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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.

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.

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.

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.

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.