Long-term in vitro culture of Plasmodium vivax isolates from Madagascar maintained in Saimiri boliviensis blood.

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.

Pharmacogenomic variation in the Malagasy population: implications for the antimalarial drug primaquine metabolism.

Aim: Antimalarial primaquine (PQ) eliminates liver hypnozoites of Plasmodium vivax. CYP2D6 gene variation contributes to PQ therapeutic failure. Additional gene variation may contribute to PQ efficacy. Information on pharmacogenomic variation in Madagascar, with vivax malaria and a unique population admixture, is scanty. Methods: The authors performed genome-wide genotyping of 55 Malagasy samples and analyzed data with a focus on a set of 28 pharmacogenes most relevant to PQ. Results: Mainly, the study identified 110 coding or splicing variants, including those that, based on previous studies in other populations, may be implicated in PQ response and copy number variation, specifically in chromosomal regions that contain pharmacogenes. Conclusion: With this pilot information, larger genome-wide association analyses with PQ metabolism and response are substantially more feasible.

Duffy antigen is expressed during erythropoiesis in Duffy-negative individuals.

The erythrocyte silent Duffy blood group phenotype in Africans is thought to confer resistance to Plasmodium vivax blood-stage infection. However, recent studies report P. vivax infections across Africa in Fy-negative individuals. This suggests that the globin transcription factor 1 (GATA-1) SNP underlying Fy negativity does not entirely abolish Fy expression or that P. vivax has developed a Fy-independent red blood cell (RBC) invasion pathway. We show that RBCs and erythroid progenitors from in vitro differentiated CD34 cells and from bone marrow aspirates from Fy-negative samples express a functional Fy on their surface. This suggests that the GATA-1 SNP does not entirely abolish Fy expression. Given these results, we developed an in vitro culture system for P. vivax and show P. vivax can invade erythrocytes from Duffy-negative individuals. This study provides evidence that Fy is expressed in Fy-negative individuals and explains their susceptibility to P. vivax with major implications and challenges for P. vivax malaria eradication.

CYP2D6 gene resequencing in the Malagasy, a population at the crossroads between Asia and Africa: a pilot study.

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.

CYP2D6 Genetic Variation and Its Implication for Vivax Malaria Treatment 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.

Spatiotemporal mapping of malaria prevalence in Madagascar using routine surveillance and health survey data.

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.

A Novel Assay for Simultaneous Assessment of Mammalian Host Blood, Mosquito Species, and Plasmodium spp. in the Medically Important Anopheles Mosquitoes of Madagascar.

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 falciparum Parasitemia and Band Sensitivity of the SD Bioline Malaria Ag P.f/Pan Rapid Diagnostic Test in Madagascar.

Current malaria rapid diagnostic tests (RDTs) contain antibodies against Plasmodium falciparum-specific histidine-rich protein 2 (PfHRP2), Plasmodium lactate dehydrogenase (pLDH), and aldolase in various combinations. Low or high parasite densities/target antigen concentrations may influence the accuracy and sensitivity of PfHRP2-detecting RDTs. We analyzed the SD Bioline Malaria Ag P.f/Pan RDT performance in relation to P. falciparum parasitemia in Madagascar, where clinical Plasmodium vivax malaria exists alongside P. falciparum. Nine hundred sixty-three samples from patients seeking care for suspected malaria infection were analyzed by RDT, microscopy, and Plasmodium species-specific, ligase detection reaction-fluorescent microsphere assay (LDR-FMA). Plasmodium infection positivity by these diagnostics was 47.9%, 46.9%, and 58%, respectively. Plasmodium falciparum-only infections were predominant (microscopy, 45.7%; LDR-FMA, 52.3%). In all, 16.3% of P. falciparum, 70% of P. vivax, and all of Plasmodium malariae, Plasmodium ovale, and mixed-species infections were submicroscopic. In 423 P. falciparum mono-infections, confirmed by microscopy and LDR-FMA, the parasitemia in those who were positive for both the PfHRP2 and pan-pLDH test bands was significantly higher than that in those who were positive only for the PfHRP2 band (P < 0.0001). Plasmodium falciparum parasitemia in those that were detected as P. falciparum-only infections by microscopy but P. falciparum mixed infections by LDR-FMA also showed similar outcome by the RDT band positivity. In addition, we used varying parasitemia (3-0.0001%) of the laboratory-maintained 3D7 strain to validate this observation. A positive pLDH band in high P. falciparum-parasitemic individuals may complicate diagnosis and treatment, particularly when the microscopy is inconclusive for P. vivax, and the two infections require different treatments.

Anopheles mosquito surveillance in Madagascar reveals multiple blood feeding behavior and Plasmodium infection.

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.

Insights into the Performance of SD Bioline Malaria Ag P.f/Pan Rapid Diagnostic Test and Plasmodium falciparum Histidine-Rich Protein 2 Gene Variation in Madagascar.

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.

[Rapid diagnostic test for malaria: preliminary study in Madagascar in 2003]. / Utilisation du test de diagnostic rapide du paludisme à Madagascar: étude préliminaire en 2003.

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