In silico characterisation of putative Plasmodium falciparum vaccine candidates in African malaria populations.

Genetic diversity of surface exposed and stage specific Plasmodium falciparum immunogenic proteins pose a major roadblock to developing an effective malaria vaccine with broad and long-lasting immunity. We conducted a prospective genetic analysis of candidate antigens (msp1, ama1, rh5, eba175, glurp, celtos, csp, lsa3, Pfsea, trap, conserved chrom3, hyp9, hyp10, phistb, surfin8.2, and surfin14.1) for malaria vaccine development on 2375 P. falciparum sequences from 16 African countries. We described signatures of balancing selection inferred from positive values of Tajima's D for all antigens across all populations except for glurp. This could be as a result of immune selection on these antigens as positive Tajima's D values mapped to regions with putative immune epitopes. A less diverse phistb antigen was characterised with a transmembrane domain, glycophosphatidyl anchors between the N and C- terminals, and surface epitopes that could be targets of immune recognition. This study demonstrates the value of population genetic and immunoinformatic analysis for identifying and characterising new putative vaccine candidates towards improving strain transcending immunity, and vaccine efficacy across all endemic populations.

Virgibacillus doumboii sp. nov., a halophilic bacterium isolated from the stool of a healthy child in Mali.

A moderately halophilic and strictly aerobic bacterium was isolated from a human stool as part of a study on the diagnosis of childhood malnutrition in Mali. Strain Marseille-Q1616T is a Gram-stain-positive, rod-shaped, catalase-positive and oxidase-negative bacterium. It has a genome size of 3.91 Mbp with 39.79% G+C content, which contains 3954 protein-coding genes including genes encoding phosphomycin resistance and Listeria monocytogenes, 16 rRNA genes and 64 tRNA genes. Strain Marseille-Q1616T exhibited a 96.3% 16S rRNA gene sequence similarity and shared an OrthoANI value of 70.64% (the highest observed) with Virgibacillus kekensis, the phylogenetically closest validly published species. Based on phenotypic and phylogenetic evidence and genomic average nucleotide identity values, we suggest the creation of a new species within the Virgibacillus genus, named Virgibacillus doumboii sp. nov., type strain Marseille-Q1616T (= CSURQ1616).

Building a center of excellence in biomedical research in an unfavorable environment: the Malaria Research and Training Center in Mali.

By choosing a capacity-building strategy based on human resources, the late Professor Ogobara Doumbo and Professor Yeya Toure have succeeded in establishing a center devoted to malaria research in the economically unfavorable environment of Mali. By taking advantage of the advent of a pluralist democracy in Mali in 1991, the Malaria Research and Training Centre (MRTC) has become a model of excellence in training in biomedical research and a renowned clinical research center. Since 2003, MRTC researchers have conducted more than 20 phase-1 and -2 clinical trials of antimalarial vaccines, thus becoming a reference both in Africa and globally. The MRTC owes its success to several factors. While the focus on human capacity building has been critical for sustainability, the quality of the partnerships and the density of the partnership network have also played a critical role. The NIH partnership enabled us to construct new buildings to house modern laboratories. These facilities made it possible to conduct leading-edge research programs, the results of which in turn provided access to significant other funding sources, not only to finance new projects, but also to purchase modern heavy equipment. Lastly, it has been possible to set up a policy for training Malian researchers at the Masters and PhD levels, with the aim of fueling a critical mass of scientific expertise. The combination of each of these factors has created an environment conducive to sustainable research and whose recent results have heightened expectations for a rich future.

[Prevalence of Plasmodium falciparum, anemia and molecular markers of chloroquine and sulfadoxine-pyriméthamine resistance in delivered women in Fana, Mali]. / Prévalence de Plasmodium falciparum, de l'anémie et des marqueurs moléculaires de la résistance à la chloroquine et à la sulfadoxine-pyrim éthamine chez les femmes accouchées à Fana, Mali.

The aim of this study was to describe the malaria morbidity and the frequencies of molecular markers of resistance to chloroquine and sulfadoxine-pyrimethamine in pregnant women at delivery in Mali. Two hundred pregnant women have been included at the delivery clinic in Fana. The age group of 14-19 years was predominant. Fifty two per cent (52.3%: 104/200) were malaria slides positive in their peripheral blood and 15% (30/200) of the women carried parasite in their placenta. The prevalence rate of anemia was 44.5% (89/200). PCR technique was successfully performed on 16 paired samples. The frequency of the Pfcrt K76T mutants in Plasmodium falciparum infections in peripheral blood was 68.8% (11/16) and 100% (16/16) in the placenta (p = 0.004). The frequency in peripheral blood of the DHFR N51I mutation was 12.5% (2/16) and 18.8% (3/16) in the placenta (p=0.12). The frequencies of the DHPS A437G mutants were similar in both sites 25% (4/16). No DHPS K540E and DHFR 164L mutations were found in the Fana pregnancy women samples.

[Epidemiology of cutaneous leishmaniasis in five villages of Dogon country, Mali]. / Épidémiologie clinique et parasitologique de la leishmaniose cutanée dans cinq villages du Pays Dogon, Mali.

The epidemiology of the cutaneous leishmaniasis (CL) with Leishmania major is poorly documented in Mali. Following reports of CL in the tourist areas of the Dogon country (Bandiagara Escarpment), a joint French and Malian bio-clinical team conducted a field study from 16 to 27 January, 2010. The population of 5 villages has been examined by a dermato-infectiologist and cases were selected by visual inspection of skin lesions. Smears and biopsies (from the lesions) and venous blood were obtained from suspected cases of CL. Diagnosis was performed by light microscopy, in vitro cultures, serology and molecular biology. Fifty patients with skin lesions have been examined. Twenty-one have been suspected as CL. At least one sample was obtained from 18 patients. The lesions were predominantly old, more or less scarring and secondary infected. A skin smear was performed for 15 patients, a skin biopsy for 14 patients: smears and cultures were all negative. The PCR (Leishmania spp.) made on 14 biopsies was positive for 12 patients (86%). The low amount of amplified DNA obtained did not allow the sequencing and identification of the species of Leishmania. Western blot (WB) serology was positive in 11 cases out of 12 (92%). This investigation showed the presence of cutaneous leishmaniasis in Bandiagara. A further investigation is required during transmission period (September-October) to confirm the presence of Leishmania major epidemic in Dogon country.

[Seasonal variability of intestinal helminths and Schistosoma haematobium in a rural area of the Sahel in Mali]. / Variations saisonnières de l'infestation par les helminthes intestinaux et Schistosoma haematobium en zone rurale sahélienne au Mali

OBJECTIVE: The objective of this study was to determine the prevalence of intestinal helminths and Schistosoma haematobium before and after the rainy season in Pongonon, Mali. METHODS: Volunteers aged one year and above were included. The Kato-Katz method was used to detect eggs and cysts in stool samples, and Wattman filtration to detect S. haematobium eggs in urine samples. Two cross-sectional surveys were conducted in July and November 2007. RESULTS: In July (beginning of the rainy season), 304 volunteers were included; 278 were seen again in November (at the end of the rainy season). We found more intestinal helminths at the end of the rainy season (8.3%) compared to the beginning of the season (2.9%) (P = 0.01). There was no infection with S. haematobium in July but 7.6% in November (P < 0.001). The prevalence of intestinal helminths in children and adults was similar (P > 0.05), but the prevalence of infection with S. haematobium was higher in children aged 6 to 16 years (17/153) than in adults (2/74) (P = 0.02). CONCLUSION: Infections with helminth and S. Haematobium were both more prevalent at the end of the rainy season. Adults were infected as well as children and may constitute potential reservoirs of parasites. Effective control of these parasitic infections requires mass drug administration programs that take place during the seasons of high parasite egg excretion and that also include adult populations in some areas.

[Development of anti-malarial vaccines and need for clinical trials in accordance with international standards in South Africa]. / Le développement de vaccins antipaludiques et la nécessité des essais cliniques conformes aux normes internationales en Afrique.

In the 20th century malaria remains a major problem of public health in sub-Saharan Africa. This haemosporidium discovered in Africa by Laveran in 1880, kills one child every 30 seconds which amounts to three "tsunami" flowing each year into the African continent. The current international solidarity raises new hopes as regards the possibility to suppress the morbidity effects on the population's health condition. In order to be efficient, today's strategies (impregnated mosquito nets, intermittent preventive treatments, artemisinin based combination therapy) should reach at least 80% of the targeted population (pregnant women and children). By 2025, the uncontrolled urbanization of the African population and the social disorders will make a new population a target for malaria. The new data of functional genomics and proteonics open new avenues of research for new mechanisms, new therapeutics and vaccine targets and new tools of diagnosis and prognosis. The current candidate vaccines of the first generation have allowed the development of African competences in clinical trials of international standard. Although they represent scientific advances they will not resolve the problem of public health. Research on candidate vaccines of 2nd and 3rd generation remains a challenge for the international scientific community. Africa should play a determining role in this process. Scientific information on the field remains essential for these generations of new anti-malarial vaccines. The ethical aspects regarding those clinical trials and actions of public health and research remain an universal necessity Deontology and ethics are two complementary approaches for the good practice of medicine and research of a good practitioner. For the protection and advantages of the patient and/or volunteer of the research are the cornerstones of the ethical approach. The scientific quality of a research protocol submitted to an independent research ethics committee and the volunteer 's informed consent are universal ethical obligations. For the quality of ethics observance in a country reflects best the quality of the efficiency of its research system and its democracy.

[In vitro cultivation of fields isolates of Plasmodium falciparum in Mali]. / Culture in vitro d'isolats de terrain de Plasmodium falciparum au Mali.

Malaria immunology, molecular biology and pathogenicity studies often require the adaptation of Plasmodium falciparum field isolates to continuous in vitro cultivation. For this purpose we have established propagation protocols of asexual erythrocytic stages of P. falciparum samples from malaria patients or asymptomatic carriers in Mali. The parasites were grown in standard culture medium supplemented by human serum and in a culture medium without human serum but supplemented by AlbuMax 1. The candle jar environment and tissue culture flasks gassed with 5% CO2, 5% O2 and 90% N2 obtained from a portable gas mixer were used. Protocols for parasite cultivation in a resource-poor setting were developed. These protocols were successfully applied to fresh isolates in Mali as well as to blood samples frozen in liquid nitrogen and shipped to a laboratory in U.S.A.

Conservation of a novel vacuolar transporter in Plasmodium species and its central role in chloroquine resistance of P. falciparum.

Chloroquine resistance in Plasmodium falciparum has recently been shown to result from mutations in the novel vacuolar transporter, PfCRT. Field studies have demonstrated the importance of these mutations in clinical resistance. Although a pfcrt ortholog has been identified in Plasmodiumvivax, there is no association between in vivo chloroquine resistance and codon mutations in the P. vivax gene. This is consistent with lines of evidence that suggest alternative mechanisms of chloroquine resistance among various malaria parasite species.

Chloroquine treatment of uncomplicated Plasmodium falciparum malaria in Mali: parasitologic resistance versus therapeutic efficacy.

Whether and when to replace chloroquine with other antimalarial drugs is an urgent public health question in much of Africa, where Plasmodium falciparum, which is increasingly resistant to chloroquine, continues to kill millions each year. Antimalarial drug efficacy has traditionally been measured as parasitologic resistance, but recent guidelines use both clinical and parasitologic criteria to monitor therapeutic efficacy. To assess the new efficacy protocol, we measured parasitologic and therapeutic outcomes in 514 patients treated with chloroquine for uncomplicated P. falciparum malaria in Mali. There was a general agreement between parasitologic and therapeutic outcomes at two sites, with 13-17% parasitologic resistance rates and 10-15% treatment failure rates. However, the new protocol overestimated early treatment failure rates (21-71% of cases classified as early treatment failure had sensitive or RI parasitologic responses), particularly where resistance was rare, and missed low-level parasitologic resistance. Modifications of the protocol for monitoring antimalarial therapeutic efficacy are recommended.

A molecular marker for chloroquine-resistant falciparum malaria.

BACKGROUND: Chloroquine-resistant Plasmodium falciparum malaria is a major health problem, particularly in sub-Saharan Africa. Chloroquine resistance has been associated in vitro with point mutations in two genes, pfcrt and pfmdr 1, which encode the P. falciparum digestive-vacuole transmembrane proteins PfCRT and Pgh1, respectively. METHODS: To assess the value of these mutations as markers for clinical chloroquine resistance, we measured the association between the mutations and the response to chloroquine treatment in patients with uncomplicated falciparum malaria in Mali. The frequencies of the mutations in patients before and after treatment were compared for evidence of selection of resistance factors as a result of exposure to chloroquine. RESULTS: The pfcrt mutation resulting in the substitution of threonine (T76) for lysine at position 76 was present in all 60 samples from patients with chloroquine-resistant infections (those that persisted or recurred after treatment), as compared with a base-line prevalence of 41 percent in samples obtained before treatment from 116 randomly selected patients (P<0.001), indicating absolute selection for this mutation. The pfmdr 1 mutation resulting in the substitution of tyrosine for asparagine at position 86 was also selected for, since it was present in 48 of 56 post-treatment samples from patients with chloroquine-resistant infections (86 percent), as compared with a base-line prevalence of 50 percent in 115 samples obtained before treatment (P<0.001). The presence of pfcrt T76 was more strongly associated with the development of chloroquine resistance (odds ratio, 18.8; 95 percent confidence interval, 6.5 to 58.3) than was the presence of pfmdr 1 Y86 (odds ratio, 3.2; 95 percent confidence interval, 1.5 to 6.8) or the presence of both mutations (odds ratio, 9.8; 95 percent confidence interval, 4.4 to 22.1). CONCLUSIONS: This study shows an association between the pfcrt T76 mutation in P. falciparum and the development of chloroquine resistance during the treatment of malaria. This mutation can be used as a marker in surveillance for chloroquine-resistant falciparum malaria.

Hemoglobin C associated with protection from severe malaria in the Dogon of Mali, a West African population with a low prevalence of hemoglobin S.

The malaria hypothesis proposes a survival advantage for individuals with hemoglobin variants in areas of endemic Plasmodium falciparum malaria. Hemoglobin C (HbC) is a possible example in West Africa, where this hemoglobin has a centric distribution with high frequencies among certain populations including the Dogon ethnic group. To test whether HbC is associated with protection from malaria, we performed a case-control study in the Dogon of Bandiagara, Mali. HbC was present in 68 of 391 (17.4%) of uncomplicated malaria control cases, whereas it was detected in only 3 of 67 cases (4.5%) of severe malaria (odds ratio [OR], 0.22; P =. 01). Further, HbC was present in only 1 of 34 cases (2.9%) with cerebral manifestations, the most common presentation of severe malaria in this population (OR, 0.14; P =.03). Episodes of uncomplicated malaria and parasitemias (4800-205 050/microL) were identified in cases of homozygous HbC (HbCC), which indicates that P falciparum parasites are able to efficiently replicate within HbCC erythrocytes in vivo. These findings suggest that HbC does not protect against infection or uncomplicated malaria but can protect against severe malaria in the Dogon population of Bandiagara, Mali. The data also suggest that the protective effect associated with HbC may be greater than that of HbS in this population.

Rapid selection of Plasmodium falciparum dihydrofolate reductase mutants by pyrimethamine prophylaxis.

A prospective study was conducted to measure the selective effect of pyrimethamine prophylaxis on point mutations in Plasmodium falciparum dihydrofolate reductase (DHFR). A total of 109 Malian children were given pyrimethamine weekly for 5 weeks. P. falciparum infections were analyzed by polymerase chain reaction for DHFR mutations, which were dramatically more frequent among prophylaxis-breakthrough infections than at baseline: the prevalence of Asn-108 rose from 13% to 100%, Ile-51 from 4% to 50%, and Arg-59 from 11% to 90%. Eight persistent infections lacking detectable DHFR mutations at baseline developed multiple mutations within 1 week of the patients' starting pyrimethamine prophylaxis. Microsatellite analysis found no evidence of clonal identity among baseline and breakthrough infections. Analysis of these data demonstrates that under prophylaxis conditions, pyrimethamine is strongly selective for DHFR mutations, which arise extremely rapidly under drug pressure, even when undetectable in the initial infection. These findings have implications for prophylaxis regimens with other antifolate drugs.

Pyrimethamine-sulfadoxine efficacy and selection for mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase in Mali.

To assess pyrimethamine-sulfadoxine (PS) efficacy in Mali, and the role of mutations in Plasmodium falciparum dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) in in vivo PS resistance, 190 patients with uncomplicated P. falciparum malaria were treated with PS and monitored for 56 days. Mutation-specific polymerase chain reactions and digestion with restriction endonucleases were used to detect DHFR and DHPS mutations on filter paper blood samples from pretreatment and post-treatment infections. Only one case each of RI and RII level resistance and no cases of RIII resistance or therapeutic failure were observed. Post-PS treatment infections had significantly higher rates of DHFR mutations at codons 108 and 59. No significant selection for DHPS mutations was seen. Pyrimethamine-sulfadoxine is highly efficacious in Mali, and while the low level of resistance precludes assessing the utility of molecular assays for in vivo PS resistance, rapid selection of DHFR mutations supports their role in PS failure.

Use of antimalarial drugs in Mali: policy versus reality.

Inappropriate use of antimalarial drugs undermines therapeutic efficacy and promotes the emergence and spread of drug-resistant malaria. Strategies for improving compliance require accurate information about current practices. Here we describe Knowledge-Attitude-Practice surveys conducted among health providers and consumers in two Malian villages, one rural and one periurban. All sanctioned providers limited their first choices of antimalarial drug to those recommended by the national malaria control program and reported using correct dosing regimens. However, the majority of consumers in the two villages chose non-recommended treatments for malaria and reported suboptimal treatment regimens when they did use recommended drugs. Antimalarial drugs were also widely available from unsanctioned sources, often accompanied by erroneous advice on dosing regimens. This study demonstrates that even when the most peripheral health providers are well-trained in correct use of antimalarial drugs, additional measures directly targeting consumers will be required to improve drug use practices.

Mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase and epidemiologic patterns of pyrimethamine-sulfadoxine use and resistance.

To assess the relationship between mutations in Plasmodium falciparum dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) and clinical pyrimethamine-sulfadoxine resistance, polymerase chain reaction surveys and analyses for new mutations were conducted in four countries with increasing levels of pyrimethamine-sulfadoxine resistance: Mali, Kenya, Malawi, and Bolivia. Prevalence of mutations at DHFR codon 108 and a new mutation at DHPS 540 correlated with increased pyrimethamine-sulfadoxine resistance (P < .05). Mutations at DHFR 51, DHFR 59, and DHPS 437 correlated with resistance without achieving statistical significance. Mutations at DHFR 164 and DHPS 581 were common in Bolivia, where pyrimethamine-sulfadoxine resistance is widespread, but absent in African sites. Two new DHFR mutations, a point mutation at codon 50 and an insert at codon 30, were found only in Bolivia. DHFR and DHPS mutations occur in a progressive, stepwise fashion. Identification of specific sets of mutations causing in vivo drug failure may lead to the development of molecular surveillance methods for pyrimethamine-sulfadoxine resistance.

Resistance to antifolates in Plasmodium falciparum monitored by sequence analysis of dihydropteroate synthetase and dihydrofolate reductase alleles in a large number of field samples of diverse origins.

Resistance of Plasmodium falciparum to antifolate chemotherapy is a significant problem where combinations such as Fansidar (pyrimethamine-sulfadoxine; PYR-SDX) are used in the treatment of chloroquine-resistant malaria. Antifolate resistance has been associated with variant sequences of dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS), the targets of PYR and SDX respectively. However, while the nature and distribution of mutations in the dhfr gene are well established, this is not yet the case for dhps. We have thus examined by DNA sequence analysis 141 field samples from several geographical regions with differing Fansidar usage (West and East Africa, the Middle East and Viet Nam) to establish a database of the frequency and repertoire of dhps mutations, which were found in 60% of the samples. We have also simultaneously determined from all samples their dhfr sequences, to better understand the relationship of both types of mutation to Fansidar resistance. Whilst the distribution of mutations was quite different across the regions surveyed, it broadly mirrored our understanding of relative Fansidar usage. In samples taken from individual patients before and after drug treatment, we found an association between the more highly mutated forms of dhps and/or dhfr and parasites that were not cleared by antifolate therapy. We also report a novel mutation in a Pakistani sample at position 16 of DHFR (A16S) that is combined with the familiar C59R mutation, but is wild-type at position 108. This is the first observation in a field sample of a mutant dhfr allele where the 108 codon is unchanged.

Community pyrimethamine-sulfadoxine use and prevalence of resistant Plasmodium falciparum genotypes in Mali: a model for deterring resistance.

Pyrimethamine-sulfadoxine (PS, Fansidar; Hoffman-LaRoche, Basel, Switzerland) is now the first-line antimalarial therapy in parts of Africa with high rates of chloroquine-resistant Plasmodium falciparum. With PS resistance increasing and no suitably inexpensive and effective third antimalarial drug available, strategies for delaying the spread of PS resistance in Africa are needed. Community PS usage was measured in two Malian villages, one rural and one periurban, and prevalence of pyrimethamine-resistant P. falciparum genotypes was determined at these sites and two urban sites. The prevalence of resistant genotypes was 22.6% (n = 84) in the periurban village where PS was available from multiple sources and large stocks of PS were observed, and 13.5% (n = 89) and 23.4% (n = 77) in a large town and a city, respectively, where PS is widely available. No pyrimethamine-resistant genotypes (n = 58) were detected in Kolle, a rural village with a community-supported dispensary and clinic where PS is used sparingly and no PS was available in pharmacies or markets. The high rates of pyrimethamine resistant genotypes concurrent with higher PS usage argue for a policy of judicious PS use in Mali and in similar settings. A possible model for slowing the spread of drug-resistant malaria is illustrated by the example of the Kolle clinic.