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Background: Plasmodium vivax malaria is endemic in Madagascar, where populations have genetic inheritance from Southeast Asia and East Africa. Primaquine, a drug of choice for vivax malaria, is metabolized principally via CYP2D6. CYP2D6 variation was characterized by locus-specific gene sequencing and was compared with TaqMan™ genotype data. Materials & methods: Long-range PCR amplicons were generated from 96 Malagasy samples and subjected to next-generation sequencing. Results: The authors observed high concordance between TaqMan™-based CYP2D6 genotype calls and the base calls from sequencing. In addition, there are new variants and haplotypes present in the Malagasy. Conclusion: Sequencing unique admixed populations provides more detailed and accurate insights regarding CYP2D6 variability, which may help optimize primaquine treatment across human genetic diversity.

Background Gametocytes are the sexual stages ensuring continuity of the development cycle of the parasite, as well as its transmission to humans. The efficacy of artemisinin-based anti-malarials against asexual stages of Plasmodium has been reported in Madagascar, but their effects on gametocytes are not well documented. The present study aims to determine the emergence of gametocyte and gametocyte clearance after artesunate-amodiaquine (ASAQ) or artemether-lumefantrine (AL) treatment in children with uncomplicated Plasmodium falciparum malaria in 5 regions of Madagascar. Methods 558 children with uncomplicated P. falciparum malaria, aged between 1 and 15 years, were assigned randomly to AL or ASAQ treatment. They come from 5 regions of Madagascar with different epidemiological facies related to malaria: Ankilivalo, Benenitra, Ampanihy, Ankazomborona and Matanga. Gametocytes were identified by microscopy, from t blood smears at day 1, day 2, day 3, day 7, day 14, day 21 and day 28 after treatment. Results At baseline, 9.7% (54/558) children [95% CI: 7.4–12.5%] had detectable gametocyte by microscopy. Among the 54 enrolled children, gametocytes emergence rate was high during the first days of treatment in both treatment arms (AL and ASAQ), especially on day 1. Gametocytes were undetectable from day 14 for AL arm while for ASAQ arm, gametocyte carriage was gradually decreased but persisted until day 21. Conclusion This study demonstrates that AL has a more rapid effect on gametocyte clearance compared to ASAQ in children with uncomplicated Plasmodium falciparum malaria.

Contexte. La surveillance de l’efficacité thérapeutique et des marqueurs génétiques de la résistance aux antipaludiques est primordiale afin de détecter le plus tôt possible l’émergence des parasites potentiellement résistants. Dans ce contexte, notre étude a pour objectif de réaliser le typage du gène Plasmodium falciparum chloroquine resistance transporter ou Pfcrt et Plasmodium falciparum multidrug resistant gene 1 ou Pfmdr1 sur des isolats provenant des enfants du district de Vatomandry. Méthodologies. Au total, 142 isolats de P. falciparum collectés lors d’un dépistage actif du paludisme chez des enfants âgés de moins de 15 ans, entre février et mars des années 2016 et 2017 à Vatomandry, ont été analysés. Le typage du codon K76T du gène Pfcrt et du codon N86Y du gène Pfmdr1 a été ensuite effectué par la technique de polymérisation en chaîne suivie de digestion enzymatique (restriction fragment length polymorphism) ou PCR-RFLP. Résultats. Le taux de succès d’amplification des gènes Pfcrt et Pfmdr1 était faible, de l’ordre de 27 % et 39 % respectivement. La prévalence des isolats mutants pour le codon K76T de Pfcrt était de 2,6 % [intervalle de confiance à 95 % (IC95%) : 0,1 - 15,0 %] et de 36 % [IC95% : 23,7 - 49,7 %] pour le codon N86Y du gène Pfmdr1. Conclusion. Notre étude a mis en évidence la présence d’isolats portant à la fois la mutation au niveau du codon K76T de Pfcrt et N86Y de Pfmdr1. Bien que le taux de mutation que nous avons observé soit faible, d’autres études méritent d’être effectuées afin de suivre l’évolution de ces marqueurs dans le temps et dans l’espace à Madagascar. PCR-RFLP genotyping of Pfcrt and Pfmdr1 in Plasmodium falciparum isolates from children Background. Malaria is a parasitic disease caused by a hematozoan of the genus Plasmodium. Early diagnosis followed by effective treatment is one of the keys to control this disease. In Madagascar, after more than 60 years of use for the treatment of uncomplicated malaria, chloroquine (CQ) was abandoned in favor of artesunate + amodiaquine (ASAQ) combination because of high prevalence of CQ treatment failure. Surveillance based on the assessment of therapeutic efficacy and genetic markers of resistance to antimalarials is therefore essential in order to detect the emergence of potentially resistant parasites as early as possible. In this context, our study aimed to genotype the Plasmodium falciparum chloroquine resistance transporter gene or Pfcrt and Plasmodium falciparum multidrug resistance gene 1 or Pfmdr1 in isolates collected from children in the district of Vatomandry. Methods. A total of 142 P. falciparum isolates collected during active case detection of malaria in children under 15 years old, between February and March of 2016 and 2017 in Vatomandry district, were analyzed. Pfcrt (K76T codon) and Pfmdr1 (N86Y codon) genotyping was carried out by polymerase chain reaction followed by enzymatic digestion (restriction fragment length polymorphism) or PCR-RFLP. Results. The successful rates of amplification of Pfcrt and Pfmdr1 genes were low, around 27% and 39% respectively. The prevalence of isolates carrying the mutant Pfcrt K76T codon and the mutant Pfmdr1 N86Y codon was 2.6% [95% confidence interval (95% CI): 0.1 - 15.0%] and 36% [95% CI: 23.7 - 49.7%] respectively. Conclusion. Despite the limited number of samples analyzed, our study highlighted the circulation of isolates carrying both the mutant Pfcrt K76T and Pfmdr1 N86Y alleles. Although the prevalence of mutations in Pfcrt and Pfmdr1 genes that we observed was low, other studies should be carried out in order to follow the evolution of these markers in time and space. The use of more sensitive methods will better characterize P. falciparum strains circulating in Madagascar. Artesunate-amodiaquine is used as a first-line treatment for uncomplicated malaria in the country; it is also crucial to monitor the other codons, i.e. 184 and 1246 of the Pfmdr1 gene, implicated in the resistance of P. falciparum to amodiaquine in Africa., MTSI, Vol. 2 No 2 (2022): MTSI-Revue

Histidine-rich protein 2 (HRP2)-based rapid diagnostic tests detect Plasmodium falciparum malaria and are used throughout sub-Saharan Africa. However, deletions in the pfhrp2 and related pfhrp3 (pfhrp2/3) genes threaten use of these tests. Therapeutic efficacy studies (TESs) enroll persons with symptomatic P. falciparum infection. We screened TES samples collected during 2016-2018 in Ethiopia, Kenya, Rwanda, and Madagascar for HRP2/3, pan-Plasmodium lactate dehydrogenase, and pan-Plasmodium aldolase antigen levels and selected samples with low levels of HRP2/3 for pfhrp2/3 genotyping. We observed deletion of pfhrp3 in samples from all countries except Kenya. Single-gene deletions in pfhrp2 were observed in 1.4% (95% CI 0.2%-4.8%) of Ethiopia samples and in 0.6% (95% CI 0.2%-1.6%) of Madagascar samples, and dual pfhrp2/3 deletions were noted in 2.0% (95% CI 0.4%-5.9%) of Ethiopia samples. Although this study was not powered for precise prevalence estimates, evaluating TES samples revealed a low prevalence of pfhrp2/3 deletions in most sites.

Background There is currently no validated, effective, safe treatment for severe illness caused by SARS-CoV-2. CVO PLUS CURATIF (CVO+C) is a capsule formulation of two compounds of plant origin with anti-inflammatory and antiviral activities in vitro: artemisinin and 1,8-cineole. These compounds have been repurposed for possible use as an oral treatment against COVID-19. Methods We performed a phase 3 randomized clinical trials on patients over the age of 18 years with SARS-CoV-2 infection confirmed by RT-PCR and mild-to-moderate symptoms. Patients were randomly assigned to receive CVO+C (3 capsules per day) or placebo for 15 days. The primary outcome was the proportion of patients testing negative for SARS-CoV-2 by RT-PCR on day 28 and an absence of severe and serious adverse events. Recovery time, and biological parameters on days 7, 14, 21 and 28 of the trial were considered as secondary outcomes. The safety outcomes considered were adverse events on treatment. Results In total, 1,576 individuals underwent RT-PCR screening for SARS-CoV-2 infection during the study period. Positive test results were obtained for 591 subjects, 339 of whom met the inclusion criteria for this study. The final analysis included 339 subjects: 132 from the CVO+C arm and 144 from the placebo arm. Treatment efficacy differed significantly (p=0.011) between the CVO+C arm (87.1%, 95% CI: 81.3%-92.9%, with 70.45% of patients cured by day 14) and the placebo arm (75.0%, 95% CI: 67.8% - 82.1%), with an OR of 2.25. The median time to recovery was 14 days for the CVO+C group and 21 days for the placebo group. A total of 72 incidences of mild and moderate adverse events, 14 severe adverse events and no serious adverse events were observed in both groups. ConclusionCVO+C was effective for the treatment of mild-to-moderate COVID-19. None of the patients in the CVO+C arm displayed progression to the severe form of COVID-19. Liver kidney and metabolic functions were preserved in all patients.Trial registration: Registered at Pan African Clinical Trials Registry: (No. PACTR202103601407640, date of approval: 24/03/2021) and approved by the ethics committee of the Ministry of Public Health of Madagascar (approval No. 216 MINSANP/SG/AGMED/CERBM, 17/12/2020)

Background Rapid diagnostic tests (RDT) are widely used for malaria diagnosis in Madagascar, where Plasmodium falciparum is the predominant species. Molecular diagnosis is essential for malaria surveillance, but requires additional blood samples for DNA extraction. Used RDTs is an attractive alternative that can be used as a source of DNA. Plasmodium falciparum genetic diversity and multiplicity of infection, usually determined by the genotyping of polymorphic regions of merozoite surface proteins 1 and 2 genes (msp1, msp2), and the repeated region RII of the glutamate-rich protein gene (glurp) have been associated with malaria transmission levels and subsequently with the impact of the deployed control strategies. Thus, the study aims to use RDT as DNA source to detect Plasmodium species, to characterize Plasmodium falciparum genetic diversity and determine the multiplicity of infection. Methods A pilot study was conducted in two sites with different epidemiological patterns: Ankazomborona (low transmission area) and Matanga (high transmission area). On May 2018, used RDT (SD BIOLINE Malaria Ag P.f/Pan, 05FK63) were collected as DNA source. Plasmodium DNA was extracted by simple elution with nuclease free water. Nested-PCR were performed to confirm Plasmodium species and to analyse P. falciparum msp1, msp2 and glurp genes polymorphisms. Results Amongst the 170 obtained samples (N = 74 from Ankazomborona and N = 96 from Matanga), Plasmodium positivity rate was 23.5% (40/170) [95% CI 17.5–30.8%] by nested-PCR with 92.2% (37/40) positive to P. falciparum, 5% (2/40) to Plasmodium vivax and 2.5% (1/40) to P. falciparum/P. vivax mixed infection. Results showed high polymorphisms in P. falciparum msp1, msp2 and glurp genes. Multiple infection rate was 28.6% [95% CI 12.2–52.3%]. The mean of MOI was 1.79 ± 0.74. Conclusion This pilot study highlighted that malaria diagnosis and molecular analysis are possible by using used malaria RDT. A large-scale study needs to be conducted to assess more comprehensively malaria parasites transmission levels and provide new data for guiding the implementation of local strategies for malaria control and elimination. Trial registration Retrospectively registered

Malaria is a parasitic disease caused by a hematozoan of the genusA total of 142The successful rates of amplification ofDespite the limited number of samples analyzed, our study highlighted the circulation of isolates carrying both the mutant

Additional file 2: AL dosage.

International audience; Introduction: Epilepsy is a chronic disease of the brain that affects approximately 50 million people globally, with over 80 % of them living in low- and middle-income countries (LMICs). In Madagascar, as in most LMICs, one of the main obstacles to treatment is the stigma and discrimination experienced by patients. Beliefs and prejudices regarding this disease are common, especially among children. "Ao Tsara" is an educational comic book regarding epilepsy, which has been translated in Malagasy from a French version, and which objective is to raise awareness and fight epilepsy related stigma and discrimination. Comic books have indeed been used successfully to raise awareness and change behaviors in several areas of public health. Methods: We conducted a study to evaluate the effect of a single reading of this comic book on epilepsy related knowledge, attitudes and practices (KAP) in schoolchildren in Madagascar. This quasi-experimental study compared data collected before and immediately after reading "Ao Tsara". It was conducted both in a school in an urban area and in a school in a rural area. Results: We recruited 244 children with a mean age of 11.4 (±1.5) in this study. We noted a significant improvement in the global KAP score after reading the comic book, overall as well as both in the urban school and the rural school. Out of a maximum score of twenty, the global KAP score increased from 9.4 to 11.2 (p < 0.001). Although the increase in knowledge was reasonable (10.2-12.9, p < 0.001) and the corresponding subscore after reading the comic book was at a satisfactory level, that was not the case for attitudes & practices, where the sub-score despite a significant increase remained low (8.7-9.5 out of a maximum score of twenty, p < 0.001). The comic book was much appreciated by the children with more than 50.0 % giving it the top rating, and 66.4 % stating they had learned a lot from it. Conclusion: A single reading of the comic book has demonstrated a positive effect on the knowledge, attitudes and practices of primary school children in Madagascar. This educational tool, which was much enjoyed by the children, could be of great value to raise awareness about epilepsy in Madagascar. By targeting a slightly older age group and adjusting the reading approach, the outcomes could be optimized especially in terms of attitudes and practices.

Anopheles mosquitoes vary in habitat preference, feeding pattern, and susceptibility to various measures of vector control. Consequently, it is important that we identify reservoirs of disease, identify vectors, and characterize feeding patterns to effectively implement targeted control measures. Using 467 anopheline mosquito abdomen squashes captured in Madagascar, we designed a novel ligase detection reaction and fluorescent microsphere assay, dubbed Bloodmeal Detection Assay for Regional Transmission (BLOODART), to query the bloodmeal content, identify five Anopheles mosquito species, and detect Plasmodium infection. Validation of mammalian bloodspots was achieved by preparation and analysis of known hosts (singular and mixed), sensitivity to degradation and storage method were assessed through mosquito feeding experiments, and quantification was explored by altering ratios of two mammal hosts. BLOODART identifications were validated by comparison with mosquito samples identified by sequenced portions of the internal transcribed spacer 2. BLOODART identification of control mammal bloodspots was 100% concordant for singular and mixed mammalian blood. BLOODART was able to detect hosts up to 42 hours after digestion when mosquito samples were stored in ethanol. A mammalian host was identified in every field-collected, blood-fed female Anopheles mosquito by BLOODART. The predominant mosquito host was cow (n = 451), followed by pig (n = 26) and human (n = 25). Mixed species bloodmeals were commonly observed (n = 33). A BLOODART molecular identification was successful for 318/467 mosquitoes, with an overall concordance of 60% with all field-captured, morphologically identified Anopheles specimens. BLOODART enables characterization of large samples and simultaneous pathogen detection to monitor and incriminate disease vectors in Madagascar.

Plasmodium vivax is one of the five human malaria parasite species, which has a wide geographical distribution and can cause severe disease and fatal outcomes. It has the ability to relapse from dormant liver stages (hypnozoites), weeks to months after clearance of the acute blood-stage infection. An 8-aminoquinoline drug primaquine (PQ) can clear the hypnozoites, and thus can be used as an anti-relapse therapeutic agent. Recently, a number of studies have found that its efficacy is compromised by polymorphisms in the cytochrome P450 2D6 (CYP2D6) gene; decreased or absence of CYP2D6 activity contributes to PQ therapeutic failure. The present study sought to characterize CYP2D6 genetic variation in Madagascar, where populations originated from admixture between Asian and African populations, vivax malaria is endemic, and PQ can be deployed soon to achieve national malaria elimination. In a total of 211 samples collected from two health districts, CYP2D6 decreased function alleles CYP2D6*10, *17, *29, *36+*10, and *41 were observed at frequencies of 3.55–17.06%. In addition, nonfunctional alleles were observed, the most common of which were CYP2D6*4 (2.13%), *5 (1.66%), and the *4x2 gene duplication (1.42%). Given these frequencies, 34.6% of the individuals were predicted to be intermediate metabolizers (IM) with an enzyme activity score (AS) ≤ 1.0; both the IM phenotype and AS ≤ 1.0 have been found to be associated with PQ therapeutic failure. Furthermore, the allele and genotype frequency distributions add to the archaeological and genomic evidence of Malagasy populations constituting a unique, Asian-African admixed origin. The results from this exploratory study provide fresh insights about genomic characteristics that could affect the metabolism of PQ into its active state, and may enable optimization of PQ treatment across human genetic diversity, which is critical for achieving P. vivax elimination.

Background Assessment of the genetic diversity of Plasmodium falciparum parasites from various malaria transmission settings could help to define tailored local strategies for malaria control and elimination. Such assessments are currently scarce in Madagascar. The study presented here aimed to bridge this gap by investigating the genetic diversity of P. falciparum populations in three epidemiological strata (Equatorial, Tropical and Fringes) in Madagascar. Methods Two-hundred and sixty-six P. falciparum isolates were obtained from patients with uncomplicated malaria enrolled in clinical drug efficacy studies conducted at health centres in Tsaratanana (Equatorial stratum), Antanimbary (Tropical stratum) and Anjoma Ramartina (Fringes) in 2013 and 2016. Parasite DNA was extracted from blood samples collected before anti-malarial treatment. Plasmodium species were identified by nested PCR targeting the 18 S rRNA gene. The genetic profiles of P. falciparum parasites were defined by allele-specific nested PCR on the polymorphic regions of the msp-1 and msp-2 genes. Results Fifty-eight alleles were detected in the P. falciparum samples tested: 18 alleles for msp-1 and 40 for msp-2. K1 (62.9%, 139/221) and FC27 (69.5%, 114/164) were the principal msp-1 and msp-2 allele families detected, although the proportions of the msp-1 and msp-2 alleles varied significantly between sites. Polyclonal infections were more frequent at sites in the Equatorial stratum (69.8%) than at sites in the Tropical stratum (60.5%) or Fringes (58.1%). Population genetics analyses showed that genetic diversity was similar between sites and that parasite flow within sites was limited. Conclusions This study provides recent information about the genetic diversity of P. falciparum populations in three transmission strata in Madagascar, and valuable baseline data for further evaluation of the impact of the control measures implemented in Madagascar.

International audience; Background: In Madagascar, prevalence of epilepsy is 27.3/1000 and the lifetime prevalence of mental disorders 34.0. From 2013 to 2018, interventions were carried out by the MoH to train GPs on these diseases and raise awareness among the population.Objectives: The objective was to compare the availability and cost of drugs between intervention and control areas in 8 regions of Madagascar.Methods: Data were collected from all types of points of sale (POS) for medicines (public and private). All psychotropic and antiepileptic drugs were included. The study received ethical approval from the Madagascar MoH.Results: 218 POS (82.5%) agreed to be investigated. Regarding the overall availability of AEDs, there was no significant difference between intervention and control areas (95.9% vs 94.4% of POS stocking at least one AED). There were however differences for carbamazepine (79.7% vs 53.4%) and sodium valproate (27.0% vs 11.8%) with these being available in a greater number of POS in the intervention areas (p

Background In low-malaria–transmission areas of Madagascar, annual parasite incidence (API) from routine data has been used to target indoor residual spraying at subdistrict commune level. To assess validity of this approach, we conducted school-based serological surveys and health facility (HF) data quality assessments in 7 districts to compare API to gold-standard commune-level serological measures. Methods At 2 primary schools in each of 93 communes, 60 students were randomly selected with parents and teachers. Capillary blood was drawn for rapid diagnostic tests (RDTs) and serology. Multiplex bead-based immunoassays to detect antibodies to 5 Plasmodium falciparum antigens were conducted, and finite mixture models used to characterize seronegative and seropositive populations. Reversible catalytic models generated commune-level annual seroconversion rates (SCRs). HF register data were abstracted to assess completeness and accuracy. Results RDT positivity from 12 770 samples was 0.5%. Seroprevalence to tested antigens ranged from 17.9% (MSP-1) to 59.7% (PF13). Median commune-level SCR was 0.0108 (range, 0.001–0.075). Compared to SCRs, API identified 71% (95% confidence interval, 51%–87%) of the 30% highest-transmission communes; sensitivity declined at lower levels. Routine data accuracy did not substantially affect API performance. Conclusions API performs reasonably well at identifying higher-transmission communes but sensitivity declined at lower transmission levels.

Malaria transmission in Madagascar is highly heterogeneous, exhibiting spatial, seasonal and long-term trends. Previous efforts to map malaria risk in Madagascar used prevalence data from Malaria Indicator Surveys. These cross-sectional surveys, conducted during the high transmission season most recently in 2013 and 2016, provide nationally representative prevalence data but cover relatively short time frames. Conversely, monthly case data are collected at health facilities but suffer from biases, including incomplete reporting and low rates of treatment seeking. We combined survey and case data to make monthly maps of prevalence between 2013 and 2016. Health facility catchment populations were estimated to produce incidence rates from the case data. Smoothed incidence surfaces, environmental and socioeconomic covariates, and survey data informed a Bayesian prevalence model, in which a flexible incidence-to-prevalence relationship was learned. Modelled spatial trends were consistent over time, with highest prevalence in the coastal regions and low prevalence in the highlands and desert south. Prevalence was lowest in 2014 and peaked in 2015 and seasonality was widely observed, including in some lower transmission regions. These trends highlight the utility of monthly prevalence estimates over the four year period. By combining survey and case data using this two-step modelling approach, we were able to take advantage of the relative strengths of each metric while accounting for potential bias in the case data. Similar modelling approaches combining large datasets of different malaria metrics may be applicable across sub-Saharan Africa.

BackgroundThe Madagascar National Strategic Plan for Malaria Control 2018 (NSP) outlines malaria control pre-elimination strategies that include detailed goals for mosquito control. Primary surveillance protocols and mosquito control interventions focus on indoor vectors of malaria, while many potential vectors feed and rest outdoors. Here we describe the application of novel tools that advance our understanding of diversity, host choice, and Plasmodium infection in the Anopheline mosquitoes of the Western Highland Fringe of Madagascar.Methodology/Principal FindingsWe employed a novel outdoor trap, the QUadrant Enabled Screen Trap (QUEST), in conjunction with the recently developed multiplex BLOOdmeal Detection Assay for Regional Transmission (BLOODART). We captured a total of 1252 female Anopheles mosquitoes (10 species), all of which were subjected to BLOODART analysis. QUEST collection captured a heterogenous distribution of mosquito density, diversity, host choice, and Plasmodium infection. Concordance between Anopheles morphology and BLOODART species identifications ranged from 93-99%. Mosquito feeding behavior in this collection frequently exhibited multiple blood meal hosts (single host = 53.6%, two hosts = 42.1%, three hosts = 4.3%). The overall percentage of human positive bloodmeals increased between the December 2017 and the April 2018 timepoints (27% to 44%). Plasmodium positivity was found primarily in vectors considered to be of secondary importance, with an overall prevalence of 6%.Conclusions/SignificanceThe QUEST was an efficient tool for sampling Anopheline mosquitoes. Vectors considered to be of secondary importance were commonly found with Plasmodium DNA in their abdomens, indicating a need to account for these species in routine surveillance efforts. Mosquitoes exhibited multiple blood feeding behavior within a gonotrophic cycle, with predominantly non-human hosts in the bloodmeal. Taken together, this complex feeding behavior could enhance the role of multiple Anopheline species in malaria transmission, possibly tempered by zoophilic feeding tendencies.Author SummaryMalaria continues to be a significant threat to public health in Madagascar. Elimination of this disease is impeded by numerous factors, such as vector surveillance that does little to account for the potential role of secondary malaria vectors, which rest and feed outdoors. In this study, we designed a novel, low cost QUadrant Enabled Screen Trap (QUEST) to address the lack of traps for outdoor mosquitoes. We used this in conjunction with our novel BLOOdmeal Detection Assay for Regional Transmission (BLOODART) to assess mosquito feeding behavior in the Western Highland Fringe of Madagascar. Our analysis revealed significant variability in mosquito density, diversity, host choice, and Plasmodium infection across traps placed within and between two nearby villages at two timepoints; indicating a strong, small-scale spatial component to disease transmission that warrants further investigation. Many of the mosquitoes in this sample (46.4%) fed on two or three host species, indicating complex feeding behaviors that could influence malaria transmission. Further, Plasmodium DNA was detected in the abdomens of numerous vectors of supposed secondary importance, indicating a neglected parasite reservoir and an increased need to account for these species in routine surveillance efforts.

Background The Madagascar National Strategic Plan for Malaria Control 2018 (NSP) outlines malaria control pre-elimination strategies that include detailed goals for mosquito control. Primary surveillance protocols and mosquito control interventions focus on indoor vectors of malaria, while many potential vectors feed and rest outdoors. Here we describe the application of tools that advance our understanding of diversity, host choice, and Plasmodium infection in the Anopheline mosquitoes of the Western Highland Fringe of Madagascar. Methodology/Principal findings We employed a modified barrier screen trap, the QUadrant Enabled Screen Trap (QUEST), in conjunction with the recently developed multiplex BLOOdmeal Detection Assay for Regional Transmission (BLOODART). We captured a total of 1252 female Anopheles mosquitoes (10 species), all of which were subjected to BLOODART analysis. QUEST collection captured a heterogenous distribution of mosquito density, diversity, host choice, and Plasmodium infection. Concordance between Anopheles morphology and BLOODART species identifications ranged from 93–99%. Mosquito feeding behavior in this collection frequently exhibited multiple blood meal hosts (single host = 53.6%, two hosts = 42.1%, three hosts = 4.3%). The overall percentage of human positive bloodmeals increased between the December 2017 and the April 2018 timepoints (27% to 44%). Plasmodium positivity was frequently observed in the abdomens of vectors considered to be of secondary importance, with an overall prevalence of 6%. Conclusions/Significance The QUEST was an efficient tool for sampling exophilic Anopheline mosquitoes. Vectors considered to be of secondary importance were commonly found with Plasmodium DNA in their abdomens, indicating a need to account for these species in routine surveillance efforts. Mosquitoes exhibited multiple blood feeding behavior within a gonotrophic cycle, with predominantly non-human hosts in the bloodmeal. Taken together, this complex feeding behavior could enhance the role of multiple Anopheline species in malaria transmission, possibly tempered by zoophilic feeding tendencies., Author summary Malaria continues to be a significant threat to public health in Madagascar. Elimination of this disease is impeded by numerous factors, such as vector surveillance that does little to account for the potential role of secondary malaria vectors, which tend to rest and feed outdoors. In this study, we designed a low cost, modified barrier screen trap, called the QUadrant Enabled Screen Trap (QUEST). We used this in conjunction with the recently developed BLOOdmeal Detection Assay for Regional Transmission (BLOODART) to assess mosquito feeding behavior in the Western Highland Fringe of Madagascar. Our analysis revealed significant variability in mosquito density, diversity, host choice, and Plasmodium infection across traps placed within and between two nearby villages at two timepoints; indicating a strong, small-scale spatial component to disease transmission that warrants further investigation. Many of the mosquitoes in this sample (46.4%) fed on two or three host species, indicating complex feeding behaviors that could influence malaria transmission. Further, Plasmodium DNA was detected in the abdomens of numerous vectors of supposed secondary importance, indicating a neglected parasite reservoir and an increased need to account for these species in routine surveillance efforts.

Background Plasmodium vivax is the most prevalent human malaria parasite and is likely to increase proportionally as malaria control efforts more rapidly impact the prevalence of Plasmodium falciparum. Despite the prominence of P. vivax as a major human pathogen, vivax malaria qualifies as a neglected and under-studied tropical disease. Significant challenges bringing P. vivax into the laboratory, particularly the capacity for long-term propagation of well-characterized strains, have limited the study of this parasite’s red blood cell (RBC) invasion mechanism, blood-stage development, gene expression, and genetic manipulation. Methods and results Patient isolates of P. vivax have been collected and cryopreserved in the rural community of Ampasimpotsy, located in the Tsiroanomandidy Health District of Madagascar. Periodic, monthly overland transport of these cryopreserved isolates to the country’s National Malaria Control Programme laboratory in Antananarivo preceded onward sample transfer to laboratories at Case Western Reserve University, USA. There, the P. vivax isolates have been cultured through propagation in the RBCs of Saimiri boliviensis. For the four patient isolates studied to-date, the median time interval between sample collection and in vitro culture has been 454 days (range 166–961 days). The median time in culture, continually documented by light microscopy, has been 159 days; isolate AMP2014.01 was continuously propagated for 233 days. Further studies show that the P. vivax parasites propagated in Saimiri RBCs retain their ability to invade human RBCs, and can be cryopreserved, thawed and successfully returned to productive in vitro culture. Conclusions/significance Long-term culture of P. vivax is possible in the RBCs of Saimiri boliviensis. These studies provide an alternative to propagation of P. vivax in live animals that are becoming more restricted. In vitro culture of P. vivax in Saimiri RBCs provides an opening to stabilize patient isolates, which would serve as precious resources to apply new strategies for investigating the molecular and cellular biology of this important malaria parasite. Electronic supplementary material The online version of this article (10.1186/s12936-017-2090-7) contains supplementary material, which is available to authorized users.

Introduction: cette etude se propose d’evaluer l’adherence des professionnels de sante du secteur prive a l'utilisation du TDR-palu (Test de Diagnostic Rapide-palu) et a la prescription de l’ACT (Artemisinin-based combination therapy) en cas de paludisme simple. Methodes: une approche evaluative retrospective et transversale a ete menee en septembre et octobre 2015 aupres de 11 districts sanitaires repartis dans les quatre facies epidemiologiques existant a Madagascar. Au total, 43 prestataires de soins issus de 39 formations sanitaires privees (FSP) ont ete interviewes et visites. Resultats: les prestataires declarent avoir lu le manuel de prise en charge du paludisme dans 16,3% des cas (4/43). Seul le quart (25,6%) des prestataires dispose de TDR dans leur bureau. L’ACT a ete cite par 83,7% des prestataires comme medicament de premiere intention pour traiter le paludisme simple. Dans la pratique, 55,6% des prestataires emettent des doutes sur les resultats des TDR. L’utilisation des antipaludeens malgre les resultats negatifs des TDR (38,2%) est plus frequente chez ceux ayant emis des doutes (p = 0,03). Inversement, malgre un TDR positif, la moitie des prestataires ne prescrit pas d’ACT (50%). La non-participation aux revues periodiques du District sanitaire (p = 0,05) influence negativement l’adherence aux politiques. Conclusion: la faible adherence des prestataires de soin du secteur prive aux directives nationales sur la prise en charge des cas de paludisme simple interpelle sur l’interet d’encadrer davantage ce secteur.

Community prevalence of infection is a widely used, standardized metric for evaluating malaria endemicity. Conventional methods for measuring prevalence include light microscopy and rapid diagnostic tests (RDTs), but their detection thresholds are inadequate for diagnosing low-density infections. The significance of submicroscopic malaria infections is poorly understood in Madagascar, a country of heterogeneous malaria epidemiology. A cross-sectional community survey in the western foothills of Madagascar during the March 2014 transmission season found malaria infection to be predominantly submicroscopic and asymptomatic. Prevalence of Plasmodium infection diagnosed by microscopy, RDT, and molecular diagnosis was 2.4%, 4.1%, and 13.8%, respectively. This diagnostic discordance was greatest for Plasmodium vivax infection, which was 98.5% submicroscopic. Village location, insecticide-treated bednet ownership, and fever were significantly associated with infection outcomes, as was presence of another infected individual in the household. Duffy-negative individuals were diagnosed with P. vivax, but with reduced odds relative to Duffy-positive hosts. The observation of high proportions of submicroscopic infections calls for a wider assessment of the parasite reservoir in other regions of the island, particularly given the country's current focus on malaria elimination and the poorly documented distribution of the non-P. falciparum parasite species.

International audience; Background: Reliable measures of disease burden over time are necessary to evaluate the impact of interventions and assess sub-national trends in the distribution of infection. Three Malaria Indicator Surveys (MISs) have been conducted in Madagascar since 2011. They provide a valuable resource to assess changes in burden that is complementary to the country's routine case reporting system. Methods: A Bayesian geostatistical spatio-temporal model was developed in an integrated nested Laplace approximation framework to map the prevalence of Plasmodium falciparum malaria infection among children from 6 to 59 months in age across Madagascar for 2011, 2013 and 2016 based on the MIS datasets. The model was informed by a suite of environmental and socio-demographic covariates known to influence infection prevalence. Spatio-temporal trends were quantified across the country.METHODS:A Bayesian geostatistical spatio-temporal model was developed in an integrated nested Laplace approximation framework to map the prevalence of Plasmodium falciparum malaria infection among children from 6 to 59 months in age across Madagascar for 2011, 2013 and 2016 based on the MIS datasets. The model was informed by a suite of environmental and socio-demographic covariates known to influence infection prevalence. Spatio-temporal trends were quantified across the country.RESULTS:Despite a relatively small decrease between 2013 and 2016, the prevalence of malaria infection has increased substantially in all areas of Madagascar since 2011. In 2011, almost half (42.3%) of the country's population lived in areas of very low malaria risk (20%) increased from only 2.2% in 2011 to 9.2% in 2016. A comparison of the model-based estimates with the raw MIS results indicates there was an underestimation of the situation in 2016, since the raw figures likely associated with survey timings were delayed until after the peak transmission season.CONCLUSIONS:Malaria remains an important health problem in Madagascar. The monthly and annual prevalence maps developed here provide a way to evaluate the magnitude of change over time, taking into account variability in survey input data. These methods can contribute to monitoring sub-national trends of malaria prevalence in Madagascar as the country aims for geographically progressive elimination.

Plasmodium falciparum histidine-rich protein 2 (PfHRP2) forms the basis of many current malaria rapid diagnostic tests (RDTs). However, the parasites lacking part or all of the pfhrp2 gene do not express the PfHRP2 protein and are, therefore, not identifiable by PfHRP2-detecting RDTs. We evaluated the performance of the SD Bioline Malaria Ag P.f/Pan RDT together with pfhrp2 variation in Madagascar. Genomic DNA isolated from 260 patient blood samples were polymerase chain reaction (PCR)–amplified for the parasite 18S rRNA and pfhrp2 genes. Post-PCR ligation detection reaction-fluorescent microsphere assay (LDR-FMA) was performed for the identification of parasite species. Plasmodium falciparum histidine-rich protein 2 amplicons were sequenced. Polymerase chain reaction diagnosis of patient samples showed that 29% (75/260) were infected and P. falciparum was present in 95% (71/75) of these PCR-positive samples. Comparing RDT and P. falciparum detection by LDR-FMA, eight samples were RDT negative but P. falciparum positive (false negatives), all of which were pfhrp2 positive. The sensitivity and specificity of the RDT were 87% and 90%, respectively. Seventy-three samples were amplified for pfhrp2, from which nine randomly selected amplicons were sequenced, yielding 13 sequences. Amplification of pfhrp2, combined with RDT analysis and P. falciparum detection by LDR-FMA, showed that there was no indication of pfhrp2 deletion. Sequence analysis of pfhrp2 showed that the correlation between pfhrp2 sequence structure and RDT detection rates was unclear. Although the observed absence of pfhrp2 deletion from the samples screened here is encouraging, continued monitoring of the efficacy of the SD Bioline Malaria Ag P.f/Pan RDT for malaria diagnosis in Madagascar is warranted.

International audience; Background: Since 2006, the artemisinin-based combination therapy (ACT) are recommended to treat uncomplicated malaria including non Plasmodium falciparum malaria in Madagascar. Artesunate-amodiaquine (ASAQ) and artemether-lumefantrine are the first- and second-line treatment in uncomplicated falciparum malaria, respectively. No clinical drug efficacy study has been published since 2009 to assess the efficacy of these two artemisinin-based combinations in Madagascar, although the incidence of malaria cases has increased from 2010 to 2016. In this context, new data about the efficacy of the drug combinations currently used to treat malaria are needed.Methods: Therapeutic efficacy studies evaluating the efficacy of ASAQ were conducted in 2012, 2013 and 2016 among falciparum malaria-infected patients aged between 6 months and 56 years, in health centres in 6 sites representing different epidemiological patterns. The 2009 World Health Organization protocol for monitoring anti-malarial drug efficacy was followed.Results: A total of 348 enrolled patients met the inclusion criteria including 108 patients in 2012 (n = 64 for Matanga, n = 44 for Ampasipotsy), 123 patients in 2013 (n = 63 for Ankazomborona, n = 60 for Anjoma Ramartina) and 117 patients in 2016 (n = 67 for Tsaratanana, n = 50 for Antanimbary). The overall cumulative PCR-corrected day 28 cure rate was 99.70% (95% IC 98.30-99.95). No significant difference in cure rates was observed overtime: 99.02% (95% IC 94.65-99.83) in 2012; 100% (95% IC 96.8-100) in 2013 and 100% (95% IC 96.65-100) in 2016.Conclusion: The ASAQ combination remains highly effective for the treatment of uncomplicated falciparum malaria in Madagascar.

Introduction A global strategy of mass drug administration (MDA) has greatly reduced the burden of lymphatic filariasis (LF) in endemic countries. In Madagascar, the National Programme to eliminate LF has scaled-up annual MDA of albendazole and diethylcarbamazine across the country in the last decade, but its impact on LF transmission has never been reported. The objective of this study was to evaluate progress towards LF elimination in southeastern Madagascar. Methods Three different surveys were carried out in parallel in four health districts of the Vatovavy Fitovinany region in 2016: i) a school-based transmission assessment survey (TAS) in the districts of Manakara Atsimo, Mananjary, and Vohipeno (following a successful pre-TAS in 2013); ii) a district-representative community prevalence survey in Ifanadiana district; and iii) a community prevalence survey in sentinel and spot-check sites of these four districts. LF infection was assessed using the Alere Filariasis Test Strips, which detect circulating filarial antigens (CFA) of adult worms. A brief knowledge, attitudes and practices questionnaire was included in the community surveys. Principal findings None of the 1,825 children sampled in the TAS, and only one in 1,306 children from sentinel and spot-check sites, tested positive to CFA. However, CFA prevalence rate in individuals older than 15 years was still high in two of these three districts, at 3.5 and 9.7% in Mananjary and Vohipeno, respectively. Overall CFA prevalence in sentinel and spot-check sites of these three districts was 2.80% (N = 2,707), but only two individuals had detectable levels of microfilaraemia (0.06%). Prevalence rate estimates for Ifanadiana were substantially higher in the district-representative survey (15.8%; N = 545) than in sentinel and spot-check sites (0.8%; N = 618). Only 51.2% of individuals surveyed in these four districts reported taking MDA in the last year, and 42.2% reported knowing about LF. Conclusions Although TAS results suggest that MDA can be stopped in three districts of southeastern Madagascar, the adult population still presents high CFA prevalence levels. This discordance raises important questions about the TAS procedures and the interpretation of their results.

Malaria still persists in underdeveloped countries, including Madagascar, where it is the eighth leading cause of morbidity in Madagascar (SSS, 2011). Thanks to the application of the two insecticide-impregnated mosquito nets (ITN) and the Intra-Home Spraying Campaign (IDAC) systems, this rate has risen from 21.57% in 2003 to 5% in 2011 for children under 5 years of age and from 17.57% to 2.3% in 2011 for children over 5 years of age. The malaria mortality rate increased from 25.92% to 19% for children under 5 years of age and from 13.5% to 5% for those over 5 years of age (SSS, 2011). However, these control methods are based on the use of pesticides and require great caution. Indeed, due to poor practice, organophosphorus (insecticide used in CAID) and pyrethrinoid (insecticide impregnated on ITNs) were detected after analysis The instrument used was an Infinity 1290 Agilent Technologies HPLC Infinity 1290 system (Santa Clara, CA, USA) coupled with an Agilent Technologies 6460 triple quadrupole mass spectrometer in soil, surface water and groundwater from the three villages (Tanandava, Vohitramba and Savagna) in the south-eastern region of Madagascar where sampling takes place.

Introduction Cette étude se propose d’évaluer l’adhérence des professionnels de santé du secteur privé à l'utilisation du TDR-palu (Test de Diagnostic Rapide-palu) et à la prescription de l’ACT (Artemisinin-based combination therapy) en cas de paludisme simple. Méthodes Une approche évaluative rétrospective et transversale a été menée en septembre et octobre 2015 auprès de 11 districts sanitaires repartis dans les quatre faciès épidémiologiques existant à Madagascar. Au total, 43 prestataires de soins issus de 39 formations sanitaires privées (FSP) ont été interviewés et visités. Résultats Les prestataires déclarent avoir lu le manuel de prise en charge du paludisme dans 16,3% des cas (4/43). Seul le quart (25,6%) des prestataires dispose de TDR dans leur bureau. L’ACT a été cité par 83,7% des prestataires comme médicament de première intention pour traiter le paludisme simple. Dans la pratique, 55,6% des prestataires émettent des doutes sur les résultats des TDR. L’utilisation des antipaludéens malgré les résultats négatifs des TDR (38,2%) est plus fréquente chez ceux ayant émis des doutes (p = 0,03). Inversement, malgré un TDR positif, la moitié des prestataires ne prescrit pas d’ACT (50%). La non-participation aux revues périodiques du District sanitaire (p = 0,05) influence négativement l’adhérence aux politiques. Conclusion La faible adhérence des prestataires de soin du secteur privé aux directives nationales sur la prise en charge des cas de paludisme simple interpelle sur l’intérêt d’encadrer davantage ce secteur.

Background The prevalence and variants of G6PD deficiency in the P. vivax-endemic zones of Madagascar remain unknown. The admixed African-Austronesian origins of the Malagasy population make it probable that a heterogeneous mix of genetic variants with a spectrum of clinical severity will be circulating. This would have implications for the widespread use of P. vivax radical cure therapy. Two study populations in the P. vivax-endemic western foothills region of Madagascar were selected for G6PD screening. Both the qualitative fluorescent spot test and G6PD genotyping were used to screen all participants. Results A total of 365 unrelated male volunteers from the Tsiroanomandidy, Mandoto, and Miandrivazo districts of Madagascar were screened and 12.9% were found to be phenotypically G6PD deficient. Full gene sequencing of 95 samples identified 16 single nucleotide polymorphisms (SNPs), which were integrated into a genotyping assay. Genotyping (n=291) found one individual diagnosed with the severe G6PD MediterraneanC563T mutation, while the remaining G6PD deficient samples had mutations of African origin, G6PD A- and G6PD A. Conclusion Deployment of P. vivax radical cure in Madagascar must be considerate of the risks presented by the observed prevalence of G6PDd prevalence. The potential morbidity associated with cumulative episodes of P. vivax clinical relapses requires a strategy for increasing access to safe radical cure. The observed dominance of African G6PDd haplotypes is surprising given the known mixed African-Austronesian origins of the Malagasy population; more widespread surveying of G6PDd epidemiology across the island would be required to characterise the distribution of G6PD haplotypes across Madagascar.

International audience; The aim of this study was to provide a comprehensive analysis of the worldwide genetic polymorphism of ms4760 alleles of the pfnhe-1 gene and to discuss their usefulness as molecular marker of quinine resistance (QNR). A new numbering of ms4760 allele, classification grouping ms4760 alleles according to the number of DNNND and DDNHNDNHNND repeat motifs in blocks II and V was also proposed. A total of 1508 ms4760 sequences from isolates, culture-adapted parasites or reference strains from various geographical regions were retrieved from GenBank (last update on 15th June 2012) or from publications and were used for genetic analyses. The association of different alleles of pfnhe-1 with resistance to quinoline antimalarial drugs showed marked geographic disparities. The validity and reliability of candidate polymorphisms in pfnhe-1 gene as molecular markers of QNR appeared restricted to endemic areas from South Asia or possibly East African countries and needs to be confirmed.

Objectives. To monitor the sensitivity of Plasmodium falciparum to the drugs used to treat severe malaria and to prevent malaria in Comoros and Madagascar. Design. We used the in vitro isotopic method to test the sensitivity of P. falciparum to quinine, mefloquine and cycloguanil.Results. 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 mefloquineresistant (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, p < 10 6 ), but in vitro mefloquine was 6 -16 times more potent than quinine. No correlation was noticed between the activities of quinine and cydoguanil or between the activities of mefloquine and cycloguaniL Conclusion. 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.

Malaria therapy, experimental, and epidemiological studies have shown that erythrocyte Duffy blood group-negative people, largely of African ancestry, are resistant to erythrocyte Plasmodium vivax infection. These findings established a paradigm that the Duffy antigen is required for P. vivax erythrocyte invasion. P. vivax is endemic in Madagascar, where admixture of Duffy-negative and Duffy-positive populations of diverse ethnic backgrounds has occurred over 2 millennia. There, we investigated susceptibility to P. vivax blood-stage infection and disease in association with Duffy blood group polymorphism. Duffy blood group genotyping identified 72% Duffy-negative individuals ( FY*B ES /*B ES ) in community surveys conducted at eight sentinel sites. Flow cytometry and adsorption–elution results confirmed the absence of Duffy antigen expression on Duffy-negative erythrocytes. P. vivax PCR positivity was observed in 8.8% (42/476) of asymptomatic Duffy-negative people. Clinical vivax malaria was identified in Duffy-negative subjects with nine P. vivax monoinfections and eight mixed Plasmodium species infections that included P. vivax (4.9 and 4.4% of 183 participants, respectively). Microscopy examination of blood smears confirmed blood-stage development of P. vivax , including gametocytes. Genotyping of polymorphic surface and microsatellite markers suggested that multiple P. vivax strains were infecting Duffy-negative people. In Madagascar, P. vivax has broken through its dependence on the Duffy antigen for establishing human blood-stage infection and disease. Further studies are necessary to identify the parasite and host molecules that enable this Duffy-independent P. vivax invasion of human erythrocytes.

The aim of this study was to provide the first comprehensive spatiotemporal picture of Plasmodium falciparum resistance in various geographic areas in Madagascar. Additional data about the antimalarial resistance in the neighboring islands of the Comoros archipelago were also collected. We assessed the prevalence of pfcrt , pfmdr-1 , pfdhfr , and pfdhps mutations and the pfmdr-1 gene copy number in 1,596 P. falciparum isolates collected in 26 health centers (20 in Madagascar and 6 in the Comoros Islands) from 2006 to 2008. The in vitro responses to a panel of drugs by 373 of the parasite isolates were determined. The results showed (i) unusual profiles of chloroquine susceptibility in Madagascar, (ii) a rapid rise in the frequency of parasites with both the pfdhfr and the pfdhps mutations, (iii) the alarming emergence of the single pfdhfr 164L genotype, and (iv) the progressive loss of the most susceptible isolates to artemisinin derivatives. In the context of the implementation of the new national policy for the fight against malaria, continued surveillance for the detection of P. falciparum resistance in the future is required.

The aim of this study was to provide baseline information of the epidemiological situation of malaria in Madagascar using serological markers. We carried out cross-sectional studies in schoolchildren from eight sites in the four different malarious epidemiological strata of Madagascar. We studied the prevalence of anti-MSP1 antibodies to assess the burden, and anti-CSP antibodies to estimate the transmission intensity, of malaria. The overall prevalence of each antibody tested was 46.1% for anti-PfMSP-1, 15.2% for anti-PvMSP-1, 14.9% for anti-PfCSP, 4.9% for anti-PvCSP and 2.4% for anti-PmCSP. The prevalence of the five antibodies varied significantly between the sites (P10(-6)). We also found significant effects of ethnic origin on the prevalence of anti-PfMSP1 antibodies. With regular testing in the same target populations, this data will be particularly useful for managing the elimination strategy supported by the Malagasy Government.

Summary The performance and the reliability of a SYBR Green I fluorescence-based assay to assess drug susceptibility in routine monitoring were evaluated in 138 Plasmodium falciparum clinical samples. Blood samples were studied for susceptibility to four antimalarial drugs by the SYBR Green I based assay, with the traditional [3H]-hypoxanthine isotopic assay as a reference. The two methods were observed to have similar geometric means of IC50s and IC90s, and high correlation (r = 0.93 for IC50s and r = 0.94 for IC90s) for the drugs tested. The strength of agreement estimated by using concordance coefficient correlation was from almost perfect to substantial for IC50s. Our data demonstrate (i) the reliability of a simple, rapid and easy to use fluorescence-based assay for the routine monitoring of susceptibility of P. falciparum clinical isolates, and (ii) the possible switch from the traditional in vitro drug sensitivity assay to the SYBR Green I method, because previous data acquired by the isotopic assay were comparable with those obtained by the SYBR Green I method. We conclude that this assay will provide an easier method for testing drug susceptibility of malaria parasites, especially in malaria-endemic countries, where there is massive implementation of new artemisinin-based combination therapies.

The main purpose of this study was to assess the accuracy of various techniques available for diagnosis of malaria. Blood samples were collected from 313 patients with clinical suspicion of uncomplicated malaria in 2 primary health centers in Madagascar. The presence of Plasmodium parasites was assessed by conventional microscopy, 2 rapid diagnostic tests (one HRP2-based test, PALUTOP +4 , and one pLDH-based test, OptiMAL-IT), and real-time poly- merase chain reaction (PCR), which is used as the "gold standard" method. The degree of agreement observed was very high for microscopy (0.99) and the HRP2-based test (0.93) and high for the pLDH-based test (0.82). Public-health implications are also discussed in this paper.

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).

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