Minority-Variant pfcrt K76T Mutations and Chloroquine Resistance, Malawi

Jonathan J Juliano,Jesse J Kwiek,Steven R Meshnick,Victor Mwapasa,Kathryn Cappell

doi:10.3201/eid1306.061182

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-specific 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 confirmed by subcloning and sequencing. This finding 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 drug resistant mutations may be useful in making antimalarial drug policy.

Highlights

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
We describe a new multiple site-specific heteroduplex tracking assay (MSS-Heteroduplex Tracking Assay (HTA)) for detecting the pfcrt (Plasmodium falciparum chloroquine resistance transporter gene) K76T mutation
The multiple site–specific heteroduplex tracking assay (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 [6,7,8,9]

Summary

Introduction

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. 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 difficult in highly malaria-endemic areas, where infections are almost always polyclonal [3,4]. We describe a new multiple site-specific 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 [6,7,8,9]

Methods

Results

Conclusion