Evolution of Plasmodium falciparum drug resistance genes following artemisinin combination therapy in Sudan.

BACKGROUND: Malaria control efforts in Sudan rely heavily on case management. In 2004, health authorities adopted artemisinin-based combination therapies (ACTs) for the treatment of uncomplicated malaria. However, some recent surveys have reported ACT failure and a prevalent irrational malaria treatment practice. Here we examine whether the widespread use of ACT and failure to adhere to national guidelines have led to the evolution of drug resistance genes. METHODS: We genotyped known drug resistance markers (Pfcrt, Pfmdr-1, Pfdhfr, Pfdhps, Pfk13 propeller) and their flanking microsatellites among Plasmodium falciparum isolates obtained between 2009 and 2016 in different geographical regions in Sudan. Data were then compared with published findings pre-ACT (1992-2003). RESULTS: A high prevalence of Pfcrt76T, Pfmdr-1-86Y, Pfdhfr51I, Pfdhfr108N, Pfdhps37G was observed in all regions, while no Pfk13 mutations were detected. Compared with pre-ACT data, Pfcrt-76T and Pfmdr-1-86Y have decayed, while Pfdhfr-51I, Pfdhfr-108N and Pfdhps-437G strengthened. Haplotypes Pfcrt-CVIET, Pfmdr-1-NFSND/YFSND, Pfdhfr-ICNI and Pfdhps-SGKAA predominated in all sites. Microsatellites flanking drug resistance genes showed lower diversity than neutral ones, signifying high ACT pressure/selection. CONCLUSIONS: Evaluation of P. falciparum drug resistance genes in Sudan matches the drug deployment pattern. Regular monitoring of these genes, coupled with clinical response, should be considered to combat the spread of ACT resistance.

High gametocyte complexity and mosquito infectivity of Plasmodium falciparum in the Gambia.

The purpose of this work was to determine the infectivity to mosquitoes of genetically diverse Plasmodium falciparum clones seen in natural infections in the Gambia. Two principal questions were addressed: (i) how infectious are gametocytes of sub-patent infections, particularly at the end of the dry season; and (ii) are all clones in multiclonal infections equally capable of infecting mosquitoes? The work was carried out with two cohorts of infected individuals. Firstly, a group of 31 P. falciparum-infected people were recruited in the middle of the dry season (May, 2003), then examined for P. falciparum at the beginning (August 2003) and middle (October, 2003) of the transmission season. On each occasion, we examined the genotypes of asexual forms and gametocytes by PCR and RT-PCR, as well as their infectivity to Anopheles gambiae using membrane feeds. One individual gave rise to infected mosquitoes in May, and two in August. Different gametocyte genotypes co-existed in the same infection and fluctuated over time. The mean multiplicity of infection was 1.4, 1.7 and 1.5 clones in May, August and October, respectively. Second, a group of patients undergoing drug-treatment during August 2003 was tested for asexual and gametocyte genotypes and their infectivity to mosquitoes. Forty-three out of 100 feeds produced infections. The genetic complexity of the parasites in mosquitoes was sometimes greater than that detectable in the blood on which the mosquitoes had fed. This suggested that gametocytes of clones existing in the blood below PCR detection limits at the time of the feed were at least as infectious to the mosquitoes as the more abundant clones. These findings emphasise the crucial role of gametocyte complexity and infectivity in generating the remarkable diversity of P. falciparum genotypes seen in infected people, even in an area of seasonal transmission.

Impact of genetic complexity on longevity and gametocytogenesis of Plasmodium falciparum during the dry and transmission-free season of eastern Sudan.

Malaria in eastern Sudan is characterised by limited seasonal transmission, with the majority of the year remaining transmission-free. Some inhabitants who contract malaria during the transmission season retain long-lasting sub-patent infections, which probably initiate transmission the following year. Here we have monitored Plasmodium falciparum infection prevalence and gametocyte production during the dry season, and examined the impact of parasite genetic multiplicity on infection longevity. A cohort of 38 individuals who were infected with P. falciparum in November 2001 was monitored monthly by microscopy and PCR until December 2002. Reverse transcriptase polymerase chain reaction of the pfg377 gene was used to detect sub-patent gametocytes. In addition, all isolates were examined for msp-2 alleles and the mean number of parasite clones per infection was estimated. We found that a large proportion (40%) of the cohort retained gametocytes throughout the dry season. The majority of patients retained asexual infection for at least 7 months. Genetic multiplicity of P. falciparum significantly influenced longevity of asexual infection and its gametocyte production. Gametocytes from mixed genotype P. falciparum infections persisted three times longer than those from single genotype infections, suggesting that genetic diversity promotes persistence. These findings are discussed in the context of the parasite biology and malaria epidemiology in the study area.

Transmission and cross-mating of high-level resistance Plasmodium falciparum dihydrofolate reductase haplotypes in The Gambia.

A high-level pyrimethamine resistance Plasmodium falciparum lineage with triple dihydrofolate reductase (dhfr) mutations prevails across Africa. However, additional minority lineages were seen. We examined transmission success of mutant dhfr haplotypes among 22 children in The Gambia and 60 infected Anopheles gambiae mosquitoes fed on their blood. Additional polymorphic genes of the gametocyte-specific protein (pfg377) and merozoite surface protein-1 (MSP-1) were examined. Similarities were seen between pfg377 and MSP-1 alleles in children and mosquitoes and evidence of cross-mating between different parasite genotypes was seen in some infected mosquitoes, reflecting high transmission success of existing clones. With regard to dhfr, 16 haplotypes were seen among the children: 2 carried double mutations and 14 carried triple mutations. However, only nine haplotypes, all with triple mutations, were detected among mosquitoes. A single triple-mutant dhfr haplotype, similar to that in other countries in Africa, predominated among children (42%) and mosquitoes (60%), supporting the hypothesis of migration of this haplotype across Africa. However, evidence of cross-mating between the above haplotypes signifies the role of local evolution.

Distinct haplotypes of dhfr and dhps among Plasmodium falciparum isolates in an area of high level of sulfadoxine-pyrimethamine (SP) resistance in eastern Sudan.

Typing of polymorphic microsatellites that are linked to drug resistance genes has shed light on the origin and pattern of spread of some anti-malarial drugs. Recent surveys revealed spread of a high-level pyrimethemine resistant lineage of Plasmodium falciparum, of Asian origin, across Africa. Here, we examined mutations in dihydrofolate reductase, dhfr [chromsosome 4], the dihydropteroate synthase, dhps [chromosome 8] associated with resistance to sulfadoxine-pyrimethamine (SP), and neighboring microsatellites among P. falciparum isolates in Asar village, eastern Sudan. This area lies at the fringes of malaria endemicity, where the remote P. falciparum parasites have some distinct genetic characteristics. Overall, 89% (84/94) of the examined isolates carried double mutations at dhfr (N51I and S108N), but the 59R and I164L mutations were not seen. Similarly, the majority, 43% (35/81) of the isolates carried double mutations at dhps (437G, 540E). Analysis of neighboring microsatellites revealed one major dhfr haplotype with mutations (51I, 108N) and one dhps haplotype with mutations (436S, 437G, 540E). These haplotypes differ from the major ones thought to drive resistance to SP across Africa. The resistant haplotypes of dhfr and dhps, in Asar, share some microsatellites with the wild genotypes suggesting that they were generated locally. Among isolates successfully examined, 40% shared identical haplotypes of the 2 loci, comprising a dominant resistant lineage. Undoubtedly, this lineage plays an important role in clinical failure to SP in this area.