Analysis of Pfmdr1 and Pfcrt Drug Resistant Markers in Plasmodium falciparum Following Treatments of Patients in Nyando and Mbita, Nyanza Region with Artemisinin-Based Combination Therapy

Abstract

The multidrug resistant Plasmodium falciparum is a major public health concern in the global management of malaria, especially in developing countries. Currently, malaria treatment is primarily dependent on the sustained efficacy of artemisinin-based combination therapy (ACT). Since the introduction of ACT, Plasmodium falciparum resistance has been reported in various malaria endemic sites globally. For instance, recent reports indicating decline in efficacy of ACT and artesunate monotherapy in Southeast Asia are a major threat to global malaria control achievements. Initially, mutations in Plasmodium falciparum multidrug resistance-1 (Pfmdr1) gene were associated with chloroquine and amodiaquine resistance only, but have recently been associated with conferring tolerance to ACT. This study used Restriction Fragment Length Polymorphism (RFLP) to evaluate the prevalence of genetic polymorphisms of Plasmodium falciparum multidrug resistance (Pfmdr1) and Plasmodium falciparum chloroquine-resistant transporter (Pfcrt) gene mutations in Mbita and Nyando field isolates between 2008 and 2015. A small proportion (24.7%) of the samples were sequenced using the Sanger sequencing technique, “ChromasPro software” (Version 2.6.6) was used to clean/trim sequences while the “Muscle tool” was used to perform the multiple sequence alignment to confirm the presence or absence of mutations. The findings report that the prevalence of Pfcrt K76T mutation in Plasmodium falciparum field isolates declined significantly from 76.3% (95% CI 21.0-31.0) in 2008 to 51.9% (95% CI 19.0-25.0) in Mbita (2015), a reduction of 24.4% (χ2df =1 = 4.709, p = 0.0000). In Nyando, the prevalence stood at 66.8% (95% CI 12.0-14.0) in 2015 down from 73.7% (95% CI 27.0-48.0) in 2008, insignificant reduction of 6.9% (χ2df =1 = 17.699, p = 0.0913). Prevalence of the Pfmdr1 86Y mutation reduced to 21.9% (95% CI 31.0-49.0) in Mbita in 2015 from 67.3% (95% CI 37.0-46) in 2008, a significant reduction of 38.2% (χ2df =1 = 6.491, p = 0.001). While in Nyando, mutation was 38.1% (95% CI 27.0-18.9) in 2015 down from 40.0% (95% CI 33.0-38) in 2008, insignificant reduction of 1.9% (χ2df =1 = 6.231, p = 0.0933). The sequencing results confirmed 12/20(60.0%) were mutant strain 76T, 8/20(40.0%) were wild-type K76 and there was zero mixed mutation (76K/T) for Pfcrt gene in Mbita. Nyando field isolates recorded 8/15(53.3%) mutant (76T), 3/15(20.0%) wild-type (K76) while 4/15(26.7%) harbored CVIET/CVMNK mixed infection. For both Mbita and Nyando, Pfmdr1 prevalence recorded prevalence variation across all the 5 target codons (86, 184, 1034, 1042 and 1246). Recorded mixed infection ranged from 0.0% to 35.5% (N1042D). Findings in this study confirm a common trend reported by previous studies that there has been a small decline in the prevalence of the Pfcrt mutation between 2008 and 2015 since the withdrawal of Chloroquine as the first line of treatment for uncomplicated cases of malaria in Kenya. In addition, the findings also reveal potential Pfmdr1 markers tolerance to ACT as exhibited by the elevated mutation prevalences at codon 86Y, 184F, 1042D and 1246Y.