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We report the results of clinical studies carried out at six sites in Madagascar, between January and October 2006. The aims were (i) to update our knowledge of the burden of Plasmodium malariae infection and (ii) to assess the therapeutic efficacy of chloroquine for uncomplicated quartan malaria. Our findings confirm that P. malariae is the third leading cause of malaria, accounting for 1.1% of all malarial infections. They also demonstrate that chlo- roquine—currently recommended for the home management of presumed malaria in children under the age of five years and commonly used by adults—remains highly effective in patients with uncomplicated P. malariae infection. Plasmodium malariae, one of the four species of Plasmo- dium affecting humans, is found in tropical and subtropical regions, often in sympatry with other Plasmodium species, as in Madagascar. Its reported prevalence varies from less than 4% to more than 20% in endemic regions. 1-4 No accurate estimate of the prevalence of P. malariae infection worldwide is currently available, but it has been calculated that there are probably at least 60 million infections per year, based on the prevalence of P. falciparum 5,6 and known underestimation of the prevalence of P. malariae. 7,8 The clinical features associ- ated with febrile bouts of P. malariae are generally milder than those caused by other species. 9 Fever displays quartan (4-day) periodicity, parasite density is usually considerably below 1000 parasites per ml of blood, and infection is rarely life-threatening in the absence of complications, such as neph

International audience; Home management of malaria is recommended for prompt, effective antimalarial treatment in children less than five years of age. Compliance, safety, and effectiveness of the new fixed-dose artesunate-amodiaquine regimen used to treat suspected malaria were assessed in febrile children enrolled in a 24-month cohort study in two settings in Madagascar. Children with fever were asked to visit community health workers. Presumptive antimalarial treatment was given and further visits were scheduled for follow-up. The primary endpoint was the risk of clinical/parasitologic treatment failure. Secondary outcomes included fever/parasite clearance, change in hemoglobin levels, and frequency of adverse events. The global clinical cure rate was 98.4% by day 28 and 97.9% by day 42. Reported compliance was 83.4%. No severe adverse effects were observed. This study provides comprehensive data concerning the clinical cure rate obtained with artesunate-amodiaquine and evidence supporting the scaling up of home management of malaria.

Intermittent preventive treatment (IPT) of pregnant women with sulfadoxine-pyrimethamine (SP) is being considered as a routine practice in Madagascar, mainly to decrease the risks of malaria-associated severe anaemia in the women, and of low birthweight in their babies. There is, however, relatively little information available on the efficacy of SP when used, in Madagascar, to treat cases of Plasmodium falciparum malaria. In a preliminary study, carried out in 2003 in the village of Saharevo, 36 uncomplicated cases were each treated with a standard dose of SP and with paracetamol and then followed up for 28 days. No case of therapeutic failure occurred and all the asexual parasitaemias cleared by day 3. It therefore appears that SP is effective against P. falciparum in Saharevo (and probably in the whole, rural district of Moramanga in which the village lies). This is an encouraging observation to make before IPT is initiated throughout the country.

Use of mefloquine against P. falciparum jeopardizes its future use against P. vivax., Numerous studies have indicated a strong association between amplification of the multidrug resistance-1 gene and in vivo and in vitro mefloquine resistance of Plasmodium falciparum. Although falciparum infection usually is not treated with mefloquine, incorrect diagnosis, high frequency of undetected mixed infections, or relapses of P. vivax infection triggered by P. falciparum infections expose non–P. falciparum parasites to mefloquine. To assess the consequences of such unintentional treatments on P. vivax, we studied variations in number of Pvmdr-1 (PlasmoDB accession no. PVX_080100, NCBI reference sequence NC_009915.1) copies worldwide in 607 samples collected in areas with different histories of mefloquine use from residents and from travelers returning to France. Number of Pvmdr-1 copies correlated with drug use history. Treatment against P. falciparum exerts substantial collateral pressure against sympatric P. vivax, jeopardizing future use of mefloquine against P. vivax. A drug policy is needed that takes into consideration all co-endemic species of malaria parasites.

Plasmodium vivax is the geographically most widespread human malaria parasite. To analyze patterns of microsatellite diversity and population structure across countries of different transmission intensity, genotyping data from 11 microsatellite markers was either generated or compiled from 841 isolates from four continents collected in 1999–2008. Diversity was highest in South-East Asia (mean allelic richness 10.0–12.8), intermediate in the South Pacific (8.1–9.9) Madagascar and Sudan (7.9–8.4), and lowest in South America and Central Asia (5.5–7.2). A reduced panel of only 3 markers was sufficient to identify approx. 90% of all haplotypes in South Pacific, African and SE-Asian populations, but only 60–80% in Latin American populations, suggesting that typing of 2–6 markers, depending on the level of endemicity, is sufficient for epidemiological studies. Clustering analysis showed distinct clusters in Peru and Brazil, but little sub-structuring was observed within Africa, SE-Asia or the South Pacific. Isolates from Uzbekistan were exceptional, as a near-clonal parasite population was observed that was clearly separated from all other populations (F ST>0.2). Outside Central Asia F ST values were highest (0.11–0.16) between South American and all other populations, and lowest (0.04–0.07) between populations from South-East Asia and the South Pacific. These comparisons between P. vivax populations from four continents indicated that not only transmission intensity, but also geographical isolation affect diversity and population structure. However, the high effective population size results in slow changes of these parameters. This persistency must be taken into account when assessing the impact of control programs on the genetic structure of parasite populations., Author Summary Plasmodium vivax is the predominant malaria parasite in Latin America, Asia and the South Pacific. Different factors are expected to shape diversity and population structure across continents, e.g. transmission intensity which is much lower in South America as compared to Southeast-Asia and the South Pacific, or geographical isolation of P. vivax populations in the South Pacific. We have compiled data from 841 isolates from South and Central America, Africa, Central Asia, Southeast-Asia and the South Pacific typed with a panel of 11 microsatellite markers. Diversity was highest in Southeast-Asia, where transmission is intermediate-high and migration of infected hosts is high, and lowest in South America and Central Asia where malaria transmission is low and focal. Reducing the panel of microsatellites showed that 2–6 markers are sufficient for genotyping for most drug trials and epidemiological studies, as these markers can identify >90% of all haplotypes. Parasites clustered according to continental origin, with high population differentiation between South American and Central Asian populations and the other populations, and lowest differences between Southeast-Asia and the South Pacific. Current attempts to reduce malaria transmission might change this pattern, but only after transmission is reduced for an extended period of time.

In Madagascar, although chloroquine (CQ) remains the first-line treatment of choice for malaria, the gradual spread of resistance to this antimalarial drug is of increasing concern. As part of a la...

Background Plasmodium vivax is the most prevalent human malaria parasite, causing serious public health problems in malaria-endemic countries. Until recently the Duffy-negative blood group phenotype was considered to confer resistance to vivax malaria for most African ethnicities. We and others have reported that P. vivax strains in African countries from Madagascar to Mauritania display capacity to cause clinical vivax malaria in Duffy-negative people. New insights must now explain Duffy-independent P. vivax invasion of human erythrocytes. Methods/Principal Findings Through recent whole genome sequencing we obtained ≥70× coverage of the P. vivax genome from five field-isolates, resulting in ≥93% of the Sal I reference sequenced at coverage greater than 20×. Combined with sequences from one additional Malagasy field isolate and from five monkey-adapted strains, we describe here identification of DNA sequence rearrangements in the P. vivax genome, including discovery of a duplication of the P. vivax Duffy binding protein (PvDBP) gene. A survey of Malagasy patients infected with P. vivax showed that the PvDBP duplication was present in numerous locations in Madagascar and found in over 50% of infected patients evaluated. Extended geographic surveys showed that the PvDBP duplication was detected frequently in vivax patients living in East Africa and in some residents of non-African P. vivax-endemic countries. Additionally, the PvDBP duplication was observed in travelers seeking treatment of vivax malaria upon returning home. PvDBP duplication prevalence was highest in west-central Madagascar sites where the highest frequencies of P. vivax-infected, Duffy-negative people were reported. Conclusions/Significance The highly conserved nature of the sequence involved in the PvDBP duplication suggests that it has occurred in a recent evolutionary time frame. These data suggest that PvDBP, a merozoite surface protein involved in red cell adhesion is rapidly evolving, possibly in response to constraints imposed by erythrocyte Duffy negativity in some human populations., Author Summary Malaria results from infection of human red blood cells (RBC) by Plasmodium parasite's merozoite. For Plasmodium vivax the process of RBC invasion has been hypothesized to depend on interactions between the parasite's Duffy binding protein (PvDBP) and human Duffy blood group antigen because Duffy-negative people (most often people of African descent) were shown to be highly resistant to RBC infection and disease. Over the past five years, researchers are reporting with increasing frequency that Duffy-negative individuals are infected with P. vivax. This raises new questions as to how P. vivax infects the RBC when the Duffy blood group antigen is not available. Here we show that the parasite's Duffy binding protein gene has been duplicated in multiple P. vivax strains, especially at high prevalence in Madagascar. The specificity and prevalence of this polymorphism suggest that the parasite genome has responded to the barrier of Duffy negativity through the duplication of the PvDBP gene. Our results indicate that the PvDBP duplication is a recent event and provide novel research avenues to understand alternative pathways for P. vivax RBC invasion.

To date, 11 studies conducted in different countries to test the association between Plasmodium falciparum Na(+)/H(+) exchanger gene (pfnhe-1; PF13_0019) polymorphisms and in vitro susceptibility to quinine have generated conflicting data. In this context and to extend our knowledge of the genetic polymorphism of Pfnhe gene, we have sequenced the ms4760 locus from 595 isolates collected in the Comoros (N = 250; an area with a high prevalence of chloroquine and sulfadoxine-pyrimethamine resistance) and Madagascar (N = 345; a low drug-resistance area). Among them, 29 different alleles were observed, including 8 (27%) alleles not previously described. Isolates from the Comoros showed more repeats in block II (DNNND), which some studies have found to be positively associated with in vitro resistance to quinine, compared with isolates from Madagascar. Additional studies are required to better define the mechanisms underlying quinine resistance, which involve multiple gene interactions.

Background Early diagnosis, as well as prompt and effective treatment of uncomplicated malaria, are essential components of the anti-malaria strategy in Madagascar to prevent severe malaria, reduce mortality and limit malaria transmission. The purpose of this study was to assess the performance of the malaria rapid diagnostic tests (RDTs) used by community health workers (CHWs) by comparing RDT results with two reference methods (microscopy and Polymerase Chain Reaction, PCR). Methods Eight CHWs in two districts, each with a different level of endemic malaria transmission, were trained to use RDTs in the management of febrile children under five years of age. RDTs were performed by CHWs in all febrile children who consulted for fever. In parallel, retrospective parasitological diagnoses were made by microscopy and PCR. The results of these different diagnostic methods were analysed to evaluate the diagnostic performance of the RDTs administered by the CHWs. The stability of the RDTs stored by CHWs was also evaluated. Results Among 190 febrile children with suspected malaria who visited CHWs between February 2009 and February 2010, 89.5% were found to be positive for malaria parasites by PCR, 51.6% were positive by microscopy and 55.8% were positive by RDT. The performance accuracy of the RDTs used by CHWs in terms of sensitivity, specificity, positive and negative predictive values was greater than 85%. Concordance between microscopy and RDT, estimated by the Kappa value was 0.83 (95% CI: 0.75-0.91). RDTs stored by CHWs for 24 months were capable of detecting Plasmodium falciparum in blood at a level of 200 parasites/μl. Conclusion Introduction of easy-to-use diagnostic tools, such as RDTs, at the community level appears to be an effective strategy for improving febrile patient management and for reducing excessive use of anti-malarial drugs.

Molecular investigations performed following the emergence of sulfadoxine-pyrimethamine (SP) resistance in Plasmodium falciparum have allowed the identification of the dihydrofolate reductase (DHFR) enzyme as the target of pyrimethamine. Although clinical cases of Plasmodium malariae are not usually treated with antifolate therapy, incorrect diagnosis and the high frequency of undetected mixed infections has probably exposed non- P. falciparum parasites to antifolate therapy in many areas. In this context, we aimed to assess the worldwide genetic diversity of the P. malariae dhfr gene in 123 samples collected in Africa and Asia, areas with different histories of SP use. Among the 10 polymorphic sites found, we have observed 7 new mutations (K55E, S58R, S59A, F168S, N194S, D207G, and T221A), which led us to describe 6 new DHFR proteins. All isolates from African countries were classified as wild type, while new mutations and haplotypes were recognized as exclusive to Madagascar (except for the double mutations at nucleotides 341 and 342 [S114N] found in one Cambodian isolate). Among these nonsynonymous mutations, two were likely related to pyrimethamine resistance: S58R (corresponding to C59R in P. falciparum and S58R in Plasmodium vivax ; observed in one Malagasy sample) and S114N (corresponding to S108N in P. falciparum and S117N in P. vivax ; observed in three Cambodian samples).

International audience; The purpose of this review was to provide up-to-date information on the resistance of Plasmodium falciparum to the main antimalarials used in Madagascar and to assist implementation of the malaria control and elimination program. In 2006, the failure rate for chloroquine treatment was 44% (n = 300) and was comparable to the rate observed in continental Africa. Most treatment failures occurred after the first week of follow-up. P. falciparum resistance to chloroquine appeared to be special in Madagascar with only 3.2% of isolates showing in vitro resistance (n = 372, 7 sentinel sites) and less than 1% harbouring mutant parasites within the Pfcrt gene. Conversely, the Pfmdr1 N86Y point mutation was found in 64.3% (n = 174) of isolates in 2006 and in 51.7% (n = 343) in 2007. Failure of combined sulfadoxine-pyrimethamine therapy, i.e., the recommended intermittent preventive treatment for malaria during pregnancy, and in vitro resistance to pyrimethamine were rare. However, the Pfdhfr 51I/59R/108N allele showed consistently high prevalence levels reaching 33.3% in 2008. Moreover, the single Pfdhfr 164L mutant allele, a haplotype unique to Madagascar, was discovered in 2006 and showed prevalence rates up to 30% in some locations (southeast) in 2008. Up to now, the quadruple mutant allele Pfdhfr 51I/59R/108N/164L has not been observed. Susceptibility to the other antimalarials tested appeared excellent but the number of isolates showing in vitro susceptibility to artemisinin derivatives has been fallen in recent years and this decline may herald a decrease in the efficacy of these drugs.; Cette revue a pour objectif de faire le point sur la résistance actuelle de P. falciparum aux principaux antipaludiques utilisés à Madagascar et d’apporter des données récentes pouvant être utiles à la mise en œuvre du programme de contrôle et de l’élimination du paludisme. En 2006, le taux d’échec global à la chloroquine s’élevait à 44 % (n=300), taux comparable à ceux observés sur le continent Africain. Les échecs observés étaient majoritairement de type tardif. La résistance de P. falciparum à la chloroquine à Madagascar apparaît particulière avec seulement 3,2 % d’isolats ayant un phénotype résistant (n=372,7 sites sentinelles) et moins de 1 % d’isolats présentant des parasites mutants sur le gène Pfcrt. Par contre la mutation Pfmdr1N86Y, a été trouvée chez 64,3 %des isolats (n=174) en 2006 et chez 51,7 % des isolats (n=343) en 2007. Les échecs thérapeutiques de l’association sulfadoxine-pyrimethamine, recommandée pour le traitement préventif intermittent des femmes enceintes, et la résistance in vitro à la pyriméthamine sont rares. Cependant, nous rapportons une fréquence importante de triples mutants Pfdhfr 51I/59R/108N allant jusqu’à 33,3 % en 2008. De plus, un haplotype unique à Madagascar, l’allèle simple mutant Pfdhfr 164L, a été trouvé en 2006 avec une fréquence allant jusqu’à 30 % des isolats en certains sites en 2008 (Sud-Est). Pour l’instant nous n’avons pas trouvé de quadruples mutants Pfdhfr 51I/59R/108N/164L. La sensibilité aux autres antipaludiques testés apparaît excellente mais la perte des isolats les plus sensibles in vitro aux dérivés de l’artémisinine, observée ces dernières années, pourrait annoncer le début d’une moindre efficacité de ces molécules.

The combination of sulfadoxine-pyrimethamine is recommended for use as intermittent preventive treatment of malaria during pregnancy and is deployed in Africa. The emergence and the spread of resistant parasites are major threats to such an intervention. We have characterized the Plasmodium falciparum dhfr ( pfdhfr ) haplotypes and flanking microsatellites in 322 P. falciparum isolates collected from the Comoros Islands and Madagascar. One hundred fifty-six (48.4%) carried the wild-type pfdhfr allele, 19 (5.9%) carried the S108N single-mutation allele, 30 (9.3%) carried the I164L single-mutation allele, 114 (35.4%) carried the N51I/C59R/S108N triple-mutation allele, and 3 (1.0%) carried the N51I/C59R/S108N/I164L quadruple-mutation allele. Microsatellite analysis showed the introduction from the Comoros Islands of the ancestral pfdhfr triple mutant allele of Asian origin and its spread in Madagascar. Evidence for the emergence on multiple occasions of the I164L single-mutation pfdhfr allele in Madagascar was also obtained. Thus, the conditions required to generate mutants with quadruple mutations are met in Madagascar, representing a serious threat to current drug policy.

Background The use of artemisinin derivative-based combination therapy (ACT) such as artesunate plus amodiaquine is currently recommended for the treatment of uncomplicated Plasmodium falciparum malaria. Fixed-dose combinations are more adapted to patients than regimens involving multiple tablets and improve treatment compliance. A fixed-dose combination of artesunate + amodiaquine (ASAQ) was recently developed. To assess the efficacy and safety of this new combination and to define its optimum dosage regimen (once or twice daily) in the treatment of uncomplicated P. falciparum malaria, a multicentre clinical study was conducted. Methods A multicentre, randomized, controlled, investigator-blinded, parallel-group study was conducted in five African centers in Cameroon, Madagascar, Mali and Senegal from March to December 2006. Efficacy and safety of ASAQ were assessed compared to those of artemether + lumefantrine (AL). The WHO protocol with a 28-day follow-up for assessing the drug therapeutic efficacy was used. Patients suffering from uncomplicated P. falciparum malaria were randomized to receive ASAQ orally once daily (ASAQ1), ASAQ twice daily (ASAQ2) or AL twice daily (AL) for three days. The primary outcome was PCR-corrected parasitological cure rate and clinical response. Results Of 941 patients initially randomized and stratified into two age groups ( Conclusion The non-inferiority of ASAQ compared with AL was demonstrated. The fixed-dose combination artesunate + amodiaquine (ASAQ) is safe and efficacious even in young children under 5 years of age. Whilst administration on a twice-a-day basis does not improve the efficacy of ASAQ significantly, a once-a-day intake of this new combination clearly appears as an effective and safe therapy in the treatment of uncomplicated P. falciparum malaria both in adults and children. Implications of such findings are of primary importance in terms of public health especially in African countries. As most national policies plan to strengthen malaria control to reach the elimination of this disease, anti-malarial drugs such as the artesunate + amodiaquine fixed-dose ACT will play a pivotal role in this process. Trial registration The protocol was registered with the www.clinicaltrials.gov open clinical trial registry under the identifier number NCT00316329.

We have developed a High-Resolution DNA Melting method to detect mutations related to Plasmodium falciparum resistance. This method is based on real-time PCR followed by High Resolution Melting ramping from 67 degrees C to 80 degrees C with fluorescence data acquisition set at 0.1 degrees C increments. The accuracy of the technique was assessed using 177 P. falciparum clinical isolates and two reference strains. Results perfectly matched those obtained by DNA sequencing for some important genetic markers of P. falciparum resistance. This technique could be of great value for epidemiological studies, especially in developing countries.

Background Madagascar has been known for having bio-geo-ecological diversity which is reflected by a complex malaria epidemiology ranging from hyperendemic to malaria-free areas. Malaria-related attacks and infection are frequently recorded both in children and adults living in areas of low malaria transmission. To integrate this variability in the national malaria control policy, extensive epidemiological studies are required to up-date previous records and adjust strategies. Methods A longitudinal malaria survey was conducted from July 1996 to June 2005 among an average cohort of 214 villagers in Saharevo, located at 900 m above the sea. Saharevo is a typical eastern foothill site at the junction between a costal wet tropical area (equatorial malaria pattern) and a drier high-altitude area (low malaria transmission). Results Passive and active malaria detection revealed that malaria transmission in Saharevo follows an abrupt seasonal variation. Interestingly, malaria was confirmed in 45% (1,271/2,794) of malaria-presumed fevers seen at the health centre. All four Plasmodia that infect humans were also found: Plasmodium falciparum; Plasmodium vivax, Plasmodium malariae and Plasmodium ovale. Half of the malaria-presumed fevers could be confirmed over the season with the highest malaria transmission level, although less than a quarter in lower transmission time, highlighting the importance of diagnosis prior to treatment intake. P. falciparum malaria has been predominant (98%). The high prevalence of P. falciparum malaria affects more particularly under 10 years old children in both symptomatic and asymptomatic contexts. Children between two and four years of age experienced an average of 2.6 malaria attacks with P. falciparum per annum. Moreover, estimated incidence of P. falciparum malaria tends to show that half of the attacks (15 attacks) risk to occur during the first 10 years of life for a 60-year-old adult who would have experienced 32 malaria attacks. Conclusion The incidence of malaria decreased slightly with age but remained important among children and adults in Saharevo. These results support that a premunition against malaria is slowly acquired until adolescence. However, this claims for a weak premunition among villagers in Saharevo and by extension in the whole eastern foothill area of Madagascar. While the Malagasy government turns towards malaria elimination plans nowadays, choices and expectations to up-date and adapt malaria control strategies in the foothill areas are discussed in this paper.

We assessed the performance of two new commercially available rapid diagnostic tests (RDTs) for malaria (SD Bioline Malaria Ag Pf test and Ag Pf/Pan test) in 200 patients with uncomplicated malaria between August and October 2007 in Madagascar. Results of the two RDTs were compared with those obtained by microscopy and real-time polymerase chain reaction. The sensitivity and specificity for detectio no fPlasmodium falciparum were 93% and 98.9%, respectively, for the SD Bioline Malaria Ag Pf test and 92.9% and 98.9% for the SD Bioline Malaria Ag Pf/Pan ® test. The sensitivity of the SD Bioline Malaria Ag Pf/Pan test was much lower for detection of other species (63.6%). The sensitivity of the two new assays decreased to 77.3% at parasitemia levels < 100 parasites/L for detection of P. falciparum. In most malaria-endemic countries, since the introduction of more effective but more expensive antimalarial drug com- binations, such as artemisinin combination therapy as first- line treatment, parasitologic confirmation has become essen- tial in routine malaria case management. This medical prac- tice ensures that antimalarial drugs are administered to patients who need them. This is considered as a public health priority by the World Health Organization, in particular in limiting the unnecessary use of inappropriate treatments and thereby avoiding selection and spread of drug-resistant Plas- modium falciparum parasites. Over the past two decades, malaria rapid diagnostic tests (RDTs) have been developed for use in any situation where the only realistic alternative was the clinical diagnosis of ma- laria. These diagnostic tests are fast and easy to perform, and do not require electricity or specific equipment. 1-3 Currently, 86 malaria RDT products from 28 different manufacturers are available. 4 They are all based on the same principle and use antibodies that detect only three groups of antigen. Most products are based on the detection of a P. falciparum- specific protein, either P. falciparum histidine-rich protein 2 (PfHRP2) or P. falciparum lactate dehydrogenase (pfLDH). Some tests detect P. falciparum-specific and pan-specific an- tigens (aldolase or pan-malaria pLDH) and distinguish a non- falciparum infection from P. falciparum or P. falciparum/ mixed infections. The purpose of this study was to assess the performance of two new commercially available immunochromatographic as- says: the SD Bioline Malaria Ag Pf (ref. 05FK50) test and the SD Bioline Malaria Ag Pf/Pan (ref. 05FK60) test (Stan- dard Diagnostics Inc., Suwon City, South Korea). These tests both contain a membrane strip encased in a flat plastic hous- ing. The strip is precoated with two antibodies: one that is specific for P. falciparum HRP2 (both kits) and one that is pan-specific for pLDH from Plasmodium species (SD Bioline Malaria Ag Pf/Pan). Our study was carried out between August and October during the season of low malaria transmission at the primary health center in Ampasimpotsy, a rural area in the western foothill of the central highlands in Madagascar. Malaria trans- mission in this area is low and predominantly seasonal. The main vector is Anopheles funestus and the number of infective bites associated with P. falciparum is estimated to be 1-2 per person each year. 5,6 Patients with a fever, or who have had a fever within the past 24 hours, and with typical malaria symp- toms were invited to participate in the study. Pregnant women and patients with signs of severe and complicated P. falci- parum malaria, as defined by the World Health Organization (2001), were excluded. 7 The study protocol was reviewed and

No data were available concerning Plasmodium vivax resistance to chloroquine (CQ) in Madagascar. We investigated the therapeutic efficacy of CQ in P. vivax malaria, the prevalence of mutations in the pvcrt-o and pvmdr1 genes before treatment, and the association between mutant parasites and the clinical response of the patients to CQ treatment. Clinical isolates were collected at six sentinel sites located in the three epidemiological strata for malaria throughout Madagascar in 2006. Patients were enrolled, treated, and followed up according to the WHO 2001 guidelines for P. vivax infections. Sequencing was used to analyze polymorphisms of the pvcrt-o (exons 1 to 6) and pvmdr1 genes. The treatment failure rate, after adjustment for genotyping, was estimated at 5.1% for the 105 patients included, ranging from zero in the South to 14.8% in the foothills of the Central Highlands. All samples were wild type for pvcrt-o but mutant for the pvmdr1 gene. Ten nonsynonymous mutations were found in the pvmdr1 gene, including five new mutations, four of which were present at low frequencies (1.3% to 7.5%) while the S513R mutation was present at a much higher frequency (96.3%). The other five mutations, including Y976F, had been described before and had frequencies of 97.8% to 100%. Our findings suggest that CQ-resistant P. vivax isolates are present in Madagascar, particularly in the foothills of the Central Highlands. The 976Y pvmdr1 mutation was found not to be useful for monitoring CQ resistance. Further efforts are required to develop suitable tools for monitoring drug resistance in P. vivax malaria.

We report the results of clinical studies carried out at six sites in Madagascar, between January and October 2006. The aims were (i) to update our knowledge of the burden of Plasmodium malariae infection and (ii) to assess the therapeutic efficacy of chloroquine for uncomplicated quartan malaria. Our findings confirm that P. malariae is the third leading cause of malaria, accounting for 1.1% of all malarial infections. They also demonstrate that chloroquine-currently recommended for the home management of presumed malaria in children under the age of five years and commonly used by adults-remains highly effective in patients with uncomplicated P. malariae infection.

This study was conducted in 2003 as part of the training of laboratory technicians in the use of rapid diagnostic tests (RDTs) for malaria and to evaluate these tests in Madagascar in field conditions for the first time. Two types of RDT were used separately. The dipstick (Optimal-I) that detects circulating pLDH was tested in 168 patients with clinically suspected malaria (fever or recent history of fever) at primary health centers. Microscopy confirmed malaria in 93/168 (55.4%) cases. Monoparasitic P. falciparum infection was identified in 86/93, P. malariae in 3/93, P. vivax in 3/93 and P. ovale in 1/93. A positive Optimal-I test was a highly sensitive indicator of P. falciparum infection with parasitemia exceeding 500 trophozoites/mul (sensitivity of 97.2%; with a specificity of 100%); it also confirmed 6/7 cases of non-P. falciparum malaria. A community malaria survey used the Malaria Hexagon dipstick (detecting P. falciparum-specific HRP2) for 273 patients: 17 (6.2%) RDT tests were positive, and 16 (5.9%) microscopic tests. Although this dipstick did not detect the only case of infection with P. vivax, its specificity was 100% for detection of P. falciparum infection. Installing microscopes and qualified microscopists in the health centers of the one hundred and eleven districts in Madagascar would be extremely difficult, but our results show that RDT is an effective alternative diagnostic tool for daily use as well as for sporadic malaria epidemics. The revised antimalarial treatment policy, involving a drug ten to twenty times more expensive than chloroquine, demonstrates the need to improve malaria diagnosis: presumptive treatment has become prohibitively expensive. RDT can be used to improve malaria case management at the primary heath centers in Madagascar. We discuss the choice of RDTs.

Field and laboratory studies were carried out in October and November 2005 to provide a comparative evaluation of the performance of three rapid malaria detection tests, two of which were recently introduced (the CareStart™ Malaria test and the SD Malaria Antigen Bioline™ test) and the well-known OptiMAL-IT™ test. Com- pared with microscopy, the sensitivity of the three tests to detect Plasmodium falciparum malaria was 97% for the CareStart™ Malaria test, 89.4% for the SD Malaria Antigen Bioline™ test, and 92.6% for the OptiMAL-IT™ test. The three tests were less sensitive in detecting non-P. falciparum infections, and the sensitivity decreased at levels of parasitemia 500 parasites/L for P. falciparum and 5,000 parasites/L for other Plasmodium spp. On the basis of World Health Organization recommendations, only the CareStart™ Malaria test and the OptiMAL-IT™ test had sensitivities greater than 95% for samples with parasitemias 100 parasites/L. those of other febrile illnesses, 3 a malaria diagnosis based on clinical grounds alone is unreliable. Microscopic examination of a thick blood film is the standard method for malaria di- agnosis and it is only one currently used in provincial or dis- trict hospitals in Madagascar or in some private laboratories in the capital of Antananarivo. This method is relatively simple and has low direct costs, but it requires microscopes and trained microscopists; its reliability is also questionable, particularly at low levels of parasitemia and in the interpre- tation of mixed infections. 4,5 The new Madagascar national policy for the fight against malaria was elaborated in 2005, and it will be supported by resources from the Global Fund. Its main goal nationally is to reduce morbidity and mortality caused by malaria, especially by reducing transmission in unstable malaria-endemic areas such as in the central highlands and the semi-arid southern region, by reducing mortality in areas of stable malaria in the coastal zones, by improving malaria case management through the implementation of biologic diagnosis based on rapid diagnosis tests (RDTs), and by rapid treatment of posi- tive cases with combinations of artesunate plus amodiaquine. At the end of 2005 at the request of the National Malaria Control Program (NMCP), the principal recipient of the Global Fund project round 4 for Madagascar launched an invitation for acquisition of an RDT based on the detection of Plasmodium-specific lactate dehydrogenase (pLDH), accord- ing to the technical choice of the NMCP. Three RDT sup- pliers responded to the invitation with products that target Plasmodium falciparum pLDH and pan-malaria pLDH: the CareStart™ Malaria test, the SD Malaria Antigen Bioline™ test, and the OptiMAL-IT™ test. References in peer- reviewed journals were not found for the CareStart™ Malaria test and the SD Malaria Antigen Bioline™ test. Only the SD Malaria Antigen Bioline™ test and the OptiMAL-IT™ test were found on the list of Manufacturers and Distributors of all known commercially available malaria RDTs from the World Health Organization website (www.wpro.who.int/rdt) at the beginning of the study in October 2005. The Malaria Research Unit of Institut Pasteur de Mada- gascar was asked to evaluate the performances of three RDTs for malaria diagnosis. The study was designed to assess sen- sitivity, specificity, and positive and negative predictive values in field conditions of the three RDTs in symptomatically di- agnosed malaria patients compared with microscopy of thick or thin blood films. A laboratory study was also carried out to test absolute parasite detection limits of the RDTs.

To alleviate the insufficient number of experienced medical teams invited to and accepting to monitor the effectiveness of drugs prescribed to patients with a diagnosis of uncomplicated malaria and to insure the surveillance of the susceptibility of P. falciparum to current antimalarials used in Madagascar, there is a need to draw a feasible study protocol carefully discussed with them. We carried out a preliminary study in two rural areas and assessed the efficacy of sulfadoxine-pyrimethamine (SP) for curing uncomplicated P. falciparum malaria, with a simplified protocol based on the principle of observational study. A single dose of SP was given on day 0 with paracetamol. The persons to whom the drugs were administered accepted two other interventions of one member of the medical teams on day 14 and day 28. Nineteen patients, 3-63 years old, fulfilled the follow-up. The efficacy of this combination was noted for the 19 persons. Our results show that P. falciparum strains are susceptible to SP. Since SP will be used in intermittent preventive treatment in pregnant women in Madagascar, one way to delay the occurrence of SP resistant parasites will be (a) to avoid massive use of SP for the non pregnant persons and (b) to monitor susceptibility of P. falciparum to SP as part of pilot studies using standard WHO protocol (which is not really easy for most of the peripheral health facilities--with the follow-up procedures with clinical examination and parasitological control at Days 0, 1, 2, 3, 7, 14, 21 and 28), and routinely with simplified protocol such as the analytical observational study illustrated in this present study. Limit and advantage of observational study are discussed.

To monitor the sensitivity of Plasmodium falciparum to the drugs used to treat severe malaria and to prevent malaria in Comoros and Madagascar.We used the in vitro isotopic method to test the sensitivity of P. falciparum to quinine, mefloquine and cycloguanil.We tested fresh isolates of P. falciparum, collected from patients living in urban, suburban and rural areas and suffering from uncomplicated malaria in 2001, against at least one of the antimalarials cited above. In both countries all of the successfully tested isolates were sensitive to quinine (N = 243) and to cycloguanil (N = 67). The mean IC50 ranged from 85.7 to 133.7 nM for quinine. For cycloguanil, the mean IC50 ranged from 1.4 to 20.2 nM and the highest IC50 value (102.5 nM) was recorded in Comoros. Only 0.9% (1/110) of the informative isolates from Madagascar were mefloquine-resistant (0/18 in Comoros). The mefloquine mean IC50s were 8.2 nM, 14.1 nM and 11.6 nM respectively in the rural, suburban and urban areas of Madagascar, and 5.9 nM in Comoros. A positive correlation was found between quinine and mefloquine IC50s (N = 127, r = 0.48, p10(-6)), but in vitro mefloquine was 6-16 times more potent than quinine. No correlation was noticed between the activities of quinine and cycloguanil or between the activities of mefloquine and cycloguanil.We therefore advocate the use of a full-course regimen of quinine, as recommended by the World Health Organisation (WHO), to treat above all severe malaria in Madagascar and Comoros. Our results also demonstrate that the use of mefloquine- and cycloguanil-based antimalarials is still justified to prevent malaria in both countries, mainly in the case of travellers.

To redefine strategy and policy to cure or to prevent malaria, there is a need to get relevant and updated data on Plasmodium sp sensitivity level to antimalarial drugs. Thus, in September 1999, the Madagascan Ministry of Health and the Institut Pasteur de Madagascar (IPM) formed a network named RER for malaria resistance surveillance. To alleviate the lack of experienced medical teams within the health centres, and due to technical and logistic matters, as part of the network activities, it was decided to give a start with the in vitro studies which are carried out at IPM. In vitro sensitivity testing is done by use of the isotopic method. Results from the study done in 2001 demonstrate that the Madagascan P. falciparum isolates are susceptible to amodiaquine (n = 215), to cycloguanil (n = 56), to pyrimethamine (n = 98) and to quinine (n = 214). One isolate (1/110 i.e. 0.9%) of mefloquine-resistant phenotype is detected from the Eastern region. P. falciparum susceptibility to chloroquine is satisfactory with 95.4% (206/216) of in vitro sensitive isolates. RER arises from the partnership and collaboration between the Madagascan Ministry of Health and the IPM. The network set-up is presented. The usefulness of the in vivo approach, and the in vitro investigations (chemosusceptibility test and screening of mutations accounting for resistance to chloroquine) to monitor the emergence and the dissemination of drug-resistant parasites in Madagascar as well as in the subregion of the Indian Ocean is discussed.

To determine how sensitive Plasmodium falciparum is to the major antimalarial drugs in Madagascar.Assessment of Plasmodium falciparum isolates sensitivity to antimalarials, by use of the in-vitro radioisotope method.Ankazobe and Saharevo in the foothill areas; and Toamasina and Tolagnaro in the coastal areas (between January 1998 and November 1999).Primary Plasmodium falciparum isolates from patients with uncomplicated malaria attack.Between January 1998 and November 1999, of the 293 in-vitro tests done with at least one antimalarial, 70% (205/293) were interpretable. As there was no significant difference between results from the four study sites, the data have been expressed as a whole. All of the successfully tested isolates were sensitive to halofantrine (n = 56) and to quinine (n = 199), 5.8% (12/205) of the isolates were resistant to chloroquine and 2% (4/199) to mefloquine. The geometric mean IC50 was 0.3 microg/L for halofantrine (95% CI = 0.1-0.4 microg/L); 9.4 microg/L for chloroquine (95% CI = 7.3-10.8 microg/L); 3.8 microg/L for mefloquine (95% CI = 3.3-4.3 microg/L); and 26.8 microg/L for quinine (95% CI = 24.3-29.4 microg/L). The low positive correlation found between halofantrine and chloroquine IC50s (n = 56; r = 0.41, P = 0.002) suggests a risk of cross-resistance between these two drugs.The degree and frequency of chloroquine resistance in-vitro is stationary in Madagascar compared to previous results during the last decade. The in-vitro sensitivity of P. falciparum to quinine, mefloquine and halofantrine encourages the use of these drugs as alternative in case of chloroquine treatment failure. Nevertheless, it is important to maintain and to extend malaria and drug sensitivity surveillance in Madagascar.

Objective: To determine how sensitive Plasmodium falciparum is to the major antimalarial drugs in Madagascar. Design: Assessment of Plasmodium falciparum isolates sensitivity to antimalarials, by use of the in-vitro radioisotope method. Setting: Ankazobe and Saharevo in the foothill areas; and Toamasina and Tolagnaro in the coastal areas (between January 1998 and November 1999). Subjects: Primary Plasmodium falciparum isolates from patients with uncomplicated malaria attack. Results: Between January 1998 and November 1999, of the 293 in-vitro tests done with at least one antimalarial, 70% (205/293) were interpretable. As there was no significant difference between results from the four study sites, the data have been expressed as a whole. All of the successfully tested isolates were sensitive to halofantrine (n = 56) and to quinine (n = 199), 5.8% (12/205) of the isolates were resistant to chloroquine and 2% (4/199) to mefloquine. The geometric mean IC50 was 0.3 µg/L for halofantrine (95% CI = 0.1 - 0.4 µg/L); 9.4 µg/L for chloroquine (95% CI = 7.3 - 10.8 µg/L); 3.8 µg/L for mefloquine (95% CI = 3.3 - 4.3 µg/L); and 26.8 µg/L for quinine (95% CI = 24.3 - 29.4 µg/L). The low positive correlation found between halofantrine and chloroquine IC50s (n=56; r = 0.41, P = 0.002) suggests a risk of cross-resistance between these two drugs. Conclusion: The degree and frequency of chloroquine resistance in-vitro is stationary in Madagascar compared to previous results during the last decade. The in-vitro sensitivity of P. falciparum to quinine, mefloquine and halofantrine encourages the use of these drugs as alternative in case of chloroquine treatment failure. Nevertheless, it is important to maintain and to extend malaria and drug sensitivity surveillance in Madagascar. (East African Medical Journal: 2002 79(5): 237-241))

Objective : To determine how sensitive Plasmodium falciparum is to the major antimalarial drugs in Madagascar. Design : Assessment of Plasmodium falciparum isolates sensitivity to antimalarials, by use of the in-vitro radioisotope method. Setting : Ankazobe and Saharevo in the foothill areas; and Toamasina and Tolagnaro in the coastal areas (between January 1998 and November 1999). Subjects : Primary Plasmodium falciparum isolates from patients with uncomplicated malaria attack. Results : Between January 1998 and November 1999, of the 293 in-vitro tests done with at least one antimalarial, 70% (205/293) were interpretable. As there was no significant difference between results from the four study sites, the data have been expressed as a whole. All of the successfully tested isolates were sensitive to halofantrine (n = 56) and to quinine (n = 199), 5.8% (12/205) of the isolates were resistant to chloroquine and 2% (4/199) to mefloquine. The geometric mean IC50 was 0.3 µg/L for halofantrine (95% CI = 0.1 - 0.4 µg/L); 9.4 µg/L for chloroquine (95% CI = 7.3 - 10.8 µg/L); 3.8 µg/L for mefloquine (95% CI = 3.3 - 4.3 µg/L); and 26.8 µg/L for quinine (95% CI = 24.3 - 29.4 µg/L). The low positive correlation found between halofantrine and chloroquine IC50s (n=56; r = 0.41, P = 0.002) suggests a risk of cross-resistance between these two drugs. Conclusion : The degree and frequency of chloroquine resistance in-vitro is stationary in Madagascar compared to previous results during the last decade. The in-vitro sensitivity of P. falciparum to quinine, mefloquine and halofantrine encourages the use of these drugs as alternative in case of chloroquine treatment failure. Nevertheless, it is important to maintain and to extend malaria and drug sensitivity surveillance in Madagascar. (East African Medical Journal: 2002 79(5): 237-241))

In Madagascar, although chloroquine (CQ) remains the first-line treatment of choice for malaria, the gradual spread of resistance to this antimalarial drug is of increasing concern. As part of a larger investigation of the effectiveness of the second- and third-line drugs used to treat malaria, the in-vitro susceptibilities of Plasmodium falciparum collected in Madagascar to CQ, mefloquine (MQ) and artemether (ART) were therefore investigated. Median inhibitory concentrations (IC(50)) were determined for isolates collected from residents of two villages in the foothills of the central highlands. The IC(50) for ART ranged from 0.23-17.50 nM [N = 51; geometric mean = 4.02 nM; 95% confidence interval (CI) = 2.99-5.05 nM], four isolates exhibiting IC(50) (12 nM) indicative of resistance to this drug. The artemether IC(50) were found to be correlated with those of CQ (N = 46; Spearman's r = 0.51; P = 0.0002), which varied widely (0.4-254.3 nM; mean = 23.4 nM; CI = 7.1-39.7 nM; N = 46). Five (11%) of the 46 isolates exposed to CQ in vitro were considered resistant to this drug (i.e. to have IC(50)100 nM), with IC(50) ranging from 109-245.3 nM (mean = 171.6 nM; CI = 110.4-232.8 nM). However, all the CQ-resistant isolates were considered sensitive to ART and vice versa. All the isolates tested also appeared sensitive to MQ (IC(50) = 2.21-43.1 nM; mean = 10.5 nM; CI = 7.95-13.07 nM; N = 46), the IC(50) for MQ being correlated with those for CQ (N = 46; Spearman's r =0.46; P = 0.001). There was no significant correlation between ART and MQ activities. Although the sample was fairly small, the present results indicate that P. falciparum in Madagascar is generally becoming less sensitive to CQ and ART. The observation of a correlation between the IC(50) for these two drugs perhaps indicates that artemisinin derivatives would be better used in combination with antimalarial drugs other than 4-aminoquinolines.

Background An estimated 2.85 billion people live at risk of Plasmodium vivax transmission. In endemic countries vivax malaria causes significant morbidity and its mortality is becoming more widely appreciated, drug-resistant strains are increasing in prevalence, and an increasing number of reports indicate that P. vivax is capable of breaking through the Duffy-negative barrier long considered to confer resistance to blood stage infection. Absence of robust in vitro propagation limits our understanding of fundamental aspects of the parasite's biology, including the determinants of its dormant hypnozoite phase, its virulence and drug susceptibility, and the molecular mechanisms underlying red blood cell invasion. Methodology/Principal Findings Here, we report results from whole genome sequencing of five P. vivax isolates obtained from Malagasy and Cambodian patients, and of the monkey-adapted Belem strain. We obtained an average 70–400 X coverage of each genome, resulting in more than 93% of the Sal I reference sequence covered by 20 reads or more. Our study identifies more than 80,000 SNPs distributed throughout the genome which will allow designing association studies and population surveys. Analysis of the genome-wide genetic diversity in P. vivax also reveals considerable allele sharing among isolates from different continents. This observation could be consistent with a high level of gene flow among parasite strains distributed throughout the world. Conclusions Our study shows that it is feasible to perform whole genome sequencing of P. vivax field isolates and rigorously characterize the genetic diversity of this parasite. The catalogue of polymorphisms generated here will enable large-scale genotyping studies and contribute to a better understanding of P. vivax traits such as drug resistance or erythrocyte invasion, partially circumventing the lack of laboratory culture that has hampered vivax research for years., Author Summary Plasmodium vivax is the most frequently transmitted and widely distributed cause of malaria in the world. Each year P. vivax is responsible for approximately 250 million clinical cases of malaria and its global economic burden, placed largely on the poor, has been estimated to exceed US$1.4 billion. In contrast to P. falciparum, P. vivax cannot be propagated in continuous in vitro culture and this limits our understanding of the parasite’s biology. In this study, we sequenced the entire genome of five P. vivax isolates directly from blood samples of infected patients. Our data indicated that each patient was infected with multiple P. vivax strains. We also identified more than 80,000 DNA polymorphisms distributed throughout the genome that will enable future studies of the P. vivax population and association mapping studies. Our study illustrates the potential of genomic studies for better understanding P. vivax biology and how the parasite successfully evades malaria elimination efforts worldwide.

International audience; BACKGROUND: In Madagascar, very little is known about the etiology and prevalence of acute respiratory infections (ARIs) in a rural tropical area. Recent data are needed to determine the viral and atypical bacterial etiologies in children with defined clinical manifestations of ARIs. METHODS: During one year, we conducted a prospective study on ARIs in children between 2 to 59 months in the community hospital of Ampasimanjeva, located in the south-east of Madagascar. Respiratory samples were analyzed by multiplex real-time RT-PCR, including 18 viruses and 2 atypical bacteria. The various episodes of ARI were grouped into four clinical manifestations with well-documented diagnosis: "Community Acquired Pneumonia"(CAP, group I), "Other acute lower respiratory infections (Other ALRIs, group II)", "Upper respiratory tract infections with cough (URTIs with cough, group III)"and "Upper respiratory tract infections without cough (URTIs without cough, group IV)". RESULTS: 295 children were included in the study between February 2010 and February 2011. Viruses and/or atypical bacteria respiratory pathogens were detected in 74.6% of samples, the rate of co-infection was 27.3%. Human rhinovirus (HRV; 20.5%), metapneumovirus (HMPV A/B, 13.8%), coronaviruses (HCoV, 12.5%), parainfluenza virus (HPIV, 11.8%) and respiratory syncytial virus A and B (RSV A/B, 11.8%) were the most detected. HRV was predominantly single detected (23.8%) in all the clinical groups while HMPV A/B (23.9%) was mainly related to CAP (group I), HPIV (17.3%) to the "Other ALRIs" (group II), RSV A/B (19.5%) predominated in the group "URTIs with cough" (group III) and Adenovirus (HAdV, 17.8%) was mainly detected in the "without cough" (group IV). INTERPRETATION: This study describes for the first time the etiology of respiratory infections in febrile children under 5 years in a malaria rural area of Madagascar and highlights the role of respiratory viruses in a well clinically defined population of ARIs.