Minority-Variant pfcrt K76T Mutations and Chloroquine Resistance, Malawi

Abstract

Genotyping of the chloroquine-resistance biomarker pfcrt (Plasmodium falciparum chloroquine resistance transporter gene) suggests that, in the absence of chloroquine pressure, Plasmodium falciparum parasites in Malawi have reverted to chloroquine sensitivity. However, malaria infections in Africa are commonly polyclonal, and standard PCRs cannot detect minority genotypes if present in <20% of the parasites in an individual host. We have developed a multiple site-specifi c heteroduplex tracking assay (MSS-HTA) that can detect pfcrt 76T mutant parasites consisting of as little as 1% of the parasite population. In clinical samples, no pfcrt 76T was detected in 87 pregnant Malawian women by standard PCR. However, 22 (25%) contained minority-variant resistant genotypes detected by the MSS-HTA. These results were confi rmed by subcloning and sequencing. This fi nding suggests that the chloroquine-resistant genotype remains common in Malawians and that PCR-undetectable drug-resistant genotypes may be present in disease-endemic populations. Surveillance for minority-variant drugresistant mutations may be useful in making antimalarial drug policy. D rug-resistant Plasmodium falciparum malaria continues to be a growing health problem throughout most of the world (1). To combat this threat, governments and aid agencies need accurate drug resistance surveillance data. The World Health Organization has stressed the need for methods of detecting molecular markers of drug resistance that will be useful in predicting responses to both clinical and public health interventions (2). This has been diffi cult in highly malaria-endemic areas, where infections are almost always polyclonal (3,4). In patients with polyclonal infections, small drug-resistant parasite populations (minority variants) may be masked by larger drug-sensitive populations because standard PCRs are relatively insensitive to minority variants (2). Therefore, new methods capable of detecting these subpopulations may lead to better drug resistance surveillance and provide a better tool to predict outcome.We describe a new multiple site-specifi c heteroduplex tracking assay (MSS-HTA) for detecting the pfcrt (Plasmodium falciparum chloroquine resistance transporter gene) K76T mutation. This mutation in a putative transporter gene is well-associated with chloroquine resistance in P. falciparum (5). The MSS-HTA was compared with a standard allele-restricted PCR (ARPCR) in clinical samples from Malawi, a country where standard PCR analyses and a recent clinical trial have suggested that chloroquine-resistant malaria has disappeared (6-9). Materials and Methods Study SamplesInformed consent, as approved by the ethics committees of the University of North Carolina and the Malawi College of Medicine/Ministry of Health, was obtained from all participants in this research study. The Malaria and HIV in Pregnancy Study (MHP) patient samples originated from a study of pregnant women attending Queen Elizabeth Central Hospital, an urban hospital in Blantyre. The complete characte...

Exposing malaria in-host diversity and estimating population diversity by capture-recapture using massively parallel pyrosequencing

“…However, in the past several years, it has been suggested that the amount of previous reports of diversity have described only the "tip of the iceberg" (10,11,16). Here we expose the extent of in-host diversity by using MPP to quantify and identify rare variants in polyclonal malaria infections in a high-throughput manner.…”

“…However, nPCR with gel electrophoresis detects only size polymorphisms and cannot detect sequence polymorphisms. Furthermore, it has been shown that nPCR methods are insensitive to low-abundance variants (3,16). Last, the method is not quantitative for relative population sizes of different variants.…”

The evolution of drug resistance and the curious orthodoxy of aggressive chemotherapy

“…Mixed infections arise from inoculations of genetically diverse parasites by a single mosquito or contemporaneous bites by multiple mosquitoes infected with different parasites. Consequently, the coexistence of drug-sensitive and drug-resistant parasites is common, and indeed may even be the rule (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22).…”

Plasmodium vivax Isolates from Cambodia and Thailand Show High Genetic Complexity and Distinct Patterns of P. vivax Multidrug Resistance Gene 1 (pvmdr1) Polymorphisms

“…8 The heteroduplex tracking assay (HTA) is a genotyping technique that has proven adept at uncovering minority variants within malaria infections and in infections with other agents. [13][14][15][16] Thus, it provides a more accurate picture of within-host genetic diversity. 17 It also facilitates the tracking of clones within hosts over time, potentially providing a nuanced portrait of relapsing vivax populations.…”