite a conceptually attractive therapeutic target. Progress in the functional and molecular characterization of P. falciparum merozoite proteins, however, has been hampered by the technical challenges associated with expressing these proteins in a biochemically active recombinant form. This challenge is particularly acute for extracellular proteins, which are the likely targets of host antibody responses, because they contain structurally critical post-translational modifications that are not added by some recombinant expression systems. Here, we report the development of a method that uses a mammalian expression system to compile a protein resource containing the entire ectodomains of 42 P. falciparum merozoite secreted and cell surface proteins, many of which have not previously been characterized. Importantly, we are able to recapitulate known biochemical activities by showing that recombinant MSP1-MSP7 and P12-P41 directly interact, and that both recombinant EBA175 and EBA140 can bind human erythrocytes in a sialic acid-dependent manner. Finally, we use sera from malaria-exposed immune adults to profile the relative immunoreactivity of the proteins and show that the majority of the antigens contain conformational (heat-labile) epitopes. We envisage that this resource of recombinant proteins will make a valuable contribution toward a molecular understanding of the blood stage of P. falciparum infections and facilitate the comparative screening of antigens as blood-stage vaccine candidates.

Rono J, Osier FH, Olsson D, Montgomery S, Mhoja L, Rooth I, Marsh K, Färnert A. 2013. Breadth of anti-merozoite antibody responses is associated with the genetic diversity of asymptomatic Plasmodium falciparum infections and protection against clinical malaria. Clin Infect Dis, 57 (10), pp. 1409-1416. | Show Abstract | Read more

BACKGROUND: Elucidating the mechanisms of naturally acquired immunity to Plasmodium falciparum infections would be highly valuable for malaria vaccine development. Asymptomatic multiclonal infections have been shown to predict protection from clinical malaria in a transmission-dependent manner, but the mechanisms underlying this are unclear. We assessed the breadth of antibody responses to several vaccine candidate merozoite antigens in relation to the infecting parasite population and clinical immunity. METHODS: In a cohort study in Tanzania, 320 children aged 1-16 years who were asymptomatic at baseline were included. We genotyped P. falciparum infections by targeting the msp2 gene using polymerase chain reaction and capillary electrophoresis and measured antibodies to 7 merozoite antigens using a multiplex assay. We assessed the correlation between the number of clones and the breadth of the antibody response, and examined their effects on the risk of malaria during 40 weeks of follow-up using age-adjusted multivariate regression models. RESULTS: The antibody breadth was positively correlated with the number of clones (RR [risk ratio], 1.63; 95% confidence interval [CI], 1.32-2.02). Multiclonal infections were associated with a nonsignificant reduction in the risk of malaria in the absence of antibodies (RR, 0.83; 95% CI, .29-2.34). The breadth of the antibody response was significantly associated with a reduced risk of malaria in the absence of infections (RR, 0.25; 95% CI, .09-.66). In combination, these factors were associated with a lower risk of malaria than they were individually (RR, 0.14; 95% CI, .04-.48). CONCLUSIONS: These data suggest that malaria vaccines mimicking naturally acquired immunity should ideally induce antibody responses that can be boosted by natural infections.

Lang T, Marsh K, Peeling R, Farrar J. 2013. Sharing methods for global health research: an assessment of methodology LANCET, 382 pp. 5-5.

Warimwe GM, Fletcher HA, Olotu A, Agnandji ST, Hill AV, Marsh K, Bejon P. 2013. Peripheral blood monocyte-to-lymphocyte ratio at study enrollment predicts efficacy of the RTS,S malaria vaccine: analysis of pooled phase II clinical trial data. BMC Med, 11 (1), pp. 184. | Show Abstract | Read more

BACKGROUND: RTS,S is the most advanced candidate malaria vaccine but it is only partially protective and the causes of inter-individual variation in efficacy are poorly understood. Here, we investigated whether peripheral blood monocyte-to-lymphocyte ratios (ML ratio), previously shown to correlate with clinical malaria risk, could account for differences in RTS,S efficacy among phase II trial participants in Africa. METHODS: Of 11 geographical sites where RTS,S has been evaluated, pre-vaccination ML ratios were only available for trial participants in Kilifi, Kenya (N = 421) and Lambarene, Gabon (N = 189). Using time to first clinical malaria episode as the primary endpoint we evaluated the effect of accounting for ML ratio on RTS,S vaccine efficacy against clinical malaria by Cox regression modeling. RESULTS: The unadjusted efficacy of RTS,S in this combined dataset was 47% (95% confidence interval (CI) 26% to 62%, P <0.001). However, RTS,S efficacy decreased with increasing ML ratio, ranging from 67% (95% CI 64% to 70%) at an ML ratio of 0.1 to 5% (95% CI -3% to 13%) at an ML ratio of 0.6. The statistical interaction between RTS,S vaccination and ML ratio was still evident after adjustment for covariates associated with clinical malaria risk in this dataset. CONCLUSION: The results suggest that stratification of study participants by ML ratio, easily measured from full differential blood counts before vaccination, might help identify children who are highly protected and those that are refractory to protection with the RTS,S vaccine. Identifying causes of low vaccine efficacy among individuals with high ML ratio could inform strategies to improve overall RTS,S vaccine efficacy. TRIAL REGISTRATION: ClinicalTrials.Gov numbers NCT00380393 and NCT00436007.

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Murungi LM, Kamuyu G, Lowe B, Bejon P, Theisen M, Kinyanjui SM, Marsh K, Osier FHA. 2013. A threshold concentration of anti-merozoite antibodies is required for protection from clinical episodes of malaria Vaccine, 31 (37), pp. 3936-3942. | Show Abstract | Read more

Antibodies to selected Plasmodium falciparum merozoite antigens are often reported to be associated with protection from malaria in one epidemiological cohort, but not in another. Here, we sought to understand this paradox by exploring the hypothesis that a threshold concentration of antibodies is necessary for protection. We analyzed data from two independent cohorts along the Kenyan coast, one in which antibodies to AMA1, MSP-2 and MSP-3 were associated with protection from malaria (Chonyi) and another in which this association was not observed (Junju). We used a malaria reference reagent to standardize antibody measurements across both cohorts, and applied statistical methods to derive the threshold concentration of antibodies against each antigen that best correlated with a reduced risk of malaria (the protective threshold), in the Chonyi cohort. We then tested whether antibodies in Junju reached the protective threshold concentrations observed in the Chonyi cohort. Except for children under 3 years, the age-matched proportions of children achieving protective threshold concentrations of antibodies against AMA1 and MSP-2 were significantly lower in Junju compared to Chonyi (Fishers exact test, P < 0.01). For MSP-3, this difference was significant only among 4-5 year olds. We conclude that although antibodies are commonly detected in malaria endemic populations, they may be present in concentrations that are insufficient for protection. Our results have implications for the analysis and interpretation of similar data from immuno-epidemiological studies. © 2013 The Authors.

Persson KE, Fowkes FJ, McCallum FJ, Gicheru N, Reiling L, Richards JS, Wilson DW, Lopaticki S, Cowman AF, Marsh K, Beeson JG. 2013. Erythrocyte-binding antigens of Plasmodium falciparum are targets of human inhibitory antibodies and function to evade naturally acquired immunity. J Immunol, 191 (2), pp. 785-794. | Show Abstract | Read more

Abs that inhibit Plasmodium falciparum invasion of erythrocytes form an important component of human immunity against malaria, but key target Ags are largely unknown. Phenotypic variation by P. falciparum mediates the evasion of inhibitory Abs, contributing to the capacity of P. falciparum to cause repeat and chronic infections. However, Ags involved in mediating immune evasion have not been defined, and studies of the function of human Abs are limited. In this study, we used novel approaches to determine the importance of P. falciparum erythrocyte-binding Ags (EBAs), which are important invasion ligands, as targets of human invasion-inhibitory Abs and define their role in contributing to immune evasion through variation in function. We evaluated the invasion-inhibitory activity of acquired Abs from malaria-exposed children and adults from Kenya, using P. falciparum with disruption of genes encoding EBA140, EBA175, and EBA181, either individually or combined as EBA140/EBA175 or EBA175/EBA181 double knockouts. Our findings provide important new evidence that variation in the expression and function of the EBAs plays an important role in evasion of acquired Abs and that a substantial amount of phenotypic diversity results from variation in expression of different EBAs that contributes to immune evasion by P. falciparum. All three EBAs were identified as important targets of naturally acquired inhibitory Abs demonstrated by differential inhibition of parental parasites greater than EBA knockout lines. This knowledge will help to advance malaria vaccine development and suggests that multiple invasion ligands need to be targeted to overcome the capacity of P. falciparum for immune evasion.

Mugyenyi CK, Elliott SR, McCallum FJ, Anders RF, Marsh K, Beeson JG. 2013. Antibodies to polymorphic invasion-inhibitory and non-Inhibitory epitopes of Plasmodium falciparum apical membrane antigen 1 in human malaria. PLoS One, 8 (7), pp. e68304. | Show Abstract | Read more

BACKGROUND: Antibodies to P. falciparum apical membrane protein 1 (AMA1) may contribute to protective immunity against clinical malaria by inhibiting blood stage growth of P. falciparum, and AMA1 is a leading malaria vaccine candidate. Currently, there is limited knowledge of the acquisition of strain-specific and cross-reactive antibodies to AMA1 in humans, or the acquisition of invasion-inhibitory antibodies to AMA1. METHODOLOGY/FINDINGS: We examined the acquisition of human antibodies to specific polymorphic invasion-inhibitory and non-inhibitory AMA1 epitopes, defined by the monoclonal antibodies 1F9 and 2C5, respectively. Naturally acquired antibodies were measured in cohorts of Kenyan children and adults. Antibodies to the invasion-inhibitory 1F9 epitope and non-inhibitory 2C5 epitope were measured indirectly by competition ELISA. Antibodies to the 1F9 and 2C5 epitopes were acquired by children and correlated with exposure, and higher antibody levels and prevalence were observed with increasing age and with active P. falciparum infection. Of note, the prevalence of antibodies to the inhibitory 1F9 epitope was lower than antibodies to AMA1 or the 2C5 epitope. Antibodies to AMA1 ectodomain, the 1F9 or 2C5 epitopes, or a combination of responses, showed some association with protection from P. falciparum malaria in a prospective longitudinal study. Furthermore, antibodies to the invasion-inhibitory 1F9 epitope were positively correlated with parasite growth-inhibitory activity of serum antibodies. CONCLUSIONS/SIGNIFICANCE: Individuals acquire antibodies to functional, polymorphic epitopes of AMA1 that may contribute to protective immunity, and these findings have implications for AMA1 vaccine development. Measuring antibodies to the 1F9 epitope by competition ELISA may be a valuable approach to assessing human antibodies with invasion-inhibitory activity in studies of acquired immunity and vaccine trials of AMA1.

Murungi LM, Kamuyu G, Lowe B, Bejon P, Theisen M, Kinyanjui SM, Marsh K, Osier FH. 2013. A threshold concentration of anti-merozoite antibodies is required for protection from clinical episodes of malaria. Vaccine, 31 (37), pp. 3936-3942. | Show Abstract | Read more

Antibodies to selected Plasmodium falciparum merozoite antigens are often reported to be associated with protection from malaria in one epidemiological cohort, but not in another. Here, we sought to understand this paradox by exploring the hypothesis that a threshold concentration of antibodies is necessary for protection. We analyzed data from two independent cohorts along the Kenyan coast, one in which antibodies to AMA1, MSP-2 and MSP-3 were associated with protection from malaria (Chonyi) and another in which this association was not observed (Junju). We used a malaria reference reagent to standardize antibody measurements across both cohorts, and applied statistical methods to derive the threshold concentration of antibodies against each antigen that best correlated with a reduced risk of malaria (the protective threshold), in the Chonyi cohort. We then tested whether antibodies in Junju reached the protective threshold concentrations observed in the Chonyi cohort. Except for children under 3 years, the age-matched proportions of children achieving protective threshold concentrations of antibodies against AMA1 and MSP-2 were significantly lower in Junju compared to Chonyi (Fishers exact test, P<0.01). For MSP-3, this difference was significant only among 4-5 year olds. We conclude that although antibodies are commonly detected in malaria endemic populations, they may be present in concentrations that are insufficient for protection. Our results have implications for the analysis and interpretation of similar data from immuno-epidemiological studies.

Abdulla S, Alonso P, Binka F, Graves P, Greenwood B, Leke R, Malik E, Marsh K, Meek S, Mendis K et al. 2013. Malaria Policy Advisory Committee to the WHO: conclusions and recommendations of March 2013 meeting MALARIA JOURNAL, 12 (1), pp. 213-213. | Read more

Band G, Le QS, Jostins L, Pirinen M, Kivinen K, Jallow M, Sisay-Joof F, Bojang K, Pinder M, Sirugo G et al. 2013. Imputation-based meta-analysis of severe malaria in three African populations. PLoS Genet, 9 (5), pp. e1003509. | Show Abstract | Read more

Combining data from genome-wide association studies (GWAS) conducted at different locations, using genotype imputation and fixed-effects meta-analysis, has been a powerful approach for dissecting complex disease genetics in populations of European ancestry. Here we investigate the feasibility of applying the same approach in Africa, where genetic diversity, both within and between populations, is far more extensive. We analyse genome-wide data from approximately 5,000 individuals with severe malaria and 7,000 population controls from three different locations in Africa. Our results show that the standard approach is well powered to detect known malaria susceptibility loci when sample sizes are large, and that modern methods for association analysis can control the potential confounding effects of population structure. We show that pattern of association around the haemoglobin S allele differs substantially across populations due to differences in haplotype structure. Motivated by these observations we consider new approaches to association analysis that might prove valuable for multicentre GWAS in Africa: we relax the assumptions of SNP-based fixed effect analysis; we apply Bayesian approaches to allow for heterogeneity in the effect of an allele on risk across studies; and we introduce a region-based test to allow for heterogeneity in the location of causal alleles.

Olotu A, Fegan G, Wambua J, Nyangweso G, Awuondo KO, Leach A, Lievens M, Leboulleux D, Njuguna P, Peshu N et al. 2013. Four-year efficacy of RTS,S/AS01E and its interaction with malaria exposure. N Engl J Med, 368 (12), pp. 1111-1120. | Show Abstract | Read more

BACKGROUND: The candidate malaria vaccine RTS,S/AS01E has entered phase 3 trials, but data on long-term outcomes are limited. METHODS: For 4 years, we followed children who had been randomly assigned, at 5 to 17 months of age, to receive three doses of RTS,S/AS01E vaccine (223 children) or rabies vaccine (224 controls). The end point was clinical malaria (temperature of ≥37.5°C and Plasmodium falciparum parasitemia density of >2500 parasites per cubic millimeter). Each child's exposure to malaria was estimated with the use of the distance-weighted local prevalence of malaria. RESULTS: Over a period of 4 years, 118 of 223 children who received the RTS,S/AS01E vaccine and 138 of 224 of the controls had at least 1 episode of clinical malaria. Vaccine efficacies in the intention-to-treat and per-protocol analyses were 29.9% (95% confidence interval [CI], 10.3 to 45.3; P=0.005) and 32.1% (95% CI, 11.6 to 47.8; P=0.004), respectively, calculated by Cox regression. Multiple episodes were common, with 551 and 618 malarial episodes in the RTS,S/AS01E and control groups, respectively; vaccine efficacies in the intention-to-treat and per-protocol analyses were 16.8% (95% CI, -8.6 to 36.3; P=0.18) and 24.3% (95% CI, 1.9 to 41.6; P=0.04), respectively, calculated by the Andersen-Gill extension of the Cox model. For every 100 vaccinated children, 65 cases of clinical malaria were averted. Vaccine efficacy declined over time (P=0.004) and with increasing exposure to malaria (P=0.001) in the per-protocol analysis. Vaccine efficacy was 43.6% (95% CI, 15.5 to 62.3) in the first year but was -0.4% (95% CI, -32.1 to 45.3) in the fourth year. Among children with a malaria-exposure index that was average or lower than average, the vaccine efficacy was 45.1% (95% CI, 11.3 to 66.0), but among children with a malaria-exposure index that was higher than average it was 15.9% (95% CI, -11.0 to 36.4). CONCLUSIONS: The efficacy of RTS,S/AS01E vaccine over the 4-year period was 16.8%. Efficacy declined over time and with increasing malaria exposure. (Funded by the PATH Malaria Vaccine Initiative and Wellcome Trust; ClinicalTrials.gov number, NCT00872963.).

Borrmann S, Straimer J, Mwai L, Abdi A, Rippert A, Okombo J, Muriithi S, Sasi P, Kortok MM, Lowe B et al. 2013. Genome-wide screen identifies new candidate genes associated with artemisinin susceptibility in Plasmodium falciparum in Kenya. Sci Rep, 3 (1), pp. 3318. | Show Abstract | Read more

Early identification of causal genetic variants underlying antimalarial drug resistance could provide robust epidemiological tools for timely public health interventions. Using a novel natural genetics strategy for mapping novel candidate genes we analyzed >75,000 high quality single nucleotide polymorphisms selected from high-resolution whole-genome sequencing data in 27 isolates of Plasmodium falciparum. We identified genetic variants associated with susceptibility to dihydroartemisinin that implicate one region on chromosome 13, a candidate gene on chromosome 1 (PFA0220w, a UBP1 ortholog) and others (PFB0560w, PFB0630c, PFF0445w) with putative roles in protein homeostasis and stress response. There was a strong signal for positive selection on PFA0220w, but not the other candidate loci. Our results demonstrate the power of full-genome sequencing-based association studies for uncovering candidate genes that determine parasite sensitivity to artemisinins. Our study provides a unique reference for the interpretation of results from resistant infections.

Tetteh KK, Osier FH, Salanti A, Kamuyu G, Drought L, Failly M, Martin C, Marsh K, Conway DJ. 2013. Analysis of antibodies to newly described Plasmodium falciparum merozoite antigens supports MSPDBL2 as a predicted target of naturally acquired immunity. Infect Immun, 81 (10), pp. 3835-3842. | Show Abstract | Read more

Prospective studies continue to identify malaria parasite genes with particular patterns of polymorphism which indicate they may be under immune selection, and the encoded proteins require investigation. Sixteen new recombinant protein reagents were designed to characterize three such polymorphic proteins expressed in Plasmodium falciparum schizonts and merozoites: MSPDBL1 (also termed MSP3.4) and MSPDBL2 (MSP3.8), which possess Duffy binding-like (DBL) domains, and SURFIN4.2, encoded by a member of the surface-associated interspersed (surf) multigene family. After testing the antigenicities of these reagents by murine immunization and parasite immunofluorescence, we analyzed naturally acquired antibody responses to the antigens in two cohorts in coastal Kenya in which the parasite was endemic (Chonyi [n = 497] and Ngerenya [n = 461]). As expected, the prevalence and levels of serum antibodies increased with age. We then investigated correlations with subsequent risk of clinical malaria among children <11 years of age during 6 months follow-up surveillance. Antibodies to the polymorphic central region of MSPDBL2 were associated with reduced risk of malaria in both cohorts, with statistical significance remaining for the 3D7 allelic type after adjustment for individuals' ages in years and antibody reactivity to whole-schizont extract (Chonyi, risk ratio, 0.51, and 95% confidence interval [CI], 0.28 to 0.93; Ngerenya, risk ratio, 0.38, and 95% CI, 0.18 to 0.82). For the MSPDBL1 Palo Alto allelic-type antigen, there was a protective association in one cohort (Ngerenya, risk ratio, 0.53, and 95% CI, 0.32 to 0.89), whereas the other antigens showed no protective associations after adjustment. These findings support the prediction that antibodies to the polymorphic region of MSPDBL2 contribute to protective immunity.

Warimwe GM, Recker M, Kiragu EW, Buckee CO, Wambua J, Musyoki JN, Marsh K, Bull PC. 2013. Plasmodium falciparum var gene expression homogeneity as a marker of the host-parasite relationship under different levels of naturally acquired immunity to malaria. PLoS One, 8 (7), pp. e70467. | Show Abstract | Read more

Acquired immunity to Plasmodium falciparum infection causes a change from frequent, sometimes life-threatening, malaria in young children to asymptomatic, chronic infections in older children and adults. Little is known about how this transition occurs but antibodies to the extremely diverse PfEMP1 parasite antigens are thought to play a role. PfEMP1 is encoded by a family of 60 var genes that undergo clonal antigenic variation, potentially creating an antigenically heterogeneous infecting population of parasites within the host. Previous theoretical work suggests that antibodies to PfEMP1 may play a role in "orchestrating" their expression within infections leading to sequential, homogeneous expression of var genes, and prolonged infection chronicity. Here, using a cloning and sequencing approach we compare the var expression homogeneity (VEH) between isolates from children with asymptomatic and clinical infections. We show that asymptomatic infections have higher VEH than clinical infections and a broader host antibody response. We discuss this in relation to the potential role of host antibodies in promoting chronicity of infection and parasite survival through the low transmission season.

Lundblom K, Murungi L, Nyaga V, Olsson D, Rono J, Osier F, Ogada E, Montgomery S, Scott JA, Marsh K, Färnert A. 2013. Plasmodium falciparum infection patterns since birth and risk of severe malaria: a nested case-control study in children on the coast of Kenya. PLoS One, 8 (2), pp. e56032. | Show Abstract | Read more

Children in malaria endemic areas acquire immunity to severe malaria faster than to mild malaria. Only a minority of children suffers from severe malaria and it is not known what determines this. The aim of this study was to establish how P. falciparum infections during the first years of life affect the risk of severe malaria. A matched case-control study was nested within a large birth cohort set up to study the immunoepidemiology of pneumococci on the Kenyan coast. Infection patterns in three-monthly blood samples in cohort children admitted to hospital with severe malaria were compared to controls matched on age, residential location and time of sampling. P. falciparum detected at least once from birth conferred an increased risk of severe malaria and particularly if multiclonal infections, as characterized by genotyping of a polymorphic antigen gene, were ever detected. The results show for the first time that children with severe malaria have more infections early in life compared to community controls. These findings provide important insights on the immunity to severe disease, knowledge essential for the development of a vaccine against severe malaria.

Warimwe GM, Murungi LM, Kamuyu G, Nyangweso GM, Wambua J, Naranbhai V, Fletcher HA, Hill AV, Bejon P, Osier FH, Marsh K. 2013. The ratio of monocytes to lymphocytes in peripheral blood correlates with increased susceptibility to clinical malaria in Kenyan children. PLoS One, 8 (2), pp. e57320. | Show Abstract | Read more

BACKGROUND: Plasmodium falciparum malaria remains a major cause of illness and death in sub-Saharan Africa. Young children bear the brunt of the disease and though older children and adults suffer relatively fewer clinical attacks, they remain susceptible to asymptomatic P. falciparum infection. A better understanding of the host factors associated with immunity to clinical malaria and the ability to sustain asymptomatic P. falciparum infection will aid the development of improved strategies for disease prevention. METHODS AND FINDINGS: Here we investigate whether full differential blood counts can predict susceptibility to clinical malaria among Kenyan children sampled at five annual cross-sectional surveys. We find that the ratio of monocytes to lymphocytes, measured in peripheral blood at the time of survey, directly correlates with risk of clinical malaria during follow-up. This association is evident among children with asymptomatic P. falciparum infection at the time the cell counts are measured (Hazard ratio (HR)  =  2.7 (95% CI 1.42, 5.01, P  =  0.002) but not in those without detectable parasitaemia (HR  =  1.0 (95% CI 0.74, 1.42, P  =  0.9). CONCLUSIONS: We propose that the monocyte to lymphocyte ratio, which is easily derived from routine full differential blood counts, reflects an individual's capacity to mount an effective immune response to P. falciparum infection.

Roetynck S, Olotu A, Simam J, Marsh K, Stockinger B, Urban B, Langhorne J. 2013. Phenotypic and functional profiling of CD4 T cell compartment in distinct populations of healthy adults with different antigenic exposure. PLoS One, 8 (1), pp. e55195. | Show Abstract | Read more

BACKGROUND: Multiparameter flow cytometry has revealed extensive phenotypic and functional heterogeneity of CD4 T cell responses in mice and humans, emphasizing the importance of assessing multiple aspects of the immune response in correlation with infection or vaccination outcome. The aim of this study was to establish and validate reliable and feasible flow cytometry assays, which will allow us to characterize CD4 T cell population in humans in field studies more fully. METHODOLOGY/PRINCIPAL FINDINGS: We developed polychromatic flow cytometry antibody panels for immunophenotyping the major CD4 T cell subsets as well as broadly characterizing the functional profiles of the CD4 T cells in peripheral blood. We then validated these assays by conducting a pilot study comparing CD4 T cell responses in distinct populations of healthy adults living in either rural or urban Kenya. This study revealed that the expression profile of CD4 T cell activation and memory markers differed significantly between African and European donors but was similar amongst African individuals from either rural or urban areas. Adults from rural Kenya had, however, higher frequencies and greater polyfunctionality among cytokine producing CD4 T cells compared to both urban populations, particularly for "Th1" type of response. Finally, endemic exposure to malaria in rural Kenya may have influenced the expansion of few discrete CD4 T cell populations with specific functional signatures. CONCLUSION/SIGNIFICANCE: These findings suggest that environmentally driven T cell activation does not drive the dysfunction of CD4 T cells but is rather associated with greater magnitude and quality of CD4 T cell response, indicating that the level or type of microbial exposure and antigenic experience may influence and shape the functionality of CD4 T cell compartment. Our data confirm that it is possible and mandatory to assess multiple functional attributes of CD4 T cell response in the context of infection.

Illingworth J, Butler NS, Roetynck S, Mwacharo J, Pierce SK, Bejon P, Crompton PD, Marsh K, Ndungu FM. 2013. Chronic exposure to Plasmodium falciparum is associated with phenotypic evidence of B and T cell exhaustion. J Immunol, 190 (3), pp. 1038-1047. | Show Abstract | Read more

Naturally acquired immunity to malaria develops slowly, requiring several years of repeated exposure to be effective. The cellular and molecular factors underlying this observation are only partially understood. Recent studies suggest that chronic Plasmodium falciparum exposure may induce functional exhaustion of lymphocytes, potentially impeding optimal control of infection. However, it remains unclear whether the "atypical" memory B cells (MBCs) and "exhausted" CD4 T cells described in humans exposed to endemic malaria are driven by P. falciparum per se or by other factors commonly associated with malaria, such as coinfections and malnutrition. To address this critical question we took advantage of a "natural" experiment near Kilifi, Kenya, and compared profiles of B and T cells of children living in a rural community where P. falciparum transmission is ongoing to the profiles of age-matched children living under similar conditions in a nearby community where P. falciparum transmission ceased 5 y prior to this study. We found that continuous exposure to P. falciparum drives the expansion of atypical MBCs. Persistent P. falciparum exposure was associated with an increased frequency of CD4 T cells expressing phenotypic markers of exhaustion, both programmed cell death-1 (PD-1) alone and PD-1 in combination with lymphocyte-activation gene-3 (LAG-3). This expansion of PD-1-expressing and PD-1/LAG-3-coexpressing CD4 T cells was largely confined to CD45RA(+) CD4 T cells. The percentage of CD45RA(+)CD27(+) CD4 T cells coexpressing PD-1 and LAG-3 was inversely correlated with frequencies of activated and classical MBCs. Taken together, these results suggest that P. falciparum infection per se drives the expansion of atypical MBCs and phenotypically exhausted CD4 T cells, which has been reported in other endemic areas.

RTS,S Clinical Trials Partnership, Agnandji ST, Lell B, Fernandes JF, Abossolo BP, Methogo BG, Kabwende AL, Adegnika AA, Mordmüller B, Issifou S et al. 2012. A phase 3 trial of RTS,S/AS01 malaria vaccine in African infants. N Engl J Med, 367 (24), pp. 2284-2295. | Show Abstract | Read more

BACKGROUND: The candidate malaria vaccine RTS,S/AS01 reduced episodes of both clinical and severe malaria in children 5 to 17 months of age by approximately 50% in an ongoing phase 3 trial. We studied infants 6 to 12 weeks of age recruited for the same trial. METHODS: We administered RTS,S/AS01 or a comparator vaccine to 6537 infants who were 6 to 12 weeks of age at the time of the first vaccination in conjunction with Expanded Program on Immunization (EPI) vaccines in a three-dose monthly schedule. Vaccine efficacy against the first or only episode of clinical malaria during the 12 months after vaccination, a coprimary end point, was analyzed with the use of Cox regression. Vaccine efficacy against all malaria episodes, vaccine efficacy against severe malaria, safety, and immunogenicity were also assessed. RESULTS: The incidence of the first or only episode of clinical malaria in the intention-to-treat population during the 14 months after the first dose of vaccine was 0.31 per person-year in the RTS,S/AS01 group and 0.40 per person-year in the control group, for a vaccine efficacy of 30.1% (95% confidence interval [CI], 23.6 to 36.1). Vaccine efficacy in the per-protocol population was 31.3% (97.5% CI, 23.6 to 38.3). Vaccine efficacy against severe malaria was 26.0% (95% CI, -7.4 to 48.6) in the intention-to-treat population and 36.6% (95% CI, 4.6 to 57.7) in the per-protocol population. Serious adverse events occurred with a similar frequency in the two study groups. One month after administration of the third dose of RTS,S/AS01, 99.7% of children were positive for anti-circumsporozoite antibodies, with a geometric mean titer of 209 EU per milliliter (95% CI, 197 to 222). CONCLUSIONS: The RTS,S/AS01 vaccine coadministered with EPI vaccines provided modest protection against both clinical and severe malaria in young infants. (Funded by GlaxoSmithKline Biologicals and the PATH Malaria Vaccine Initiative; RTS,S ClinicalTrials.gov number, NCT00866619.).

Offeddu V, Thathy V, Marsh K, Matuschewski K. 2012. Naturally acquired immune responses against Plasmodium falciparum sporozoites and liver infection (vol 42, pg 535, 2012) INTERNATIONAL JOURNAL FOR PARASITOLOGY, 42 (10), pp. 961-961. | Read more

Chan JA, Howell KB, Reiling L, Ataide R, Mackintosh CL, Fowkes FJ, Petter M, Chesson JM, Langer C, Warimwe GM et al. 2012. Targets of antibodies against Plasmodium falciparum-infected erythrocytes in malaria immunity. J Clin Invest, 122 (9), pp. 3227-3238. | Show Abstract | Read more

Plasmodium falciparum is the major cause of malaria globally and is transmitted by mosquitoes. During parasitic development, P. falciparum-infected erythrocytes (P. falciparum-IEs) express multiple polymorphic proteins known as variant surface antigens (VSAs), including the P. falciparum erythrocyte membrane protein 1 (PfEMP1). VSA-specific antibodies are associated with protection from symptomatic and severe malaria. However, the importance of the different VSA targets of immunity to malaria remains unclear, which has impeded an understanding of malaria immunity and vaccine development. In this study, we developed assays using transgenic P. falciparum with modified PfEMP1 expression to quantify serum antibodies to VSAs among individuals exposed to malaria. We found that the majority of the human antibody response to the IE targets PfEMP1. Furthermore, our longitudinal studies showed that individuals with PfEMP1-specific antibodies had a significantly reduced risk of developing symptomatic malaria, whereas antibodies to other surface antigens were not associated with protective immunity. Using assays that measure antibody-mediated phagocytosis of IEs, an important mechanism in parasite clearance, we identified PfEMP1 as the major target of these functional antibodies. Taken together, these data demonstrate that PfEMP1 is a key target of humoral immunity. These findings advance our understanding of the targets and mediators of human immunity to malaria and have major implications for malaria vaccine development.

Manske M, Miotto O, Campino S, Auburn S, Almagro-Garcia J, Maslen G, O'Brien J, Djimde A, Doumbo O, Zongo I et al. 2012. Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing. Nature, 487 (7407), pp. 375-379. | Show Abstract | Read more

Malaria elimination strategies require surveillance of the parasite population for genetic changes that demand a public health response, such as new forms of drug resistance. Here we describe methods for the large-scale analysis of genetic variation in Plasmodium falciparum by deep sequencing of parasite DNA obtained from the blood of patients with malaria, either directly or after short-term culture. Analysis of 86,158 exonic single nucleotide polymorphisms that passed genotyping quality control in 227 samples from Africa, Asia and Oceania provides genome-wide estimates of allele frequency distribution, population structure and linkage disequilibrium. By comparing the genetic diversity of individual infections with that of the local parasite population, we derive a metric of within-host diversity that is related to the level of inbreeding in the population. An open-access web application has been established for the exploration of regional differences in allele frequency and of highly differentiated loci in the P. falciparum genome.

Ndungu FM, Olotu A, Mwacharo J, Nyonda M, Apfeld J, Mramba LK, Fegan GW, Bejon P, Marsh K. 2012. Memory B cells are a more reliable archive for historical antimalarial responses than plasma antibodies in no-longer exposed children. Proc Natl Acad Sci U S A, 109 (21), pp. 8247-8252. | Show Abstract | Read more

Humans respond to foreign antigen by generating plasma Abs and memory B cells (MBCs). The Ab response then declines, sometimes to below the limit of detection. In contrast, MBCs are generally thought to be long-lived. We tested and compared Plasmodium falciparum (Pf)-specific Ab and MBC responses in two populations of children: (i) previously exposed children who had documented Pf infections several years ago, but minimal exposure since then; and (ii) persistently exposed children living in a separate but nearby endemic area. We found that although Pf-specific plasma Abs were lower in previously exposed children compared with persistently exposed children, their cognate MBCs were maintained at similar frequencies. We conclude that serological analysis by itself would greatly underestimate the true memory of Pf-specific Ab responses in previously exposed children living in areas where Pf transmission has been reduced or eliminated.

Abdulla S, Alonso P, Binka F, Graves P, Greenwood B, Leke R, Malik E, Marsh K, Meek S, Mendis K et al. 2012. Inaugural meeting of the malaria policy advisory committee to the WHO: conclusions and recommendations MALARIA JOURNAL, 11 (1), pp. 137-137. | Read more

Scott JA, Bauni E, Moisi JC, Ojal J, Gatakaa H, Nyundo C, Molyneux CS, Kombe F, Tsofa B, Marsh K et al. 2012. Profile: The Kilifi Health and Demographic Surveillance System (KHDSS). Int J Epidemiol, 41 (3), pp. 650-657. | Show Abstract | Read more

The Kilifi Health and Demographic Surveillance System (KHDSS), located on the Indian Ocean coast of Kenya, was established in 2000 as a record of births, pregnancies, migration events and deaths and is maintained by 4-monthly household visits. The study area was selected to capture the majority of patients admitted to Kilifi District Hospital. The KHDSS has 260 000 residents and the hospital admits 4400 paediatric patients and 3400 adult patients per year. At the hospital, morbidity events are linked in real time by a computer search of the population register. Linked surveillance was extended to KHDSS vaccine clinics in 2008. KHDSS data have been used to define the incidence of hospital presentation with childhood infectious diseases (e.g. rotavirus diarrhoea, pneumococcal disease), to test the association between genetic risk factors (e.g. thalassaemia and sickle cell disease) and infectious diseases, to define the community prevalence of chronic diseases (e.g. epilepsy), to evaluate access to health care and to calculate the operational effectiveness of major public health interventions (e.g. conjugate Haemophilus influenzae type b vaccine). Rapport with residents is maintained through an active programme of community engagement. A system of collaborative engagement exists for sharing data on survival, morbidity, socio-economic status and vaccine coverage.

Warimwe GM, Fegan G, Musyoki JN, Newton CR, Opiyo M, Githinji G, Andisi C, Menza F, Kitsao B, Marsh K, Bull PC. 2012. Prognostic indicators of life-threatening malaria are associated with distinct parasite variant antigen profiles. Sci Transl Med, 4 (129), pp. 129ra45. | Show Abstract | Read more

PfEMP1 is a family of cytoadhesive surface antigens expressed on erythrocytes infected with Plasmodium falciparum, the parasite that causes the most severe form of malaria. These surface antigens play a role in immune evasion and are thought to contribute to the pathogenesis of the malaria parasite. Previous studies have suggested a role for a specific subset of PfEMP1 called "group A" in severe malaria. To explore the role of group A PfEMP1 in disease, we measured the expression of the var genes that encode them in parasites from clinical isolates collected from children suffering from malaria. We also looked at the ability of these clinical isolates to induce rosetting of erythrocytes, which indicates a cytoadhesion phenotype that is thought to be important in pathogenesis. These two sets of data were correlated with the presence of two life-threatening manifestations of severe malaria in the children: impaired consciousness and respiratory distress. Using regression analysis, we show that marked rosetting was associated with respiratory distress, whereas elevated expression of group A-like var genes without elevated rosetting was associated with impaired consciousness. The results suggest that manifestations of malarial disease may reflect the distribution of cytoadhesion phenotypes expressed by the infecting parasite population.

Ibison F, Olotu A, Muema DM, Mwacharo J, Ohuma E, Kimani D, Marsh K, Bejon P, Ndungu FM. 2012. Lack of avidity maturation of merozoite antigen-specific antibodies with increasing exposure to Plasmodium falciparum amongst children and adults exposed to endemic malaria in Kenya. PLoS One, 7 (12), pp. e52939. | Show Abstract | Read more

BACKGROUND: Although antibodies are critical for immunity to malaria, their functional attributes that determine protection remain unclear. We tested for associations between antibody avidities to Plasmodium falciparum (Pf) antigens and age, asymptomatic parasitaemia, malaria exposure index (a distance weighted local malaria prevalence) and immunity to febrile malaria during 10-months of prospective follow up. METHODS: Cross-sectional antibody levels and avidities to Apical Membrane Antigen 1 (AMA1), Merozoite Surface Protein 1(42) (MSP1) and Merozoite Surface Protein 3 (MSP3) were measured by Enzyme Linked Immunosorbent Assay in 275 children, who had experienced at least one episode of clinical malaria by the time of this study, as determined by active weekly surveillance. RESULTS: Antibody levels to AMA1, MSP1 and MSP3 increased with age. Anti-AMA1 and MSP1 antibody avidities were (respectively) positively and negatively associated with age, while anti-MSP3 antibody avidities did not change. Antibody levels to all three antigens were elevated in the presence of asymptomatic parasitaemia, but their associated avidities were not. Unlike antibody levels, antibody avidities to the three-merozoite antigens did not increase with exposure to Pf malaria. There were no consistent prospective associations between antibody avidities and malaria episodes. CONCLUSION: We found no evidence that antibody avidities to Pf-merozoite antigens are associated with either exposure or immunity to malaria.

Offeddu V, Thathy V, Marsh K, Matuschewski K. 2012. Erratum to ‘‘Naturally acquired immune responses against Plasmodium falciparum sporozoites and liver infection” [Int. J. Parasitol. 42 (2012) 535–548] International Journal for Parasitology, 42 (10), pp. 961-961. | Read more

Offeddu V, Thathy V, Marsh K, Matuschewski K. 2012. Erratum to ''Naturally acquired immune responses against Plasmodium falciparum sporozoites and liver infection" [Int. J. Parasitol. 42 (2012) 535-548] (DOI:10.1016/j.ijpara.2012.03.011) International Journal for Parasitology,

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Midega JT, Smith DL, Olotu A, Mwangangi JM, Nzovu JG, Wambua J, Nyangweso G, Mbogo CM, Christophides GK, Marsh K, Bejon P. 2012. Wind direction and proximity to larval sites determines malaria risk in Kilifi District in Kenya Nature Communications, 3 | Show Abstract | Read more

Studies of the fine-scale spatial epidemiology of malaria consistently identify malaria hotspots, comprising clusters of homesteads at high transmission intensity. These hotspots sustain transmission, and may be targeted by malaria-control programmes. Here we describe the spatial relationship between the location of Anopheles larval sites and human malaria infection in a cohort study of 642 children, aged 1-10-years-old. Our data suggest that proximity to larval sites predict human malaria infection, when homesteads are upwind of larval sites, but not when homesteads are downwind of larval sites. We conclude that following oviposition, female Anophelines fly upwind in search for human hosts and, thus, malaria transmission may be disrupted by targeting vector larval sites in close proximity, and downwind to malaria hotspots. © 2012 Macmillan Publishers Limited. All rights reserved.

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Offeddu V, Thathy V, Marsh K, Matuschewski K. 2012. Naturally acquired immune responses against Plasmodium falciparum sporozoites and liver infection International Journal for Parasitology, 42 (6), pp. 535-548. | Show Abstract | Read more

Malaria is a vector-borne infectious disease caused by infection with eukaryotic pathogens termed . Plasmodium. Epidemiological hallmarks of . Plasmodium falciparum malaria are continuous re-infections, over which time the human host may experience several clinical malaria episodes, slow acquisition of partial protection against infection, and its partial decay upon migration away from endemic regions. To overcome the exposure-dependence of naturally acquired immunity and rapidly elicit robust long-term protection are ultimate goals of malaria vaccine development. However, cellular and molecular correlates of naturally acquired immunity against either parasite infection or malarial disease remain elusive. Sero-epidemiological studies consistently suggest that acquired immunity is primarily directed against the asexual blood stages. Here, we review available data on the relationship between immune responses against the . Anopheles mosquito-transmitted sporozoite and exo-erythrocytic liver stages and the incidence of malaria. We discuss current limitations and research opportunities, including the identification of additional sporozoite antigens and the use of systematic immune profiling and functional studies in longitudinal cohorts to look for pre-erythrocytic signatures of naturally acquired immunity. © 2012 Australian Society for Parasitology Inc..

Offeddu V, Thathy V, Marsh K, Matuschewski K. 2012. Naturally acquired immune responses against Plasmodium falciparum sporozoites and liver infection. Int J Parasitol, 42 (6), pp. 535-548. | Show Abstract | Read more

Malaria is a vector-borne infectious disease caused by infection with eukaryotic pathogens termed Plasmodium. Epidemiological hallmarks of Plasmodium falciparum malaria are continuous re-infections, over which time the human host may experience several clinical malaria episodes, slow acquisition of partial protection against infection, and its partial decay upon migration away from endemic regions. To overcome the exposure-dependence of naturally acquired immunity and rapidly elicit robust long-term protection are ultimate goals of malaria vaccine development. However, cellular and molecular correlates of naturally acquired immunity against either parasite infection or malarial disease remain elusive. Sero-epidemiological studies consistently suggest that acquired immunity is primarily directed against the asexual blood stages. Here, we review available data on the relationship between immune responses against the Anopheles mosquito-transmitted sporozoite and exo-erythrocytic liver stages and the incidence of malaria. We discuss current limitations and research opportunities, including the identification of additional sporozoite antigens and the use of systematic immune profiling and functional studies in longitudinal cohorts to look for pre-erythrocytic signatures of naturally acquired immunity.

Snow RW, Amratia P, Kabaria CW, Noor AM, Marsh K. 2012. The changing limits and incidence of malaria in Africa: 1939-2009. Adv Parasitol, 78 pp. 169-262. | Show Abstract | Read more

Understanding the historical, temporal changes of malaria risk following control efforts in Africa provides a unique insight into what has been and might be archived towards a long-term ambition of elimination on the continent. Here, we use archived published and unpublished material combined with biological constraints on transmission accompanied by a narrative on malaria control to document the changing incidence of malaria in Africa since earliest reports pre-second World War. One result is a more informed mapped definition of the changing margins of transmission in 1939, 1959, 1979, 1999 and 2009.

Olotu A, Fegan G, Wambua J, Nyangweso G, Ogada E, Drakeley C, Marsh K, Bejon P. 2012. Estimating individual exposure to malaria using local prevalence of malaria infection in the field. PLoS One, 7 (3), pp. e32929. | Show Abstract | Read more

BACKGROUND: Heterogeneity in malaria exposure complicates survival analyses of vaccine efficacy trials and confounds the association between immune correlates of protection and malaria infection in longitudinal studies. Analysis may be facilitated by taking into account the variability in individual exposure levels, but it is unclear how exposure can be estimated at an individual level. METHOD AND FINDINGS: We studied three cohorts (Chonyi, Junju and Ngerenya) in Kilifi District, Kenya to assess measures of malaria exposure. Prospective data were available on malaria episodes, geospatial coordinates, proximity to infected and uninfected individuals and residence in predefined malaria hotspots for 2,425 individuals. Antibody levels to the malaria antigens AMA1 and MSP1(142) were available for 291 children from Junju. We calculated distance-weighted local prevalence of malaria infection within 1 km radius as a marker of individual's malaria exposure. We used multivariable modified Poisson regression model to assess the discriminatory power of these markers for malaria infection (i.e. asymptomatic parasitaemia or clinical malaria). The area under the receiver operating characteristic (ROC) curve was used to assess the discriminatory power of the models. Local malaria prevalence within 1 km radius and AMA1 and MSP1(142) antibodies levels were independently associated with malaria infection. Weighted local malaria prevalence had an area under ROC curve of 0.72 (95%CI: 0.66-0.73), 0.71 (95%CI: 0.69-0.73) and 0.82 (95%CI: 0.80-0.83) among cohorts in Chonyi, Junju and Ngerenya respectively. In a small subset of children from Junju, a model incorporating weighted local malaria prevalence with AMA1 and MSP1(142) antibody levels provided an AUC of 0.83 (95%CI: 0.79-0.88). CONCLUSION: We have proposed an approach to estimating the intensity of an individual's malaria exposure in the field. The weighted local malaria prevalence can be used as individual marker of malaria exposure in malaria vaccine trials and longitudinal studies of natural immunity to malaria.

Mwai L, Diriye A, Masseno V, Muriithi S, Feltwell T, Musyoki J, Lemieux J, Feller A, Mair GR, Marsh K et al. 2012. Genome wide adaptations of Plasmodium falciparum in response to lumefantrine selective drug pressure. PLoS One, 7 (2), pp. e31623. | Show Abstract | Read more

The combination therapy of the Artemisinin-derivative Artemether (ART) with Lumefantrine (LM) (Coartem®) is an important malaria treatment regimen in many endemic countries. Resistance to Artemisinin has already been reported, and it is feared that LM resistance (LMR) could also evolve quickly. Therefore molecular markers which can be used to track Coartem® efficacy are urgently needed. Often, stable resistance arises from initial, unstable phenotypes that can be identified in vitro. Here we have used the Plasmodium falciparum multidrug resistant reference strain V1S to induce LMR in vitro by culturing the parasite under continuous drug pressure for 16 months. The initial IC(50) (inhibitory concentration that kills 50% of the parasite population) was 24 nM. The resulting resistant strain V1S(LM), obtained after culture for an estimated 166 cycles under LM pressure, grew steadily in 378 nM of LM, corresponding to 15 times the IC(50) of the parental strain. However, after two weeks of culturing V1S(LM) in drug-free medium, the IC(50) returned to that of the initial, parental strain V1S. This transient drug tolerance was associated with major changes in gene expression profiles: using the PFSANGER Affymetrix custom array, we identified 184 differentially expressed genes in V1S(LM). Among those are 18 known and putative transporters including the multidrug resistance gene 1 (pfmdr1), the multidrug resistance associated protein and the V-type H+ pumping pyrophosphatase 2 (pfvp2) as well as genes associated with fatty acid metabolism. In addition we detected a clear selective advantage provided by two genomic loci in parasites grown under LM drug pressure, suggesting that all, or some of those genes contribute to development of LM tolerance--they may prove useful as molecular markers to monitor P. falciparum LM susceptibility.

Weiss GE, Ndungu FM, McKittrick N, Li S, Kimani D, Crompton PD, Marsh K, Pierce SK. 2012. High efficiency human memory B cell assay and its application to studying Plasmodium falciparum-specific memory B cells in natural infections Journal of Immunological Methods, 375 (1-2), pp. 68-74. | Show Abstract | Read more

Memory B cells (MBCs) are a key component of long term humoral immunity to many human infectious diseases. Despite their importance, we know little about the generation or maintenance of antigen-(Ag)-specific MBCs in humans in response to infection. A frequently employed method for quantifying Ag-specific MBCs in human peripheral blood (Crotty et al., 2004) relies on the ability of MBCs but not naïve B cells to differentiate into antibody secreting cells (ASCs) in response to polyclonal activators and Toll-like receptor agonists in vitro and the measurement of Ag-specific ASCs by ELISPOT assays. Here we report on studies to optimize the efficiency of this ELISPOT-based assay and to apply this assay to the detection of Plasmodium falciparum (Pf)-specific MBCs in adults living in a malaria endemic area where immunity to Pf is acquired through natural infection. We show that the addition of IL-10 to in vitro cultures of human peripheral blood mononuclear cells increased the efficiency of the assay from 10% to over 90% without increasing the ASC burst size and without any substantial increase in background from naïve B cells or plasma cells (PCs). Using this assay we were able to quantify the frequency of Pf-specific MBCs in peripheral blood of adults living in a malaria endemic area. Thus, this highly efficient assay appears to be well suited to field studies of the generation and maintenance of MBCs where the volumes of blood obtainable are often limiting. © 2011 Elsevier B.V.

Gitau EN, Tuju J, Stevenson L, Kimani E, Karanja H, Marsh K, Bull PC, Urban BC. 2012. T-cell responses to the DBLα-tag, a short semi-conserved region of the Plasmodium falciparum membrane erythrocyte protein 1. PLoS One, 7 (1), pp. e30095. | Show Abstract | Read more

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a variant surface antigen expressed on mature forms of infected erythrocytes. It is considered an important target of naturally acquired immunity. Despite its extreme sequence heterogeneity, variants of PfEMP1 can be stratified into distinct groups. Group A PfEMP1 have been independently associated with low host immunity and severe disease in several studies and are now of potential interest as vaccine candidates. Although antigen-specific antibodies are considered the main effector mechanism in immunity to malaria, the induction of efficient and long-lasting antibody responses requires CD4+ T-cell help. To date, very little is known about CD4+ T-cell responses to PfEMP1 expressed on clinical isolates. The DBLα-tag is a small region from the DBLα-domain of PfEMP1 that can be amplified with universal primers and is accessible in clinical parasite isolates. We identified the dominant expressed PfEMP1 in 41 individual clinical parasite isolates and expressed the corresponding DBLα-tag as recombinant antigen. Individual DBLα-tags were then used to activate CD4+ T-cells from acute and convalescent blood samples in children who were infected with the respective clinical parasite isolate. Here we show that CD4+ T-cell responses to the homologous DBLα-tag were induced in almost all children during acute malaria and maintained in some for 4 months. Children infected with parasites that dominantly expressed group A-like PfEMP1 were more likely to maintain antigen-specific IFNγ-producing CD4+ T-cells than children infected with parasites dominantly expressing other PfEMP1. These results suggest that group A-like PfEMP1 may induce long-lasting effector memory T-cells that might be able to provide rapid help to variant-specific B cells. Furthermore, a number of children induced CD4+ T-cell responses to heterologous DBLα-tags, suggesting that CD4+ T-cells may recognise shared epitopes between several DBLα-tags.

Midega JT, Smith DL, Olotu A, Mwangangi JM, Nzovu JG, Wambua J, Nyangweso G, Mbogo CM, Christophides GK, Marsh K, Bejon P. 2012. Wind direction and proximity to larval sites determines malaria risk in Kilifi District in Kenya. Nat Commun, 3 pp. 674. | Show Abstract | Read more

Studies of the fine-scale spatial epidemiology of malaria consistently identify malaria hotspots, comprising clusters of homesteads at high transmission intensity. These hotspots sustain transmission, and may be targeted by malaria-control programmes. Here we describe the spatial relationship between the location of Anopheles larval sites and human malaria infection in a cohort study of 642 children, aged 1-10-years-old. Our data suggest that proximity to larval sites predict human malaria infection, when homesteads are upwind of larval sites, but not when homesteads are downwind of larval sites. We conclude that following oviposition, female Anophelines fly upwind in search for human hosts and, thus, malaria transmission may be disrupted by targeting vector larval sites in close proximity, and downwind to malaria hotspots.

RTS,S Clinical Trials Partnership, Agnandji ST, Lell B, Soulanoudjingar SS, Fernandes JF, Abossolo BP, Conzelmann C, Methogo BG, Doucka Y, Flamen A et al. 2011. First results of phase 3 trial of RTS,S/AS01 malaria vaccine in African children. N Engl J Med, 365 (20), pp. 1863-1875. | Show Abstract | Read more

BACKGROUND: An ongoing phase 3 study of the efficacy, safety, and immunogenicity of candidate malaria vaccine RTS,S/AS01 is being conducted in seven African countries. METHODS: From March 2009 through January 2011, we enrolled 15,460 children in two age categories--6 to 12 weeks of age and 5 to 17 months of age--for vaccination with either RTS,S/AS01 or a non-malaria comparator vaccine. The primary end point of the analysis was vaccine efficacy against clinical malaria during the 12 months after vaccination in the first 6000 children 5 to 17 months of age at enrollment who received all three doses of vaccine according to protocol. After 250 children had an episode of severe malaria, we evaluated vaccine efficacy against severe malaria in both age categories. RESULTS: In the 14 months after the first dose of vaccine, the incidence of first episodes of clinical malaria in the first 6000 children in the older age category was 0.32 episodes per person-year in the RTS,S/AS01 group and 0.55 episodes per person-year in the control group, for an efficacy of 50.4% (95% confidence interval [CI], 45.8 to 54.6) in the intention-to-treat population and 55.8% (97.5% CI, 50.6 to 60.4) in the per-protocol population. Vaccine efficacy against severe malaria was 45.1% (95% CI, 23.8 to 60.5) in the intention-to-treat population and 47.3% (95% CI, 22.4 to 64.2) in the per-protocol population. Vaccine efficacy against severe malaria in the combined age categories was 34.8% (95% CI, 16.2 to 49.2) in the per-protocol population during an average follow-up of 11 months. Serious adverse events occurred with a similar frequency in the two study groups. Among children in the older age category, the rate of generalized convulsive seizures after RTS,S/AS01 vaccination was 1.04 per 1000 doses (95% CI, 0.62 to 1.64). CONCLUSIONS: The RTS,S/AS01 vaccine provided protection against both clinical and severe malaria in African children. (Funded by GlaxoSmithKline Biologicals and the PATH Malaria Vaccine Initiative; RTS,S ClinicalTrials.gov number, NCT00866619 .).

Moïsi JC, Gatakaa H, Berkley JA, Maitland K, Mturi N, Newton CR, Njuguna P, Nokes J, Ojal J, Bauni E et al. 2011. Excess child mortality after discharge from hospital in Kilifi, Kenya: a retrospective cohort analysis. Bull World Health Organ, 89 (10), pp. 725-732A. | Show Abstract | Read more

OBJECTIVE: To explore excess paediatric mortality after discharge from Kilifi District Hospital, Kenya, and its duration and risk factors. METHODS: Hospital and demographic data were used to describe post-discharge mortality and survival probability in children aged < 15 years, by age group and clinical syndrome. Cox regression models were developed to identify risk factors. FINDINGS: In 2004-2008, approximately 111,000 children were followed for 555,000 person-years. We analysed 14,971 discharges and 535 deaths occurring within 365 days of discharge. Mortality was higher in the post-discharge cohort than in the community cohort (age-adjusted rate ratio, RR: 7.7; 95% confidence interval, CI: 6.6-8.9) and declined little over time. An increased post-discharge mortality hazard was found in children aged < 5 years with the following: weight-for-age Z score < -4 (hazard ratio, HR: 6.5); weight-for-age Z score > -4 but < -3 (HR: 3.4); hypoxia (HR: 2.3); bacteraemia (HR: 1.8); hepatomegaly (HR: 2.3); jaundice (HR: 1.8); hospital stay > 13 days (HR: 1.8). Older age was protective (reference < 1 month): 6-23 months, HR: 0.8; 2-4 years, HR: 0.6. Children with at least one risk factor accounted for 545 (33%) of the 1655 annual discharges and for 39 (47%) of the 83 discharge-associated deaths. CONCLUSION: Hospital admission selects vulnerable children with a sustained increased risk of dying. The risk factors identified provide an empiric basis for effective outpatient follow-up.

Nduati E, Gwela A, Karanja H, Mugyenyi C, Langhorne J, Marsh K, Urban BC. 2011. The plasma concentration of the B cell activating factor is increased in children with acute malaria. J Infect Dis, 204 (6), pp. 962-970. | Show Abstract | Read more

Malaria-specific antibody responses in children often appear to be short-lived but the mechanisms underlying this phenomenon are not well understood. In this study, we investigated the relationship between the B-cell activating factor (BAFF) and its receptors expressed on B cells with antibody responses during and after acute malaria in children. Our results demonstrate that BAFF plasma levels increased during acute malarial disease and reflected disease severity. The expression profiles for BAFF receptors on B cells agreed with rapid activation and differentiation of a proportion of B cells to plasma cells. However, BAFF receptor (BAFF-R) expression was reduced on all peripheral blood B cells during acute infection, but those children with the highest level of BAFF-R expression on B cells maintained schizont-specific immunoglobin G (IgG) over a period of 4 months, indicating that dysregulation of BAFF-R expression on B cells may contribute to short-lived antibody responses to malarial antigens in children. In summary, this study suggests a potential role for BAFF during malaria disease, both as a marker for disease severity and in shaping the differentiation pattern of antigen-specific B cells.

Vekemans J, Marsh K, Greenwood B, Leach A, Kabore W, Soulanoudjingar S, Asante KP, Ansong D, Evans J, Sacarlal J et al. 2011. Assessment of severe malaria in a multicenter, phase III, RTS, S/AS01 malaria candidate vaccine trial: case definition, standardization of data collection and patient care. Malar J, 10 (1), pp. 221. | Show Abstract | Read more

BACKGROUND: An effective malaria vaccine, deployed in conjunction with other malaria interventions, is likely to substantially reduce the malaria burden. Efficacy against severe malaria will be a key driver for decisions on implementation. An initial study of an RTS, S vaccine candidate showed promising efficacy against severe malaria in children in Mozambique. Further evidence of its protective efficacy will be gained in a pivotal, multi-centre, phase III study. This paper describes the case definitions of severe malaria used in this study and the programme for standardized assessment of severe malaria according to the case definition. METHODS: Case definitions of severe malaria were developed from a literature review and a consensus meeting of expert consultants and the RTS, S Clinical Trial Partnership Committee, in collaboration with the World Health Organization and the Malaria Clinical Trials Alliance. The same groups, with input from an Independent Data Monitoring Committee, developed and implemented a programme for standardized data collection.The case definitions developed reflect the typical presentations of severe malaria in African hospitals. Markers of disease severity were chosen on the basis of their association with poor outcome, occurrence in a significant proportion of cases and on an ability to standardize their measurement across research centres. For the primary case definition, one or more clinical and/or laboratory markers of disease severity have to be present, four major co-morbidities (pneumonia, meningitis, bacteraemia or gastroenteritis with severe dehydration) are excluded, and a Plasmodium falciparum parasite density threshold is introduced, in order to maximize the specificity of the case definition. Secondary case definitions allow inclusion of co-morbidities and/or allow for the presence of parasitaemia at any density. The programmatic implementation of standardized case assessment included a clinical algorithm for evaluating seriously sick children, improvements to care delivery and a robust training and evaluation programme for clinicians. CONCLUSIONS: The case definition developed for the pivotal phase III RTS, S vaccine study is consistent with WHO recommendations, is locally applicable and appropriately balances sensitivity and specificity in the diagnosis of severe malaria. Processes set up to standardize severe malaria data collection will allow robust assessment of the efficacy of the RTS, S vaccine against severe malaria, strengthen local capacity and benefit patient care for subjects in the trial. TRIAL REGISTRATION: Clinicaltrials.gov NCT00866619.

Bejon P, Cook J, Bergmann-Leitner E, Olotu A, Lusingu J, Mwacharo J, Vekemans J, Njuguna P, Leach A, Lievens M et al. 2011. Effect of the pre-erythrocytic candidate malaria vaccine RTS,S/AS01E on blood stage immunity in young children. J Infect Dis, 204 (1), pp. 9-18. | Show Abstract | Read more

BACKGROUND: RTS,S/AS01(E) is the lead candidate malaria vaccine and confers pre-erythrocytic immunity. Vaccination may therefore impact acquired immunity to blood-stage malaria parasites after natural infection. METHODS: We measured, by enzyme-linked immunosorbent assay, antibodies to 4 Plasmodium falciparum merozoite antigens (AMA-1, MSP-1(42), EBA-175, and MSP-3) and by growth inhibitory activity (GIA) using 2 parasite clones (FV0 and 3D7) at 4 times on 860 children who were randomized to receive with RTS,S/AS01(E) or a control vaccine. RESULTS:  Antibody concentrations to AMA-1, EBA-175, and MSP-1(42) decreased with age during the first year of life, then increased to 32 months of age. Anti-MSP-3 antibody concentrations gradually increased, and GIA gradually decreased up to 32 months. Vaccination with RTS,S/AS01(E) resulted in modest reductions in AMA-1, EBA-175, MSP-1(42), and MSP-3 antibody concentrations and no significant change in GIA. Increasing anti-merozoite antibody concentrations and GIA were prospectively associated with increased risk of clinical malaria. CONCLUSIONS: Vaccination with RTS,S/AS01E reduces exposure to blood-stage parasites and, thus, reduces anti-merozoite antigen antibody concentrations. However, in this study, these antibodies were not correlates of clinical immunity to malaria. Instead, heterogeneous exposure led to confounded, positive associations between increasing antibody concentration and increasing risk of clinical malaria.

Kariuki SM, Ikumi M, Ojal J, Sadarangani M, Idro R, Olotu A, Bejon P, Berkley JA, Marsh K, Newton CR. 2011. Acute seizures attributable to falciparum malaria in an endemic area on the Kenyan coast. Brain, 134 (Pt 5), pp. 1519-1528. | Show Abstract | Read more

Falciparum malaria is an important cause of acute symptomatic seizures in children admitted to hospitals in sub-Saharan Africa, and these seizures are associated with neurological disabilities and epilepsy. However, it is difficult to determine the proportion of seizures attributable to malaria in endemic areas since a significant proportion of asymptomatic children have malaria parasitaemia. We studied children aged 0-13 years who had been admitted with a history of seizures to a rural Kenyan hospital between 2002 and 2008. We examined the changes in the incidence of seizures with the reduction of malaria. Logistic regression was used to model malaria-attributable fractions for seizures (the proportion of seizures caused by malaria) to determine if the observed decrease in acute symptomatic seizures was a measure of seizures that are attributable to malaria. The overall incidence of acute symptomatic seizures over the period was 651/100,000/year (95% confidence interval 632-670) and it was 400/100,000/year (95% confidence interval 385-415) for acute complex symptomatic seizures (convulsive status epilepticus, repetitive or focal) and 163/100,000/year (95% confidence interval 154-173) for febrile seizures. From 2002 to 2008, the incidence of all acute symptomatic seizures decreased by 809/100,000/year (69.2%) with 93.1% of this decrease in malaria-associated seizures. The decrease in the incidence of acute complex symptomatic seizures during the period was 111/100,000/year (57.2%) for convulsive status epilepticus, 440/100,000/year (73.7%) for repetitive seizures and 153/100,000/year (80.5%) for focal seizures. The adjusted malaria-attributable fractions for seizures with parasitaemia were 92.9% (95% confidence interval 90.4-95.1%) for all acute symptomatic seizures, 92.9% (95% confidence interval 89.4-95.5%) for convulsive status epilepticus, 93.6% (95% confidence interval 90.9-95.9%) for repetitive seizures and 91.8% (95% confidence interval 85.6-95.5%) for focal seizures. The adjusted malaria-attributable fractions for seizures in children above 6 months of age decreased with age. The observed decrease in all acute symptomatic seizures (809/100 000/year) was similar to the predicted decline (794/100,000/year) estimated by malaria-attributable fractions at the beginning of the study. In endemic areas, falciparum malaria is the most common cause of seizures and the risk for seizures in malaria decreases with age. The reduction in malaria has decreased the burden of seizures that are attributable to malaria and this could lead to reduced neurological disabilities and epilepsy in the area.

Ochola LB, Siddondo BR, Ocholla H, Nkya S, Kimani EN, Williams TN, Makale JO, Liljander A, Urban BC, Bull PC et al. 2011. Specific receptor usage in Plasmodium falciparum cytoadherence is associated with disease outcome. PLoS One, 6 (3), pp. e14741. | Show Abstract | Read more

Our understanding of the basis of severe disease in malaria is incomplete. It is clear that pathology is in part related to the pro-inflammatory nature of the host response but a number of other factors are also thought to be involved, including the interaction between infected erythrocytes and endothelium. This is a complex system involving several host receptors and a major parasite-derived variant antigen (PfEMP1) expressed on the surface of the infected erythrocyte membrane. Previous studies have suggested a role for ICAM-1 in the pathology of cerebral malaria, although these have been inconclusive. In this study we have examined the cytoadherence patterns of 101 patient isolates from varying clinical syndromes to CD36 and ICAM-1, and have used variant ICAM-1 proteins to further characterise this adhesive phenotype. Our results show that increased binding to CD36 is associated with uncomplicated malaria while ICAM-1 adhesion is raised in parasites from cerebral malaria cases.

Liljander A, Bejon P, Mwacharo J, Kai O, Ogada E, Peshu N, Marsh K, Färnert A. 2011. Clearance of asymptomatic P. falciparum Infections Interacts with the number of clones to predict the risk of subsequent malaria in Kenyan children. PLoS One, 6 (2), pp. e16940. | Show Abstract | Read more

BACKGROUND: Protective immunity to malaria is acquired after repeated infections in endemic areas. Asymptomatic multiclonal P. falciparum infections are common and may predict host protection. Here, we have investigated the effect of clearing asymptomatic infections on the risk of clinical malaria. METHODS: Malaria episodes were continuously monitored in 405 children (1-6 years) in an area of moderate transmission, coastal Kenya. Blood samples collected on four occasions were assessed by genotyping the polymorphic P. falciparum merozoite surface protein 2 using fluorescent PCR and capillary electrophoresis. Following the second survey, asymptomatic infections were cleared with a full course of dihydroartemisinin. RESULTS: Children who were parasite negative by PCR had a lower risk of subsequent malaria regardless of whether treatment had been given. Children with ≥ 2 clones had a reduced risk of febrile malaria compared with 1 clone after clearance of asymptomatic infections, but not if asymptomatic infections were not cleared. Multiclonal infection was associated with an increased risk of re-infection after drug treatment. However, among the children who were re-infected, multiclonal infections were associated with a shift from clinical malaria to asymptomatic parasitaemia. CONCLUSION: The number of clones was associated with exposure as well as blood stage immunity. These effects were distinguished by clearing asymptomatic infection with anti-malarials. Exposure to multiple P. falciparum infections is associated with protective immunity, but there appears to be an additional effect in untreated multiclonal infections that offsets this protective effect.

Makani J, Cox SE, Soka D, Komba AN, Oruo J, Mwamtemi H, Magesa P, Rwezaula S, Meda E, Mgaya J et al. 2011. Mortality in sickle cell anemia in Africa: a prospective cohort study in Tanzania. PLoS One, 6 (2), pp. e14699. | Show Abstract | Read more

BACKGROUND: The World Health Organization has declared Sickle Cell Anemia (SCA) a public health priority. There are 300,000 births/year, over 75% in Africa, with estimates suggesting that 6 million Africans will be living with SCA if average survival reaches half the African norm. Countries such as United States of America and United Kingdom have reduced SCA mortality from 3 to 0.13 per 100 person years of observation (PYO), with interventions such as newborn screening, prevention of infections and comprehensive care, but implementation of interventions in African countries has been hindered by lack of locally appropriate information. The objective of this study was to determine the incidence and factors associated with death from SCA in Dar-es-Salaam. METHODS AND FINDINGS: A hospital-based cohort study was conducted, with prospective surveillance of 1,725 SCA patients recruited from 2004 to 2009, with 209 (12%) lost to follow up, while 86 died. The mortality rate was 1.9 (95%CI 1.5, 2.9) per 100 PYO, highest under 5-years old [7.3 (4.8-11.0)], adjusting for dates of birth and study enrollment. Independent risk factors, at enrollment to the cohort, predicting death were low hemoglobin (<5 g/dL) [3.8 (1.8-8.2); p = 0.001] and high total bilirubin (≥102 µmol/L) [1.7 (1.0-2.9); p = 0.044] as determined by logistic regression. CONCLUSIONS: Mortality in SCA in Africa is high, with the most vulnerable period being under 5-years old. This is most likely an underestimate, as this was a hospital cohort and may not have captured SCA individuals with severe disease who died in early childhood, those with mild disease who are undiagnosed or do not utilize services at health facilities. Prompt and effective treatment for anemia in SCA is recommended as it is likely to improve survival. Further research is required to determine the etiology, pathophysiology and the most appropriate strategies for management of anemia in SCA.

Chen DS, Barry AE, Leliwa-Sytek A, Smith TA, Peterson I, Brown SM, Migot-Nabias F, Deloron P, Kortok MM, Marsh K et al. 2011. A molecular epidemiological study of var gene diversity to characterize the reservoir of Plasmodium falciparum in humans in Africa. PLoS One, 6 (2), pp. e16629. | Show Abstract | Read more

BACKGROUND: The reservoir of Plasmodium infection in humans has traditionally been defined by blood slide positivity. This study was designed to characterize the local reservoir of infection in relation to the diverse var genes that encode the major surface antigen of Plasmodium falciparum blood stages and underlie the parasite's ability to establish chronic infection and transmit from human to mosquito. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the molecular epidemiology of the var multigene family at local sites in Gabon, Senegal and Kenya which differ in parasite prevalence and transmission intensity. 1839 distinct var gene types were defined by sequencing DBLα domains in the three sites. Only 76 (4.1%) var types were found in more than one population indicating spatial heterogeneity in var types across the African continent. The majority of var types appeared only once in the population sample. Non-parametric statistical estimators predict in each population at minimum five to seven thousand distinct var types. Similar diversity of var types was seen in sites with different parasite prevalences. CONCLUSIONS/SIGNIFICANCE: Var population genomics provides new insights into the epidemiology of P. falciparum in Africa where malaria has never been conquered. In particular, we have described the extensive reservoir of infection in local African sites and discovered a unique var population structure that can facilitate superinfection through minimal overlap in var repertoires among parasite genomes. Our findings show that var typing as a molecular surveillance system defines the extent of genetic complexity in the reservoir of infection to complement measures of malaria prevalence. The observed small scale spatial diversity of var genes suggests that var genetics could greatly inform current malaria mapping approaches and predict complex malaria population dynamics due to the import of var types to areas where no widespread pre-existing immunity in the population exists.

Olotu A, Lusingu J, Leach A, Lievens M, Vekemans J, Msham S, Lang T, Gould J, Dubois MC, Jongert E et al. 2011. Efficacy of RTS,S/AS01E malaria vaccine and exploratory analysis on anti-circumsporozoite antibody titres and protection in children aged 5-17 months in Kenya and Tanzania: a randomised controlled trial. Lancet Infect Dis, 11 (2), pp. 102-109. | Show Abstract | Read more

BACKGROUND: RTS,S/AS01E is the lead candidate malaria vaccine. We recently showed efficacy against clinical falciparum malaria in 5-17 month old children, during an average of 8 months follow-up. We aimed to assess the efficacy of RTS,S/AS01E during 15 months of follow-up. METHODS: Between March, 2007, and October, 2008, we enrolled healthy children aged 5-17 months in Kilifi, Kenya, and Korogwe, Tanzania. Computer-generated block randomisation was used to randomly assign participants (1:1) to receive three doses (at month 0, 1, and 2) of either RTS,S/AS01E or human diploid-cell rabies vaccine. The primary endpoint was time to first clinical malaria episode, defined as the presence of fever (temperature ≥37·5°C) and a Plasmodium falciparum density of 2500/μL or more. Follow-up was 12 months for children from Korogwe and 15 months for children from Kilifi. Primary analysis was per protocol. In a post-hoc modelling analysis we characterised the associations between anti-circumsporozoite antibodies and protection against clinical malaria episodes. This study is registered with ClinicalTrials.gov, number NCT00380393. FINDINGS: 894 children were assigned, 447 in each treatment group. In the per-protocol analysis, 82 of 415 children in the RTS,S/AS01E group and 125 of 420 in the rabies vaccine group had first or only clinical malaria episode by 12 months, vaccine efficacy 39·2% (95% CI 19·5-54·1, p=0·0005). At 15 months follow-up, 58 of 209 children in the RTS,S/AS01E group and 85 of 206 in the rabies vaccine group had first or only clinical malaria episode, vaccine efficacy 45·8% (24·1-61·3, p=0·0004). At 12 months after the third dose, anti-circumsporozoite antibody titre data were available for 390 children in the RTS,S/AS01E group and 391 in the rabies group. A mean of 15 months (range 12-18 months) data were available for 172 children in the RTS,S/AS01E group and 155 in the rabies group. These titres at 1 month after the third dose were not associated with protection, but titres at 6·5 months were. The level of protection increased abruptly over a narrow range of antibody concentrations. The most common adverse events were pneumonia, febrile convulsion, gastroenteritis, and P falciparum malaria. INTERPRETATION: RTS,S/AS01E confers sustained efficacy for at least 15 months and shows promise as a potential public health intervention against childhood malaria in malaria endemic countries. FUNDING: PATH Malaria Vaccine Initiative (MVI), GlaxoSmithKline.

Klein Klouwenberg P, Sasi P, Bashraheil M, Awuondo K, Bonten M, Berkley J, Marsh K, Borrmann S. 2011. Temporal association of acute hepatitis A and Plasmodium falciparum malaria in children. PLoS One, 6 (7), pp. e21013. | Show Abstract | Read more

BACKGROUND: In sub-Saharan Africa, Plasmodium falciparum and hepatitis A (HAV) infections are common, especially in children. Co-infections with these two pathogens may therefore occur, but it is unknown if temporal clustering exists. MATERIALS AND METHODS: We studied the pattern of co-infection of P. falciparum malaria and acute HAV in Kenyan children under the age of 5 years in a cohort of children presenting with uncomplicated P. falciparum malaria. HAV status was determined during a 3-month follow-up period. DISCUSSION: Among 222 cases of uncomplicated malaria, 10 patients were anti-HAV IgM positive. The incidence of HAV infections during P. falciparum malaria was 1.7 (95% CI 0.81-3.1) infections/person-year while the cumulative incidence of HAV over the 3-month follow-up period was 0.27 (95% CI 0.14-0.50) infections/person-year. Children with or without HAV co-infections had similar mean P. falciparum asexual parasite densities at presentation (31,000/µL vs. 34,000/µL, respectively), largely exceeding the pyrogenic threshold of 2,500 parasites/µL in this population and minimizing risk of over-diagnosis of malaria as an explanation. CONCLUSION: The observed temporal association between acute HAV and P. falciparum malaria suggests that co-infections of these two hepatotrophic human pathogens may result from changes in host susceptibility. Testing this hypothesis will require larger prospective studies.

Bejon P, Warimwe G, Mackintosh CL, Mackinnon MJ, Kinyanjui SM, Musyoki JN, Bull PC, Marsh K. 2011. Analysis of Immunity to Febrile Malaria in Children That Distinguishes Immunity from Lack of Exposure (vol 77, pg 1917, 2009) INFECTION AND IMMUNITY, 79 (4), pp. 1804-1804. | Read more

Olotu A, Moris P, Mwacharo J, Vekemans J, Kimani D, Janssens M, Kai O, Jongert E, Lievens M, Leach A et al. 2011. Circumsporozoite-specific T cell responses in children vaccinated with RTS,S/AS01E and protection against P falciparum clinical malaria. PLoS One, 6 (10), pp. e25786. | Show Abstract | Read more

BACKGROUND: RTS,S/AS01(E) is the lead candidate pre-erythrocytic malaria vaccine. In Phase IIb field trials the safety profile was acceptable and the efficacy was 53% (95%CI 31%-72%) for protecting children against clinical malaria caused by P. falciparum. We studied CS-specific T cell responses in order to identify correlates of protection. METHODS AND FINDINGS: We used intracellular cytokine staining (for IL2, IFNγ, and TNFα), ex-vivo ELISPOTs (IFNγ and IL2) and IFNγ cultured ELISPOT assays to characterize the CS-specific cellular responses in 407 children (5-17 months of age) in a phase IIb randomized controlled trial of RTS,S/AS01(E) (NCT00380393). RTS,S/ AS01(E) vaccinees had higher frequencies of CS-specific CD4+ T cells producing IFNγ, TNFα or IL2 compared to control vaccinees. In a multivariable analysis TNFα(+) CD4(+) T cells were independently associated with a reduced risk for clinical malaria among RTS,S/AS01(E) vaccinees (HR = 0.64, 95%CI 0.49-0.86, p = 0.002). There was a non-significant tendency towards reduced risk among control vaccinees (HR = 0.80, 95%CI 0.62-1.03, p = 0.084), albeit with lower CS-specific T cell frequencies and higher rates of clinical malaria. When data from both RTS,S/AS01(E) vaccinees and control vaccinees were combined (with adjusting for vaccination group), the HR was 0.74 (95%CI 0.62-0.89, p = 0.001). After a Bonferroni correction for multiple comparisons (n-18), the finding was still significant at p = 0.018. There was no significant correlation between cultured or ex vivo ELISPOT data and protection from clinical malaria. The combination of TNFα(+) CD4(+) T cells and anti-CS antibody statistically accounted for the protective effect of vaccination in a Cox regression model. CONCLUSIONS: RTS,S/AS01(E) induces CS-specific Th1 T cell responses in young children living in a malaria endemic area. The combination of anti-CS antibody concentrations titers and CS-specific TNFα(+) CD4(+) T cells could account for the level of protection conferred by RTS,S/AS01(E). The correlation between CS-specific TNFα(+) CD4(+) T cells and protection needs confirmation in other datasets.

Borrmann S, Sasi P, Mwai L, Bashraheil M, Abdallah A, Muriithi S, Frühauf H, Schaub B, Pfeil J, Peshu J et al. 2011. Declining responsiveness of Plasmodium falciparum infections to artemisinin-based combination treatments on the Kenyan coast. PLoS One, 6 (11), pp. e26005. | Show Abstract | Read more

BACKGROUND: The emergence of artemisinin-resistant P. falciparum malaria in South-East Asia highlights the need for continued global surveillance of the efficacy of artemisinin-based combination therapies. METHODS: On the Kenyan coast we studied the treatment responses in 474 children 6-59 months old with uncomplicated P. falciparum malaria in a randomized controlled trial of dihydroartemisinin-piperaquine vs. artemether-lumefantrine from 2005 to 2008. (ISRCTN88705995). RESULTS: The proportion of patients with residual parasitemia on day 1 rose from 55% in 2005-2006 to 87% in 2007-2008 (odds ratio, 5.4, 95%CI, 2.7-11.1; P<0.001) and from 81% to 95% (OR, 4.1, 95%CI, 1.7-9.9; P = 0.002) in the DHA-PPQ and AM-LM groups, respectively. In parallel, Kaplan-Meier estimated risks of apparent recrudescent infection by day 84 increased from 7% to 14% (P = 0.1) and from 6% to 15% (P = 0.05) with DHA-PPQ and AM-LM, respectively. Coinciding with decreasing transmission in the study area, clinical tolerance to parasitemia (defined as absence of fever) declined between 2005-2006 and 2007-2008 (OR body temperature >37.5°C, 2.8, 1.9-4.1; P<0.001). Neither in vitro sensitivity of parasites to DHA nor levels of antibodies against parasite extract accounted for parasite clearance rates or changes thereof. CONCLUSIONS: The significant, albeit small, decline through time of parasitological response rates to treatment with ACTs may be due to the emergence of parasites with reduced drug sensitivity, to the coincident reduction in population-level clinical immunity, or both. Maintaining the efficacy of artemisinin-based therapy in Africa would benefit from a better understanding of the mechanisms underlying reduced parasite clearance rates. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN88705995.

Weiss GE, Ndungu FM, McKittrick N, Li S, Kimani D, Crompton PD, Marsh K, Pierce SK. 2012. High efficiency human memory B cell assay and its application to studying Plasmodium falciparum-specific memory B cells in natural infections. J Immunol Methods, 375 (1-2), pp. 68-74. | Show Abstract | Read more

Memory B cells (MBCs) are a key component of long term humoral immunity to many human infectious diseases. Despite their importance, we know little about the generation or maintenance of antigen-(Ag)-specific MBCs in humans in response to infection. A frequently employed method for quantifying Ag-specific MBCs in human peripheral blood (Crotty et al., 2004) relies on the ability of MBCs but not naïve B cells to differentiate into antibody secreting cells (ASCs) in response to polyclonal activators and Toll-like receptor agonists in vitro and the measurement of Ag-specific ASCs by ELISPOT assays. Here we report on studies to optimize the efficiency of this ELISPOT-based assay and to apply this assay to the detection of Plasmodium falciparum (Pf)-specific MBCs in adults living in a malaria endemic area where immunity to Pf is acquired through natural infection. We show that the addition of IL-10 to in vitro cultures of human peripheral blood mononuclear cells increased the efficiency of the assay from 10% to over 90% without increasing the ASC burst size and without any substantial increase in background from naïve B cells or plasma cells (PCs). Using this assay we were able to quantify the frequency of Pf-specific MBCs in peripheral blood of adults living in a malaria endemic area. Thus, this highly efficient assay appears to be well suited to field studies of the generation and maintenance of MBCs where the volumes of blood obtainable are often limiting.

Cox SE, Makani J, Fulford AJ, Komba AN, Soka D, Williams TN, Newton CR, Marsh K, Prentice AM. 2011. Nutritional status, hospitalization and mortality among patients with sickle cell anemia in Tanzania. Haematologica, 96 (7), pp. 948-953. | Show Abstract | Read more

BACKGROUND: Reduced growth is common in children with sickle cell anemia, but few data exist on associations with long-term clinical course. Our objective was to determine the prevalence of malnutrition at enrollment into a hospital-based cohort and whether poor nutritional status predicted morbidity and mortality within an urban cohort of Tanzanian sickle cell anemia patients. DESIGN AND METHODS: Anthropometry was conducted at enrollment into the sickle cell anemia cohort (n=1,618; ages 0.5-48 years) and in controls who attended screening (siblings, walk-ins and referrals) but who were found not to have sickle cell anemia (n=717; ages 0.5-64 years). Prospective surveillance recorded hospitalization at Muhimbili National Hospital and mortality between March 2004 and September 2009. RESULTS: Sickle cell anemia was associated with stunting (OR=1.92, P<0.001, 36.2%) and wasting (OR=1.66, P=0.002, 18.4%). The greatest growth deficits were observed in adolescents and in boys. Independent of age and sex, lower hemoglobin concentration was associated with increased odds of malnutrition in sickle cell patients. Of the 1,041 sickle cell anemia patients with a body mass index z-score at enrollment, 92% were followed up until September 2009 (n=908) or death (n=50). Body mass index and weight-for-age z-score predicted hospitalization (hazard ratio [HZR]=0.90, P=0.04 and HZR=0.88, P=0.02) but height-for-age z-score did not (HZR=0.93, NS). The mortality rate of 2.5 per 100 person-years was not associated with any of the anthropometric measures. CONCLUSIONS: In this non-birth-cohort of sickle cell anemia with significant associated undernutrition, wasting predicted an increased risk of hospital admission. Targeted nutritional interventions should prioritize treatment and prevention of wasting.

Campino S, Auburn S, Kivinen K, Zongo I, Ouedraogo JB, Mangano V, Djimde A, Doumbo OK, Kiara SM, Nzila A et al. 2011. Population genetic analysis of Plasmodium falciparum parasites using a customized Illumina GoldenGate genotyping assay. PLoS One, 6 (6), pp. e20251. | Show Abstract | Read more

The diversity in the Plasmodium falciparum genome can be used to explore parasite population dynamics, with practical applications to malaria control. The ability to identify the geographic origin and trace the migratory patterns of parasites with clinically important phenotypes such as drug resistance is particularly relevant. With increasing single-nucleotide polymorphism (SNP) discovery from ongoing Plasmodium genome sequencing projects, a demand for high SNP and sample throughput genotyping platforms for large-scale population genetic studies is required. Low parasitaemias and multiple clone infections present a number of challenges to genotyping P. falciparum. We addressed some of these issues using a custom 384-SNP Illumina GoldenGate assay on P. falciparum DNA from laboratory clones (long-term cultured adapted parasite clones), short-term cultured parasite isolates and clinical (non-cultured isolates) samples from East and West Africa, Southeast Asia and Oceania. Eighty percent of the SNPs (n = 306) produced reliable genotype calls on samples containing as little as 2 ng of total genomic DNA and on whole genome amplified DNA. Analysis of artificial mixtures of laboratory clones demonstrated high genotype calling specificity and moderate sensitivity to call minor frequency alleles. Clear resolution of geographically distinct populations was demonstrated using Principal Components Analysis (PCA), and global patterns of population genetic diversity were consistent with previous reports. These results validate the utility of the platform in performing population genetic studies of P. falciparum.

Scott JA, Berkley JA, Mwangi I, Ochola L, Uyoga S, Macharia A, Ndila C, Lowe BS, Mwarumba S, Bauni E et al. 2011. Relation between falciparum malaria and bacteraemia in Kenyan children: a population-based, case-control study and a longitudinal study. Lancet, 378 (9799), pp. 1316-1323. | Show Abstract | Read more

BACKGROUND: Many investigators have suggested that malaria infection predisposes individuals to bacteraemia. We tested this hypothesis with mendelian randomisation studies of children with the malaria-protective phenotype of sickle-cell trait (HbAS). METHODS: This study was done in a defined area around Kilifi District Hospital, Kilifi, Kenya. We did a matched case-control study to identify risk factors for invasive bacterial disease, in which cases were children aged 3 months to 13 years who were admitted to hospital with bacteraemia between Sept 16, 1999, and July 31, 2002. We aimed to match two controls, by age, sex, location, and time of recruitment, for every case. We then did a longitudinal case-control study to assess the relation between HbAS and invasive bacterial disease as malaria incidence decreased. Cases were children aged 0-13 years who were admitted to hospital with bacteraemia between Jan 1, 1999, and Dec 31, 2007. Controls were born in the study area between Jan 1, 2006, and June 23, 2009. Finally, we modelled the annual incidence of bacteraemia against the community prevalence of malaria during 9 years with Poisson regression. RESULTS: In the matched case-control study, we recruited 292 cases-we recruited two controls for 236, and one for the remaining 56. Sickle-cell disease, HIV, leucocyte haemozoin pigment, and undernutrition were positively associated with bacteraemia and HbAS was strongly negatively associated with bacteraemia (odds ratio 0·36; 95% CI 0·20-0·65). In the longitudinal case-control study, we assessed data from 1454 cases and 10,749 controls. During the study period, the incidence of admission to hospital with malaria per 1000 child-years decreased from 28·5 to 3·45, with a reduction in protection afforded by HbAS against bacteraemia occurring in parallel (p=0·0008). The incidence of hospital admissions for bacteraemia per 1000 child-years also decreased from 2·59 to 1·45. The bacteraemia incidence rate ratio associated with malaria parasitaemia was 6·69 (95% CI 1·31-34·3) and, at a community parasite prevalence of 29% in 1999, 62% (8·2-91) of bacteraemia cases were attributable to malaria. INTERPRETATION: Malaria infection strongly predisposes individuals to bacteraemia and can account for more than half of all cases of bacteraemia in malaria-endemic areas. Interventions to control malaria will have a major additional benefit by reducing the burden of invasive bacterial disease. FUNDING: Wellcome Trust.

Bejon P, Turner L, Lavstsen T, Cham G, Olotu A, Drakeley CJ, Lievens M, Vekemans J, Savarese B, Lusingu J et al. 2011. Serological evidence of discrete spatial clusters of Plasmodium falciparum parasites. PLoS One, 6 (6), pp. e21711. | Show Abstract | Read more

BACKGROUND: Malaria transmission may be considered to be homogenous with well-mixed parasite populations (as in the classic Ross/Macdonald models). Marked fine-scale heterogeneity of transmission has been observed in the field (i.e., over a few kilometres), but there are relatively few data on the degree of mixing. Since the Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) is highly polymorphic, the host's serological responses may be used to infer exposure to parasite sub-populations. METHODS AND FINDINGS: We measured the antibody responses to 46 individual PfEMP1 domains at four time points among 450 children in Kenya, and identified distinct spatial clusters of antibody responses to individual domains. 35 domains showed strongly significant sero-clusters at p = 0.001. Individuals within the high transmission hotspot showed the greatest diversity of anti-PfEMP1 responses. Individuals outside the hotspot had a less diverse range of responses, even if as individuals they were at relatively intense exposure. CONCLUSIONS: We infer that antigenically distinct sub-populations of parasites exist on a fine spatial scale in a study area of rural Kenya. Further studies should examine antigenic variation over longer periods of time and in different study areas.

Klouwenberg PK, Sasi P, Bashraheil M, Awuondo K, Bonten M, Berkley J, Marsh K, Borrmann S. 2011. Temporal Association of Acute Hepatitis A and Plasmodium falciparum Malaria in Children PLOS ONE, 6 (7), pp. e21013-e21013. | Read more

Douglas AD, Williams AR, Illingworth JJ, Kamuyu G, Biswas S, Goodman AL, Wyllie DH, Crosnier C, Miura K, Wright GJ et al. 2011. The blood-stage malaria antigen PfRH5 is susceptible to vaccine-inducible cross-strain neutralizing antibody. Nat Commun, 2 (1), pp. 601. | Show Abstract | Read more

Current vaccine strategies against the asexual blood stage of Plasmodium falciparum are mostly focused on well-studied merozoite antigens that induce immune responses after natural exposure, but have yet to induce robust protection in any clinical trial. Here we compare human-compatible viral-vectored vaccines targeting ten different blood-stage antigens. We show that the full-length P. falciparum reticulocyte-binding protein homologue 5 (PfRH5) is highly susceptible to cross-strain neutralizing vaccine-induced antibodies, out-performing all other antigens delivered by the same vaccine platform. We find that, despite being susceptible to antibody, PfRH5 is unlikely to be under substantial immune selection pressure; there is minimal acquisition of anti-PfRH5 IgG antibodies in malaria-exposed Kenyans. These data challenge the widespread beliefs that any merozoite antigen that is highly susceptible to immune attack would be subject to significant levels of antigenic polymorphism, and that erythrocyte invasion by P. falciparum is a degenerate process involving a series of parallel redundant pathways.

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Scopus

Nduati E, Gwela A, Karanja H, Mugyenyi C, Langhorne J, Marsh K, Urban BC. 2011. The plasma concentration of the B cell activating factor is increased in children with acute malaria Journal of Infectious Diseases, 204 (6), pp. 962-970. | Show Abstract | Read more

Malaria-specific antibody responses in children often appear to be short-lived but the mechanisms underlying this phenomenon are not well understood. In this study, we investigated the relationship between the B-cell activating factor (BAFF) and its receptors expressed on B cells with antibody responses during and after acute malaria in children. Our results demonstrate that BAFF plasma levels increased during acute malarial disease and reflected disease severity. The expression profiles for BAFF receptors on B cells agreed with rapid activation and differentiation of a proportion of B cells to plasma cells. However, BAFF receptor (BAFF-R) expression was reduced on all peripheral blood B cells during acute infection, but those children with the highest level of BAFF-R expression on B cells maintained schizont-specific immunoglobin G (IgG) over a period of 4 months, indicating that dysregulation of BAFF-R expression on B cells may contribute to short-lived antibody responses to malarial antigens in children. In summary, this study suggests a potential role for BAFF during malaria disease, both as a marker for disease severity and in shaping the differentiation pattern of antigen-specific B cells. © The Author 2011. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved.

Olotu A, Fegan G, Williams TN, Sasi P, Ogada E, Bauni E, Wambua J, Marsh K, Borrmann S, Bejon P. 2010. Defining clinical malaria: the specificity and incidence of endpoints from active and passive surveillance of children in rural Kenya. PLoS One, 5 (12), pp. e15569. | Show Abstract | Read more

BACKGROUND: Febrile malaria is the most common clinical manifestation of P. falciparum infection, and is often the primary endpoint in clinical trials and epidemiological studies. Subjective and objective fevers are both used to define the endpoint, but have not been carefully compared, and the relative incidence of clinical malaria by active and passive case detection is unknown. METHODS: We analyzed data from cohorts under active and passive surveillance, including 19,462 presentations with fever and 5,551 blood tests for asymptomatic parasitaemia. A logistic regression model was used to calculate Malaria Attributable Fractions (MAFs) for various case definitions. Incidences of febrile malaria by active and passive surveillance were compared in a subset of children matched for age and location. RESULTS: Active surveillance identified three times the incidence of clinical malaria as passive surveillance in a subset of children matched for age and location. Objective fever (temperature≥37.5°C) gave consistently higher MAFs than case definitions based on subjective fever. CONCLUSION: The endpoints from active and passive surveillance have high specificity, but the incidence of endpoints is lower on passive surveillance. Subjective fever had low specificity and should not be used in primary endpoint. Passive surveillance will reduce the power of clinical trials but may cost-effectively deliver acceptable sensitivity in studies of large populations.

Lusingu J, Olotu A, Leach A, Lievens M, Vekemans J, Olivier A, Benns S, Olomi R, Msham S, Lang T et al. 2010. Safety of the malaria vaccine candidate, RTS,S/AS01E in 5 to 17 month old Kenyan and Tanzanian Children. PLoS One, 5 (11), pp. e14090. | Show Abstract | Read more

The malaria vaccine candidate, RTS,S/AS01(E), showed promising protective efficacy in a trial of Kenyan and Tanzanian children aged 5 to 17 months. Here we report on the vaccine's safety and tolerability. The experimental design was a Phase 2b, two-centre, double-blind (observer- and participant-blind), randomised (1∶1 ratio) controlled trial. Three doses of study or control (rabies) vaccines were administered intramuscularly at 1 month intervals. Solicited adverse events (AEs) were collected for 7 days after each vaccination. There was surveillance and reporting for unsolicited adverse events for 30 days after each vaccination. Serious adverse events (SAEs) were recorded throughout the study period which lasted for 14 months after dose 1 in Korogwe, Tanzania and an average of 18 months post-dose 1 in Kilifi, Kenya. Blood samples for safety monitoring of haematological, renal and hepatic functions were taken at baseline, 3, 10 and 14 months after dose 1. A total of 894 children received RTS,S/AS01(E) or rabies vaccine between March and August 2007. Overall, children vaccinated with RTS,S/AS01(E) had fewer SAEs (51/447) than children in the control group (88/447). One SAE episode in a RTS,S/AS01(E) recipient and nine episodes among eight rabies vaccine recipients met the criteria for severe malaria. Unsolicited AEs were reported in 78% of subjects in the RTS,S/AS01(E) group and 74% of subjects in the rabies vaccine group. In both vaccine groups, gastroenteritis and pneumonia were the most frequently reported unsolicited AE. Fever was the most frequently observed solicited AE and was recorded after 11% of RTS,S/AS01(E) doses compared to 31% of doses of rabies vaccine. The candidate vaccine RTS,S/AS01(E) showed an acceptable safety profile in children living in a malaria-endemic area in East Africa. More data on the safety of RTS,S/AS01(E) will become available from the Phase 3 programme.

Marsh K. 2010. Research priorities for malaria elimination. Lancet, 376 (9753), pp. 1626-1627. | Read more

Ochola LI, Tetteh KK, Stewart LB, Riitho V, Marsh K, Conway DJ. 2010. Allele frequency-based and polymorphism-versus-divergence indices of balancing selection in a new filtered set of polymorphic genes in Plasmodium falciparum. Mol Biol Evol, 27 (10), pp. 2344-2351. | Show Abstract | Read more

Signatures of balancing selection operating on specific gene loci in endemic pathogens can identify candidate targets of naturally acquired immunity. In malaria parasites, several leading vaccine candidates convincingly show such signatures when subjected to several tests of neutrality, but the discovery of new targets affected by selection to a similar extent has been slow. A small minority of all genes are under such selection, as indicated by a recent study of 26 Plasmodium falciparum merozoite-stage genes that were not previously prioritized as vaccine candidates, of which only one (locus PF10_0348) showed a strong signature. Therefore, to focus discovery efforts on genes that are polymorphic, we scanned all available shotgun genome sequence data from laboratory lines of P. falciparum and chose six loci with more than five single nucleotide polymorphisms per kilobase (including PF10_0348) for in-depth frequency-based analyses in a Kenyan population (allele sample sizes >50 for each locus) and comparison of Hudson-Kreitman-Aguade (HKA) ratios of population diversity (π) to interspecific divergence (K) from the chimpanzee parasite Plasmodium reichenowi. Three of these (the msp3/6-like genes PF10_0348 and PF10_0355 and the surf(4.1) gene PFD1160w) showed exceptionally high positive values of Tajima's D and Fu and Li's F indices and have the highest HKA ratios, indicating that they are under balancing selection and should be prioritized for studies of their protein products as candidate targets of immunity. Combined with earlier results, there is now strong evidence that high HKA ratio (as well as the frequency-independent ratio of Watterson's /K) is predictive of high values of Tajima's D. Thus, the former offers value for use in genome-wide screening when numbers of genome sequences within a species are low or in combination with Tajima's D as a 2D test on large population genomic samples.

Osier FH, Weedall GD, Verra F, Murungi L, Tetteh KK, Bull P, Faber BW, Remarque E, Thomas A, Marsh K, Conway DJ. 2010. Allelic diversity and naturally acquired allele-specific antibody responses to Plasmodium falciparum apical membrane antigen 1 in Kenya. Infect Immun, 78 (11), pp. 4625-4633. | Show Abstract | Read more

Although Plasmodium falciparum apical membrane antigen 1 (AMA1) is a leading malaria vaccine candidate, extensive allelic diversity may compromise its vaccine potential. We have previously shown that naturally acquired antibodies to AMA1 were associated with protection from clinical malaria in this Kenyan population. To assess the impact of allelic diversity on naturally acquired immunity, we first sequenced the ectodomain-encoding region of P. falciparum ama1 from subjects with asymptomatic, mild, and severe malaria and measured allele frequency distributions. We then measured antibodies to three allelic AMA1 proteins (AMA1_3D7, AMA1_FVO, and AMA1_HB3) and used competition enzyme-linked immunosorbent assays (ELISAs) to analyze allele-specific antibodies. Seventy-eight unique haplotypes were identified from 129 alleles sampled. No clustering of allelic haplotypes with disease severity or year of sampling was observed. Differences in nucleotide frequencies in clinical (severe plus mild malaria) versus asymptomatic infections were observed at 16 polymorphic positions. Allele frequency distributions were indicative of balancing selection, with the strongest signature being identified in domain III (Tajima's D = 2.51; P < 0.05). Antibody reactivities to each of the three allelic AMA1 proteins were highly correlated (P < 0.001 for all pairwise comparisons). Although antibodies to conserved epitopes were abundant, 48% of selected children with anti-AMA1 IgG (n = 106) had detectable reactivity to allele-specific epitopes as determined by a competition ELISA. Antibodies to both conserved and allele-specific epitopes in AMA1 may contribute to clinical protection.

Bejon P, Williams TN, Liljander A, Noor AM, Wambua J, Ogada E, Olotu A, Osier FH, Hay SI, Färnert A, Marsh K. 2010. Stable and unstable malaria hotspots in longitudinal cohort studies in Kenya. PLoS Med, 7 (7), pp. e1000304. | Show Abstract | Read more

BACKGROUND: Infectious diseases often demonstrate heterogeneity of transmission among host populations. This heterogeneity reduces the efficacy of control strategies, but also implies that focusing control strategies on "hotspots" of transmission could be highly effective. METHODS AND FINDINGS: In order to identify hotspots of malaria transmission, we analysed longitudinal data on febrile malaria episodes, asymptomatic parasitaemia, and antibody titres over 12 y from 256 homesteads in three study areas in Kilifi District on the Kenyan coast. We examined heterogeneity by homestead, and identified groups of homesteads that formed hotspots using a spatial scan statistic. Two types of statistically significant hotspots were detected; stable hotspots of asymptomatic parasitaemia and unstable hotspots of febrile malaria. The stable hotspots were associated with higher average AMA-1 antibody titres than the unstable clusters (optical density [OD] = 1.24, 95% confidence interval [CI] 1.02-1.47 versus OD = 1.1, 95% CI 0.88-1.33) and lower mean ages of febrile malaria episodes (5.8 y, 95% CI 5.6-6.0 versus 5.91 y, 95% CI 5.7-6.1). A falling gradient of febrile malaria incidence was identified in the penumbrae of both hotspots. Hotspots were associated with AMA-1 titres, but not seroconversion rates. In order to target control measures, homesteads at risk of febrile malaria could be predicted by identifying the 20% of homesteads that experienced an episode of febrile malaria during one month in the dry season. That 20% subsequently experienced 65% of all febrile malaria episodes during the following year. A definition based on remote sensing data was 81% sensitive and 63% specific for the stable hotspots of asymptomatic malaria. CONCLUSIONS: Hotspots of asymptomatic parasitaemia are stable over time, but hotspots of febrile malaria are unstable. This finding may be because immunity offsets the high rate of febrile malaria that might otherwise result in stable hotspots, whereas unstable hotspots necessarily affect a population with less prior exposure to malaria.

Idro R, Marsh K, John CC, Newton CR. 2010. Cerebral malaria: mechanisms of brain injury and strategies for improved neurocognitive outcome. Pediatr Res, 68 (4), pp. 267-274. | Show Abstract | Read more

Cerebral malaria is the most severe neurological complication of infection with Plasmodium falciparum. With >575,000 cases annually, children in sub-Saharan Africa are the most affected. Surviving patients have an increased risk of neurological and cognitive deficits, behavioral difficulties, and epilepsy making cerebral malaria a leading cause of childhood neurodisability in the region. The pathogenesis of neurocognitive sequelae is poorly understood: coma develops through multiple mechanisms and there may be several mechanisms of brain injury. It is unclear how an intravascular parasite causes such brain injury. Understanding these mechanisms is important to develop appropriate neuroprotective interventions. This article examines possible mechanisms of brain injury in cerebral malaria, relating this to the pathogenesis of the disease, and explores prospects for improved neurocognitive outcome.

McAuley CF, Webb C, Makani J, Macharia A, Uyoga S, Opi DH, Ndila C, Ngatia A, Scott JA, Marsh K, Williams TN. 2010. High mortality from Plasmodium falciparum malaria in children living with sickle cell anemia on the coast of Kenya. Blood, 116 (10), pp. 1663-1668. | Show Abstract | Read more

Although malaria is widely considered a major cause of death in young children born with sickle cell anemia (SCA) in sub-Saharan Africa, this is poorly quantified. We attempted to investigate this question through 4 large case-control analyses involving 7164 children living on the coast of Kenya. SCA was associated with an increased risk of admission to hospital both with nonmalaria diseases in general (odds ratio [OR] = 4.17; 95% confidence interval [CI], 1.95-8.92; P < .001) and with invasive bacterial diseases in particular (OR = 8.73; 95% CI, 4.51-16.89; P < .001). We found no evidence for a strongly increased risk of either uncomplicated malaria (OR = 0.43; 95% CI, 0.09-2.10; P = .30) or malaria complicated by a range of well-described clinical features of severity (OR = 0.80; 95% CI, 0.25-2.51; P = .70) overall; nevertheless, mortality was considerably higher among SCA than non-SCA children hospitalized with malaria. Our findings highlight both the central role that malaria plays in the high early mortality seen in African children with SCA and the urgent need for better quantitative data. Meanwhile, our study confirms the importance of providing all children living with SCA in malaria-endemic areas with effective prophylaxis.

Khor CC, Vannberg FO, Chapman SJ, Guo H, Wong SH, Walley AJ, Vukcevic D, Rautanen A, Mills TC, Chang KC et al. 2010. CISH and susceptibility to infectious diseases. N Engl J Med, 362 (22), pp. 2092-2101. | Show Abstract | Read more

BACKGROUND: The interleukin-2-mediated immune response is critical for host defense against infectious pathogens. Cytokine-inducible SRC homology 2 (SH2) domain protein (CISH), a suppressor of cytokine signaling, controls interleukin-2 signaling. METHODS: Using a case-control design, we tested for an association between CISH polymorphisms and susceptibility to major infectious diseases (bacteremia, tuberculosis, and severe malaria) in blood samples from 8402 persons in Gambia, Hong Kong, Kenya, Malawi, and Vietnam. We had previously tested 20 other immune-related genes in one or more of these sample collections. RESULTS: We observed associations between variant alleles of multiple CISH polymorphisms and increased susceptibility to each infectious disease in each of the study populations. When all five single-nucleotide polymorphisms (SNPs) (at positions -639, -292, -163, +1320, and +3415 [all relative to CISH]) within the CISH-associated locus were considered together in a multiple-SNP score, we found an association between CISH genetic variants and susceptibility to bacteremia, malaria, and tuberculosis (P=3.8x10(-11) for all comparisons), with -292 accounting for most of the association signal (P=4.58x10(-7)). Peripheral-blood mononuclear cells obtained from adult subjects carrying the -292 variant, as compared with wild-type cells, showed a muted response to the stimulation of interleukin-2 production--that is, 25 to 40% less CISH expression. CONCLUSIONS: Variants of CISH are associated with susceptibility to diseases caused by diverse infectious pathogens, suggesting that negative regulators of cytokine signaling have a role in immunity against various infectious diseases. The overall risk of one of these infectious diseases was increased by at least 18% among persons carrying the variant CISH alleles.

Mackinnon MJ, Marsh K. 2010. The selection landscape of malaria parasites. Science, 328 (5980), pp. 866-871. | Show Abstract | Read more

Malaria parasites have to survive and transmit within a highly selective and ever-changing host environment. Because immunity to malaria is nonsterilizing and builds up slowly through repeated infections, commonly the parasite invades a host that is immunologically and physiologically different from its previous host. During the course of infection, the parasite must also keep pace with changes in host immune responses and red-blood-cell physiology. Here, we describe the "selection landscape" of the most virulent of the human malaria parasites, Plasmodium falciparum, and the adaptive mechanisms it uses to navigate through that landscape. Taking a cost-benefit view of parasite fitness, we consider the evolutionary outcomes of the most important forces of selection operating on the parasite, namely immunity, host death, drugs, mosquito availability, and coinfection. Given the huge potential for malaria parasite evolution in the context of the recently renewed effort to eradicate malaria, a deeper understanding of P. falciparum adaptation is essential.

Snow RW, Marsh K. 2010. Malaria in Africa: progress and prospects in the decade since the Abuja Declaration. Lancet, 376 (9735), pp. 137-139. | Read more

Osier FH, Murungi LM, Fegan G, Tuju J, Tetteh KK, Bull PC, Conway DJ, Marsh K. 2010. Allele-specific antibodies to Plasmodium falciparum merozoite surface protein-2 and protection against clinical malaria. Parasite Immunol, 32 (3), pp. 193-201. | Show Abstract | Read more

IgG and IgG3 antibodies to merozoite surface protein-2 (MSP-2) of Plasmodium falciparum have been associated with protection from clinical malaria in independent studies. We determined whether this protection was allele-specific by testing whether children who developed clinical malaria lacked IgG/IgG3 antibodies specific to the dominant msp2 parasite genotypes detected during clinical episodes. We analysed pre-existing IgG and IgG1/IgG3 antibodies to antigens representing the major dimorphic types of MSP-2 by ELISA. We used quantitative real-time PCR to determine the dominant msp2 alleles in parasites detected in clinical episodes. Over half (55%, 80/146) of infections contained both allelic types. Single or dominant IC1- and FC27-like alleles were detected in 46% and 42% of infections respectively, and both types were equally dominant in 12%. High levels of IgG/IgG3 antibodies to the FC27-like antigen were not significantly associated with a lower likelihood of clinical episodes caused by parasites bearing FC27-like compared to IC1-like alleles, and vice versa for IgG/IgG3 antibodies to the IC1-like antigen. These findings were supported by competition ELISAs which demonstrated the presence of IgG antibodies to allele-specific epitopes within both antigens. Thus, even for this well-studied antigen, the importance of an allele-specific component of naturally acquired protective immunity to malaria remains to be confirmed.

Makani J, Komba AN, Cox SE, Oruo J, Mwamtemi K, Kitundu J, Magesa P, Rwezaula S, Meda E, Mgaya J et al. 2010. Malaria in patients with sickle cell anemia: burden, risk factors, and outcome at the outpatient clinic and during hospitalization. Blood, 115 (2), pp. 215-220. | Show Abstract | Read more

Approximately 280,000 children are born with sickle cell anemia (SCA) in Africa annually, yet few survive beyond childhood. Falciparum malaria is considered a significant cause of this mortality. We conducted a 5-year prospective surveillance study for malaria parasitemia, clinical malaria, and severe malarial anemia (SMA) in Dar-es-Salaam, Tanzania, between 2004 and 2009. We recorded 10,491 visits to the outpatient clinic among 1808 patients with SCA and 773 visits among 679 patients without SCA. Similarly, we recorded 691 hospital admissions among 497 patients with SCA and 2017 in patients without SCA. Overall, the prevalence of parasitemia was lower in patients with SCA than in patients without SCA both at clinic (0.7% vs 1.6%; OR, 0.53; 95% CI, 0.32-0.86; P = .008) and during hospitalization (3.0% vs 5.6%; OR, 0.46; 95% CI, 0.25-0.94; P = .01). Furthermore, patients with SCA had higher rates of malaria during hospitalization than at clinic, the ORs being 4.29 (95% CI, 2.63-7.01; P < .001) for parasitemia, 17.66 (95% CI, 5.92-52.71; P < .001) for clinical malaria, and 21.11 (95% CI, 8.46-52.67; P < .001) for SMA. Although malaria was rare among patients with SCA, parasitemia during hospitalization was associated with both severe anemia and death. Effective treatment for malaria during severe illness episodes and further studies to determine the role chemoprophylaxis are required.

Mwaniki MK, Gatakaa HW, Mturi FN, Chesaro CR, Chuma JM, Peshu NM, Mason L, Kager P, Marsh K, English M et al. 2010. An increase in the burden of neonatal admissions to a rural district hospital in Kenya over 19 years. BMC Public Health, 10 (1), pp. 591. | Show Abstract | Read more

BACKGROUND: Most of the global neonatal deaths occur in developing nations, mostly in rural homes. Many of the newborns who receive formal medical care are treated in rural district hospitals and other peripheral health centres. However there are no published studies demonstrating trends in neonatal admissions and outcome in rural health care facilities in resource poor regions. Such information is critical in planning public health interventions. In this study we therefore aimed at describing the pattern of neonatal admissions to a Kenyan rural district hospital and their outcome over a 19 year period, examining clinical indicators of inpatient neonatal mortality and also trends in utilization of a rural hospital for deliveries. METHODS: Prospectively collected data on neonates is compared to non-neonatal paediatric (≤ 5 years old) admissions and deliveries' in the maternity unit at Kilifi District Hospital from January 1(st) 1990 up to December 31(st) 2008, to document the pattern of neonatal admissions, deliveries and changes in inpatient deaths. Trends were examined using time series models with likelihood ratios utilised to identify indicators of inpatient neonatal death. RESULTS: The proportion of neonatal admissions of the total paediatric ≤ 5 years admissions significantly increased from 11% in 1990 to 20% by 2008 (trend 0.83 (95% confidence interval 0.45-1.21). Most of the increase in burden was from neonates born in hospital and very young neonates aged < 7 days. Hospital deliveries also increased significantly. Clinical diagnoses of neonatal sepsis, prematurity, neonatal jaundice, neonatal encephalopathy, tetanus and neonatal meningitis accounted for over 75% of the inpatient neonatal admissions. Inpatient case fatality for all ≤ 5 years declined significantly over the 19 years. However, neonatal deaths comprised 33% of all inpatient death among children aged ≤ 5 years in 1990, this increased to 55% by 2008. Tetanus 256/390 (67%), prematurity 554/1,280(43%) and neonatal encephalopathy 253/778(33%) had the highest case fatality. A combination of six indicators: irregular respiration, oxygen saturation of <90%, pallor, neck stiffness, weight < 1.5 kg, and abnormally elevated blood glucose > 7 mmol/l predicted inpatient neonatal death with a sensitivity of 81% and a specificity of 68%. CONCLUSIONS: There is clear evidence of increasing burden in neonatal admissions at a rural district hospital in contrast to reducing numbers of non-neonatal paediatrics' admissions aged ≤ 5 years. Though the inpatient case fatality for all admissions aged ≤ 5 years declined significantly, neonates now comprise close to 60% of all inpatient deaths. Simple indicators may identify neonates at risk of death.

Lang TA, White NJ, Tran HT, Farrar JJ, Day NP, Fitzpatrick R, Angus BJ, Denis E, Merson L, Cheah PY et al. 2010. Clinical research in resource-limited settings: enhancing research capacity and working together to make trials less complicated. PLoS Negl Trop Dis, 4 (6), pp. e619. | Read more

Mwaniki MK, Talbert AW, Mturi FN, Berkley JA, Kager P, Marsh K, Newton CR. 2010. Congenital and neonatal malaria in a rural Kenyan district hospital: an eight-year analysis. Malar J, 9 (1), pp. 313. | Show Abstract | Read more

BACKGROUND: Malaria remains a significant burden in sub-Saharan Africa. However, data on burden of congenital and neonatal malaria is scarce and contradictory, with some recent studies reporting a high burden. Using prospectively collected data on neonatal admissions to a rural district hospital in a region of stable malaria endemicity in Kenya, the prevalence of congenital and neonatal malaria was described. METHODS: From 1st January 2002 to 31st December 2009, admission and discharge information on all neonates admitted to Kilifi District Hospital was collected. At admission, blood was also drawn for routine investigations, which included a full blood count, blood culture and blood slide for malaria parasites. RESULTS: Of the 5,114 neonates admitted during the eight-year surveillance period, blood slide for malaria parasites was performed in 4,790 (93.7%). 18 (0.35%) neonates with Plasmodium falciparum malaria parasitaemia, of whom 11 were admitted within the first week of life and thus classified as congenital parasitaemia, were identified. 7/18 (39%) had fever. Parasite densities were low, ≤50 per μl in 14 cases. The presence of parasitaemia was associated with low haemoglobin (Hb) of <10 g/dl (χ² 10.9 P = 0.001). The case fatality rate of those with and without parasitaemia was similar. Plasmodium falciparum parasitaemia was identified as the cause of symptoms in four neonates. CONCLUSION: Congenital and neonatal malaria are rare in this malaria endemic region. Performing a blood slide for malaria parasites among sick neonates in malaria endemic regions is advisable. This study does not support routine treatment with anti-malarial drugs among admitted neonates with or without fever even in a malaria endemic region.

Olotu A, Fegan G, Williams TN, Sasi P, Ogada E, Bauni E, Wambua J, Marsh K, Borrmann S, Bejon P. 2010. Defining clinical malaria: the specificity and incidence of endpoints from active and passive surveillance of children in rural Kenya. PloS one, 5 (12), | Show Abstract

Febrile malaria is the most common clinical manifestation of P. falciparum infection, and is often the primary endpoint in clinical trials and epidemiological studies. Subjective and objective fevers are both used to define the endpoint, but have not been carefully compared, and the relative incidence of clinical malaria by active and passive case detection is unknown. We analyzed data from cohorts under active and passive surveillance, including 19,462 presentations with fever and 5,551 blood tests for asymptomatic parasitaemia. A logistic regression model was used to calculate Malaria Attributable Fractions (MAFs) for various case definitions. Incidences of febrile malaria by active and passive surveillance were compared in a subset of children matched for age and location. Active surveillance identified three times the incidence of clinical malaria as passive surveillance in a subset of children matched for age and location. Objective fever (temperature≥37.5°C) gave consistently higher MAFs than case definitions based on subjective fever. The endpoints from active and passive surveillance have high specificity, but the incidence of endpoints is lower on passive surveillance. Subjective fever had low specificity and should not be used in primary endpoint. Passive surveillance will reduce the power of clinical trials but may cost-effectively deliver acceptable sensitivity in studies of large populations.

Bejon P, Ogada E, Peshu N, Marsh K. 2009. Interactions between age and ITN use determine the risk of febrile malaria in children. PLoS One, 4 (12), pp. e8321. | Show Abstract | Read more

BACKGROUND: Control measures which reduce individual exposure to malaria are expected to reduce disease, but also to eventually reduce immunity. Reassuringly, long term data following community wide ITN distribution show sustained benefits at a population level. However, the more common practice in Sub-Saharan Africa is to target ITN distribution on young children. There are few data on the long term outcomes of this practice. METHODOLOGY/PRINCIPAL FINDINGS: Episodes of febrile malaria were identified by active surveillance in 383 children over 18 months of follow up. In order to compare the short and long term outcomes of ITN use, we examined interactions between ITN use and age (12-42 months of age versus 42-80 months) in determining the risk of febrile malaria. ITN use and older age protected against the first or only episode of malaria (Hazard Ratio [HR] = 0.33, 95%CI 0.17-0.65 and HR = 0.30, 95%CI 0.17-0.51, respectively). The interaction term between ITN use and older age was HR = 2.91, 95%CI 1.02-8.3, p = 0.045, indicating that ITNs did not protect older children. When multiple episodes were included in analysis, ITN use and older age were again protective against malaria episodes (Incident Rate Ratio [IRR] = 0.43 95%CI 0.27-0.7) and IRR = 0.23, 95%CI 0.13-0.42, respectively) and the interaction term indicated that ITNs did not protect older children (IRR = 2.71, 95%CI 1.3-5.7, p = 0.008). CONCLUSIONS/SIGNIFICANCE: These data on age interactions with ITN use suggest that larger scale studies on the long term individual outcomes should be undertaken if the policy of targeted ITN use for vulnerable groups is to continue.

Warimwe GM, Keane TM, Fegan G, Musyoki JN, Newton CR, Pain A, Berriman M, Marsh K, Bull PC. 2009. Plasmodium falciparum var gene expression is modified by host immunity. Proc Natl Acad Sci U S A, 106 (51), pp. 21801-21806. | Show Abstract | Read more

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a potentially important family of immune targets, which play a central role in the host-parasite interaction by binding to various host molecules. They are encoded by a diverse family of genes called var, of which there are approximately 60 copies in each parasite genome. In sub-Saharan Africa, although P. falciparum infection occurs throughout life, severe malarial disease tends to occur only in childhood. This could potentially be explained if (i) PfEMP1 variants differ in their capacity to support pathogenesis of severe malaria and (ii) this capacity is linked to the likelihood of each molecule being recognized and cleared by naturally acquired antibodies. Here, in a study of 217 Kenyan children with malaria, we show that expression of a group of var genes "cys2," containing a distinct pattern of cysteine residues, is associated with low host immunity. Expression of cys2 genes was associated with parasites from young children, those with severe malaria, and those with a poorly developed antibody response to parasite-infected erythrocyte surface antigens. Cys-2 var genes form a minor component of all genomic var repertoires analyzed to date. Therefore, the results are compatible with the hypothesis that the genomic var gene repertoire is organized such that PfEMP1 molecules that confer the most virulence to the parasite tend also to be those that are most susceptible to the development of host immunity. This may help the parasite to adapt effectively to the development of host antibodies through modification of the host-parasite relationship.

Kingsley RA, Msefula CL, Thomson NR, Kariuki S, Holt KE, Gordon MA, Harris D, Clarke L, Whitehead S, Sangal V et al. 2009. Epidemic multiple drug resistant Salmonella Typhimurium causing invasive disease in sub-Saharan Africa have a distinct genotype. Genome Res, 19 (12), pp. 2279-2287. | Show Abstract | Read more

Whereas most nontyphoidal Salmonella (NTS) are associated with gastroenteritis, there has been a dramatic increase in reports of NTS-associated invasive disease in sub-Saharan Africa. Salmonella enterica serovar Typhimurium isolates are responsible for a significant proportion of the reported invasive NTS in this region. Multilocus sequence analysis of invasive S. Typhimurium from Malawi and Kenya identified a dominant type, designated ST313, which currently is rarely reported outside of Africa. Whole-genome sequencing of a multiple drug resistant (MDR) ST313 NTS isolate, D23580, identified a distinct prophage repertoire and a composite genetic element encoding MDR genes located on a virulence-associated plasmid. Further, there was evidence of genome degradation, including pseudogene formation and chromosomal deletions, when compared with other S. Typhimurium genome sequences. Some of this genome degradation involved genes previously implicated in virulence of S. Typhimurium or genes for which the orthologs in S. Typhi are either pseudogenes or are absent. Genome analysis of other epidemic ST313 isolates from Malawi and Kenya provided evidence for microevolution and clonal replacement in the field.

Mwai L, Kiara SM, Abdirahman A, Pole L, Rippert A, Diriye A, Bull P, Marsh K, Borrmann S, Nzila A. 2009. In vitro activities of piperaquine, lumefantrine, and dihydroartemisinin in Kenyan Plasmodium falciparum isolates and polymorphisms in pfcrt and pfmdr1. Antimicrob Agents Chemother, 53 (12), pp. 5069-5073. | Show Abstract | Read more

We have analyzed the in vitro chemosensitivity profiles of 115 Kenyan isolates for chloroquine (CQ), piperaquine, lumefantrine (LM), and dihydroartemisinin in association with polymorphisms in pfcrt at codon 76 and pfmdr1 at codon 86, as well as with variations of the copy number of pfmdr1. The median drug concentrations that inhibit 50% of parasite growth (IC(50)s) were 41 nM (interquartile range [IQR], 18 to 73 nM), 50 nM (IQR, 29 to 96 nM), 32 nM (IQR, 17 to 46 nM), and 2 nM (IQR, 1 to 3 nM) for CQ, LM, piperaquine, and dihydroartemisinin, respectively. The activity of CQ correlated inversely with that of LM (r(2) = -0.26; P = 0.02). Interestingly, parasites for which LM IC(50)s were higher were wild type for pfcrt-76 and pfmdr1-86. All isolates had one pfmdr1 copy. Thus, the decrease in LM activity is associated with the selection of wild-type pfcrt-76 and pfmdr1-86 parasites, a feature that accounts for the inverse relationship between CQ and LM. Therefore, the use of LM-artemether is likely to lead to the selection of more CQ-susceptible parasites.

Kinyanjui SM, Bejon P, Osier FH, Bull PC, Marsh K. 2009. What you see is not what you get: implications of the brevity of antibody responses to malaria antigens and transmission heterogeneity in longitudinal studies of malaria immunity. Malar J, 8 (1), pp. 242. | Show Abstract | Read more

BACKGROUND: A major handicap in developing a malaria vaccine is the difficulty in pinpointing the immune responses that protect against malaria. The protective efficacy of natural or vaccine-induced immune responses against malaria is normally assessed by relating the level of the responses in an individual at the beginning of a follow-up period and the individual's experience of malaria infection or disease during the follow-up. This approach has identified a number of important responses against malaria, but their protective efficacies vary considerably between studies. HYPOTHESIS: It is likely that apart from differences in study methodologies, differences in exposure among study subjects within each study and brevity of antibody responses to malaria antigen are important sources of the variation in protective efficacy of anti-malaria immune responses mentioned above. Since malaria immunity is not complete, anyone in an area of stable malaria transmission who does not become asymptomatically or symptomatically infected during follow-up subsequent to treatment is most likely unexposed rather than immune. TESTING THE HYPOTHESIS: It is proposed that individuals involved in a longitudinal study of malaria immunity should be treated for malaria prior to the start of the study and only those who present with at least an asymptomatic infection during the follow-up should be included in the analysis. In addition, it is proposed that more closely repeated serological survey should be carried out during follow-up in order to get a better picture of an individual's serological status. IMPLICATIONS OF THE HYPOTHESIS: Failure to distinguish between individuals who do not get a clinical episode during follow-up because they were unexposed and those who are genuinely immune undermines our ability to assign a protective role to immune responses against malaria. The brevity of antibodies responses makes it difficult to assign the true serological status of an individual at any given time, i.e. those positive at a survey may be negative by the time they encounter the next infection.

Williams TN, Uyoga S, Macharia A, Ndila C, McAuley CF, Opi DH, Mwarumba S, Makani J, Komba A, Ndiritu MN et al. 2009. Bacteraemia in Kenyan children with sickle-cell anaemia: a retrospective cohort and case-control study. Lancet, 374 (9698), pp. 1364-1370. | Show Abstract | Read more

BACKGROUND: In sub-Saharan Africa, more than 90% of children with sickle-cell anaemia die before the diagnosis can be made. The causes of death are poorly documented, but bacterial sepsis is probably important. We examined the risk of invasive bacterial diseases in children with sickle-cell anaemia. METHODS: This study was undertaken in a rural area on the coast of Kenya, with a case-control approach. We undertook blood cultures on all children younger than 14 years who were admitted from within a defined study area to Kilifi District Hospital between Aug 1, 1998, and March 31, 2008; those with bacteraemia were defined as cases. We used two sets of controls: children recruited by random sampling in the same area into several studies undertaken between Sept 1, 1998, and Nov 30, 2005; and those born consecutively within the area between May 1, 2006, and April 30, 2008. Cases and controls were tested for sickle-cell anaemia retrospectively. FINDINGS: We detected 2157 episodes of bacteraemia in 38 441 admissions (6%). 1749 of these children with bacteraemia (81%) were typed for sickle-cell anaemia, of whom 108 (6%) were positive as were 89 of 13 492 controls (1%). The organisms most commonly isolated from children with sickle-cell anaemia were Streptococcus pneumoniae (44/108 isolates; 41%), non-typhi Salmonella species (19/108; 18%), Haemophilus influenzae type b (13/108; 12%), Acinetobacter species (seven of 108; 7%), and Escherichia coli (seven of 108; 7%). The age-adjusted odds ratio for bacteraemia in children with sickle-cell anaemia was 26.3 (95% CI 14.5-47.6), with the strongest associations for S pneumoniae (33.0, 17.4-62.8), non-typhi Salmonella species (35.5, 16.4-76.8), and H influenzae type b (28.1, 12.0-65.9). INTERPRETATION: The organisms causing bacteraemia in African children with sickle-cell anaemia are the same as those in developed countries. Introduction of conjugate vaccines against S pneumoniae and H influenzae into the childhood immunisation schedules of African countries could substantially affect survival of children with sickle-cell anaemia. FUNDING: Wellcome Trust, UK.

Mackinnon MJ, Li J, Mok S, Kortok MM, Marsh K, Preiser PR, Bozdech Z. 2009. Comparative transcriptional and genomic analysis of Plasmodium falciparum field isolates. PLoS Pathog, 5 (10), pp. e1000644. | Show Abstract | Read more

Mechanisms for differential regulation of gene expression may underlie much of the phenotypic variation and adaptability of malaria parasites. Here we describe transcriptional variation among culture-adapted field isolates of Plasmodium falciparum, the species responsible for most malarial disease. It was found that genes coding for parasite protein export into the red cell cytosol and onto its surface, and genes coding for sexual stage proteins involved in parasite transmission are up-regulated in field isolates compared with long-term laboratory isolates. Much of this variability was associated with the loss of small or large chromosomal segments, or other forms of gene copy number variation that are prevalent in the P. falciparum genome (copy number variants, CNVs). Expression levels of genes inside these segments were correlated to that of genes outside and adjacent to the segment boundaries, and this association declined with distance from the CNV boundary. This observation could not be explained by copy number variation in these adjacent genes. This suggests a local-acting regulatory role for CNVs in transcription of neighboring genes and helps explain the chromosomal clustering that we observed here. Transcriptional co-regulation of physical clusters of adaptive genes may provide a way for the parasite to readily adapt to its highly heterogeneous and strongly selective environment.

Färnert A, Williams TN, Mwangi TW, Ehlin A, Fegan G, Macharia A, Lowe BS, Montgomery SM, Marsh K. 2009. Transmission-dependent tolerance to multiclonal Plasmodium falciparum infection. J Infect Dis, 200 (7), pp. 1166-1175. | Show Abstract | Read more

Whether the number of concurrent clones in asymptomatic Plasmodium falciparum infections reflects the degree of host protection was investigated in children living in areas with different levels of transmission on the coast of Kenya. The number of concurrent clones was determined on the basis of polymorphism in msp2, which encodes the vaccine candidate antigen merozoite surface protein 2. In a low-transmission area, most children had monoclonal infections, and diversity did not predict a risk of clinical malaria. In an area of moderate transmission, asymptomatic infections with 2 clones were, compared with 1 clone, associated with an increased risk of subsequent malaria. In a comparative assessment in a high-transmission area in Tanzania, multiclonal infections conferred a reduced risk. The different nonlinear associations between the number of clones and malaria morbidity suggest that levels of tolerance to multiclonal infections are transmission dependent as a result of cumulative exposure to antigenically diverse P. falciparum infections.

Sadarangani M, Makani J, Komba AN, Ajala-Agbo T, Newton CR, Marsh K, Williams TN. 2009. An observational study of children with sickle cell disease in Kilifi, Kenya. Br J Haematol, 146 (6), pp. 675-682. | Show Abstract | Read more

Globally, sickle cell disease (SCD) has its highest prevalence and worst prognosis in sub-Saharan Africa. Nevertheless, relatively few studies describe the clinical characteristics of children with SCD in this region. We conducted a prospective observational study of children with SCD attending a specialist out-patient clinic in Kilifi, Kenya. A total of 124 children (median age 6.3 years) were included in the study. Splenomegaly was present in 41 (33%) subjects and hepatomegaly in 25 (20%), both being common in all age groups. A positive malaria slide was found at 6% of clinic visits. The mean haemoglobin concentration was 73 g/l, compared to 107 g/l in non-SCD controls (P < 0.001). Liver function tests were elevated; plasma bilirubin concentrations were 46 micromol/l and aspartate aminotransferase was 124 iu/l. Forty-eight (39%) children were admitted to hospital and two died. Children with SCD in Kilifi have a similar degree of anaemia and liver function derangement to patients living in developed countries, but splenomegaly persists into later childhood. The prevalence of malaria was lower than expected given the prevalence in the local community. This study provides valuable data regarding the clinical characteristics of children living with SCD in a rural setting in East Africa.

Berkley JA, Bejon P, Mwangi T, Gwer S, Maitland K, Williams TN, Mohammed S, Osier F, Kinyanjui S, Fegan G et al. 2009. HIV infection, malnutrition, and invasive bacterial infection among children with severe malaria. Clin Infect Dis, 49 (3), pp. 336-343. | Show Abstract | Read more

BACKGROUND: Human immunodeficiency virus (HIV) infection, malnutrition, and invasive bacterial infection (IBI) are reported among children with severe malaria. However, it is unclear whether their cooccurrence with falciparum parasitization and severe disease happens by chance or by association among children in areas where malaria is endemic. METHODS: We examined 3068 consecutive children admitted to a Kenyan district hospital with clinical features of severe malaria and 592 control subjects from the community. We performed multivariable regression analysis, with each case weighted for its probability of being due to falciparum malaria, using estimates of the fraction of severe disease attributable to malaria at different parasite densities derived from cross-sectional parasitological surveys of healthy children from the same community. RESULTS: HIV infection was present in 133 (12%) of 1071 consecutive parasitemic admitted children (95% confidence interval [CI], 11%-15%). Parasite densities were higher in HIV-infected children. The odds ratio for admission associated with HIV infection for admission with true severe falciparum malaria was 9.6 (95% CI, 4.9-19); however, this effect was restricted to children aged 1 year. Malnutrition was present in 507 (25%) of 2048 consecutive parasitemic admitted children (95% CI, 23%-27%). The odd ratio associated with malnutrition for admission with true severe falciparum malaria was 4.0 (95% CI, 2.9-5.5). IBI was detected in 127 (6%) of 2048 consecutive parasitemic admitted children (95% CI, 5.2%-7.3%). All 3 comorbidities were associated with increased case fatality. CONCLUSIONS: HIV, malnutrition and IBI are biologically associated with severe disease due to falciparum malaria rather than being simply alternative diagnoses in co-incidentally parasitized children in an endemic area.

Komba AN, Makani J, Sadarangani M, Ajala-Agbo T, Berkley JA, Newton CR, Marsh K, Williams TN. 2009. Malaria as a cause of morbidity and mortality in children with homozygous sickle cell disease on the coast of Kenya. Clin Infect Dis, 49 (2), pp. 216-222. | Show Abstract | Read more

BACKGROUND: To date, it has been widely assumed that malaria is a common cause of morbidity and mortality in children with sickle cell disease (SCD) in malaria-endemic countries, and as a result, malarial prophylaxis is commonly recommended. Nevertheless, few data are available that support this practice. METHODS: We conducted a retrospective analysis of the data collected prospectively from children aged 0-13 years who were admitted to Kilifi District Hospital during the period from July 1998 through June 2005. We studied the prevalence, clinical features, and outcome of malarial infections in these children, stratified by SCD status. RESULTS: Although we estimated the prevalence of SCD in children to be only 0.8% (71 of 8531 children) during the period from August 2006 through September 2008 in the community surrounding the hospital, 555 (1.6%) of 34,529 children admitted to the hospital during the study period (i.e., from July 1998 through June 2005) were children with SCD; in fact, a total of 309 children with SCD were admitted 555 times. The prevalence of Plasmodium falciparum parasitemia was lower among children with SCD than it was among children without SCD (86 [15.6%] of 551 children vs. 13,835 [41.3%] of 33,500 children; P < .001). Similarly, among those infected with P. falciparum parasites, the mean parasite density was significantly lower among children with SCD than it was among children without SCD (2205 vs. 23,878 parasites/microL; P < .001). Fourteen (16.3%) of 86 parasitemic patients with SCD had features consistent with severe malaria, compared with 3424 (24.7%) of 13,835 parasitemic patients without SCD (odds ratio, 0.59; P < .07). We found no association between malarial parasitemia and death. CONCLUSIONS: We found no evidence to support the conclusion that the risk of malaria is higher among children with SCD than it is among children without SCD in a rural area on the coast of Kenya. Further studies should be undertaken to help policy makers develop appropriate guidelines regarding malarial prophylaxis for patients with SCD in malaria-endemic regions.

Fry AE, Ghansa A, Small KS, Palma A, Auburn S, Diakite M, Green A, Campino S, Teo YY, Clark TG et al. 2009. Positive selection of a CD36 nonsense variant in sub-Saharan Africa, but no association with severe malaria phenotypes. Hum Mol Genet, 18 (14), pp. 2683-2692. | Show Abstract | Read more

The prevalence of CD36 deficiency in East Asian and African populations suggests that the causal variants are under selection by severe malaria. Previous analysis of data from the International HapMap Project indicated that a CD36 haplotype bearing a nonsense mutation (T1264G; rs3211938) had undergone recent positive selection in the Yoruba of Nigeria. To investigate the global distribution of this putative selection event, we genotyped T1264G in 3420 individuals from 66 populations. We confirmed the high frequency of 1264G in the Yoruba (26%). However, the 1264G allele is less common in other African populations and absent from all non-African populations without recent African admixture. Using long-range linkage disequilibrium, we studied two West African groups in depth. Evidence for recent positive selection at the locus was demonstrable in the Yoruba, although not in Gambians. We screened 70 variants from across CD36 for an association with severe malaria phenotypes, employing a case-control study of 1350 subjects and a family study of 1288 parent-offspring trios. No marker was significantly associated with severe malaria. We focused on T1264G, genotyping 10,922 samples from four African populations. The nonsense allele was not associated with severe malaria (pooled allelic odds ratio 1.0; 95% confidence interval 0.89-1.12; P = 0.98). These results suggest a range of possible explanations including the existence of alternative selection pressures on CD36, co-evolution between host and parasite or confounding caused by allelic heterogeneity of CD36 deficiency.

Blythe JE, Niang M, Marsh K, Holder AA, Langhorne J, Preiser PR. 2009. Characterization of the repertoire diversity of the Plasmodium falciparum stevor multigene family in laboratory and field isolates. Malar J, 8 (1), pp. 140. | Show Abstract | Read more

BACKGROUND: The evasion of host immune response by the human malaria parasite Plasmodium falciparum has been linked to expression of a range of variable antigens on the infected erythrocyte surface. Several genes are potentially involved in this process with the var, rif and stevor multigene families being the most likely candidates and coding for rapidly evolving proteins. The high sequence diversity of proteins encoded by these gene families may have evolved as an immune evasion strategy that enables the parasite to establish long lasting chronic infections. Previous findings have shown that the hypervariable region (HVR) of STEVOR has significant sequence diversity both within as well as across different P. falciparum lines. However, these studies did not address whether or not there are ancestral stevor that can be found in different parasites. METHODS: DNA and RNA sequences analysis as well as phylogenetic approaches were used to analyse the stevor sequence repertoire and diversity in laboratory lines and Kilifi (Kenya) fresh isolates. RESULTS: Conserved stevor genes were identified in different P. falciparum isolates from different global locations. Consistent with previous studies, the HVR of the stevor gene family was found to be highly divergent both within and between isolates. Importantly phylogenetic analysis shows some clustering of stevor sequences both within a single parasite clone as well as across different parasite isolates. CONCLUSION: This indicates that the ancestral P. falciparum parasite genome already contained multiple stevor genes that have subsequently diversified further within the different P. falciparum populations. It also confirms that STEVOR is under strong selection pressure.

Dudareva M, Andrews L, Gilbert SC, Bejon P, Marsh K, Mwacharo J, Kai O, Nicosia A, Hill AV. 2009. Prevalence of serum neutralizing antibodies against chimpanzee adenovirus 63 and human adenovirus 5 in Kenyan children, in the context of vaccine vector efficacy. Vaccine, 27 (27), pp. 3501-3504. | Show Abstract | Read more

Vaccination against Plasmodium falciparum malaria could reduce the worldwide burden of this disease, and decrease its high mortality in children. Replication-defective recombinant adenovirus vectors carrying P. falciparum epitopes may be useful as part of a vaccine that raises cellular immunity to the pre-erythrocytic stage of malaria infection. However, existing immunity to the adenovirus vector results in antibody-mediated neutralization of the vaccine vector, and reduced vaccine immunogenicity. Our aim was to examine a population of children who are at risk from P. falciparum malaria for neutralizing immunity to replication-deficient recombinant chimpanzee adenovirus 63 vector (AdC63), compared to human adenovirus 5 vector (AdHu5). We measured 50% and 90% vector neutralization titers in 200 individual sera, taken from a cohort of children from Kenya, using a secreted alkaline phosphatase neutralization assay. We found that 23% of the children (aged 1-6 years) had high-titer neutralizing antibodies to AdHu5, and 4% had high-titer neutralizing antibodies to AdC63. Immunity to both vectors was age-dependent. Low-level neutralization of AdC63 was significantly less frequent than AdHu5 neutralization at the 90% neutralization level. We conclude that AdC63 may be a useful vector as part of a prime-boost malaria vaccine in children.

Jallow M, Teo YY, Small KS, Rockett KA, Deloukas P, Clark TG, Kivinen K, Bojang KA, Conway DJ, Pinder M et al. 2009. Genome-wide and fine-resolution association analysis of malaria in West Africa. Nat Genet, 41 (6), pp. 657-665. | Show Abstract | Read more

We report a genome-wide association (GWA) study of severe malaria in The Gambia. The initial GWA scan included 2,500 children genotyped on the Affymetrix 500K GeneChip, and a replication study included 3,400 children. We used this to examine the performance of GWA methods in Africa. We found considerable population stratification, and also that signals of association at known malaria resistance loci were greatly attenuated owing to weak linkage disequilibrium (LD). To investigate possible solutions to the problem of low LD, we focused on the HbS locus, sequencing this region of the genome in 62 Gambian individuals and then using these data to conduct multipoint imputation in the GWA samples. This increased the signal of association, from P = 4 × 10(-7) to P = 4 × 10(-14), with the peak of the signal located precisely at the HbS causal variant. Our findings provide proof of principle that fine-resolution multipoint imputation, based on population-specific sequencing data, can substantially boost authentic GWA signals and enable fine mapping of causal variants in African populations.

Mwai L, Ochong E, Abdirahman A, Kiara SM, Ward S, Kokwaro G, Sasi P, Marsh K, Borrmann S, Mackinnon M, Nzila A. 2009. Chloroquine resistance before and after its withdrawal in Kenya. Malar J, 8 (1), pp. 106. | Show Abstract | Read more

BACKGROUND: The spread of resistance to chloroquine (CQ) led to its withdrawal from use in most countries in sub-Saharan Africa in the 1990s. In Malawi, this withdrawal was followed by a rapid reduction in the frequency of resistance to the point where the drug is now considered to be effective once again, just nine years after its withdrawal. In this report, the polymorphisms of markers associated with CQ-resistance against Plasmodium falciparum isolates from coastal Kenya (Kilifi) were investigated, from 1993, prior to the withdrawal of CQ, to 2006, seven years after its withdrawal. Changes to those that occurred in the dihydrofolate reductase gene (dhfr) that confers resistance to the replacement drug, pyrimethamine/sulphadoxine were also compared. METHODS: Mutations associated with CQ resistance, at codons 76 of pfcrt, at 86 of pfmdr1, and at codons 51, 59 and 164 of dhfr were analysed using PCR-restriction enzyme methods. In total, 406, 240 and 323 isolates were genotyped for pfcrt-76, pfmdr1-86 and dhfr, respectively. RESULTS: From 1993 to 2006, the frequency of the pfcrt-76 mutant significantly decreased from around 95% to 60%, while the frequency of pfmdr1-86 did not decline, remaining around 75%. Though the frequency of dhfr mutants was already high (around 80%) at the start of the study, this frequency increased to above 95% during the study period. Mutation at codon 164 of dhfr was analysed in 2006 samples, and none of them had this mutation. CONCLUSION: In accord with the study in Malawi, a reduction in resistance to CQ following official withdrawal in 1999 was found, but unlike Malawi, the decline of resistance to CQ in Kilifi was much slower. It is estimated that, at current rates of decline, it will take 13 more years for the clinical efficacy of CQ to be restored in Kilifi. In addition, CQ resistance was declining before the drug's official withdrawal, suggesting that, prior to the official ban, the use of CQ had decreased, probably due to its poor clinical effectiveness.

Bejon P, Warimwe G, Mackintosh CL, Mackinnon MJ, Kinyanjui SM, Musyoki JN, Bull PC, Marsh K. 2009. Analysis of immunity to febrile malaria in children that distinguishes immunity from lack of exposure. Infect Immun, 77 (5), pp. 1917-1923. | Show Abstract | Read more

In studies of immunity to malaria, the absence of febrile malaria is commonly considered evidence of "protection." However, apparent "protection" may be due to a lack of exposure to infective mosquito bites or due to immunity. We studied a cohort that was given curative antimalarials before monitoring began and documented newly acquired asymptomatic parasitemia and febrile malaria episodes during 3 months of surveillance. With increasing age, there was a shift away from febrile malaria to acquiring asymptomatic parasitemia, with no change in the overall incidence of infection. Antibodies to the infected red cell surface were associated with acquiring asymptomatic infection rather than febrile malaria or remaining uninfected. Bed net use was associated with remaining uninfected rather than acquiring asymptomatic infection or febrile malaria. These observations suggest that most uninfected children were unexposed rather than "immune." Had they been immune, we would have expected the proportion of uninfected children to rise with age and that the uninfected children would have been distinguished from children with febrile malaria by the protective antibody response. We show that removing the less exposed children from conventional analyses clarifies the effects of immunity, transmission intensity, bed nets, and age. Observational studies and vaccine trials will have increased power if they differentiate between unexposed and immune children.

Makani J, Kirkham FJ, Komba A, Ajala-Agbo T, Otieno G, Fegan G, Williams TN, Marsh K, Newton CR. 2009. Risk factors for high cerebral blood flow velocity and death in Kenyan children with Sickle Cell Anaemia: role of haemoglobin oxygen saturation and febrile illness. Br J Haematol, 145 (4), pp. 529-532. | Show Abstract | Read more

High cerebral blood flow velocity (CBFv) and low haemoglobin oxygen saturation (SpO(2)) predict neurological complications in sickle cell anaemia (SCA) but any association is unclear. In a cross-sectional study of 105 Kenyan children, mean CBFv was 120 +/- 34.9 cm/s; 3 had conditional CBFv (170-199 cm/s) but none had abnormal CBFv (>200 cm/s). After adjustment for age and haematocrit, CBFv > or =150 cm/s was predicted by SpO(2) < or = 95% and history of fever. Four years later, 10 children were lost to follow-up, none had suffered neurological events and 11/95 (12%) had died, predicted by history of fever but not low SpO(2). Natural history of SCA in Africa may be different from North America and Europe.

Clark TG, Diakite M, Auburn S, Campino S, Fry AE, Green A, Richardson A, Small K, Teo YY, Wilson J et al. 2009. Tumor necrosis factor and lymphotoxin-alpha polymorphisms and severe malaria in African populations. J Infect Dis, 199 (4), pp. 569-575. | Show Abstract | Read more

The tumor necrosis factor gene (TNF) and lymphotoxin-alpha gene (LTA) have long attracted attention as candidate genes for susceptibility traits for malaria, and several of their polymorphisms have been found to be associated with severe malaria (SM) phenotypes. In a large study involving >10,000 individuals and encompassing 3 African populations, we found evidence to support the reported associations between the TNF -238 polymorphism and SM in The Gambia. However, no TNF/LTA polymorphisms were found to be associated with SM in cohorts in Kenya and Malawi. It has been suggested that the causal polymorphisms regulating the TNF and LTA responses may be located some distance from the genes. Therefore, more-detailed mapping of variants across TNF/LTA genes and their flanking regions in the Gambian and allied populations may need to be undertaken to find any causal polymorphisms.

Lyons EJ, Amos W, Berkley JA, Mwangi I, Shafi M, Williams TN, Newton CR, Peshu N, Marsh K, Scott JA, Hill AV. 2009. Homozygosity and risk of childhood death due to invasive bacterial disease. BMC Med Genet, 10 (1), pp. 55. | Show Abstract | Read more

BACKGROUND: Genetic heterozygosity is increasingly being shown to be a key predictor of fitness in natural populations, both through inbreeding depression, inbred individuals having low heterozygosity, and also through chance linkage between a marker and a gene under balancing selection. One important component of fitness that is often highlighted is resistance to parasites and other pathogens. However, the significance of equivalent loci in human populations remains unclear. Consequently, we performed a case-control study of fatal invasive bacterial disease in Kenyan children using a genome-wide screen with microsatellite markers. METHODS: 148 cases, comprising children aged <13 years who died of invasive bacterial disease, (variously, bacteraemia, bacterial meningitis or neonatal sepsis) and 137 age-matched, healthy children were sampled in a prospective study conducted at Kilifi District Hospital, Kenya. Samples were genotyped for 134 microsatellite markers using the ABI LD20 marker set and analysed for an association between homozygosity and mortality. RESULTS: At five markers homozygosity was strongly associated with mortality (odds ratio range 4.7 - 12.2) with evidence of interactions between some markers. Mortality was associated with different non-overlapping marker groups in Gram positive and Gram negative bacterial disease. Homozygosity at susceptibility markers was common (prevalence 19-49%) and, with the large effect sizes, this suggests that bacterial disease mortality may be strongly genetically determined. CONCLUSION: Balanced polymorphisms appear to be more widespread in humans than previously appreciated and play a critical role in modulating susceptibility to infectious disease. The effect sizes we report, coupled with the stochasticity of exposure to pathogens suggests that infection and mortality are far from random due to a strong genetic basis.

Bejon P, Ogada E, Peshu N, Marsh K. 2009. Interactions between age and ITN use determine the risk of febrile malaria in children PLoS ONE, 4 (12), | Show Abstract | Read more

Background: Control measures which reduce individual exposure to malaria are expected to reduce disease, but also to eventually reduce immunity. Reassuringly, long term data following community wide ITN distribution show sustained benefits at a population level. However, the more common practice in Sub-Saharan Africa is to target ITN distribution on young children. There are few data on the long term outcomes of this practice.Methodology/Principal Findings: Episodes of febrile malaria were identified by active surveillance in 383 children over 18 months of follow up. In order to compare the short and long term outcomes of ITN use, we examined interactions between ITN use and age (12-42 months of age versus 42-80 months) in determining the risk of febrile malaria. ITN use and older age protected against the first or only episode of malaria (Hazard Ratio [HR] =0.33, 95%CI 0.17-0.65 and HR =0.30, 95%CI0.17-0.51, respectively). The interaction term between ITN use and older age was HR =2.91, 95%CI 1.02-8.3, p=0.045, indicating that ITNs did not protect older children. When multiple episodes were included in analysis, ITN use and older age were again protective against malaria episodes (Incident Rate Ratio [IRR] =0.43 95%CI 0.27-0.7) and IRR = 0.23, 95%CI 0.13-0.42, respectively) and the interaction term indicated that ITNs did not protect older children (IRR =2.71, 95%CI 1.3-5.7, p=0.008). Conclusions/Significance: These data on age interactions with ITN use suggest that larger scale studies on the long term individual outcomes should be undertaken if the policy of targeted ITN use for vulnerable groups is to continue. © 2009 Bejon et al.

Mangano VD, Clark TG, Auburn S, Campino S, Diakite M, Fry AE, Green A, Richardson A, Jallow M, Sisay-Joof F et al. 2009. Lack of association of interferon regulatory factor 1 with severe malaria in affected child-parental trio studies across three African populations. PLoS One, 4 (1), pp. e4206. | Show Abstract | Read more

Interferon Regulatory Factor 1 (IRF-1) is a member of the IRF family of transcription factors, which have key and diverse roles in the gene-regulatory networks of the immune system. IRF-1 has been described as a critical mediator of IFN-gamma signalling and as the major player in driving TH1 type responses. It is therefore likely to be crucial in both innate and adaptive responses against intracellular pathogens such as Plasmodium falciparum. Polymorphisms at the human IRF1 locus have been previously found to be associated with the ability to control P. falciparum infection in populations naturally exposed to malaria. In order to test whether genetic variation at the IRF1 locus also affects the risk of developing severe malaria, we performed a family-based test of association for 18 Single Nucleotide Polymorphisms (SNPs) across the gene in three African populations, using genotype data from 961 trios consisting of one affected child and his/her two parents (555 from The Gambia, 204 from Kenya and 202 from Malawi). No significant association with severe malaria or severe malaria subphenotypes (cerebral malaria and severe malaria anaemia) was observed for any of the SNPs/haplotypes tested in any of the study populations. Our results offer no evidence that the molecular pathways regulated by the transcription factor IRF-1 are involved in the immune-based pathogenesis of severe malaria.

Tetteh KK, Stewart LB, Ochola LI, Amambua-Ngwa A, Thomas AW, Marsh K, Weedall GD, Conway DJ. 2009. Prospective identification of malaria parasite genes under balancing selection. PLoS One, 4 (5), pp. e5568. | Show Abstract | Read more

BACKGROUND: Endemic human pathogens are subject to strong immune selection, and interrogation of pathogen genome variation for signatures of balancing selection can identify important target antigens. Several major antigen genes in the malaria parasite Plasmodium falciparum have shown such signatures in polymorphism-versus-divergence indices (comparing with the chimpanzee parasite P. reichenowi), and in allele frequency based indices. METHODOLOGY/PRINCIPAL FINDINGS: To compare methods for prospective identification of genes under balancing selection, 26 additional genes known or predicted to encode surface-exposed proteins of the invasive blood stage merozoite were first sequenced from a panel of 14 independent P. falciparum cultured lines and P. reichenowi. Six genes at the positive extremes of one or both of the Hudson-Kreitman-Aguade (HKA) and McDonald-Kreitman (MK) indices were identified. Allele frequency based analysis was then performed on a Gambian P. falciparum population sample for these six genes and three others as controls. Tajima's D (TjD) index was most highly positive for the msp3/6-like PF10_0348 (TjD = 1.96) as well as the positive control ama1 antigen gene (TjD = 1.22). Across the genes there was a strong correlation between population TjD values and the relative HKA indices (whether derived from the population or the panel of cultured laboratory isolates), but no correlation with the MK indices. CONCLUSIONS/SIGNIFICANCE: Although few individual parasite genes show significant evidence of balancing selection, analysis of population genomic and comparative sequence data with the HKA and TjD indices should discriminate those that do, and thereby identify likely targets of immunity.

Moorthy VS, Diggs C, Ferro S, Good MF, Herrera S, Hill AV, Imoukhuede EB, Kumar S, Loucq C, Marsh K et al. 2009. Report of a consultation on the optimization of clinical challenge trials for evaluation of candidate blood stage malaria vaccines, 18-19 March 2009, Bethesda, MD, USA. Vaccine, 27 (42), pp. 5719-5725. | Show Abstract | Read more

Development and optimization of first generation malaria vaccine candidates has been facilitated by the existence of a well-established Plasmodium falciparum clinical challenge model in which infectious sporozoites are administered to human subjects via mosquito bite. While ideal for testing pre-erythrocytic stage vaccines, some researchers believe that the sporozoite challenge model is less appropriate for testing blood stage vaccines. Here we report a consultation, co-sponsored by PATH MVI, USAID, EMVI and WHO, where scientists from all institutions globally that have conducted such clinical challenges in recent years and representatives from regulatory agencies and funding agencies met to discuss clinical malaria challenge models. Participants discussed strengthening and harmonizing the sporozoite challenge model and considered the pros and cons of further developing a blood stage challenge possibly better suited for evaluating the efficacy of blood stage vaccines. This report summarizes major findings and recommendations, including an update on the Plasmodium vivax clinical challenge model, the prospects for performing experimental challenge trials in malaria endemic countries and an update on clinical safety data. While the focus of the meeting was on the optimization of clinical challenge models for evaluation of blood stage candidate malaria vaccines, many of the considerations are relevant for the application of challenge trials to other purposes.

O'Meara WP, Noor A, Gatakaa H, Tsofa B, McKenzie FE, Marsh K. 2009. The impact of primary health care on malaria morbidity--defining access by disease burden. Trop Med Int Health, 14 (1), pp. 29-35. | Show Abstract | Read more

OBJECTIVES: Primary care facilities are increasingly becoming the focal point for distribution of malaria intervention strategies, but physical access to these facilities may limit the extent to which communities can be reached. To investigate the impact of travel time to primary care on the incidence of hospitalized malaria episodes in a rural district in Kenya. METHODS: The incidence of hospitalized malaria in a population under continuous demographic surveillance was recorded over 3 years. The time to travel to the nearest primary health care facility was calculated for every child between birth and 5 years of age and trends in incidence of hospitalized malaria as a function of travel time were evaluated. RESULTS: The incidence of hospitalized malaria more than doubled as travel time to the nearest primary care facility increased from 10 min to 2 h. Good access to primary health facilities may reduce the burden of disease by as much as 66%. CONCLUSIONS: Our results highlight both the potential of the primary health care system in reaching those most at risk and reducing the disease burden. Insufficient access is an important risk factor, one that may be inequitably distributed to the poorest households.

Bejon P, Lusingu J, Olotu A, Leach A, Lievens M, Vekemans J, Mshamu S, Lang T, Gould J, Dubois MC et al. 2008. Efficacy of RTS,S/AS01E vaccine against malaria in children 5 to 17 months of age. N Engl J Med, 359 (24), pp. 2521-2532. | Show Abstract | Read more

BACKGROUND: Plasmodium falciparum malaria is a pressing global health problem. A previous study of the malaria vaccine RTS,S (which targets the circumsporozoite protein), given with an adjuvant system (AS02A), showed a 30% rate of protection against clinical malaria in children 1 to 4 years of age. We evaluated the efficacy of RTS,S given with a more immunogenic adjuvant system (AS01E) in children 5 to 17 months of age, a target population for vaccine licensure. METHODS: We conducted a double-blind, randomized trial of RTS,S/AS01E vaccine as compared with rabies vaccine in children in Kilifi, Kenya, and Korogwe, Tanzania. The primary end point was fever with a falciparum parasitemia density of more than 2500 parasites per microliter, and the mean duration of follow-up was 7.9 months (range, 4.5 to 10.5). RESULTS: A total of 894 children were randomly assigned to receive the RTS,S/AS01E vaccine or the control (rabies) vaccine. Among the 809 children who completed the study procedures according to the protocol, the cumulative number in whom clinical malaria developed was 32 of 402 assigned to receive RTS,S/AS01E and 66 of 407 assigned to receive the rabies vaccine; the adjusted efficacy rate for RTS,S/AS01E was 53% (95% confidence interval [CI], 28 to 69; P<0.001) on the basis of Cox regression. Overall, there were 38 episodes of clinical malaria among recipients of RTS,S/AS01E, as compared with 86 episodes among recipients of the rabies vaccine, with an adjusted rate of efficacy against all malarial episodes of 56% (95% CI, 31 to 72; P<0.001). All 894 children were included in the intention-to-treat analysis, which showed an unadjusted efficacy rate of 49% (95% CI, 26 to 65; P<0.001). There were fewer serious adverse events among recipients of RTS,S/AS01E, and this reduction was not only due to a difference in the number of admissions directly attributable to malaria. CONCLUSIONS: RTS,S/AS01E shows promise as a candidate malaria vaccine. (ClinicalTrials.gov number, NCT00380393.)

Auburn S, Diakite M, Fry AE, Ghansah A, Campino S, Richardson A, Jallow M, Sisay-Joof F, Pinder M, Griffiths MJ et al. 2008. Association of the GNAS locus with severe malaria. Hum Genet, 124 (5), pp. 499-506. | Show Abstract | Read more

Functional studies have demonstrated an interaction between the stimulatory G protein alpha subunit (G-alpha-s) and the malaria parasite at a cellular level. Obstruction of signal transduction via the erythrocyte G-alpha-s subunit reduced invasion by Plasmodium falciparum parasites. We sought to determine whether this signal pathway had an impact at the disease level by testing polymorphisms in the gene encoding G-alpha-s (GNAS) for association with severe malaria in a large multi-centre study encompassing family and case-control studies from The Gambia, Kenya and Malawi, and a case-control study from Ghana. We gained power to detect association using meta-analysis across the seven studies, with an overall sample size approximating 4,000 cases and 4,000 controls. Out of 12 SNPs investigated in the 19 kb GNAS region, four presented signals of association (P < 0.05) with severe malaria. The strongest single-locus association demonstrated an odds ratio of 1.13 (1.05-1.21), P = 0.001. Three of the loci presenting significant associations were clustered at the 5-prime end of the GNAS gene. Accordingly, haplotypes constructed from these loci demonstrated significant associations with severe malaria [OR = 0.88 (0.81-0.96), P = 0.005 and OR = 1.12 (1.03-1.20), P = 0.005]. The evidence presented here indicates that the influence of G-alpha-s on erythrocyte invasion efficacy may, indeed, alter individual susceptibility to disease.

O'Meara WP, Bejon P, Mwangi TW, Okiro EA, Peshu N, Snow RW, Newton CR, Marsh K. 2008. Effect of a fall in malaria transmission on morbidity and mortality in Kilifi, Kenya. Lancet, 372 (9649), pp. 1555-1562. | Show Abstract | Read more

BACKGROUND: As efforts to control malaria are expanded across the world, understanding the role of transmission intensity in determining the burden of clinical malaria is crucial to the prediction and measurement of the effectiveness of interventions to reduce transmission. Furthermore, studies comparing several endemic sites led to speculation that as transmission decreases morbidity and mortality caused by severe malaria might increase. We aimed to assess the epidemiological characteristics of malaria in Kilifi, Kenya, during a period of decreasing transmission intensity. METHODS: We analyse 18 years (1990-2007) of surveillance data from a paediatric ward in a malaria-endemic region of Kenya. The hospital has a catchment area of 250 000 people. Clinical data and blood-film results for more than 61 000 admissions are reported. FINDINGS: Hospital admissions for malaria decreased from 18.43 per 1000 children in 2003 to 3.42 in 2007. Over 18 years of surveillance, the incidence of cerebral malaria initially increased; however, malaria mortality decreased overall because of a decrease in incidence of severe malarial anaemia since 1997 (4.75 to 0.37 per 1000 children) and improved survival among children admitted with non-severe malaria. Parasite prevalence, the mean age of children admitted with malaria, and the proportion of children with cerebral malaria began to change 10 years before hospitalisation for malaria started to fall. INTERPRETATION: Sustained reduction in exposure to infection leads to changes in mean age and presentation of disease similar to those described in multisite studies. Changes in transmission might not lead to immediate reductions in incidence of clinical disease. However, longitudinal data do not indicate that reductions in transmission intensity lead to transient increases in morbidity and mortality.

McCallum FJ, Persson KE, Mugyenyi CK, Fowkes FJ, Simpson JA, Richards JS, Williams TN, Marsh K, Beeson JG. 2008. Acquisition of growth-inhibitory antibodies against blood-stage Plasmodium falciparum. PLoS One, 3 (10), pp. e3571. | Show Abstract | Read more

BACKGROUND: Antibodies that inhibit the growth of blood-stage Plasmodium falciparum may play an important role in acquired and vaccine-induced immunity in humans. However, the acquisition and activity of these antibodies is not well understood. METHODS: We tested dialysed serum and purified immunoglobulins from Kenyan children and adults for inhibition of P. falciparum blood-stage growth in vitro using different parasite lines. Serum antibodies were measured by ELISA to blood-stage parasite antigens, extracted from P. falciparum schizonts, and to recombinant merozoite surface protein 1 (42 kDa C-terminal fragment, MSP1-42). RESULTS: Antibodies to blood-stage antigens present in schizont protein extract and to recombinant MSP1-42 significantly increased with age and were highly correlated. In contrast, growth-inhibitory activity was not strongly associated with age and tended to decline marginally with increasing age and exposure, with young children demonstrating the highest inhibitory activity. Comparison of growth-inhibitory activity among samples collected from the same population at different time points suggested that malaria transmission intensity influenced the level of growth-inhibitory antibodies. Antibodies to recombinant MSP1-42 were not associated with growth inhibition and high immunoglobulin G levels were poorly predictive of inhibitory activity. The level of inhibitory activity against different isolates varied. CONCLUSIONS: Children can acquire growth-inhibitory antibodies at a young age, but once they are acquired they do not appear to be boosted by on-going exposure. Inhibitory antibodies may play a role in protection from early childhood malaria.

Mackintosh CL, Mwangi T, Kinyanjui SM, Mosobo M, Pinches R, Williams TN, Newbold CI, Marsh K. 2008. Failure to respond to the surface of Plasmodium falciparum infected erythrocytes predicts susceptibility to clinical malaria amongst African children. Int J Parasitol, 38 (12), pp. 1445-1454. | Show Abstract | Read more

Following infection with Plasmodium falciparum malaria, children in endemic areas develop antibodies specific to antigens on the parasite-infected red cell surface of the infecting isolate, antibodies associated with protection against subsequent infection with that isolate. In some circumstances induction of antibodies to heterologous parasite isolates also occurs and this has been suggested as evidence for cross-reactivity of responses against the erythrocyte surface. The role of these relatively cross-reactive antibodies in protection from clinical malaria is currently unknown. We studied the incidence of clinical malaria amongst children living on the coast of Kenya through one high transmission season. By categorising individuals according to their pre-season parasite status and antibody response to the surface of erythrocytes infected with four parasite isolates we were able to identify a group of children, those who failed to make a concomitant antibody response in the presence of an asymptomatic parasitaemia, at increased susceptibility to clinical malaria in the subsequent 6 months. The fact that this susceptible group was identified regardless of the parasite isolate tested infers a cross-reactive or conserved target is present on the surface of infected erythrocytes. Identification of this target will significantly aid understanding of naturally acquired immunity to clinical malaria amongst children in endemic areas.

Lairumbi GM, Molyneux S, Snow RW, Marsh K, Peshu N, English M. 2008. Promoting the social value of research in Kenya: examining the practical aspects of collaborative partnerships using an ethical framework. Soc Sci Med, 67 (5), pp. 734-747. | Show Abstract | Read more

The ethics of research continue to attract considerable debate, particularly when that research is sponsored by partners from the North and carried out in the South. Ethical research should contribute to social value in the country where research is being carried out, but there is significant debate around how this might be achieved and who is responsible. The literature suggests that researchers might employ two inter-related strategies to maximise social value: collaborative partnerships with policy makers and communities from the outset of research, and dissemination of research results to participants, policy makers and implementers once the research is over. These areas have received relatively little empirical attention. In this study, we carried out 40 in-depth interviews to explore the role of collaborative partnerships in health research priority setting, and the way in which research findings are disseminated to aid policy making and implementation in Kenya. Interviewees included policy makers, researchers, policy implementers and representatives of organisations funding health reforms in Kenya. Two policy issues were drawn upon as tracers wherever possible: (1) the introduction of Artemesinin-based Combination Therapies (ACTs), an anti-malarial treatment policy; and (2) Haemophilus influenzae (Hib) vaccine for the prevention of pneumococcal diseases among children. The findings point to significant gaps in the 'research to policy to practice' pathway, particularly for national research institutions with a focus on clinical/biomedical research. These gaps reflect poorly effective partnerships among stakeholders and limit the potential social value of much research. While more investment is needed to establish strong structures for promoting and directing collaboration and partnership, how to target this investment is not entirely clear, especially in the context of the considerable power of the global health agenda and the research financing tied to it.

Mackintosh CL, Christodoulou Z, Mwangi TW, Kortok M, Pinches R, Williams TN, Marsh K, Newbold CI. 2008. Acquisition of naturally occurring antibody responses to recombinant protein domains of Plasmodium falciparum erythrocyte membrane protein 1. Malar J, 7 (1), pp. 155. | Show Abstract | Read more

BACKGROUND: Antibodies targeting variant antigens expressed on the surface of Plasmodium falciparum infected erythrocytes have been associated with protection from clinical malaria. The precise target for these antibodies is unknown. The best characterized and most likely target is the erythrocyte surface-expressed variant protein family Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). METHODS: Using recombinant proteins corresponding to five domains of the expressed A4 var gene, A4 PfEMP1, the naturally occurring antibody response was assessed, by ELISA, to each domain in serum samples obtained from individuals resident in two communities of differing malaria transmission intensity on the Kenyan coast. Using flow cytometry, the correlation in individual responses to each domain with responses to intact A4-infected erythrocytes expressing A4 PfEMP1 on their surface as well as responses to two alternative parasite clones and one clinical isolate was assessed. RESULTS: Marked variability in the prevalence of responses between each domain and between each transmission area was observed, as wasa strong correlation between age and reactivity with some but not all domains. Individual responses to each domain varied strikingly, with some individuals showing reactivity to all domains and others with no reactivity to any, this was apparent at all age groups. Evidence for possible cross-reactivity in responses to the domain DBL4gamma was found. CONCLUSION: Individuals acquire antibodies to surface expressed domains of a highly variant protein. The finding of potential cross-reactivity in responses to one of these domains is an important initial finding in the consideration of potential vaccine targets.

O'Meara WP, Mwangi TW, Williams TN, McKenzie FE, Snow RW, Marsh K. 2008. Relationship between exposure, clinical malaria, and age in an area of changing transmission intensity. Am J Trop Med Hyg, 79 (2), pp. 185-191. | Show Abstract

The relationship between malaria transmission intensity and clinical disease is important for predicting the outcome of control measures that reduce transmission. Comparisons of hospital data between areas of differing transmission intensity suggest that the mean age of hospitalized clinical malaria is higher under relatively lower transmission, but the total number of episodes is similar until transmission drops below a threshold, where the risks of hospitalized malaria decline. These observations have rarely been examined longitudinally in a single community where transmission declines over time. We reconstructed 16 years (1991-2006) of pediatric hospital surveillance data and infection prevalence surveys from a circumscribed geographic area on the Kenyan coast. The incidence of clinical malaria remained high, despite sustained reductions in exposure to infection. However, the age group experiencing the clinical attacks of malaria increased steadily as exposure declined and may precede changes in the number of episodes in an area with declining transmission.

Langhorne J, Ndungu FM, Sponaas AM, Marsh K. 2008. Immunity to malaria: more questions than answers. Nat Immunol, 9 (7), pp. 725-732. | Show Abstract | Read more

Malaria is one of the main health problems facing developing countries today. At present, preventative and treatment strategies are continuously hampered by the issues of the ever-emerging parasite resistance to newly introduced drugs, considerable costs and logistical problems. The main hope for changing this situation would be the development of effective malaria vaccines. An important part of this process is understanding the mechanisms of naturally acquired immunity to malaria. This review will highlight key aspects of immunity to malaria, about which surprisingly little is known and which will prove critical in the search for effective malaria vaccines.

Blythe JE, Yam XY, Kuss C, Bozdech Z, Holder AA, Marsh K, Langhorne J, Preiser PR. 2008. Plasmodium falciparum STEVOR proteins are highly expressed in patient isolates and located in the surface membranes of infected red blood cells and the apical tips of merozoites. Infect Immun, 76 (7), pp. 3329-3336. | Show Abstract | Read more

The human parasite Plasmodium falciparum has the potential to express a vast repertoire of variant proteins on the surface of the infected red blood cell (iRBC). Variation in the expression pattern of these proteins is linked to antigenic variation and thereby evasion of host antibody-mediated immunity. The genes in the stevor multigene family code for small variant antigens that are expressed in blood-stage parasites where they can be detected in membranous structures called Maurer's clefts (MC). Some studies have indicated that STEVOR protein may also be trafficked to the iRBC membrane. To address the location of STEVOR protein in more detail, we have analyzed expression in several cultured parasite lines and in parasites obtained directly from patients. We detected STEVOR expression in a higher proportion of parasites recently isolated from patients than in cultured parasite lines and show that STEVOR is trafficked in schizont-stage parasites from the MC to the RBC cytosol and the iRBC membrane. Furthermore, STEVOR protein is also detected at the apical end of merozoites. Importantly, we show that culture-adapted parasites do not require STEVOR for survival. These findings provide new insights into the role of the stevor multigene family during both the schizont and merozoite stages of the parasite and highlight the importance of studying freshly isolated parasites, rather than parasite lines maintained in culture, when investigating potential mediators of host-parasite interactions.

Fry AE, Auburn S, Diakite M, Green A, Richardson A, Wilson J, Jallow M, Sisay-Joof F, Pinder M, Griffiths MJ et al. 2008. Variation in the ICAM1 gene is not associated with severe malaria phenotypes. Genes Immun, 9 (5), pp. 462-469. | Show Abstract | Read more

Evidence from autopsy and in vitro binding studies suggests that adhesion of erythrocytes infected with Plasmodium falciparum to the human host intercellular adhesion molecule (ICAM)-1 receptor is important in the pathogenesis of severe malaria. Previous association studies between polymorphisms in the ICAM1 gene and susceptibility to severe malarial phenotypes have been inconclusive and often contradictory. We performed genetic association studies with 15 single nucleotide polymorphisms (SNPs) around the ICAM1 locus. All SNPs were screened in a family study of 1071 trios from The Gambia, Malawi and Kenya. Two key non-synonymous SNPs with previously reported associations, rs5491 (K56M or 'ICAM-1(Kilifi)') and rs5498 (K469E), were tested in an additional 708 Gambian trios and a case-control study of 4058 individuals. None of the polymorphisms were associated with severe malaria phenotypes. Pooled results across our studies for ICAM-1(Kilifi) were, in severe malaria, odds ratio (OR) 1.02, 95% confidence interval (CI) 0.96-1.09, P=0.54, and cerebral malaria OR 1.07, CI 0.97-1.17, P=0.17. We assess the available epidemiological, population genetic and functional evidence that links ICAM-1(Kilifi) to severe malaria susceptibility.

Bull PC, Buckee CO, Kyes S, Kortok MM, Thathy V, Guyah B, Stoute JA, Newbold CI, Marsh K. 2008. Plasmodium falciparum antigenic variation. Mapping mosaic var gene sequences onto a network of shared, highly polymorphic sequence blocks. Mol Microbiol, 68 (6), pp. 1519-1534. | Show Abstract | Read more

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a potentially important family of immune targets, encoded by an extremely diverse gene family called var. Understanding of the genetic organization of var genes is hampered by sequence mosaicism that results from a long history of non-homologous recombination. Here we have used software designed to analyse social networks to visualize the relationships between large collections of short var sequences tags sampled from clinical parasite isolates. In this approach, two sequences are connected if they share one or more highly polymorphic sequence blocks. The results show that the majority of analysed sequences including several var-like sequences from the chimpanzee parasite Plasmodium reichenowi can be either directly or indirectly linked together in a single unbroken network. However, the network is highly structured and contains putative subgroups of recombining sequences. The major subgroup contains the previously described group A var genes, previously proposed to be genetically distinct. Another subgroup contains sequences found to be associated with rosetting, a parasite virulence phenotype. The mosaic structure of the sequences and their division into subgroups may reflect the conflicting problems of maximizing antigenic diversity and minimizing epitope sharing between variants while maintaining their host cell binding functions.

Mwangi TW, Fegan G, Williams TN, Kinyanjui SM, Snow RW, Marsh K. 2008. Evidence for over-dispersion in the distribution of clinical malaria episodes in children. PLoS One, 3 (5), pp. e2196. | Show Abstract | Read more

BACKGROUND: It may be assumed that patterns of clinical malaria in children of similar age under the same level of exposure would follow a Poisson distribution with no over-dispersion. Longitudinal studies that have been conducted over many years suggest that some children may experience more episodes of clinical malaria than would be expected. The aim of this study was to identify this group of children and investigate possible causes for this increased susceptibility. METHODOLOGY AND PRINCIPAL FINDINGS: Using Poisson regression, we chose a group of children whom we designated as 'more susceptible' to malaria from 373 children under 10 years of age who were followed up for between 3 to 5 years from 1998-2003. About 21% of the children were categorized as 'more susceptible' and although they contributed only 23% of the person-time of follow-up, they experienced 55% of total clinical malaria episodes. Children that were parasite negative at all cross-sectional survey were less likely to belong to this group [AOR = 0.09, (95% CI: 0.14-0.61), p = 0.001]. CONCLUSIONS AND SIGNIFICANCE: The pattern of clinical malaria episodes follows a negative binomial distribution. Use of lack of a clinical malaria episode in a certain time period as endpoints for intervention or immunological studies may not adequately distinguish groups who are more or less immune. It may be useful in such studies, in addition to the usual endpoint of the time to first episode, to include end points which take into account the total number of clinical episodes experienced per child.

Osier FH, Fegan G, Polley SD, Murungi L, Verra F, Tetteh KK, Lowe B, Mwangi T, Bull PC, Thomas AW et al. 2008. Breadth and magnitude of antibody responses to multiple Plasmodium falciparum merozoite antigens are associated with protection from clinical malaria. Infect Immun, 76 (5), pp. 2240-2248. | Show Abstract | Read more

Individuals living in areas where malaria is endemic are repeatedly exposed to many different malaria parasite antigens. Studies on naturally acquired antibody-mediated immunity to clinical malaria have largely focused on the presence of responses to individual antigens and their associations with decreased morbidity. We hypothesized that the breadth (number of important targets to which antibodies were made) and magnitude (antibody level measured in a random serum sample) of the antibody response were important predictors of protection from clinical malaria. We analyzed naturally acquired antibodies to five leading Plasmodium falciparum merozoite-stage vaccine candidate antigens, and schizont extract, in Kenyan children monitored for uncomplicated malaria for 6 months (n = 119). Serum antibody levels to apical membrane antigen 1 (AMA1) and merozoite surface protein antigens (MSP-1 block 2, MSP-2, and MSP-3) were inversely related to the probability of developing malaria, but levels to MSP-1(19) and erythrocyte binding antigen (EBA-175) were not. The risk of malaria was also inversely associated with increasing breadth of antibody specificities, with none of the children who simultaneously had high antibody levels to five or more antigens experiencing a clinical episode (17/119; 15%; P = 0.0006). Particular combinations of antibodies (AMA1, MSP-2, and MSP-3) were more strongly predictive of protection than others. The results were validated in a larger, separate case-control study whose end point was malaria severe enough to warrant hospital admission (n = 387). These findings suggest that under natural exposure, immunity to malaria may result from high titers antibodies to multiple antigenic targets and support the idea of testing combination blood-stage vaccines optimized to induce similar antibody profiles.

Agak GW, Bejon P, Fegan G, Gicheru N, Villard V, Kajava AV, Marsh K, Corradin G. 2008. Longitudinal analyses of immune responses to Plasmodium falciparum derived peptides corresponding to novel blood stage antigens in coastal Kenya. Vaccine, 26 (16), pp. 1963-1971. | Show Abstract | Read more

We have recently described 95 predicted alpha-helical coiled-coil peptides derived from putative Plasmodium falciparum erythrocytic stage proteins. Seventy peptides recognized with the highest level of prevalence by sera from three endemic areas were selected for further studies. In this study, we sequentially examined antibody responses to these synthetic peptides in two cohorts of children at risk of clinical malaria in Kilifi district in coastal Kenya, in order to characterize the level of peptide recognition by age, and the role of anti-peptide antibodies in protection from clinical malaria. Antibody levels from 268 children in the first cohort (Chonyi) were assayed against 70 peptides. Thirty-nine peptides were selected for further study in a second cohort (Junju). The rationale for the second cohort was to confirm those peptides identified as protective in the first cohort. The Junju cohort comprised of children aged 1-6 years old (inclusive). Children were actively followed up to identify episodes of febrile malaria in both cohorts. Of the 70 peptides examined, 32 showed significantly (p<0.05) increased antibody recognition in older children and 40 showed significantly increased antibody recognition in parasitaemic children. Ten peptides were associated with a significantly reduced odds ratio (OR) for an episode of clinical malaria in the first cohort of children and two of these peptides (LR146 and AS202.11) were associated with a significantly reduced OR in both cohorts. LR146 is derived from hypothetical protein PFB0145c in PlasmoDB. Previous work has identified this protein as a target of antibodies effective in antibody dependent cellular inhibition (ADCI). The current study substantiates further the potential of protein PFB0145c and also identifies protein PF11_0424 as another likely target of protective antibodies against P. falciparum malaria.

Jenkins N, Wu Y, Chakravorty S, Kai O, Marsh K, Craig A. 2008. Plasmodium falciparum Icam-1-based cytoadherence-related signalling in endothelical cells JOURNAL OF INFECTION, 56 (4), pp. 299-299. | Read more

Fry AE, Griffiths MJ, Auburn S, Diakite M, Forton JT, Green A, Richardson A, Wilson J, Jallow M, Sisay-Joof F et al. 2008. Common variation in the ABO glycosyltransferase is associated with susceptibility to severe Plasmodium falciparum malaria. Hum Mol Genet, 17 (4), pp. 567-576. | Show Abstract | Read more

There is growing epidemiological and molecular evidence that ABO blood group affects host susceptibility to severe Plasmodium falciparum infection. The high frequency of common ABO alleles means that even modest differences in susceptibility could have a significant impact on the health of people living in malaria endemic regions. We performed an association study, the first to utilize key molecular genetic variation underlying the ABO system, genotyping >9000 individuals across three African populations. Using population- and family-based tests, we demonstrated that alleles producing functional ABO enzymes are associated with greater risk of severe malaria phenotypes (particularly malarial anemia) in comparison with the frameshift deletion underlying blood group O: case-control allelic odds ratio (OR), 1.2; 95% confidence interval (CI), 1.09-1.32; P = 0.0003; family-studies allelic OR, 1.19; 95% CI, 1.08-1.32; P = 0.001; pooled across all studies allelic OR, 1.18; 95% CI, 1.11-1.26; P = 2 x 10(-7). We found suggestive evidence of a parent-of-origin effect at the ABO locus by analyzing the family trios. Non-O haplotypes inherited from mothers, but not fathers, are significantly associated with severe malaria (likelihood ratio test of Weinberg, P = 0.046). Finally, we used HapMap data to demonstrate a region of low F(ST) (-0.001) between the three main HapMap population groups across the ABO locus, an outlier in the empirical distribution of F(ST) across chromosome 9 (approximately 99.5-99.9th centile). This low F(ST) region may be a signal of long-standing balancing selection at the ABO locus, caused by multiple infectious pathogens including P. falciparum.

Bejon P, Mwangi TW, Lowe B, Peshu N, Hill AV, Marsh K. 2008. Helminth infection and eosinophilia and the risk of Plasmodium falciparum malaria in 1- to 6-year-old children in a malaria endemic area. PLoS Negl Trop Dis, 2 (1), pp. e164. | Show Abstract | Read more

BACKGROUND: Helminth infection is common in malaria endemic areas, and an interaction between the two would be of considerable public health importance. Animal models suggest that helminth infections may increase susceptibility to malaria, but epidemiological data has been limited and contradictory. METHODOLOGY/PRINCIPAL FINDINGS: In a vaccine trial, we studied 387 one- to six-year-old children for the effect of helminth infections on febrile Plasmodium falciparum malaria episodes. Gastrointestinal helminth infection and eosinophilia were prevalent (25% and 50% respectively), but did not influence susceptibility to malaria. Hazard ratios were 1 for gastrointestinal helminth infection (95% CI 0.6-1.6) and 0.85 and 0.85 for mild and marked eosinophilia, respectively (95% CI 0.56-1.76 and 0.69-1.96). Incident rate ratios for multiple episodes were 0.83 for gastro-intestinal helminth infection (95% CI 0.5-1.33) and 0.86 and 0.98 for mild and marked eosinophilia (95% CI 0.5-1.4 and 0.6-1.5). CONCLUSIONS/SIGNIFICANCE: There was no evidence that infection with gastrointestinal helminths or urinary schistosomiasis increased susceptibility to Plasmodium falciparum malaria in this study. Larger studies including populations with a greater prevalence of helminth infection should be undertaken.

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Achidi EA, Agbenyega T, Allen S, Amodu O, Bojang K, Conway D, Corran P, Deloukas P, Djimde A, Dolo A et al. 2008. A global network for investigating the genomic epidemiology of malaria Nature, 456 (7223), pp. 732-737. | Show Abstract | Read more

Large-scale studies of genomic variation could assist efforts to eliminate malaria. But there are scientific, ethical and practical challenges to carrying out such studies in developing countries, where the burden of disease is greatest. The Malaria Genomic Epidemiology Network (MalariaGEN) is now working to overcome these obstacles, using a consortial approach that brings together researchers from 21 countries. © 2008 Macmillan Publishers Limited. All rights reserved.

Gwer S, Punt J, Idro R, Mwamuye I, Gatakaa H, Charles RJCN, Marsh K. 2008. Biphasic clinical course among Kenyan children with cerebral malaria African Journal of Neurological Sciences, 27 (1), | Show Abstract

Background: Cerebral malaria is the most severe neurological complication of Falciparum malaria. It is associated with a significant risk of death and neurological sequelae. A biphasic clinical picture is associated with an even greater risk of neurological sequelae. Objective: To examine the incidence and clinical characteristics of a biphasic clinical course in children with cerebral malaria and to study its relationship with outcome Method: We undertook a retrospective study of children admitted to Kilifi District Hospital with a history of impaired consciousness and Falciparum infection between January 1994 and December 2004. We identified children with a biphasic clinical course and examined their clinical characteristics and outcome against that of those with a single clinical course. Results: Out of 587 children with cerebral malaria, 11 were found to have a biphasic clinical course often heralded by recurrence of seizures. This clinical pattern was associated with a greater incidence of neurological sequelae but no death. Conclusion: We speculate that a biphasic clinical course may occur due to recurrent seizures, co-morbidity and reperfusion of cerebral areas previously clogged by parasitized red blood cells. A prospective examination of this group may shed more light on causality and enlighten further on pathogenesis of cerebral malaria. © 2002 African Journal of Neurological Sciences. All rights reserved.

Todryk SM, Bejon P, Mwangi T, Plebanski M, Urban B, Marsh K, Hill AV, Flanagan KL. 2008. Correlation of memory T cell responses against TRAP with protection from clinical malaria, and CD4 CD25 high T cells with susceptibility in Kenyans. PLoS One, 3 (4), pp. e2027. | Show Abstract | Read more

BACKGROUND: Immunity to malaria develops naturally in endemic regions, but the protective immune mechanisms are poorly understood. Many vaccination strategies aim to induce T cells against diverse pre-erythrocytic antigens, but correlates of protection in the field have been limited. The objective of this study was to investigate cell-mediated immune correlates of protection in natural malaria. Memory T cells reactive against thrombospondin-related adhesive protein (TRAP) and circumsporozoite (CS) protein, major vaccine candidate antigens, were measured, as were frequencies of CD4(+) CD25(high) T cells, which may suppress immunity, and CD56(+) NK cells and gammadelta T cells, which may be effectors or may modulate immunity. METHODOLOGY AND PRINCIPAL FINDINGS: 112 healthy volunteers living in rural Kenya were entered in the study. Memory T cells reactive against TRAP and CS were measured using a cultured IFNgamma ELISPOT approach, whilst CD4(+) CD25(high) T cells, CD56(+) NK cells, and gammadelta T cells were measured by flow cytometry. We found that T cell responses against TRAP were established early in life (<5 years) in contrast to CS, and cultured ELISPOT memory T cell responses did not correlate with ex-vivo IFNgamma ELISPOT effector responses. Data was examined for associations with risk of clinical malaria for a period of 300 days. Multivariate logistic analysis incorporating age and CS response showed that cultured memory T cell responses against TRAP were associated with a significantly reduced incidence of malaria (p = 0.028). This was not seen for CS responses. Higher numbers of CD4(+) CD25(high) T cells, potentially regulatory T cells, were associated with a significantly increased risk of clinical malaria (p = 0.039). CONCLUSIONS: These data demonstrate a role for central memory T cells in natural malarial immunity and support current vaccination strategies aimed at inducing durable protective T cell responses against the TRAP antigen. They also suggest that CD4(+) CD25(high) T cells may negatively affect naturally acquired malarial immunity.

Bejon P, Mwangi TW, Lowe B, Peshu N, Hill AVS, Marsh K. 2008. Helminth infection and eosinophilia and the risk of Plasmodium falciparum malaria in 1- to 6-year-old children in a malaria endemic area PLoS Neglected Tropical Diseases, 2 (2), | Show Abstract | Read more

Background: Helminth infection is common in malaria endemic areas, and an interaction between the two would be of considerable public health importance. Animal models suggest that helminth infections may increase susceptibility to malaria, but epidemiological data has been limited and contradictory. Methodology/Principal Findings: In a vaccine trial, we studied 387 one- to six-year-old children for the effect of helminth infections on febrile Plasmodium falciparum malaria episodes. Gastrointestinal helminth infection and eosinophilia were prevalent (25% and 50% respectively), but did not influence susceptibility to malaria. Hazard ratios were 1 for gastrointestinal helminth infection (95% CI 0.6-1.6) and 0.85 and 0.85 for mild and marked eosinophilia, respectively (95% CI 0.56-1.76 and 0.69-1.96). Incident rate ratios for multiple episodes were 0.83 for gastro-intestinal helminth infection (95% CI 0.5-1.33) and 0.86 and 0.98 for mild and marked eosinophilia (95% CI 0.5-1.4 and 0.6-1.5). Conclusions/Significance: There was no evidence that infection with gastrointestinal helminths or urinary schistosomiasis increased susceptibility to Plasmodium falciparum malaria in this study. Larger studies including populations with a greater prevalence of helminth infection should be undertaken. © 2008 Bejon et al.

Bejon P, Mwangi TW, Lowe B, Peshu N, Hill AVS, Marsh K. 2008. Helminth infection and eosinophilia and the risk of Plasmodium falciparum malaria in 1- to 6-year-old children in a malaria endemic area. PLoS neglected tropical diseases, 2 (1), | Show Abstract

BACKGROUND: Helminth infection is common in malaria endemic areas, and an interaction between the two would be of considerable public health importance. Animal models suggest that helminth infections may increase susceptibility to malaria, but epidemiological data has been limited and contradictory. METHODOLOGY/PRINCIPAL FINDINGS: In a vaccine trial, we studied 387 one- to six-year-old children for the effect of helminth infections on febrile Plasmodium falciparum malaria episodes. Gastrointestinal helminth infection and eosinophilia were prevalent (25% and 50% respectively), but did not influence susceptibility to malaria. Hazard ratios were 1 for gastrointestinal helminth infection (95% CI 0.6-1.6) and 0.85 and 0.85 for mild and marked eosinophilia, respectively (95% CI 0.56-1.76 and 0.69-1.96). Incident rate ratios for multiple episodes were 0.83 for gastro-intestinal helminth infection (95% CI 0.5-1.33) and 0.86 and 0.98 for mild and marked eosinophilia (95% CI 0.5-1.4 and 0.6-1.5). CONCLUSIONS/SIGNIFICANCE: There was no evidence that infection with gastrointestinal helminths or urinary schistosomiasis increased susceptibility to Plasmodium falciparum malaria in this study. Larger studies including populations with a greater prevalence of helminth infection should be undertaken.

Persson KE, McCallum FJ, Reiling L, Lister NA, Stubbs J, Cowman AF, Marsh K, Beeson JG. 2008. Variation in use of erythrocyte invasion pathways by Plasmodium falciparum mediates evasion of human inhibitory antibodies. J Clin Invest, 118 (1), pp. 342-351. | Show Abstract | Read more

Antibodies that inhibit Plasmodium falciparum invasion of erythrocytes are believed to be an important component of immunity against malaria. During blood-stage infection, P. falciparum can use different pathways for erythrocyte invasion by varying the expression and/or utilization of members of 2 invasion ligand families: the erythrocyte-binding antigens (EBAs) and reticulocyte-binding homologs (PfRhs). Invasion pathways can be broadly classified into 2 groups based on the use of sialic acid (SA) on the erythrocyte surface by parasite ligands. We found that inhibitory antibodies are acquired by malaria-exposed Kenyan children and adults against ligands of SA-dependent and SA-independent invasion pathways, and the ability of antibodies to inhibit erythrocyte invasion depended on the pathway used by P. falciparum isolates. Differential inhibition of P. falciparum lines that varied in their use of specific EBA and PfRh proteins pointed to these ligand families as major targets of inhibitory antibodies. Antibodies against recombinant EBA and PfRh proteins were acquired in an age-associated manner, and inhibitory antibodies against EBA175 appeared prominent among some individuals. These findings suggest that variation in invasion phenotype might have evolved as a mechanism that facilitates immune evasion by P. falciparum and that a broad inhibitory response against multiple ligands may be required for effective immunity.

Bejon P, Mwangi T, Lowe B, Peshu N, Hill AV, Marsh K. 2007. Clearing asymptomatic parasitaemia increases the specificity of the definition of mild febrile malaria. Vaccine, 25 (48), pp. 8198-8202. | Show Abstract | Read more

In clinical trials, the specificity of the disease endpoint is critical to an accurate estimate of vaccine efficacy. We used a logistic regression model to analyse parasite densities among children before and after treatment with antimalarials, in order to estimate the impact that clearing asymptomatic parasitaemia had on the specificity of the endpoint of febrile malaria. The malaria attributable fever fraction was higher after antimalarial treatment (i.e. fever and parasitaemia were more likely to be causally related), implying that drug treatment prior to monitoring decreased the misclassification of febrile malaria. In intervention studies with febrile malaria as an endpoint, clearing asymptomatic parasitaemia increases the study's power more effectively than raising the threshold parasitaemia.

Okiro EA, Hay SI, Gikandi PW, Sharif SK, Noor AM, Peshu N, Marsh K, Snow RW. 2007. The decline in paediatric malaria admissions on the coast of Kenya. Malar J, 6 (1), pp. 151. | Show Abstract | Read more

BACKGROUND: There is only limited information on the health impact of expanded coverage of malaria control and preventative strategies in Africa. METHODS: Paediatric admission data were assembled over 8.25 years from three District Hospitals; Kilifi, Msambweni and Malindi, situated along the Kenyan Coast. Trends in monthly malaria admissions between January 1999 and March 2007 were analysed using several time-series models that adjusted for monthly non-malaria admission rates and the seasonality and trends in rainfall. RESULTS: Since January 1999 paediatric malaria admissions have significantly declined at all hospitals. This trend was observed against a background of rising or constant non-malaria admissions and unaffected by long-term rainfall throughout the surveillance period. By March 2007 the estimated proportional decline in malaria cases was 63% in Kilifi, 53% in Kwale and 28% in Malindi. Time-series models strongly suggest that the observed decline in malaria admissions was a result of malaria-specific control efforts in the hospital catchment areas. CONCLUSION: This study provides evidence of a changing disease burden on the Kenyan coast and that the most parsimonious explanation is an expansion in the coverage of interventions such as the use of insecticide-treated nets and the availability of anti-malarial medicines. While specific attribution to intervention coverage cannot be computed what is clear is that this area of Kenya is experiencing a malaria epidemiological transition.

Rowe JA, Handel IG, Thera MA, Deans AM, Lyke KE, Koné A, Diallo DA, Raza A, Kai O, Marsh K et al. 2007. Blood group O protects against severe Plasmodium falciparum malaria through the mechanism of reduced rosetting. Proc Natl Acad Sci U S A, 104 (44), pp. 17471-17476. | Show Abstract | Read more

Malaria has been a major selective force on the human population, and several erythrocyte polymorphisms have evolved that confer resistance to severe malaria. Plasmodium falciparum rosetting, a parasite virulence phenotype associated with severe malaria, is reduced in blood group O erythrocytes compared with groups A, B, and AB, but the contribution of the ABO blood group system to protection against severe malaria has received little attention. We hypothesized that blood group O may confer resistance to severe falciparum malaria through the mechanism of reduced rosetting. In a matched case-control study of 567 Malian children, we found that group O was present in only 21% of severe malaria cases compared with 44-45% of uncomplicated malaria controls and healthy controls. Group O was associated with a 66% reduction in the odds of developing severe malaria compared with the non-O blood groups (odds ratio 0.34, 95% confidence interval 0.19-0.61, P < 0.0005, severe cases versus uncomplicated malaria controls). In the same sample set, P. falciparum rosetting was reduced in parasite isolates from group O children compared with isolates from the non-O blood groups (P = 0.003, Kruskal-Wallis test). Statistical analysis indicated a significant interaction between host ABO blood group and parasite rosette frequency that supports the hypothesis that the protective effect of group O operates through the mechanism of reduced P. falciparum rosetting. This work provides insights into malaria pathogenesis and suggests that the selective pressure imposed by malaria may contribute to the variable global distribution of ABO blood groups in the human population.

Khor CC, Vannberg FO, Chapman SJ, Walley A, Aucan C, Loke H, White NJ, Peto T, Khor LK, Kwiatkowski D et al. 2007. Positive replication and linkage disequilibrium mapping of the chromosome 21q22.1 malaria susceptibility locus. Genes Immun, 8 (7), pp. 570-576. | Show Abstract | Read more

Four cytokine receptor genes are located on Chr21q22.11, encoding the alpha and beta subunits of the interferon-alpha receptor (IFNAR1 and IFNAR2), the beta subunit of the interleukin 10 receptor (IL10RB) and the second subunit of the interferon-gamma receptor (IFNGR2). We previously reported that two variants in IFNAR1 were associated with susceptibility to malaria in Gambians. We now present an extensive fine-scale mapping of the associated region utilizing 45 additional genetic markers obtained from public databases and by sequencing a 44 kb region in and around the IFNAR1 gene in 24 Gambian children (12 cases/12 controls). Within the IFNAR1 gene, a newly studied C --> G single-nucleotide polymorphism (IFNAR1 272354c-g) at position -576 relative to the transcription start was found to be more strongly associated with susceptibility to severe malaria. Association was observed in three populations: in Gambian (P=0.002), Kenyan (P=0.022) and Vietnamese (P=0.005) case-control studies. When all three studies were combined, using the Mantel-Haenszel test, the presence of IFNAR1 -576G was associated with a substantially elevated risk of severe malaria (N=2444, OR=1.38, 95% CI: 1.17-1.64; P=1.7 x 10(-4)). This study builds on previous work to further highlight the importance of the type-I interferon pathway in malaria susceptibility and illustrates the utility of typing SNPs within regions of high linkage disequilibrium in multiple populations to confirm initial positive associations.

Idro R, Crawley J, Marsh K, Newton CRJC, Neville BGR. 2007. Neurological involvement in acute falciparum malaria in Kenyan children - Reply JAMA-JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION, 298 (11), pp. 1274-1274. | Read more

Bejon P, Mwacharo J, Kai O, Todryk S, Keating S, Lowe B, Lang T, Mwangi TW, Gilbert SC, Peshu N et al. 2007. The induction and persistence of T cell IFN-gamma responses after vaccination or natural exposure is suppressed by Plasmodium falciparum. J Immunol, 179 (6), pp. 4193-4201. | Show Abstract

Epidemiological observations suggest that T cell immunity may be suppressed in malaria-endemic areas. In vitro studies, animal models, and limited data in humans link immunosuppression with malaria, malnutrition, and other parasitic infections. However, there are no data to determine whether malaria-induced immunosuppression is significant in the long-term, or relative data comparing it with other factors in malaria-endemic areas, so as to measure the impact of malaria, other parasitic disease, nutritional status, age. and location on the acquisition and longevity of IFN-gamma responses in children in Kenya. We studied these factors in two cohorts of 1- to 6-year-old children in a malaria-endemic area. T cell responses were induced by vaccination in one cohort, and acquired as a result of natural exposure in a second cohort. Serial ELISPOT assays conducted over a 1-year period measured the induction and kinetics of IFN-gamma production in response to the malaria Ag thrombospondin-related adhesion protein. Induced responses in both cohorts and the longevity of response in the vaccinated cohort were fitted to potential explanatory variables. Parasitemia was prospectively associated with reduced IFN-gamma-producing T cells in both cohorts (by 15-25%), and both parasitemia and episodes of febrile malaria were associated with 19 and 31% greater attrition of T cell responses, respectively. Malaria may reduce the efficacy vaccinations such as bacillus Calmette-Guérin and investigational T cell-inducing vaccines, and may delay the acquisition of immunity following natural exposure to malaria and other pathogens.

Bejon P, Ogada E, Mwangi T, Milligan P, Lang T, Fegan G, Gilbert SC, Peshu N, Marsh K, Hill AV. 2007. Extended follow-up following a phase 2b randomized trial of the candidate malaria vaccines FP9 ME-TRAP and MVA ME-TRAP among children in Kenya. PLoS One, 2 (8), pp. e707. | Show Abstract | Read more

BACKGROUND: "FFM ME-TRAP" is sequential immunisation with two attenuated poxvirus vectors (FP9 and modified vaccinia virus Ankara) delivering the pre-erythrocytic malaria antigen ME-TRAP. Over nine months follow-up in our original study, there was no evidence that FFM ME-TRAP provided protection against malaria. The incidence of malaria was slightly higher in children who received FFM ME-TRAP, but this was not statistically significant (hazard ratio 1.5, 95% CI 1.0-2.3). Although the study was unblinded, another nine months follow-up was planned to monitor the incidence of malaria and other serious adverse events. METHODS AND FINDINGS: 405 children aged 1-6 yrs were initially randomized to vaccination with either FFM ME-TRAP or control (rabies vaccine). 380 children were still available for follow-up after the first nine months. Children were seen weekly and whenever they were unwell for nine months monitoring. The axillary temperature was measured, and blood films taken when febrile. The primary analysis was time to parasitaemia >2,500/microl. During the second nine months monitoring, 49 events met the primary endpoint (febrile malaria with parasites >2,500/microl) in the Intention To Treat (ITT) group. 23 events occurred among the 189 children in the FFM ME-TRAP group, and 26 among the 194 children in the control group. In the full 18 months of monitoring, there were 63 events in the FFM ME-TRAP group and 60 in the control group (HR = 1.2, CI 0.84-1.73, p = 0.35). There was no evidence that the HR changed over the 18 months (test for interaction between time and vaccination p = 0.11). CONCLUSIONS: Vaccination with FFM ME-TRAP was not protective against malaria in this study. Malaria incidence during 18 months of surveillance was similar in both vaccine groups. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN88335123.

Cumberland P, Shulman CE, Maple PA, Bulmer JN, Dorman EK, Kawuondo K, Marsh K, Cutts FT. 2007. Maternal HIV infection and placental malaria reduce transplacental antibody transfer and tetanus antibody levels in newborns in Kenya. J Infect Dis, 196 (4), pp. 550-557. | Show Abstract | Read more

BACKGROUND: In clinical trials, maternal tetanus toxoid (TT) vaccination is effective in protecting newborns against tetanus infection, but inadequate placental transfer of tetanus antibodies may contribute to lower-than-expected rates of protection in routine practice. We studied the effect of placental malaria and maternal human immunodeficiency virus (HIV) infection on placental transfer of antibodies to tetanus. METHODS: A total of 704 maternal-cord paired serum samples were tested by ELISA for antibodies to tetanus. The HIV status of all women was determined by an immunoglobulin G antibody-capture particle-adherence test, and placental malaria was determined by placental biopsy. Maternal history of TT vaccination was recorded. RESULTS: Tetanus antibody levels were reduced by 52% (95% confidence interval [CI], 30%-67%) in newborns of HIV-infected women and by 48% (95% CI, 26%-62%) in newborns whose mothers had active-chronic or past placental malaria. Thirty-seven mothers (5.3%) and 55 newborns (7.8%) had tetanus antibody levels <0.1 IU/mL (i.e., were seronegative). Mothers' self-reported history of lack of tetanus immunization was the strongest predictor of seronegativity and of tetanus antibody levels in maternal and cord serum. CONCLUSION: Malarial and HIV infections may hinder efforts to eliminate maternal and neonatal tetanus, making implementation of the current policy for mass vaccination of women of childbearing age an urgent priority.

Bejon P, Berkley JA, Mwangi T, Ogada E, Mwangi I, Maitland K, Williams T, Scott JA, English M, Lowe BS et al. 2007. Defining childhood severe falciparum malaria for intervention studies. PLoS Med, 4 (8), pp. e251. | Show Abstract | Read more

BACKGROUND: Clinical trials of interventions designed to prevent severe falciparum malaria in children require a clear endpoint. The internationally accepted definition of severe malaria is sensitive, and appropriate for clinical purposes. However, this definition includes individuals with severe nonmalarial disease and coincident parasitaemia, so may lack specificity in vaccine trials. Although there is no "gold standard" individual test for severe malaria, malaria-attributable fractions (MAFs) can be estimated among groups of children using a logistic model, which we use to test the suitability of various case definitions as trial endpoints. METHODS AND FINDINGS: A total of 4,583 blood samples were taken from well children in cross-sectional surveys and from 1,361 children admitted to a Kenyan District hospital with severe disease. Among children under 2 y old with severe disease and over 2,500 parasites per microliter of blood, the MAFs were above 85% in moderate- and low-transmission areas, but only 61% in a high-transmission area. HIV and malnutrition were not associated with reduced MAFs, but gastroenteritis with severe dehydration (defined by reduced skin turgor), lower respiratory tract infection (clinician's final diagnosis), meningitis (on cerebrospinal fluid [CSF] examination), and bacteraemia were associated with reduced MAFs. The overall MAF was 85% (95% confidence interval [CI] 83.8%-86.1%) without excluding these conditions, 89% (95% CI 88.4%-90.2%) after exclusions, and 95% (95% CI 94.0%-95.5%) when a threshold of 2,500 parasites/mul was also applied. Applying a threshold and exclusion criteria reduced sensitivity to 80% (95% CI 77%-83%). CONCLUSIONS: The specificity of a case definition for severe malaria is improved by applying a parasite density threshold and by excluding children with meningitis, lower respiratory tract infection (clinician's diagnosis), bacteraemia, and gastroenteritis with severe dehydration, but not by excluding children with HIV or malnutrition.

Osier FH, Polley SD, Mwangi T, Lowe B, Conway DJ, Marsh K. 2007. Naturally acquired antibodies to polymorphic and conserved epitopes of Plasmodium falciparum merozoite surface protein 3. Parasite Immunol, 29 (8), pp. 387-394. | Show Abstract | Read more

Many studies on the role of merozoite surface protein 3 (MSP3) in immunity against malaria have focused on a conserved section of MSP3. New evidence suggests that polymorphic sequences within MSP3 are under immune selection. We report a detailed analysis of naturally-acquired antibodies to allele-specific and conserved parts of MSP3 in a Kenyan cohort. Indirect and competition ELISA to heterologous recombinant MSP3 proteins were used for antibody assays, and parasites were genotyped for msp3 alleles. Antibody reactivity to allele-specific and conserved epitopes of MSP3 was heterogeneous between individuals. Overall, the prevalence of allele-specific antibody reactivity was significantly higher (3D7-specific 54%, K1-specific 41%) than that to a recombinant protein representing a conserved portion of C-terminal MSP3 (24%, P < 0.01). The most abundant IgG subclass was IgG3, followed by IgG1. Allele-specific reactivity to the K1-type of MSP3 was associated with a lower risk of clinical malaria episodes during a 6-month follow-up in individuals who were parasitized at the start of the malaria transmission season (Relative risk 0.41 with 95% confidence interval 0.20-0.81, P = 0.011). The potential importance of allele-specific immunity to MSP3 should be considered in addition to immunity to conserved epitopes, in the development of an MSP3 malaria vaccine.

Jenkins N, Wu Y, Chakravorty S, Kai O, Marsh K, Craig A. 2007. Plasmodium falciparum intercellular adhesion molecule-1-based cytoadherence-related signaling in human endothelial cells. J Infect Dis, 196 (2), pp. 321-327. | Show Abstract | Read more

BACKGROUND: Cytoadherence of Plasmodium falciparum-infected erythrocytes to host endothelium has been associated with pathology in severe malaria, but, despite extensive information on the primary processes involved in the adhesive interactions, the mechanisms underlying disease are poorly understood. METHODS: We compared parasite lines varying in their binding properties to human endothelial cells for their ability to stimulate signaling activity. RESULTS: In human umbilical vein endothelial cells (HUVECs), which rely on adhesion to intercellular adhesion molecule (ICAM)-1 for binding, signaling is related to the avidity of the parasite line for ICAM-1 and can be blocked either through the use of anti-ICAM-1 monoclonal antibodies or HUVECs with altered ICAM-1 binding properties (i.e., ICAM-1(Kilifi)). Human dermal microvascular endothelial cells (HDMECs), which can bind infected erythrocytes via ICAM-1 and CD36, have a more complex pattern of signaling behavior, but this is also dependent on adhesive interactions rather than merely contact between cells. CONCLUSIONS: Signaling via apposition of P. falciparum-infected erythrocytes with host endothelium is dependent, at least in part, on the cytoadherence characteristics of the invading isolate. An understanding of the postadhesive processes produced by cytoadherence may help us to understand the variable pathologies seen in malaria disease.

Bull PC, Kyes S, Buckee CO, Montgomery J, Kortok MM, Newbold CI, Marsh K. 2007. An approach to classifying sequence tags sampled from Plasmodium falciparum var genes. Mol Biochem Parasitol, 154 (1), pp. 98-102. | Read more

Beeson JG, Ndungu F, Persson KE, Chesson JM, Kelly GL, Uyoga S, Hallamore SL, Williams TN, Reeder JC, Brown GV, Marsh K. 2007. Antibodies among men and children to placental-binding Plasmodium falciparum-infected erythrocytes that express var2csa. Am J Trop Med Hyg, 77 (1), pp. 22-28. | Show Abstract

During pregnancy, specific variants of Plasmodium falciparum-infected erythrocytes (IEs) can accumulate in the placenta through adhesion to chondroitin sulfate A (CSA) mediated by expression of PfEMP1 encoded by var2csa-type genes. Antibodies against these variants are associated with protection from maternal malaria. We evaluated antibodies among Kenyan, Papua New Guinean, and Malawian men and Kenyan children against two different CSA-binding P. falciparum isolates expressing var2csa variants. Specific IgG was present at significant levels among some men and children from each population, suggesting exposure to these variants is not exclusive to pregnancy. However, the level and prevalence of antibodies was substantially lower overall than exposed multigravidas. IgG-binding was specific and did not represent antibodies to subpopulations of non-CSA-binding IEs, and some sera inhibited IE adhesion to CSA. These findings have significant implications for understanding malaria pathogenesis and immunity and may be significant for understanding the acquisition of immunity to maternal malaria.

Deans AM, Nery S, Conway DJ, Kai O, Marsh K, Rowe JA. 2007. Invasion pathways and malaria severity in Kenyan Plasmodium falciparum clinical isolates. Infect Immun, 75 (6), pp. 3014-3020. | Show Abstract | Read more

The invasion of erythrocytes by Plasmodium falciparum occurs through multiple pathways that can be studied in vitro by examining the invasion of erythrocytes treated with enzymes such as neuraminidase, trypsin, and chymotrypsin. We have studied the invasion pathways used by 31 Kenyan P. falciparum isolates from children with uncomplicated or severe malaria. Six distinct invasion profiles were detected, out of eight possible profiles. The majority of isolates (23 of 31) showed neuraminidase-resistant, trypsin-sensitive invasion, characteristic of the pathway mediated by an unknown parasite ligand and erythrocyte receptor "X." The neuraminidase-sensitive, trypsin-sensitive phenotype consistent with invasion mediated by the binding of parasite ligand erythrocyte binding antigen 175 to glycophorin A, the most common invasion profile in a previous study of Gambian field isolates, was seen in only 3 of 31 Kenyan isolates. No particular invasion profile was associated with severe P. falciparum malaria, and there was no significant difference in the levels of inhibition by the various enzyme treatments between isolates from children with severe malaria and those from children with uncomplicated malaria (P, >0.1 for all enzymes; Mann-Whitney U test). These results do not support the hypothesis that differences in invasion phenotypes play an important role in malaria virulence and indicate that considerable gaps remain in our knowledge of the molecular basis of invasion pathways in natural P. falciparum infections.

Idro R, Ndiritu M, Ogutu B, Mithwani S, Maitland K, Berkley J, Crawley J, Fegan G, Bauni E, Peshu N et al. 2007. Burden, features, and outcome of neurological involvement in acute falciparum malaria in Kenyan children. JAMA, 297 (20), pp. 2232-2240. | Show Abstract | Read more

CONTEXT: Plasmodium falciparum appears to have a particular propensity to involve the brain but the burden, risk factors, and full extent of neurological involvement have not been systematically described. OBJECTIVES: To determine the incidence and describe the clinical phenotypes and outcomes of neurological involvement in African children with acute falciparum malaria. DESIGN, SETTING, AND PATIENTS: A review of records of all children younger than 14 years admitted to a Kenyan district hospital with malaria from January 1992 through December 2004. Neurological involvement was defined as convulsive seizures, agitation, prostration, or impaired consciousness or coma. MAIN OUTCOME MEASURES: The incidence, pattern, and outcome of neurological involvement. RESULTS: Of 58,239 children admitted, 19,560 (33.6%) had malaria as the primary clinical diagnosis. Neurological involvement was observed in 9313 children (47.6%) and manifested as seizures (6563/17,517 [37.5%]), agitation (316/11,193 [2.8%]), prostration (3223/15,643 [20.6%]), and impaired consciousness or coma (2129/16,080 [13.2%]). In children younger than 5 years, the mean annual incidence of admissions with malaria was 2694 per 100,000 persons and the incidence of malaria with neurological involvement was 1156 per 100,000 persons. However, readmissions may have led to a 10% overestimate in incidence. Children with neurological involvement were older (median, 26 [interquartile range {IQR}, 15-41] vs 21 [IQR, 10-40] months; P<.001), had a shorter duration of illness (median, 2 [IQR, 1-3] vs 3 [IQR, 2-3] days; P<.001), and a higher geometric mean parasite density (42.0 [95% confidence interval {CI}, 40.0-44.1] vs 30.4 [95% CI, 29.0-31.8] x 10(3)/microL; P<.001). Factors independently associated with neurological involvement included past history of seizures (adjusted odds ratio [AOR], 3.50; 95% CI, 2.78-4.42), fever lasting 2 days or less (AOR, 2.02; 95% CI, 1.64-2.49), delayed capillary refill time (AOR, 3.66; 95% CI, 2.40-5.56), metabolic acidosis (AOR, 1.55; 95% CI, 1.29-1.87), and hypoglycemia (AOR, 2.11; 95% CI, 1.31-3.37). Mortality was higher in patients with neurological involvement (4.4% [95% CI, 4.2%-5.1%] vs 1.3% [95% CI, 1.1%-1.5%]; P<.001). At discharge, 159 (2.2%) of 7281 patients had neurological deficits. CONCLUSIONS: Neurological involvement is common in children in Kenya with acute falciparum malaria, and is associated with metabolic derangements, impaired perfusion, parasitemia, and increased mortality and neurological sequelae. This study suggests that falciparum malaria exposes many African children to brain insults.

Molyneux M. 2007. UK doctors are already put off by changes in training. BMJ, 334 (7596), pp. 709-710. | Read more

Khor CC, Chapman SJ, Vannberg FO, Dunne A, Murphy C, Ling EY, Frodsham AJ, Walley AJ, Kyrieleis O, Khan A et al. 2007. A Mal functional variant is associated with protection against invasive pneumococcal disease, bacteremia, malaria and tuberculosis. Nat Genet, 39 (4), pp. 523-528. | Show Abstract | Read more

Toll-like receptors (TLRs) and members of their signaling pathway are important in the initiation of the innate immune response to a wide variety of pathogens. The adaptor protein Mal (also known as TIRAP), encoded by TIRAP (MIM 606252), mediates downstream signaling of TLR2 and TLR4 (refs. 4-6). We report a case-control study of 6,106 individuals from the UK, Vietnam and several African countries with invasive pneumococcal disease, bacteremia, malaria and tuberculosis. We genotyped 33 SNPs, including rs8177374, which encodes a leucine substitution at Ser180 of Mal. We found that heterozygous carriage of this variant associated independently with all four infectious diseases in the different study populations. Combining the study groups, we found substantial support for a protective effect of S180L heterozygosity against these infectious diseases (N = 6,106; overall P = 9.6 x 10(-8)). We found that the Mal S180L variant attenuated TLR2 signal transduction.

Cordery DV, Kishore U, Kyes S, Shafi MJ, Watkins KR, Williams TN, Marsh K, Urban BC. 2007. Characterization of a Plasmodium falciparum macrophage-migration inhibitory factor homologue. J Infect Dis, 195 (6), pp. 905-912. | Show Abstract | Read more

BACKGROUND: Macrophage-migration inhibitory factor (MIF), one of the first cytokines described, has a broad range of proinflammatory properties. The genome sequencing project of Plasmodium falciparum identified a parasite homologue of MIF. The protein is expressed during the asexual blood stages of the parasite life cycle that cause malarial disease. The identification of a parasite homologue of MIF raised the question of whether it affects monocyte function in a manner similar to its human counterpart. METHODS: Recombinant P. falciparum MIF (PfMIF) was generated and used in vitro to assess its influence on monocyte function. Antibodies generated against PfMIF were used to determine the expression profile and localization of the protein in blood-stage parasites. Antibody responses to PfMIF were determined in Kenyan children with acute malaria and in control subjects. RESULTS: PfMIF protein was expressed in asexual blood-stage parasites, localized to the Maurer's cleft. In vitro treatment of monocytes with PfMIF inhibited random migration and reduced the surface expression of Toll-like receptor (TLR) 2, TLR4, and CD86. CONCLUSIONS: These results indicate that PfMIF is released during blood-stage malaria and potentially modulates the function of monocytes during acute P. falciparum infection.

Atkinson SH, Mwangi TW, Uyoga SM, Ogada E, Macharia AW, Marsh K, Prentice AM, Williams TN. 2007. The haptoglobin 2-2 genotype is associated with a reduced incidence of Plasmodium falciparum malaria in children on the coast of Kenya. Clin Infect Dis, 44 (6), pp. 802-809. | Show Abstract | Read more

BACKGROUND: Haptoglobin (Hp) genotype determines the efficiency of hemoglobin clearance after malaria-induced hemolysis and alters antioxidant and immune functions. The Hp2 allele is thought to have spread under strong selection pressure, but it is unclear whether this is due to protection from malaria or other diseases. METHODS: We monitored the incidence of febrile malaria and other childhood illnesses with regard to Hp genotype in a prospective cohort of 312 Kenyan children during 558.3 child-years of follow-up. We also conducted 7 cross-sectional surveys to determine the prevalence of Plasmodium falciparum parasitemia. RESULTS: The Hp2/2 genotype was associated with a 30% reduction in clinical malarial episodes (adjusted incidence rate ratio, 0.67; P=.008 for Hp2/2 vs. Hp1/1 and Hp2/1 combined). Protection increased with age; there was no protection in the first 2 years of life, 30% protection at > or = 2 years of age, and 50% protection from 4-10 years of age. Children with the Hp1/1 genotype had a significantly lower rate of nonmalarial fever (P=.001). CONCLUSIONS: Balancing selection pressures may have influenced the spread of the Hp gene. Our observations suggest that the Hp2 allele may have spread as a result of protection from malaria, and the Hp1 allele may be sustained by protection from other infections.

Ostrowski SR, Shulman CE, Peshu N, Staalsøe T, Høyer-Hansen G, Pedersen BK, Marsh K, Ullum H. 2007. Elevated plasma urokinase receptor predicts low birth weight in maternal malaria. Parasite Immunol, 29 (1), pp. 37-46. | Show Abstract | Read more

The blood level of soluble urokinase receptor (suPAR) is increased and associated with a poor clinical or fatal outcome in children with acute malaria. This study hypothesized that the suPAR level would be associated with foetal outcome in maternal malaria. suPAR was measured by ELISA in maternal and cord plasma samples taken during delivery in 253 pregnant Kenyan women stratified according to placental histology: no malaria infection (non-infected), active or active-chronic infection (actively infected) or past-chronic infection (past-infected). Maternal-suPAR was higher in actively infected women (median 3.93 (IQR 2.92-5.29) ng/mL) compared with non-infected (median 2.78 (IQR 1.86-3.87) ng/mL, P = 0.001) and past-infected (median 2.67 (IQR 1.94-3.7) ng/mL, P = 0.012) women. Cord-suPAR was comparable across the groups (median 2.98 (IQR 2.38-3.77) ng/mL). In actively infected women, maternal-suPAR and gestational age were the only independent predictors of birth weight in multivariate linear regression adjusted for maternal-suPAR, HIV-1 infection, age, BMI, haemoglobin, peripheral parasitaemia, parity and gestational age; 1 ng/mL higher maternal-suPAR predicted -56 g (95% CI -100 to -12, P = 0.016) reduced birth weight. Cord-suPAR could not predict birth weight after adjusting for gestational age. Future studies are warranted to investigate whether the maternal suPAR level is increased earlier in pregnancy in women with active placental malaria infection and whether early maternal suPAR measurements can predict birth weight. If so, measurements of maternal suPAR early in pregnancy might then potentially identify women with increased needs for antenatal care and intervention.

Bejon P, Ogada E, Mwangi T, Milligan P, Lang T, Fegan G, Gilbert SC, Peshu N, Marsh K, Hill AVS. 2007. Extended Follow-Up Following a Phase 2b Randomized Trial of the Candidate Malaria Vaccines FP9 ME-TRAP and MVA ME-TRAP among Children in Kenya PLOS ONE, 2 (8), | Show Abstract | Read more

BACKGROUND: "FFM ME-TRAP" is sequential immunisation with two attenuated poxvirus vectors (FP9 and modified vaccinia virus Ankara) delivering the pre-erythrocytic malaria antigen ME-TRAP. Over nine months follow-up in our original study, there was no evidence that FFM ME-TRAP provided protection against malaria. The incidence of malaria was slightly higher in children who received FFM ME-TRAP, but this was not statistically significant (hazard ratio 1.5, 95% CI 1.0-2.3). Although the study was unblinded, another nine months follow-up was planned to monitor the incidence of malaria and other serious adverse events. METHODS AND FINDINGS: 405 children aged 1-6 yrs were initially randomized to vaccination with either FFM ME-TRAP or control (rabies vaccine). 380 children were still available for follow-up after the first nine months. Children were seen weekly and whenever they were unwell for nine months monitoring. The axillary temperature was measured, and blood films taken when febrile. The primary analysis was time to parasitaemia >2,500/microl. During the second nine months monitoring, 49 events met the primary endpoint (febrile malaria with parasites >2,500/microl) in the Intention To Treat (ITT) group. 23 events occurred among the 189 children in the FFM ME-TRAP group, and 26 among the 194 children in the control group. In the full 18 months of monitoring, there were 63 events in the FFM ME-TRAP group and 60 in the control group (HR = 1.2, CI 0.84-1.73, p = 0.35). There was no evidence that the HR changed over the 18 months (test for interaction between time and vaccination p = 0.11). CONCLUSIONS: Vaccination with FFM ME-TRAP was not protective against malaria in this study. Malaria incidence during 18 months of surveillance was similar in both vaccine groups. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN88335123.

Verra F, Simpore J, Warimwe GM, Tetteh KK, Howard T, Osier FH, Bancone G, Avellino P, Blot I, Fegan G et al. 2007. Haemoglobin C and S role in acquired immunity against Plasmodium falciparum malaria. PLoS One, 2 (10), pp. e978. | Show Abstract | Read more

A recently proposed mechanism of protection for haemoglobin C (HbC; beta6Glu-->Lys) links an abnormal display of PfEMP1, an antigen involved in malaria pathogenesis, on the surface of HbC infected erythrocytes together with the observation of reduced cytoadhesion of parasitized erythrocytes and impaired rosetting in vitro. We investigated the impact of this hypothesis on the development of acquired immunity against Plasmodium falciparum variant surface antigens (VSA) encoding PfEMP1 in HbC in comparison with HbA and HbS carriers of Burkina Faso. We measured: i) total IgG against a single VSA, A4U, and against a panel of VSA from severe malaria cases in human sera from urban and rural areas of Burkina Faso of different haemoglobin genotypes (CC, AC, AS, SC, SS); ii) total IgG against recombinant proteins of P. falciparum asexual sporozoite, blood stage antigens, and parasite schizont extract; iii) total IgG against tetanus toxoid. Results showed that the reported abnormal cell-surface display of PfEMP1 on HbC infected erythrocytes observed in vitro is not associated to lower anti- PfEMP1 response in vivo. Higher immune response against the VSA panel and malaria antigens were observed in all adaptive genotypes containing at least one allelic variant HbC or HbS in the low transmission urban area whereas no differences were detected in the high transmission rural area. In both contexts the response against tetanus toxoid was not influenced by the beta-globin genotype. These findings suggest that both HbC and HbS affect the early development of naturally acquired immunity against malaria. The enhanced immune reactivity in both HbC and HbS carriers supports the hypothesis that the protection against malaria of these adaptive genotypes might be at least partially mediated by acquired immunity against malaria.

Kinyanjui SM, Conway DJ, Lanar DE, Marsh K. 2007. IgG antibody responses to Plasmodium falciparum merozoite antigens in Kenyan children have a short half-life. Malar J, 6 (1), pp. 82. | Show Abstract | Read more

BACKGROUND: Data suggest that antibody responses to malaria parasites merozoite antigens are generally short-lived and this has implications for serological studies and malaria vaccine designs. However, precise data on the kinetics of these responses is lacking. METHODS: IgG1 and IgG3 responses to five recombinant Plasmodium falciparum merozoite antigens (MSP-119, MSP-2 type A and B, AMA-1 ectodomain and EBA-175 region II) among Kenyan children were monitored using ELISA for 12 weeks after an acute episode of malaria and their half-lives estimated using an exponential decay model. RESULTS: The responses peaked mainly at week 1 and then decayed rapidly to very low levels within 6 weeks. Estimation of the half-lives of 40 IgG1 responses yielded a mean half-life of 9.8 days (95% CI: 7.6-12.0) while for 16 IgG3 responses it was 6.1 days (95% CI: 3.7-8.4), periods that are shorter than those normally described for the catabolic half-life of these antibody subclasses. CONCLUSION: This study indicates antibodies against merozoite antigens have very short half-lives and this has to be taken into account when designing serological studies and vaccines based on the antigens.

Idro R, Crawley J, Marsh K, Newton CRJC, Neville BGR. 2007. In reply [2] Journal of the American Medical Association, 298 (11), pp. 1274.

Makani J, Williams TN, Marsh K. 2007. Sickle cell disease in Africa: burden and research priorities. Ann Trop Med Parasitol, 101 (1), pp. 3-14. | Show Abstract | Read more

Sickle cell disease (SCD) has recently been recognised as a problem of major public-health significance by the World Health Organization. Despite the fact that >70% of sufferers live in Africa, expenditure on the related care and research in the continent is negligible, and most advances in the understanding and management of this condition have been based on research conducted in the North. In order to target limited resources, African countries need to focus research and interventions on areas that will lead to the maximum impact. This review details the epidemiological and clinical background of SCD, with an emphasis on Africa, before identifying the research priorities that will provide the necessary evidence base for improving the management of African patients. Malaria, bacterial and viral infections and cerebrovascular accidents are areas in which further research may lead to a significant improvement in SCD-related morbidity and mortality. As patients with high concentrations of foetal haemoglobin (HbF) appear to be protected from all but mild SCD, the various factors and pharmacological agents that might increase HbF levels need to be assessed in Africa, as options for interventions that would improve quality of life and reduce mortality.

Urban BC, Cordery D, Shafi MJ, Bull PC, Newbold CI, Williams TN, Marsh K. 2006. The frequency of BDCA3-positive dendritic cells is increased in the peripheral circulation of Kenyan children with severe malaria. Infect Immun, 74 (12), pp. 6700-6706. | Show Abstract | Read more

The ability of Plasmodium falciparum-infected erythrocytes to adhere to host endothelial cells via receptor molecules such as ICAM-1 and CD36 is considered a hallmark for the development of severe malaria syndromes. These molecules are also expressed on leukocytes such as dendritic cells. Dendritic cells are antigen-presenting cells that are crucial for the initiation of adaptive immune responses. In many human diseases, their frequency and function is perturbed. We analyzed the frequency of peripheral blood dendritic cell subsets and the plasma concentrations of interleukin-10 (IL-10) and IL-12 in Kenyan children with severe malaria and during convalescence and related these parameters to the adhesion phenotype of the acute parasite isolates. The frequency of CD1c(+) dendritic cells in children with acute malaria was comparable to that in healthy controls, but the frequency of BDCA3(+) dendritic cells was significantly increased. Analysis of the adhesion phenotypes of parasite isolates revealed that adhesion to ICAM-1 was associated with the frequency of peripheral blood CD1c(+) dendritic cells, whereas the adhesion of infected erythrocytes to CD36 correlated with high concentrations of IL-10 and low concentrations of IL-12 in plasma.

Beeson JG, Kelly G, Hallamore S, Persson K, Chesson J, Cortes A, Rogerson S, Reeder J, Brown G, Marsh K. 2006. Limited global diversity of antibody epitopes expressed by placental binding Plasmodium falciparum variants AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, 75 (5), pp. 104-104.

Persson KE, McCallum FJ, Reiling L, Stubbs J, Lister N, Williams T, Marsh K, Cowman AF, Beeson JG. 2006. Phenotypic variation in P-falciparum invasion of erythrocytes is a mechanism of immune evasion AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, 75 (5), pp. 283-283.

Golding M, Williams T, Marsh K, Hill A. 2006. Single nucleotide polymorphisms in trap associate with severe malarial disease: A novel parasite virulence gene AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, 75 (5), pp. 151-151.

Jenkins NE, Chakravorty SJ, Urban BC, Kai OK, Marsh K, Craig AG. 2006. The effect of Plasmodium falciparum infection on expression of monocyte surface molecules. Trans R Soc Trop Med Hyg, 100 (11), pp. 1007-1012. | Show Abstract | Read more

Plasmodium falciparum infection may result in severe malaria in susceptible individuals. The pathogenesis of severe disease is probably a combination of the sequestration of infected erythrocytes and overstimulation of the immune response. Monocytes are a key source of many of the pro-inflammatory agents implicated but also are found sequestered in blood vessels. However, little is known about the monocyte phenotype in malaria disease. Flow cytometry was performed on fresh whole blood to determine surface expression of four receptors during acute severe and non-severe malaria and again during convalescence when uninfected. Three hundred and fifty-six children with P. falciparum infection were studied and were found to show increased expression of intercellular adhesion molecule-1 (ICAM-1), urokinase plasminogen activator receptor (uPAR), CD23 and chemokine receptor 5 (CCR5) (P<0.001) during acute disease compared with convalescent levels. Using multivariate analysis, it was found that large increases in expression of ICAM-1 (odds ratio (OR) 2.44, 95% CI 1.80-3.32) and uPAR (OR 3.14, 95% CI 1.93-5.09) but small increases in expression of CD23 (OR 0.82, 95% CI 0.68-0.96) were independently associated with severe malaria. These results give an insight into the cellular processes occurring in severe malaria and suggest that pathology is based on a complex repertoire of pro- and anti-inflammatory processes.

Bejon P, Keating S, Mwacharo J, Kai OK, Dunachie S, Walther M, Berthoud T, Lang T, Epstein J, Carucci D et al. 2006. Early gamma interferon and interleukin-2 responses to vaccination predict the late resting memory in malaria-naïve and malaria-exposed individuals. Infect Immun, 74 (11), pp. 6331-6338. | Show Abstract | Read more

Two different cell populations respond to potent T-cell-inducing vaccinations. The induction and loss of effector cells can be seen using an ex vivo enzyme-linked immunospot (ELISPOT) assay, but the more durable resting memory response is demonstrable by a cultured ELISPOT assay. The relationship of the early effector response to durable resting memory is incompletely understood. Effector phenotype is usually identified by gamma interferon (IFN-gamma) production, but interleukin-2 (IL-2) has been specifically linked to the differentiation of memory cells. Here, IFN-gamma- and IL-2-secreting effector cells were identified by an ex vivo ELISPOT assay 1 week after vaccination and compared with the resting memory responses detected by a cultured ELISPOT assay 3 months later. The different kinetics and induction of IL-2 by different vaccines and natural exposure are described. Furthermore, both early IFN-gamma and IL-2 production independently predicted subsequent memory responses at 3 months in malaria-naïve volunteers, but only IFN-gamma predicted memory in malaria-exposed volunteers. However, dual ELISPOT assays were also performed on malaria-exposed volunteers to identify cells producing both cytokines simultaneously. This demonstrated that double-cytokine-producing cells were highly predictive of memory. This assay may be useful in predicting vaccinations most likely to generate stable, long-term memory responses.

Bejon P, Mwacharo J, Kai O, Mwangi T, Milligan P, Todryk S, Keating S, Lang T, Lowe B, Gikonyo C et al. 2006. A phase 2b randomised trial of the candidate malaria vaccines FP9 ME-TRAP and MVA ME-TRAP among children in Kenya. PLoS Clin Trials, 1 (6), pp. e29. | Show Abstract | Read more

OBJECTIVE: The objective was to measure the efficacy of the vaccination regimen FFM ME-TRAP in preventing episodes of clinical malaria among children in a malaria endemic area. FFM ME-TRAP is sequential immunisation with two attenuated poxvirus vectors (FP9 and modified vaccinia virus Ankara), which both deliver the pre-erythrocytic malaria antigen construct multiple epitope-thrombospondin-related adhesion protein (ME-TRAP). DESIGN: The trial was randomised and double-blinded. SETTING: The setting was a rural, malaria-endemic area of coastal Kenya. PARTICIPANTS: We vaccinated 405 healthy 1- to 6-year-old children. INTERVENTIONS: Participants were randomised to vaccination with either FFM ME-TRAP or control (rabies vaccine). OUTCOME MEASURES: Following antimalarial drug treatment children were seen weekly and whenever they were unwell during nine months of monitoring. The axillary temperature was measured, and blood films taken when febrile. The primary analysis was time to a parasitaemia of over 2,500 parasites/mul. RESULTS: The regime was moderately immunogenic, but the magnitude of T cell responses was lower than in previous studies. In intention to treat (ITT) analysis, time to first episode was shorter in the FFM ME-TRAP group. The cumulative incidence of febrile malaria was 52/190 (27%) for FFM ME-TRAP and 40/197 (20%) among controls (hazard ratio = 1.52). This was not statistically significant (95% confidence interval [CI] 1.0-2.3; p = 0.14 by log-rank). A group of 346 children were vaccinated according to protocol (ATP). Among these children, the hazard ratio was 1.3 (95% CI 0.8-2.1; p = 0.55 by log-rank). When multiple malaria episodes were included in the analyses, the incidence rate ratios were 1.6 (95% CI 1.1-2.3); p = 0.017 for ITT, and 1.4 (95% CI 0.9-2.1); p = 0.16 for ATP. Haemoglobin and parasitaemia in cross-sectional surveys at 3 and 9 mo did not differ by treatment group. Among children vaccinated with FFM ME-TRAP, there was no correlation between immunogenicity and malaria incidence. CONCLUSIONS: No protection was induced against febrile malaria by this vaccine regimen. Future field studies will require vaccinations with stronger immunogenicity in children living in malarious areas.

Verra F, Chokejindachai W, Weedall GD, Polley SD, Mwangi TW, Marsh K, Conway DJ. 2006. Contrasting signatures of selection on the Plasmodium falciparum erythrocyte binding antigen gene family. Mol Biochem Parasitol, 149 (2), pp. 182-190. | Show Abstract | Read more

Erythrocyte binding antigens of Plasmodium falciparum are involved in erythrocyte invasion, and may be targets of acquired immunity. Of the five eba genes, protein products have been detected for eba-175, eba-181 and eba-140, but not for psieba-165 or ebl-1, providing opportunity for comparative analysis of genetic variation to identify selection. Region II of each of these genes was sequenced from a cross-sectional sample of parasites in an endemic Kenyan population, and the frequency distributions of polymorphisms analysed. A positive value of Tajima's D was observed for eba-175 (D=1.13) indicating an excess of intermediate frequency polymorphisms, while all other genes had negative values, the most negative being ebl-1 (D=-2.35) followed by psieba-165 (D=-1.79). The eba-175 and ebl-1 genes were then studied in a sample of parasites from Thailand, for which a positive Tajima's D value was again observed for eba-175 (D=1.79), and a negative value for ebl-1 (D=-1.85). This indicates that eba-175 is under balancing selection in each population, in strong contrast to the other members of the gene family, particularly ebl-1 and psieba-165 that may have been under recent directional selection. Population expansion simulations were performed under a neutral model, further supporting the departures from neutrality of these genes.

Nery S, Deans AM, Mosobo M, Marsh K, Rowe JA, Conway DJ. 2006. Expression of Plasmodium falciparum genes involved in erythrocyte invasion varies among isolates cultured directly from patients. Mol Biochem Parasitol, 149 (2), pp. 208-215. | Show Abstract | Read more

Plasmodium falciparum merozoites invade erythrocytes using a range of alternative ligands that includes erythrocyte binding antigenic proteins (EBAs) and reticulocyte binding protein homologues (Rh). Variation in the expression of some of these genes among culture-adapted parasite lines correlates with the use of different erythrocyte receptors. Here, expression profiles of four Rh genes and eba175 are analysed in a sample of 42 isolates cultured from malaria patients in Kenya. The profiles cluster into distinct groups, largely because of very strong negative correlations between the levels of expression of particular gene pairs (Rh1 versus Rh2b, eba175 versus Rh2b, and eba175 versus Rh4), previously associated with alternative invasion pathways in culture-adapted parasite lines. High levels of eba175 are seen in isolates in expression profile group I, and may be associated with sialic acid-dependent invasion. Groups II and III are, respectively, characterized by high levels of Rh2b and Rh4, and are more likely to be associated with sialic acid-independent invasion.

Bejon P, Kai OK, Mwacharo J, Keating S, Lang T, Gilbert SC, Peshu N, Marsh K, Hill AV. 2006. Alternating vector immunizations encoding pre-erythrocytic malaria antigens enhance memory responses in a malaria endemic area. Eur J Immunol, 36 (8), pp. 2264-2272. | Show Abstract | Read more

A heterologous prime-boost strategy has been developed to potently induce T cell responses to pre-erythrocytic malaria antigens. Efficacy in the field is likely to depend on both peak immunogenicity and the durability of responses. To improve both immunogenicity and durability of responses, 54 adult males from a malaria endemic area were immunized with different vaccination regimens, systematically varying antigenic insert and the number and sequence of component vaccinations. The component vaccinations were recombinant attenuated viruses, either fowlpox (FP) 9 or modified vaccinia virus Ankara (MVA). These were recombinant for either of two pre-erythrocytic malaria antigens (multiple epitope-thrombospondin-related adhesion protein, ME-TRAP, or circumsporozoite antigen (CS). ELISPOT assays were used to measure the effector and resting memory T cell responses. Sequence, antigen insert and number of vaccinations influenced immunogenicity, but the novel alternating vector immunizations generated the largest resting memory T cell populations. Effector responses were maintained at 84% of the peak response after 270 days. This durability of response is unprecedented. Classical prime-boost vaccination responses were at 5% of the peak after 270 days. Vaccines administered by heterologous prime-boost regimes are being developed for diverse pathogens and cancer. These data suggest these vaccines should also be administered by alternating vector regimens in clinical development.

Bejon P, Mwacharo J, Kai OK, Todryk S, Keating S, Lang T, Gilbert SC, Peshu N, Marsh K, Hill AV. 2006. Immunogenicity of the candidate malaria vaccines FP9 and modified vaccinia virus Ankara encoding the pre-erythrocytic antigen ME-TRAP in 1-6 year old children in a malaria endemic area. Vaccine, 24 (22), pp. 4709-4715. | Show Abstract | Read more

In a phase 1 trial, 22 children in a malaria endemic area were immunised with candidate malaria vaccination regimes. The regimes used two recombinant viral vectors, attenuated fowlpox strain FP9 and modified vaccinia virus Ankara (MVA). Both encoded the pre-erythrocytic malaria antigen construct ME-TRAP. Strong T cell responses were detected by both ex vivo and cultured ELISpot assays. Data from phase 1 trials in adults on anti-vector responses raised by FP9 is presented. These responses partially cross-reacted with MVA, and detectably reduced the immunogenicity of vaccination with MVA. This prompted the comparison of half dose and full dose FP9 priming vaccinations in children. Regimes using half dose FP9 priming tended to be more immunogenic than full dose. The potential for enhanced immunogenicity with half doses of priming vectors warrants further investigation, and larger studies to determine protection against malaria in children are required.

Polley SD, Conway DJ, Cavanagh DR, McBride JS, Lowe BS, Williams TN, Mwangi TW, Marsh K. 2006. High levels of serum antibodies to merozoite surface protein 2 of Plasmodium falciparum are associated with reduced risk of clinical malaria in coastal Kenya. Vaccine, 24 (19), pp. 4233-4246. | Show Abstract | Read more

The merozoite surface protein (MSP) 2 is a vaccine candidate antigen of Plasmodium falciparum that is polymorphic in natural populations. In a prospective cohort study in two coastal populations of Kenya using recombinant proteins derived from the two major allelic types of MSP2, high serum levels of IgG to MSP2 were associated with protection from clinical malaria. This protection was independent of that associated with antibodies to another vaccine candidate antigen (AMA1) in these populations. However, low antibody levels to MSP2 appeared to be associated with increased susceptibility to malaria within people who were parasite negative at the time of serum collection. These data suggest that an MSP2 based vaccine should be designed to induce high level antibody responses against the different MSP2 types present globally in P. falciparum populations and that MSP2 could be combined with other P. falciparum antigens to form a multi-component malaria vaccine.

Ndungu FM, Sanni L, Urban B, Stephens R, Newbold CI, Marsh K, Langhorne J. 2006. CD4 T cells from malaria-nonexposed individuals respond to the CD36-Binding Domain of Plasmodium falciparum erythrocyte membrane protein-1 via an MHC class II-TCR-independent pathway. J Immunol, 176 (9), pp. 5504-5512. | Show Abstract

We have studied the human CD4 T cell response to a functionally conserved domain of Plasmodium falciparum erythrocyte membrane protein-1, cysteine interdomain region-1alpha (CIDR-1alpha). Responses to CIDR-1alpha were striking in that both exposed and nonexposed donors responded. The IFN-gamma response to CIDR-1alpha in the nonexposed donors was partially independent of TCR engagement of MHC class II and peptide. Contrastingly, CD4 T cell and IFN-gamma responses in malaria-exposed donors were MHC class II restricted, suggesting that the CD4 T cell response to CIDR-1alpha in malaria semi-immune adults also has a TCR-mediated component, which may represent a memory response. Dendritic cells isolated from human peripheral blood were activated by CIDR-1alpha to produce IL-12, IL-10, and IL-18. IL-12 was detectable only between 6 and 12 h of culture, whereas the IL-10 continued to increase throughout the 24-h time course. These data strengthen previous observations that P. falciparum interacts directly with human dendritic cells, and suggests that the interaction between CIDR-1alpha and the host cell may be responsible for regulation of the CD4 T cell and cytokine responses to P. falciparum-infected erythrocytes reported previously.

Persson KE, Lee CT, Marsh K, Beeson JG. 2006. Development and optimization of high-throughput methods to measure Plasmodium falciparum-specific growth inhibitory antibodies. J Clin Microbiol, 44 (5), pp. 1665-1673. | Show Abstract | Read more

Antibodies that inhibit replication of Plasmodium falciparum in erythrocytes are thought to be important both in acquired immunity to malaria and as mediators of immunity generated by candidate blood-stage vaccines. However, several constraints have limited the study of these functional antibodies in population studies and vaccine trials. We report the development and optimization of high-throughput growth inhibition assays with improved sensitivity that use minimal volumes of test serum. The major inhibitory activity of serum from exposed donors was antibody mediated, but nonspecific inhibitory factors were found in untreated serum. Culture volumes could be effectively reduced to 25 microl to limit amounts of test serum or inhibitors used in assays. Performing inhibition assays over two cycles of parasite replication gave greater sensitivity than single-cycle assays, and a simple two-cycle inhibition assay was developed that yielded highly reproducible results. Determination of parasite growth by flow cytometry was most suitable for high-throughput assays using small culture volumes and was more sensitive than parasite lactate dehydrogenase assays and less prone to error and variation than microscopy. We evaluated and optimized methods to remove antimalarials and nonspecific inhibitory factors from serum that are suitable for use with small volumes of samples that are typically obtained from clinical studies. Both microdialysis and immunoglobulin purification by ammonium sulfate precipitation were effective and practical. These methods should facilitate evaluation of vaccine trials and clinical studies of immunity and are also suitable for testing drugs and other compounds for antimalarial activity.

Wambua S, Mwangi TW, Kortok M, Uyoga SM, Macharia AW, Mwacharo JK, Weatherall DJ, Snow RW, Marsh K, Williams TN. 2006. The effect of alpha+-thalassaemia on the incidence of malaria and other diseases in children living on the coast of Kenya. PLoS Med, 3 (5), pp. e158. | Show Abstract | Read more

BACKGROUND: The alpha-thalassaemias are the commonest genetic disorders of humans. It is generally believed that this high frequency reflects selection through a survival advantage against death from malaria; nevertheless, the epidemiological description of the relationships between alpha-thalassaemia, malaria, and other common causes of child mortality remains incomplete. METHODS AND FINDINGS: We studied the alpha+-thalassaemia-specific incidence of malaria and other common childhood diseases in two cohorts of children living on the coast of Kenya. We found no associations between alpha+-thalassaemia and the prevalence of symptomless Plasmodium falciparum parasitaemia, the incidence of uncomplicated P. falciparum disease, or parasite densities during mild or severe malaria episodes. However, we found significant negative associations between alpha+-thalassaemia and the incidence rates of severe malaria and severe anaemia (haemoglobin concentration < 50 g/l). The strongest associations were for severe malaria anaemia (> 10,000 P. falciparum parasites/mul) and severe nonmalaria anaemia; the incidence rate ratios and 95% confidence intervals (CIs) for alpha+-thalassaemia heterozygotes and homozygotes combined compared to normal children were, for severe malaria anaemia, 0.33 (95% CI, 0.15,0.73; p = 0.006), and for severe nonmalaria anaemia, 0.26 (95% CI, 0.09,0.77; p = 0.015). CONCLUSIONS: Our observations suggest, first that selection for alpha+-thalassaemia might be mediated by a specific effect against severe anaemia, an observation that may lead to fresh insights into the aetiology of this important condition. Second, although alpha+-thalassaemia is strongly protective against severe and fatal malaria, its effects are not detectable at the level of any other malaria outcome; this result provides a cautionary example for studies aimed at testing malaria interventions or identifying new malaria-protective genes.

Bejon P, Peshu N, Gilbert SC, Lowe BS, Molyneux CS, Forsdyke J, Lang T, Hill AV, Marsh K. 2006. Safety profile of the viral vectors of attenuated fowlpox strain FP9 and modified vaccinia virus Ankara recombinant for either of 2 preerythrocytic malaria antigens, ME-TRAP or the circumsporozoite protein, in children and adults in Kenya. Clin Infect Dis, 42 (8), pp. 1102-1110. | Show Abstract | Read more

BACKGROUND: We are developing a heterologous prime-boost vaccine strategy against malaria. This approach uses sequential immunization with different vectors to deliver a common preerythrocytic malaria antigen. Preliminary evidence of efficacy and safety has been previously documented in studies from an area where malaria is nonendemic. Additional safety data from an area where malaria is endemic are now required before larger-scale studies are undertaken to determine the efficacy of this vaccine strategy in the field. Other modified vaccinia virus Ankara (MVA) recombinants and prime-boost immunizations are being developed as vaccines against human immunodeficiency virus (HIV) infection, tuberculosis, and cancer, and MVA is a candidate attenuated smallpox vaccine. METHODS: Candidate vaccines against malaria were intradermally administered to 73 adults (7 of whom were HIV positive) and 22 children in Kenya. These vaccines used the attenuated fowlpox strain FP9 and the MVA recombinant for either of 2 preerythrocytic malaria antigens, multiple preerythrocytic-stage epitopes joined with the preerythrocytic-stage antigen TRAP (ME-TRAP) and the circumsporozoite protein (CS). Adverse events were recorded. RESULTS: Reactogenicity was mild. MVA caused less frequent and less severe cutaneous reaction if given after FP9 priming. Half doses reduced the frequency and the severity of systemic reactogenicity, and particular vaccine lots were associated with different reactogenicities. Unexpectedly, prior immunity to the ME-TRAP antigen appeared to be protective against local reactions after immunization. CONCLUSIONS: Where the final intention is to use MVA after FP9 priming, previous testing of MVA alone overestimates reactogenicity. These recombinant vectors appear to be safe and suitable for use in larger-scale studies of children in Africa and of HIV-positive individuals.

Urban BC, Shafi MJ, Cordery DV, Macharia A, Lowe B, Marsh K, Williams TN. 2006. Frequencies of peripheral blood myeloid cells in healthy Kenyan children with alpha+ thalassemia and the sickle cell trait. Am J Trop Med Hyg, 74 (4), pp. 578-584. | Show Abstract

The high frequencies of both alpha+ thalassemia and the sickle cell trait (hemoglobin AS [HbAS]) found in many tropical populations are thought to reflect selection pressure from Plasmodium falciparum malaria. For HbAS, but not for alpha+ thalassemia, protection appears to be mediated by the enhanced phagocytic clearance of ring-infected erythrocytes. We have investigated the genotype-specific distributions of peripheral blood leukocyte populations in two groups of children living on the coast of Kenya: a group of healthy P. falciparum parasite-negative children sampled at cross-sectional survey during a period of low malaria transmission, and a group of children attending the hospital with acute malaria. We report distinctive distributions of peripheral blood myeloid dendritic cells and monocytes in children with alpha+ thalassemia and HbAS during healthy periods and disease, and suggest ways in which these might relate to the mechanisms of protection afforded by these conditions.

Deans AM, Lyke KE, Thera MA, Plowe CV, Koné A, Doumbo OK, Kai O, Marsh K, Mackinnon MJ, Raza A, Rowe JA. 2006. Low multiplication rates of African Plasmodium falciparum isolates and lack of association of multiplication rate and red blood cell selectivity with malaria virulence. Am J Trop Med Hyg, 74 (4), pp. 554-563. | Show Abstract

Two potential malaria virulence factors, parasite multiplication rate (PMR) and red blood cell selectivity (measured as selectivity index [SI]), were assessed in Plasmodium falciparum clinical isolates from Mali and Kenya. At both sites, PMRs were low (Kenya median = 2.2, n = 33; Mali median = 2.6, n = 61) and did not differ significantly between uncomplicated and severe malaria cases. Malian isolates from hyperparasitemic patients had significantly lower PMRs (median = 1.8, n = 19) than other Malian isolates (uncomplicated malaria median = 3.1, n = 23; severe malaria median = 2.8, n = 19; P = 0.03, by Kruskal-Wallis test). Selective invasion occurred at both sites (Kenya geometric mean SI = 1.9, n = 98; Mali geometric mean SI = 1.6, n = 104), and there was no significant association between the SI and malaria severity. Therefore, in contrast to previous results from Thailand, we found no association of PMR and SI with malaria severity in African children. This raises the possibility of differences in the mechanisms of malaria virulence between sub-Saharan Africa and Asia.

Flanagan KL, Plebanski M, Odhiambo K, Sheu E, Mwangi T, Gelder C, Hart K, Kortok M, Lowe B, Robson KJ et al. 2006. Cellular reactivity to the p. Falciparum protein trap in adult kenyans: novel epitopes, complex cytokine patterns, and the impact of natural antigenic variation. Am J Trop Med Hyg, 74 (3), pp. 367-375. | Show Abstract

Malaria vaccines based on thrombospondin-related adhesive protein of Plasmodium falciparum (Pf TRAP) are currently undergoing clinical trials in humans. This study was designed to investigate naturally acquired cellular immunity to Pf TRAP in adults from a target population for future trials of TRAP-based vaccines in Kilifi, Kenya. We first tested reactivity to a panel of 53 peptides spanning Pf TRAP and identified 26 novel T-cell epitopes. A panel of naturally occurring polymorphic variant epitope peptides were made to the most commonly recognized epitope regions and tested for ability to elicit IFN-gamma, IL-4, and IL-10 production. These data provide for the first time a complex cytokine matrix mapping naturally induced T-cell responses to TRAP and suggest that T-cell responses boosted by vaccination with Pf TRAP could stimulate the release of competing pro- and anti-inflammatory cytokines. They further define polymorphic variants able to boost specific Th1, Th2, and possibly Tr1 reactivity.

Ajala-Agbo T, Komba A, Makani J, Williams T, Marsh K, Newton C, Kirkham F. 2006. Spectrum of cerebral blood flow velocities measured by transcranial Doppler ultrasonography in children with sickle cell disease in Africa DEVELOPMENTAL MEDICINE AND CHILD NEUROLOGY, 48 pp. 13-14.

Jenkins NE, Mwangi TW, Kortok M, Marsh K, Craig AG, Williams TN. 2005. A polymorphism of intercellular adhesion molecule-1 is associated with a reduced incidence of nonmalarial febrile illness in Kenyan children. Clin Infect Dis, 41 (12), pp. 1817-1819. | Show Abstract | Read more

An intercellular adhesion molecule-1 polymorphism (ICAM-1(Kilifi)) is present at a high frequency across sub-Saharan Africa, and its presence may increase susceptibility to cerebral malaria. Here, we report that, compared with children in whom wild-type intercellular adhesion molecule-1 is present, the incidence of nonmalarial fever is significantly lower among those homozygous for ICAM-1(Kilifi). We propose that ICAM-1(Kilifi) may be associated with reduced rates of tissue damage and of death due to sepsis.

Mackinnon MJ, Mwangi TW, Snow RW, Marsh K, Williams TN. 2005. Heritability of malaria in Africa. PLoS Med, 2 (12), pp. e340. | Show Abstract | Read more

BACKGROUND: While many individual genes have been identified that confer protection against malaria, the overall impact of host genetics on malarial risk remains unknown. METHODS AND FINDINGS: We have used pedigree-based genetic variance component analysis to determine the relative contributions of genetic and other factors to the variability in incidence of malaria and other infectious diseases in two cohorts of children living on the coast of Kenya. In the first, we monitored the incidence of mild clinical malaria and other febrile diseases through active surveillance of 640 children 10 y old or younger, living in 77 different households for an average of 2.7 y. In the second, we recorded hospital admissions with malaria and other infectious diseases in a birth cohort of 2,914 children for an average of 4.1 y. Mean annual incidence rates for mild and hospital-admitted malaria were 1.6 and 0.054 episodes per person per year, respectively. Twenty-four percent and 25% of the total variation in these outcomes was explained by additively acting host genes, and household explained a further 29% and 14%, respectively. The haemoglobin S gene explained only 2% of the total variation. For nonmalarial infections, additive genetics explained 39% and 13% of the variability in fevers and hospital-admitted infections, while household explained a further 9% and 30%, respectively. CONCLUSION: Genetic and unidentified household factors each accounted for around one quarter of the total variability in malaria incidence in our study population. The genetic effect was well beyond that explained by the anticipated effects of the haemoglobinopathies alone, suggesting the existence of many protective genes, each individually resulting in small population effects. While studying these genes may well provide insights into pathogenesis and resistance in human malaria, identifying and tackling the household effects must be the more efficient route to reducing the burden of disease in malaria-endemic areas.

Heinrichs V, Mundorff E, Lohre JA, West LL, Kuznetsova MA, Howard TA, Kortok MM, Marsh K, Makobongo MO, Liu X et al. 2005. Improvement of Plasmodium Falciparum erythrocyte membrane protein-1 (PfEMP-1) vaccine antigens using directed molecular evolution AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, 73 (6), pp. 339-339.

Williams TN, Mwangi TW, Wambua S, Peto TE, Weatherall DJ, Gupta S, Recker M, Penman BS, Uyoga S, Macharia A et al. 2005. Negative epistasis between the malaria-protective effects of alpha+-thalassemia and the sickle cell trait. Nat Genet, 37 (11), pp. 1253-1257. | Show Abstract | Read more

The hemoglobinopathies, disorders of hemoglobin structure and production, protect against death from malaria. In sub-Saharan Africa, two such conditions occur at particularly high frequencies: presence of the structural variant hemoglobin S and alpha(+)-thalassemia, a condition characterized by reduced production of the normal alpha-globin component of hemoglobin. Individually, each is protective against severe Plasmodium falciparum malaria, but little is known about their malaria-protective effects when inherited in combination. We investigated this question by studying a population on the coast of Kenya and found that the protection afforded by each condition inherited alone was lost when the two conditions were inherited together, to such a degree that the incidence of both uncomplicated and severe P. falciparum malaria was close to baseline in children heterozygous with respect to the mutation underlying the hemoglobin S variant and homozygous with respect to the mutation underlying alpha(+)-thalassemia. Negative epistasis could explain the failure of alpha(+)-thalassemia to reach fixation in any population in sub-Saharan Africa.

Bull PC, Berriman M, Kyes S, Quail MA, Hall N, Kortok MM, Marsh K, Newbold CI. 2005. Plasmodium falciparum variant surface antigen expression patterns during malaria. PLoS Pathog, 1 (3), pp. e26. | Show Abstract | Read more

The variant surface antigens expressed on Plasmodium falciparum-infected erythrocytes are potentially important targets of immunity to malaria and are encoded, at least in part, by a family of var genes, about 60 of which are present within every parasite genome. Here we use semi-conserved regions within short var gene sequence "tags" to make direct comparisons of var gene expression in 12 clinical parasite isolates from Kenyan children. A total of 1,746 var clones were sequenced from genomic and cDNA and assigned to one of six sequence groups using specific sequence features. The results show the following. (1) The relative numbers of genomic clones falling in each of the sequence groups was similar between parasite isolates and corresponded well with the numbers of genes found in the genome of a single, fully sequenced parasite isolate. In contrast, the relative numbers of cDNA clones falling in each group varied considerably between isolates. (2) Expression of sequences belonging to a relatively conserved group was negatively associated with the repertoire of variant surface antigen antibodies carried by the infected child at the time of disease, whereas expression of sequences belonging to another group was associated with the parasite "rosetting" phenotype, a well established virulence determinant. Our results suggest that information on the state of the host-parasite relationship in vivo can be provided by measurements of the differential expression of different var groups, and need only be defined by short stretches of sequence data.

Ochola LB, Marsh K, Lowe B, Gal S, Pluschke G, Smith T. 2005. Estimation of the sequestered parasite load in severe malaria patients using both host and parasite markers. Parasitology, 131 (Pt 4), pp. 449-458. | Show Abstract | Read more

The virulence of the malaria parasite Plasmodium falciparum is due, in part, to its ability to cytoadhere in deep vascular beds. Our inability to quantify the load of sequestered parasites hampers our understanding of the pathophysiological mechanisms involved in disease progression and complicates diagnosis. In this study we evaluate potential biochemical markers of sequestered load by comparing them with estimates of the sequestered load from a statistical model fitted to longitudinal patterns of peripheral parasite densities in a series of 22 patients with severe Plasmodium falciparum malaria. The markers comprised the host factors: haematocrit, circulating host DNA, sTNF-R75 and parasite derived products HRP2, pLDH, pigments and circulating parasite DNA. We investigated the suitability of these markers in determining sequestered loads in patients on quinine treatment. Observed peripheral parasitaemia, plasma levels of sTNF-R75 and circulating parasite DNA were most strongly correlated with estimates of sequestered loads on admission. However the dynamics of both sTNF-R75 and circulating parasite DNA during follow-up were very different from those of the estimated sequestered mass. These analyses suggest that none of the markers gave reliable estimates of the current sequestered load, though they may reflect the history of infection. Longitudinal analyses are needed that allow for the clearance rates of the marker molecules and for variations between hosts in the history of parasitaemia.

Ndungu FM, Urban BC, Marsh K, Langhorne J. 2005. Regulation of immune response by Plasmodium-infected red blood cells. Parasite Immunol, 27 (10-11), pp. 373-384. | Show Abstract | Read more

During the asexual blood stage infection of the human malaria parasite, Plasmodium falciparum, parasite-derived proteins are inserted onto the surface of the host red blood cell membrane. These proteins are highly variable and were originally thought only to mediate antigenic variation, and sequestration of parasites from peripheral circulation, thus enabling immune evasion. Recent studies have revealed that PfEMP-1 and other molecules on the P. falciparum-infected red blood cell (PfRBC) activate and modulate the immune response. In this review, we discuss how PfRBCs interact with antigen-presenting cells (APCs) and other cells of the immune system, and how such interactions could modulate the host response to Plasmodium infections.

Molyneux CS, Peshu N, Marsh K. 2005. Trust and informed consent: insights from community members on the Kenyan coast. Soc Sci Med, 61 (7), pp. 1463-1473. | Show Abstract | Read more

Trust is an important theme running through the literature on the ethics of biomedical research, but it is rarely given centre stage. In this paper, we present data gathered from a study aimed at exploring community views regarding the informed consent processes carried out by a large research centre on the Kenyan Coast. The findings point to the centrality of trust and elements of mistrust in general community views, in parents' (mis)understanding of studies they consent their children to be involved in, in refusals and concerns, and in community members' views about whether informed consent is a relevant and practical model to follow. Tentative ideas on how trust and a healthy mistrust might be balanced highlight the importance of strengthening communication surrounding basic health care as well as research, and of fostering 'an inner generated ethic of service'. The latter is particularly fundamental, but cannot be built and regulated through the laws, policies and guidelines that currently govern biomedical research practice.

Bull PC, Pain A, Ndungu FM, Kinyanjui SM, Roberts DJ, Newbold CI, Marsh K. 2005. Plasmodium falciparum antigenic variation: relationships between in vivo selection, acquired antibody response, and disease severity. J Infect Dis, 192 (6), pp. 1119-1126. | Show Abstract | Read more

BACKGROUND: Variant surface antigens (VSA) on Plasmodium falciparum-infected erythrocytes are potentially important targets of immunity to malaria. We previously identified a VSA phenotype--VSA with a high frequency of antibody recognition (VSA(FoRH))--that is associated with young host age and severe malaria. We hypothesized that VSA(FoRH) are positively selected by host molecules such as intercellular adhesion molecule 1 (ICAM1) and CD36 and dominate in the absence of an effective immune response. Here, we assessed, in 115 Kenyan children, the potential role played by in vivo selection pressures in either favoring or selecting against VSA(FoRH) among parasites that cause malaria. METHODS: We tested for associations between VSA(FoRH) and (1) the repertoire of VSA antibodies carried by children at the time of acute malaria and (2) polymorphisms in ICAM1 (K29M) and CD36 (T188G) that could potentially reduce the positive selection of VSA(FoRH). RESULTS: An expected negative association between VSA antibody repertoire and VSA(FoRH) was observed in children with nonsevere malaria. However, this association did not extend to children with severe malaria, many of whom apparently had well-developed VSA antibody responses despite being infected by parasites expressing VSA(FoRH). There was no evidence for involvement of CD36 or ICAM1 in positive selection of VSA(FoRH). On the contrary, a weak positive association between carriage of the CD36 (T188G) allele and VSA(FoRH) was observed in children with severe malaria. CONCLUSION: The association between the VSA(FoRH) parasite phenotype and severe malaria cannot be explained simply in terms of the total repertoire of VSA antibodies carried at the time of acute disease.

Nzila A, Ward SA, Marsh K, Sims PF, Hyde JE. 2005. Comparative folate metabolism in humans and malaria parasites (part II): activities as yet untargeted or specific to Plasmodium. Trends Parasitol, 21 (7), pp. 334-339. | Show Abstract | Read more

The folate pathway represents a powerful target for combating rapidly dividing systems such as cancer cells, bacteria and malaria parasites. Whereas folate metabolism in mammalian cells and bacteria has been studied extensively, it is understood less well in malaria parasites. In two articles, we attempt to reconstitute the malaria folate pathway based on available information from mammalian and microbial systems, in addition to Plasmodium-genome-sequencing projects. In part I, we focused on folate enzymes that are already used clinically as anticancer drug targets or that are under development in drug-discovery programs. In this article, we discuss mammalian folate enzymes that have not yet been exploited as potential drug targets, and enzymes that function in the de novo folate-synthesis pathway of the parasite--a particularly attractive area of attack because of its absence from the mammalian host.

Molyneux CS, Wassenaar DR, Peshu N, Marsh K. 2005. 'Even if they ask you to stand by a tree all day, you will have to do it (laughter)...!': community voices on the notion and practice of informed consent for biomedical research in developing countries. Soc Sci Med, 61 (2), pp. 443-454. | Show Abstract | Read more

Ethical dilemmas in biomedical research, especially in vulnerable populations, often spark heated debate. Despite recommendations and guidelines, many issues remain controversial, including the relevance, prioritisation and application of individual voluntary informed consent in non-Western settings. The voices of the people likely to be the subjects of research have been notably absent from the debate. We held discussions with groups of community members living in the rural study area of a large research unit in Kenya. Discussions were facilitated by three research study vignettes outlining one field-based and two hospital-based studies being planned or taking place at the time. In addition to gathering general views about the aims and activities of the research unit, questions focused on whether consent should be sought for studies, and if so from whom (chiefs, elders, men/women, children), and on ascertaining whether there are any special concerns about the physical act of signing consent forms. The findings revealed the community's difficulty in distinguishing research from clinical investigations conducted in clinical settings. There was a spectrum of views regarding perceived appropriateness of consent procedures, in part because of difficulty in disentangling clinical from research aims, and because of other challenges to applying consent in practice. Debates between community members highlight the inadequacy of simplistic assumptions about community members' views on informed consent, and the complexity of incorporating lay opinions into biomedical research. Failure to appreciate these issues risks exaggerating differences between settings, and underestimating the time and resources required to ensure meaningful community involvement in research processes. Ultimately, it risks inadequately responding to the needs and values of those on whom the success of most biomedical research depends. Although compliance with community views does not necessarily make the research more ethical, it is argued that community opinions on local issues and practices should inform ethical decision-making in health research.

Bejon P, Mwangi I, Ngetsa C, Mwarumba S, Berkley JA, Lowe BS, Maitland K, Marsh K, English M, Scott JA. 2005. Invasive Gram-negative bacilli are frequently resistant to standard antibiotics for children admitted to hospital in Kilifi, Kenya. J Antimicrob Chemother, 56 (1), pp. 232-235. | Show Abstract | Read more

OBJECTIVES: To determine the pattern of resistance among Gram-negative bacilli causing invasive bacterial disease for the antibiotics that are already in common use in Kilifi, Kenya and for two potential alternatives, ciprofloxacin and cefotaxime. Also, to determine whether prevalence and severity of resistance was increasing over time, to identify patients who are particularly at risk of resistant infections, and to explore which factors are associated with the development of resistance in our setting. METHODS: We used Etest to study antibiotic susceptibility patterns of 90 Gram-negative bacilli cultured in blood or CSF from paediatric inpatients over 8 years. RESULTS: Susceptibility to amoxicillin 28%, cefotaxime 95% and ciprofloxacin 99% did not vary significantly with age. Susceptibilities for isolates from children aged less than 14 days were: chloramphenicol, 81%; trimethoprim/sulfamethoxazole, 71%; and gentamicin, 91%. From older children, susceptibilities were: chloramphenicol, 62%; trimethoprim/sulfamethoxazole, 39%; and gentamicin, 73%. Chloramphenicol susceptibility was significantly more common among non-typhi salmonellae than other species (79% versus 53%, P < 0.0005). The combination of gentamicin and chloramphenicol covered 91% of all isolates. The prevalence of resistance did not increase over time and was not more common in patients with HIV or malnutrition. Age was the only clinical feature that predicted resistance. CONCLUSIONS: Gentamicin or chloramphenicol alone was suboptimal therapy for Gram-negative sepsis, although in this retrospective study, there was no association between resistance and mortality.

Scott JA, Mwarumba S, Ngetsa C, Njenga S, Lowe BS, Slack MP, Berkley JA, Mwangi I, Maitland K, English M, Marsh K. 2005. Progressive increase in antimicrobial resistance among invasive isolates of Haemophilus influenzae obtained from children admitted to a hospital in Kilifi, Kenya, from 1994 to 2002. Antimicrob Agents Chemother, 49 (7), pp. 3021-3024. | Show Abstract | Read more

Etest susceptibilities to amoxicillin, chloramphenicol, and trimethoprim-sulfamethoxazole of 240 invasive isolates of Haemophilus influenzae cultured from children in rural Kenya were 66%, 66%, and 38%, respectively. Resistance increased markedly over 9 years and was concentrated among serotype b isolates. In Africa, the increasing cost of treating resistant infections supports economic arguments for prevention through conjugate H. influenzae type b immunization.

Williams TN, Mwangi TW, Wambua S, Alexander ND, Kortok M, Snow RW, Marsh K. 2005. Sickle cell trait and the risk of Plasmodium falciparum malaria and other childhood diseases. J Infect Dis, 192 (1), pp. 178-186. | Show Abstract | Read more

BACKGROUND: The gene for sickle hemoglobin (HbS) is a prime example of natural selection. It is generally believed that its current prevalence in many tropical populations reflects selection for the carrier form (sickle cell trait [HbAS]) through a survival advantage against death from malaria. Nevertheless, >50 years after this hypothesis was first proposed, the epidemiological description of the relationships between HbAS, malaria, and other common causes of child mortality remains incomplete. METHODS: We studied the incidence of falciparum malaria and other childhood diseases in 2 cohorts of children living on the coast of Kenya. RESULTS: The protective effect of HbAS was remarkably specific for falciparum malaria, having no significant impact on any other disease. HbAS had no effect on the prevalence of symptomless parasitemia but was 50% protective against mild clinical malaria, 75% protective against admission to the hospital for malaria, and almost 90% protective against severe or complicated malaria. The effect of HbAS on episodes of clinical malaria was mirrored in its effect on parasite densities during such episodes. CONCLUSIONS: The present data are useful in that they confirm the mechanisms by which HbAS confers protection against malaria and shed light on the relationships between HbAS, malaria, and other childhood diseases.

Aldhous P, Butler D, Giles J, Hopkin M, Peplow M, Schiermeier Q, Mugabe J, Marsh K, Binka F, Murenzi R et al. 2005. A message to the G8 summit NATURE, 435 (7046), pp. 1146-1149. | Read more

Mwangi TW, Ross A, Snow RW, Marsh K. 2005. Case definitions of clinical malaria under different transmission conditions in Kilifi District, Kenya. J Infect Dis, 191 (11), pp. 1932-1939. | Show Abstract | Read more

BACKGROUND: Clear case definitions of malaria are an essential means of evaluating the effectiveness of present and proposed interventions in malaria. The clinical signs of malaria are nonspecific, and parasitemia accompanied by a fever may not be sufficient to define an episode of clinical malaria in endemic areas. We defined and quantified cases of malaria in people of different age groups from 2 areas with different rates of transmission of malaria. METHODS: A total of 1602 people were followed up weekly for 2 years, and all the cases of fever accompanied by parasitemia were identified. Logistic regression methods were used to derive case definitions of malaria. RESULTS: Two case definitions of malaria were derived: 1 for children 1-14 years old and 1 for infants (<1 year old) and older children and adults (> or =15 years old). We also found a higher number of episodes of clinical malaria per person per year in people from an area of low transmission of malaria, compared with the number of episodes in those from an area of higher transmission (0.84 vs. 0.55 episodes/person/year; incidence rate ratio, 0.66 [95% confidence interval, 0.61-0.72]; P<.001). CONCLUSIONS: Case definitions of malaria are bound to be altered by factors that affect immunity, such as age and transmission. Case definitions may, however, be affected by other immunity-altering factors, such as HIV and vaccination status, and this needs to be borne in mind during vaccine trials.

Mwangi TW, Mohammed M, Dayo H, Snow RW, Marsh K. 2005. Clinical algorithms for malaria diagnosis lack utility among people of different age groups. Trop Med Int Health, 10 (6), pp. 530-536. | Show Abstract | Read more

We conducted a study to determine whether clinical algorithms would be useful in malaria diagnosis among people living in an area of moderate malaria transmission within Kilifi District in Kenya. A total of 1602 people of all age groups participated. We took smears and recorded clinical signs and symptoms (prompted or spontaneous) of all those presenting to the study clinic with a history of fever. A malaria case was defined as a person presenting to the clinic with a history of fever and concurrent parasitaemia. A set of clinical signs and symptoms (algorithms) with the highest sensitivity and specificity for diagnosing a malaria case was selected for the age groups </=5 years, 6-14 years and >/=15 years. These age-optimized derived algorithms were able to identify about 66% of the cases among those <15 years of age but only 23% of cases among adults. Were these algorithms to be used as a basis for a decision on treatment among those presenting to the clinic, 16% of children </=5 years, 44% of those 6-14 years of age and 66% of the adults who had a history of fever and parasitaemia >/=5000 parasites/microl of blood would be sent home without treatment. Clinical algorithms therefore appear to have little utility in malaria diagnosis, performing even worse in the older age groups, where avoiding unnecessary use of anti-malarials would make more drugs available to the really needy population of children under 5 years of age.

Nzila A, Ward SA, Marsh K, Sims PF, Hyde JE. 2005. Comparative folate metabolism in humans and malaria parasites (part I): pointers for malaria treatment from cancer chemotherapy. Trends Parasitol, 21 (6), pp. 292-298. | Show Abstract | Read more

New inhibitors are urgently needed to overcome the burgeoning problem of drug resistance in the treatment of Plasmodium falciparum infection. Targeting the folate pathway has proved to be a powerful strategy for drug development against rapidly multiplying systems such as cancer cells and microorganisms. Antifolates have long been used for malaria treatment but, despite their success, much less is known about parasite folate metabolism than about that of the human host. In this article, we focus on folate enzymes used clinically as anticancer drug targets, in addition to those that have potential to be used as drug targets, for which there are inhibitors at various stages of development. We discuss how this information could lead to the identification of new targets in malaria parasites.

Scott S, Cumberland P, Shulman CE, Cousens S, Cohen BJ, Brown DW, Bulmer JN, Dorman EK, Kawuondo K, Marsh K, Cutts F. 2005. Neonatal measles immunity in rural Kenya: the influence of HIV and placental malaria infections on placental transfer of antibodies and levels of antibody in maternal and cord serum samples. J Infect Dis, 191 (11), pp. 1854-1860. | Show Abstract | Read more

INTRODUCTION: Young infants are protected from measles infection by maternal measles antibodies. The level of these antibodies at birth depends on the level of antibodies in the mother and the extent of placental transfer. We investigated predictors of levels of measles antibodies in newborns in rural Kenya. METHODS: A total of 747 paired maternal-cord serum samples (91 from human immunodeficiency virus [HIV]-infected and 656 from HIV-uninfected mothers) were tested for measles immunoglobulin G antibodies. Placental malaria infection was determined by biopsy. Data on pregnancy history, gestational age, and anthropometric and socioeconomic status were collected. RESULTS: Infants born to HIV-infected mothers were more likely (odds ratio, 4.6 [95% confidence interval {CI}, 2.2-9.7]) to be seronegative and had 35.1% (95% CI, 9.8%-53.2%) lower levels of measles antibodies than did those born to HIV-uninfected mothers. Preterm delivery, early maternal age, and ethnic group were also associated with reduced levels of measles antibodies. There was little evidence that placental malaria infection was associated with levels of measles antibodies in newborns. CONCLUSION: Our results suggest that maternal HIV infection may reduce levels of measles antibodies in newborns. Low levels of measles antibodies at birth render children susceptible to measles infection at an early age. This is of concern in sub-Saharan African countries, where not only is the prevalence of HIV high, but measles is the cause of much morbidity and mortality.

Dorfman JR, Bejon P, Ndungu FM, Langhorne J, Kortok MM, Lowe BS, Mwangi TW, Williams TN, Marsh K. 2005. B cell memory to 3 Plasmodium falciparum blood-stage antigens in a malaria-endemic area. J Infect Dis, 191 (10), pp. 1623-1630. | Show Abstract | Read more

To gain insight into why antibody responses to malarial antigens tend to be short lived, we studied antigen-specific memory B cells from donors in an area where malaria is endemic. We compared antibody and memory B cell responses to tetanus toxoid with those to 3 Plasmodium falciparum candidate vaccine antigens: the C-terminal portion of merozoite surface protein 1 (MSP1(19)), apical membrane antigen 1 (AMA1), and the cysteine-rich interdomain region 1 alpha (CIDR1 alpha ) of a protein from the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family. These data are the first to be generated on memory B cells in children who are in the process of acquiring antimalarial immunity, and they reveal defects in B cell memory to P. falciparum antigens. Compared with the results for tetanus toxoid, more donors who were positive for antibody to AMA1 and CIDR1 alpha were negative for memory B cells. These data imply that some exposures to malaria do not result in the establishment of stable populations of circulating antigen-specific memory B cells, suggesting possible mechanisms for the short-lived nature of many anti-malarial antibody responses.

Griffiths MJ, Shafi MJ, Popper SJ, Hemingway CA, Kortok MM, Wathen A, Rockett KA, Mott R, Levin M, Newton CR et al. 2005. Genomewide analysis of the host response to malaria in Kenyan children. J Infect Dis, 191 (10), pp. 1599-1611. | Show Abstract | Read more

Malaria is a global problem, and there is a critical need for further understanding of the disease process. When malarial parasites invade and develop within the bloodstream, they stimulate a profound host response whose main clinical sign is fever. To explore this response, we measured host gene expression in whole blood from Kenyan children hospitalized with either acute malaria or other febrile illnesses. Genomewide analysis of expression identified 2 principal gene-expression profiles related to neutrophil and erythroid activity. In addition to these general acute responses, a third gene-expression profile was associated with host parasitemia; mediators of erythrophagocytosis and cellular stress were notable components of this response. The delineation of subjects on the basis of patterns of gene expression provides a molecular perspective of the host response to malaria and further functional insight into the underlying processes of pathogenesis.

Williams TN, Mwangi TW, Roberts DJ, Alexander ND, Weatherall DJ, Wambua S, Kortok M, Snow RW, Marsh K. 2005. An immune basis for malaria protection by the sickle cell trait. PLoS Med, 2 (5), pp. e128. | Show Abstract | Read more

BACKGROUND: Malaria resistance by the sickle cell trait (genotype HbAS) has served as the prime example of genetic selection for over half a century. Nevertheless, the mechanism of this resistance remains the subject of considerable debate. While it probably involves innate factors such as the reduced ability of Plasmodium falciparum parasites to grow and multiply in HbAS erythrocytes, recent observations suggest that it might also involve the accelerated acquisition of malaria-specific immunity. METHODS AND FINDINGS: We studied the age-specific protection afforded by HbAS against clinical malaria in children living on the coast of Kenya. We found that protection increased with age from only 20% in the first 2 y of life to a maximum of 56% by the age of 10 y, returning thereafter to 30% in participants greater than 10 y old. CONCLUSIONS: Our observations suggest that malaria protection by HbAS involves the enhancement of not only innate but also of acquired immunity to the parasite. A better understanding of the underlying mechanisms might yield important insights into both these processes.

Ogutu BR, Nzila AM, Ochong E, Mithwani S, Wamola B, Olola CH, Lowe B, Kokwaro GO, Marsh K, Newton CR. 2005. The role of sequential administration of sulphadoxine/pyrimethamine following quinine in the treatment of severe falciparum malaria in children. Trop Med Int Health, 10 (5), pp. 484-488. | Show Abstract | Read more

Sulphadoxine/pyrimethamine (SP) is often administered with quinine in the treatment of severe falciparum malaria to shorten the course of quinine. The efficacy of SP alone in the treatment of non-severe malaria has been declining rapidly in East Africa, raising concerns of the usefulness of a shortened course of quinine followed SP. We audited the efficacy of quinine/SP in the treatment of severe malaria in Kenyan children. Children with severe falciparum malaria were treated with parenteral quinine followed by a single oral dose of SP. A clinical evaluation was performed 3 weeks later in which a blood sample was obtained for full haemogram, blood slide and analysis of the parasite dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) codons, mutations of which are associated with resistance to SP. A total of 452 children were enrolled, of whom 374 completed the study. Fifty-two (13.9%) children were parasitaemic by 3 weeks of whom 17 (4.5%) had fever as well. The treatment failure group had a significantly higher parasitaemia (129 061 vs. 43 339; P<0.001) and haemoglobin on admission, but only admission parasitaemia independently predicted treatment failure. Those with treatment failure had a significantly lower rise in haemoglobin at 3 weeks compared with treatment successes (9.0 vs. 10.0 g/dl). Of the 76 parasite isolates collected before treatment, 40 (53%) were triple mutant DHFR-double DHPS (Tp-Db), the genotype most associated with SP resistance. Three weeks after SP treatment, the proportion of Tp-Db increased to 72% (31/43). The high treatment failure rate and proportion of parasites with Tp-Db negate the use of SP to shorten the course of quinine treatment in East Africa.

Nzila A, Ochong E, Nduati E, Gilbert K, Winstanley P, Ward S, Marsh K. 2005. Why has the dihydrofolate reductase 164 mutation not consistently been found in Africa yet? Trans R Soc Trop Med Hyg, 99 (5), pp. 341-346. | Show Abstract | Read more

Resistance to the antifolate sulfadoxine-pyrimethamine (SP), the current mass-treatment antimalarial drug, is associated with selection of point mutations in dihydrofolate reductase and dihydropteroate synthase. Among these mutations, the leucine 164 dihydrofolate reductase mutation (Leu-164) is associated with higher levels of SP resistance; this mutation is also associated with a decrease in the efficacy of chlorproguanil/dapsone, a newly developed antifolate antimalarial drug. Leu-164 has been detected in Southeast Asia and South America, regions where SP is no longer effective. Surprisingly, this mutation has not yet been detected in Africa, using the standard protocol based on PCR-RFLP, despite high SP resistance. In this paper, we discuss briefly the reasons why Leu-164 has not yet been selected in Africa and we propose a means that may slow down the selection of this mutation.

Berkley JA, Maitland K, Mwangi I, Ngetsa C, Mwarumba S, Lowe BS, Newton CR, Marsh K, Scott JA, English M. 2005. Use of clinical syndromes to target antibiotic prescribing in seriously ill children in malaria endemic area: observational study. BMJ, 330 (7498), pp. 995. | Show Abstract | Read more

OBJECTIVES: To determine how well antibiotic treatment is targeted by simple clinical syndromes and to what extent drug resistance threatens affordable antibiotics. DESIGN: Observational study involving a priori definition of a hierarchy of syndromic indications for antibiotic therapy derived from World Health Organization integrated management of childhood illness and inpatient guidelines and application of these rules to a prospectively collected dataset. SETTING: Kilifi District Hospital, Kenya. PARTICIPANTS: 11,847 acute paediatric admissions. MAIN OUTCOME MEASURES: Presence of invasive bacterial infection (bacteraemia or meningitis) or Plasmodium falciparum parasitaemia; antimicrobial sensitivities of isolated bacteria. RESULTS: 6254 (53%) admissions met criteria for syndromes requiring antibiotics (sick young infants; meningitis/encephalopathy; severe malnutrition; very severe, severe, or mild pneumonia; skin or soft tissue infection): 672 (11%) had an invasive bacterial infection (80% of all invasive bacterial infections identified), and 753 (12%) died (93% of all inpatient deaths). Among P falciparum infected children with a syndromic indication for parenteral antibiotics, an invasive bacterial infection was detected in 4.0-8.8%. For the syndrome of meningitis/encephalopathy, 96/123 (76%) isolates were fully sensitive in vitro to penicillin or chloramphenicol. CONCLUSIONS: Simple clinical syndromes effectively target children admitted with invasive bacterial infection and those at risk of death. Malaria parasitaemia does not justify withholding empirical parenteral antibiotics. Lumbar puncture is critical to the rational use of antibiotics.

Nyakeriga AM, Williams TN, Marsh K, Wambua S, Perlmann H, Perlmann P, Grandien A, Troye-Blomberg M. 2005. Cytokine mRNA expression and iron status in children living in a malaria endemic area. Scand J Immunol, 61 (4), pp. 370-375. | Show Abstract | Read more

Iron deficiency has been reported to affect both malaria pathogenesis and cell-mediated immune responses; however, it is unclear whether the protection afforded by iron deficiency is mediated through direct effects on the parasite, through immune effector functions or through both. We have determined cytokine mRNA expression levels in 59 children living in a malaria endemic area on the coast of Kenya who we selected on the basis of their biochemical iron status. Real-time quantitative reverse transcriptase polymerase chain reaction analysis of cytokine mRNA levels of peripheral blood mononuclear cells (PBMC) obtained from these children showed an association between interleukin-4 (IL-4) mRNA levels and all the biochemical indices of iron that we measured. Furthermore, IL-10 mRNA was higher in parasite blood smear-positive children than in blood smear-negative children irrespective of their iron status. This study suggests that IL-4 expression by PBMC may be affected by iron status.

Maitland K, Pamba A, English M, Peshu N, Marsh K, Newton C, Levin M. 2005. Randomized trial of volume expansion with albumin or saline in children with severe malaria: preliminary evidence of albumin benefit. Clin Infect Dis, 40 (4), pp. 538-545. | Show Abstract | Read more

BACKGROUND: Metabolic acidosis is the best predictor of death in children with severe falciparum malaria; however, its treatment presents a therapeutic dilemma, because acidosis and hypovolemia may coexist with coma, which can be associated with elevated intracranial pressure. We postulated that volume resuscitation with albumin might correct acidosis and hypovolemia with a lower risk of precipitating cerebral edema than crystalloid. In an open-label, randomized, controlled trial, we compared the safety of resuscitation with albumin to saline in Kenyan children with severe malaria. METHODS: We randomly assigned children with severe malaria and metabolic acidosis (base deficit, >8 mmol/L) to receive fluid resuscitation with either 4.5% albumin or normal saline. A control (maintenance only) group was only included for patients with a base deficit of <15 mmol/L. The primary outcome measure was the percentage reduction in base deficit at 8 h. Secondary end points included death, the requirement for rescue therapies, and neurological sequelae in survivors. RESULTS: Of 150 children recruited for the trial, 61 received saline, 56 received albumin, and 33 served as control subjects. There was no significant difference in the resolution of acidosis between the groups; however, the mortality rate was significantly lower among patients who received albumin (3.6% [2 of 56 patients]) than among those who received saline (18% [11 of 61]; relative risk, 5.5; 95% confidence interval, 1.2-24.8; P=.013). CONCLUSIONS: In high-risk children with severe malaria and acidosis, fluid resuscitation with albumin may reduce mortality. Our study design did not enable us to determine whether saline administration is preferable to fluid restriction or whether saline administration is actually hazardous. Further studies are needed to confirm our findings before definitive treatment recommendations can be made.

Maitland K, Pamba A, English M, Peshu N, Levin M, Marsh K, Newton CR. 2005. Pre-transfusion management of children with severe malarial anaemia: a randomised controlled trial of intravascular volume expansion. Br J Haematol, 128 (3), pp. 393-400. | Show Abstract | Read more

Symptomatic severe malarial anaemia (SMA) has a high fatality rate of 30-40%; most deaths occur in children awaiting blood transfusion. Blood transfusion services in most of Africa are not capable of delivering adequate supplies of safe blood in a timely manner to critically ill children with SMA. Contrary to widely held belief, hypovolaemia, rather than heart failure, has emerged as a common complication in such children. We examined the safety of pre-transfusion management (PTM) by volume expansion, aimed at stabilizing children and obviating the urgency for blood transfusion. Kenyan children with severe falciparum anaemia (haemoglobin <5 g/dl) and respiratory distress were randomly assigned to 20 ml/kg of 4.5% albumin or 0.9% saline or maintenance only (control) while awaiting blood transfusion. PTM was apparently safe since it did not lead to the development of pulmonary oedema or other adverse events. There was no significant difference in the primary outcome [mean percentage reduction in base excess between admission and 8 h (95% confidence interval)] between the control group 42% (19-66%) albumin group 44% (32-57%) and saline group 36% (16-57%); adjusted analysis of variance F=0.31, P=0.7. However, the number of children requiring emergency interventions was significantly greater in the control group, four of 18 (22%) than the saline group 0 of 20 (P=0.03). We have established the safety of this PTM in children with SMA whilst awaiting blood transfusion at a hospital with an adequate blood-banking program. The impact on mortality should be assessed where blood transfusion services are unable to supply emergency transfusions.

Berkley JA, Lowe BS, Mwangi I, Williams T, Bauni E, Mwarumba S, Ngetsa C, Slack MP, Njenga S, Hart CA et al. 2005. Bacteremia among children admitted to a rural hospital in Kenya. N Engl J Med, 352 (1), pp. 39-47. | Show Abstract | Read more

BACKGROUND: There are few epidemiologic data on invasive bacterial infections among children in sub-Saharan Africa. We studied every acute pediatric admission to a rural district hospital in Kenya to examine the prevalence, incidence, types, and outcome of community-acquired bacteremia. METHODS: Between August 1998 and July 2002, we cultured blood on admission from 19,339 inpatients and calculated the incidence of bacteremia on the basis of the population served by the hospital. RESULTS: Of a total of 1783 infants who were under 60 days old, 228 had bacteremia (12.8 percent), as did 866 of 14,787 children who were 60 or more days of age (5.9 percent). Among infants who were under 60 days old, Escherichia coli and group B streptococci predominated among a broad range of isolates (14 percent and 11 percent, respectively). Among infants who were 60 or more days of age, Streptococcus pneumoniae, nontyphoidal salmonella species, Haemophilus influenzae, and E. coli accounted for more than 70 percent of isolates. The minimal annual incidence of community-acquired bacteremia was estimated at 1457 cases per 100,000 children among infants under a year old, 1080 among children under 2 years, and 505 among children under 5 years. Of all in-hospital deaths, 26 percent were in children with community-acquired bacteremia. Of 308 deaths in children with bacteremia, 103 (33.4 percent) occurred on the day of admission and 217 (70.5 percent) within two days. CONCLUSIONS: Community-acquired bacteremia is a major cause of death among children at a rural sub-Saharan district hospital, a finding that highlights the need for prevention and for overcoming the political and financial barriers to widespread use of existing vaccines for bacterial diseases.

Cited:

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Scopus

Williams TN, Mwangi TW, Roberts DJ, Alexander ND, Weatherall DJ, Wambua S, Kortok M, Snow RW, Marsh K. 2005. An immune basis for malaria protection by the sickle cell trait PLoS Medicine, 2 (5), pp. 0441-0445. | Show Abstract | Read more

Background: Malaria resistance by the sickle cell trait (genotype HbAS) has served as the prime example of genetic selection for over half a century. Nevertheless, the mechanism of this resistance remains the subject of considerable debate. While it probably involves innate factors such as the reduced ability of Plasmodium falciparum parasites to grow and multiply in HbAS erythrocytes, recent observations suggest that it might also involve the accelerated acquisition of malaria-specific immunity. Methods and Findings: We studied the age-specific protection afforded by HbAS against clinical malaria in children living on the coast of Kenya. We found that protection increased with age from only 20% in the first 2 y of life to a maximum of 56% by the age of 10 y, returning thereafter to 30% in participants greater than 10 y old. Conclusions: Our observations suggest that malaria protection by HbAS involves the enhancement of not only innate but also of acquired immunity to the parasite. A better understanding of the underlying mechanisms might yield important insights into both these processes. © 2005 Williams et al.

Maitland K, Newton C, Marsh K, Levin M. 2005. Volume status in severe malaria: no evidence provided for the degree of filling of the intravascular compartment. PLoS Med, 2 (1), pp. e27. | Read more

Maitland K, Newton C, Marsh K, Levin M, Krishna S, Planche T. 2005. Volume status in severe malaria: No evidence provided for the degree of filling of the intravascular compartment [1] (multiple letters) PLoS Medicine, 2 pp. 0079. | Read more

Polley SD, Mwangi T, Kocken CH, Thomas AW, Dutta S, Lanar DE, Remarque E, Ross A, Williams TN, Mwambingu G et al. 2004. Human antibodies to recombinant protein constructs of Plasmodium falciparum Apical Membrane Antigen 1 (AMA1) and their associations with protection from malaria. Vaccine, 23 (5), pp. 718-728. | Show Abstract | Read more

Serum antibodies from 1071 people in two Kenyan villages were assayed using eight different recombinant Apical Membrane Antigen 1 (AMA1) protein constructs to investigate their role in naturally acquired immunity. In both communities, antibodies against the full-length ectodomain (both FVO and 3D7 allele constructs) prior to a malaria transmission season were significantly associated with protection from malaria in the following 6 months, even after adjusting for age and antibody reactivity to whole parasite (schizont) extract. However, these protective associations of antibodies were only seen among subjects that were parasite slide positive at the time of pre-season serum sampling. Competition ELISAs with the FVO and 3D7 allele constructs showed that antibodies can recognise either conserved or allele-specific epitopes in AMA1. Results encourage the development of an AMA1 vaccine based on the full-length ectodomain, and indicate that the function of human antibodies to allele-specific and conserved epitopes in AMA1 should be studied further.

Mackintosh CL, Beeson JG, Marsh K. 2004. Clinical features and pathogenesis of severe malaria. Trends Parasitol, 20 (12), pp. 597-603. | Show Abstract | Read more

A major change in recent years has been the recognition that severe malaria, predominantly caused by Plasmodium falciparum, is a complex multi-system disorder presenting with a range of clinical features. It is becoming apparent that syndromes such as cerebral malaria, which were previously considered relatively clear cut, are not homogenous conditions with a single pathological correlate or pathogenic process. This creates challenges both for elucidating key mechanisms of disease and for identifying suitable targets for adjunctive therapy. The development of severe malaria probably results from a combination of parasite-specific factors, such as adhesion and sequestration in the vasculature and the release of bioactive molecules, together with host inflammatory responses. These include cytokine and chemokine production and cellular infiltrates. This review summarizes progress in several areas presented at a recent meeting.

Nyakeriga AM, Troye-Blomberg M, Chemtai AK, Marsh K, Williams TN. 2004. Malaria and nutritional status in children living on the coast of Kenya. Am J Clin Nutr, 80 (6), pp. 1604-1610. | Show Abstract

BACKGROUND: The relation between malnutrition and malaria is controversial. On the one hand, malaria may cause malnutrition, whereas on the other hand, malnutrition itself may modulate susceptibility to the disease. OBJECTIVE: The objective was to investigate the association between Plasmodium falciparum malaria and malnutrition in a cohort of Kenyan children. DESIGN: The study involved the longitudinal follow-up of children aged 0-95 [corrected] mo for clinical malaria episodes and anthropometric measurements through 4 cross-sectional surveys. We used Poisson regression analysis to investigate the association between malaria and nutritional status. RESULTS: The crude incidence rate ratios (IRRs) for malaria during the 6-mo period before assessment in children defined as malnourished on the basis of low height-for-age or low weight-for-age z scores (<-2) were 1.17 (95% CI: 0.91, 1.50; P=0.21) and 0.94 (0.71, 1.25; P=0.67), respectively, which suggests no association between malaria and the subsequent development of protein-energy malnutrition. However, we found that age acted as an effect modifier in the association between malaria episodes and malnutrition on prospective follow-up. The IRR for malaria in children aged 0-2 y, who were subsequently characterized as underweight, was 1.65 (1.10, 2.20; P=0.01), and a significant overall relation between malaria and stunting was found on regression analysis after adjustment for the interaction with age (IRR: 1.91; 1.01, 3.58; P=0.04). CONCLUSION: Although children living on the coast of Kenya continue to experience clinical episodes of uncomplicated malaria throughout the first decade of life, the effect of malaria on nutritional status appears to be greatest during the first 2 y of life.

Berkley JA, Brent A, Mwangi I, English M, Maitland K, Marsh K, Peshu N, Newton CR. 2004. Mortality among Kenyan children admitted to a rural district hospital on weekends as compared with weekdays. Pediatrics, 114 (6), pp. 1737-1738. | Read more

Molyneux CS, Peshu N, Marsh K. 2004. Understanding of informed consent in a low-income setting: three case studies from the Kenyan Coast. Soc Sci Med, 59 (12), pp. 2547-2559. | Show Abstract | Read more

In our research unit on the Kenyan Coast, parents sign consent for over 4000 children to be involved in research activities every year. Children are recruited into studies ranging from purely observational research to the testing of new procedures and drugs. Thousands more community members consent verbally or in writing to the interviews and sometimes invasive procedures required in community-based research. Although every study and consent form is reviewed in advance by independent national and international committees, the views and understanding of the 'subjects' of these activities had not been documented before this study. In this paper, we focus on participant understanding of one field-based and two hospital-based studies, all of which involve blood sampling. The findings highlight a range of inter-related issues for consideration in the study setting and beyond, including conceptual and linguistic barriers to communicating effectively about research, the critical and complex role of communicators (fieldworkers and nurses) in consent procedures, features of research unit-community relations which impact on these processes, and the special sensitivity of certain issues such as blood sampling. These themes and emerging recommendations are expected to be relevant to, and would benefit from, experiences and insights of researchers working elsewhere.

Salanti A, Dahlbäck M, Turner L, Nielsen MA, Barfod L, Magistrado P, Jensen AT, Lavstsen T, Ofori MF, Marsh K et al. 2004. Evidence for the involvement of VAR2CSA in pregnancy-associated malaria. J Exp Med, 200 (9), pp. 1197-1203. | Show Abstract | Read more

In Plasmodium falciparum-endemic areas, pregnancy-associated malaria (PAM) is an important health problem. The condition is precipitated by accumulation of parasite-infected erythrocytes (IEs) in the placenta, and this process is mediated by parasite-encoded variant surface antigens (VSA) binding to chondroitin sulfate A (CSA). Parasites causing PAM express unique VSA types, VSAPAM, which can be serologically classified as sex specific and parity dependent. It is sex specific because men from malaria-endemic areas do not develop VSAPAM antibodies; it is parity dependent because women acquire anti-VSAPAM immunoglobulin (Ig) G as a function of parity. Previously, it was shown that transcription of var2csa is up-regulated in placental parasites and parasites selected for CSA binding. Here, we show the following: (a) that VAR2CSA is expressed on the surface of CSA-selected IEs; (b) that VAR2CSA is recognized by endemic plasma in a sex-specific and parity-dependent manner; (c) that high anti-VAR2CSA IgG levels can be found in pregnant women from both West and East Africa; and (d) that women with high plasma levels of anti-VAR2CSA IgG give birth to markedly heavier babies and have a much lower risk of delivering low birth weight children than women with low levels.

Kinyanjui SM, Mwangi T, Bull PC, Newbold CI, Marsh K. 2004. Protection against clinical malaria by heterologous immunoglobulin G antibodies against malaria-infected erythrocyte variant surface antigens requires interaction with asymptomatic infections. J Infect Dis, 190 (9), pp. 1527-1533. | Show Abstract | Read more

Erythrocytes infected with mature stages of Plasmodium falciparum express variant surface antigens (VSAs) of parasite origin, including P. falciparum erythrocyte membrane protein 1. Anti-VSA antibodies protect against clinical malaria caused by parasites bearing VSAs to which they are specific (homologous), but their role in protecting against heterologous infection is unclear. Here, we report that, among 256 Kenyan children involved in a 1-year active case surveillance study, asymptomatic parasitemia was associated with an enlarged repertoire of anti-VSA immunoglobulin G (IgG) antibodies specific to apparently heterologous parasite isolates, as measured by flow cytometry. Together, asymptomatic infection and anti-VSA IgG were associated with reduced odds of experiencing an episode of clinical malaria during follow-up, whereas, independently, they were associated with increased susceptibility. These results support previous findings and underline the importance of considering the parasitological status of study participants when examining the role that immune responses to VSAs and other malaria antigens play.

Ommeh S, Nduati E, Mberu E, Kokwaro G, Marsh K, Rosowsky A, Nzila A. 2004. In vitro activities of 2,4-diaminoquinazoline and 2,4-diaminopteridine derivatives against Plasmodium falciparum ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 48 (10), pp. 3711-3714. | Show Abstract | Read more

The activities of 28 6-substituted 2,4-diaminoquinazolines, 2,4-diamino-5,6,7,8-tetrahydroquinazolines, and 2,4-diaminopteridines against Plasmodium falciparum were tested. The 50% inhibitory concentrations (IC 50s) of six compounds were <50 nM, and the most potent compound was 2,4-diamino-5-chloro-6-[N-(2,5-dimethoxybenzyl)amino]quinazoline (compound 1), with an IC50 of 9 nM. The activity of compound 1 was potentiated by the dihydropteroate synthase inhibitor dapsone, an indication that these compounds are inhibitors of dihydrofolate reductase. Further studies are warranted to assess the therapeutic potential of this combination in vivo.

Smith T, Dietz K, Vounatsou P, Muller I, English M, Marsh K. 2004. Bayesian age-stage modelling of Plasmodium falciparum sequestered parasite loads in severe malaria patients. Parasitology, 129 (Pt 3), pp. 289-299. | Show Abstract | Read more

A discrete-time age-stage model is proposed for estimating the number of sequestered parasites in severe malaria patients. A Bayesian Markov chain Monte Carlo (MCMC) approach is used to model the dynamics of Plasmodium falciparum parasitaemia in 107 paediatric patients in a randomized controlled trial of quinine and artemether in Kenya, in whom 4-hourly peripheral parasitaemia determinations were made. The MCMC approach allows the model to be fitted simultaneously to the entire dataset, providing point and interval estimates for both population and individual patient parameters. Analysis of a simulated dataset indicated that the models gave good estimates of the distribution of parasites between different stages on enrolment, for patients with a wide range of initial states. The analysis of the Kenyan patients suggested that there is considerable variation between patients within the same centre, in both the proportion of sequestered parasites and the intrinsic rate of increase of the parasite population in the absence of treatment. The resulting models should prove a useful tool for cross-validating biochemical approaches for estimating the sequestered load.

Staalsoe T, Shulman CE, Dorman EK, Kawuondo K, Marsh K, Hviid L. 2004. Intermittent preventive sulfadoxine-pyrimethamine treatment of primigravidae reduces levels of plasma immunoglobulin G, which protects against pregnancy-associated Plasmodium falciparum malaria. Infect Immun, 72 (9), pp. 5027-5030. | Show Abstract | Read more

Pregnancy-associated malaria (PAM) is an important cause of maternal and neonatal suffering. It is caused by Plasmodium falciparum capable of inhabiting the placenta through expression of particular variant surface antigens (VSA) with affinity for proteoglycans such as chondroitin sulfate A. Protective immunity to PAM develops following exposure to parasites inhabiting the placenta, and primigravidae are therefore particularly susceptible to PAM. The adverse consequences of PAM in primigravidae are preventable by intermittent preventive treatment (IPTp), where women are given antimalarials at specified intervals during pregnancy, but this may interfere with acquisition of protective PAM immunity. We found that Kenyan primigravidae receiving sulfadoxine-pyrimethamine IPTp had significantly lower levels of immunoglobulin G (IgG) with specificity for the type of parasite-encoded VSA-called VSA(PAM)-that specifically mediate protection against PAM than did women receiving a placebo. VSA(PAM)-specific IgG levels depended on the number of IPTp doses received and were sufficiently low to be of clinical concern among multidose recipients. Our data suggest that IPTp should be extended to women of all parities, in line with current World Health Organization recommendations.

Nyakeriga AM, Troye-Blomberg M, Dorfman JR, Alexander ND, Bäck R, Kortok M, Chemtai AK, Marsh K, Williams TN. 2004. Iron deficiency and malaria among children living on the coast of Kenya. J Infect Dis, 190 (3), pp. 439-447. | Show Abstract | Read more

Both iron deficiency and malaria are common in much of sub-Saharan Africa, and the interaction between these conditions is complex. To investigate the association between nutritional iron status, immunoglobulins, and clinical Plasmodium falciparum malaria, we determined the incidence of malaria in a cohort of children between the ages of 8 months and 8 years who were living on the Kenyan coast. Biochemical iron status and malaria-specific immune responses were determined during 2 cross-sectional surveys. We found that the incidence of clinical malaria was significantly lower among iron-deficient children (incidence-rate ratio [IRR], 0.70; 95% confidence interval [CI], 0.51-0.99; P<.05), that the incidence of malaria was significantly associated with plasma ferritin concentration (IRR for log ferritin concentration, 1.48; 95% CI, 1.01-2.17; P<.05), and that iron status was strongly associated with a range of malaria-specific immunoglobulins. We conclude that iron deficiency was associated with protection from mild clinical malaria in our cohort of children in coastal Kenya and discuss possible mechanisms for this protection.

English M, Mohammed S, Ross A, Ndirangu S, Kokwaro G, Shann F, Marsh K. 2004. A randomised, controlled trial of once daily and multi-dose daily gentamicin in young Kenyan infants. Arch Dis Child, 89 (7), pp. 665-669. | Show Abstract | Read more

AIMS: To test the suitability of a simple once daily (OD) gentamicin regimen for use in young infants where routine therapeutic drug monitoring is not possible. METHODS: In an open, randomised, controlled trial, infants with suspected severe sepsis admitted to a Kenyan, rural district hospital received a novel, OD gentamicin regimen or routine multi-dose (MD) regimens. RESULTS: A total of 297 infants (over 40% < or =7 days) were randomised per protocol; 292 contributed at least some data for analysis of pharmacological endpoints. One hour after the first dose, 5% (7/136) and 28% (35/123) of infants in OD and MD arms respectively had plasma gentamicin concentrations <4 microg/ml (a surrogate of treatment inadequacy). Geometric mean gentamicin concentrations at this time were 9.0 microg/ml (95% CI 8.3 to 9.9) and 4.7 microg/ml (95% CI 4.2 to 5.3) respectively. By the fourth day, pre-dose concentrations > or =2 microg/ml (a surrogate of potential treatment toxicity) were found in 6% (5/89) and 24% (21/86) of infants respectively. Mortality was similar in both groups and clinically insignificant, although potential gentamicin induced renal toxicity was observed in <2% infants. CONCLUSIONS: A "two, four, six, eight" OD gentamicin regime, appropriate for premature infants and those in the first days and weeks of life, seems a suitable, safe prescribing guide in resource poor settings.

Recker M, Nee S, Bull PC, Kinyanjui S, Marsh K, Newbold C, Gupta S. 2004. Transient cross-reactive immune responses can orchestrate antigenic variation in malaria. Nature, 429 (6991), pp. 555-558. | Show Abstract | Read more

The malaria parasite Plasmodium falciparum has evolved to prolong its duration of infection by antigenic variation of a major immune target on the surface of the infected red blood cell. This immune evasion strategy depends on the sequential, rather than simultaneous, appearance of immunologically distinct variants. Although the molecular mechanisms by which a single organism switches between variants are known in part, it remains unclear how an entire population of parasites within the host can synchronize expression to avoid rapidly exhausting the variant repertoire. Here we show that short-lived, partially cross-reactive immune responses to parasite-infected erythrocyte surface antigens can produce a cascade of sequentially dominant antigenic variants, each of which is the most immunologically distinct from its preceding types. This model reconciles several previously unexplained and apparently conflicting epidemiological observations by demonstrating that individuals with stronger cross-reactive immune responses can, paradoxically, be more likely to sustain chronic infections. Antigenic variation has always been seen as an adaptation of the parasite to evade host defence: we show that the coordination necessary for the success of this strategy might be provided by the host.

Kinyanjui SM, Howard T, Williams TN, Bull PC, Newbold CI, Marsh K. 2004. The use of cryopreserved mature trophozoites in assessing antibody recognition of variant surface antigens of Plasmodium falciparum-infected erythrocytes. J Immunol Methods, 288 (1-2), pp. 9-18. | Show Abstract | Read more

Mature stages of Plasmodium falciparum insert variant antigens (VSA) into the surface of infected erythrocytes, and antibodies against such antigen provide variant-specific protection against malaria. Because mature P. falciparum trophozoites normally sequester away from the peripheral circulation, parasites for anti-VSA antibody studies are obtained from patients as ring trophozoites, cryopreserved, and cultured to maturity when required. However, this process is associated with problems of poor recovery from cryopreservation, growth failure and variations in time different isolates take to mature after recovery. We therefore assessed the use of cryopreserved mature trophozoites in anti-VSA assays. Cryopreservation of parasites did not alter their anti-VSA antibody reactivity phenotype as determined by agglutination assays or flow cytometry. We have therefore demonstrated that cryopreserved mature trophozoites are suitable for use in anti-VSA antibody assays. The use of cryopreserved mature trophozoites could help to circumvent the problems associated with recovery of cryopreserved ring trophozoites.

Marsh VM, Mutemi WM, Willetts A, Bayah K, Were S, Ross A, Marsh K. 2004. Improving malaria home treatment by training drug retailers in rural Kenya. Trop Med Int Health, 9 (4), pp. 451-460. | Show Abstract | Read more

Recent global malaria control initiatives highlight the potential role of drug retailers to improve access to early effective malaria treatment. We report on the findings and discuss the implications of an educational programme for rural drug retailers and communities in Kenya between 1998 and 2001 in a study population of 70,000. Impact was evaluated through annual household surveys of over-the-counter (OTC) drug use and simulated retail client surveys in an early (1999) and a late (2000) implementation area. The programme achieved major improvements in drug selling practices. The proportion of OTC anti-malarial drug users receiving an adequate dose rose from 8% (n = 98) to 33% (n = 121) between 1998 and 1999 in the early implementation area. By 2001, and with the introduction of sulphadoxine pyrimethamine group drugs in accordance with national policy, this proportion rose to 64% (n = 441) across the early and late implementation areas. Overall, the proportion of shop-treated childhood fevers receiving an adequate dose of a recommended anti-malarial drug within 24 h rose from 1% (n = 681) to 28% (n = 919) by 2001. These findings strongly support the inclusion of private drug retailers in control strategies aiming to improve prompt effective treatment of malaria.

Maitland K, Marsh K. 2004. Pathophysiology of severe malaria in children. Acta Trop, 90 (2), pp. 131-140. | Show Abstract | Read more

Over the past decade there has been a growing recognition that the rationalization of severe malaria in children into the two major syndromes of cerebral malaria and severe malaria anaemia is much too simplistic. Indeed, it has become apparent that death from severe malaria may arise from a wider spectrum of pathophysiological disorders with many features in common with the derangements seen in sepsis syndromes. Amongst these derangements acidosis has emerged as a central feature of severe malaria and the major predictor of a fatal outcome. We review the improved understanding of the pathophysiology of severe malaria through a series of clinical scenarios that reflect more accurately the clinical diversity of severe malaria in African children. Current therapeutic challenges are discussed and research priorities are highlighted.

Nzila AM, Kokwaro G, Winstanley PA, Marsh K, Ward SA. 2004. Therapeutic potential of folate uptake inhibition in Plasmodium falciparum. Trends Parasitol, 20 (3), pp. 109-112. | Show Abstract | Read more

Plasmodium falciparum parasites resistant to the combination sulfadoxine-pyrimethamine are spreading in Africa, particularly in East Africa. This is a matter of concern because there are no other affordable drugs available. This article provides the evidence indicating that sulfadoxine-pyrimethamine resistance can be reversed in vitro and discusses how this information might be exploited to extend the therapeutic lifetime of sulfadoxine-pyrimethamine in vivo.

Mithwani S, Aarons L, Kokwaro GO, Majid O, Muchohi S, Edwards G, Mohamed S, Marsh K, Watkins W. 2004. Population pharmacokinetics of artemether and dihydroartemisinin following single intramuscular dosing of artemether in African children with severe falciparum malaria. Br J Clin Pharmacol, 57 (2), pp. 146-152. | Show Abstract | Read more

AIMS: To determine the population pharmacokinetics of artemether and dihydroartemisinin in African children with severe malaria and acidosis associated with respiratory distress following an intramuscular injection of artemether. METHODS: Following a single intramuscular (i.m.) injection of 3.2 mg kg-1 artemether, blood samples were withdrawn at various times over 24 h after the dose. Plasma was assayed for artemether and dihydroartemisinin by gas chromatography-mass spectrometry. The software program NONMEM was used to fit the concentration-time data and investigate the influence of a range of clinical characteristics (respiratory distress and metabolic acidosis, demographic features and disease) on the pharmacokinetics of artemether and dihydroartemisinin. RESULTS: A total of 100 children with a median age of 36.4 (range 5-108) months were recruited into the study and data from 90 of these children (30 with respiratory distress and 60 with no respiratory distress) were used in the population pharmacokinetic analysis. The best model to describe the disposition of artemether was a one-compartment model with first-order absorption and elimination. The population estimate of clearance (clearance/bioavailability, CL/F) was 14.3 l h-1 with 53% intersubject variability and that of the terminal half-life was 18.5 h. If it was assumed that artemisin displays "flip-flop" kinetics, the elimination half-life was estimated to be 21 min and the corresponding volume of distribution was 8.44 l, with an intersubject variability of 104%. None of the covariates could be identified as having any influence on the disposition of artemether. The disposition of dihydroartemisinin was fitted separately using a one-compartment linear model in which the volume of distribution was fixed to the same value as that of artemether. Assuming that artemether is completely converted to dihydroartemisinin, the estimated value of CL/F for dihydroartemisinin was 93.5 l h-1, with an intersubject variability of 90.2%. The clearance of dihydroartemisinin was formation rate limited. CONCLUSIONS: Administration of a single 3.2 mg kg-1 i.m. dose of artemether to African children with severe malaria and acidosis is characterized by variable absorption kinetics, probably related to drug formulation characteristics rather than to pathophysiological factors. Use of i.m. artemether in such children needs to be reconsidered.

Staalsoe T, Shulman CE, Bulmer JN, Kawuondo K, Marsh K, Hviid L. 2004. Variant surface antigen-specific IgG and protection against clinical consequences of pregnancy-associated Plasmodium falciparum malaria. Lancet, 363 (9405), pp. 283-289. | Show Abstract | Read more

BACKGROUND: Pregnancy-associated malaria caused by Plasmodium falciparum adherence to chondroitin sulfate A in the placental intervillous space is a major cause of low birthweight and maternal anaemia in areas of endemic P falciparum transmission. Adhesion-blocking antibodies that specifically recognise parasite-encoded variant surface antigens (VSA) are associated with resistance to pregnancy-associated malaria. We looked for a possible relation between VSA-specific antibody concentrations, placental infection, and protection from low birthweight and maternal anaemia. METHODS: We used flow cytometry to measure VSA-specific IgG concentrations in plasma samples taken during child birth from 477 Kenyan women selected from a cohort of 910 women on the basis of HIV-1 status, gravidity, and placental histology. We measured VSA expressed by one placental P falciparum isolate and two isolates selected or not selected for chondroitin sulfate A adhesiveness in-vitro. FINDINGS: Concentrations of plasma IgG specific for VSA, expressed by chondroitin sulfate A-adhering parasites (VSA in pregnancy-associated malaria or vsa-pam), increased with gravidity and were associated with placental histological findings. Women with chronic pregnancy-associated malaria and low or absent VSA-PAM-specific IgG had lower haemoglobin values (reduced by 17 g/L; 95% CI 8.1-25.2) and delivered smaller babies (birthweight reduced by 0.26 kg; 0.10-0.55) than did corresponding women with high VSA-PAM-specific IgG. No such relation was shown for concentrations of IgG with specificity for non-pregnancy-associated malaria VSA. INTERPRETATION: VSA-PAM-specific IgG protects against low birthweight and maternal anaemia. Our data indicate an important mechanism of clinical protection against malaria and raise hope for the clinical effectiveness of a potential VSA-based vaccine against pregnancy-associated malaria.

White N, Nosten F, Björkman A, Marsh K, Snow RW. 2004. WHO, the Global Fund, and medical malpractice in malaria treatment. Lancet, 363 (9415), pp. 1160. | Read more

Ochong E, Nzila A, Kimani S, Kokwaro G, Mutabingwa T, Watkins W, Marsh K. 2003. Molecular monitoring of the Leu-164 mutation of dihydrofolate reductase in a highly sulfadoxine/pyrimethamine-resistant area in Africa. Malar J, 2 (1), pp. 46. | Show Abstract | Read more

The selection of point mutation at codon 164 (from isoleucine to leucine) of the dihydrofolate reductase (DHFR) enzyme in Plasmodium falciparum is associated with high sulfadoxine /pyrimethamine (SP) resistance. Using the yeast expression system that allows the detection of dhfr allele present at low level, the presence of this mutation had previously been reported between 1998-1999 in Muheza, Tanzania, an area of high SP resistance. Eighty five P. falciparum isolates, obtained from the same area between 2002 and 2003, were analysed for the presence of Leu-164 mutation, using standard protocol based on PCR-RFLP. None of the isolates had the Leu-164 mutation.

Nzila A, Mberu E, Bray P, Kokwaro G, Winstanley P, Marsh K, Ward S. 2003. Chemosensitization of Plasmodium falciparum by probenecid in vitro. Antimicrob Agents Chemother, 47 (7), pp. 2108-2112. | Show Abstract | Read more

Resistance to drugs can result from changes in drug transport, and this resistance can sometimes be overcome by a second drug that modifies the transport mechanisms of the cell. This strategy has been exploited to partly reverse resistance to chloroquine in Plasmodium falciparum. Studies with human tumor cells have shown that probenecid can reverse resistance to the antifolate methotrexate, but the potential for reversal of antifolate resistance has not been studied in P. falciparum. In the present study we tested the ability of probenecid to reverse antifolate resistance in P. falciparum in vitro. Probenecid, at concentrations that had no effect on parasite viability alone (50 microM), was shown to increase the sensitivity of a highly resistant parasite isolate to the antifolates pyrimethamine, sulfadoxine, chlorcycloguanil, and dapsone by seven-, five-, three-, and threefold, respectively. The equivalent effects against an antifolate-sensitive isolate were activity enhancements of approximately 3-, 6-, 1.2-, and 19-fold, respectively. Probenecid decreased the level of uptake of radiolabeled folic acid, suggesting a transport-based mechanism linked to folate salvage. When probenecid was tested with chloroquine, it chemosensitized the resistant isolate to chloroquine (i.e., enhanced the activity of chloroquine). This enhancement of activity was associated with increased levels of chloroquine accumulation. In conclusion, we have shown that probenecid can chemosensitize malaria parasites to antifolate compounds via a mechanism linked to reduced folate uptake. Notably, this effect is observed in both folate-sensitive and -resistant parasites. In contrast to the activities of antifolate compounds, the effect of probenecid on chloroquine sensitivity was selective for chloroquine-resistant parasites (patent P407595GB [W. P. Thompson & Co., Liverpool, United Kingdom] has been filed to protect this intellectual property).

Mwangi TW, Ross A, Marsh K, Snow RW. 2003. The effects of untreated bednets on malaria infection and morbidity on the Kenyan coast. Trans R Soc Trop Med Hyg, 97 (4), pp. 369-372. | Show Abstract | Read more

A study was conducted in order to determine whether children that slept under untreated bednets were protected against both malaria infection and clinical disease compared with children not sleeping under bednets. The study was conducted in Kilifi District, Kenya, during the malaria season (June-August, 2000) and involved 416 children aged < or = 10 years. Data collected from a cross-sectional survey showed evidence of protection against malaria infection among children sleeping under untreated bednets in good condition compared with those not using nets (adjusted odds ratio [AOR] = 0.4, 95% CI 0.22-0.72, P = 0.002). There was no evidence of a protective effect against infection when comparing those that used untreated bednets that were worn and those not using nets (AOR = 0.75, 95% CI 0.34-1.63, P = 0.47). When these same children were followed-up during the malaria season, there was evidence of a lower rate of clinical malaria among those that used untreated nets in good condition (adjusted incidence rate ratio = 0.65, 95% CI 0.45-0.94, P = 0.022), while the rate of clinical malaria among those that used untreated bednets that were worn was similar to that of those that did not use bednets. In the face of persistent failure of communities to take up net retreatment, there is hope that untreated nets will offer some protection against malaria infection and disease compared with not using nets at all.

Scott JA, Marston EL, Hall AJ, Marsh K. 2003. Diagnosis of pneumococcal pneumonia by psaA PCR analysis of lung aspirates from adult patients in Kenya. J Clin Microbiol, 41 (6), pp. 2554-2559. | Show Abstract | Read more

psaA is the gene encoding pneumococcal surface adhesin A (PsaA), a 37-kDa protein expressed on the surface of Streptococcus pneumoniae. PCR primers for psaA have been shown to amplify the target DNA sequence in all 90 serotypes of S. pneumoniae and in none of 67 heterologous pathogens and colonizing bacteria of the upper respiratory tract. Pathogenic bacteria identified in lung aspirate specimens cannot normally be dismissed as contaminants or colonizers, which limit the assay specificity of other respiratory tract specimens. psaA PCR analysis was evaluated in 171 lung aspirates from Kenyan adults with acute pneumonia. The limit of detection was one genome equivalent. Sensitivity, estimated in 35 culture-positive lung aspirates, was 0.83 (95% confidence interval, 0.70 to 0.95). psaA PCR analysis extended the number of identifications of S. pneumoniae in lung aspirates from 35 on culture to 61 by both methods. Of 26 new pneumococcal diagnoses, 19 were corroborated by results of blood culture or urine antigen detection. Sequences of the PCR products from 12 positive samples were identical to the psaA target gene fragment. Using an internal control for the PCR, inhibition of psaA PCR was demonstrated in 17% (8 of 47) of false-negative specimens. The results of a control PCR for the human gene beta-actin suggested that false-negative psaA PCR results are attributable to problems of specimen collection, processing, or DNA extraction in 30% of cases (14 of 47). psaA PCR analysis is a sensitive tool for diagnosis of pneumococcal pneumonia in adults.

Maitland K, Levin M, English M, Mithwani S, Peshu N, Marsh K, Newton CR. 2003. Severe P. falciparum malaria in Kenyan children: evidence for hypovolaemia. QJM, 96 (6), pp. 427-434. | Show Abstract | Read more

BACKGROUND: The role of volume resuscitation in severe Plasmodium falciparum malaria is controversial. AIM: To examine the role of hypovolaemia in severe childhood malaria. STUDY DESIGN: Retrospective review. METHODS: We studied 515 children admitted with severe malaria to a high-dependency unit (HDU) in Kilifi, Kenya. On admission to the HDU, children underwent a further assessment of vital signs and a standard clinical examination. RESULTS: Factors associated with a fatal outcome included deep breathing or acidosis (base excess below -8), hypotension (systolic blood pressure <80 mmHg), raised plasma creatinine (>80 micro mol/l), low oxygen saturation (<90%), dehydration and hypoglycaemia (<2.5 mmol/l). Shock was present in 212/372 (57%) children, of whom 37 (17.5%) died, and was absent in 160, of who only 7 (4.4%) died (chi(2) = 14.9; p = 0.001). DISCUSSION: Impaired tissue perfusion may play a role in the mortality of severe malaria. Moreover, volume resuscitation, an important life-saving intervention in children with hypovolaemia, should be considered in severe malaria with evidence of impaired tissue perfusion.

Makani J, Matuja W, Liyombo E, Snow RW, Marsh K, Warrell DA. 2003. Admission diagnosis of cerebral malaria in adults in an endemic area of Tanzania: implications and clinical description. QJM, 96 (5), pp. 355-362. | Show Abstract | Read more

BACKGROUND: Cerebral malaria is commonly diagnosed in adults in endemic areas in Africa, both in hospitals and in the community. This presents a paradox inconsistent with the epidemiological understanding that the development of immunity during childhood confers protection against severe disease in adult life. AIM: To establish the contribution of Plasmodium falciparum infection in adults admitted with neurological dysfunction in an endemic area, to assess the implications of an admission clinical diagnosis of 'cerebral malaria' on the treatment and clinical outcome, and to describe the clinical features of patients with malaria parasitaemia. DESIGN: Prospective observational study. METHODS: We studied adult patients admitted with neurological dysfunction to Muhimbili National Hospital, Dar-es-Salaam, Tanzania from October 2000 to July 2001. A full blood count was done and serum creatinine, blood glucose and P. falciparum parasite load were measured. RESULTS: Of 199 patients (median age 34.6 years), 38% were diagnosed as 'cerebral malaria' on admission, but only 7.5% had detectable parasitaemia, giving a positive predictive value of 13.3%. Only 1% fulfilled the WHO criteria for cerebral malaria. The prevalence of parasitaemia (7.5%) was less than that observed in a group of asymptomatic controls (9.3%), but distribution of parasite densities was higher in the patients. Mortality was higher in patients with no parasitaemia (22.3%) than in those with parasitaemia (13%). DISCUSSION: Cerebral malaria was grossly overdiagnosed, resulting in unnecessary treatment and insufficient investigation of other possible diagnoses, which could lead to higher mortality. Extension of this misperception to the assessment of cause of death in community surveys may lead to an overestimation of the impact of malaria in adults.

Marsh K. 2003. Management of severe malaria: implications for research. Br J Clin Pharmacol, 55 (5), pp. 460-463. | Read more

Flanagan KL, Mwangi T, Plebanski M, Odhiambo K, Ross A, Sheu E, Kortok M, Lowe B, Marsh K, Hill AV. 2003. Ex vivo interferon-gamma immune response to thrombospondin-related adhesive protein in coastal Kenyans: longevity and risk of Plasmodium falciparum infection. Am J Trop Med Hyg, 68 (4), pp. 421-430. | Show Abstract

Thrombospondin-related adhesive protein (TRAP) of Plasmodium falciparum is currently being tested in human vaccine studies. However, its natural reactivity in the field remains poorly characterized. More than 40% of 217 Kenyan donors responded in an ex vivo interferon-gamma (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay to at least one of 14 20mer peptides spanning 42% of the antigen. Reactivity was comparable from early childhood (>1 year of age) to old age, and the maximal precursor frequency of TRAP-specific cells to all 14 peptides was 1 in 4,000. Prospective follow-up for one year indicated that these low-level ex vivo responses to TRAP did not protect against the subsequent development of malaria. Retesting of selected donors after one year showed a complete change in the reactivity pattern, suggesting that malaria-specific ex vivo IFN-gamma ELISPOT assay responses are short lived in naturally exposed donors, even to conserved epitopes. This study provides important information regarding natural reactivity to a key malaria antigen.

Kinyanjui SM, Bull P, Newbold CI, Marsh K. 2003. Kinetics of antibody responses to Plasmodium falciparum-infected erythrocyte variant surface antigens. J Infect Dis, 187 (4), pp. 667-674. | Show Abstract | Read more

The kinetics of antibody responses to the Plasmodium falciparum malaria parasite-induced erythrocyte surface antigens (PIESAs) in 26 Kenyan children were examined by use of flow cytometry and agglutination assays. Although 19 of the 26 children mounted a primary antibody response to PIESAs within 2 weeks of experiencing an acute episode and maintained high antibody levels for at least 12 weeks, the remaining 7 children had responses that were weak and brief. Resistance to reparasitization was decreased in the children with short-lived responses. Isotype profiles of responses in 11 of the children studied suggest that they may have failed to switch to IgG after the initial IgM response. These data suggest that children vary widely in their ability to respond to PIESAs and that, in some individuals or with certain PIESA variants, short-lived antibody responses are induced that may be associated with poor antibody class switching.

Berkley JA, Ross A, Mwangi I, Osier FH, Mohammed M, Shebbe M, Lowe BS, Marsh K, Newton CR. 2003. Prognostic indicators of early and late death in children admitted to district hospital in Kenya: cohort study. BMJ, 326 (7385), pp. 361. | Show Abstract | Read more

OBJECTIVES: To identify clinical indicators of immediate, early, and late mortality in children at admission to a sub-Saharan district hospital and to develop prognostic scores. DESIGN: Prospective cohort study. SETTING: One district hospital in Kenya. PARTICIPANTS: Children aged over 90 days admitted to hospital from 1 July 1998 to 30 June 2001. MAIN OUTCOME MEASURES: Prognostic indicators of mortality. RESULTS: Of 8091 children admitted up to 1 June 2000, 436 (5%) died. Sixty (14%) died within four hours after admission (immediate), 193 (44%) after 4-48 hours (early), and 183 (42%) after 48 hours (late). There were marked differences in the clinical features associated with immediate, early, and late death. Seven indicators (neurological status, respiratory distress (subcostal indrawing or deep breathing), nutritional status (wasting or kwashiorkor), severe anaemia, jaundice, axillary temperature, and length of history) were included in simplified prognostic scores. Data from 4802 children admitted from 1 July 2000 to 30 June 2001 were used to validate the scores. For simplified prognostic scores the areas under the receiver operating characteristic curves were 0.93 (95% confidence interval 0.92 to 0.94), 0.82 (0.80 to 0.83), and 0.82 (0.81 to 0.84) for immediate, early, and late death, respectively. CONCLUSION: In children admitted to a sub-Saharan hospital, the prognostic indicators of early and late deaths differ but a small number of simple clinical signs predict outcome well.

English M, Berkley J, Mwangi I, Mohammed S, Ahmed M, Osier F, Muturi N, Ogutu B, Marsh K, Newton CR. 2003. Hypothetical performance of syndrome-based management of acute paediatric admissions of children aged more than 60 days in a Kenyan district hospital. Bull World Health Organ, 81 (3), pp. 166-173. | Show Abstract

OBJECTIVE: To investigate whether the outpatient, syndrome-based approach of the Integrated Management of Childhood Illness (IMCI) protocol could be extended to the inpatient arena to give clear and simple minimum standards of care for poorly resourced facilities. METHODS: A prospective, one-year admission cohort retrospectively compared hypothetical performance of syndrome-based management with paediatrician-defined final diagnosis. Admission syndrome definitions were based on local adaptations to the IMCI protocol that encompassed 20 clinical features, measurement of oxygen saturation, and malaria microscopy. FINDINGS: After 315 children with clinically obvious diagnoses (e.g. sickle cell disease and burns) were excluded, 3705 admission episodes were studied. Of these, 2334 (63%) met criteria for at least one severe syndrome (mortality 8% vs <1% for "non-severe" cases), and half of these had features of two or more severe syndromes. No cases of measles were seen. Syndrome-based treatment would have been appropriate (sensitivity >95%) for severe pneumonia, severe malaria, and diarrhoea with severe dehydration, and probably for severe malnutrition (sensitivity 71%). Syndrome-directed treatment suggested the use of broad-spectrum antibiotics in 75/133 (56% sensitivity) children with bacteraemic and 63/71 (89% sensitivity) children with meningitis. CONCLUSIONS: Twenty clinical features, oxygen saturation measurements, and results of malaria blood slides could be used for inpatient, syndrome-based management of acute paediatric admissions. The addition of microscopy of the cerebrospinal fluid and haemoglobin measurements would improve syndrome-directed treatment considerably. This approach might rationalize admission policy and standardize inpatient paediatric care in resource-poor countries, although the clinical detection of bacteraemia remains a problem.

Morahan G, Boutlis CS, Huang D, Pain A, Saunders JR, Hobbs MR, Granger DL, Weinberg JB, Peshu N, Mwaikambo ED et al. 2002. A promoter polymorphism in the gene encoding interleukin-12 p40 (IL12B) is associated with mortality from cerebral malaria and with reduced nitric oxide production. Genes Immun, 3 (7), pp. 414-418. | Show Abstract | Read more

Interleukin-12 (IL-12) is an important regulatory cytokine in infection and immunity. Administration of IL-12 may reduce complications of severe malaria in rodents. Polymorphisms in IL12B, the gene encoding the IL-12 p40 subunit, influence the secretion of IL-12 and susceptibility to Type 1 diabetes. We therefore investigated whether IL12B polymorphisms may affect the outcome of severe malaria. Homozygosity for a polymorphism in the IL12B promoter was associated with increased mortality in Tanzanian children having cerebral malaria but not in Kenyan children with severe malaria. Furthermore, homozygotes for the IL12B promotor polymorphism had decreased production of nitric oxide, which is in part regulated by IL-12 activity. These studies suggest that IL12B polymorphisms, via regulation of IL-12 production, may influence the outcome of malaria infection in at least one African population.

Mwangi I, Berkley J, Lowe B, Peshu N, Marsh K, Newton CR. 2002. Acute bacterial meningitis in children admitted to a rural Kenyan hospital: increasing antibiotic resistance and outcome. Pediatr Infect Dis J, 21 (11), pp. 1042-1048. | Show Abstract | Read more

BACKGROUND: Acute bacterial meningitis (ABM) is an important cause of mortality in Africa, but most studies are based in urban referral hospitals. Poor laboratory facilities make diagnosis difficult, and treatment is limited to inexpensive antibiotics. METHODS: We retrospectively reviewed data from children admitted with ABM to a Kenyan district hospital from 1994 through 2000. We calculated the minimum incidence in children admitted from a defined area. We also examined the antibiotic susceptibility patterns. RESULTS: We identified 390 cases (1.3% of all admissions) of whom 88% were <5 years old. The apparent minimum annual incidence in children younger than 5 years of age increased from 120 to 202 per 100,000 between 1995 and 2000 (P < 0.001). Increasing the lumbar punctures performed by including prostrated or convulsing children significantly increased the number of cases detected (P < 0.005). The most common organisms in infants <3 months were streptococci and Enterobacteriaceae. Streptococcus pneumoniae (43.1%) and Haemophilus influenzae (41.9%) were predominant in the postneonatal period. The overall mortality was 30.1%, and 23.5% of survivors developed neurologic sequelae. Chloramphenicol resistance of H. influenzae rose from 8% in 1994 to 80% in 2000 (P < 0.0001) accompanied by an apparent increase in mortality. A short history, impaired consciousness and hypoglycemia were associated with death. Prolonged coma and low cerebrospinal fluid glucose were associated with neurologic sequelae. CONCLUSION: ABM in rural Kenya is a severe illness with substantial mortality and morbidity. Prognosis could be improved by broadening the criteria for lumbar puncture and use of appropriate antibiotics.

Sulo J, Chimpeni P, Hatcher J, Kublin JG, Plowe CV, Molyneux ME, Marsh K, Taylor TE, Watkins WM, Winstanley PA. 2002. Chlorproguanil-dapsone versus sulfadoxine-pyrimethamine for sequential episodes of uncomplicated falciparum malaria in Kenya and Malawi: a randomised clinical trial. Lancet, 360 (9340), pp. 1136-1143. | Show Abstract | Read more

BACKGROUND: Chlorproguanil-dapsone exerts lower resistance pressure on Plasmodium falciparum than does sulfadoxine-pyrimethamine, but is rapidly eliminated. We aimed to find out whether chlorproguanil-dapsone results in a higher retreatment rate for malaria than sulfadoxine-pyrimethamine. METHODS: In a randomised trial of paediatric outpatients with uncomplicated falciparum malaria, patients received either chlorproguanil-dapsone or sulfadoxine-pyrimethamine and were followed up for up to 1 year. Sites were in Kenya (n=410) and Malawi (n=500). We used per-protocol analysis to assess the primary outcome of annual malaria incidence. FINDINGS: Drop-outs were 117 of 410 (28.5%) in Kenya, and 342 of 500 (68.4%) in Malawi. Follow-up was for a median of 338 days (IQR 128-360) and 342 days (152-359) in Kilifi (chlorproguanil-dapsone and sulfadoxine-pyrimethamine, respectively), and for 120 days (33-281) and 84 days (26-224) in Blantyre. Mean annual malaria incidence was 2.5 versus 2.1 in Kenya (relative risk 1.16, 95% CI 0.98-1.37), and 2.2 versus 2.8 in Malawi (0.77, 0.63-0.94). 4.3% versus 12.8%, and 5.4% versus 20.1%, of patients were withdrawn for treatment failure in Kenya and Malawi, respectively. In Kenya haemoglobin concentration of 50 g/L or less caused exit in 6.9% of chlorproguanil-dapsone patients and 1.5% of sulfadoxine-pyrimethamine patients, but most anaemia occurred before re-treatment. In Malawi only one patient exited because of anaemia. INTERPRETATION: Despite the rapid elimination of chlorproguanil-dapsone, children treated with this drug did not have a higher incidence of malaria episodes than those treated with sulfadoxine-pyrimethamine. Treatment failure was more common with sulfadoxine-pyrimethamine. Cause of anaemia in Kenya was probably not adverse reaction to chlorproguanil-dapsone, but this observation requires further study.

Gravenor MB, Lloyd AL, Kremsner PG, Missinou MA, English M, Marsh K, Kwiatkowski D. 2002. A model for estimating total parasite load in falciparum malaria patients. J Theor Biol, 217 (2), pp. 137-148. | Show Abstract | Read more

We describe an age-structured mathematical model of the malaria parasite life cycle that uses clinical observations of peripheral parasitaemia to estimate population dynamics of sequestered parasites, which are hidden from the clinical investigator. First, the model was tested on parasite populations cultured in vitro, and was found to account for approximately 72% of the variation in that sub-population of parasites that would have been sequestered in vivo. Next, the model was applied to patients undergoing antimalarial therapy. Using individual data sets we found that although the model fitted the peripheral parasite curves very well, unique solutions for the fit could not be obtained; therefore, robust estimates of sequestered parasite dynamics remained unavailable. We conclude that even given detailed data on individual parasitaemia, estimates of sequestered numbers may be difficult to obtain. However, if data on individuals undergoing similar therapy are collected at equal time intervals, some of these problems may be overcome by estimating specific parameters over groups of patients. In this manner we estimated sequestered parasite density in a group of patients sampled at identical time points following antimalarial treatment. Using this approach we found significant relationships between changes in parasite density, age structure and temperature that were not apparent from the analysis of peripheral parasitaemia only.

Rowe JA, Shafi J, Kai OK, Marsh K, Raza A. 2002. Nonimmune IgM, but not IgG binds to the surface of Plasmodium falciparum-infected erythrocytes and correlates with rosetting and severe malaria. Am J Trop Med Hyg, 66 (6), pp. 692-699. | Show Abstract | Read more

Recent work suggests that IgG and IgM from nonimmune human serum (natural antibodies) bind to the surface of Plasmodium falciparum-infected erythrocytes and contribute to rosette formation by stabilizing the interaction between infected and uninfected erythrocytes. Here we show, in both laboratory clones and field isolates, that only IgM but not IgG is detected on the surface of infected cells. In field isolates, there was a strong positive correlation between IgM binding and rosette formation (Spearman's rank correlation coefficient p = 0.804, P < 0.001). Both rosette formation and IgM binding were associated with severe malaria, although statistical analysis indicates that rosette formation is the more strongly associated variable. Rosette formation, but not IgM binding, was also associated with malarial anemia. We conclude that IgM is the predominant class of natural antibodies binding to the surface of infected erythrocytes. However, we could not confirm previous suggestions that infected erythrocytes are coated with nonimmune IgG, which could lead to their interaction with host Fcgamma receptors.

Rowe JA, Obiero J, Marsh K, Raza A. 2002. Short report: Positive correlation between rosetting and parasitemia in Plasmodium falciparum clinical isolates. Am J Trop Med Hyg, 66 (5), pp. 458-460. | Show Abstract | Read more

Plasmodium falciparum isolates that form rosettes with uninfected red cells are associated with severe malaria in African children, although the mechanism by which rosetting contributes to severe disease is unknown. Here we have analyzed the relationship between rosetting and parasitemia in two samples of clinical isolates from children with malaria in Kilifi, Kenya. A consistent positive correlation was found between rosetting and parasitemia (Spearman's rank correlation coefficent p = 0.467, P < .001, n = 154, for 1993 study; p = .407, P < .001, n = 74, for 2000 study). Rosetting may enhance parasite growth and survival by facilitating invasion or promoting immune evasion, thus allowing higher parasitemia to develop and increasing the likelihood of severe malaria.

Dondorp AM, Nyanoti M, Kager PA, Mithwani S, Vreeken J, Marsh K. 2002. The role of reduced red cell deformability in the pathogenesis of severe falciparum malaria and its restoration by blood transfusion. Trans R Soc Trop Med Hyg, 96 (3), pp. 282-286. | Show Abstract | Read more

As reduced red cell deformability (RCD) can contribute to derangement of the microcirculation, a central feature in the pathogenesis of severe malaria, RCD was measured with a laser diffraction technique in 232 consecutive patients with falciparum malaria on the Kenyan coast, of whom 99 had severe disease. RCD on admission (measured as elongation index [EI] at shear stress = 1.7 Pa) was reduced in proportion with severity of disease (fatal outcome: EI = 0.182 (SD = 0.048), survivors from severe disease: EI = 0.217 (SD = 0.043), uncomplicated malaria: EI = 0.249 (SD = 0.030), healthy controls: EI = 0.268 (SD = 0.022). All but 2 survivors with severe malaria and rigid erythrocytes received a blood transfusion restoring RCD. Reduced RCD may contribute to impaired microcirculatory flow and a fatal outcome in falciparum malaria. RCD can be improved by blood transfusion. Since severely reduced RCD has a strong predictive value for mortality, blood transfusion possibly improves disease outcome not only through its beneficial effect on anaemia but also on RCD.

Allsopp CE, Sanni LA, Reubsaet L, Ndungu F, Newbold C, Mwangi T, Marsh K, Langhorne J. 2002. CD4 T cell responses to a variant antigen of the malaria parasite Plasmodium falciparum, erythrocyte membrane protein-1, in individuals living in malaria-endemic areas. J Infect Dis, 185 (6), pp. 812-819. | Show Abstract | Read more

Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1) is a variant antigen on the surface of malaria-infected red blood cells. Antibody responses to PfEMP-1 correlate with immunity, and, therefore, PfEMP-1 may be a good candidate for a malaria vaccine. However, the specificity of CD4 T cells required for a protective variant-specific antibody response is not known. We have measured the CD4 T cell response to 3 different regions that are relatively homologous among different PfEMP-1 variants. The response to the cysteine-rich interdomain region was unusual in that the majority of donors, whether malaria exposed or not, had positive CD4 T cell, interleukin-10, and interferon-gamma responses. The CD4 T cell response to the exon 2 and duffy binding-like domain proteins was significantly greater in malaria-exposed donors than in unexposed Europeans, which suggests that these regions contain peptides recognized by T cells, which thus may be useful as components of a vaccine.

Lee EA, Palmer DR, Flanagan KL, Reece WH, Odhiambo K, Marsh K, Pinder M, Gravenor MB, Keitel WA, Kester KE et al. 2002. Induction of T helper type 1 and 2 responses to 19-kilodalton merozoite surface protein 1 in vaccinated healthy volunteers and adults naturally exposed to malaria. Infect Immun, 70 (3), pp. 1417-1421. | Show Abstract | Read more

Plasmodium falciparum malaria is a major cause of death in the tropics. The 19-kDa subunit of P. falciparum merozoite surface protein 1 (MSP-1(19)), a major blood stage vaccine candidate, is the target of cellular and humoral immune responses in animals and humans. In this phase I trial of MSP-1(19), immunization of nonexposed human volunteers with either of the two allelic forms of recombinant MSP-1(19) induced high levels of antigen-specific Th1 (gamma interferon) and Th2 (interleukin 4 [IL-4] and IL-10) type lymphokines. The adjustment of the antigen dose and number of immunizations regulated the level of specificity of immune responses and Th1/Th2 bias of responses induced by vaccination. Novel conserved and allelic T-cell epitopes which induced cross-strain immune responses were identified. Importantly, responses to many of these novel epitopes were also present in adults exposed to malaria, both in east (Kenya) and west Africa (The Gambia). These data suggest that epitope-specific naturally acquired MSP-1(19) immune responses in endemic populations can be boosted by vaccination.

Miller LH, Baruch DI, Marsh K, Doumbo OK. 2002. The pathogenic basis of malaria. Nature, 415 (6872), pp. 673-679. | Show Abstract | Read more

Malaria is today a disease of poverty and underdeveloped countries. In Africa, mortality remains high because there is limited access to treatment in the villages. We should follow in Pasteur's footsteps by using basic research to develop better tools for the control and cure of malaria. Insight into the complexity of malaria pathogenesis is vital for understanding the disease and will provide a major step towards controlling it. Those of us who work on pathogenesis must widen our approach and think in terms of new tools such as vaccines to reduce disease. The inability of many countries to fund expensive campaigns and antimalarial treatment requires these tools to be highly effective and affordable.

Dorman EK, Shulman CE, Kingdom J, Bulmer JN, Mwendwa J, Peshu N, Marsh K. 2002. Impaired uteroplacental blood flow in pregnancies complicated by falciparum malaria. Ultrasound Obstet Gynecol, 19 (2), pp. 165-170. | Show Abstract | Read more

OBJECTIVE: In endemic areas, maternal malaria infection is usually asymptomatic. However, it is known that infected maternal erythrocytes sequester in the intervillous space of the placenta. There is a strong association between placental malaria infection and both low birth weight (LBW) and severe maternal anemia. We aimed to determine whether impaired uteroplacental blood flow might account for the low infant birth weight associated with maternal falciparum malaria infection. METHODS: This observational study was carried out during a large double-blind, randomized, controlled trial of an antimalarial drug intervention for primigravidae. Nine hundred and ninety-five women were recruited from the antenatal clinic at a district hospital on the Kenya coast and had at least one Doppler ultrasound scan. Uterine artery resistance index and the presence or absence of a diastolic notch were recorded. In the third trimester, blood was taken for hemoglobin and malaria film. RESULTS: Malaria infection at 32-35 weeks of gestation was associated with abnormal uterine artery flow velocity waveforms on the day of blood testing (relative risk (RR) 2.11, 95% confidence interval (CI) 1.24-3.59, P = 0.006). This association persisted after controlling for pre-eclampsia. Impaired uteroplacental blood flow in the women studied was also predictive of poor perinatal outcome, including low birth weight, preterm delivery and perinatal death. The risk of preterm delivery in women with histological evidence of past placental malaria infection was more than twice that of women without infection (RR 2.33, 95% CI 1.31-4.13, P = 0.004). CONCLUSIONS: Uteroplacental hemodynamics are altered in the presence of maternal falciparum malaria infection. This may account for some of the excess of LBW babies observed in malaria endemic areas. Strategies that prevent or clear placental malaria may confer perinatal benefit through preservation of placental function.

Ndungu FM, Bull PC, Ross A, Lowe BS, Kabiru E, Marsh K. 2002. Naturally acquired immunoglobulin (Ig)G subclass antibodies to crude asexual Plasmodium falciparum lysates: evidence for association with protection for IgG1 and disease for IgG2. Parasite Immunol, 24 (2), pp. 77-82. | Show Abstract | Read more

There is longstanding evidence for a role of immunoglobulin (Ig)G in protection against malarial disease and infection. IgG1 and IgG3 have been shown to be particularly efficient at associating with monocytes in potentially protective mechanisms (i.e. antibody-dependent cellular inhibition, opsonization and phagocytosis). Conversely, there is some evidence that IgG2 (and possibly IgG4) antibodies may be antagonistic to this protection. The protective effect of IgG subclass antibody activity present before the beginning of a malaria transmission season (preseason antibody levels) against severe malaria has not been tested in longitudinal studies. We measured IgG class and subclass antibody levels specific to crude Plasmodium falciparum lysates by enzyme linked immunosorbent assay in a case-control study of 76 children on the coast of Kenya. The mean optical density values for both IgG class and subclass antibodies were not significantly different between the children who developed severe malaria and those who remained healthy during an observation period of two malaria transmission seasons. However, elevated levels of IgG1 in relation to levels of IgG2 and IgG4 antibodies were associated with protection from severe malaria (P = 0.02). Conversely, elevated levels of IgG2 in relation to IgG1 and IgG3 antibodies were associated with a higher risk of developing severe malaria (P = 0.006).

Bull PC, Marsh K. 2002. The role of antibodies to Plasmodium falciparum-infected-erythrocyte surface antigens in naturally acquired immunity to malaria. Trends Microbiol, 10 (2), pp. 55-58. | Show Abstract | Read more

Plasmodium falciparum, the most virulent species of human malaria parasite, causes 1-3 million deaths per year. Because this parasite is susceptible to naturally acquired host immunity the main burden of diseases falls on young children. The mechanism of this immunity is still unclear. However, the parasite makes a considerable investment in the insertion of highly polymorphic antigens (parasite-infected-erythrocyte surface antigens, PIESA) on the infected erythrocyte surface, and these antigens are potentially important immune targets.

Weatherall DJ, Miller LH, Baruch DI, Marsh K, Doumbo OK, Casals-Pascual C, Roberts DJ. 2002. Malaria and the red cell. Hematology Am Soc Hematol Educ Program, 2002 (1), pp. 35-57. | Show Abstract | Read more

Because of the breakdown of malaria control programs, the constant emergence of drug resistant parasites, and, possibly, climatic changes malaria poses a major problem for the developing countries. In addition, because of the speed of international travel it is being seen with increasing frequency as an imported disease in non-tropical countries. This update explores recent information about the pathophysiology of the disease, its protean hematological manifestations, and how carrier frequencies for the common hemoglobin disorders have been maintained by relative resistance to the malarial parasite. In Section I, Dr. Louis Miller and colleagues consider recent information about the pathophysiology of malarial infection, including new information about interactions between the malarial parasite and vascular endothelium. In Section II, Dr. David Roberts discusses what is known about the complex interactions between red cell production and destruction that characterize the anemia of malaria, one of the commonest causes of anemia in tropical countries. In Section III, Dr. David Weatherall reviews recent studies on how the high gene frequencies of the thalassemias and hemoglobin variants have been maintained by heterozygote advantage against malaria and how malaria has shaped the genetic structure of human populations.

Ogutu BR, Newton CR, Crawley J, Muchohi SN, Otieno GO, Edwards G, Marsh K, Kokwaro GO. 2002. Pharmacokinetics and anticonvulsant effects of diazepam in children with severe falciparum malaria and convulsions. Br J Clin Pharmacol, 53 (1), pp. 49-57. | Show Abstract | Read more

AIMS: Convulsions are a common complication of severe malaria in children and are associated with poor outcome. Diazepam is used to terminate convulsions but its pharmacokinetics and pharmacodynamics have not been studied in this group. Accordingly, we carried out a comparative study of the pharmacokinetics of intravenous (i.v.) and rectal (p.r.) diazepam. METHODS: Twenty-five children with severe malaria and a convulsion lasting >5 min were studied. Sixteen children received diazepam intravenously (i.v.; 0.3 mg kg(-1)) and nine rectally (p.r.; 0.5 mg kg(-1)). Plasma diazepam concentrations were measured by reversed phase high-performance liquid chromatography. The duration of convulsions, depth of coma, respiratory and cardiovascular parameters were monitored. RESULTS: Median maximum plasma diazepam concentrations of 634 (range 402-1507) ng ml(-1) and 423 (range 112-1953) ng ml(-1) were achieved at 5 and 25 min following i.v. and p.r. administration, respectively. All patients except three (one i.v. and two p.r.) achieved plasma diazepam concentration >200 ng ml(-1) within 5 min. Following p.r. administration, plasma diazepam concentrations were more variable than i.v. administration. A single dose of i.v. diazepam terminated convulsions in all children but in only 6/9 after p.r. administration. However, nine children treated with i.v. and all those treated with p.r. diazepam had a recurrence of convulsions occurring at median plasma diazepam concentrations of 157 (range: 67-169) and 172 (range: 74-393) ng ml(-1) , respectively. All the children in the i.v. and four in the PR diazepam group who had recurrence of convulsions required treatment. None of the children developed respiratory depression or hypotension. CONCLUSIONS: Administration of diazepam i.v. or p.r. resulted in achievement of therapeutic concentrations of diazepam rapidly, without significant cardio-respiratory adverse effects. However, following p.r. administration, diazepam did not terminate all convulsions and plasma drug concentrations were more variable.

Snow RW, Marsh K. 2002. The consequences of reducing transmission of Plasmodium falciparum in Africa. Adv Parasitol, 52 pp. 235-264. | Show Abstract | Read more

Malaria transmission intensity in Africa varies over several log orders, from less than one infected bite per year to more than one thousand. In this review we examine the consequences in terms of age pattern, clinical spectrum and overall burden of disease and discuss the possible implications for interventions that reduce exposure to infected bites. With very low transmission intensity, all age groups are susceptible to severe malaria. With increasing transmission intensities, older children and adults suffer less severe disease and with high transmission rates the majority of severe cases occur in infants under one year of age. This pattern reflects the increasingly rapid acquisition of immune responses that limit the life-threatening effects of malaria with increasing exposure to the parasite. The clinical spectrum of severe malaria varies with transmission: with high transmission, severe malarial anaemia dominates and cerebral malaria is rare. As one moves towards lower transmission rates, cerebral malaria accounts for an increasingly large proportion of cases. Although the population risk of severe disease falls with age, the risk of death at an individual level may rise with age after an initial fall from very high case fatality rates in children aged under 6 months. Of central interest to malaria control is how the overall amount of disease in childhood varies with transmission. Data from a number of sources suggest that, with low transmission, the amount of malarial disease rises with increasing exposure but that this saturates relatively early. A key issue is whether the same pattern obtains for deaths, both those directly due to malaria and those from all causes. The methodological limitations of ecological comparisons between different areas are discussed before presenting a review of attempts to use this approach in Africa. This suggests that children living in areas of low malarial endemicity have all-cause mortality rates about half of those of children living in areas of moderate to high transmission. Deaths in the first year of life rise linearly with increasing exposure to malaria over a wide range of transmission intensities; by contrast all-cause mortality in children aged 0-4 years appears to saturate at relatively low transmission intensities. These data suggest that interventions that reduce exposure to malaria parasites, such as insecticide-treated bed nets (ITNs), will have the greatest chance of a sustained effect when used in areas where disease burdens are high but the frequency of parasite exposure is low-to-moderate. In conditions of high transmission, initial reductions in mortality may prove difficult to sustain as the reduced level of transmission may still lie on the part of the curve where mortality has saturated. However, at all levels of transmission the overall balance of benefits, including reduced load on families and health services from non-life-threatening malaria, favours the widespread introduction of ITNs in endemic areas of Africa.

Snow RW, Trape JF, Marsh K. 2001. The past, present and future of childhood malaria mortality in Africa. Trends Parasitol, 17 (12), pp. 593-597. | Show Abstract | Read more

During the past few years, there has been a historic series of declarations of renewed commitment to malaria control in Africa. Whether the burden of malaria is increasing in Africa is a moot point. This article attempts to re-construct the evidence for the trends in childhood mortality as a result of Plasmodium falciparum infection over the last century in Africa.

Berkley J, Lowe B, Mwangi I, Marsh K, Newton CRJC. 2001. Diagnosis of bacterial meningitis - Reply LANCET, 358 (9292), pp. 1549-1550. | Read more

Urban BC, Mwangi T, Ross A, Kinyanjui S, Mosobo M, Kai O, Lowe B, Marsh K, Roberts DJ. 2001. Peripheral blood dendritic cells in children with acute Plasmodium falciparum malaria. Blood, 98 (9), pp. 2859-2861. | Show Abstract | Read more

The importance of dendritic cells (DCs) for the initiation and regulation of immune responses not only to foreign organisms but also to the self has raised considerable interest in the qualitative and quantitative analysis of these cells in various human diseases. Plasmodium falciparum malaria is characterized by the poor induction of long-lasting protective immune responses. This study, therefore, investigated the percentage of peripheral blood DCs as lineage marker-negative and HLA-DR(+) or CD83(+) cells in healthy children and in children suffering from acute malaria in Kilifi, Kenya. Comparable percentages of CD83(+) DCs were found in peripheral blood of healthy children and children with malaria. However, the percentage of HLA-DR(+) peripheral blood DCs was significantly reduced in children with malaria. The results suggest that a proportion of peripheral blood DCs may be functionally impaired due to the low expression of HLA-DR on their surface.

Shulman CE, Marshall T, Dorman EK, Bulmer JN, Cutts F, Peshu N, Marsh K. 2001. Malaria in pregnancy: adverse effects on haemoglobin levels and birthweight in primigravidae and multigravidae. Trop Med Int Health, 6 (10), pp. 770-778. | Show Abstract | Read more

BACKGROUND: In areas of endemic transmission, malaria in pregnancy is associated with severe maternal anaemia and low birthweight babies. The prevalence of infection is highest in primigravidae (PG), and hence control efforts are usually geared towards this high risk group. Using a sensitive measure of placental infection, we investigated the relationship between active-acute, active-chronic and past placental infection with maternal anaemia and low birthweight in women of all gravidities. METHODS: Between January 1996 and July 1997, 912 women delivering in Kilifi District Hospital, Kenya, were recruited. Haemoglobin and peripheral malaria slides were taken prior to delivery, placental biopsies and smears were taken at the time of delivery and birthweight and maternal height and weight were measured soon after birth. Information was obtained on socio-economic and educational status. The association between placental malaria, severe anaemia and low birthweight was investigated for women of different gravidities. FINDINGS: By placental histology, the prevalence of active or past malaria in all gravidities was high, ranging from 64% in PG to 30% in gravidities 5 and above. In gravidities 1-4, active malaria infection was associated with severe maternal anaemia, adjusted OR 2.21 (95% CI 1.36, 3.61). There was a significant interaction between chronic or past malaria and severe anaemia in their effects on birthweight, whereby the risk of low birthweight was very high in women with both chronic or past placental malaria and severe anaemia: OR 4.53 (1.19, 17.2) in PG; 13.5 (4.57, 40) in gravidities 2-4. INTERPRETATION: In this area of moderate malaria transmission, women of all parities have substantially increased risk of low birthweight and severe anaemia as a result of malaria infection in pregnancy. The risk of low birthweight is likely to be particularly high in areas with a high prevalence of severe anaemia.

Pain A, Oscar K, Marsh K, Roberts DJ. 2001. A non-sense mutation and protection from severe malaria - Reply LANCET, 358 (9285), pp. 927-928. | Read more

Heddini A, Pettersson F, Kai O, Shafi J, Obiero J, Chen Q, Barragan A, Wahlgren M, Marsh K. 2001. Fresh isolates from children with severe Plasmodium falciparum malaria bind to multiple receptors. Infect Immun, 69 (9), pp. 5849-5856. | Show Abstract | Read more

The sequestration of Plasmodium falciparum-infected erythrocytes (pRBC) away from the peripheral circulation is a property of all field isolates. Here we have examined the pRBC of 111 fresh clinical isolates from children with malaria for a number of adhesive features in order to study their possible coexpression and association with severity of disease. A large number of adhesion assays were performed studying rosetting, giant rosetting, and binding to CD36, intercellular adhesion molecule 1, platelet endothelial cell adhesion molecule 1, thrombospondin, heparin, blood group A, and immunoglobulins. Suspension assays were performed at the actual parasitemia of the isolate, while all the static adhesion assays were carried out at an equal adjusted parasitemia. The ability to bind to multiple receptors, as well as the ability to form rosettes and giant rosettes, was found to be more frequent among isolates from children with severe versus mild malaria (P = 0.0015). Rosettes and giant rosettes were more frequent for children with severe malaria, and the cell aggregates were larger and tighter, than for those with mild disease (P = 0.0023). Binding of immunoglobulins (97% of isolates) and of heparin (81% of isolates) to infected erythrocytes was common, and binding to heparin and blood group A was associated with severity of disease (P = 0.011 and P = 0.031, respectively). These results support the idea that isolates that bind to multiple receptors are involved in the causation of severe malaria and that several receptor-ligand interactions work synergistically in bringing about severe disease.

Heddini A, Chen Q, Obiero J, Kai O, Fernandez V, Marsh K, Muller WA, Wahlgren M. 2001. Binding of Plasmodium falciparum-infected erythrocytes to soluble platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31): frequent recognition by clinical isolates. Am J Trop Med Hyg, 65 (1), pp. 47-51. | Show Abstract | Read more

Platelet-endothelial cell adhesion molecule-1 or CD31 (PECAM-1/CD31) is a receptor recognized by Plasmodium falciparum-parasitized erythrocytes (pRBCs). Fluorescence-labeled soluble recombinant PECAM-1/CD31 (sPECAM-1/CD31) is shown to bind to the surface of P. falciparum-infected erythrocytes on up to 70% of the cells. Binding is blocked by the addition of the unlabeled receptor in a dose-dependent fashion, but not by unrelated receptor-proteins. A significant correlation was found between the binding of sPECAM-1/CD31 to pRBCs and the binding to transfected L cells expressing the receptor as seen with six different P. falciparum lines or clones. Panning of cultures on PECAM-1/CD31 transfected L cells was paralleled by an increase in the binding of sPECAM-1/CD31. The pRBCs of 54% of fresh patient-isolates bound sPECAM-1/CD31 with a mean rate of 12.9% (range = 1.1-44%). The data suggest that PECAM-1/CD31 is a common receptor recognized by wild isolates and that the soluble PECAM-1/CD31 suspension assay is a sensitive and reliable way to study PECAM-1/CD31 binding.

Berkley JA, Mwangi I, Ngetsa CJ, Mwarumba S, Lowe BS, Marsh K, Newton CR. 2001. Diagnosis of acute bacterial meningitis in children at a district hospital in sub-Saharan Africa. Lancet, 357 (9270), pp. 1753-1757. | Show Abstract | Read more

BACKGROUND: The diagnosis of acute bacterial meningitis in children is difficult in sub-Saharan Africa, because the clinical features overlap with those of other common diseases, and laboratory facilities are inadequate in many areas. We have assessed the value of non-laboratory tests and incomplete laboratory data in diagnosing childhood acute bacterial meningitis in this setting. METHODS: We prospectively studied 905 children undergoing lumbar puncture at a rural district hospital in Kenya over 1 year. We related microbiological findings and cerebrospinal-fluid (CSF) laboratory measurements to tests that would typically be available at such a hospital. FINDINGS: Acute bacterial meningitis was proven in 45 children (5.0% [95% CI 3.7-6.6]) and probable in 26 (2.9% [1.9-4.2]). 21 of the 71 cases of proven or probable acute bacterial meningitis had neither neck stiffness nor turbid CSF. In eight of 45 children with proven disease the CSF leucocyte count was less than 10x10(6)/L or leucocyte counting was not possible because of blood-staining. The presence of either a leucocyte count of 50x10(6)/L or more or a CSF/blood glucose ratio of 0.10 or less detected all but two of the 45 children with proven acute bacterial meningitis; these two samples were grossly blood-stained. INTERPRETATION: The diagnosis of childhood acute bacterial meningitis is likely to be missed in a third of cases at district hospitals in sub-Saharan Africa without adequate and reliable laboratory resources. CSF culture facilities are expensive and difficult to maintain, and greater gains could be achieved with facilities for accurate leucocyte counting and glucose measurement.

Pain A, Urban BC, Kai O, Casals-Pascual C, Shafi J, Marsh K, Roberts DJ. 2001. A non-sense mutation in Cd36 gene is associated with protection from severe malaria. Lancet, 357 (9267), pp. 1502-1503. | Show Abstract | Read more

We sought genetic evidence for the importance of host-parasite interactions involving CD36 in severe malaria. We identified a non-sense mutation in Cd36 gene and looked at the influence of this mutation on the outcome of malaria infection in 693 African children with severe malaria and a similar number of ethnically matched controls. We showed that heterozygosity for this mutation is associated with protection from severe disease (OR 0.74, 95% CI 0.55-0.99; p=0.036). These findings suggest that this Cd36 mutation might have a complex effect on malaria infection by decreasing parasite sequestration, and also by decreasing host immune responses.

Griffiths MJ, Ndungu F, Baird KL, Muller DP, Marsh K, Newton CR. 2001. Oxidative stress and erythrocyte damage in Kenyan children with severe Plasmodium falciparum malaria. Br J Haematol, 113 (2), pp. 486-491. | Show Abstract | Read more

Anaemia causes significant morbidity in children with Plasmodium falciparum malaria, but the mechanism(s) are unclear. During malarial infection, increased reactive oxygen species (ROS) are generated that may contribute to erythrocyte damage and anaemia. This study measured the concentrations of alpha-tocopherol in plasma and erythrocyte membranes, and the percentage polyunsaturated fatty acid composition (%PUFA) (an indirect marker of ROS damage) in erythrocyte membranes in children with severe P. falciparum malaria from Kilifi, Kenya, and asymptomatic children from the same district. Malarial subjects were stratified into complicated malaria and malaria anaemia. Results demonstrated significant reductions in erythrocyte membrane alpha-tocopherol concentration (1.63 +/- 0.16 versus 3.38 +/- 0.18 micromol/mg protein; P < 0.001) and total %PUFA (30.7 +/- 0.49 versus 32.8 +/- 0.44% P < 0.005) for the malarial subjects (non-stratified) compared with controls. Malarial subjects showed a significant positive correlation between membrane alpha-tocopherol and haemoglobin concentrations (P < 0.005 r = 0.63 complicated malaria group; P < 0.05 r = 0.36 non-stratified data). There were no significant differences in plasma alpha-tocopherol concentration between malaria patients and controls. In conclusion, malarial infection may be associated with oxidative damage and reduced alpha-tocopherol reserve in the erythrocyte membrane, suggesting that local antioxidant depletion may contribute to erythrocyte loss in severe malaria. Erythrocyte membrane alpha-tocopherol appeared a better indicator of ROS exposure than plasma.

Shulman CE, Levene M, Morison L, Dorman E, Peshu N, Marsh K. 2001. Screening for severe anaemia in pregnancy in Kenya, using pallor examination and self-reported morbidity. Trans R Soc Trop Med Hyg, 95 (3), pp. 250-255. | Show Abstract | Read more

Severe anaemia in pregnancy is an important preventable cause of maternal and perinatal morbidity and mortality. Different methods of screening for severe anaemia in pregnancy were evaluated in a 2-phased study conducted in Kilifi, Kenya. In phase 1 (in 1994/95), pallor testing was evaluated alone and in addition to raised respiratory/pulse rates: 1787 pregnant women were examined by one of 2 midwives. Sensitivities for detecting severe anaemia (haemoglobin < 7 g/dL) were 62% and 69% and specificities 87% and 77%, respectively for each of the midwives. Addition of high pulse rate increased sensitivity to 77% and 81%, but specificity reduced to 60% and 51%, respectively. In phase 2, following qualitative in-depth work, a screening questionnaire was developed. An algorithm based on screening questions had 80% sensitivity and 40% specificity. Midwife pallor-assessment was conducted following the screening questionnaire. In this phase (conducted in 1997), the midwife performed very highly in detecting severe anaemia, achieving sensitivity of 84% and specificity of 92%. Spending a few minutes asking women questions may have improved the ability to interpret pallor findings. This study demonstrates the value of pallor testing and raises alternative approaches to improving it.

Yung BM, Browne-Yung K, Marsh K. 2001. Outcome of cardiopulmonary resuscitation in hospitalized African children. J Trop Pediatr, 47 (2), pp. 108-110. | Show Abstract

The outcome of cardiopulmonary resuscitation at the research ward of the Kenya Medical Research Institute is reviewed. The outcome for respiratory arrest was 15 per cent (95 per cent CI 6.6-27) to discharge, and worse for cardiorespiratory arrest with no survival. The illnesses leading to cardiopulmonary arrest and causes for the disappointing outcome are discussed.

Crawley J, Smith S, Muthinji P, Marsh K, Kirkham F. 2001. Electroencephalographic and clinical features of cerebral malaria. Arch Dis Child, 84 (3), pp. 247-253. | Show Abstract | Read more

BACKGROUND: Seizures are a prominent feature of childhood cerebral malaria, and are associated with an increased risk of death and neurological sequelae. We present the electroencephalographic (EEG) findings from a detailed clinical and electrophysiological study. METHODS: Children with cerebral malaria had EEGs recorded within six hours of admission, and at 12 hourly intervals until recovery of consciousness. Ten deeply comatose children underwent intracranial pressure monitoring. Children were not mechanically ventilated, which made it possible to directly correlate the clinical and EEG findings. RESULTS: Of 65 children aged 9 months and above, 40 had one or more seizures, and 18 had an episode of status epilepticus. Most seizures were partial motor, and spike wave activity consistently arose from the posterior temporo-parietal region, a border zone area lying between territories supplied by the carotid and vertebrobasilar circulations. Fifteen children had seizures that were clinically subtle or electrographic. Clinical seizures were associated with an abrupt rise in intracranial pressure. Fifty children recovered fully, seven died, and eight had persistent neurological sequelae. Initial EEG recordings of very slow frequency, or with background asymmetry, burst suppression, or interictal discharges, were associated with an adverse outcome. CONCLUSIONS: Serial EEG recording has uncovered a range of clinical, subtle, and electrographic seizures complicating childhood cerebral malaria, and has emphasised their importance in the pathogenesis of coma. Further work is required to determine the most appropriate regimen for the prophylaxis and treatment of seizures in cerebral malaria, in order to improve outcome.

Lee EA, Flanagan KL, Odhiambo K, Reece WH, Potter C, Bailey R, Marsh K, Pinder M, Hill AV, Plebanski M. 2001. Identification of frequently recognized dimorphic T-cell epitopes in plasmodium falciparum merozoite surface protein-1 in West and East Africans: lack of correlation of immune recognition and allelic prevalence. Am J Trop Med Hyg, 64 (3-4), pp. 194-203. | Show Abstract | Read more

The merozoite surface protein-1 (MSP1) is the most studied malaria blood-stage vaccine candidate. Lymphokines such as interferon gamma (IFN-gamma) and interleukin 4 (IL-4) may mediate blood-stage specific protection. Here we identify Plasmodiumfalciparum MSP1 T-cell epitopes capable of rapid induction of IFN-gamma and/or IL-4 from peripheral blood mononuclear cells of East and West African donors. Both allelic forms of these novel MSP1 T-cell epitopes were stimulatory. An unusually high numbers of Gambian responders (> 80%) to these epitopes were observed, suggesting that MSPI reactivity may have been underestimated previously in this population. Surprisingly, IFN-gamma responses to allelic T-cell epitopes failed to correlate with differential antigenic exposure in The Gambia compared to Kenya. These results suggest an unexpected level of immunoregulation of IFN-gamma response with variable allelic T-cell reactivity independent of the level of antigenic exposure. Further analysis of the mechanisms determining this response pattern may be required if vaccines are to overcome this allelic reactivity bias in malaria-exposed populations.

Pain A, Ferguson DJ, Kai O, Urban BC, Lowe B, Marsh K, Roberts DJ. 2001. Platelet-mediated clumping of Plasmodium falciparum-infected erythrocytes is a common adhesive phenotype and is associated with severe malaria. Proc Natl Acad Sci U S A, 98 (4), pp. 1805-1810. | Show Abstract | Read more

Sequestration of malaria-infected erythrocytes in the peripheral circulation has been associated with the virulence of Plasmodium falciparum. Defining the adhesive phenotypes of infected erythrocytes may therefore help us to understand how severe disease is caused and how to prevent or treat it. We have previously shown that malaria-infected erythrocytes may form apparent autoagglutinates of infected erythrocytes. Here we show that such autoagglutination of a laboratory line of P. falciparum is mediated by platelets and that the formation of clumps of infected erythrocytes and platelets requires expression of the platelet surface glycoprotein CD36. Platelet-dependent clumping is a distinct adhesive phenotype, expressed by some but not all CD36-binding parasite lines, and is common in field isolates of P. falciparum. Finally, we have established that platelet-mediated clumping is strongly associated with severe malaria. Precise definition of the molecular basis of this intriguing adhesive phenotype may help to elucidate the complex pathophysiology of malaria.

Cavanagh DR, Dobaño C, Elhassan IM, Marsh K, Elhassan A, Hviid L, Khalil EA, Theander TG, Arnot DE, McBride JS. 2001. Differential patterns of human immunoglobulin G subclass responses to distinct regions of a single protein, the merozoite surface protein 1 of Plasmodium falciparum. Infect Immun, 69 (2), pp. 1207-1211. | Show Abstract | Read more

Comparisons of immunoglobulin G (IgG) subclass responses to the major polymorphic region and to a conserved region of MSP-1 in three cohorts of African villagers exposed to Plasmodium falciparum revealed that responses to Block 2 are predominantly IgG3 whereas antibodies to MSP-1(19) are mainly IgG1. The striking dominance of IgG3 to Block 2 may explain the short duration of this response and also the requirement for continuous stimulation by malaria infection to maintain clinical immunity.

Konotey-Ahulu FI. 2001. A non-sense mutation and protection from severe malaria. Lancet, 358 (9285), pp. 927-928. | Read more

Crawley J, Kokwaro G, Ouma D, Watkins W, Marsh K. 2000. Chloroquine is not a risk factor for seizures in childhood cerebral malaria. Trop Med Int Health, 5 (12), pp. 860-864. | Show Abstract | Read more

OBJECTIVES: There are a number of case reports in the medical literature suggesting an association between the ingestion of chloroquine and subsequent seizure activity. Our study was designed to investigate the relationship between blood levels of chloroquine (CQ), its metabolite desethylchloroquine (DCQ), and seizures in children admitted to hospital with cerebral malaria. METHODS: Serial blood levels of CQ and DCQ were measured over the first 24 h of hospital admission in children with cerebral malaria. The number and duration of all seizures was recorded, and statistical analysis subsequently performed to determine the relationship between seizure activity and blood concentrations of CQ and DCQ. RESULTS: Chloroquine was detected in 92% (100/109) of admission blood samples. 54% (59/109) of the patients had one or more seizures after admission, while 8% (9/109) had an episode of status epilepticus. Median (interquartile range) baseline concentrations of CQ and DCQ were, respectively, 169.4 microg/ml (75.1-374.9) and 352.3 microg/ml (81.9-580.1) for those children who had seizures after admission, compared to CQ 227.5 microg/ml (79.4-430.2) and DCQ 364.0 microg/ml (131.3-709.4) for those who did not have seizures (P > 0.5 for all comparisons). Baseline concentrations of CQ and DCQ were not significantly associated with the occurrence of seizures lasting for 5 min or more. The nine children who had an episode of status epilepticus had significantly lower median admission levels of CQ than those without status epilepticus: 75.1 microg/l (7.4-116.5) vs. 227.5 microg/l (85.6-441.2), P = 0.02. Multivariate logistic regression analysis, taking into account factors likely to affect the risk of seizures in hospital, failed to change the significance of these results. CONCLUSIONS: These findings suggest that chloroquine does not play an important role in the aetiology of seizures in childhood cerebral malaria.

Rowe JA, Rogerson SJ, Raza A, Moulds JM, Kazatchkine MD, Marsh K, Newbold CI, Atkinson JP, Miller LH. 2000. Mapping of the region of complement receptor (CR) 1 required for Plasmodium falciparum rosetting and demonstration of the importance of CR1 in rosetting in field isolates. J Immunol, 165 (11), pp. 6341-6346. | Show Abstract

The malaria parasite Plasmodium falciparum induces a number of novel adhesion properties in the erythrocytes that it infects. One of these properties, the ability of infected erythrocytes to bind uninfected erythrocytes to form rosettes, is associated with severe malaria and may play a direct role in the pathogenesis of disease. Previous work has shown that erythrocytes deficient in complement receptor (CR) 1 (CR1, CD35; C3b/C4b receptor) have greatly reduced rosetting capacity, indicating an essential role for CR1 in rosette formation. Using deletion mutants and mAbs, we have localized the region of CR1 required for the formation of P. falciparum rosettes to the area of long homologous repeat regions B and C that also acts as the binding site for the activated complement component C3b. This result raises the possibility that C3b could be an intermediary in rosetting, bridging between the infected erythrocyte and CR1. We were able to exclude this hypothesis, however, as parasites grown in C3-deficient human serum formed rosettes normally. We have also shown in this report that rosettes can be reversed by mAb J3B11 that recognizes the C3b binding site of CR1. This rosette-reversing activity was demonstrated in a range of laboratory-adapted parasite strains and field isolates from Kenya and Malawi. Thus, we have mapped the region of CR1 required for rosetting and demonstrated that the CR1-dependent rosetting mechanism occurs commonly in P. falciparum isolates, and could therefore be a potential target for future therapeutic interventions to treat severe malaria.

Brown GV, Beck HP, Molyneux M, Marsh K. 2000. Molecular approaches to epidemiology and clinical aspects of malaria. Parasitol Today, 16 (10), pp. 448-451. | Show Abstract | Read more

Malaria is a problem of global importance, responsible for 1-2 million deaths per year, mainly in African children, as well as considerable morbidity manifested as severe anaemia and encephalopathy in young children. Fundamental to the development of new tools for malaria control in humans is an increased understanding of key features of malaria infection, such as the diversity of outcome in different individuals, the understanding of different manifestations of the disease and of the mechanisms of immunity that allow clinical protection in the face of ongoing low-grade infection (concomitant immunity or premunition). Here, Graham Brown and colleagues review some of the ways in which molecular approaches might be used to increase our understanding of the epidemiology and clinical manifestations of malaria, as discussed at the Molecular Approaches to Malaria conference (MAM2000), Lorne, Australia, 2-5 February 2000.

Crawley J, Waruiru C, Winstanley P, Peto T, Marsh K. 2000. Phenobarbital for children with cerebral malaria - Reply LANCET, 356 (9225), pp. 256-257. | Read more

Snow RW, Howard SC, Mung'Ala-Odera V, English M, Molyneux CS, Waruiru C, Mwangi I, Roberts DJ, Donnelly CA, Marsh K. 2000. Paediatric survival and re-admission risks following hospitalization on the Kenyan coast. Trop Med Int Health, 5 (5), pp. 377-383. | Show Abstract | Read more

The district general hospital (DGH) is a common feature of health service provision in many developing countries. We have used linked demographic and clinical surveillance in a rural community located close to a DGH on the Kenyan coast to define the use and public health significance of essential clinical services provided by it. Of a birth cohort of over 4000 children followed for approximately 6 years, about a third were admitted to hospital at least once. Significantly more children admitted with major infectious diseases such as malaria and acute respiratory tract infections were readmitted with the same condition during the surveillance period than would have been expected by chance. Among surviving admissions, mortality post-discharge was significantly higher than in the cohort which had not been admitted within 3, 6 and 12 months. Most of the patients who died after discharge had been admitted with a diagnosis of gastroenteritis. Most children admitted to the DGH survive hospitalization and the remaining period of childhood. Despite no clinical trial evidence to support the claim, it seems reasonable to assume that in the absence of intensive clinical management provided by a DGH, a significant proportion of these children would not have survived. However, the DGH is able to define a group of at-risk children who re-present with severe complications of infectious disease, and of these several may have underlying conditions not amenable to DGH intervention and continue to have a poor prognosis. Both groups of children represent statistically significant subsets of a rural paediatric community and the future organization and co-ordination of DGH and primary care services need to work in unison to strengthen the service needs of children at risk.

Roberts DJ, Pain A, Kai O, Kortok M, Marsh K. 2000. Autoagglutination of malaria-infected red blood cells and malaria severity. Lancet, 355 (9213), pp. 1427-1428. | Show Abstract | Read more

Red blood cells infected with Plasmodium falciparum can adhere to each other and so form large autoagglutinates. We show that this phenotype is common in field isolates and is strongly associated with severe malaria.

Scott JA, Hall AJ, Muyodi C, Lowe B, Ross M, Chohan B, Mandaliya K, Getambu E, Gleeson F, Drobniewski F, Marsh K. 2000. Aetiology, outcome, and risk factors for mortality among adults with acute pneumonia in Kenya. Lancet, 355 (9211), pp. 1225-1230. | Show Abstract | Read more

BACKGROUND: Despite a substantial disease burden, there is little descriptive epidemiology of acute pneumonia in sub-Saharan Africa. We did this study to define the aetiology of acute pneumonia, to estimate mortality at convalescence, and to analyse mortality risk-factors. METHODS: We studied 281 Kenyan adults who presented to two public hospitals (one urban and one rural) with acute radiologically confirmed pneumonia during 1994-96. We did blood and lung-aspirate cultures, mycobacterial cultures, serotype-specific pneumococcal antigen detection, and serology for viral and atypical agents. FINDINGS: Aetiology was defined in 182 (65%) patients. Streptococcus pneumoniae was the most common causative agent, being found in 129 (46%) cases; Mycobacterium tuberculosis was found in 26 (9%). Of 255 patients followed up for at least 3 weeks, 25 (10%) died at a median age of 33 years. In multivariate analyses, risk or protective factors for mortality were age (odds ratio 1.51 per decade [95% CI 1.04-2.19]), unemployment (4.42 [1.21-16.1]), visiting a traditional healer (5.26 [1.67-16.5]), visiting a pharmacy (0.30 [0.10-0.91]), heart rate (1.64 per 10 beats [1.24-2.16]), and herpes labialis (15.4 [2.22-107]). HIV-1 seropositivity, found in 52%, was not associated with mortality. Death or failure to recover after 3 weeks was more common in patients with pneumococci of intermediate resistance to benzylpenicillin, which comprised 28% of pneumococcal isolates, than in those infected with susceptible pneumococci (5.60 [1.33-23.6]). INTERPRETATION: We suggest that tuberculosis is a sufficiently common cause of acute pneumonia in Kenyan adults to justify routine sputum culture, and that treatment with benzylpenicillin remains appropriate for clinical failure due to M. tuberculosis, intermediate-resistant pneumococci, and other bacterial pathogens. However, interventions restricted to hospital management will fail to decrease mortality associated with socioeconomic, educational, and behavioural factors.

Crawley J, Waruiru C, Mithwani S, Mwangi I, Watkins W, Ouma D, Winstanley P, Peto T, Marsh K. 2000. Effect of phenobarbital on seizure frequency and mortality in childhood cerebral malaria: a randomised, controlled intervention study. Lancet, 355 (9205), pp. 701-706. | Show Abstract | Read more

BACKGROUND: Seizures commonly complicate cerebral malaria and are associated with an increased risk of death and neurological sequelae. We undertook a randomised study to assess the efficacy of intramuscular phenobarbital in preventing seizures in childhood cerebral malaria. METHODS: Children with cerebral malaria admitted to one hospital in Kilifi, Kenya, were randomly assigned a single intramuscular dose of phenobarbital (20 mg/kg) or identical placebo. Clinical tolerance was assessed at the start of the trial, with particular reference to respiratory depression and hypotension. Seizures were timed and recorded, and treated in a standard way. Plasma phenobarbital concentrations were measured. Analyses were by intention to treat. FINDINGS: 440 children with cerebral malaria were admitted to the hospital; 100 were not recruited to the study. Of the remaining 340, 170 received phenobarbital and 170 placebo. The drug was adequately absorbed and well tolerated. Seizure frequency was significantly lower in the phenobarbital group than in the placebo group (18 [11%] vs 46 [27%] children had three or more seizures of any duration; odds ratio 0.32 [95% CI 0.18-0.58]) but mortality was doubled (30 [18%] vs 14 [8%] deaths; 2.39 [1.28-4.64]). The frequency of respiratory arrest was higher in the phenobarbital group than in the placebo group, and mortality was greatly increased in children who received phenobarbital plus three or more doses of diazepam (odds ratio 31.7 [1.2-814]). INTERPRETATION: In children with cerebral malaria, phenobarbital 20 mg/kg provides highly effective seizure prophylaxis but is associated with an unacceptable increase in mortality. Use of this dose cannot, therefore, be recommended.

Dobbie M, Crawley J, Waruiru C, Marsh K, Surtees R. 2000. Cerebrospinal fluid studies in children with cerebral malaria: an excitotoxic mechanism? Am J Trop Med Hyg, 62 (2), pp. 284-290. | Show Abstract | Read more

The pathogenesis of cerebral malaria is poorly understood. One hypothesis is that activation of microglia and astrocytes in the brain might cause the cerebral symptoms by excitotoxic mechanisms. Cerebrospinal fluid was sampled in 97 Kenyan children with cerebral malaria, 85% within 48 hr of admission. When compared with an age-matched reference range, there were large increases in concentrations of the excitotoxin quinolinic acid (geometric mean ratio cerebral malaria/reference population [95% confidence limits] = 14.1 [9.8-20.4], P < 0.001) and total neopterin (10.9 [9.1-13.0], P < 0.001) and lesser increases in tetra-hydrobiopterin, di-hydrobiopterin, and 5-hydroxyindoleacetic acid. There was no change in tryptophan concentration. In contrast, nitrate plus nitrite concentrations were decreased (geometric mean ratio = 0.45 [0.35-0.59], P < 0.001). There was a graded increment in quinolinic acid concentration across outcome groups of increasing severity. The increased concentration of quinolinic acid suggests that excitotoxic mechanisms may contribute to the pathogenesis of cerebral malaria.

Beales PF, Brabin B, Dorman E, Gilles HM, Loutain L, Marsh K, Molyneux ME, Olliaro P, Schapira A, Touze JE et al. 2000. Severe falciparum malaria. World Health Organization, Communicable Diseases Cluster. Trans R Soc Trop Med Hyg, 94 Suppl 1 (SUPPL.1), pp. S1-90.

Holding PA, Stevenson J, Peshu N, Marsh K. 1999. Cognitive sequelae of severe malaria with impaired consciousness. Trans R Soc Trop Med Hyg, 93 (5), pp. 529-534. | Show Abstract | Read more

Although cerebral malaria is the most common acute encephalopathy arising in children in Africa little is known of its effect upon the longer-term cognitive development of survivors. In Kenya, we compared the performance of 87 survivors of severe malaria with impaired consciousness to matched community controls on a wide range of tasks, not less than 42 months post illness episode. The presence of cognitive impairment was then related to both the pattern of symptoms at the time of the acute illness and the presence of gross neurological impairment on discharge. Significant group differences were found in areas of cognitive functioning suggestive of widespread impairment in the development of the ability to initiate, plan and carry out tasks (the executive functions). On tasks of more discrete cognitive skills (information processing) there were no significant group differences, although impaired performance was found more frequently in the severe malaria group. The odds ratio associated with the development of cognitive impairment following severe malaria with impaired consciousness was found to be 4.48 (95% CI 1.22, 16.47). A combination of 4 signs (coma, hypoglycaemia, seizures, and absence of hyperpyrexia) proved to have greater accuracy than the presence of gross neurological sequelae in predicting cognitive impairment (95% vs 93% specificity, 67% vs 58% sensitivity).

Marsh K, Snow RW. 1999. Malaria transmission and morbidity. Parassitologia, 41 (1-3), pp. 241-246. | Show Abstract

Stable malaria endemicity is maintained over a wide range of transmission intensities in sub-Saharan Africa. This paper considers variations in the clinical manifestations and their consequences with differences in transmission intensity. Epidemiological approaches to malarial disease have concentrated on two clinical syndromes, severe malarial anaemia and cerebral malaria. Within an area the mean age of children with severe malarial anaemia is always lower than that of those with cerebral malaria. In areas of higher malaria transmission children, on average, encounter malaria at a younger age and the mean age of clinical cases is lower. Malarial anaemia tends therefore to be relatively more important under high transmission settings and cerebral malaria tends to gain in importance under lower transmission settings. In a number of studies the total load of malaria morbidity, whether measured as none severe malaria in the community or as severe malaria admitted to hospital, is low under stable low transmission conditions but is at its highest under moderate intensities of transmission. Thereafter it reaches a plateau, or even falls, at the highest transmission intensities. It is not known whether the same is true for mortality in communities living under different transmission settings. Possible implications for changes in patterns of morbidity and mortality following interventions which lower malaria transmission are discussed. It is concluded that such interventions should play an important role in integrated malaria control programmes but that these should involve concomitant introduction of other interventions, in order to minimise the possible risks of a reduced effect as the immune response of the population re-equilibrates in the face of reduced challenge.

White NJ, Nosten F, Looareesuwan S, Watkins WM, Marsh K, Snow RW, Kokwaro G, Ouma J, Hien TT, Molyneux ME et al. 1999. Averting a malaria disaster. Lancet, 353 (9168), pp. 1965-1967. | Read more

Knight JC, Udalova I, Hill AV, Greenwood BM, Peshu N, Marsh K, Kwiatkowski D. 1999. A polymorphism that affects OCT-1 binding to the TNF promoter region is associated with severe malaria. Nat Genet, 22 (2), pp. 145-150. | Show Abstract | Read more

Genetic variation in cytokine promoter regions is postulated to influence susceptibility to infection, but the molecular mechanisms by which such polymorphisms might affect gene regulation are unknown. Through systematic DNA footprinting of the TNF (encoding tumour necrosis factor, TNF) promoter region, we have identified a single nucleotide polymorphism (SNP) that causes the helix-turn-helix transcription factor OCT-1 to bind to a novel region of complex protein-DNA interactions and alters gene expression in human monocytes. The OCT-1-binding genotype, found in approximately 5% of Africans, is associated with fourfold increased susceptibility to cerebral malaria in large case-control studies of West African and East African populations, after correction for other known TNF polymorphisms and linked HLA alleles.

Berkley J, Mwarumba S, Bramham K, Lowe B, Marsh K. 1999. Bacteraemia complicating severe malaria in children. Trans R Soc Trop Med Hyg, 93 (3), pp. 283-286. | Show Abstract | Read more

Bacteraemia associated with severe malaria in childhood is a sporadically reported phenomenon but its incidence and clinical importance are unknown. We have reviewed clinical and laboratory data from 783 Kenyan children sequentially admitted with a primary diagnosis of severe malaria. The overall incidence of bacteraemia in children with severe malaria was 7.8% (95% CI 5.5-10.0); however, in children under 30 months of age the incidence was 12.0% (95% CI 8.3-15.7). The presence of bacteraemia was associated with a 3-fold increase in mortality (33.3% vs. 10.4%, P < 0.001). We conclude that invasive bacterial disease may contribute to the pathophysiology of the clinical syndrome of severe malaria in an important subgroup of children. We recommend that young children with severe malaria be treated with broad-spectrum antibiotics in addition to antimalarial drugs.

Marsh VM, Mutemi WM, Muturi J, Haaland A, Watkins WM, Otieno G, Marsh K. 1999. Changing home treatment of childhood fevers by training shop keepers in rural Kenya. Trop Med Int Health, 4 (5), pp. 383-389. | Show Abstract | Read more

BACKGROUND: Malaria control in Africa relies primarily on early effective treatment for clinical disease, but most early treatments for fever occur through self-medication with shop-bought drugs. Lack of information to community members on over-the-counter drug use has led to widespread ineffective treatment of fevers, increased risks of drug toxicity and accelerating drug resistance. We examined the feasibility and measured the likely impact of training shop keepers in rural Africa on community drug use. METHODS: In a rural area of coastal Kenya, we implemented a shop keeper training programme in 23 shops serving a population of approximately 3500, based on formative research within the community. We evaluated the training by measuring changes in the proportions of drug sales where an adequate amount of chloroquine was purchased and in the percentage of home-treated childhood fevers given an adequate amount of chloroquine. The programme was assessed qualitatively in the community following the shop keeper training. RESULTS: The percentage of drug sales for children with fever which included an antimalarial drug rose from 34.3% (95% CI 28.9%-40.1%) before the training to a minimum of 79.3% (95% CI 71.8%-85.3%) after the training. The percentage of antimalarial drug sales where an adequate amount of drug was purchased rose from 31.8% (95% CI 26.6%-37.6%) to a minimum of 82.9% (95% CI 76.3%-87.3%). The percentage of childhood fevers where an adequate dose of chloroquine was given to the child rose from 3.7% (95% CI 1.2%-9.7%) before the training to a minimum of 65.2% (95% CI 57.7%-72.0%) afterwards, which represents an increase in the appropriate use of over-the-counter chloroquine by at least 62% (95% CI 53.7%-69.3%). Shop keepers and community members were strongly supportive of the aims and outcome of the programme. CONCLUSIONS: The large shifts in behaviour observed indicate that the approach of training shop keepers as a channel for information to the community is both feasible and likely to have a significant impact. Whilst some of the impact seen may be attributable to research effects in a relatively small scale pilot study, the magnitude of the changes support further investigation into this approach as a potentially important new strategy in malaria control.

Brooker S, Peshu N, Warn PA, Mosobo M, Guyatt HL, Marsh K, Snow RW. 1999. The epidemiology of hookworm infection and its contribution to anaemia among pre-school children on the Kenyan coast. Trans R Soc Trop Med Hyg, 93 (3), pp. 240-246. | Show Abstract | Read more

Intestinal nematode infections are recognized as a major public health problem, and helminth control is currently being directed towards school-aged children who are known to harbour the heaviest infections and are most likely to suffer from associated morbidity. However, few data are available for the epidemiology of intestinal nematodes in pre-school children in Africa, and the contribution of hookworm infection to the aetiology and severity of anaemia among pre-school children remains poorly understood. This paper investigates the epidemiology of parasitic infections in 460 pre-school children who were part of a larger case-control study of severe malaria in Kilifi on the Kenyan coast. Almost one-third (28.7%) were infected with hookworm, 20.2% with Ascaris lumbricoides and 15.0% with Trichuris trichiura. Infection prevalence of each species rose with age, and the prevalence of heavy infection with hookworm and mean intensity of hookworm were markedly age-dependent. One-third (34.3%) of children had malaria. Overall, 76.3% of children were anaemic (haemoglobin < 110 g/L), with the prevalence decreasing with age. Anaemia was significantly worst in children with heavy hookworm infection (> 200 eggs per gram). This relationship held for all ages, both sexes, and was independent of socioeconomic factors. The application of attributable morbidity methods confirmed the contribution of hookworm infection to anaemia.

Mbogo CN, Kabiru EW, Glass GE, Forster D, Snow RW, Khamala CP, Ouma JH, Githure JI, Marsh K, Beier JC. 1999. Vector-related case-control study of severe malaria in Kilifi District, Kenya. Am J Trop Med Hyg, 60 (5), pp. 781-785. | Show Abstract | Read more

A case-control study examined vector-related and environmental parameters associated with severe malaria in Kilifi District along the coast of Kenya. Over an 11-month period, 119 children identified with severe malaria infections at the Kilifi District Hospital were matched by age with control children who reported to the outpatient clinic with nonsevere infections. Intensive mosquito sampling was done in each of the case-control houses over a four-day period, beginning within a week of index case admission. A total of 109 environmental, demographic, behavioral, and animal husbandry variables were characterized for each household. Vector species (Anopheles gambiae s.l. and An. funestus) were detected in 40.1% and 36.1% of case and control houses, respectively. The relative abundance of vectors in individual houses was stable over the two-week resampling periods (r = 0.9). Both the overall abundance of anopheline mosquitoes (odds ratio [OR] = 1.5) and P. falciparum sporozoite rates (OR = 1.5) were not significantly different between case and control houses. In a matched analysis, 11 of 109 house variables associated significantly with severe malaria were also associated with vector abundance, as determined by chi-square linear trend analysis. Under conditions of year-round, low-level transmission on the coast of Kenya, the risk of severe disease in children is multifactorial and not governed strictly by transmission intensity or environmental heterogeneity affecting vector abundance and distributions. This suggests that current interventions that appear to be achievable only in areas where transmission is already low to moderate should be appropriate. However, such interventions should be monitored so that inappropriate and possibly disastrous control activities can be avoided in Africa.

Berkley JA, Mwangi I, Mellington F, Mwarumba S, Marsh K. 1999. Cerebral malaria versus bacterial meningitis in children with impaired consciousness. QJM, 92 (3), pp. 151-157. | Show Abstract | Read more

Cerebral malaria (CM) and acute bacterial meningitis (ABM) are the two common causes of impaired consciousness in children presenting to hospital in sub-Sahara Africa. Since the clinical features of the two diseases may be very similar, treatment is often guided by the initial laboratory findings. However, no detailed studies have examined the extent to which the laboratory findings in these two diseases may overlap. We reviewed data from 555 children with impaired consciousness admitted to Kilifi District Hospital, Kenya. Strictly defined groups were established based on the malaria slide, cerebrospinal fluid (CSF) leucocyte count and the results of blood and CSF culture and CSF bacterial antigen testing. Our data suggests significant overlap in the initial CSF findings between CM and ABM. The absolute minimum proportions of children with impaired consciousness and malaria parasitaemia who also had definite bacterial meningitis were 4% of all children and 14% of children under 1 year of age. The estimated maximum proportion of all children with impaired consciousness and malaria parasitaemia in whom the diagnosis was dual or unclear was at least 13%. The finding of malaria parasites in the blood of an unconscious child in sub-Saharan Africa is not sufficient to establish a diagnosis of cerebral malaria, and acute bacterial meningitis must be actively excluded in all cases.

Gupta S, Snow RW, Donnelly CA, Marsh K, Newbold C. 1999. Immunity to non-cerebral severe malaria is acquired after one or two infections. Nat Med, 5 (3), pp. 340-343. | Show Abstract | Read more

In areas of stable transmission, clinical immunity to mild malaria is acquired slowly, so it is not usually effective until early adolescence. Life-threatening disease is, however, restricted to a much younger age group, indicating that resistance to the severe clinical consequences of infection is acquired more quickly. Understanding how rapidly immunity develops to severe malaria is essential, as severe malaria should be the primary target of intervention strategies, and predicting the result of interventions that reduce host exposure will require consideration of these dynamics. Severe disease in childhood is less frequent in areas where transmission is the greatest. One explanation for this is that infants experience increased exposure to infection while they are protected from disease, possibly by maternal antibody. They therefore emerge from this period of clinical protection with considerably more immunity than those who experience lower transmission intensities. Here we use this data, assuming a period of clinical protection, to estimate the number of prior infections needed to reduce the risk of severe disease to negligible levels. Contrary to expectations, one or two successful infective bites seem to be all that is necessary across a broad range of transmission intensities.

Shulman CE, Dorman EK, Cutts F, Kawuondo K, Bulmer JN, Peshu N, Marsh K. 1999. Intermittent sulphadoxine-pyrimethamine to prevent severe anaemia secondary to malaria in pregnancy: a randomised placebo-controlled trial. Lancet, 353 (9153), pp. 632-636. | Show Abstract | Read more

BACKGROUND: In areas of endemic transmission, malaria in pregnancy is associated with severe maternal anaemia and low-birthweight babies. We studied the efficacy of intermittent treatment doses of sulphadoxine-pyrimethamine in preventing malaria and severe anaemia in pregnancy in a double-blind placebo-controlled trial among primigravid women living in Kilifi District, Kenya. METHODS: Between January, 1996, and April, 1997, 1264 primigravid women were recruited when they attended for antenatal care, and randomly assigned sulphadoxine-pyrimethamine (640) or placebo (624). Women received one, two, or three doses of study medication depending on the duration of gestation at enrolment. Primary outcome measures were severe anaemia (haemoglobin <8 g/dL) and malaria parasitaemia, assessed at 34 weeks of gestation. Analyses were based on intention to treat among women who had study blood tests at 34 weeks. FINDINGS: 30 (5.3%) of 567 women in the sulphadoxine-pyrimethamine group and 199 (35.3%) of 564 in the placebo group had peripheral parasitaemia (protective efficacy 85% [95% CI 78-90], p<0.0001). 82 (14.5%) and 134 (23.7%) had severe anaemia (protective efficacy 39% [22-52], p<0.0001). Even women who booked late and received only one dose of sulphadoxine-pyrimethamine benefited significantly from the intervention. The effects were seen both in women who owned insecticide-treated bednets and in women who did not. INTERPRETATION: Intermittent presumptive treatment with sulphadoxine-pyrimethamine is an effective, practicable strategy to decrease the risk of severe anaemia in primigravidae living in malarious areas.

Bull PC, Lowe BS, Kortok M, Marsh K. 1999. Antibody recognition of Plasmodium falciparum erythrocyte surface antigens in Kenya: evidence for rare and prevalent variants. Infect Immun, 67 (2), pp. 733-739. | Show Abstract

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is the name given to a family of parasite proteins that are inserted into the infected erythrocyte surface. Studies using agglutination assays have shown previously that PfEMP1 epitopes are extremely diverse. In a study in Kenya, 21 parasite isolates, including nine from children with severe malaria, were tested for agglutination by 33 pairs of plasma, 21 of which were from the corresponding children. Each plasma pair consisted of a sample taken at the time of disease (acute) and one taken 3 weeks later (convalescent). In agreement with previous studies, infection was generally followed by the induction of antibodies specific to the homologous parasite isolate. In addition however, the results show that (i) some isolates were agglutinated very frequently by heterologous plasma; (ii) unexpectedly, these frequently agglutinated isolates tended to be from individuals with severe malaria; (iii) an inverse relationship existed between the agglutination frequency of each parasite isolate in heterologous plasma and the agglutinating antibody repertoire of the homologous child at the time of disease; and (iv) A 3-month-old child apparently still carrying maternal antibodies was infected by a rarely agglutinated isolate. This child's plasma agglutinated all isolates at the time of disease, apart from the homologous isolate. These results support the idea that preexisting anti-PfEMP1 antibodies can select the variants that are expressed during a new infection and may suggest the existence of a dominant subset of PfEMP1 variants.

Lewallen S, Harding SP, Ajewole J, Schulenburg WE, Molyneux ME, Marsh K, Usen S, White NJ, Taylor TE. 1999. A review of the spectrum of clinical ocular fundus findings in P. falciparum malaria in African children with a proposed classification and grading system. Trans R Soc Trop Med Hyg, 93 (6), pp. 619-622. | Show Abstract | Read more

Ocular fundus pathology in Plasmodium falciparum malaria is common and has prognostic significance. We have made a collaborative effort to document the ocular features in several populations. Based on examination of 735 patients in Malawi, Kenya and The Gambia by direct and indirect ophthalmoscopy with dilated pupils, we have determined that the 5 distinct clinical features (in order of frequency) include retinal whitening, haemorrhages, unique vessel abnormalities, papilloedema, and cotton wool spots. Photographs and descriptions of these are presented, along with a proposed grading scheme.

Snow RW, Craig M, Deichmann U, Marsh K. 1999. Estimating mortality, morbidity and disability due to malaria among Africa's non-pregnant population. Bull World Health Organ, 77 (8), pp. 624-640. | Show Abstract

The contribution of malaria to morbidity and mortality among people in Africa has been a subject of academic interest, political advocacy, and speculation. National statistics for much of sub-Saharan Africa have proved to be an unreliable source of disease-specific morbidity and mortality data. Credible estimates of disease-specific burdens are required for setting global and national priorities for health in order to rationalize the use of limited resources and lobby for financial support. We have taken an empirical approach to defining the limits of Plasmodium falciparum transmission across the continent and interpolated the distributions of projected populations in 1995. By combining a review of the literature on malaria in Africa and models of acquired functional immunity, we have estimated the age-structured rates of the fatal, morbid and disabling sequelae following exposure to malaria infection under different epidemiological conditions.

Scott JA, Hall AJ, Hannington A, Edwards R, Mwarumba S, Lowe B, Griffiths D, Crook D, Marsh K. 1998. Serotype distribution and prevalence of resistance to benzylpenicillin in three representative populations of Streptococcus pneumoniae isolates from the coast of Kenya. Clin Infect Dis, 27 (6), pp. 1442-1450. | Show Abstract | Read more

As surveillance data from sub-Saharan Africa are few, three representative populations of Streptococcus pneumoniae isolates were examined in Kenya for serotype distribution and Etest minimum inhibitory concentrations (MICs) of benzylpenicillin: (1) 75 lung aspirate or blood culture isolates from 301 consecutive adult patients with pneumonia, (2) 112 invasive isolates from continuous pediatric inpatient surveillance over 4 years, and (3) 97 nasopharyngeal isolates from systematically selected sick children. The proportions with benzylpenicillin MICs of > or = 0.1 microgram/mL were 0.27, 0.29, and 0.47, respectively. Vaccine-related serotypes accounted for 96% of invasive isolates from children and 90% of those from human immunodeficiency virus (HIV)-seropositive adults. Serotype 1 accounted for 44% of pneumococci from HIV-seronegative patients but only 5% of those from HIV-seropositive patients (P = .0002). Of serotype 1 isolates, 98% were susceptible to benzylpenicillin, but serogroups 13, 14, 19, and 23 were strongly associated with an MIC of > or = 0.1 microgram/mL.

Marsh K. 1998. Malaria disaster in Africa. Lancet, 352 (9132), pp. 924. | Show Abstract | Read more

Chloroquine resistance is spreading across Africa. Trape et al. examined mortality patterns in 3 areas of Senegal over a period of 11 years, during which chloroquine resistance first emerged there. There has been a considerable increase in malaria-specific mortality in all 3 areas, areas which differ in levels of malaria endemicity and health care provision. In each case, the increase began when chloroquine resistance was first noted. The risk of death from malaria in 2 of the areas more than doubled, while in the 3rd area, Mlomp, a moderate transmission area with unusually good health care, the risk of malaria mortality among children under age 5 years rose 8-fold. Trape et al. attribute these increases in mortality exclusively to chloroquine resistance. These data demonstrate that chloroquine resistance is already a problem in West Africa, while other epidemiologists believe that malaria mortality is also on the rise in East Africa. Guidelines are needed on the levels of resistance at which countries should consider a switch to alternate first-line drugs. The data also indicate that case treatment is central to controlling malaria in Africa. Effective, affordable drugs capable of delaying the onset of resistance need to be used particularly where the core of the problem is, in rural communities with minimum access to health facilities.

Snow RW, Peshu N, Forster D, Bomu G, Mitsanze E, Ngumbao E, Chisengwa R, Schellenberg JR, Hayes RJ, Newbold CI, Marsh K. 1998. Environmental and entomological risk factors for the development of clinical malaria among children on the Kenyan coast. Trans R Soc Trop Med Hyg, 92 (4), pp. 381-385. | Show Abstract | Read more

Several malariometric studies have examined the impact on human-vector contact of house construction, demographics, bed net and insect repellent use. However, few studies have documented the significance of these proximate determinants on the risks of clinical disease. We undertook a matched case-control study of the risks of both mild clinical malaria and severe life-threatening malaria according to a range of putative factors which would influence the frequency of child-vector encounters in Kilifi district on the Kenyan coast. Among 394 severe disease cases, 380 age-matched mild disease cases, and their respective location and age-matched community controls, we were unable to demonstrate any statistically significant effect upon disease outcome of house construction, presence of domestic animals, or bed net use. Higher population density within a 250 m radius of the homes conferred significant protection from the risks of developing severe malaria compared to community controls. The risks of developing severe malaria compared to the community controls and the transition from mild to severe disease were statistically significantly lower in those who reported use of mosquito coils, local repellents or aerosol insecticides. We concluded that it is likely that the impact of household features on disease outcome is dependent upon both the density of infecting mosquitoes and acquired immunity within a given locality.

Kyes S, Taylor H, Craig A, Marsh K, Newbold C. 1998. Genomic representation of var gene sequences in Plasmodium falciparum field isolates from different geographic regions (vol 87, pg 235, 1997) MOLECULAR AND BIOCHEMICAL PARASITOLOGY, 93 (1), pp. 159-159.

Crawley J, English M, Waruiru C, Mwangi I, Marsh K. 1998. Abnormal respiratory patterns in childhood cerebral malaria. Trans R Soc Trop Med Hyg, 92 (3), pp. 305-308. | Show Abstract | Read more

Of 295 children with cerebral malaria, 117 (40%) had an abnormal respiratory pattern; 15 children exhibited more than one pattern during their clinical course. Four distinct patterns were seen. (i) Deep breathing (80 children); this was associated with severe metabolic acidosis, and resolved following treatment with intravenous fluids and/or blood. (ii) Hypoventilation with nystagmus and salivation (18 children); simultaneous electroencephalographic recording revealed continuous electrical seizure activity, demonstrating that these children were in subtle status epilepticus; anticonvulsant treatment resulted in return to normal of blood gases and recovery of consciousness. (iii) Hyperventilation with extensor posturing (20 children), which was associated with varying degrees of intracranial hypertension. (iv) Periodic respiration (14 children); all had clinical features suggestive of transtentorial herniation, and died following a respiratory arrest. Abnormal respiratory patterns can alert the clinician to complications of cerebral malaria that require treatment. Recognition of these patterns and rapid initiation of appropriate supportive therapy may help to reduce the high mortality rate of this disease.

Schellenberg JA, Newell JN, Snow RW, Mung'ala V, Marsh K, Smith PG, Hayes RJ. 1998. An analysis of the geographical distribution of severe malaria in children in Kilifi District, Kenya. Int J Epidemiol, 27 (2), pp. 323-329. | Show Abstract | Read more

BACKGROUND: Although malaria is known to be a major cause of child mortality and morbidity throughout sub-Saharan Africa there are few detailed studies of malaria mortality rates and incidence of severe malarial disease in defined communities. We have studied the geographical pattern of admissions to hospital with severe malaria and the stability of this pattern over time in Kilifi District on the Kenyan Coast. METHODS: Over a 2-year period all children under 5 years of age with severe malaria admitted to the district hospital and living in a rural study population of about 50,000 people were identified. Annual censuses were carried out in the study area, and all households were mapped using a hand-held satellite navigation system. The resulting databases were linked using a geographical information system (GIS). RESULTS: Using methods originally developed for the study of the geographical distribution of childhood leukaemia we assessed the spatial pattern of hospital admission rates for severe malaria. As expected, admission rates were significantly higher in children with easier access to the hospital. For example, those living more than 25 km from the hospital had admission rates which were about one-fifth of those for children living within 5 km of the hospital. Those living more than 2.5 km from the nearest road had admission rates that were about half of those for children living within 0.5 km of a road. We also investigated short-term local fluctuations in severe malaria and found evidence of space-time clustering of severe malaria. CONCLUSIONS: Hospital admission rates for severe malaria are higher in households with better access to hospital than in those further away. The finding of space-time clusters of severe malaria suggests that it would be of value to conduct case-control studies of environmental, genetic and human behavioural factors involved in the aetiology of the disease.

Shulman CE, Dorman EK, Talisuna AO, Lowe BS, Nevill C, Snow RW, Jilo H, Peshu N, Bulmer JN, Graham S, Marsh K. 1998. A community randomized controlled trial of insecticide-treated bednets for the prevention of malaria and anaemia among primigravid women on the Kenyan coast. Trop Med Int Health, 3 (3), pp. 197-204. | Show Abstract | Read more

The effectiveness of insecticide-treated bednets (ITBN) in preventing malaria and anaemia among primigravidae living in Kilifi District, Kenya, was assessed by a randomized controlled trial between September 1994 and November 1995. All residents within 28 community clusters received ITBN in July 1993, whilst residents of another 28 clusters served as contemporaneous controls. All resident primigravid women with singleton pregnancies attending antenatal care at Kilifi District Hospital were eligible for recruitment. 503 primigravidae were recruited. 91.4% were anaemic antenatally (Hb < 11 g/dl): 91.0% from the intervention arm and 92.0% from the control arm. Severe anaemia (Hb < 7 g/dl) was found among 15.1% of intervention women and 20.1% of control women (P = 0.28). No significant differences were observed in reports of febrile illness or the presence of chloroquine in the serum or peripheral parasitaemia during the third trimester between the two groups. In the women delivering in hospital (n = 130), there was no association between placental malaria infection and the intervention: 77.4% of placentas from control women had evidence of past or active infection, compared with 72.0% of placentas from intervention women (P = 0.76). Similarly, in the women delivering in hospital, ITBN did not improve birth weight, and there were no differences in perinatal mortality between the two study groups. Despite ITBN having a great impact on paediatric severe malaria and mortality in this transmission setting, there was very little impact of ITBN on the morbidity associated with malaria infection in primigravidae. Alternative strategies are required to tackle this continued public health problem for pregnant women living in endemic areas similar to the Kenyan Coast.

English M, Wale S, Binns G, Mwangi I, Sauerwein H, Marsh K. 1998. Hypoglycaemia on and after admission in Kenyan children with severe malaria. QJM, 91 (3), pp. 191-197. | Show Abstract | Read more

We investigated the pathophysiology of hypoglycaemia in severe malaria in African children, especially the potential importance of glycerol as a substrate for gluconeogenesis, and whether substrate limitation contributes to hypoglycaemia in severe disease. Of 171 children with moderate or severe malaria, 16% were hypoglycaemic on admission, while at least 9% of children with severe malaria treated with quinine and a concurrent 4% dextrose infusion had a definite episode of hypoglycaemia after admission. Blood levels of gluconeogenic precursors are as high (alanine and lactate) or higher (glycerol) in those with either hypoglycaemia on or after admission as they are in children never having an episode of hypoglycaemia. Among children with severe malaria, however, those having a definite episode of hypoglycaemia at some stage are more acidotic and have greater evidence of renal impairment than those who are never hypoglycaemic (mean base excess -14.4 vs. -7.2, p < 0.001, mean creatinine 97 vs. 64, p < 0.001 and mean urea 8.1 vs. 5.8, p = 0.03, respectively). These data do not support a role for reduced gluconeogenic substrate supply in the pathogenesis of hypoglycaemia in severe childhood malaria, but do support the hypothesis that gluconeogenesis is impaired. Commonly-used bedside blood glucose monitoring devices may overestimate blood glucose measurements in the normal range, and paradoxically may also seriously overestimate the frequency of hypoglycaemia.

Bull PC, Lowe BS, Kortok M, Molyneux CS, Newbold CI, Marsh K. 1998. Parasite antigens on the infected red cell surface are targets for naturally acquired immunity to malaria. Nat Med, 4 (3), pp. 358-360. | Show Abstract | Read more

The feasibility of a malaria vaccine is supported by the fact that children in endemic areas develop naturally acquired immunity to disease. Development of disease immunity is characterized by a decrease in the frequency and severity of disease episodes over several years despite almost continuous infection, suggesting that immunity may develop through the acquisition of a repertoire of specific, protective antibodies directed against polymorphic target antigens. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a potentially important family of target antigens, because these proteins are inserted into the red cell surface and are prominently exposed and because they are highly polymorphic and undergo clonal antigenic variation, a mechanism of immune evasion maintained by a large family of var genes. In a large prospective study of Kenyan children, we have used the fact that anti-PfEMP1 antibodies agglutinate infected erythrocytes in a variant-specific manner, to show that the PfEMP1 variants expressed during episodes of clinical malaria were less likely to be recognized by the corresponding child's own preexisting antibody response than by that of children of the same age from the same community. In contrast, a heterologous parasite isolate was just as likely to be recognized. The apparent selective pressure exerted by established anti-PfEMP1 antibodies on infecting parasites supports the idea that such responses provide variant-specific protection against disease.

Haruki K, Winstanley PA, Watkins WM, Marsh K. 1998. Quinine sensitivity of isolates of Plasmodium falciparum from the coast of Kenya. Trans R Soc Trop Med Hyg, 92 (2), pp. 195-196. | Read more

Snow RW, Nahlen B, Palmer A, Donnelly CA, Gupta S, Marsh K. 1998. Risk of severe malaria among African infants: direct evidence of clinical protection during early infancy. J Infect Dis, 177 (3), pp. 819-822. | Show Abstract | Read more

Little empirical evidence from field-based studies exists on the relative magnitude or duration of clinical protection from Plasmodium falciparum malaria in infancy. A prospective study was undertaken to examine the age distribution of hospital admissions in four geographically and demographically well-defined areas with differing intensities of P. falciparum transmission. Where transmission was perennial, significant clinical protection from severe morbidity was observed up to the third month of life; in the seasonal transmission area, disease rates rose after the sixth month of life. Infants exposed to the highest rates of P. falciparum exposure demonstrated significant declines in the risks of severe malaria from 6 months of age. These data provide direct evidence for the very early acquisition of clinical immunity and for the existence of a period of clinical protection, which together may explain why, in these communities, the cumulative risk of malarial disease throughout childhood appears to decline with increasing transmission intensity.

Snow RW, Marsh K. 1998. New insights into the epidemiology of malaria relevant for disease control. Br Med Bull, 54 (2), pp. 293-309. | Show Abstract | Read more

Despite over 100 years of scientific investigation, malaria remains the leading cause of death among children living in sub-Saharan Africa. Our understanding of the epidemiology of clinical malaria has, until recently, been hampered by a paucity of empirical data from endemic settings. A striking feature of Plasmodium falciparum malaria is that, compared to infection and mild disease, severe complications and death are rare. Perhaps the single most important factor which ameliorates the risk of asymptomatic infection progressing to life-threatening pathology is the development of clinical immunity. Examination of recent epidemiological evidence suggests that the speed with which clinical immunity is acquired is dependent upon the frequency of parasite exposure from birth. Consequently, the age at which disease presentation peaks, the clinical spectrum of disease and the life-time risks of disease appear to be a function of the intensity of transmission within a given community. These observations are discussed in relation to control measures aimed at reducing P. falciparum exposure and the need to understand better the processes by which children naturally acquire clinical immunity before more rational statements can be made about their wide-spread use in Africa.

Snow RW, Marsh K. 1998. The epidemiology of clinical malaria among African children. Bull Inst Pasteur, 96 (1), pp. 15-23. | Show Abstract | Read more

There is a resurgence of interest in the clinical epidemiology of malaria among African children. This renewed interest follows fifty years of failure to eradicate infection in Africa and redirected efforts toward disease control and prevention. We have a poor understanding of the mechanisms by which clinical immunity is acquired; however, several recent studies have provided new insights into how fast clinical protection is acquired under the varied transmission intensities common to Africa. What is clear is that the frequency with which individuals encounter infection from birth will determine the speed with which they become clinically immune and the patterns of severe pathology they are likely to experience. There remains doubt and concerns over the long-term consequences of reducing natural parasite exposure in several areas of Africa. New field studies are urgently required to tackle these issues so that control may be guided by an improved understanding of malaria as a disease that can lead to death.

Kwiatkowski D, Marsh K. 1997. Development of a malaria vaccine. Lancet, 350 (9092), pp. 1696-1701. | Show Abstract | Read more

Development of an effective malaria vaccine poses a major scientific challenge both in the laboratory and in the field. Such a vaccine is necessary because of the massive disease burden of malaria in the developing world, the global spread of drug resistance, and the difficulty of sustainable control of the mosquito vector. Animal models have shown the immunological feasibility of vaccines targeted against different stages of parasite development, and studies in human volunteers have shown that a recombinant protein vaccine can protect against challenge with the homologous strain of parasite. However, both natural and vaccine-induced immunity are hampered by the remarkable capacity of the parasites to vary critical antigenic structures; large field trials of a synthetic peptide vaccine gave equivocal results. In an attempt to overcome the dual difficulty of poor immunogenicity and parasite diversity, much experimental work is now focused on complex antigenic constructs, delivered as DNA vaccines or in live vectors such as vaccinia, with multiple targets at each stage of parasite development.

Amukoye E, Winstanley PA, Watkins WM, Snow RW, Hatcher J, Mosobo M, Ngumbao E, Lowe B, Ton M, Minyiri G, Marsh K. 1997. Chlorproguanil-dapsone: effective treatment for uncomplicated falciparum malaria. Antimicrob Agents Chemother, 41 (10), pp. 2261-2264. | Show Abstract

Pyrimethamine-sulfadoxine, the first choice for uncomplicated falciparum malaria in Africa, exerts strong selection pressure for resistance because of its slow elimination. It is likely that resistance will emerge rapidly, and there is no widely affordable replacement. Chlorproguanil-dapsone is cheap, rapidly eliminated, more potent than pyrimethamine-sulfadoxine, and could be introduced in the near future to delay the onset of antifolate resistance and as "salvage therapy" for pyrimethamine-sulfadoxine failure. A total of 448 children were randomly allocated (double blind) to either a single dose of pyrimethamine-sulfadoxine or to one of two chlorproguanil-dapsone regimens: a single dose or three doses at 24-h intervals. Reinfections are clinically indistinguishable from recrudescence and are more likely after treatment with rapidly eliminated drugs; we measured the incidence of parasitemia in 205 initially aparasitemic children to allow comparison with the three treatment groups. The patients and a community surveillance group were followed up for 28 days. At the study end point, 31.2% (95% confidence interval, 24.9-38.0) of the community surveillance group subjects were parasitemic, compared with subjects in the treatment groups, whose rates of parasitemia were 40.8% (32.9-49.0; relative risk [RR], 1.31 [0.99-1.73]) after triple-dose chlorproguanil-dapsone, 19.7% (13.5-27.2; RR, 0.63 [0.43-0.93]) after pyrimethamine-sulfadoxine, and 65.6% (57.5-73.0; RR, 2.10 [1.66-2.65]) after single-dose chlorproguanil-dapsone. Pyrimethamine-sulfadoxine and triple-dose chlorproguanil-dapsone were effective treatments. Pyrimethamine-sulfadoxine provided chemoprophylaxis during follow-up because of its slow elimination. Triple-dose chlorproguanil-dapsone should now be developed in an attempt to reduce the rate of emergence of antifolate resistance in Africa and for affordable salvage therapy in cases of pyrimethamine-sulfadoxine failure.