Full-Length Sequence Analysis of Chloroquine Resistance Transporter Gene inPlasmodium falciparumIsolates from Sabah, Malaysia

Abstract

Chloroquine resistance (CQR) in falciparum malaria was identified to be associated with several mutations in the chloroquine resistance transporter gene (pfcrt) that encodes the transmembrane transporter in digestive vacuole membrane of the parasite. This study aimed to investigate the point mutations across the full-length pfcrt in Plasmodium falciparum isolates in Sabah, Malaysia. A total of 31 P. falciparum positive samples collected from Keningau, Kota Kinabalu, and Kudat, Sabah, were analyzed. pfcrt was PCR amplified and cloned prior to sequence analysis. This study showed that all the previously described 10 point mutations associated with CQR at codons 72, 74, 75, 76, 97, 220, 271, 326, 356, and 371 were found with different prevalence. Besides, two novel point mutations, I166V and H273N, were identified with 22.5% and 19.3%, respectively. Three haplotypes, namely, CVMNK (29%), CVIET (3.2%), and SVMNT (67.7%), were identified. High prevalence of SVMNT among P. falciparum isolates from Sabah showed that these isolates are closer to the P. falciparum isolates from Papua New Guinea rather than to the more proximal Southeast Asian CVIET haplotype. Full-length analysis of pfcrt showed that chloroquine resistant P. falciparum in Sabah is still prevalent despite the withdrawal of chloroquine usage since 1979.

Prevalence of Plasmodium falciparum Molecular Markers of Antimalarial Drug Resistance in a Residual Malaria Focus Area in Sabah, Malaysia

“…falciparum CQ, PYR and SDX resistant previously reported in Pahang [9], Lundu, Sarawak [8] and Sabah: Kalabakan (Tawau Division) [6, 30], Keningau & Nabawan (Interior Division) [39], Kota Kinabalu (West Coast Division) [35], a Kota Marudu—present study, b Kudat [35] (Kudat Division). This map was generated by using SimpleMappr online software [40].…”

“…falciparum isolates in Kudat, Sabah (Fig 1 and Table 3). In contrast, a high prevalence rate of pfcrt K76T mutation was previously observed in Kalabakan and Kota Kinabalu, located in Tawau division and West Coast Division of Sabah, respectively (Fig 1 and Table 3) [6, 35]. A hundred percent prevalence of K76T mutation was reported in Lundu, Sarawak in a study conducted in 1999 and 2000 (Fig 1 and Table 3) [8].…”

Functional Comparison of 45 Naturally Occurring Isoforms of the Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT)

“…27,38 Some of these isoforms, such as GadIII and KT099, harbor only a single CQR-associated mutation in an otherwise wild-type background (Table 2). While each of these isoforms was found to transport CQ at levels similar to that of HB3, their PQ transport was found to be substantially mitigated relative to HB3, especially in the case of KT099 (Figure 7, middle).…”

Combinatorial Genetic Modeling ofpfcrt-Mediated Drug Resistance Evolution inPlasmodium falciparum

“…In regions in Africa ( Mwai et al 2009 ; Laufer et al 2010 ; Mekonnen et al 2014 ) and Asia ( Wang et al 2005 ; Chen et al 2008 ), where the CVIET PfCRT haplotype (residues 72–76) predominates among CQ-resistant parasites, removal of CQ pressure promoted regional resurgences of CQ-sensitive parasites encoding the fitter, wild-type (CVMNK) PfCRT haplotype. In contrast, in South America ( Vieira et al 2004 ; Gama et al 2009 ; Griffing et al 2010 ; Adhin et al 2013 ) and in select regions elsewhere ( de Almeida et al 2009 ; Mallick et al 2012 ; Khattak et al 2013 ; Tan et al 2014 ), the predominant mutant PfCRT haplotype, SVMNT, persists despite a decline in CQ use. The prospect that this haplotype is less deleterious to parasites than CVIET ( Sa and Twu 2010 ) is supported by recent in vitro studies, in which isogenic intraerythrocytic parasites expressing the CVIET-encoding Dd2 pfcrt allele demonstrated a higher fitness cost than parasites expressing the SVMNT-encoding 7G8 pfcrt allele ( Petersen et al 2015 ).…”