Pf7: an open dataset of Plasmodium falciparum genome variation in 20,000 worldwide samples.

We describe the MalariaGEN Pf7 data resource, the seventh release of Plasmodium falciparum genome variation data from the MalariaGEN network.  It comprises over 20,000 samples from 82 partner studies in 33 countries, including several malaria endemic regions that were previously underrepresented.  For the first time we include dried blood spot samples that were sequenced after selective whole genome amplification, necessitating new methods to genotype copy number variations.  We identify a large number of newly emerging crt mutations in parts of Southeast Asia, and show examples of heterogeneities in patterns of drug resistance within Africa and within the Indian subcontinent.  We describe the profile of variations in the C-terminal of the csp gene and relate this to the sequence used in the RTS,S and R21 malaria vaccines.  Pf7 provides high-quality data on genotype calls for 6 million SNPs and short indels, analysis of large deletions that cause failure of rapid diagnostic tests, and systematic characterisation of six major drug resistance loci, all of which can be freely downloaded from the MalariaGEN website.

Comparative effect of artemether-lumefantrine and artesunate-amodiaquine on gametocyte clearance in children with uncomplicated Plasmodium falciparum malaria in Madagascar.

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.

Proactive community case management decreased malaria prevalence in rural Madagascar: results from a cluster randomized trial.

BACKGROUND: Malaria remains a leading cause of morbidity and mortality worldwide, with progress in malaria control stalling in recent years. Proactive community case management (pro-CCM) has been shown to increase access to diagnosis and treatment and reduce malaria burden. However, lack of experimental evidence may hinder the wider adoption of this intervention. We conducted a cluster randomized community intervention trial to assess the efficacy of pro-CCM at decreasing malaria prevalence in rural endemic areas of Madagascar. METHODS: Twenty-two fokontany (smallest administrative unit) of the Mananjary district in southeast Madagascar were selected and randomized 1:1 to pro-CCM (intervention) or conventional integrated community case management (iCCM). Residents of all ages in the intervention arm were visited by a community health worker every 2 weeks from March to October 2017 and screened for fever; those with fever were tested by a rapid diagnostic test (RDT) and treated if positive. Malaria prevalence was assessed using RDTs on all consenting study area residents prior to and following the intervention. Hemoglobin was measured among women of reproductive age. Intervention impact was assessed via difference-in-differences analyses using logistic regressions in generalized estimating equations. RESULTS: A total of 27,087 and 20,475 individuals participated at baseline and endline, respectively. Malaria prevalence decreased from 8.0 to 5.4% in the intervention arm for individuals of all ages and from 6.8 to 5.7% in the control arm. Pro-CCM was associated with a significant reduction in the odds of malaria positivity in children less than 15 years (OR = 0.59; 95% CI [0.38-0.91]), but not in older age groups. There was no impact on anemia among women of reproductive age. CONCLUSION: This trial suggests that pro-CCM approaches could help reduce malaria burden in rural endemic areas of low- and middle-income countries, but their impact may be limited to younger age groups with the highest malaria burden. TRIAL REGISTRATION: NCT05223933. Registered on February 4, 2022.

[Pcr-rflp genotyping of pfcrt and pfmdr1 in plasmodium falciparum isolates from children in Vatomandry, Madagascar]. / Génotypage par pcr-rflp de pfcrt et pfmdr1 sur des isolats de plasmodium falciparum collectés chez des enfants de Vatomandry, Madagascar.

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.

DNA recovery from used malaria RDT to detect Plasmodium species and to assess Plasmodium falciparum genetic diversity: a pilot study in Madagascar.

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.

An open dataset of Plasmodium vivax genome variation in 1,895 worldwide samples.

This report describes the MalariaGEN Pv4 dataset, a new release of curated genome variation data on 1,895 samples of Plasmodium vivax collected at 88 worldwide locations between 2001 and 2017. It includes 1,370 new samples contributed by MalariaGEN and VivaxGEN partner studies in addition to previously published samples from these and other sources. We provide genotype calls at over 4.5 million variable positions including over 3 million single nucleotide polymorphisms (SNPs), as well as short indels and tandem duplications. This enlarged dataset highlights major compartments of parasite population structure, with clear differentiation between Africa, Latin America, Oceania, Western Asia and different parts of Southeast Asia. Each sample has been classified for drug resistance to sulfadoxine, pyrimethamine and mefloquine based on known markers at the dhfr, dhps and mdr1 loci. The prevalence of all of these resistance markers was much higher in Southeast Asia and Oceania than elsewhere. This open resource of analysis-ready genome variation data from the MalariaGEN and VivaxGEN networks is driven by our collective goal to advance research into the complex biology of P. vivax and to accelerate genomic surveillance for malaria control and elimination.

An open dataset of Plasmodium falciparum genome variation in 7,000 worldwide samples.

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed.  Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination.

Effects of mefloquine use on Plasmodium vivax multidrug resistance.

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.

Monitoring parasite diversity for malaria elimination in sub-Saharan Africa.

The African continent continues to bear the greatest burden of malaria and the greatest diversity of parasites, mosquito vectors, and human victims. The evolutionary plasticity of malaria parasites and their vectors is a major obstacle to eliminating the disease. Of current concern is the recently reported emergence of resistance to the front-line drug, artemisinin, in South-East Asia in Plasmodium falciparum, which calls for preemptive surveillance of the African parasite population for genetic markers of emerging drug resistance. Here we describe the Plasmodium Diversity Network Africa (PDNA), which has been established across 11 countries in sub-Saharan Africa to ensure that African scientists are enabled to work together and to play a key role in the global effort for tracking and responding to this public health threat.

Activation of minority-variant Plasmodium vivax hypnozoites following artesunate + amodiaquine treatment in a 23-year old man with relapsing malaria in Antananarivo, Madagascar.

In endemic areas, Plasmodium vivax relapses are difficult to distinguish from new infections. Genotyping of patients who experience relapse after returning to a malaria-free area can be used to explore the nature of hypnozoite activation and relapse. This paper describes a person who developed P. vivax malaria for the first time after travelling to Boriziny in the malaria endemic coastal area of Madagascar, then suffered two P. vivax relapses 11 weeks and 21 weeks later despite remaining in Antananarivo in the malaria-free central highlands area. He was treated with the combination artesunate + amodiaquine according to the national malaria policy in Madagascar. Genotyping by PCR-RFLP at pvmsp-3α as well as pvmsp1 heteroduplex tracking assay (HTA) showed the same dominant genotype at each relapse. Multiple recurring minority variants were also detected at each relapse, highlighting the propensity for multiple hypnozoite clones to activate simultaneously to cause relapse.

The origins of African Plasmodium vivax; insights from mitochondrial genome sequencing.

Plasmodium vivax, the second most prevalent of the human malaria parasites, is estimated to affect 75 million people annually. It is very rare, however, in west and central Africa, due to the high prevalence of the Duffy negative phenotype in the human population. Due to its rarity in Africa, previous studies on the phylogeny of world-wide P. vivax have suffered from insufficient samples of African parasites. Here we compare the mitochondrial sequence diversity of parasites from Africa with those from other areas of the world, in order to investigate the origin of present-day African P. vivax. Mitochondrial genome sequencing revealed relatively little polymorphism within the African population compared to parasites from the rest of the world. This, combined with sequence similarity with parasites from India, suggests that the present day African P. vivax population in humans may have been introduced relatively recently from the Indian subcontinent. Haplotype network analysis also raises the possibility that parasites currently found in Africa and South America may be the closest extant relatives of the ancestors of the current world population. Lines of evidence are adduced that this ancestral population may be from an ancient stock of P. vivax in Africa.

Plasmodium falciparum drug resistance in Madagascar: facing the spread of unusual pfdhfr and pfmdr-1 haplotypes and the decrease of dihydroartemisinin susceptibility.

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.