Prevalence and patterns of antifolate and chloroquine drug resistance markers in Plasmodium vivax across Pakistan

Abstract

BackgroundPlasmodium vivax is the most prevalent malaria species in Pakistan, with a distribution that coincides with Plasmodium falciparum in many parts of the country. Both species are likely exposed to drug pressure from a number of anti-malarials including chloroquine, sulphadoxine-pyrimethamine (SP), and artemisinin combination therapy, yet little is known regarding the effects of drug pressure on parasite genes associated with drug resistance. The aims of this study were to determine the prevalence of polymorphisms in the SP resistance-associated genes pvdhfr, pvdhps and chloroquine resistance-associated gene pvmdr1 in P. vivax isolates collected from across the country.MethodsIn 2011, 801 microscopically confirmed malaria-parasite positive filter paper blood samples were collected at 14 sites representing four provinces and the capital city of Islamabad. Species-specific polymerase chain reaction (PCR) was used to identify human Plasmodium species infection. PCR-positive P. vivax isolates were subjected to sequencing of pvdhfr, pvdhps and pvmdr1 and to real-time PCR analysis to assess pvmdr1 copy number variation.ResultsOf the 801 samples, 536 were determined to be P. vivax, 128 were P. falciparum, 43 were mixed vivax/falciparum infections and 94 were PCR-negative for Plasmodium infection. Of PCR-positive P. vivax samples, 372 were selected for sequence analysis. Seventy-six of the isolates (23%) were double mutant at positions S58R and S117N in pvdhfr. Additionally, two mutations at positions N50I and S93H were observed in 55 (15%) and 24 (7%) of samples, respectively. Three 18 base pair insertion-deletions (indels) were observed in pvdhfr, with two insertions at different nucleotide positions in 36 isolates and deletions in 10. Ninety-two percent of samples contained the pvdhps (S382/A383G/K512/A553/V585) SAKAV wild type haplotype. For pvmdr1, all isolates were wild type at position Y976F and 335 (98%) carried the mutation at codon F1076L. All isolates harboured single copies of the pvmdr1 gene.ConclusionsThe prevalence of mutations associated with SP resistance in P. vivax is low in Pakistan. The high prevalence of P. vivax mutant pvmdr1 codon F1076L indicates that efficacy of chloroquine plus primaquine could be in danger of being compromised, but further studies are required to assess the clinical relevance of this observation. These findings will serve as a baseline for further monitoring of drug-resistant P. vivax malaria in Pakistan.

Distribution of Mutations Associated with Antifolate and Chloroquine Resistance among Imported Plasmodium vivax in the State of Qatar

“…Our observation corroborates the previous reports from Pakistan, Nepal, and Sudan. [39][40][41] However, no triple/quadruple mutations were detected, which is in contrast with previous findings from India 16,36 as India has different degrees of malaria endemicity, and the levels of mutations from the different geographical state is also varied. 42 With regard to the Pvdhps gene, majority of our isolates (77.2%) carried wild type alleles and frequency of single mutant (383G) and double mutant genotypes (383G/553G) was 10.2% and 12.6%, respectively, which have been found to be directly related to sulphadoxine resistance 43 and associated with reduced sensitivity to both sulfa drugs and sulfones.…”

Molecular Detection of Antimalarial Drug Resistance in Plasmodium vivax from Returned Travellers to NSW, Australia during 2008–2018

“…In addition, the high prevalence of the L1076 mutation in the South Asian region was consistent with other recent studies in India and Pakistan with respective mutations of 54.5% and 98.0% [34,35]. Only two isolates carried the F976:L1076 mutations in the Indian study [34], whereas the previous study in Pakistan did not find either the F976 mutation or the F976:L1076 mutations [35]. Furthermore, if the hypothesis of a two-step mutation trajectory is true, then the L1076 mutation occurs first, followed by the F976 mutation before the emergence of chloroquine resistance [17,36,37].…”

“…This suggests that there was a rise in the number of chloroquine resistance markers since the first reported case of resistant P. vivax in the region, even though chloroquine has not been recommended in the antimalarial drug policy since 2009 [33]. In addition, the high prevalence of the L1076 mutation in the South Asian region was consistent with other recent studies in India and Pakistan with respective mutations of 54.5% and 98.0% [34,35]. Only two isolates carried the F976:L1076 mutations in the Indian study [34], whereas the previous study in Pakistan did not find either the F976 mutation or the F976:L1076 mutations [35].…”

Selective sweep and phylogenetic models for the emergence and spread of pyrimethamine resistance mutations inPlasmodium vivax

“…Subsequent work has consistently shown that F57L/I (TT A / A T A ), S58R (AG A ), S117N/T (A A C/A C C) single mutations and S58R (AG A ) /S117N/T (A A C/A C C) double mutations are widely distributed in different geographical regions of south Asia (Auliff et al, 2006; Brega et al, 2004; de Pecoulas et al, 1998; Hastings et al, 2005; Imwong et al, 2003; Kaur et al, 2006; Kuesap et al, 2011; Lu et al, 2012; Mint Lekweiry et al, 2012; Ranjitkar et al, 2011; Schunk et al, 2006). Previous studies in Pakistan have shown that the S58R (AG A )/S117N (A A C) double mutation and S117N (A A C) single mutation conferring pyrimethamine resistance were present in different cities of the Punjab, Sindh and KPK provinces (Khattak et al, 2013; Zakeri et al, 2011). In the present study, the S58R (AG A )/ S117N (A A C) double mutation was also present in 10/38 populations.…”