register interest

Professor Nicholas PJ Day FMedSci FRCP

Research Area: Global Health
Technology Exchange: Mass spectrometry
Scientific Themes: Tropical Medicine & Global Health
Keywords: malaria, melioidosis, Rickettsia, Leptospirosis, influenza and scrub typhus
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Nick Day's particular interests include the epidemiology, pathophysiology and treatment of malaria, melioidosis, leptospirosis, rickettsial infections, Staphylococcus aureus infections, influenza, dengue and other communicable diseases afflicting rural populations throughout Asia and beyond.

Name Department Institution Country
Professor Nicholas J White FRS Tropical Medicine University of Oxford United Kingdom
Professor Adrianus Dondorp Tropical Medicine University of Oxford United Kingdom
Professor François H Nosten Tropical Medicine University of Oxford United Kingdom
Professor Jeremy Farrar Tropical Medicine University of Oxford United Kingdom
Professor Paul Newton Tropical Medicine University of Oxford United Kingdom
Dr Gareth Turner Tropical Medicine University of Oxford United Kingdom
Professor Daniel H Paris Tropical Medicine University of Oxford United Kingdom

Miotto O, Amato R, Ashley EA, MacInnis B, Almagro-Garcia J, Amaratunga C, Lim P, Mead D et al. 2015. Genetic architecture of artemisinin-resistant Plasmodium falciparum. Nat Genet, 47 (3), pp. 226-234. Read abstract | Read more

We report a large multicenter genome-wide association study of Plasmodium falciparum resistance to artemisinin, the frontline antimalarial drug. Across 15 locations in Southeast Asia, we identified at least 20 mutations in kelch13 (PF3D7_1343700) affecting the encoded propeller and BTB/POZ domains, which were associated with a slow parasite clearance rate after treatment with artemisinin derivatives. Nonsynonymous polymorphisms in fd (ferredoxin), arps10 (apicoplast ribosomal protein S10), mdr2 (multidrug resistance protein 2) and crt (chloroquine resistance transporter) also showed strong associations with artemisinin resistance. Analysis of the fine structure of the parasite population showed that the fd, arps10, mdr2 and crt polymorphisms are markers of a genetic background on which kelch13 mutations are particularly likely to arise and that they correlate with the contemporary geographical boundaries and population frequencies of artemisinin resistance. These findings indicate that the risk of new resistance-causing mutations emerging is determined by specific predisposing genetic factors in the underlying parasite population. Hide abstract

Mok S, Ashley EA, Ferreira PE, Zhu L, Lin Z, Yeo T, Chotivanich K, Imwong M et al. 2015. Drug resistance. Population transcriptomics of human malaria parasites reveals the mechanism of artemisinin resistance. Science, 347 (6220), pp. 431-435. Read abstract | Read more

Artemisinin resistance in Plasmodium falciparum threatens global efforts to control and eliminate malaria. Polymorphisms in the kelch domain-carrying protein K13 are associated with artemisinin resistance, but the underlying molecular mechanisms are unknown. We analyzed the in vivo transcriptomes of 1043 P. falciparum isolates from patients with acute malaria and found that artemisinin resistance is associated with increased expression of unfolded protein response (UPR) pathways involving the major PROSC and TRiC chaperone complexes. Artemisinin-resistant parasites also exhibit decelerated progression through the first part of the asexual intraerythrocytic development cycle. These findings suggest that artemisinin-resistant parasites remain in a state of decelerated development at the young ring stage, whereas their up-regulated UPR pathways mitigate protein damage caused by artemisinin. The expression profiles of UPR-related genes also associate with the geographical origin of parasite isolates, further suggesting their role in emerging artemisinin resistance in the Greater Mekong Subregion. Hide abstract

Maude RR, Maude RJ, Ghose A, Amin MR, Islam MB, Ali M, Bari MS, Majumder MI et al. 2014. Serosurveillance of Orientia tsutsugamushi and Rickettsia typhi in Bangladesh. Am J Trop Med Hyg, 91 (3), pp. 580-583. Read abstract | Read more

Scrub and murine typhus infections are under-diagnosed causes of febrile illness across the tropics, and it is not known how common they are in Bangladesh. We conducted a prospective seroepidemiologic survey across six major teaching hospitals in Bangladesh by using an IgM enzyme-linked immunosorbent assay. Results indicated recent exposure (287 of 1,209, 23.7% seropositive for Orientia tsutsugamushi and 805 of 1,209, 66.6% seropositive for Rickettsia typhi). Seropositive rates were different in each region. However, there was no geographic clustering of seropositive results for both organisms. There was no difference between those from rural or urban areas. Rickettsia typhi seroreactivity was positively correlated with age. Scrub typhus and murine typhus should be considered as possible causes of infection in Bangladesh. Hide abstract

Lubell Y, White L, Varadan S, Drake T, Yeung S, Cheah PY, Maude RJ, Dondorp A, Day NP, White NJ, Parker M. 2014. Ethics, economics, and the use of primaquine to reduce falciparum malaria transmission in asymptomatic populations. PLoS Med, 11 (8), pp. e1001704. Read abstract | Read more

Yoel Lubell and colleagues consider ethical and economic perspectives on mass drug administration of primaquine to limit transmission of P. falciparum malaria. Please see later in the article for the Editors' Summary. Hide abstract

Ashley EA, Dhorda M, Fairhurst RM, Amaratunga C, Lim P, Suon S, Sreng S, Anderson JM et al. 2014. Spread of artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med, 371 (5), pp. 411-423. Read abstract | Read more

BACKGROUND: Artemisinin resistance in Plasmodium falciparum has emerged in Southeast Asia and now poses a threat to the control and elimination of malaria. Mapping the geographic extent of resistance is essential for planning containment and elimination strategies. METHODS: Between May 2011 and April 2013, we enrolled 1241 adults and children with acute, uncomplicated falciparum malaria in an open-label trial at 15 sites in 10 countries (7 in Asia and 3 in Africa). Patients received artesunate, administered orally at a daily dose of either 2 mg per kilogram of body weight per day or 4 mg per kilogram, for 3 days, followed by a standard 3-day course of artemisinin-based combination therapy. Parasite counts in peripheral-blood samples were measured every 6 hours, and the parasite clearance half-lives were determined. RESULTS: The median parasite clearance half-lives ranged from 1.9 hours in the Democratic Republic of Congo to 7.0 hours at the Thailand-Cambodia border. Slowly clearing infections (parasite clearance half-life >5 hours), strongly associated with single point mutations in the "propeller" region of the P. falciparum kelch protein gene on chromosome 13 (kelch13), were detected throughout mainland Southeast Asia from southern Vietnam to central Myanmar. The incidence of pretreatment and post-treatment gametocytemia was higher among patients with slow parasite clearance, suggesting greater potential for transmission. In western Cambodia, where artemisinin-based combination therapies are failing, the 6-day course of antimalarial therapy was associated with a cure rate of 97.7% (95% confidence interval, 90.9 to 99.4) at 42 days. CONCLUSIONS: Artemisinin resistance to P. falciparum, which is now prevalent across mainland Southeast Asia, is associated with mutations in kelch13. Prolonged courses of artemisinin-based combination therapies are currently efficacious in areas where standard 3-day treatments are failing. (Funded by the U.K. Department of International Development and others; number, NCT01350856.). Hide abstract

Onyamboko MA, Fanello CI, Wongsaen K, Tarning J, Cheah PY, Tshefu KA, Dondorp AM, Nosten F, White NJ, Day NP. 2014. Randomized comparison of the efficacies and tolerabilities of three artemisinin-based combination treatments for children with acute Plasmodium falciparum malaria in the Democratic Republic of the Congo. Antimicrob Agents Chemother, 58 (9), pp. 5528-5536. Read abstract | Read more

An open-label, randomized controlled trial was carried out in 2011-2012 in the Democratic Republic of the Congo to test the efficacy, safety, and tolerability of the artemisinin-based combination treatments dihydroartemisinin-piperaquine, amodiaquine-artesunate, and artemether-lumefantrine. Six hundred eighty-four children aged 3 to 59 months with uncomplicated Plasmodium falciparum malaria were randomly allocated to each study arm. Children were hospitalized for 3 days, given supervised treatment, and followed up weekly for 42 days. All regimens were well tolerated and rapidly effective. The median parasitemia clearance half-life was 2.2 h, and half-lives were similar between arms (P=0.19). The PCR-uncorrected cure rates by day 42 were 73.0% for amodiaquine-artesunate, 70.2% for artemether-lumefantrine, and 86.3% for dihydroartemisinin-piperaquine (P=0.001). Early treatment failure occurred in three patients (0.5%), one in each arm. The PCR-corrected cure rates were 93.4% for amodiaquine-artesunate, 92.7% for artemether-lumefantrine, and 94.3% for dihydroartemisinin-piperaquine (P=0.78). The last provided a longer posttreatment prophylactic effect than did the other two treatments. The day 7 plasma concentration of piperaquine was below 30 ng/ml in 47% of the children treated with dihydroartemisinin-piperaquine, and the day 7 lumefantrine concentration was below 280 ng/ml in 37.0% of children who received artemether-lumefantrine. Thus, although cure rates were all satisfactory, they could be improved by increasing the dose. (This study has been registered with the International Standard Randomized Controlled Trial Number Register [] under registration no. ISRCTN20984426.). Hide abstract

Chotivanich K, Tripura R, Das D, Yi P, Day NP, Pukrittayakamee S, Chuor CM, Socheat D, Dondorp AM, White NJ. 2014. Laboratory detection of artemisinin-resistant Plasmodium falciparum. Antimicrob Agents Chemother, 58 (6), pp. 3157-3161. Read abstract | Read more

Conventional 48-h in vitro susceptibility tests have low sensitivity in identifying artemisinin-resistant Plasmodium falciparum, defined phenotypically by low in vivo parasite clearance rates. We hypothesized originally that this discrepancy was explained by a loss of ring-stage susceptibility and so developed a simple field-adapted 24-h trophozoite maturation inhibition (TMI) assay focusing on the ring stage and compared it to the standard 48-h schizont maturation inhibition (WHO) test. In Pailin, western Cambodia, where artemisinin-resistant P. falciparum is prevalent, the TMI test mean (95% confidence interval) 50% inhibitory concentration (IC50) for artesunate was 6.8 (5.2 to 8.3) ng/ml compared with 1.5 (1.2 to 1.8) ng/ml for the standard 48-h WHO test (P = 0.001). TMI IC50s correlated significantly with the in vivo responses to artesunate (parasite clearance time [r = 0.44, P = 0.001] and parasite clearance half-life [r = 0.46, P = 0.001]), whereas the standard 48-h test values did not. On continuous culture of two resistant isolates, the artemisinin-resistant phenotype was lost after 6 weeks (IC50s fell from 10 and 12 ng/ml to 2.7 and 3 ng/ml, respectively). Slow parasite clearance in falciparum malaria in western Cambodia results from reduced ring-stage susceptibility. Hide abstract

Tarning J, Lindegardh N, Lwin KM, Annerberg A, Kiricharoen L, Ashley E, White NJ, Nosten F, Day NP. 2014. Population pharmacokinetic assessment of the effect of food on piperaquine bioavailability in patients with uncomplicated malaria. Antimicrob Agents Chemother, 58 (4), pp. 2052-2058. Read abstract | Read more

Previously published literature reports various impacts of food on the oral bioavailability of piperaquine. The aim of this study was to use a population modeling approach to investigate the impact of concomitant intake of a small amount of food on piperaquine pharmacokinetics. This was an open, randomized comparison of piperaquine pharmacokinetics when administered as a fixed oral formulation once daily for 3 days with (n=15) and without (n=15) concomitant food to patients with uncomplicated Plasmodium falciparum malaria in Thailand. Nonlinear mixed-effects modeling was used to characterize the pharmacokinetics of piperaquine and the influence of concomitant food intake. A modified Monte Carlo mapped power approach was applied to evaluate the relationship between statistical power and various degrees of covariate effect sizes of the given study design. Piperaquine population pharmacokinetics were described well in fasting and fed patients by a three-compartment distribution model with flexible absorption. The final model showed a 25% increase in relative bioavailability per dose occasion during recovery from malaria but demonstrated no clinical impact of concomitant intake of a low-fat meal. Body weight and age were both significant covariates in the final model. The novel power approach concluded that the study was adequately powered to detect a food effect of at least 35%. This modified Monte Carlo mapped power approach may be a useful tool for evaluating the power to detect true covariate effects in mixed-effects modeling and a given study design. A small amount of food does not affect piperaquine absorption significantly in acute malaria. Hide abstract

Jittamala P, Pukrittayakamee S, Tarning J, Lindegardh N, Hanpithakpong W, Taylor WR, Lawpoolsri S, Charunwattana P, Panapipat S, White NJ, Day NP. 2014. Pharmacokinetics of orally administered oseltamivir in healthy obese and nonobese Thai subjects. Antimicrob Agents Chemother, 58 (3), pp. 1615-1621. Read abstract | Read more

Oseltamivir is the most widely used anti-influenza drug. In the 2009 H1N1 pandemic, in which the influenza viruses were oseltamivir sensitive, obesity was identified as a risk factor for severe disease and unfavorable outcomes. The aim of this study was to investigate the pharmacokinetic properties of oseltamivir and its active metabolite, oseltamivir carboxylate, in obese and nonobese healthy subjects. A single-dose, randomized, two-sequence crossover study was conducted in 12 obese and 12 nonobese healthy Thai volunteers. Each volunteer was given 75 mg and 150 mg oseltamivir orally with an intervening washout period of more than 3 days. The pharmacokinetic properties of oseltamivir and oseltamivir carboxylate were evaluated using a noncompartmental approach. The median (range) body mass indexes (BMIs) for obese subjects were 33.8 kg/m(2) (30.8 to 43.2) and 22.2 (18.8 to 24.2) for nonobese subjects. The pharmacokinetic parameters of oseltamivir carboxylate, the active metabolite of oseltamivir, were not significantly different between obese and nonobese subjects for both 75-mg and 150-mg doses. Both doses were well tolerated. Despite the lower dose per kilogram body weight in obese subjects, there was no significant difference in the exposure of oseltamivir carboxylate between the obese and nonobese groups. Standard dosing is appropriate for obese subjects. (The study was registered at under registration no. NCT 01049763.). Hide abstract

Chetchotisakd P, Chierakul W, Chaowagul W, Anunnatsiri S, Phimda K, Mootsikapun P, Chaisuksant S, Pilaikul J et al. 2014. Trimethoprim-sulfamethoxazole versus trimethoprim-sulfamethoxazole plus doxycycline as oral eradicative treatment for melioidosis (MERTH): a multicentre, double-blind, non-inferiority, randomised controlled trial. Lancet, 383 (9919), pp. 807-814. Read abstract | Read more

BACKGROUND: Melioidosis, an infectious disease caused by the Gram-negative bacillus Burkholderia pseudomallei, is difficult to cure. Antimicrobial treatment comprises intravenous drugs for at least 10 days, followed by oral drugs for at least 12 weeks. The standard oral regimen based on trial evidence is trimethoprim-sulfamethoxaxole (TMP-SMX) plus doxycycline. This regimen is used in Thailand but is associated with side-effects and poor adherence by patients, and TMP-SMX alone is recommended in Australia. We compared the efficacy and side-effects of TMP-SMX with TMP-SMX plus doxycycline for the oral phase of melioidosis treatment. METHODS: For this multi-centre, double-blind, non-inferiority, randomised placebo-controlled trial, we enrolled patients (aged ≥15 years) from five centres in northeast Thailand with culture-confirmed melioidosis who had received a course of parenteral antimicrobial drugs. Using a computer-generated sequence, we randomly assigned patients to receive TMP-SMX plus placebo or TMP-SMX plus doxycycline for 20 weeks (1:1; block size of ten, stratified by study site). We followed patients up every 4 months for 1 year and annually thereafter to the end of the study. The primary endpoint was culture-confirmed recurrent melioidosis, and the non-inferiority margin was a hazard ratio (HR) of 1.7. This study is registered with, number ISRCTN86140460. FINDINGS: We enrolled and randomly assigned 626 patients: 311 to TMP-SMX plus placebo and 315 to TMP-SMX plus doxycycline. 16 patients (5%) in the TMP-SMX plus placebo group and 21 patients (7%) in the TMP-SMX plus doxycycline group developed culture-confirmed recurrent melioidosis (HR 0.81; 95% CI 0.42-1.55). The criterion for non-inferiority was met (p=0.01). Adverse drug reactions were less common in the TMP-SMX plus placebo group than in the TMP-SMX plus doxycycline group (122 [39%] vs 167 [53%]). INTERPRETATION: Our findings suggest that TMP-SMX is not inferior to TMP-SMX plus doxycycline for the oral phase of melioidosis treatment, and is preferable on the basis of safety and tolerance by patients. FUNDING: Thailand Research Fund, the Melioidosis Research Center, the Center of Excellence in Specific Health Problems in Greater Mekong Sub-region cluster, and the Wellcome Trust. Hide abstract

Miotto O, Almagro-Garcia J, Manske M, Macinnis B, Campino S, Rockett KA, Amaratunga C, Lim P et al. 2013. Multiple populations of artemisinin-resistant Plasmodium falciparum in Cambodia. Nat Genet, 45 (6), pp. 648-655. Read abstract | Read more

We describe an analysis of genome variation in 825 P. falciparum samples from Asia and Africa that identifies an unusual pattern of parasite population structure at the epicenter of artemisinin resistance in western Cambodia. Within this relatively small geographic area, we have discovered several distinct but apparently sympatric parasite subpopulations with extremely high levels of genetic differentiation. Of particular interest are three subpopulations, all associated with clinical resistance to artemisinin, which have skewed allele frequency spectra and high levels of haplotype homozygosity, indicative of founder effects and recent population expansion. We provide a catalog of SNPs that show high levels of differentiation in the artemisinin-resistant subpopulations, including codon variants in transporter proteins and DNA mismatch repair proteins. These data provide a population-level genetic framework for investigating the biological origins of artemisinin resistance and for defining molecular markers to assist in its elimination. Hide abstract

Day NPJ. 2013. Panton-Valentine leucocidin and staphylococcal disease The Lancet Infectious Diseases, 13 (1), pp. 5-6. | Read more

Blacksell S, Richards A, Paris DH, Tanganuchitcharnchai A, Day N. 2012. Validation of a semi-quantitative IgM ELISA for the detection of Orientia tsutsugamushi IgM antibodies for use in seroepidemiology studies in Thailand INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES, 16 pp. E390-E390. | Read more

Phyo AP, Nkhoma S, Stepniewska K, Ashley EA, Nair S, McGready R, ler Moo C, Al-Saai S et al. 2012. Emergence of artemisinin-resistant malaria on the western border of Thailand: a longitudinal study. Lancet, 379 (9830), pp. 1960-1966. Read abstract | Read more

BACKGROUND: Artemisinin-resistant falciparum malaria has arisen in western Cambodia. A concerted international effort is underway to contain artemisinin-resistant Plasmodium falciparum, but containment strategies are dependent on whether resistance has emerged elsewhere. We aimed to establish whether artemisinin resistance has spread or emerged on the Thailand-Myanmar (Burma) border. METHODS: In malaria clinics located along the northwestern border of Thailand, we measured six hourly parasite counts in patients with uncomplicated hyperparasitaemic falciparum malaria (≥4% infected red blood cells) who had been given various oral artesunate-containing regimens since 2001. Parasite clearance half-lives were estimated and parasites were genotyped for 93 single nucleotide polymorphisms. FINDINGS: 3202 patients were studied between 2001 and 2010. Parasite clearance half-lives lengthened from a geometric mean of 2·6 h (95% CI 2·5-2·7) in 2001, to 3·7 h (3·6-3·8) in 2010, compared with a mean of 5·5 h (5·2-5·9) in 119 patients in western Cambodia measured between 2007 and 2010. The proportion of slow-clearing infections (half-life ≥6·2 h) increased from 0·6% in 2001, to 20% in 2010, compared with 42% in western Cambodia between 2007 and 2010. Of 1583 infections genotyped, 148 multilocus parasite genotypes were identified, each of which infected between two and 13 patients. The proportion of variation in parasite clearance attributable to parasite genetics increased from 30% between 2001 and 2004, to 66% between 2007 and 2010. INTERPRETATION: Genetically determined artemisinin resistance in P falciparum emerged along the Thailand-Myanmar border at least 8 years ago and has since increased substantially. At this rate of increase, resistance will reach rates reported in western Cambodia in 2-6 years. FUNDING: The Wellcome Trust and National Institutes of Health. Hide abstract

Blacksell SD, Paris DH, Chierakul W, Wuthiekanun V, Teeratakul A, Kantipong P, Day NP. 2012. Prospective evaluation of commercial antibody-based rapid tests in combination with a loop-mediated isothermal amplification PCR assay for detection of Orientia tsutsugamushi during the acute phase of scrub typhus infection. Clin Vaccine Immunol, 19 (3), pp. 391-395. Read abstract | Read more

Samples from 160 prospectively recruited febrile patients with typhus-like illness in an area of Thailand (Chiang Rai, northern Thailand) where scrub typhus is endemic were used to evaluate the diagnostic capabilities of four rapid immunochromatographic tests (ICTs) for the detection of Orientia tsutsugamushi IgM and total antibodies during acute scrub typhus infection. Of the 160 cases, 54 (34%) had been confirmed to have scrub typhus using the reference scrub typhus infection criteria (STIC), i.e., positive cell culture isolation, an admission IgM antibody titer of ≥1:12,800, a 4-fold rising IgM antibody titer, and/or positivity for ≥2 out of 3 PCR gene targets). The ICTs gave the following sensitivities and specificities: the Panbio IgM ICT, 46% (95% confidence interval [CI], 33 to 60) and 95% (95% CI, 89 to 98), respectively; the Standard Diagnostics IgM ICT, 68% (95% CI, 60 to 75) and 73% (95% CI, 68 to 78), respectively; the AccessBio IgM ICT, 56% (95% CI, 48 to 63) and 90% (95% CI, 87 to 94), respectively; and the AccessBio total antibody ABt ICT, 61% (95% CI, 53 to 68) and 68% (95% CI, 63 to 73), respectively. An isothermal loop amplification (LAMP) PCR assay for scrub typhus demonstrated a sensitivity of 52% (95% CI, 38 to 66) and a specificity of 94% (95% CI, 88 to 98). This study has revealed the diagnostic limitations of antibody-based assays in an acute care setting. However, the combination of ICTs with LAMP usually increased sensitivity with a minimal reduction in specificity. The best combination, the Panbio IgM ICT and LAMP, resulted in a sensitivity of 67% (95% CI, 53 to 79) and a specificity of 91% (95% CI, 83 to 95). The combination of antibody-based assays with DNA- or antigen-based tests shows promise for improved diagnostic sensitivity. Hide abstract

White LJ, Newton PN, Maude RJ, Pan-ngum W, Fried JR, Mayxay M, Maude RR, Day NP. 2012. Defining disease heterogeneity to guide the empirical treatment of febrile illness in resource poor settings. PLoS One, 7 (9), pp. e44545. Read abstract | Read more

BACKGROUND: Malaria incidence is in decline in many parts of SE Asia leading to a decreasing proportion of febrile illness that is attributable to malaria. However in the absence of rapid, affordable and accurate diagnostic tests, the non-malaria causes of these illnesses cannot be reliably identified. Studies on the aetiology of febrile illness have indicated that the causes are likely to vary by geographical location within countries (i.e. be spatially heterogeneous) and that national empirical treatment policies based on the aetiology measured in a single location could lead to inappropriate treatment. METHODS: Using data from Vientiane as a reference for the incidence of major febrile illnesses in the Lao People's Democratic Republic (Laos) and estimated incidences, plausible incidence in other Lao provinces were generated using a mathematical model for a range of national and local scale variations. For a range of treatment protocols, the mean number of appropriate treatments was predicted and the potential impact of a spatially explicit national empirical treatment protocol assessed. FINDINGS: The model predicted a negative correlation between number of appropriate treatments and the level of spatial heterogeneity. A spatially explicit national treatment protocol was predicted to increase the number of appropriate treatments by 50% for intermediate levels of spatial heterogeneity. CONCLUSIONS: The results suggest that given even only moderate spatial variation, a spatially explicit treatment algorithm will result in a significant improvement in the outcome of undifferentiated fevers in Laos and other similar resource poor settings. Hide abstract

Paris DH, Chansamouth V, Nawtaisong P, Löwenberg EC, Phetsouvanh R, Blacksell SD, Lee SJ, Dondorp AM et al. 2012. Coagulation and inflammation in scrub typhus and murine typhus--a prospective comparative study from Laos. Clin Microbiol Infect, 18 (12), pp. 1221-1228. Read abstract | Read more

Scrub typhus (caused by Orientia tsutsugamushi) and murine typhus (caused by Rickettsia typhi) cause up to 28% of febrile episodes in Thailand and Laos. The current understanding of coagulation and inflammation in the pathogenesis of these clinically very similar vasculotropic diseases is limited. This study compared human in vivo changes in 15 coagulation, inflammation and endothelial activation markers in prospectively collected admission and follow-up samples of 121 patients (55 scrub typhus, 55 murine typhus, and 11 typhus-like illness) and 51 healthy controls from Laos. As compared with controls, all but one of the markers assessed were significantly affected in typhus patients; however, the activation patterns differed significantly between scrub and murine typhus patients. The levels of markers of coagulation activation and all inflammatory cytokines, except for interleukin-12, were significantly higher in patients with scrub typhus than in those with murine typhus. In patients with murine typhus, however, the levels of endothelium-derived markers were significantly higher. Anticoagulant factors were inhibited in both typhus patient groups. This is the first study demonstrating that, in scrub typhus, in vivo coagulation activation is prominent and is related to a strong proinflammatory response, whereas in murine typhus, changes in coagulant and fibrinolytic pathways are suggestive of endothelial cell perturbation. These data suggest that, although late-stage endothelial infection is common in both diseases, the in vivo pathogenic mechanisms of R. typhi and O. tsutsugamushi could differ in the early phase of infection and may contribute to disease differentiation. Hide abstract

Blacksell SD, Jarman RG, Bailey MS, Tanganuchitcharnchai A, Jenjaroen K, Gibbons RV, Paris DH, Premaratna R, de Silva HJ, Lalloo DG, Day NP. 2011. Evaluation of six commercial point-of-care tests for diagnosis of acute dengue infections: the need for combining NS1 antigen and IgM/IgG antibody detection to achieve acceptable levels of accuracy. Clin Vaccine Immunol, 18 (12), pp. 2095-2101. Read abstract | Read more

Six assays were evaluated in this study to determine their suitability for the diagnosis of acute dengue infection using samples from 259 Sri Lankan patients with acute fevers (99 confirmed dengue cases and 160 patients with other confirmed acute febrile illnesses): (i) the Merlin dengue fever IgG & IgM combo device (Merlin), (ii) the Standard Diagnostics Dengue Duo nonstructural 1 (NS1) antigen and IgG/IgM combo device (Standard Diagnostics, South Korea), (iii) the Biosynex Immunoquick dengue fever IgG and IgM (Biosynex, France) assay, (iv) the Bio-Rad NS1 antigen strip (Bio-Rad, France), (v) the Panbio Dengue Duo IgG/IgM Cassette (Inverness, Australia), and (vi) the Panbio dengue NS1 antigen strip (Inverness, Australia). The median number of days of fever prior to admission sample collection was 5 days (interquartile range, 3 to 7 days). Sensitivity and specificity of the NS1 antigen tests ranged from 49 to 59% and from 93 to 99%, respectively, and sensitivity and sensitivity of the IgM antibody test ranged from 71 to 80% and from 46 to 90%, respectively. Combining the NS1 antigen and IgM antibody results from the Standard Diagnostics Dengue Duo test gave the best compromise of sensitivity and specificity (93% and 89%, respectively) and provided the best sensitivity in patients presenting at different times after fever onset. The Merlin IgM/IgG antibody tests correctly classified 64% and 86% of the primary and secondary dengue infection cases, respectively, and the Standard Diagnostics IgM/IgG antibody tests correctly classified 71% and 83% of the primary and secondary dengue infection cases, respectively. This study provides strong evidence of the value of combining dengue antigen- and antibody-based test results in the rapid diagnostic test (RDT) format for the acute diagnosis of dengue. Hide abstract

Paris DH, Blacksell SD, Nawtaisong P, Jenjaroen K, Teeraratkul A, Chierakul W, Wuthiekanun V, Kantipong P, Day NP. 2011. Diagnostic accuracy of a loop-mediated isothermal PCR assay for detection of Orientia tsutsugamushi during acute Scrub Typhus infection. PLoS Negl Trop Dis, 5 (9), pp. e1307. Read abstract | Read more

BACKGROUND: There is an urgent need to develop rapid and accurate point-of-care (POC) technologies for acute scrub typhus diagnosis in low-resource, primary health care settings to guide clinical therapy. METHODOLOGY/PRINCIPAL FINDINGS: In this study we present the clinical evaluation of loop-mediated isothermal PCR assay (LAMP) in the context of a prospective fever study, including 161 patients from scrub typhus-endemic Chiang Rai, northern Thailand. A robust reference comparator set comprising following 'scrub typhus infection criteria' (STIC) was used: a) positive cell culture isolate and/or b) an admission IgM titer ≥1∶12,800 using the 'gold standard' indirect immunofluorescence assay (IFA) and/or c) a 4-fold rising IFA IgM titer and/or d) a positive result in at least two out of three PCR assays. Compared to the STIC criteria, all PCR assays (including LAMP) demonstrated high specificity ranging from 96-99%, with sensitivities varying from 40% to 56%, similar to the antibody based rapid test, which had a sensitivity of 47% and a specificity of 95%. CONCLUSIONS/SIGNIFICANCE: The diagnostic accuracy of the LAMP assay was similar to realtime and nested conventional PCR assays, but superior to the antibody-based rapid test in the early disease course. The combination of DNA- and antibody-based detection methods increased sensitivity with minimal reduction of specificity, and expanded the timeframe of adequate diagnostic coverage throughout the acute phase of scrub typhus. Hide abstract

Lindegardh N, Hanpithakpong W, Kamanikom B, Pattayaso J, Singhasivanon P, White NJ, Day NP. 2011. Quantification of dihydroartemisinin, artesunate and artemisinin in human blood: overcoming the technical challenge of protecting the peroxide bridge. Bioanalysis, 3 (14), pp. 1613-1624. Read abstract | Read more

BACKGROUND: Quantification of artemisinin (ARN) and its derivatives in whole blood has hitherto been thought impossible. RESULTS: A LC-MS/MS method for the analysis of artesunate (ARS), its metabolite dihydroartemisinin (DHA) and artemisinin in human whole blood has been developed and successfully validated. The method includes stabilization of the blood matrix at the time of collection and at the time of analysis. Addition of potassium dichromate to the blood samples deactivated the Fe(2+) core in hemoglobin, while deferoxamine chelated Fe(3+) and prevented back conversion into Fe(2+). A pilot study showed that the blood:plasma ratio for ARS and DHA is approximately 0.75, indicating a significantly lower uptake in red blood cells than had previously been estimated using radiolabeled drug methodology. CONCLUSIONS: The developed LC-MS/MS assay is the first method available for quantification of ARN and its derivatives in blood and opens up new possibilities of studying these drugs inside infected red blood cells. Hide abstract

Nickerson EK, Wuthiekanun V, Kumar V, Amornchai P, Wongdeethai N, Chheng K, Chantratita N, Putchhat H, Thaipadungpanit J, Day NP, Peacock SJ. 2011. Emergence of community-associated methicillin-resistant Staphylococcus aureus carriage in children in Cambodia. Am J Trop Med Hyg, 84 (2), pp. 313-317. Read abstract | Read more

We previously described the first reported isolation of methicillin-resistant Staphylococcus aureus (MRSA) (a case series of pediatric community-associated MRSA infections) in Cambodia. We define the rate of pediatric MRSA carriage in the same population and characterize the associated bacterial genotypes by using pulsed-field gel electrophoresis and multilocus sequence typing. A prospective cohort study of MRSA carriage conducted over one month at the Angkor Hospital for Children, Siem Reap, Cambodia, identified MRSA carriage in 87 (3.5%) of 2,485 children who came to the outpatient department, and 6 (4.1%) of 145 inpatients, including at least two with cases of nosocomial acquisition. Genotyping of all 93 MRSA isolates resolved 5 genotypes. Most (91%) isolates were assigned to sequence type 834. Only 28 (32%) of 87 MRSA carriers identified in the outpatient department had no history of recent healthcare contact. The study findings have important implications for healthcare in a setting where diagnostic microbiology and access to antimicrobial drugs with efficacy against MRSA are limited. Hide abstract

Nguyen HP, Hanson J, Bethell D, Nguyen TH, Tran TH, Ly VC, Pham PL, Sinh DX et al. 2011. A retrospective analysis of the haemodynamic and metabolic effects of fluid resuscitation in vietnamese adults with severe falciparum malaria PLoS ONE, 6 (10), Read abstract | Read more

Background: Optimising the fluid resuscitation of patients with severe malaria is a simple and potentially cost-effective intervention. Current WHO guidelines recommend central venous pressure (CVP) guided, crystalloid based, resuscitation in adults. Methods: Prospectively collected haemodynamic data from intervention trials in Vietnamese adults with severe malaria were analysed retrospectively to assess the responses to fluid resuscitation. Results: 43 patients were studied of whom 24 received a fluid load. The fluid load resulted in an increase in cardiac index (mean increase: 0.75 L/min/m2 (95% Confidence interval (CI): 0.41 to 1.1)), but no significant change in acid-base status post resuscitation (mean increase base deficit 0.6 mmol/L (95% CI: -0.1 to 1.3). The CVP and PAoP (pulmonary artery occlusion pressure) were highly inter-correlated (rs = 0.7, p&0.0001), but neither were correlated with acid-base status (arterial pH, serum bicarbonate, base deficit) or respiratory status (PaO2/FiO2 ratio). There was no correlation between the oxygen delivery (DO2) and base deficit at the 63 time-points where they were assessed simultaneously (rs=-0.09, p=0.46). Conclusions: In adults with severe falciparum malaria there was no observed improvement in patient outcomes or acid-base status with fluid loading. Neither CVP nor PAoP correlated with markers of end-organ perfusion or respiratory status, suggesting these measures are poor predictors of their fluid resuscitation needs. © 2011 Phu et al. Hide abstract

Medana IM, Day NP, Sachanonta N, Mai NT, Dondorp AM, Pongponratn E, Hien TT, White NJ, Turner GD. 2011. Coma in fatal adult human malaria is not caused by cerebral oedema. Malar J, 10 (1), pp. 267. Read abstract | Read more

BACKGROUND: The role of brain oedema in the pathophysiology of cerebral malaria is controversial. Coma associated with severe Plasmodium falciparum malaria is multifactorial, but associated with histological evidence of parasitized erythrocyte sequestration and resultant microvascular congestion in cerebral vessels. To determine whether these changes cause breakdown of the blood-brain barrier and resultant perivascular or parenchymal cerebral oedema, histology, immunohistochemistry and image analysis were used to define the prevalence of histological patterns of oedema and the expression of specific molecular pathways involved in water balance in the brain in adults with fatal falciparum malaria. METHODS: The brains of 20 adult Vietnamese patients who died of severe malaria were examined for evidence of disrupted vascular integrity. Immunohistochemistry and image analysis was performed on brainstem sections for activation of the vascular endothelial growth factor (VEGF) receptor 2 and expression of the aquaporin 4 (AQP4) water channel protein. Fibrinogen immunostaining was assessed as evidence of blood-brain barrier leakage and perivascular oedema formation. Correlations were performed with clinical, biochemical and neuropathological parameters of severe malaria infection. RESULTS: The presence of oedema, plasma protein leakage and evidence of VEGF signalling were heterogeneous in fatal falciparum malaria and did not correlate with pre-mortem coma. Differences in vascular integrity were observed between brain regions with the greatest prevalence of disruption in the brainstem, compared to the cortex or midbrain. There was a statistically non-significant trend towards higher AQP4 staining in the brainstem of cases that presented with coma (P = .02). CONCLUSIONS: Histological evidence of cerebral oedema or immunohistochemical evidence of localised loss of vascular integrity did not correlate with the occurrence of pre-mortem coma in adults with fatal falciparum malaria. Enhanced expression of AQP4 water channels in the brainstem may, therefore, reflect a mix of both neuropathological or attempted neuroprotective responses to oedema formation. Hide abstract

Lindegardh N, Hanpithakpong W, Kamanikom B, Farrar J, Hien TT, Singhasivanon P, White NJ, Day NP. 2011. Quantification of the anti-influenza drug zanamivir in plasma using high-throughput HILIC-MS/MS. Bioanalysis, 3 (2), pp. 157-165. Read abstract | Read more

BACKGROUND: parenteral zanamivir is a promising drug for the treatment of severe influenza. However, quantification of this polar drug in biological matrices has traditionally been difficult and the methods developed have been relatively insensitive. RESULTS: a high-throughput bioanalytical method for the analysis of zanamivir in human plasma using SPE in the 96-well plate format and LC coupled to positive MS/MS has been developed and validated according to US FDA guidelines. The method uses 50 microl of plasma and covers a large working range from 1-50, 000 ng/ml with a LOD of 0.50 ng/ml. CONCLUSION: this new LC-MS/MS assay is more sensitive than previous methods despite using a small plasma volume sample. It is particularly suitable for clinical studies on both parenteral and inhaled zanamivir. Hide abstract

Saralamba S, Pan-Ngum W, Maude RJ, Lee SJ, Tarning J, Lindegårdh N, Chotivanich K, Nosten F et al. 2011. Intrahost modeling of artemisinin resistance in Plasmodium falciparum. Proc Natl Acad Sci U S A, 108 (1), pp. 397-402. Read abstract | Read more

Artemisinin-resistant Plasmodium falciparum malaria has emerged in western Cambodia. Resistance is characterized by prolonged in vivo parasite clearance times (PCTs) following artesunate treatment. The biological basis is unclear. The hypothesis that delayed parasite clearance results from a stage-specific reduction in artemisinin sensitivity of the circulating young asexual parasite ring stages was examined. A mathematical model was developed, describing the intrahost parasite stage-specific pharmacokinetic-pharmacodynamic relationships. Model parameters were estimated using detailed pharmacokinetic and parasite clearance data from 39 patients with uncomplicated falciparum malaria treated with artesunate from Pailin (western Cambodia) where artemisinin resistance was evident and 40 patients from Wang Pha (northwestern Thailand) where efficacy was preserved. The mathematical model reproduced the observed parasite clearance for each patient with an accurate goodness of fit (rmsd: 0.03-0.67 in log(10) scale). The parameter sets that provided the best fits with the observed in vivo data consist of a highly conserved concentration-effect relationship for the trophozoite and schizont parasite stages, but a variable relationship for the ring stages. The model-derived assessment suggests that the efficacy of artesunate on ring stage parasites is reduced significantly in Pailin. This result supports the hypothesis that artemisinin resistance mainly reflects reduced ring-stage susceptibility and predicts that doubling the frequency of dosing will accelerate clearance of artemisinin-resistant parasites. Hide abstract

Dondorp AM, Fanello CI, Hendriksen IC, Gomes E, Seni A, Chhaganlal KD, Bojang K, Olaosebikan R et al. 2010. Artesunate versus quinine in the treatment of severe falciparum malaria in African children (AQUAMAT): an open-label, randomised trial. Lancet, 376 (9753), pp. 1647-1657. Read abstract | Read more

BACKGROUND: Severe malaria is a major cause of childhood death and often the main reason for paediatric hospital admission in sub-Saharan Africa. Quinine is still the established treatment of choice, although evidence from Asia suggests that artesunate is associated with a lower mortality. We compared parenteral treatment with either artesunate or quinine in African children with severe malaria. METHODS: This open-label, randomised trial was undertaken in 11 centres in nine African countries. Children (<15 years) with severe falciparum malaria were randomly assigned to parenteral artesunate or parenteral quinine. Randomisation was in blocks of 20, with study numbers corresponding to treatment allocations kept inside opaque sealed paper envelopes. The trial was open label at each site, and none of the investigators or trialists, apart from for the trial statistician, had access to the summaries of treatment allocations. The primary outcome measure was in-hospital mortality, analysed by intention to treat. This trial is registered, number ISRCTN50258054. FINDINGS: 5425 children were enrolled; 2712 were assigned to artesunate and 2713 to quinine. All patients were analysed for the primary outcome. 230 (8·5%) patients assigned to artesunate treatment died compared with 297 (10·9%) assigned to quinine treatment (odds ratio [OR] stratified for study site 0·75, 95% CI 0·63-0·90; relative reduction 22·5%, 95% CI 8·1-36·9; p=0·0022). Incidence of neurological sequelae did not differ significantly between groups, but the development of coma (65/1832 [3·5%] with artesunate vs 91/1768 [5·1%] with quinine; OR 0·69 95% CI 0·49-0·95; p=0·0231), convulsions (224/2712 [8·3%] vs 273/2713 [10·1%]; OR 0·80, 0·66-0·97; p=0·0199), and deterioration of the coma score (166/2712 [6·1%] vs 208/2713 [7·7%]; OR 0·78, 0·64-0·97; p=0·0245) were all significantly less frequent in artesunate recipients than in quinine recipients. Post-treatment hypoglycaemia was also less frequent in patients assigned to artesunate than in those assigned to quinine (48/2712 [1·8%] vs 75/2713 [2·8%]; OR 0·63, 0·43-0·91; p=0·0134). Artesunate was well tolerated, with no serious drug-related adverse effects. INTERPRETATION: Artesunate substantially reduces mortality in African children with severe malaria. These data, together with a meta-analysis of all trials comparing artesunate and quinine, strongly suggest that parenteral artesunate should replace quinine as the treatment of choice for severe falciparum malaria worldwide. FUNDING: The Wellcome Trust. Hide abstract

Sonthayanon P, Peacock SJ, Chierakul W, Wuthiekanun V, Blacksell SD, Holden MT, Bentley SD, Feil EJ, Day NP. 2010. High rates of homologous recombination in the mite endosymbiont and opportunistic human pathogen Orientia tsutsugamushi. PLoS Negl Trop Dis, 4 (7), pp. e752. Read abstract | Read more

Orientia tsutsugamushi is an intracellular alpha-proteobacterium which resides in trombiculid mites, and is the causative agent of scrub typhus in East Asia. The genome sequence of this species has revealed an unprecedented number of repeat sequences, most notably of the genes encoding the conjugative properties of a type IV secretion system (T4SS). Although this observation is consistent with frequent intragenomic recombination, the extent of homologous recombination (gene conversion) in this species is unknown. To address this question, and to provide a protocol for the epidemiological surveillance of this important pathogen, we have developed a multilocus sequence typing (MLST) scheme based on 7 housekeeping genes (gpsA, mdh, nrdB, nuoF, ppdK, sucD, sucB). We applied this scheme to the two published genomes, and to DNA extracted from blood taken from 84 Thai scrub typhus patients, from 20 cultured Thai patient isolates, 1 Australian patient sample, and from 3 cultured type strains. These data demonstrated that the O. tsutsugamushi population was both highly diverse [Simpson's index (95% CI) = 0.95 (0.92-0.98)], and highly recombinogenic. These results are surprising given the intracellular life-style of this species, but are broadly consistent with results obtained for Wolbachia, which is an alpha-proteobacterial reproductive parasite of arthropods. We also compared the MLST data with ompA sequence data and noted low levels of consistency and much higher discrimination by MLST. Finally, twenty-five percent of patients in this study were simultaneously infected with multiple sequence types, suggesting multiple infection caused by either multiple mite bites, or multiple strains co-existing within individual mites. Hide abstract

Pagnarith Y, Kumar V, Thaipadungpanit J, Wuthiekanun V, Amornchai P, Sin L, Day NP, Peacock SJ. 2010. Emergence of pediatric melioidosis in Siem Reap, Cambodia. Am J Trop Med Hyg, 82 (6), pp. 1106-1112. Read abstract | Read more

We describe the first cases of pediatric melioidosis in Cambodia. Thirty-nine cases were diagnosed at the Angkor Hospital for Children, Siem Reap, between October 2005 and December 2008 after the introduction of microbiology capabilities. Median age was 7.8 years (range = 1.6-16.2 years), 15 cases were male (38%), and 4 cases had pre-existing conditions that may have pre-disposed the patient to melioidosis. Infection was localized in 27 cases (69%) and disseminated in 12 cases (31%). Eleven cases (28%) were treated as outpatients, and 28 (72%) cases were admitted. Eight children (21%) died a median of 2 days after admission; seven deaths were attributable to melioidosis, all of which occurred in children receiving suboptimal antimicrobial therapy and before bacteriological culture results were available. Our findings indicate the need for heightened awareness of melioidosis in Cambodia, and they have led us to review microbiology procedures and antimicrobial prescribing of suspected and confirmed cases. Hide abstract

Anderson TJ, Nair S, Nkhoma S, Williams JT, Imwong M, Yi P, Socheat D, Das D et al. 2010. High heritability of malaria parasite clearance rate indicates a genetic basis for artemisinin resistance in western Cambodia. J Infect Dis, 201 (9), pp. 1326-1330. Read abstract | Read more

In western Cambodia, malaria parasites clear slowly from the blood after treatment with artemisinin derivatives, but it is unclear whether this results from parasite, host, or other factors specific to this population. We measured heritability of clearance rate by evaluating patients infected with identical or nonidentical parasite genotypes, using methods analogous to human twin studies. A substantial proportion (56%-58%) of the variation in clearance rate is explained by parasite genetics. This has 2 important implications: (1) selection with artemisinin derivatives will tend to drive resistance spread and (2) because heritability is high, the genes underlying parasite clearance rate may be identified by genome-wide association. Hide abstract

Phu NH, Tuan PQ, Day N, Mai NT, Chau TT, Chuong LV, Sinh DX, White NJ, Farrar J, Hien TT. 2010. Randomized controlled trial of artesunate or artemether in Vietnamese adults with severe falciparum malaria. Malar J, 9 (1), pp. 97. Read abstract | Read more

BACKGROUND: Both artemether and artesunate have been shown to be superior to quinine for the treatment of severe falciparum malaria in Southeast Asian adults, although the magnitude of the superiority has been greater for artesunate than artemether. These two artemisinin derivatives had not been compared in a randomized trial. METHODS: A randomized double blind trial in 370 adults with severe falciparum malaria; 186 received intramuscular artesunate (2.4 mg/kg immediately followed by 1.2 mg/kg at 12 hours then 24 hours then daily) and 184 received intramuscular artemether (3.6 mg per kilogram immediately followed by 1.8 mg per kilogram daily) was conducted in Viet Nam. Both drugs were given for a minimum of 72 hours. RESULTS: There were 13 deaths in the artesunate group (7 percent) and 24 in the artemether group (13 percent); P = 0.052; relative risk of death in the patients given artesunate, 0.54; (95 percent confidence interval 0.28-1.02). Parasitaemia declined more rapidly in the artesunate group. Both drugs were very well tolerated. CONCLUSIONS: Intramuscular artesunate may be superior to intramuscular artemether for the treatment of severe malaria in adults. Hide abstract

Dondorp AM, Yeung S, White L, Nguon C, Day NP, Socheat D, von Seidlein L. 2010. Artemisinin resistance: current status and scenarios for containment. Nat Rev Microbiol, 8 (4), pp. 272-280. Read abstract | Read more

Artemisinin combination therapies are the first-line treatments for uncomplicated Plasmodium falciparum malaria in most malaria-endemic countries. Recently, partial artemisinin-resistant P. falciparum malaria has emerged on the Cambodia-Thailand border. Exposure of the parasite population to artemisinin monotherapies in subtherapeutic doses for over 30 years, and the availability of substandard artemisinins, have probably been the main driving force in the selection of the resistant phenotype in the region. A multifaceted containment programme has recently been launched, including early diagnosis and appropriate treatment, decreasing drug pressure, optimising vector control, targeting the mobile population, strengthening management and surveillance systems, and operational research. Mathematical modelling can be a useful tool to evaluate possible strategies for containment. Hide abstract

Suputtamongkol Y, Pongtavornpinyo W, Lubell Y, Suttinont C, Hoontrakul S, Phimda K, Losuwanaluk K, Suwancharoen D, Silpasakorn S, Chierakul W, Day N. 2010. Strategies for diagnosis and treatment of suspected leptospirosis: a cost-benefit analysis. PLoS Negl Trop Dis, 4 (2), pp. e610. Read abstract | Read more

BACKGROUND: Symptoms and signs of leptospirosis are non-specific. Several diagnostic tests for leptospirosis are available and in some instances are being used prior to treatment of leptospirosis-suspected patients. There is therefore a need to evaluate the cost-effectiveness of the different treatment strategies in order to avoid misuse of scarce resources and ensure best possible health outcomes for patients. METHODS: The study population was adult patients, presented with uncomplicated acute febrile illness, without an obvious focus of infection or malaria or typical dengue infection. We compared the cost and effectiveness of 5 management strategies: 1) no patients tested or given antibiotic treatment; 2) all patients given empirical doxycycline treatment; patients given doxycycline when a patient is tested positive for leptospirosis using: 3) lateral flow; 4) MCAT; 5) latex test. The framework used is a cost-benefit analysis, accounting for all direct medical costs in diagnosing and treating patients suspected of leptospirosis. Outcomes are measured in length of fever after treatment which is then converted to productivity losses to capture the full economic costs. FINDINGS: Empirical doxycycline treatment was the most efficient strategy, being both the least costly alternative and the one that resulted in the shortest duration of fever. The limited sensitivity of all three diagnostic tests implied that their use to guide treatment was not cost-effective. The most influential parameter driving these results was the cost of treating patients with complications for patients who did not receive adequate treatment as a result of incorrect diagnosis or a strategy of no-antibiotic-treatment. CONCLUSIONS: Clinicians should continue treating suspected cases of leptospirosis on an empirical basis. This conclusion holds true as long as policy makers are not prioritizing the reduction of use of antibiotics, in which case the use of the latex test would be the most efficient strategy. Hide abstract

Harris SR, Feil EJ, Holden MT, Quail MA, Nickerson EK, Chantratita N, Gardete S, Tavares A et al. 2010. Evolution of MRSA during hospital transmission and intercontinental spread. Science, 327 (5964), pp. 469-474. Read abstract | Read more

Current methods for differentiating isolates of predominant lineages of pathogenic bacteria often do not provide sufficient resolution to define precise relationships. Here, we describe a high-throughput genomics approach that provides a high-resolution view of the epidemiology and microevolution of a dominant strain of methicillin-resistant Staphylococcus aureus (MRSA). This approach reveals the global geographic structure within the lineage, its intercontinental transmission through four decades, and the potential to trace person-to-person transmission within a hospital environment. The ability to interrogate and resolve bacterial populations is applicable to a range of infectious diseases, as well as microbial ecology. Hide abstract

Dondorp AM, Fanello CI, Hendriksen IC, Gomes E, Seni A, Chhaganlal KD, Bojang K, Olaosebikan R et al. 2010. Artesunate versus quinine in the treatment of severe falciparum malaria in African children (AQUAMAT): An open-label, randomised trial The Lancet, 376 (9753), pp. 1647-1657. Read abstract | Read more

Severe malaria is a major cause of childhood death and often the main reason for paediatric hospital admission in sub-Saharan Africa. Quinine is still the established treatment of choice, although evidence from Asia suggests that artesunate is associated with a lower mortality. We compared parenteral treatment with either artesunate or quinine in African children with severe malaria. This open-label, randomised trial was undertaken in 11 centres in nine African countries. Children (<15 years) with severe falciparum malaria were randomly assigned to parenteral artesunate or parenteral quinine. Randomisation was in blocks of 20, with study numbers corresponding to treatment allocations kept inside opaque sealed paper envelopes. The trial was open label at each site, and none of the investigators or trialists, apart from for the trial statistician, had access to the summaries of treatment allocations. The primary outcome measure was in-hospital mortality, analysed by intention to treat. This trial is registered, number ISRCTN50258054. 5425 children were enrolled; 2712 were assigned to artesunate and 2713 to quinine. All patients were analysed for the primary outcome. 230 (8·5) patients assigned to artesunate treatment died compared with 297 (10·9) assigned to quinine treatment (odds ratio [OR] stratified for study site 0·75, 95 CI 0·63-0·90; relative reduction 22·5, 95 CI 8·1-36·9; p=0·0022). Incidence of neurological sequelae did not differ significantly between groups, but the development of coma (65/1832 [3·5] with artesunate vs 91/1768 [5·1] with quinine; OR 0·69 95 CI 0·49-0·95; p=0·0231), convulsions (224/2712 [8·3] vs 273/2713 [10·1]; OR 0·80, 0·66-0·97; p=0·0199), and deterioration of the coma score (166/2712 [6·1] vs 208/2713 [7·7]; OR 0·78, 0·64-0·97; p=0·0245) were all significantly less frequent in artesunate recipients than in quinine recipients. Post-treatment hypoglycaemia was also less frequent in patients assigned to artesunate than in those assigned to quinine (48/2712 [1·8] vs 75/2713 [2·8]; OR 0·63, 0·43-0·91; p=0·0134). Artesunate was well tolerated, with no serious drug-related adverse effects. Artesunate substantially reduces mortality in African children with severe malaria. These data, together with a meta-analysis of all trials comparing artesunate and quinine, strongly suggest that parenteral artesunate should replace quinine as the treatment of choice for severe falciparum malaria worldwide. The Wellcome Trust. © 2010 Elsevier Ltd. Hide abstract

White NJ, Turner GD, Medana IM, Dondorp AM, Day NP. 2010. The murine cerebral malaria phenomenon. Trends Parasitol, 26 (1), pp. 11-15. Read abstract | Read more

P.berghei ANKA infection in CBA or CB57BL/6 mice is used widely as a murine 'model' of human cerebral malaria (HCM), despite markedly different histopathological features. The pathology of the murine model is characterised by marked inflammation with little or no intracerebral sequestration of parasitised erythrocytes, whereas HCM is associated with intense intracerebral sequestration, often with little inflammatory response. There are now more than ten times as many studies each year of the murine model than on HCM. Of 48 adjunctive interventions evaluated in the murine model, 44 (92%) were successful, compared with only 1 (6%) of 17 evaluated in HCM during the same period. The value of the mouse model in identifying pathological processes or therapeutic interventions in human cerebral malaria is questionable. Hide abstract

Dondorp AM, Nosten F, Yi P, Das D, Phyo AP, Tarning J, Lwin KM, Ariey F et al. 2009. Artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med, 361 (5), pp. 455-467. Read abstract | Read more

BACKGROUND: Artemisinin-based combination therapies are the recommended first-line treatments of falciparum malaria in all countries with endemic disease. There are recent concerns that the efficacy of such therapies has declined on the Thai-Cambodian border, historically a site of emerging antimalarial-drug resistance. METHODS: In two open-label, randomized trials, we compared the efficacies of two treatments for uncomplicated falciparum malaria in Pailin, western Cambodia, and Wang Pha, northwestern Thailand: oral artesunate given at a dose of 2 mg per kilogram of body weight per day, for 7 days, and artesunate given at a dose of 4 mg per kilogram per day, for 3 days, followed by mefloquine at two doses totaling 25 mg per kilogram. We assessed in vitro and in vivo Plasmodium falciparum susceptibility, artesunate pharmacokinetics, and molecular markers of resistance. RESULTS: We studied 40 patients in each of the two locations. The overall median parasite clearance times were 84 hours (interquartile range, 60 to 96) in Pailin and 48 hours (interquartile range, 36 to 66) in Wang Pha (P<0.001). Recrudescence confirmed by means of polymerase-chain-reaction assay occurred in 6 of 20 patients (30%) receiving artesunate monotherapy and 1 of 20 (5%) receiving artesunate-mefloquine therapy in Pailin, as compared with 2 of 20 (10%) and 1 of 20 (5%), respectively, in Wang Pha (P=0.31). These markedly different parasitologic responses were not explained by differences in age, artesunate or dihydroartemisinin pharmacokinetics, results of isotopic in vitro sensitivity tests, or putative molecular correlates of P. falciparum drug resistance (mutations or amplifications of the gene encoding a multidrug resistance protein [PfMDR1] or mutations in the gene encoding sarco-endoplasmic reticulum calcium ATPase6 [PfSERCA]). Adverse events were mild and did not differ significantly between the two treatment groups. CONCLUSIONS: P. falciparum has reduced in vivo susceptibility to artesunate in western Cambodia as compared with northwestern Thailand. Resistance is characterized by slow parasite clearance in vivo without corresponding reductions on conventional in vitro susceptibility testing. Containment measures are urgently needed. ( number, NCT00493363, and Current Controlled Trials number, ISRCTN64835265.) Hide abstract

Paris DH, Aukkanit N, Jenjaroen K, Blacksell SD, Day NP. 2009. A highly sensitive quantitative real-time PCR assay based on the groEL gene of contemporary Thai strains of Orientia tsutsugamushi. Clin Microbiol Infect, 15 (5), pp. 488-495. Read abstract | Read more

Partial nucleotide sequences (459 bp) of the groEL gene (encoding the 60-kDa heat shock protein, HSP60) from 23 contemporary isolates of Orientia tsutsugamushi isolated from patients with acute scrub typhus in Thailand were compared with 16 reference strain sequences to evaluate the potential of groEL as a conserved and representative target for molecular diagnostics.. Overall nucleotide identity within all available O. tsutsugamushi isolates (n = 39) was 98.8% (range: 95.0-100), reflecting a high degree of conservation; nucleotide identities were 67.5% and 65.6%, respectively, when typhus and spotted fever group rickettsiae were included.. A highly sensitive and quantitative real-time PCR assay was designed and evaluated using 61 samples, including buffy coats from patients in Thailand and Laos. Reliable and accurate quantitation of bacterial loads allows further investigation of other diagnostic methods and may lead to an improved understanding of the pathophysiology of acute scrub typhus, an important but under-recognized disease. Hide abstract

Lindegardh N, Tarning J, Toi PV, Hien TT, Farrar J, Singhasivanon P, White NJ, Ashton M, Day NP. 2009. Quantification of artemisinin in human plasma using liquid chromatography coupled to tandem mass spectrometry. J Pharm Biomed Anal, 49 (3), pp. 768-773. Read abstract | Read more

A liquid chromatographic tandem mass spectroscopy method for the quantification of artemisinin in human heparinised plasma has been developed and validated. The method uses Oasis HLB mu-elution solid phase extraction 96-well plates to facilitate a high throughput of 192 samples a day. Artesunate (internal standard) in a plasma-water solution was added to plasma (50 microL) before solid phase extraction. Artemisinin and its internal standard artesunate were analysed by liquid chromatography and MS/MS detection on a Hypersil Gold C18 (100 mm x 2.1 mm, 5 microm) column using a mobile phase containing acetonitrile-ammonium acetate 10mM pH 3.5 (50:50, v/v) at a flow rate of 0.5 mL/min. The method has been validated according to published FDA guidelines and showed excellent performance. The within-day, between-day and total precisions expressed as R.S.D., were lower than 8% at all tested quality control levels including the upper and lower limit of quantification. The limit of detection was 0.257 ng/mL for artemisinin and the calibration range was 1.03-762 ng/mL using 50 microL plasma. The method was free from matrix effects as demonstrated both graphically and quantitatively. Hide abstract

White NJ, Pongtavornpinyo W, Maude RJ, Saralamba S, Aguas R, Stepniewska K, Lee SJ, Dondorp AM, White LJ, Day NP. 2009. Hyperparasitaemia and low dosing are an important source of anti-malarial drug resistance. Malar J, 8 (1), pp. 253. Read abstract | Read more

BACKGROUND: Preventing the emergence of anti-malarial drug resistance is critical for the success of current malaria elimination efforts. Prevention strategies have focused predominantly on qualitative factors, such as choice of drugs, use of combinations and deployment of multiple first-line treatments. The importance of anti-malarial treatment dosing has been underappreciated. Treatment recommendations are often for the lowest doses that produce "satisfactory" results. METHODS: The probability of de-novo resistant malaria parasites surviving and transmitting depends on the relationship between their degree of resistance and the blood concentration profiles of the anti-malarial drug to which they are exposed. The conditions required for the in-vivo selection of de-novo emergent resistant malaria parasites were examined and relative probabilities assessed. RESULTS: Recrudescence is essential for the transmission of de-novo resistance. For rapidly eliminated anti-malarials high-grade resistance can arise from a single drug exposure, but low-grade resistance can arise only from repeated inadequate treatments. Resistance to artemisinins is, therefore, unlikely to emerge with single drug exposures. Hyperparasitaemic patients are an important source of de-novo anti-malarial drug resistance. Their parasite populations are larger, their control of the infection insufficient, and their rates of recrudescence following anti-malarial treatment are high. As use of substandard drugs, poor adherence, unusual pharmacokinetics, and inadequate immune responses are host characteristics, likely to pertain to each recurrence of infection, a small subgroup of patients provides the particular circumstances conducive to de-novo resistance selection and transmission. CONCLUSION: Current dosing recommendations provide a resistance selection opportunity in those patients with low drug levels and high parasite burdens (often children or pregnant women). Patients with hyperparasitaemia who receive outpatient treatments provide the greatest risk of selecting de-novo resistant parasites. This emphasizes the importance of ensuring that only quality-assured anti-malarial combinations are used, that treatment doses are optimized on the basis of pharmacodynamic and pharmacokinetic assessments in the target populations, and that patients with heavy parasite burdens are identified and receive sufficient treatment to prevent recrudescence. Hide abstract

Lindegardh N, Hanpithakpong W, Kamanikom B, Singhasivanon P, Socheat D, Yi P, Dondorp AM, McGready R, Nosten F, White NJ, Day NP. 2008. Major pitfalls in the measurement of artemisinin derivatives in plasma in clinical studies. J Chromatogr B Analyt Technol Biomed Life Sci, 876 (1), pp. 54-60. Read abstract | Read more

A bioanalytical method for the analysis of artesunate (ARS) and its metabolite dihydroartemisinin (DHA) in human plasma using protein precipitation and liquid chromatography coupled to positive tandem mass spectroscopy was developed. The method was validated according to published US FDA-guidelines and showed excellent performance. However, when it was applied to clinical pharmacokinetic studies in malaria, variable degradation of the artemisinins introduced an unacceptable large source of error, rendering the assay useless. Haemolytic products related to sample collection and malaria infection degraded the compounds. Addition of organic solvents during sample processing and even low volume addition of the internal standard in an organic solvent caused degradation. A solid phase extraction method avoiding organic solvents eliminated problems arising from haemolysis induced degradation. Plasma esterases mediated only approximately 20% of ex vivo hydrolysis of ARS into DHA. There are multiple sources of major preventable error in measuring ARS and DHA in plasma samples from clinical trials. These various pitfalls have undoubtedly contributed to the large inter-subject variation in plasma concentration profiles and derived pharmacokinetic parameters for these important antimalarial drugs. Hide abstract

Paris DH, Blacksell SD, Newton PN, Day NP. 2008. Simple, rapid and sensitive detection of Orientia tsutsugamushi by loop-isothermal DNA amplification. Trans R Soc Trop Med Hyg, 102 (12), pp. 1239-1246. Read abstract | Read more

We present a loop-mediated isothermal PCR assay (LAMP) targeting the groEL gene, which encodes the 60kDa heat shock protein of Orientia tsutsugamushi. Evaluation included testing of 63 samples of contemporary in vitro isolates, buffy coats and whole blood samples from patients with fever. Detection limits for LAMP were assessed by serial dilutions and quantitation by real-time PCR assay based on the same target gene: three copies/microl for linearized plasmids, 26 copies/microl for VERO cell culture isolates, 14 copies/microl for full blood samples and 41 copies/microl for clinical buffy coats. Based on a limited sample number, the LAMP assay is comparable in sensitivity with conventional nested PCR (56kDa gene), with limits of detection well below the range of known admission bacterial loads of patients with scrub typhus. This inexpensive method requires no sophisticated equipment or sample preparation, and may prove useful as a diagnostic assay in financially poor settings; however, it requires further prospective validation in the field setting. Hide abstract

Wuthiekanun V, Pheaktra N, Putchhat H, Sin L, Sen B, Kumar V, Langla S, Peacock SJ, Day NP. 2008. Burkholderia pseudomallei antibodies in children, Cambodia. Emerg Infect Dis, 14 (2), pp. 301-303. Read abstract | Read more

Antibodies to Burkholderia pseudomallei were detected in 16% of children in Siem Reap, Cambodia. This organism was isolated from 30% of rice paddies in the surrounding vicinity. Despite the lack of reported indigenous cases, melioidosis is likely to occur in Cambodia. Hide abstract

Chierakul W, Tientadakul P, Suputtamongkol Y, Wuthiekanun V, Phimda K, Limpaiboon R, Opartkiattikul N, White NJ, Peacock SJ, Day NP. 2008. Activation of the coagulation cascade in patients with leptospirosis. Clin Infect Dis, 46 (2), pp. 254-260. Read abstract | Read more

BACKGROUND: Disseminated intravascular coagulation (DIC) is common among patients with sepsis. Leptospirosis is an important cause of sepsis in tropical areas, and pulmonary hemorrhage associated with thrombocytopenia is the major cause of death, but the coagulopathy in severe leptospirosis has not been further characterized. The aim of this study was to evaluate coagulation factors and the presence of DIC in patients with leptospirosis in northeast Thailand. METHODS: We measured plasma concentrations of fibrinogen, D-dimer, thrombin-antithrombin III complexes, and prothrombin fragment 1,2 and evaluated the DIC score in 79 patients with culture-confirmed and/or serologically confirmed leptospirosis and in 33 healthy Thai control subjects. RESULTS: The median concentrations of fibrinogen, D-dimer, thrombin-antithrombin III complexes, and prothrombin fragment 1,2 were significantly elevated in a cohort of 79 patients with leptospirosis, compared with healthy control subjects (P<or=.001 for all tests). Patients with leptospirosis had significantly longer prothrombin times, longer activated partial thromboplastin times, and lower platelet counts. Thrombocytopenia was present in 38% of case patients and occurred more frequently among patients with culture-negative leptospirosis; in multivariate analysis, it was the only hemostasis factor independently associated with clinical bleeding. Patients who were culture-negative for Leptospira species had higher Acute Physiology and Chronic Health Evaluation II and Sepsis-Related Organ Failure Assessment scores and more bleeding complications. Nearly one-half of patients with leptospirosis had overt DIC as defined by an International Society on Thrombosis and Hemostasis DIC score. CONCLUSIONS: Activation of the coagulation system is an important feature of leptospirosis. Thrombocytopenia is an indicator of severe disease and risk of bleeding. Hide abstract

Dondorp AM, Ince C, Charunwatthana P, Hanson J, van Kuijen A, Faiz MA, Rahman MR, Hasan M et al. 2008. Direct in vivo assessment of microcirculatory dysfunction in severe falciparum malaria. J Infect Dis, 197 (1), pp. 79-84. Read abstract | Read more

BACKGROUND: This study sought to describe and quantify microcirculatory changes in the mucosal surfaces of patients with severe malaria, by direct in vivo observation using orthogonal polarization spectral (OPS) imaging. METHODS: The microcirculation in the rectal mucosa of adult patients with severe malaria was assessed by use of OPS imaging, at admission and then daily. Comparison groups comprised patients with uncomplicated falciparum malaria, patients with bacterial sepsis, and healthy individuals. RESULTS: Erythrocyte velocities were measured directly in 43 adult patients with severe falciparum malaria, of whom 20 died. Microcirculatory blood flow was markedly disturbed, with heterogeneous obstruction that was proportional to severity of disease. Blocked capillaries were found in 29 patients (67%) and were associated with concurrent hyperdynamic blood flow (erythrocyte velocity, >750 mm/s) in adjacent vessels in 27 patients (93%). The proportion of blocked capillaries correlated with the base deficit in plasma and with the concentration of lactate. Abnormalities disappeared when the patients recovered. In healthy individuals and in patients with uncomplicated malaria or sepsis, no stagnant erythrocytes were detected, and, in patients with sepsis, hyperdynamic blood flow was prominent. CONCLUSION: Patients with severe falciparum malaria show extensive microvascular obstruction that is proportional to the severity of the disease. This finding underscores the prominent role that microvascular obstruction plays in the pathophysiology of severe malaria and illustrates the fundamental difference between the microvascular pathophysiology of malaria and that of bacterial sepsis. Hide abstract

Wain J, Diep TS, Bay PV, Walsh AL, Vinh H, Duong NM, Ho VA, Hien TT et al. 2008. Specimens and culture media for the laboratory diagnosis of typhoid fever. J Infect Dev Ctries, 2 (6), pp. 469-474. Read abstract

BACKGROUND: Culture of S. Typhi is necessary for the definitive diagnosis of typhoid fever and provides isolates for antibiotic susceptibility testing and epidemiological studies. However, current methods are not fully optimised and sourcing culture media and bottles for culture media may be problematic. METHODOLOGY: In two hospital laboratories in Viet Nam, comparisons of media for blood and stool culture were conducted. The effect of the volume of blood or stool on culture positivity rate was examined and direct plating of the blood buffy coat was trialed. RESULTS: For 148 suspected typhoid fever cases, ox bile broth (58 positive) and brain-heart infusion broth containing saponin (63 positive), performed equally well. For 69 confirmed adult typhoid fever cases, large-volume (15 ml) blood culture gave the same sensitivity as 1 ml of bone marrow culture. For 44 confirmed typhoid fever cases, the direct plating of the buffy coat was positive in 28 cases. For 263 positive stool cultures, selenite F and selenite mannitol performed equally well and culturing 2 g rather than 1g increased the isolation rate by 10.5%. CONCLUSIONS: For the diagnosis of typhoid fever by blood culture the medium should be a rich nutrient broth containing a lysing agent. In adults 1 ml bone marrow or 15 ml blood culture gave similar results. Where isolates are needed for susceptibility testing or epidemiological studies, but resources for culture are scarce, direct plating of the blood buffy coat can be used with a 50% fall in sensitivity compared to standard blood culture. Hide abstract

Singtoroj T, Tarning J, Annerberg A, Ashton M, Bergqvist Y, White NJ, Lindegardh N, Day NP. 2006. A new approach to evaluate regression models during validation of bioanalytical assays. J Pharm Biomed Anal, 41 (1), pp. 219-227. Read abstract | Read more

The quality of bioanalytical data is highly dependent on using an appropriate regression model for calibration curves. Non-weighted linear regression has traditionally been used but is not necessarily the optimal model. Bioanalytical assays generally benefit from using either data transformation and/or weighting since variance normally increases with concentration. A data set with calibrators ranging from 9 to 10000 ng/mL was used to compare a new approach with the traditional approach for selecting an optimal regression model. The new approach used a combination of relative residuals at each calibration level together with precision and accuracy of independent quality control samples over 4 days to select and justify the best regression model. The results showed that log-log transformation without weighting was the simplest model to fit the calibration data and ensure good predictability for this data set. Hide abstract

Faiz MA, Bin Yunus E, Rahman MR, Islam F, Hoque MG, Hasan MU, Samad R, Aung S et al. 2005. Artesunate versus quinine for treatment of severe falciparum malaria: a randomised trial LANCET, 366 (9487), pp. 717-725. | Read more

Dondorp AM, Desakorn V, Pongtavornpinyo W, Sahassananda D, Silamut K, Chotivanich K, Newton PN, Pitisuttithum P, Smithyman AM, White NJ, Day NP. 2005. Estimation of the total parasite biomass in acute falciparum malaria from plasma PfHRP2. PLoS Med, 2 (8), pp. e204. Read abstract | Read more

BACKGROUND: In falciparum malaria sequestration of erythrocytes containing mature forms of Plasmodium falciparum in the microvasculature of vital organs is central to pathology, but quantitation of this hidden sequestered parasite load in vivo has not previously been possible. The peripheral blood parasite count measures only the circulating, relatively non-pathogenic parasite numbers. P. falciparum releases a specific histidine-rich protein (PfHRP2) into plasma. Quantitative measurement of plasma PfHRP2 concentrations may reflect the total parasite biomass in falciparum malaria. METHODS AND FINDINGS: We measured plasma concentrations of PfHRP2, using a quantitative antigen-capture enzyme-linked immunosorbent assay, in 337 adult patients with falciparum malaria of varying severity hospitalised on the Thai-Burmese border. Based on in vitro production rates, we constructed a model to link this measure to the total parasite burden in the patient. The estimated geometric mean parasite burden was 7 x 10(11) (95% confidence interval [CI] 5.8 x 10(11) to 8.5 x 10(11)) parasites per body, and was over six times higher in severe malaria (geometric mean 1.7 x 10(12), 95% CI 1.3 x 10(12) to 2.3 x 10(12)) than in patients hospitalised without signs of severity (geometric mean 2.8 x 10(11), 95% CI 2.3 x 10(11) to 3.5 x 10(11); p < 0.001). Parasite burden was highest in patients who died (geometric mean 3.4 x 10(12), 95% CI 1.9 x 10(12) to 6.3 x 10(12); p = 0.03). The calculated number of sequestered parasites increased with disease severity and was higher in patients with late developmental stages of P. falciparum present on peripheral blood smears. Comparing model and laboratory estimates of the time of sequestration suggested that admission to hospital with uncomplicated malaria often follows schizogony-but in severe malaria is unrelated to stage of parasite development. CONCLUSION: Plasma PfHRP2 concentrations may be used to estimate the total body parasite biomass in acute falciparum malaria. Severe malaria results from extensive sequestration of parasitised erythrocytes. Hide abstract

Feil EJ, Cooper JE, Grundmann H, Robinson DA, Enright MC, Berendt T, Peacock SJ, Smith JM et al. 2003. How clonal is Staphylococcus aureus? J Bacteriol, 185 (11), pp. 3307-3316. Read abstract | Read more

Staphylococcus aureus is an important human pathogen and represents a growing public health burden owing to the emergence and spread of antibiotic-resistant clones, particularly within the hospital environment. Despite this, basic questions about the evolution and population biology of the species, particularly with regard to the extent and impact of homologous recombination, remain unanswered. We address these issues through an analysis of sequence data obtained from the characterization by multilocus sequence typing (MLST) of 334 isolates of S. aureus, recovered from a well-defined population, over a limited time span. We find no significant differences in the distribution of multilocus genotypes between strains isolated from carriers and those from patients with invasive disease; there is, therefore, no evidence from MLST data, which index variation within the stable "core" genome, for the existence of hypervirulent clones of this pathogen. Examination of the sequence changes at MLST loci during clonal diversification shows that point mutations give rise to new alleles at least 15-fold more frequently than does recombination. This contrasts with the naturally transformable species Neisseria meningitidis and Streptococcus pneumoniae, in which alleles change between 5- and 10-fold more frequently by recombination than by mutation. However, phylogenetic analysis suggests that homologous recombination does contribute toward the evolution of this species over the long term. Finally, we note a striking excess of nonsynonymous substitutions in comparisons between isolates belonging to the same clonal complex compared to isolates belonging to different clonal complexes, suggesting that the removal of deleterious mutations by purifying selection may be relatively slow. Hide abstract

Peacock SJ, de Silva GD, Justice A, Cowland A, Moore CE, Winearls CG, Day NP. 2002. Comparison of multilocus sequence typing and pulsed-field gel electrophoresis as tools for typing Staphylococcus aureus isolates in a microepidemiological setting. J Clin Microbiol, 40 (10), pp. 3764-3770. Read abstract | Read more

Multilocus sequence typing (MLST) of Staphylococcus aureus is well suited to the study of global or long-term epidemiology, but its role in local epidemiology has not been defined. The present study has compared MLST with pulsed-field gel electrophoresis (PFGE) by using S. aureus isolates associated with carriage and disease in a busy regional renal unit. One hundred forty-four patients were prospectively recruited, of whom 103 were receiving hemodialysis and 41 were on continuous ambulatory peritoneal dialysis. Three nasal swab specimens were obtained 1 month apart on entering the study. A nasal swab was positive for S. aureus on at least one occasion in 50 patients (35%). Typing of the 104 carriage isolates demonstrated 21 PFGE types and 21 sequence types (STs). Thirty-one carriers had two or more positive nasal swabs; of these, the isolates in all swabs from a given carrier had identical PFGE types for 29 carriers; the isolates in all of the same 29 swabs had identical STs. The carriage strain in two patients changed both PFGE type and STs during the period of swabbing. Eight patients (6%) had an episode of S. aureus bacteremia during the 12-month study period, and two of these were nasal carriers. One of these invasive isolates had the same PFGE type and ST as the carriage isolate. There were no differences between Simpson's index of diversity for PFGE and Simpson's index of diversity for MLST for both invasive and carriage isolates, suggesting that the two methods have very similar discriminatory abilities. We conclude that PFGE and MLST performed equally in this study. Hide abstract

Phu NH, Hien TT, Mai NT, Chau TT, Chuong LV, Loc PP, Winearls C, Farrar J, White N, Day N. 2002. Hemofiltration and peritoneal dialysis in infection-associated acute renal failure in Vietnam. N Engl J Med, 347 (12), pp. 895-902. Read abstract | Read more

BACKGROUND: In some parts of the world, peritoneal dialysis is widely used for renal replacement in acute renal failure. In resource-rich countries, it has been supplanted in recent years by hemodialysis and, most recently, by hemofiltration and associated techniques. The relative efficacy of peritoneal dialysis and hemofiltration is not known. METHODS: We conducted an open, randomized comparison of pumped venovenous hemofiltration and peritoneal dialysis in patients with infection-associated acute renal failure in an infectious-disease referral hospital in Vietnam. RESULTS: Seventy adult patients with severe falciparum malaria (48 patients) or sepsis (22 patients) were enrolled; 34 were assigned to hemofiltration and 36 to peritoneal dialysis. The mortality rate was 47 percent (17 patients) in the group assigned to peritoneal dialysis, as compared with 15 percent (5 patients) in the group assigned to hemofiltration (P=0.005). The rates of resolution of acidosis and of decline in the serum creatinine concentration in the group assigned to hemofiltration were more than twice those in the group assigned to peritoneal dialysis (P<0.005), and renal-replacement therapy was required for a significantly shorter period. In a multivariate analysis, the odds ratio for death was 5.1 (95 percent confidence interval, 1.6 to 16) and that for a need for future dialysis was 4.7 (95 percent confidence interval, 1.3 to 17) in the group assigned to peritoneal dialysis. The cost of hemofiltration per survivor was less than half that of peritoneal dialysis, and the cost per life saved was less than one third. CONCLUSIONS: Hemofiltration is superior to peritoneal dialysis in the treatment of infection-associated acute renal failure. Hide abstract

Peacock SJ, Moore CE, Justice A, Kantzanou M, Story L, Mackie K, O'Neill G, Day NP. 2002. Virulent combinations of adhesin and toxin genes in natural populations of Staphylococcus aureus. Infect Immun, 70 (9), pp. 4987-4996. Read abstract | Read more

Most cases of severe Staphylococcus aureus disease cannot be explained by the action of a single virulence determinant, and it is likely that a number of factors act in combination during the infective process. This study examined the relationship between disease in humans and a large number of putative virulence determinants, both individually and in combination. S. aureus isolates (n = 334) from healthy blood donors and from patients with invasive disease were compared for variation in the presence of 33 putative virulence determinants. After adjusting for the effect of clonality, seven determinants (fnbA, cna, sdrE, sej, eta, hlg, and ica) were significantly more common in invasive isolates. All seven factors contributed independently to virulence. No single factor predominated as the major predictor of virulence, their effects appearing to be cumulative. No combinations of the seven genes were either more or less likely to cause disease than others with the same number of virulence-associated genes. There was evidence of considerable horizontal transfer of genes on a background of clonality. Our findings also suggested that allelic variants of a polymorphic locus can make different contributions to the disease process, further study of which is likely to expand our understanding of staphylococcal disease pathogenesis. Hide abstract

Bethell DB, Gamble J, Pham PL, Nguyen MD, Tran TH, Ha TH, Tran TN, Dong TH, Gartside IB, White NJ, Day NP. 2001. Noninvasive measurement of microvascular leakage in patients with dengue hemorrhagic fever. Clin Infect Dis, 32 (2), pp. 243-253. Read abstract | Read more

Dengue shock syndrome (DSS) is a potentially lethal complication of dengue virus infection associated with hypotension and leakage of plasma water into the extravascular space. To determine whether the underlying pathophysiology of DSS is distinct from that in milder forms of the disease, we assessed microvascular permeability, by use of strain gauge plethysmography, in Vietnamese children with DSS (n=19), or dengue hemorrhagic fever (DHF) without shock (n=16), and in healthy control children (n=15). At admission and after fluid resuscitation, the mean coefficient of microvascular permeability (K(f)) for the patients with dengue was approximately 50% higher than that for the control patients (P=.02). There was no significant difference in K(f) between the 2 groups of patients with dengue; this suggests the same underlying pathophysiology. We hypothesize that in patients with DSS, the fluctuations in K(f) are larger than those in patients with DHF, which leads to short-lived peaks of markedly increased microvascular permeability and consequent hemodynamic shock. Hide abstract

Intracellular bacteria: understanding the molecular basis of bacteria-host cell interactions

Bacteria that live inside eukaryotic cells have an intriguing relationship with their host cells. They are dependent on them for nutrition and shelter, but should avoid killing their hosts or being killed by them. They have consequently evolved a number of remarkable mechanisms to reprogramme the host cell upon entry in order to better ensure their own survival and dissemination.Rickettsiaceae are a family of obligate intracellular bacteria that include the precursor of modern mitochondria. They ...

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Mathematical modelling of the spread of antibiotic-resistant bacteria in Southeast Asia

Antimicrobial resistance is one of the most serious public health threats in the world today. Multiply antibiotic-resistant bacteria are endemic in hospitals in South and Southeast Asia and high prevalences of community carriage of important antibiotic-resistant pathogens have also been reported. However, there are major gaps in our understanding of how such drug-resistance spreads, how this spread is affected by antibiotic use, and the ecological interactions that lead to the co-existence of an ...

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The impact of point of care tests for biomarkers of bacterial infection on antibiotic prescription in developing countries

The vast majority of febrile episodes in many parts of the malaria endemic world are caused by other causes, yet other than malaria rapid tests no diagnostic tests are readily available for use in remote settings. While in some areas antibiotics are over-prescribed, contributing to the development of antimicrobial resistance, in others many patients are under-treated. Better targeting of antibiotics in these settings could improve immediate health outcomes while reducing drug pressure and mitiga ...

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