Synergism between Pfcrt and Pfmdr1 genes could account for the slow recovery of chloroquine sensitive Plasmodium falciparum strains in Ghana after chloroquine withdrawal

Summary: Unlike other countries, the chloroquine resistant marker Pfcrt T76 mutant has remained fairly stable in Ghana several years after official disuse of chloroquine. Certain mutations in Pfmdr1 may potentiate Pfcrt T76, offering a possible explanation for this observation. To understand the phenomenon, the co-existence of mutations in Pfmdr1 with Pfcrt T76 in Ghanaian Plasmodium falciparum isolates was studied. The reported presence of parasites with reduced sensitivity to amodiaquine and quinine in the country was also studied. Blood samples collected from confirmed malaria patients presenting at health facilities in two distinct ecological zones were analyzed. The prevalence of Pfcrt K76T and the five point mutations in Pfmdr1 were determined using nested PCR followed by RFLP analysis. The association between genes was determined by chi square analysis, and synergism between the two genes was ascertained using the Jonckheere–Terptra (J–T) test followed by Monte Carlo simulation (MCS). Nearly fifty-four percent (53.7%) of the P. falciparum isolates examined had the Pfcrt T76 gene, out of which 18.3% had both K76 and T76 alleles. Mutations at codon 86, 184, 1034, 1042 and 1246 of the Pfmdr1 gene were detected in 36.0%, 87.9%, 71.0%, 91.6% and 8.4% of the isolates, respectively. The haplotypes of Pfmdr1 present were NFCDD (43.46%), YFCDD (27.57%), NFSDD (7.48%), NYSNY (5.14%) and YFSDD (4.67%). Pfcrt T76 was significantly associated with a double mutation at codon 86 and 184 of Pfmdr1 (YF; χ2 = 18.045, p = 0.006). Associations were observed between Pfcrt K76T and Pfmdr1 triple mutation at codons 86, 184 and 1034 (NFC; χ2 = 13.770, p = 0.032 and YFC; χ2 = 16.489, p = 0.011). The J–T test showed significant synergism between Pfcrt 76 and Pfmdr1 polymorphisms (p < 0.0001), which was confirmed by MCS at 99% CI. Synergism between Pfcrt and Pfmdr1 mutant genes could account for the slow recovery of chloroquine sensitive P. falciparum in Ghana. The same phenomenon could explain resistance to amodiaquine and quinine. The outcomes of this study also indicated a possible emergence of artemether-lumefantrine resistance in Ghana. Keywords: Malaria, Drug resistance, Haplotype, Recovery, Synergism, Gene, Plasmodium falciparum, Chloroquine, Amodiaquine, Quinine, Pfcrt, Pfmdr