Increased pfmdr1 gene copy number and the decline in pfcrt and pfmdr1 resistance alleles in Ghanaian Plasmodium falciparum isolates after the change of anti-malarial drug treatment policy

Abstract

BackgroundWith the introduction of artemisinin-based combination therapy (ACT) in 2005, monitoring of anti-malarial drug efficacy, which includes the use of molecular tools to detect known genetic markers of parasite resistance, is important for first-hand information on the changes in parasite susceptibility to drugs in Ghana. This study investigated the Plasmodium falciparum multidrug resistance gene (pfmdr1) copy number, mutations and the chloroquine resistance transporter gene (pfcrt) mutations in Ghanaian isolates collected in seven years to detect the trends in prevalence of mutations.MethodsArchived filter paper blood blots collected from children aged below five years with uncomplicated malaria in 2003–2010 at sentinel sites were used. Using quantitative real-time polymerase chain reaction (qRT-PCR), 756 samples were assessed for pfmdr1 gene copy number. PCR and restriction fragment length polymorphism (RFLP) were used to detect alleles of pfmdr1 86 in 1,102 samples, pfmdr1 184, 1034, 1042 and 1246 in 832 samples and pfcrt 76 in 1,063 samples. Merozoite surface protein 2 (msp2) genotyping was done to select monoclonal infections for copy number analysis.ResultsThe percentage of isolates with increased pfmdr1 copy number were 4, 27, 9, and 18% for 2003–04, 2005–06, 2007–08 and 2010, respectively. Significant increasing trends for prevalence of pfmdr1 N86 (×2 = 96.31, p <0.001) and pfcrt K76 (×2 = 64.50, p <0.001) and decreasing trends in pfmdr1 Y86 (×2 = 38.52, p <0.001) and pfcrt T76 (×2 = 43.49, p <0.001) were observed from 2003–2010. The pfmdr1 F184 and Y184 prevalence showed an increasing and decreasing trends respectively but were not significant (×2 = 7.39,p=0.060; ×2 = 7.49, p = 0.057 respectively). The pfmdr1 N86-F184-D1246 haplotype, which is alleged to be selected by artemether-lumefantrine showed a significant increasing trend (×2 = 20.75, p < 0.001).ConclusionIncreased pfmdr1 gene copy number was observed in the isolates analysed and this finding has implications for the use of ACT in the country although no resistance has been reported. The decreasing trend in the prevalence of chloroquine resistance markers after change of treatment policy presents the possibility for future introduction of chloroquine as prophylaxis for malaria risk groups such as children and pregnant women in Ghana.

Impact of Antimalarial Treatment and Chemoprevention on the Drug Sensitivity of Malaria Parasites Isolated from Ugandan Children

“…Similar results were described recently in Kenya (42), Tanzania (48), and Ghana (49). As seen previously (42,50), the wildtype alleles at pfcrt K76 and pfmdr1 N86 were associated with decreased lumefantrine sensitivity.…”

Failure of atovaquone-proguanil malaria chemoprophylaxis in a traveler to Ghana

“…Our case also supports that under circumstances of drug withdrawal at a population level, chloroquine resistant genotypes may revert back to wild type due to fitness costs [16e18], thus rendering chloroquine potentially efficacious [19]. In our patient, only a small proportion of parasites (13%) demonstrated a mutant genotype at Pfcrt position 72 by pyrosequencing, a fraction of resistance and at a position that is unlikely to translate into clinical failure of chloroquine [16].…”

“…Now, rates of chloroquine resistant P. falciparum in Ghana are approximately 59%, with predominant mutations of Pfcrt position 76 [26]. "Susceptible" Pfcrt 76 alleles have been found in 25e76% of P. falciparum isolates in Ghana as recently as 2010 [19]. Thus, with withdrawal of mass chloroquine chemoprophylaxis and formulaic treatment in many parts of Africa over a decade ago, chloroquine susceptibility may re-emerge in circulating strains of P. falciparum.…”

Temporal and Seasonal Changes of Genetic Polymorphisms Associated with Altered Drug Susceptibility to Chloroquine, Lumefantrine, and Quinine in Guinea-Bissau between 2003 and 2012

“…Alternatively, finding so few samples with increased copy numbers might well be within the margin of error when doing PCRs, suggesting that no samples exist in Bissau. Either way, P. falciparum with multiple pfmdr1 copies has not become widespread in Bissau, contrary to the situation in Ghana, where relatively high frequencies were recently reported (38).…”