Correlation of molecular markers, Pfmdr1-N86Y and Pfcrt-K76T, with in vitro chloroquine resistant Plasmodium falciparum, isolated in the malaria endemic states of Assam and Arunachal Pradesh, Northeast India.

Sandeep Kumar Shrivastava,Mohan Lal Dubey,Ravi Kumar Gupta,Jagdish Mahanta,Thomas A Smith

doi:10.1371/journal.pone.0103848

Abstract

The mechanism of chloroquine (CQ) resistance in Plasmodium falciparum is not clearly understood. However, CQ resistance has been shown to be associated with point mutations in Pfcrt and Pfmdr1. These genes encode for digestive vacuole transmembrane proteins Pfcrt and Pgh1, respectively. The present study was carried out to analyze the association of Pfcrt-K76T and Pfmdr1-N86Y mutations with CQ resistance in Northeast Indian P. falciparum isolates. 115 P. falciparum isolates were subjected to in vitro CQ sensitivity testing and PCR-RFLP analysis for the Pfmdr1-N86Y and Pfcrt-K76T mutations. 100 isolates of P. falciparum were found to be resistant to CQ by the in vitro susceptibility test (geometric mean EC50 2.21 µM/L blood) while 15 were found to be CQ sensitive (geometric mean EC50 0.32 µM/L blood). All the CQ resistant isolates showed the presence of Pfmdr1 and Pfcrt mutations. CQ sensitive isolates were negative for these mutations. Strong linkage disequilibrium was observed between the alleles at these two loci (Pfmdr1-N86Y and Pfcrt-K76T). The results indicate that Pfmdr1-N86Y and Pfcrt-K76T mutations can be used as molecular markers to identify CQ resistance in P. falciparum. The result necessitates the evaluation of CQ in vivo therapeutic efficacy in endemic areas for more effective malaria control strategies.

Highlights

Malaria is one of the major public health problems of the malaria affected countries, including India
The results indicate that Pfmdr1-N86Y and Pfcrt-K76T mutations can be used as molecular markers to identify CQ resistance in P. falciparum
All 50 isolates collected from Arunachal Pradesh showed a high level of CQ resistance with geometric mean EC50 of 2.94 mmol/L blood

Summary

Introduction

Malaria is one of the major public health problems of the malaria affected countries, including India. In India, around 1.5 million laboratory confirmed cases of malaria are reported annually, out of which 50% cases are due to Plasmodium falciparum alone. Chloroquine (CQ) has been the most effective drug in the treatment of non-complicated malaria. A sudden rise in P. falciparum cases has been caused by resistance towards CQ, which was used for a long time as the first line of treatment of malaria cases [1]. CQ resistance may lead to high morbidity and mortality in P. falciparum cases, if not treated timely. CQ acts by interfering with heme metabolism in the digestive vacuole of P. falciparum and CQ resistance results from reduced accumulation of the drug by the parasites [2,3,4]

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Conclusion