Real-Time PCR Assay for Rapid Detection and Analysis of PfCRT Haplotypes of Chloroquine-Resistant Plasmodium falciparum Isolates from India

Abstract

Chloroquine-resistant Plasmodium falciparum (CRPF) malaria isolates in Southeast Asia and sub-Saharan Africa share the same Plasmodium falciparum chloroquine resistance transporter (PfCRT) haplotype (CVIET; amino acids 72 to 76). It is believed that CRPF malaria emerged in Southeast Asia and spread to sub-Saharan Africa via the Indian subcontinent. Based on this assumption, we hypothesized that CRPF isolates in India should possess the same drug resistance haplotype (PfCRT haplotype CVIET) as P. falciparum isolates in Southeast Asia and Africa and that the prevalence of CRPF may be higher and more widespread in India than appreciated. To test this postulate, we utilized a standardized real-time PCR assay to assess the prevalence and distribution of PfCRT haplotypes in P. falciparum isolates (n ‫؍‬ 406) collected from Western, Central, and Eastern states in India and compared them to isolates from South America and Africa. Based on the proportion of isolates possessing the molecular marker K76T, the prevalence of chloroquine resistance was high in all five regions of India studied (91%), as well as in Uganda (98%) and Suriname (100%). All isolates from Suriname contained the chloroquine-resistant SVMNT haplotype typical of South American isolates, and 98% of isolates from Uganda possessed the chloroquine-resistant CVIET haplotype characteristic of Southeast Asian/African strains. However, of 246 P. falciparum isolates from across India that contained the molecular marker for chloroquine resistance, 81% contained the SVMNT haplotype. In conclusion, the prevalence of CRPF malaria was high in geographically dispersed regions of India, and the primary haplotype observed, SVMNT, did not support a presumed geographic spread from contiguous Southeast Asia.

Molecular Assessment of Plasmodium falciparum Resistance to Antimalarial Drugs in Papua New Guinea Using an Extended Ligase Detection Reaction Fluorescent Microsphere Assay

“…This high prevalence of the resistant K76T polymorphism concurred with data from studies conducted between 2000 and 2005 in Madang and East Sepik Provinces (5,28,41 (codons 72 to 76) is at fixation in the PNG parasite population, with an increase in prevalence from 83% from the early 1990s to 92.3% in 2003 to 2005 (25,27,42). Another CQ-resistant haplotype, CVIET (codons 72 to 76), found commonly in Africa and Southeast Asia (22,23,32,48), was detected in two isolates as a mixed infection with the SVMNT strain, confirming its recent emergence in PNG (10). Differences between the pfcrt intronic microsatellite diversity haplotypes of the SVMNT and CVIET parasites from the province adjacent to Madang suggest that they have been imported (10), presumably by economic migrants from nearby Asian countries (27)(28)(29).…”

Coxiella-like Endosymbionts

“…The difference in prevalence rates was attributed to the diagnostic method used. Whereas Heise et al used nested PCR to detect the Coxiella -like bacterium, Jasinskas et al used real-time quantitative PCR, which could be more sensitive than the nested PCR method (Drago et al 2004 ;Farcas et al 2004 ;Keen et al 2007 ;Genc et al 2010 ) .…”

Molecular surveillance of drug-resistant Plasmodium falciparum in two distinct geographical areas of Nigeria

“…In general, CQ-resistant isolates possess pfcrt alleles with multiple mutations corresponding to the amino acid haplotype CVIET (residues 72-76) or a SVMNT haplotype [7][8][9]. CQ-sensitive strains are characterized by the CVMNK specifi c set of pfcrt mutations [10]. Polymorphisms in pfmdr1 and amplifi cations of this gene also aff ect susceptibility to structurally unrelated antimalarial drugs, including mefl oquine, artesunate, lumefantrine and quinine [11,12].…”