register interest

Dr Climent Casals-Pascual

Research Area: Global Health
Technology Exchange: Mass spectrometry and Medical statistics
Scientific Themes: Tropical Medicine & Global Health
Keywords: Severe Malaria, Cerebral malaria, Systems Biology and Clinical Proteomics
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Severe malaria (SM) is an important cause of global mortality and morbidity. The most common clinical presentations of SM are cerebral malaria (CM), severe respiratory distress (SRD) and severe malarial anaemia (SMA). The clinical symptoms of severe malaria frequently overlap with those of other severe diseases like pneumonia, meningitis and bacteraemia. In these situations, malaria microscopy is a poor diagnostic tool for clinical management and empirical antibiotic treatment is recommended. Therefore, the lack of accurate clinical case definitions for severe malaria limits our ability to diagnose and manage adequately these cases. From a public health perspective, it limits our ability to have real estimates of the malaria burden and to monitor the efficacy of interventions and malaria control programmes. Therefore better markers of SM with high sensitivity and specificity are required.

Our research aims to study the molecules involved in the pathophysiology of severe malaria to improve its diagnosis and clinical management. To do this we plan to identify and quantitate the peptides and proteins that constitute the plasma proteome of children with Plasmodium falciparum severe malaria; assess the diagnostic performance of distinctive peptide/proteins identified; and to integrate proteomic, genomic and clinical data into different pathophysiological models that may explain differences in clinical outcome in severe malaria.

Name Department Institution Country
Professor Benedikt M Kessler Target Discovery Institute Oxford University, NDM Research Building United Kingdom
Dr Hans Ackerman National Institutes of Health United States
Dr Oliver Billker Wellcome Trust Sanger Institute United Kingdom
Professor Philip Maini Centre for Mathematical Biology University of Oxford United Kingdom
Professor Dominic Kwiatkowski Wellcome Trust Centre for Human Genetics Oxford University, Henry Wellcome Building of Genomic Medicine United Kingdom
Camprubí D, Pereira A, Rodriguez-Valero N, Almuedo A, Varo R, Casals-Pascual C, Bassat Q, Malvy D, Muñoz J. 2019. Positive direct antiglobulin test in post-artesunate delayed haemolysis: more than a coincidence? Malar J, 18 (1), pp. 123. | Show Abstract | Read more

BACKGROUND: Delayed haemolysis is a frequent adverse event after treatment with artesunate (AS). Removing once-infected "pitted" erythrocytes by the spleen is the most accepted mechanism of haemolysis in these cases. However, an increasing number of cases with positive direct antiglobulin test (DAT) haemolysis after AS have been reported. METHODS: All malaria cases seen at Hospital Clinic of Barcelona between 2015 and 2017 were retrospectively reviewed. Clinical, parasitological and laboratory data from patients treated with intravenous artesunate-specifically looking for delayed haemolysis and DAT-was collected. RESULTS: Among the 36 severe malaria patients treated with artesunate at the hospital, 10 (27.8%) developed post-artesunate delayed haemolysis. Out of these, DAT was performed in six, being positive in four of them (at least 40%). DAT was positive only for complement-without IgG-suggesting drug-dependent immune-haemolytic anaemia of the immune-complex type. Three of the four patients were treated with corticosteroids and two also received blood transfusion, with a complete recovery. CONCLUSIONS: Drug-induced auto-immune phenomena in post-artesunate delayed haemolysis may be underreported and must be considered. The role of corticosteroids should be reassessed.

Rubio E, Alejo-Cancho I, Aylagas C, Camprubí D, Ferré R, Albarracín MR, Gonzalo V, Barrachina J, Álvarez-Martínez MJ, Valls ME et al. 2019. Diagnostic Value of Platelet and Leukocyte Counts in the Differential Diagnosis of Fever in the Returning Traveler. Am J Trop Med Hyg, 100 (2), pp. 470-475. | Show Abstract | Read more

Malaria, arbovirus infection and travelers' diarrhea are among the most common etiologies of fever after a stay in the tropics. Because the initial symptoms of these diseases often overlap, the differential diagnostic remains a challenge. The aim of this study was to establish the effectiveness of platelet and leukocyte counts in the differential diagnosis of fever in the returning traveler. Between 2013 and 2016, patients with a clinical suspicion of malaria, who had thick blood smears performed were retrospectively included. The microbiological etiology of each episode was established based on molecular detection in the case of arbovirus infection, the detection of pathogens in stool samples for diarrhea and other gastrointestinal symptoms and the thick and thin blood smear results for malaria. A total of 1,218 episodes were included. Malaria, arbovirus infection, and diarrhea and other gastrointestinal symptoms caused 102 (8.4%), 68 (5.6%), and 72 (5.9%) episodes, respectively. The median platelet counts in malaria episodes were 89 × 109/L and thrombocytopenia (< 150,000 × 109 platelets/L) yielded a 98% negative predictive value to predict malaria. The median leukocyte counts in arbovirus infection episodes were 3.19 × 109/L and leucopenia (< 4 × 109 leukocytes/L) yielded a 97.9% negative predictive value to predict arbovirus infections. Platelet and leukocyte counts were not significantly altered in episodes caused by diarrhea and other gastrointestinal symptoms. Initial platelet and leukocyte counts might be useful for the clinical differential diagnosis of fever in the returning traveler. Although these results are insufficient to establish a diagnosis, they should be considered in the initial clinical assessment.

Ackerman H, Casals-Pascual C. 2018. A biomarker approach to syndrome-based treatment of severe childhood illness in malaria-endemic areas. Malar J, 17 (1), pp. 378. | Show Abstract | Read more

This opinion article deals with the diagnostic clinical challenges faced by clinicians or health care workers in malaria-endemic areas when a severely sick child presents to the clinic with fever, coma or respiratory distress. Indeed, the coexistence of malaria with other severe infections like meningitis, invasive bacterial infection or pneumonia makes appropriate treatment allocation a matter of life and death. The use of biomarkers has been proposed as a potential solution to this problem. The arrival of high-throughput technologies allowed thousands of molecules (transcripts, proteins and metabolites) to be been screened in clinical samples from large cohorts of well/characterised patients. The major aim of these studies was to identify biomarkers that inform important decisions: should this child be referred to hospital? Should antibiotics, anti-malarials, or both, be administered? There is a large discrepancy between the number of biomarker discovery studies published and the number of biomarkers that have been clinically validated, let alone implemented. This article reflects on the many opportunities and obstacles encountered in biomarker research in malaria-endemic areas.

Moussa EM, Huang H, Thézénas ML, Fischer R, Ramaprasad A, Sisay-Joof F, Jallow M, Pain A, Kwiatkowski D, Kessler BM, Casals-Pascual C. 2018. Proteomic profiling of the plasma of Gambian children with cerebral malaria. Malar J, 17 (1), pp. 337. | Show Abstract | Read more

BACKGROUND: Cerebral malaria (CM) is a severe neurological complication of Plasmodium falciparum infection. A number of pathological findings have been correlated with pediatric CM including sequestration, platelet accumulation, petechial haemorrhage and retinopathy. However, the molecular mechanisms leading to death in CM are not yet fully understood. METHODS: A shotgun plasma proteomic study was conducted using samples form 52 Gambian children with CM admitted to hospital. Based on clinical outcome, children were assigned to two groups: reversible and fatal CM. Label-free liquid chromatography-tandem mass spectrometry was used to identify and compare plasma proteins that were differentially regulated in children who recovered from CM and those who died. Candidate biomarkers were validated using enzyme immunoassays. RESULTS: The plasma proteomic signature of children with CM identified 266 proteins differentially regulated in children with fatal CM. Proteins from the coagulation cascade were consistently decreased in fatal CM, whereas the plasma proteomic signature associated with fatal CM underscored the importance of endothelial activation, tissue damage, inflammation, haemolysis and glucose metabolism. The concentration of circulating proteasomes or PSMB9 in plasma was not significantly different in fatal CM when compared with survivors. Plasma PSMB9 concentration was higher in patients who presented with seizures and was significantly correlated with the number of seizures observed in patients with CM during admission. CONCLUSIONS: The results indicate that increased tissue damage and hypercoagulability may play an important role in fatal CM. The diagnostic value of this molecular signature to identify children at high risk of dying to optimize patient referral practices should be validated prospectively.

Hoffmann T, Johnston I, Greenbury S, Cominetti O, Jallow M, Kwiatkowski D, Barahona M, Jones N, Casals-Pascual C. 2018. Precision identification and prediction of high mortality phenotypes and disease progression pathways in severe malaria without requiring longitudinal data | Show Abstract | Read more

The parasite Plasmodium falciparum is the main cause of severe malaria (SM). Despite treatment with antimalarial drugs, more than 450,000 SM deaths are reported every year, mainly in African children. The diversity of clinical presentations associated with SM indicate important differences in disease pathogenesis that often require specific treatment, and this clinical heterogeneity of SM is largely unresolved. In this study, we apply new machine learning and inference tools for large-scale data analysis to dissect the heterogeneity in patterns of clinical features associated with SM in 2,695 Gambian children admitted to hospital with Plasmodium falciparum malaria. This quantitative analysis, including the powerful HyperTraPS algorithm for inference of progressive processes, reveals pathways of SM symptom progression and features predicting the severity of individual patient outcomes. Notably, our approach allows the identification and dissection of disease progression pathways without the need for longitudinal observations. Learning these pathways and features from this rich dataset allows us to construct several quantitative measures of the mortality risk associated with a patient presenting with a given set of symptoms. By independently surveying expert practitioners, we show that this data-driven approach agrees with and expands the current state of knowledge on malaria progression, while simultaneously providing a data-supported framework for predicting clinical risk.

Vergara A, Fernández-Pittol MJ, Muñoz-Mahamud E, Morata L, Bosch J, Vila J, Soriano A, Casals-Pascual C. 2019. Evaluation of Lipocalin-2 as a Biomarker of Periprosthetic Joint Infection. J Arthroplasty, 34 (1), pp. 123-125. | Show Abstract | Read more

BACKGROUND: Periprosthetic joint infection (PJI) remains a major clinical challenge. In this study, we evaluated the diagnostic performance of lipocalin-2 (LCN2), a well-characterized neutrophil protein, in synovial fluid to discriminate PJI and aseptic implant failure. METHODS: Synovial fluid from patients with acute or chronic PJI, aseptic failure, or controls was obtained during surgery. LCN2 was quantified using a modified enzyme immunoassay coupled with chemiluminescence (Architect Urine NGAL; Abbott Laboratories). RESULTS: Synovial fluid was collected from 72 patients: 22 (30.6%) proven infections, 22 (30.6%) aseptic implant failures, and 28 (38.8%) controls. Synovial fluid was obtained from the hip in 18 (25%) and knee in 54 (75%) cases. Among infections, there were 16 (22.2%) acute and 6 (8.3%) chronic PJIs. The median (interquartile range) LCN2 concentration in synovial fluid was 1536.5 ng/mL (261.8-12,923) in the infection group, 87.0 (54.8-135) in the aseptic group, and 55 (45-67.8) in the control group (P < .001). LCN2 discriminated nearly perfectly between controls and confirmed infection (area under the receiver operating characteristic 0.98, 95% confidence interval 0.95-1.00). The optimal cut-off value for maximal sensitivity (86.3%) and specificity (77.2%) to discriminate aseptic failure versus proven infection was 152 ng/mL, with an area under the receiver operating characteristic of 0.92 (95% confidence interval 0.84-0.99). CONCLUSION: LCN2 is a potential novel biomarker that may be helpful to inform surgical teams on the potential risk of PJI and optimize specific surgical interventions as it distinguishes between septic and aseptic failure of prosthesis with high sensitivity and specificity.

Cominetti O, Smith D, Hoffman F, Jallow M, Thézénas ML, Huang H, Kwiatkowski D, Maini PK, Casals-Pascual C. 2018. Identification of a Novel Clinical Phenotype of Severe Malaria using a Network-Based Clustering Approach. Sci Rep, 8 (1), pp. 12849. | Show Abstract | Read more

The parasite Plasmodium falciparum is the main cause of severe malaria (SM). Despite treatment with antimalarial drugs, more than 400,000 deaths are reported every year, mainly in African children. The diversity of clinical presentations associated with SM highlights important differences in disease pathogenesis that often require specific therapeutic options. The clinical heterogeneity of SM is largely unresolved. Here we report a network-based analysis of clinical phenotypes associated with SM in 2,915 Gambian children admitted to hospital with Plasmodium falciparum malaria. We used a network-based clustering method which revealed a strong correlation between disease heterogeneity and mortality. The analysis identified four distinct clusters of SM and respiratory distress that departed from the WHO definition. Patients in these clusters characteristically presented with liver enlargement and high concentrations of brain natriuretic peptide (BNP), giving support to the potential role of circulatory overload and/or right-sided heart failure as a mechanism of disease. The role of heart failure is controversial in SM and our work suggests that standard clinical management may not be appropriate. We find that our clustering can be a powerful data exploration tool to identify novel disease phenotypes and therapeutic options to reduce malaria-associated mortality.

Ripa M, Morata L, Rodríguez-Núñez O, Cardozo C, Puerta-Alcalde P, Hernández-Meneses M, Ambrosioni J, Linares L, Bodro M, Valcárcel A et al. 2018. Short-Term Peripheral Venous Catheter-Related Bloodstream Infections: Evidence for Increasing Prevalence of Gram-Negative Microorganisms from a 25-Year Prospective Observational Study. Antimicrob Agents Chemother, 62 (11), | Show Abstract | Read more

The aim of this study was to describe the etiology and outcome of short-term peripheral venous catheter (PVC)-related bloodstream infections (PVCRBSI) in a 25-year period (1992 to 2016) and to identify predictive factors of Gram-negative PVCRBSI. This was a prospective observational study including all episodes of PVCRBSI. A multivariate logistic regression model adjusted for calendar year was built to explore factors associated with a Gram-negative bacterial etiology. Over the study period, 711 episodes of PVCRBSI were identified. Incidence rate of PVCRBSI increased from 0.06 to 0.13 episodes/1,000 patient-days. A Gram-negative bacterial etiology was demonstrated in 162 (22.8%) episodes. There was a significant increase in the proportion of Gram-negative infections (22.6% in 1992 to 1996 versus 33.2% in 2012 to 2016). Independent predictive factors of Gram-negative PVCRBSI were the following: being in the hospital for more than 7 days with a catheter in situ for more than 3 days (adjusted odds ratio [aOR], 1.80; 95% confidence interval [CI], 1.20 to 2.69), surgery in the previous month (aOR, 2.39; 95% CI, 1.40 to 4.09), and antimicrobial treatment with beta-lactams (aOR, 1.80; 95% CI, 1.16 to 2.78). In conclusion, we reported an increase in the prevalence of Gram-negative PVCRBSI over the last 25 years. Factors associated with a Gram-negative bacterial etiology were being in the hospital for more than 7 days with a catheter in situ for more than 3 days, having undergone surgery, and having received antimicrobial treatment with beta-lactams.

Ndila CM, Uyoga S, Macharia AW, Nyutu G, Peshu N, Ojal J, Shebe M, Awuondo KO, Mturi N, Tsofa B et al. 2018. Human candidate gene polymorphisms and risk of severe malaria in children in Kilifi, Kenya: a case-control association study. Lancet Haematol, 5 (8), pp. e333-e345. | Show Abstract | Read more

BACKGROUND: Human genetic factors are important determinants of malaria risk. We investigated associations between multiple candidate polymorphisms-many related to the structure or function of red blood cells-and risk for severe Plasmodium falciparum malaria and its specific phenotypes, including cerebral malaria, severe malaria anaemia, and respiratory distress. METHODS: We did a case-control study in Kilifi County, Kenya. We recruited as cases children presenting with severe malaria to the high-dependency ward of Kilifi County Hospital. We included as controls infants born in the local community between Aug 1, 2006, and Sept 30, 2010, who were part of a genetics study. We tested for associations between a range of candidate malaria-protective genes and risk for severe malaria and its specific phenotypes. We used a permutation approach to account for multiple comparisons between polymorphisms and severe malaria. We judged p values less than 0·005 significant for the primary analysis of the association between candidate genes and severe malaria. FINDINGS: Between June 11, 1995, and June 12, 2008, 2244 children with severe malaria were recruited to the study, and 3949 infants were included as controls. Overall, 263 (12%) of 2244 children with severe malaria died in hospital, including 196 (16%) of 1233 with cerebral malaria. We investigated 121 polymorphisms in 70 candidate severe malaria-associated genes. We found significant associations between risk for severe malaria overall and polymorphisms in 15 genes or locations, of which most were related to red blood cells: ABO, ATP2B4, ARL14, CD40LG, FREM3, INPP4B, G6PD, HBA (both HBA1 and HBA2), HBB, IL10, LPHN2 (also known as ADGRL2), LOC727982, RPS6KL1, CAND1, and GNAS. Combined, these genetic associations accounted for 5·2% of the variance in risk for developing severe malaria among individuals in the general population. We confirmed established associations between severe malaria and sickle-cell trait (odds ratio [OR] 0·15, 95% CI 0·11-0·20; p=2·61 × 10-58), blood group O (0·74, 0·66-0·82; p=6·26 × 10-8), and -α3·7-thalassaemia (0·83, 0·76-0·90; p=2·06 × 10-6). We also found strong associations between overall risk of severe malaria and polymorphisms in both ATP2B4 (OR 0·76, 95% CI 0·63-0·92; p=0·001) and FREM3 (0·64, 0·53-0·79; p=3·18 × 10-14). The association with FREM3 could be accounted for by linkage disequilibrium with a complex structural mutation within the glycophorin gene region (comprising GYPA, GYPB, and GYPE) that encodes for the rare Dantu blood group antigen. Heterozygosity for Dantu was associated with risk for severe malaria (OR 0·57, 95% CI 0·49-0·68; p=3·22 × 10-11), as was homozygosity (0·26, 0·11-0·62; p=0·002). INTERPRETATION: Both ATP2B4 and the Dantu blood group antigen are associated with the structure and function of red blood cells. ATP2B4 codes for plasma membrane calcium-transporting ATPase 4 (the major calcium pump on red blood cells) and the glycophorins are ligands for parasites to invade red blood cells. Future work should aim at uncovering the mechanisms by which these polymorphisms can result in severe malaria protection and investigate the implications of these associations for wider health. FUNDING: Wellcome Trust, UK Medical Research Council, European Union, and Foundation for the National Institutes of Health as part of the Bill & Melinda Gates Grand Challenges in Global Health Initiative.

Casals-Pascual C, Vergara A, Vila J. 2018. Intestinal microbiota and antibiotic resistance: Perspectives and solutions Human Microbiome Journal, 9 pp. 11-15. | Show Abstract | Read more

© 2018 The intestinal commensal microbiota provides a myriad of benefits to the healthy host, including colonisation resistance against pathogens. Perturbations of the intestinal microbiota (dysbiosis) may adversely affect the health status of an individual and prevent protection against colonisation. The whole range of antibiotic resistance genes (resistome) in a specific microbiota is found in pathogenic and non-pathogenic bacteria. The administration of antibiotics may cause dysbiosis, contributing to the loss of colonisation resistance followed by an increment of the resistome in the intestinal microbiota. Treatments to control the current increase of multi drug-resistant (MDR) bacteria are extremely limited. In this context, the administration of healthy faecal microbiota to restore colonisation resistance and displace MDR bacteria emerges as a promising therapeutic alternative. This brief review describes the role of the intestinal microbiota as a reservoir of MDR bacteria, the impact of different groups of antibiotics in the selection of MDR bacteria and crucially, the potential use of faecal microbiota transplantation using “healthy” or “MDR-free microbiota” to displace MDR bacteria and restore colonisation resistance.

Moussa E, Huang H, Ahras M, Lall A, Thezenas ML, Fischer R, Kessler BM, Pain A, Billker O, Casals-Pascual C. 2018. Proteomic profiling of the brain of mice with experimental cerebral malaria. J Proteomics, 180 pp. 61-69. | Show Abstract | Read more

Cerebral malaria (CM) is a severe neurological complication of malaria infection in both adults and children. In pursuit of effective treatment of CM, clinical studies, postmortem analysis and animal models have been employed to understand the pathology and identify effective interventions. In this study, a shotgun proteomics analysis was conducted to profile the proteomic signature of the brain tissue of mice with experimental cerebral malaria (ECM) in order to further understand the underlying pathology. To identify CM-associated response, proteomic signatures of the brains of C57/Bl6N mice infected with P. berghei ANKA that developed neurological syndrome were compared to those of mice infected with P. berghei NK65 that developed equally high parasite burdens without neurological signs, and to those of non-infected mice. The results show that the CM-associated response in mice that developed neurological signs comprise mainly acute-phase reaction and coagulation cascade activation, and indicate the leakage of plasma proteins into the brain parenchyma. SIGNIFICANCE: Cerebral malaria (CM) remains a major cause of death in children. The majority of these deaths occur in sub-Saharan Africa. Even with adequate access to treatment, mortality remains high and neurological sequelae can be found in up to 20% of survivors. No adjuvant treatment to date has been shown to reduce mortality and the pathophysiology of CM is largely unknown. Experimental cerebral malaria (ECM) is a well-established model that may contribute to identify and test druggable targets. In this study we have identified the disruption of the blood-brain barrier following inflammatory and vascular injury as a mechanism of disease. In this study we report a number of proteins that could be validated as potential biomarkers of ECM. Further studies, will be required to validate the clinical relevance of these biomarkers in human CM.

Naydenova E, Tsanas A, Howie S, Casals-Pascual C, De Vos M. 2016. The power of data mining in diagnosis of childhood pneumonia. J R Soc Interface, 13 (120), pp. 20160266-20160266. | Show Abstract | Read more

Childhood pneumonia is the leading cause of death of children under the age of 5 years globally. Diagnostic information on the presence of infection, severity and aetiology (bacterial versus viral) is crucial for appropriate treatment. However, the derivation of such information requires advanced equipment (such as X-rays) and clinical expertise to correctly assess observational clinical signs (such as chest indrawing); both of these are often unavailable in resource-constrained settings. In this study, these challenges were addressed through the development of a suite of data mining tools, facilitating automated diagnosis through quantifiable features. Findings were validated on a large dataset comprising 780 children diagnosed with pneumonia and 801 age-matched healthy controls. Pneumonia was identified via four quantifiable vital signs (98.2% sensitivity and 97.6% specificity). Moreover, it was shown that severity can be determined through a combination of three vital signs and two lung sounds (72.4% sensitivity and 82.2% specificity); addition of a conventional biomarker (C-reactive protein) further improved severity predictions (89.1% sensitivity and 81.3% specificity). Finally, we demonstrated that aetiology can be determined using three vital signs and a newly proposed biomarker (lipocalin-2) (81.8% sensitivity and 90.6% specificity). These results suggest that a suite of carefully designed machine learning tools can be used to support multi-faceted diagnosis of childhood pneumonia in resource-constrained settings, compensating for the shortage of expensive equipment and highly trained clinicians.

Busby GB, Band G, Si Le Q, Jallow M, Bougama E, Mangano VD, Amenga-Etego LN, Enimil A, Apinjoh T, Ndila CM et al. 2016. Admixture into and within sub-Saharan Africa. Elife, 5 | Show Abstract | Read more

Similarity between two individuals in the combination of genetic markers along their chromosomes indicates shared ancestry and can be used to identify historical connections between different population groups due to admixture. We use a genome-wide, haplotype-based, analysis to characterise the structure of genetic diversity and gene-flow in a collection of 48 sub-Saharan African groups. We show that coastal populations experienced an influx of Eurasian haplotypes over the last 7000 years, and that Eastern and Southern Niger-Congo speaking groups share ancestry with Central West Africans as a result of recent population expansions. In fact, most sub-Saharan populations share ancestry with groups from outside of their current geographic region as a result of gene-flow within the last 4000 years. Our in-depth analysis provides insight into haplotype sharing across different ethno-linguistic groups and the recent movement of alleles into new environments, both of which are relevant to studies of genetic epidemiology.

Uyoga S, Ndila CM, Macharia AW, Nyutu G, Shah S, Peshu N, Clarke GM, Kwiatkowski DP, Rockett KA, Williams TN, MalariaGEN Consortium. 2015. Glucose-6-phosphate dehydrogenase deficiency and the risk of malaria and other diseases in children in Kenya: a case-control and a cohort study. Lancet Haematol, 2 (10), pp. e437-e444. | Show Abstract | Read more

BACKGROUND: The global prevalence of X-linked glucose-6-phosphate dehydrogenase (G6PD) deficiency is thought to be a result of selection by malaria, but epidemiological studies have yielded confusing results. We investigated the relationships between G6PD deficiency and both malaria and non-malarial illnesses among children in Kenya. METHODS: We did this study in Kilifi County, Kenya, where the G6PD c.202T allele is the only significant cause of G6PD deficiency. We tested the associations between G6PD deficiency and severe and complicated Plasmodium falciparum malaria through a case-control study of 2220 case and 3940 control children. Cases were children aged younger than 14 years, who visited the high dependency ward of Kilifi County Hospital with severe malaria between March 1, 1998, and Feb 28, 2010. Controls were children aged between 3-12 months who were born within the same study area between August 2006, and September 2010. We assessed the association between G6PD deficiency and both uncomplicated malaria and other common diseases of childhood in a cohort study of 752 children aged younger than 10 years. Participants of this study were recruited from a representative sample of households within the Ngerenya and Chonyi areas of Kilifi County between Aug 1, 1998, and July 31, 2001. The primary outcome measure for the case-control study was the odds ratio for hospital admission with severe malaria (computed by logistic regression) while for the cohort study it was the incidence rate ratio for uncomplicated malaria and non-malaria illnesses (computed by Poisson regression), by G6PD deficiency category. FINDINGS: 2863 (73%) children in the control group versus 1643 (74%) in the case group had the G6PD normal genotype, 639 (16%) versus 306 (14%) were girls heterozygous for G6PD c.202T, and 438 (11%) versus 271 (12%) children were either homozygous girls or hemizygous boys. Compared with boys and girls without G6PD deficiency, we found significant protection from severe malaria (odds ratio [OR] 0·82, 95% CI 0·70-0·97; p=0·020) among G6PD c.202T heterozygous girls but no evidence for protection among G6PD c.202T hemizygous boys and homozygous girls (OR 1·18, 0·99-1·40; p=0·056). Median follow-up for the mild disease cohort study was 2·24 years (IQR 2·22-2·85). G6PD c.202T had no effect on other common diseases of childhood in heterozygous girls (incidence rate ratio 0·98, 95% CI 0·86-1·11; p=0·82) or homozygous girls or hemizygous boys (0·93, 0·82-1·04; p=0·25), with the sole exception of a marginally significant increase in the incidence of helminth infections among heterozygous girls. INTERPRETATION: Heterozygous girls might be the driving force for the positive selection of G6PD deficiency alleles. Further studies are needed to definitively establish the mechanisms by which G6PD deficiency confers an advantage against malaria in heterozygous individuals. Such studies could lead to the development of new treatments. FUNDING: Wellcome Trust, UK Medical Research Council, European Union, and Foundation for the National Institutes of Health (as part of the Bill & Melinda Gates Grand Challenges in Global Health Initiative).

Chertow JH, Alkaitis MS, Nardone G, Ikeda AK, Cunnington AJ, Okebe J, Ebonyi AO, Njie M, Correa S, Jayasooriya S et al. 2015. Plasmodium Infection Is Associated with Impaired Hepatic Dimethylarginine Dimethylaminohydrolase Activity and Disruption of Nitric Oxide Synthase Inhibitor/Substrate Homeostasis. PLoS Pathog, 11 (9), pp. e1005119. | Show Abstract | Read more

Inhibition of nitric oxide (NO) signaling may contribute to pathological activation of the vascular endothelium during severe malaria infection. Dimethylarginine dimethylaminohydrolase (DDAH) regulates endothelial NO synthesis by maintaining homeostasis between asymmetric dimethylarginine (ADMA), an endogenous NO synthase (NOS) inhibitor, and arginine, the NOS substrate. We carried out a community-based case-control study of Gambian children to determine whether ADMA and arginine homeostasis is disrupted during severe or uncomplicated malaria infections. Circulating plasma levels of ADMA and arginine were determined at initial presentation and 28 days later. Plasma ADMA/arginine ratios were elevated in children with acute severe malaria compared to 28-day follow-up values and compared to children with uncomplicated malaria or healthy children (p<0.0001 for each comparison). To test the hypothesis that DDAH1 is inactivated during Plasmodium infection, we examined DDAH1 in a mouse model of severe malaria. Plasmodium berghei ANKA infection inactivated hepatic DDAH1 via a post-transcriptional mechanism as evidenced by stable mRNA transcript number, decreased DDAH1 protein concentration, decreased enzyme activity, elevated tissue ADMA, elevated ADMA/arginine ratio in plasma, and decreased whole blood nitrite concentration. Loss of hepatic DDAH1 activity and disruption of ADMA/arginine homeostasis may contribute to severe malaria pathogenesis by inhibiting NO synthesis.

Huang H, Ideh RC, Gitau E, Thézénas ML, Jallow M, Ebruke B, Chimah O, Oluwalana C, Karanja H, Mackenzie G et al. 2014. Discovery and validation of biomarkers to guide clinical management of pneumonia in African children. Clin Infect Dis, 58 (12), pp. 1707-1715. | Show Abstract | Read more

BACKGROUND: Pneumonia is the leading cause of death in children globally. Clinical algorithms remain suboptimal for distinguishing severe pneumonia from other causes of respiratory distress such as malaria or distinguishing bacterial pneumonia and pneumonia from others causes, such as viruses. Molecular tools could improve diagnosis and management. METHODS: We conducted a mass spectrometry-based proteomic study to identify and validate markers of severity in 390 Gambian children with pneumonia (n = 204) and age-, sex-, and neighborhood-matched controls (n = 186). Independent validation was conducted in 293 Kenyan children with respiratory distress (238 with pneumonia, 41 with Plasmodium falciparum malaria, and 14 with both). Predictive value was estimated by the area under the receiver operating characteristic curve (AUC). RESULTS: Lipocalin 2 (Lpc-2) was the best protein biomarker of severe pneumonia (AUC, 0.71 [95% confidence interval, .64-.79]) and highly predictive of bacteremia (78% [64%-92%]), pneumococcal bacteremia (84% [71%-98%]), and "probable bacterial etiology" (91% [84%-98%]). These results were validated in Kenyan children with severe malaria and respiratory distress who also met the World Health Organization definition of pneumonia. The combination of Lpc-2 and haptoglobin distinguished bacterial versus malaria origin of respiratory distress with high sensitivity and specificity in Gambian children (AUC, 99% [95% confidence interval, 99%-100%]) and Kenyan children (82% [74%-91%]). CONCLUSIONS: Lpc-2 and haptoglobin can help discriminate the etiology of clinically defined pneumonia and could be used to improve clinical management. These biomarkers should be further evaluated in prospective clinical studies.

Huang H, Lamikanra AA, Alkaitis MS, Thézénas ML, Ramaprasad A, Moussa E, Roberts DJ, Casals-Pascual C. 2014. Interleukin-10 regulates hepcidin in Plasmodium falciparum malaria. PLoS One, 9 (2), pp. e88408. | Show Abstract | Read more

BACKGROUND: Acute malarial anemia remains a major public health problem. Hepcidin, the major hormone controlling the availability of iron, is raised during acute and asymptomatic parasitemia. Understanding the role and mechanism of raised hepcidin and so reduced iron availability during infection is critical to establish evidence-based guidelines for management of malaria anemia. Our recent clinical evidence suggests a potential role of IL-10 in the regulation of hepcidin in patients with acute P. falciparum malaria. METHODS: We have measured secretion of hepcidin by primary macrophages and the hepatoma cell line HepG2 stimulated with IL-10, IL-6 and Plasmodium falciparum-infected erythrocytes. FINDINGS: We have observed that IL-10 and IL-6 production increased in primary macrophages when these cells were co-cultured with Plasmodium falciparum-infected erythrocytes. We found that IL-10 induced hepcidin secretion in primary macrophages in a dose-dependent manner but not in HepG2 cells. These effects were mediated through signal transducer and activator of transcription (STAT) 3-phosphorylation and completely abrogated by a specific STAT3 inhibitor. CONCLUSION: IL-10 can directly regulate hepcidin in primary macrophages but not in HepG2 cells. This effect can be modulated by Plasmodium falciparum. The results are consistent with a role for IL-10 in modulating iron metabolism during acute phase of infection.

Kaisar M, Huang H, Akhtar ZM, Leuvenink HGD, Casals-Pascual C, Ploeg RJ. 2013. MOLECULAR MARKERS AS DIAGNOSTIC TOOLS OF DONOR ORGAN QUALITY AND PREDICTORS OF TRANSPLANTATION OUTCOME TRANSPLANT INTERNATIONAL, 26 pp. 36-36.

Thézénas ML, Huang H, Njie M, Ramaprasad A, Nwakanma DC, Fischer R, Digleria K, Walther M, Conway DJ, Kessler BM, Casals-Pascual C. 2013. PfHPRT: a new biomarker candidate of acute Plasmodium falciparum infection. J Proteome Res, 12 (3), pp. 1211-1222. | Show Abstract | Read more

Plasmodium falciparum is a protozoan parasite that causes human malaria. This parasitic infection accounts for approximately 655,000 deaths each year worldwide. Most deaths could be prevented by diagnosing and treating malaria promptly. To date, few parasite proteins have been developed into rapid diagnostic tools. We have combined a shotgun and a targeted proteomic strategy to characterize the plasma proteome of Gambian children with severe malaria (SM), mild malaria, and convalescent controls in search of new candidate biomarkers. Here we report four P. falciparum proteins with a high level of confidence in SM patients, namely, PF10_0121 (hypoxanthine phosphoribosyltransferase, pHPRT), PF11_0208 (phosphoglycerate mutase, pPGM), PF13_0141 (lactate dehydrogenase, pLDH), and PF14_0425 (fructose bisphosphate aldolase, pFBPA). We have optimized selected reaction monitoring (SRM) assays to quantify these proteins in individual patients. All P. falciparum proteins were higher in SM compared with mild cases or control subjects. SRM-based measurements correlated markedly with clinical anemia (low blood hemoglobin concentration), and pLDH and pFBPA were significantly correlated with higher P. falciparum parasitemia. These findings suggest that pHPRT is a promising biomarker to diagnose P. falciparum malaria infection. The diagnostic performance of this marker should be validated prospectively.

Jallow M, Casals-Pascual C, Ackerman H, Walther B, Walther M, Pinder M, Sisay-Joof F, Usen S, Jallow M, Abubakar I et al. 2012. Clinical features of severe malaria associated with death: a 13-year observational study in the Gambia. PLoS One, 7 (9), pp. e45645. | Show Abstract | Read more

BACKGROUND: Severe malaria (SM) is a major cause of death in sub-Saharan Africa. Identification of both specific and sensitive clinical features to predict death is needed to improve clinical management. METHODS: A 13-year observational study was conducted from 1997 through 2009 of 2,901 children with SM enrolled at the Royal Victoria Teaching Hospital in The Gambia to identify sensitive and specific predictors of poor outcome in Gambian children with severe malaria between the ages 4 months to 14 years. We have measured the sensitivity and specificity of clinical features that predict death or development of neurological sequelae. FINDINGS: Impaired consciousness (odds ratio {OR} 4.4 [95% confidence interval {CI}, 2.7-7.3]), respiratory distress (OR 2.4 [95%CI, 1.7-3.2]), hypoglycemia (OR 1.7 [95%CI, 1.2-2.3]), jaundice (OR 1.9 [95%CI, 1.2-2.9]) and renal failure (OR 11.1 [95%CI, 3.3-36.5]) were independently associated with death in children with SM. The clinical features that showed the highest sensitivity and specificity to predict death were respiratory distress (area under the curve 0.63 [95%CI, 0.60-0.65]) and impaired consciousness (AUC 0.61[95%CI, 0.59-0.63]), which were comparable to the ability of hyperlactatemia (blood lactate>5 mM) to predict death (AUC 0.64 [95%CI, 0.55-0.72]). A Blantyre coma score (BCS) of 2 or less had a sensitivity of 74% and specificity of 67% to predict death (AUC 0.70 [95% C.I. 0.68-0.72]), and sensitivity and specificity of 74% and 69%, respectively to predict development of neurological sequelae (AUC 0.72 [95% CI, 0.67-0.76]).The specificity of this BCS threshold to identify children at risk of dying improved in children less than 3 years of age (AUC 0.74, [95% C.I 0.71-0.76]). CONCLUSION: The BCS is a quantitative predictor of death. A BCS of 2 or less is the most sensitive and specific clinical feature to predict death or development of neurological sequelae in children with SM.

Casals-Pascual C, Huang H, Lakhal-Littleton S, Thezenas ML, Kai O, Newton CRJC, Roberts DJ. 2012. Hepcidin demonstrates a biphasic association with anemia in acute Plasmodium falciparum malaria. Haematologica, 97 (11), pp. 1695-1698. | Show Abstract | Read more

Hepcidin levels are high and iron absorption is limited in acute malaria. The mechanism(s) that regulate hepcidin secretion remain undefined. We have measured hepcidin concentration and cytokines in 100 Kenyan children with acute falciparum malaria and different degrees of anemia. Hepcidin was increased on admission and fell significantly one week and one month after treatment. The association of hepcidin with hemoglobin was not linear and hepcidin was very low in severe malarial anemia. Parasite density, IL-10 and IL-6 were significantly associated with hepcidin concentration. Hepcidin response to acute malaria supports the notion of iron sequestration during acute malaria infection and suggests that iron administration during acute malaria is futile. These data suggest iron supplementation policies should take into account the high hepcidin levels and probable poor utilization of iron for up to one week after treatment for the majority of patients with acute malaria.

Huang H, Mackeen MM, Cook M, Oriero E, Locke E, Thézénas ML, Kessler BM, Nwakanma D, Casals-Pascual C. 2012. Proteomic identification of host and parasite biomarkers in saliva from patients with uncomplicated Plasmodium falciparum malaria. Malar J, 11 (1), pp. 178. | Show Abstract | Read more

BACKGROUND: Malaria cases attributed to Plasmodium falciparum account for approximately 600,000 deaths yearly, mainly in African children. The gold standard method to diagnose malaria requires the visualization of the parasite in blood. The role of non-invasive diagnostic methods to diagnose malaria remains unclear. METHODS: A protocol was optimized to deplete highly abundant proteins from saliva to improve the dynamic range of the proteins identified and assess their suitability as candidate biomarkers of malaria infection. A starch-based amylase depletion strategy was used in combination with four different lectins to deplete glycoproteins (Concanavalin A and Aleuria aurantia for N-linked glycoproteins; jacalin and peanut agglutinin for O-linked glycoproteins). A proteomic analysis of depleted saliva samples was performed in 17 children with fever and a positive-malaria slide and compared with that of 17 malaria-negative children with fever. RESULTS: The proteomic signature of malaria-positive patients revealed a strong up-regulation of erythrocyte-derived and inflammatory proteins. Three P. falciparum proteins, PFL0480w, PF08_0054 and PFI0875w, were identified in malaria patients and not in controls. Aleuria aurantia and jacalin showed the best results for parasite protein identification. CONCLUSIONS: This study shows that saliva is a suitable clinical specimen for biomarker discovery. Parasite proteins and several potential biomarkers were identified in patients with malaria but not in patients with other causes of fever. The diagnostic performance of these markers should be addressed prospectively.

Ackerman HC, Carroll RW, Casals-Pascual C. 2012. A better biomarker for cerebral malaria: in the eye of the beheld?*. Crit Care Med, 40 (3), pp. 1018-1020. | Read more

Ceesay SJ, Casals-Pascual C, Nwakanma DC, Walther M, Gomez-Escobar N, Fulford AJC, Takem EN, Nogaro S, Bojang KA, Corrah T et al. 2010. Continued decline of malaria in The Gambia with implications for elimination. PLoS One, 5 (8), pp. e12242. | Show Abstract | Read more

BACKGROUND: A substantial decline in malaria was reported to have occurred over several years until 2007 in the western part of The Gambia, encouraging consideration of future elimination in this previously highly endemic region. Scale up of interventions has since increased with support from the Global Fund and other donors. METHODOLOGY/PRINCIPAL FINDINGS: We continued to examine laboratory records at four health facilities previously studied and investigated six additional facilities for a 7 year period, adding data from 243,707 slide examinations, to determine trends throughout the country until the end of 2009. We actively detected infections in a community cohort of 800 children living in rural villages throughout the 2008 malaria season, and assayed serological changes in another rural population between 2006 and 2009. Proportions of malaria positive slides declined significantly at all of the 10 health facilities between 2003 (annual mean across all sites, 38.7%) and 2009 (annual mean, 7.9%). Statistical modelling of trends confirmed significant seasonality and decline over time at each facility. Slide positivity was lowest in 2009 at all sites, except two where lowest levels were observed in 2006. Mapping households of cases presenting at the latter sites in 2007-2009 indicated that these were not restricted to a few residual foci. Only 2.8% (22/800) of a rural cohort of children had a malaria episode in the 2008 season, and there was substantial serological decline between 2006 and 2009 in a separate rural area. CONCLUSIONS: Malaria has continued to decline in The Gambia, as indicated by a downward trend in slide positivity at health facilities, and unprecedented low incidence and seroprevalence in community surveys. We recommend intensification of control interventions for several years to further reduce incidence, prior to considering an elimination programme.

Casals-Pascual C, Idro R, Picot S, Roberts DJ, Newton CRJC. 2009. Can erythropoietin be used to prevent brain damage in cerebral malaria? Trends Parasitol, 25 (1), pp. 30-36. | Show Abstract | Read more

Erythropoietin (Epo) modulates the survival of developing erythroid cells and the production of new erythrocytes in the bone marrow and is a key molecule in the adaptation to hypoxia and anaemia. Epo receptors have been found to be widely expressed on non-haematopoietic cells, and Epo has been shown to have diverse actions (in particular, preventing ischaemic damage to tissues of the central nervous system). Recently, Epo has been shown to improve the outcome in a murine model of malaria, and high plasma levels of Epo in children with cerebral malaria were associated with a better outcome. Here, we review the biological importance of Epo, its mechanisms of action and the rationale for the proposed use of Epo as an adjunct treatment in cerebral malaria.

Ceesay SJ, Casals-Pascual C, Erskine J, Anya SE, Duah NO, Fulford AJC, Sesay SSS, Abubakar I, Dunyo S, Sey O et al. 2008. Changes in malaria indices between 1999 and 2007 in The Gambia: a retrospective analysis. Lancet, 372 (9649), pp. 1545-1554. | Show Abstract | Read more

BACKGROUND: Malaria is a major cause of morbidity and mortality in Africa. International effort and funding for control has been stepped up, with substantial increases from 2003 in the delivery of malaria interventions to pregnant women and children younger than 5 years in The Gambia. We investigated the changes in malaria indices in this country, and the causes and public-health significance of these changes. METHODS: We undertook a retrospective analysis of original records to establish numbers and proportions of malaria inpatients, deaths, and blood-slide examinations at one hospital over 9 years (January, 1999-December, 2007), and at four health facilities in three different administrative regions over 7 years (January, 2001-December, 2007). We obtained additional data from single sites for haemoglobin concentrations in paediatric admissions and for age distribution of malaria admissions. FINDINGS: From 2003 to 2007, at four sites with complete slide examination records, the proportions of malaria-positive slides decreased by 82% (3397/10861 in 2003 to 337/6142 in 2007), 85% (137/1259 to 6/368), 73% (3664/16932 to 666/11333), and 50% (1206/3304 to 336/1853). At three sites with complete admission records, the proportions of malaria admissions fell by 74% (435/2530 to 69/1531), 69% (797/2824 to 89/1032), and 27% (2204/4056 to 496/1251). Proportions of deaths attributed to malaria in two hospitals decreased by 100% (seven of 115 in 2003 to none of 117 in 2007) and 90% (22/122 in 2003 to one of 58 in 2007). Since 2004, mean haemoglobin concentrations for all-cause admissions increased by 12 g/L (85 g/L in 2000-04 to 97 g/L in 2005-07), and mean age of paediatric malaria admissions increased from 3.9 years (95% CI 3.7-4.0) to 5.6 years (5.0-6.2). INTERPRETATION: A large proportion of the malaria burden has been alleviated in The Gambia. Our results encourage consideration of a policy to eliminate malaria as a public-health problem, while emphasising the importance of accurate and continuous surveillance.

Casals-Pascual C, Idro R, Gicheru N, Gwer S, Kitsao B, Gitau E, Mwakesi R, Roberts DJ, Newton CRJC. 2008. High levels of erythropoietin are associated with protection against neurological sequelae in African children with cerebral malaria. Proc Natl Acad Sci U S A, 105 (7), pp. 2634-2639. | Show Abstract | Read more

Cerebral malaria (CM) in children is associated with a high mortality and long-term neurocognitive sequelae. Both erythropoietin (Epo) and vascular endothelial growth factor (VEGF) have been shown to be neuroprotective. We hypothesized that high plasma and cerebrospinal fluid (CSF) levels of these cytokines would prevent neurological sequelae in children with CM. We measured Epo, VEGF, and tumor necrosis factor in paired samples of plasma and CSF of Kenyan children admitted with CM. Logistic regression models were used to identify risk and protective factors associated with the development of neurological sequelae. Children with CM (n = 124) were categorized into three groups: 76 without sequelae, 32 with sequelae, and 16 who died. Conditional logistic regression analysis matching the 32 patients with CM and neurological sequelae to 64 patients with CM without sequelae stratified for hemoglobin level estimated that plasma Epo (>200 units/liter) was associated with >80% reduction in the risk of developing neurological sequelae [adjusted odds ratio (OR) 0.18; 95% C.I. 0.05-0.93; P = 0.041]. Admission with profound coma (adjusted OR 5.47; 95% C.I. 1.45-20.67; P = 0.012) and convulsions after admission (adjusted OR 16.35; 95% C.I. 2.94-90.79; P = 0.001) were also independently associated with neurological sequelae. High levels of Epo were associated with reduced risk of neurological sequelae in children with CM. The age-dependent Epo response to anemia and the age-dependent protective effect may influence the clinical epidemiology of CM. These data support further study of Epo as an adjuvant therapy in CM.

Lamikanra AA, Brown D, Potocnik A, Casals-Pascual C, Langhorne J, Roberts DJ. 2007. Malarial anemia: of mice and men. Blood, 110 (1), pp. 18-28. | Show Abstract | Read more

Severe malaria is manifest by a variety of clinical syndromes dependent on properties of both the host and the parasite. In young infants, severe malarial anemia (SMA) is the most common syndrome of severe disease and contributes substantially to the considerable mortality and morbidity from malaria. There is now growing evidence, from both human and mouse studies of malaria, to show that anemia is due not only to increased hemolysis of infected and clearance of uninfected red blood cells (RBCs) but also to an inability of the infected host to produce an adequate erythroid response. In this review, we will summarize the recent clinical and experimental studies of malaria to highlight similarities and differences in human and mouse pathology that result in anemia and so inform the use of mouse models in the study of severe malarial anemia in humans.

Cheung JOP, Casals-Pascual C, Roberts DJ, Watt SM. 2007. A small-scale serum-free liquid cell culture model of erythropoiesis to assess the effects of exogenous factors. J Immunol Methods, 319 (1-2), pp. 104-117. | Show Abstract | Read more

Anaemia is an important global health problem. Therefore, it is crucial to understand its pathophysiology in various genetic or infectious diseases where dyserythropoiesis is a key pathological feature. To this effect, reproducible and reliable models of erythropoiesis in vitro are much needed as investigative tools. We have developed a serum-free liquid culture model of erythropoiesis using human umbilical cord blood CD34(+) cells cultured in the cytokine combination, interleukin-3 (IL-3), IL-6, stem cell factor (SCF) and erythropoietin (Epo), over 14 days. We found that these culture conditions favored erythroid differentiation over the expansion of the more primitive erythroid precursors. With an initiating culture density of 5x10(4) cells per ml, the nucleated cell fold expansion increased from 7.9+/-3.9 (range 4.5 to 11.1) after 4 days to 2990.2+/-1936.1 (range 626.6 to 6912.0) after 14 days in culture. Day-14 burst-forming unit-erythroid (BFU-E) frequencies peaked at day 4 (24.0+/-8.9%), with a marked decrease in BFU-E burst size as the cultures progressed. Time-course immunophenotypical profiles were characteristically erythroid with a decrease in CD34 expression (from 96.8+/-3.0% at day 0 to 0.8+/-0.8% at day 14), and a concomitant increase in the expression of erythroid-specific markers, CD36, glycophorin A (GpA) and CD71 (from 14.8+/-5.0%, 1.7+/-1.0% and 37.9+/-18.0% to 93.0+/-7.0%, 82.1+/-14.0% and 95.7+/-3.0%, respectively). Morphological studies revealed the presence of normoblasts with the complete absence of reticulocytes and mature erythrocytes after 14 days in culture. Once the culture conditions were optimized, we scaled down our culture model from 24-well plate (large-scale) to 96-well plate cultures (small-scale). We found that the small-scale cultures compared favorably with their large-scale counterpart in terms of erythroid progenitor cell proliferation and differentiation, particularly at low CD34(+) initiating cell doses. By using tumor necrosis factor-alpha (TNF-alpha), a known inhibitor of erythropoiesis, we validated our model system and showed a dose-dependent inhibition of erythroid differentiation with TNF-alpha in our cultures. Therefore, our results demonstrate a small-scale serum-free liquid culture model of erythropoiesis that is comparable with and complements our well-defined large-scale model. Our system would prove useful for screening the effects of exogenous factors on erythropoiesis in vitro.

Casals-Pascual C, Kai O, Cheung JOP, Williams S, Lowe B, Nyanoti M, Williams TN, Maitland K, Molyneux M, Newton CRJC et al. 2006. Suppression of erythropoiesis in malarial anemia is associated with hemozoin in vitro and in vivo. Blood, 108 (8), pp. 2569-2577. | Show Abstract | Read more

Malarial anemia is a global public health problem and is characterized by a low reticulocyte response in the presence of life-threatening hemolysis. Although cytokines, in particular tumor necrosis factor-alpha (TNF-alpha), can suppress erythropoiesis, the grossly abnormal bone marrow morphology indicates that other factors may contribute to ineffective erythropoiesis. We hypothesized that the cytotoxic hemozoin (Hz) residues from digested hemoglobin (Hb) significantly contribute to abnormal erythropoiesis. Here, we show that not only isolated Hz, but also delipidated Hz, inhibits erythroid development in vitro in the absence of TNF-alpha. However, when added to cultures, TNF-alpha synergizes with Hz to inhibit erythropoiesis. Furthermore, we show that, in children with malarial anemia, the proportion of circulating monocytes containing Hz is associated with anemia (P < .001) and reticulocyte suppression (P = .009), and that this is independent of the level of circulating cytokines, including TNF-alpha. Plasma Hz is also associated with anemia (P < .001) and reticulocyte suppression (P = .02). Finally, histologic examination of the bone marrow of children who have died from malaria shows that pigmented erythroid and myeloid precursors are associated with the degree of abnormal erythroid development. Taken together, these observations provide compelling evidence for inhibition of erythropoiesis by Hz.

Casals-Pascual C, Kai O, Newton CRJC, Peshu N, Roberts DJ. 2006. Thrombocytopenia in falciparum malaria is associated with high concentrations of IL-10. Am J Trop Med Hyg, 75 (3), pp. 434-436. | Show Abstract | Read more

Over the last decade, a number of observations have suggested that platelets may play a role in the pathophysiology of severe malaria. However, somewhat paradoxically, thrombocytopenia is not associated clearly with outcome. We studied the relationship between thrombocytopenia and cytokines in Kenyan children with severe malaria and showed that thrombocytopenia (platelet count < 150 x 10(9)/L) strongly correlates with high levels of interleukin (IL)-10. Several studies have shown that high levels of IL-10 are associated with a favorable outcome in severe malaria. Taken together, these data suggest why thrombocytopenia has a complex relationship with severe disease and suggest one mechanism whereby IL-10 may modify the outcome of severe disease.

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Casals-Pascual C, Kai O, Newton CRJC, Peshu N, Roberts DJ. 2006. Short report: Thrombocytopenia in falciparum malaria is associated with high concentrations of IL-10 AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, 75 (3), pp. 434-436. | Show Abstract | Read more

Over the last decade, a number of observations have suggested that platelets may play a role in the pathophysiology of severe malaria. However, somewhat paradoxically, thrombocytopenia is not associated clearly with outcome. We studied the relationship between thrombocytopenia and cytokines in Kenyan children with severe malaria and showed that thrombocytopenia (platelet count < 150 × 109/L) strongly correlates with high levels of interleukin (IL)-10. Several studies have shown that high levels of IL-10 are associated with a favorable outcome in severe malaria. Taken together, these data suggest why thrombocytopenia has a complex relationship with severe disease and suggest one mechanism whereby IL-10 may modify the outcome of severe disease. Copyright © 2006 by The American Society of Tropical Medicine and Hygiene.

Casals-Pascual C, Roberts DJ. 2006. Severe malarial anaemia. Curr Mol Med, 6 (2), pp. 155-168. | Show Abstract | Read more

This review describes the importance of severe malarial anaemia as a public health problem, and the clinical and pathophysiological aspects of this syndrome. The review also highlights the recent advances in our understanding of the epidemiological, clinical, cellular and molecular aspects of severe malarial anaemia.

Casals-Pascual C, Kai O, Lowe B, English M, Williams TN, Maitland K, Newton CRCJ, Peshu N, Roberts DJ. 2006. Lactate levels in severe malarial anaemia are associated with haemozoin-containing neutrophils and low levels of IL-12. Malar J, 5 (1), pp. 101. | Show Abstract | Read more

BACKGROUND: Hyperlactataemia is often associated with a poor outcome in severe malaria in African children. To unravel the complex pathophysiology of this condition the relationship between plasma lactate levels, parasite density, pro- and anti-inflammatory cytokines, and haemozoin-containing leucocytes was studied in children with severe falciparum malarial anaemia. METHODS: Twenty-six children with a primary diagnosis of severe malarial anaemia with any asexual Plasmodium falciparum parasite density and Hb < 5 g/dL were studied and the association of plasma lactate levels and haemozoin-containing leucocytes, parasite density, pro- and anti-inflammatory cytokines was measured. The same associations were measured in non-severe malaria controls (N = 60). RESULTS: Parasite density was associated with lactate levels on admission (r = 0.56, P < 0.005). Moreover, haemozoin-containing neutrophils and IL-12 were strongly associated with plasma lactate levels, independently of parasite density (r = 0.60, P = 0.003 and r = -0.46, P = 0.02, respectively). These associations were not found in controls with uncomplicated malarial anaemia. CONCLUSION: These data suggest that blood stage parasites, haemozoin and low levels of IL-12 may be associated with the development of hyperlactataemia in severe malarial anaemia.

Roberts DJ, Casals-Pascual C, Weatherall DJ. 2005. The clinical and pathophysiological features of malarial anaemia. Curr Top Microbiol Immunol, 295 pp. 137-167. | Show Abstract

This review will focus on the principal clinical and pathophysiological features of the anaemia of falciparum malaria, including the problems of treating malarial anaemia, and also will suggest how recent advances in genomics may help our understanding of cellular and molecular mechanisms underlying this syndrome.

Casals-Pascual C, Roberts DJ. 2004. Malaria and the red cell. Vox Sang, 87 Suppl 2 (s2), pp. 115-119. | Read more

Vargas M, Gascon J, Casals C, Schellenberg D, Urassa H, Kahigwa E, Ruiz J, Vila J. 2004. Etiology of diarrhea in children less than five years of age in Ifakara, Tanzania. Am J Trop Med Hyg, 70 (5), pp. 536-539. | Show Abstract | Read more

A total of 451 stool specimens were collected from children less than five years of age with acute diarrhea from Ifakara, Tanzania and processed to detect bacterial enteropathogens, parasites, and rotaviruses. These specimens were divided into 348 from the dry season and 103 from the rainy season. Overall, diarrheogenic Escherichia coli (35.7%) were the predominant enteropathogens, with enterotoxigenic E. coli, enteroaggregative E. coli, and enteropathogenic E. coli being the most prevalent. Moreover, enteroaggregative E. coli (63% versus 35.5%; P < 0.05), Shigella spp. (24% versus 12%; P < 0.05), and rotavirus (23% versus 4%; P < 0.05) were more prevalent in the dry season than in the rainy season and enterotoxigenic E. coli (51.6% versus 20%; P < 0.05) and Giardia lamblia (14% versus 1%; P < 0.05) were more prevalent in the rainy season.

Casals-Pascual C, Roberts DJ. 2004. Malaria and the red cell Vox Sanguinis, Supplement, 87 (2),

Ruiz J, Navia MM, Casals C, Sierra JM, Jiménez De Anta MT, Vila J. 2003. Integron-mediated antibiotic multiresistance in Acinetobacter baumannii clinical isolates from Spain. Clin Microbiol Infect, 9 (9), pp. 907-911. | Show Abstract | Read more

OBJECTIVE: To determine whether non-epidemiologically related, antibiotic-resistant isolates of Acinetobacter baumannii from different geographical origins possess common type 1 integrons. METHODS: The epidemiologic relationships between seven A. baumannii strains recovered from different Spanish hospitals were established by pulsed-field gel electrophoresis, the presence of integrons being determined by PCR and DNA sequencing. RESULTS: Integron analysis showed the presence of four different integrons, containing six different known genes (aacC1, aacA4, aadA1, aadB, oxa21 and oxa37) plus an ORF. It was found that the same integron was present in different unrelated strains and that related strains could have different integrons. CONCLUSION: These results show the potential risk of integron dissemination among different strains of A. baumannii.

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.

Casals-Pascual C, Allen S, Allen A, Kai O, Lowe B, Pain A, Roberts DJ. 2001. Short report: codon 125 polymorphism of CD31 and susceptibility to malaria. Am J Trop Med Hyg, 65 (6), pp. 736-737. | Show Abstract | Read more

Platelet-endothelial cell adhesion molecule 1 (PECAM-1/CD31) has been identified as an endothelial cell receptor of Plasmodium falciparum-infected erythrocytes. The significance of adhesion of infected erythrocytes to this receptor in malaria infection has not been determined. We have therefore studied the association of the functional mutation CTG-->GTG (Leu-->Val) in codon 125 of the Cd31 gene with severe disease in 2 case-control studies of malaria in Madang Hospital, Papua New Guinea, and in Kilifi District Hospital, Kenya. We analyzed data from 442 cases and controls from Papua New Guinea and data from 396 cases and controls from Kenya. The codon 125 polymorphism was not associated with severe malaria in either study. We conclude that the presence of CTG-->GTG (Leu-->Val) substitution in codon 125 in CD31 is not associated with protection from severe malaria, and we suggest that selective forces other than malaria may maintain this high-frequency polymorphism.

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.

Acosta CJ, Galindo CM, Kimario J, Senkoro K, Urassa H, Casals C, Corachán M, Eseko N, Tanner M, Mshinda H et al. 2001. Cholera outbreak in southern Tanzania: risk factors and patterns of transmission. Emerg Infect Dis, 7 (3 Suppl), pp. 583-587. | Show Abstract | Read more

To identify risk factors and describe the pattern of spread of the 1997 cholera epidemic in a rural area (Ifakara) in southern Tanzania, we conducted a prospective hospital-based, matched case- control study, with analysis based on the first 180 cases and 360 matched controls. Bathing in the river, long distance to water source, and eating dried fish were significantly associated with risk for cholera. Toxigenic Vibrio cholerae O1, biotype El Tor, serotype Ogawa, was isolated in samples from Ifakara's main water source and patients' stools. DNA molecular analyses showed identical patterns for all isolates.

Gascón J, Vargas M, Schellenberg D, Urassa H, Casals C, Kahigwa E, Aponte JJ, Mshinda H, Vila J. 2000. Diarrhea in children under 5 years of age from Ifakara, Tanzania: a case-control study. J Clin Microbiol, 38 (12), pp. 4459-4462. | Show Abstract

A matched case-control study was conducted in the Maternal and Child Health Clinic (MCH) in Ifakara, Tanzania, during the rainy season in order to elucidate the risk factors for and etiology of diarrheal diseases in children under 5 years of age. Cases (103) and controls (206) were matched for sex and age group. Precoded questionnaires with demographic details, clinical history, and physical signs were completed. Stools samples were collected for bacterial, parasitological, and viral studies. A high number of siblings (odds ratio [OR], 0.86; P = 0.027), the number of siblings surviving (OR, 0.82; P = 0.007), the birth order (OR, 0.85; P = 0.018) and the distance from the house to the water source (OR, 0.33; P = 0.011) were associated with the risk of diarrhea. There were high rates of enteropathogen isolates in stool samples from children without diarrhea (52.23%). Shigella species were the only enteropathogen statistically related with diarrhea (OR, 2.90; P < 0.029). Enterotoxigenic, enteropathogenic, and enteroaggregative strains of Escherichia coli were not related with diarrhea, and neither were Giardia lamblia or Salmonella species.

Vila J, Vargas M, Casals C, Urassa H, Mshinda H, Schellemberg D, Gascon J. 1999. Antimicrobial resistance of diarrheagenic Escherichia coli isolated from children under the age of 5 years from Ifakara, Tanzania. Antimicrob Agents Chemother, 43 (12), pp. 3022-3024. | Show Abstract | Read more

Diarrhea caused by multidrug-resistant bacteria is an important public health problem among children in developing countries. The prevalence and antimicrobial susceptibility of diarrheagenic Escherichia coli in 346 children under 5 years of age in Ifakara, Tanzania, were studied. Thirty-eight percent of the cases of diarrhea were due to multiresistant enterotoxigenic E. coli, enteroaggregative E. coli, or enteropathogenic E. coli. Strains of all three E. coli categories showed high-level resistance to ampicillin, tetracycline, co-trimoxazole, and chloramphenicol but were highly susceptible to quinolones. Guidelines for appropriate use of antibiotics in developing countries need updating.

Vila J, Navia M, Ruiz J, Casals C. 1997. Cloning and nucleotide sequence analysis of a gene encoding an OXA-derived beta-lactamase in Acinetobacter baumannii. Antimicrob Agents Chemother, 41 (12), pp. 2757-2759. | Show Abstract | Read more

A clinical strain of Acinetobacter baumannii (strain Ab41) that was resistant to all beta-lactam antibiotics tested except ceftazidime, ceftriaxone, ceftizoxime, and imipenem produced three beta-lactamases: a presumptive chromosomal cephalosporinase, a TEM-1-like beta-lactamase (pI 5.4), and a novel OXA-derived beta-lactamase named OXA-21 (pI 7.0). The gene encoding OXA-21 was located in an integron. The nucleotide sequence showed three mutations compared with the sequence of OXA-3, with two being silent; the nonsilent mutation generated a substitution of Ile-217 to Met.

Font C, Casals C, Kaifi T, Coll E, Cid J, Villalta J, Grau JM. 1997. Constitutional syndrome and lumbar pain. Postgrad Med J, 73 (863), pp. 599-601. | Read more

Font C, Casals C, Kaifi T, Coll E, Cid J, Villalta J, Grau JM. 1997. Constitutional syndrome and lumbar pain - Primary psoas abscess due to Haemophilus aphrophilus. POSTGRADUATE MEDICAL JOURNAL, 73 (863), pp. 599-601. | Read more

Mensa J, Almela M, Casals C, Martínez JA, Marco F, Tomás R, Vidal F, Soriano E, Jiménez de Anta T. 1997. [Yield of blood cultures in relation to the cultured blood volume in Bactec 6A bottles]. Med Clin (Barc), 108 (14), pp. 521-523. | Show Abstract

BACKGROUND: Concentration of microorganisms in blood is low in bacteremia from extravascular sources. The best yield from blood cultures is achieved by culturing a minimum of 10-20 ml, although in some processing blood culture systems such as Bactec NR-860, smaller volume is used. The objectives of the present study were to establish the frequency in which inadequate small blood volumes are employed for culturing and to analyze the relation between the cultured blood volume in Bactec 6A bottles and the yield achieved. MATERIAL AND METHODS: We weighed 2000 Bactec 6A bottles pertaining to consecutive blood cultures obtained from untreated patients with clinical suspicion of Infection. The cultured blood volume was estimated subtracting the mean empty bottle weight. RESULTS: Microorganisms were recovered from 251 bottles (12.5%). One hundred and thirty one (6.8%) isolates were considered as clinically significant and 115 (5.7%) as contaminant. The inoculated blood volume in both significant (5.532 +/- 1.587 ml) and non-significant (5.471 +/- 1.563 ml) recoveries was superior than that of bottles without microbiologic growth (5.209 +/- 1.575 ml, p = 0.016 and p = 0.06, respectively). A linear positive trend was found between the cultivated blood volume and the rate of recoveries (p = 0.008). Within the range of 1 up to 10 ml, the rate of recoveries increased 2.28% for each additional ml of cultivated blood (r = 0.953, p < 0.0001). Out of the 2,000 weighed bottles 127 (6.3%) contained less than 3 ml of blood and 576 (29%) between 3 and 5 ml. CONCLUSIONS: We have proved that the rate of recoveries from Bactec 6A bottles increased with the volume of cultured blood. In untreated patients, this increase is maintained up to volumes of 7 to 10 ml.

Martínez JA, Mensa J, Marco F, Almela M, Lopez J, Casals C, Soriano E, Jiménez de Anta MT. 1997. Risk factors for oxacillin/methicillin resistance in coagulase-negative staphylococci. J Hosp Infect, 35 (4), pp. 295-299. | Show Abstract | Read more

The clinical variables associated with isolation of oxacillin- and methicillin-resistant, coagulase-negative staphylococci (CNS) from blood cultures of hospitalized patients were studied. One hundred CNS strains (49 oxacillin-susceptible; 51 oxacillin-resistant) isolated consecutively from one of two or more sets of blood cultures were collected. Only two variables were independently associated with recovery of oxacillin/methicillin-resistant strains by a multivariate analysis: length of hospital stay > 10 days (OR 5.2, 95% CI = 1.7-15.7), and administration of antimicrobial agents in the previous 14 days (OR 4.5, 95% CI = 1.7-11.7). Analysis of the antibiotics administered indicated that only beta-lactams were associated with a statistically significant risk of resistance to oxacillin/methicillin (OR of beta-lactams vs no antibiotics = 6.94, 95% CI = 1.9-25.3; OR of non-beta-lactams vs no antibiotics = 2.64, 95% CI = 0.8-8.3). Length of hospital stay (especially > 10 days) and prior administration of antimicrobial agents (mainly beta-lactams) independently predicted the presence of oxacillin/methicillin-resistant CNS in blood cultures.

Mensa J, Almela M, Casals C, Martínez JA, Marco F, Tomás R, Vidal F, Soriano E, Jiménez De Anta T. 1997. Yield of blood cultures in relation to the cultured blood volume in Bactec 6A bottles Medicina Clinica, 108 (14), pp. 521-523. | Show Abstract

BACKGROUND: Concentration of microorganisms in blood is low in bacteremia from extravascular sources. The best yield from blood cultures is achieved by culturing a minimun of 10-20 ml, although in some processing blood culture systems such as Bactec NR-860, smaller volume is used. The objectives of the present study were to establish the frequency in which inadequate small blood volumes are employed for culturing and to analyze the relation between the cultured blood volume in Bactec 6A bottles and the yield achieved. MATERIAL AND METHODS: We weighed 2000 Bactec 6A bottles pertaining to consecutive blood cultures obtained from untreated patients with clinical suspicion of infection. The cultured blood volume was estimated subtracting the mean empty bottle weight. RESULTS: Microorganisms were recovered from 251 bottles (12.5%). One hundred and thirty one (6.8%) isolates were considered as clinically significant and 115 (5.7%) as contaminant. The inoculated blood volume in both significant (5.532 ±1.587 ml) and non-significant (5.471 ± 1.563 ml) recoveries was superior than that of bottles without microbiologic growth (5.209 ± 1.575 ml, p = 0.016 and p = 0.06, respectively). A.linear positive trend was found between the cultivated blood volume and the rate of recoveries (p = 0.008). Within the range of 1 up to 10 ml, the rate of recoveries increased 2.28% for each additional ml of cultivated blood (r = 0.953, p < 0.0001). Out of the 2,000 weighed bottles 127 (6.3%) contained less than 3 ml of blood and 576 (29%) between 3 and 5 ml. CONCLUSIONS: We have proved that the rate of recoveries from Bactec 6A bottles increased with the volume of cultured blood. In untreated patients, this increase is mantained up to volumes of 7 to 10 ml.

Vidal F, Mensa J, Almela M, Martínez JA, Marco F, Casals C, Gatell JM, Soriano E, Jimenez de Anta MT. 1996. Epidemiology and outcome of Pseudomonas aeruginosa bacteremia, with special emphasis on the influence of antibiotic treatment. Analysis of 189 episodes. Arch Intern Med, 156 (18), pp. 2121-2126. | Show Abstract | Read more

OBJECTIVE: To evaluate the trend in incidence of Pseudomonas aeruginosa bacteremia, underlying conditions of patients, mortality rate, and factors associated with poor outcome. PATIENTS AND METHODS: Medical charts of 189 consecutive episodes of P aeruginosa bacteremia, detected between January 1, 1991, and December 31, 1994, were prospectively evaluated. Associated risk factors, treatment, and outcome were recorded. RESULTS: Pseudomonas aeruginosa bacteremia represented 5.7% of the total number of bacteremias, 6.9% of nosocomial bacteremias, and 23.6% of nosocomial gram-negative bacteremias. There were 1.5 episodes per 1000 discharges. These numbers were slightly lower than those recorded at our hospital 10 years earlier. Human immunodeficiency virus infection was the most frequent underlying disease (28/189 [15%]). Overall mortality was 18% (34/189). The presence of fatal underlying disease (P < .001), surgery (P = .001), pneumonia (P = .02), and severe sepsis (P < .001) were associated with poor prognosis, the mortality of the patients with these variables being 28%, 28%, 47%, and 62%, respectively. The presence of inappropriate definitive antimicrobial treatment became an independent factor predictive of death (P = .04) only when the subset of patients with intravenous catheter-associated bacteremia was excluded from the analysis. The survival rate was no greater in patients who received 2 or more antibiotics active in vitro against P aeruginosa than in those who received only 1. Neutropenia was not associated with increased mortality. The use of colony-stimulating factors did not affect the outcome of the neutropenic patients. CONCLUSIONS: The rate of P aeruginosa bacteremia is falling slightly at our hospital. The emergence of the human immunodeficiency virus epidemic has had a considerable impact on both epidemiology and mortality. The presence of severe underlying disease, surgery, pneumonia, and, especially, severe sepsis are associated with a poor outcome. With the exclusion of patients with intravenous catheter-associated P aeruginosa bacteremia, the administration of an appropriate antimicrobial therapy is essential to a good outcome. Treatment with 1 active antibiotic seems to be sufficient.

Casals C, Almela M, Marco F, Sort P. 1995. [Pasteurella pneumotropica sepsis]. Med Clin (Barc), 104 (9), pp. 358-359.

CASALS C, ALMELA M, MARCO F, SORT P. 1995. SEPSIS BY PASTEURELLA-PNEUMOTROPICA MEDICINA CLINICA, 104 (9), pp. 358-359.

Casals C, Almela M, Marco F, Sort P. 1995. Pasteurella pneumotropica sepsis Medicina Clinica, 104 (9), pp. 358-359.

Naydenova E, Tsanas A, Howie S, Casals-Pascual C, De Vos M. 2016. The power of data mining in diagnosis of childhood pneumonia. J R Soc Interface, 13 (120), pp. 20160266-20160266. | Show Abstract | Read more

Childhood pneumonia is the leading cause of death of children under the age of 5 years globally. Diagnostic information on the presence of infection, severity and aetiology (bacterial versus viral) is crucial for appropriate treatment. However, the derivation of such information requires advanced equipment (such as X-rays) and clinical expertise to correctly assess observational clinical signs (such as chest indrawing); both of these are often unavailable in resource-constrained settings. In this study, these challenges were addressed through the development of a suite of data mining tools, facilitating automated diagnosis through quantifiable features. Findings were validated on a large dataset comprising 780 children diagnosed with pneumonia and 801 age-matched healthy controls. Pneumonia was identified via four quantifiable vital signs (98.2% sensitivity and 97.6% specificity). Moreover, it was shown that severity can be determined through a combination of three vital signs and two lung sounds (72.4% sensitivity and 82.2% specificity); addition of a conventional biomarker (C-reactive protein) further improved severity predictions (89.1% sensitivity and 81.3% specificity). Finally, we demonstrated that aetiology can be determined using three vital signs and a newly proposed biomarker (lipocalin-2) (81.8% sensitivity and 90.6% specificity). These results suggest that a suite of carefully designed machine learning tools can be used to support multi-faceted diagnosis of childhood pneumonia in resource-constrained settings, compensating for the shortage of expensive equipment and highly trained clinicians.

Huang H, Ideh RC, Gitau E, Thézénas ML, Jallow M, Ebruke B, Chimah O, Oluwalana C, Karanja H, Mackenzie G et al. 2014. Discovery and validation of biomarkers to guide clinical management of pneumonia in African children. Clin Infect Dis, 58 (12), pp. 1707-1715. | Show Abstract | Read more

BACKGROUND: Pneumonia is the leading cause of death in children globally. Clinical algorithms remain suboptimal for distinguishing severe pneumonia from other causes of respiratory distress such as malaria or distinguishing bacterial pneumonia and pneumonia from others causes, such as viruses. Molecular tools could improve diagnosis and management. METHODS: We conducted a mass spectrometry-based proteomic study to identify and validate markers of severity in 390 Gambian children with pneumonia (n = 204) and age-, sex-, and neighborhood-matched controls (n = 186). Independent validation was conducted in 293 Kenyan children with respiratory distress (238 with pneumonia, 41 with Plasmodium falciparum malaria, and 14 with both). Predictive value was estimated by the area under the receiver operating characteristic curve (AUC). RESULTS: Lipocalin 2 (Lpc-2) was the best protein biomarker of severe pneumonia (AUC, 0.71 [95% confidence interval, .64-.79]) and highly predictive of bacteremia (78% [64%-92%]), pneumococcal bacteremia (84% [71%-98%]), and "probable bacterial etiology" (91% [84%-98%]). These results were validated in Kenyan children with severe malaria and respiratory distress who also met the World Health Organization definition of pneumonia. The combination of Lpc-2 and haptoglobin distinguished bacterial versus malaria origin of respiratory distress with high sensitivity and specificity in Gambian children (AUC, 99% [95% confidence interval, 99%-100%]) and Kenyan children (82% [74%-91%]). CONCLUSIONS: Lpc-2 and haptoglobin can help discriminate the etiology of clinically defined pneumonia and could be used to improve clinical management. These biomarkers should be further evaluated in prospective clinical studies.

Huang H, Lamikanra AA, Alkaitis MS, Thézénas ML, Ramaprasad A, Moussa E, Roberts DJ, Casals-Pascual C. 2014. Interleukin-10 regulates hepcidin in Plasmodium falciparum malaria. PLoS One, 9 (2), pp. e88408. | Show Abstract | Read more

BACKGROUND: Acute malarial anemia remains a major public health problem. Hepcidin, the major hormone controlling the availability of iron, is raised during acute and asymptomatic parasitemia. Understanding the role and mechanism of raised hepcidin and so reduced iron availability during infection is critical to establish evidence-based guidelines for management of malaria anemia. Our recent clinical evidence suggests a potential role of IL-10 in the regulation of hepcidin in patients with acute P. falciparum malaria. METHODS: We have measured secretion of hepcidin by primary macrophages and the hepatoma cell line HepG2 stimulated with IL-10, IL-6 and Plasmodium falciparum-infected erythrocytes. FINDINGS: We have observed that IL-10 and IL-6 production increased in primary macrophages when these cells were co-cultured with Plasmodium falciparum-infected erythrocytes. We found that IL-10 induced hepcidin secretion in primary macrophages in a dose-dependent manner but not in HepG2 cells. These effects were mediated through signal transducer and activator of transcription (STAT) 3-phosphorylation and completely abrogated by a specific STAT3 inhibitor. CONCLUSION: IL-10 can directly regulate hepcidin in primary macrophages but not in HepG2 cells. This effect can be modulated by Plasmodium falciparum. The results are consistent with a role for IL-10 in modulating iron metabolism during acute phase of infection.

Thézénas ML, Huang H, Njie M, Ramaprasad A, Nwakanma DC, Fischer R, Digleria K, Walther M, Conway DJ, Kessler BM, Casals-Pascual C. 2013. PfHPRT: a new biomarker candidate of acute Plasmodium falciparum infection. J Proteome Res, 12 (3), pp. 1211-1222. | Show Abstract | Read more

Plasmodium falciparum is a protozoan parasite that causes human malaria. This parasitic infection accounts for approximately 655,000 deaths each year worldwide. Most deaths could be prevented by diagnosing and treating malaria promptly. To date, few parasite proteins have been developed into rapid diagnostic tools. We have combined a shotgun and a targeted proteomic strategy to characterize the plasma proteome of Gambian children with severe malaria (SM), mild malaria, and convalescent controls in search of new candidate biomarkers. Here we report four P. falciparum proteins with a high level of confidence in SM patients, namely, PF10_0121 (hypoxanthine phosphoribosyltransferase, pHPRT), PF11_0208 (phosphoglycerate mutase, pPGM), PF13_0141 (lactate dehydrogenase, pLDH), and PF14_0425 (fructose bisphosphate aldolase, pFBPA). We have optimized selected reaction monitoring (SRM) assays to quantify these proteins in individual patients. All P. falciparum proteins were higher in SM compared with mild cases or control subjects. SRM-based measurements correlated markedly with clinical anemia (low blood hemoglobin concentration), and pLDH and pFBPA were significantly correlated with higher P. falciparum parasitemia. These findings suggest that pHPRT is a promising biomarker to diagnose P. falciparum malaria infection. The diagnostic performance of this marker should be validated prospectively.

Jallow M, Casals-Pascual C, Ackerman H, Walther B, Walther M, Pinder M, Sisay-Joof F, Usen S, Jallow M, Abubakar I et al. 2012. Clinical features of severe malaria associated with death: a 13-year observational study in the Gambia. PLoS One, 7 (9), pp. e45645. | Show Abstract | Read more

BACKGROUND: Severe malaria (SM) is a major cause of death in sub-Saharan Africa. Identification of both specific and sensitive clinical features to predict death is needed to improve clinical management. METHODS: A 13-year observational study was conducted from 1997 through 2009 of 2,901 children with SM enrolled at the Royal Victoria Teaching Hospital in The Gambia to identify sensitive and specific predictors of poor outcome in Gambian children with severe malaria between the ages 4 months to 14 years. We have measured the sensitivity and specificity of clinical features that predict death or development of neurological sequelae. FINDINGS: Impaired consciousness (odds ratio {OR} 4.4 [95% confidence interval {CI}, 2.7-7.3]), respiratory distress (OR 2.4 [95%CI, 1.7-3.2]), hypoglycemia (OR 1.7 [95%CI, 1.2-2.3]), jaundice (OR 1.9 [95%CI, 1.2-2.9]) and renal failure (OR 11.1 [95%CI, 3.3-36.5]) were independently associated with death in children with SM. The clinical features that showed the highest sensitivity and specificity to predict death were respiratory distress (area under the curve 0.63 [95%CI, 0.60-0.65]) and impaired consciousness (AUC 0.61[95%CI, 0.59-0.63]), which were comparable to the ability of hyperlactatemia (blood lactate>5 mM) to predict death (AUC 0.64 [95%CI, 0.55-0.72]). A Blantyre coma score (BCS) of 2 or less had a sensitivity of 74% and specificity of 67% to predict death (AUC 0.70 [95% C.I. 0.68-0.72]), and sensitivity and specificity of 74% and 69%, respectively to predict development of neurological sequelae (AUC 0.72 [95% CI, 0.67-0.76]).The specificity of this BCS threshold to identify children at risk of dying improved in children less than 3 years of age (AUC 0.74, [95% C.I 0.71-0.76]). CONCLUSION: The BCS is a quantitative predictor of death. A BCS of 2 or less is the most sensitive and specific clinical feature to predict death or development of neurological sequelae in children with SM.

Casals-Pascual C, Huang H, Lakhal-Littleton S, Thezenas ML, Kai O, Newton CRJC, Roberts DJ. 2012. Hepcidin demonstrates a biphasic association with anemia in acute Plasmodium falciparum malaria. Haematologica, 97 (11), pp. 1695-1698. | Show Abstract | Read more

Hepcidin levels are high and iron absorption is limited in acute malaria. The mechanism(s) that regulate hepcidin secretion remain undefined. We have measured hepcidin concentration and cytokines in 100 Kenyan children with acute falciparum malaria and different degrees of anemia. Hepcidin was increased on admission and fell significantly one week and one month after treatment. The association of hepcidin with hemoglobin was not linear and hepcidin was very low in severe malarial anemia. Parasite density, IL-10 and IL-6 were significantly associated with hepcidin concentration. Hepcidin response to acute malaria supports the notion of iron sequestration during acute malaria infection and suggests that iron administration during acute malaria is futile. These data suggest iron supplementation policies should take into account the high hepcidin levels and probable poor utilization of iron for up to one week after treatment for the majority of patients with acute malaria.

Huang H, Mackeen MM, Cook M, Oriero E, Locke E, Thézénas ML, Kessler BM, Nwakanma D, Casals-Pascual C. 2012. Proteomic identification of host and parasite biomarkers in saliva from patients with uncomplicated Plasmodium falciparum malaria. Malar J, 11 (1), pp. 178. | Show Abstract | Read more

BACKGROUND: Malaria cases attributed to Plasmodium falciparum account for approximately 600,000 deaths yearly, mainly in African children. The gold standard method to diagnose malaria requires the visualization of the parasite in blood. The role of non-invasive diagnostic methods to diagnose malaria remains unclear. METHODS: A protocol was optimized to deplete highly abundant proteins from saliva to improve the dynamic range of the proteins identified and assess their suitability as candidate biomarkers of malaria infection. A starch-based amylase depletion strategy was used in combination with four different lectins to deplete glycoproteins (Concanavalin A and Aleuria aurantia for N-linked glycoproteins; jacalin and peanut agglutinin for O-linked glycoproteins). A proteomic analysis of depleted saliva samples was performed in 17 children with fever and a positive-malaria slide and compared with that of 17 malaria-negative children with fever. RESULTS: The proteomic signature of malaria-positive patients revealed a strong up-regulation of erythrocyte-derived and inflammatory proteins. Three P. falciparum proteins, PFL0480w, PF08_0054 and PFI0875w, were identified in malaria patients and not in controls. Aleuria aurantia and jacalin showed the best results for parasite protein identification. CONCLUSIONS: This study shows that saliva is a suitable clinical specimen for biomarker discovery. Parasite proteins and several potential biomarkers were identified in patients with malaria but not in patients with other causes of fever. The diagnostic performance of these markers should be addressed prospectively.

Ackerman HC, Carroll RW, Casals-Pascual C. 2012. A better biomarker for cerebral malaria: in the eye of the beheld?*. Crit Care Med, 40 (3), pp. 1018-1020. | Read more

Casals-Pascual C, Idro R, Picot S, Roberts DJ, Newton CRJC. 2009. Can erythropoietin be used to prevent brain damage in cerebral malaria? Trends Parasitol, 25 (1), pp. 30-36. | Show Abstract | Read more

Erythropoietin (Epo) modulates the survival of developing erythroid cells and the production of new erythrocytes in the bone marrow and is a key molecule in the adaptation to hypoxia and anaemia. Epo receptors have been found to be widely expressed on non-haematopoietic cells, and Epo has been shown to have diverse actions (in particular, preventing ischaemic damage to tissues of the central nervous system). Recently, Epo has been shown to improve the outcome in a murine model of malaria, and high plasma levels of Epo in children with cerebral malaria were associated with a better outcome. Here, we review the biological importance of Epo, its mechanisms of action and the rationale for the proposed use of Epo as an adjunct treatment in cerebral malaria.

Ceesay SJ, Casals-Pascual C, Erskine J, Anya SE, Duah NO, Fulford AJC, Sesay SSS, Abubakar I, Dunyo S, Sey O et al. 2008. Changes in malaria indices between 1999 and 2007 in The Gambia: a retrospective analysis. Lancet, 372 (9649), pp. 1545-1554. | Show Abstract | Read more

BACKGROUND: Malaria is a major cause of morbidity and mortality in Africa. International effort and funding for control has been stepped up, with substantial increases from 2003 in the delivery of malaria interventions to pregnant women and children younger than 5 years in The Gambia. We investigated the changes in malaria indices in this country, and the causes and public-health significance of these changes. METHODS: We undertook a retrospective analysis of original records to establish numbers and proportions of malaria inpatients, deaths, and blood-slide examinations at one hospital over 9 years (January, 1999-December, 2007), and at four health facilities in three different administrative regions over 7 years (January, 2001-December, 2007). We obtained additional data from single sites for haemoglobin concentrations in paediatric admissions and for age distribution of malaria admissions. FINDINGS: From 2003 to 2007, at four sites with complete slide examination records, the proportions of malaria-positive slides decreased by 82% (3397/10861 in 2003 to 337/6142 in 2007), 85% (137/1259 to 6/368), 73% (3664/16932 to 666/11333), and 50% (1206/3304 to 336/1853). At three sites with complete admission records, the proportions of malaria admissions fell by 74% (435/2530 to 69/1531), 69% (797/2824 to 89/1032), and 27% (2204/4056 to 496/1251). Proportions of deaths attributed to malaria in two hospitals decreased by 100% (seven of 115 in 2003 to none of 117 in 2007) and 90% (22/122 in 2003 to one of 58 in 2007). Since 2004, mean haemoglobin concentrations for all-cause admissions increased by 12 g/L (85 g/L in 2000-04 to 97 g/L in 2005-07), and mean age of paediatric malaria admissions increased from 3.9 years (95% CI 3.7-4.0) to 5.6 years (5.0-6.2). INTERPRETATION: A large proportion of the malaria burden has been alleviated in The Gambia. Our results encourage consideration of a policy to eliminate malaria as a public-health problem, while emphasising the importance of accurate and continuous surveillance.

Casals-Pascual C, Idro R, Gicheru N, Gwer S, Kitsao B, Gitau E, Mwakesi R, Roberts DJ, Newton CRJC. 2008. High levels of erythropoietin are associated with protection against neurological sequelae in African children with cerebral malaria. Proc Natl Acad Sci U S A, 105 (7), pp. 2634-2639. | Show Abstract | Read more

Cerebral malaria (CM) in children is associated with a high mortality and long-term neurocognitive sequelae. Both erythropoietin (Epo) and vascular endothelial growth factor (VEGF) have been shown to be neuroprotective. We hypothesized that high plasma and cerebrospinal fluid (CSF) levels of these cytokines would prevent neurological sequelae in children with CM. We measured Epo, VEGF, and tumor necrosis factor in paired samples of plasma and CSF of Kenyan children admitted with CM. Logistic regression models were used to identify risk and protective factors associated with the development of neurological sequelae. Children with CM (n = 124) were categorized into three groups: 76 without sequelae, 32 with sequelae, and 16 who died. Conditional logistic regression analysis matching the 32 patients with CM and neurological sequelae to 64 patients with CM without sequelae stratified for hemoglobin level estimated that plasma Epo (>200 units/liter) was associated with >80% reduction in the risk of developing neurological sequelae [adjusted odds ratio (OR) 0.18; 95% C.I. 0.05-0.93; P = 0.041]. Admission with profound coma (adjusted OR 5.47; 95% C.I. 1.45-20.67; P = 0.012) and convulsions after admission (adjusted OR 16.35; 95% C.I. 2.94-90.79; P = 0.001) were also independently associated with neurological sequelae. High levels of Epo were associated with reduced risk of neurological sequelae in children with CM. The age-dependent Epo response to anemia and the age-dependent protective effect may influence the clinical epidemiology of CM. These data support further study of Epo as an adjuvant therapy in CM.

Casals-Pascual C, Kai O, Cheung JOP, Williams S, Lowe B, Nyanoti M, Williams TN, Maitland K, Molyneux M, Newton CRJC et al. 2006. Suppression of erythropoiesis in malarial anemia is associated with hemozoin in vitro and in vivo. Blood, 108 (8), pp. 2569-2577. | Show Abstract | Read more

Malarial anemia is a global public health problem and is characterized by a low reticulocyte response in the presence of life-threatening hemolysis. Although cytokines, in particular tumor necrosis factor-alpha (TNF-alpha), can suppress erythropoiesis, the grossly abnormal bone marrow morphology indicates that other factors may contribute to ineffective erythropoiesis. We hypothesized that the cytotoxic hemozoin (Hz) residues from digested hemoglobin (Hb) significantly contribute to abnormal erythropoiesis. Here, we show that not only isolated Hz, but also delipidated Hz, inhibits erythroid development in vitro in the absence of TNF-alpha. However, when added to cultures, TNF-alpha synergizes with Hz to inhibit erythropoiesis. Furthermore, we show that, in children with malarial anemia, the proportion of circulating monocytes containing Hz is associated with anemia (P < .001) and reticulocyte suppression (P = .009), and that this is independent of the level of circulating cytokines, including TNF-alpha. Plasma Hz is also associated with anemia (P < .001) and reticulocyte suppression (P = .02). Finally, histologic examination of the bone marrow of children who have died from malaria shows that pigmented erythroid and myeloid precursors are associated with the degree of abnormal erythroid development. Taken together, these observations provide compelling evidence for inhibition of erythropoiesis by Hz.

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