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. 2011 Apr 1:10:75.
doi: 10.1186/1475-2875-10-75.

Prevalence of molecular markers of anti-malarial drug resistance in Plasmodium vivax and Plasmodium falciparum in two districts of Nepal

Samir Ranjitkar  1 , Mette L Schousboe, Thomas Thyge Thomsen, Madhav Adhikari, Christian M O Kapel, Ib C Bygbjerg, Michael Alifrangis
Affiliations

Prevalence of molecular markers of anti-malarial drug resistance in Plasmodium vivax and Plasmodium falciparum in two districts of Nepal

Samir Ranjitkar et al. Malar J. .

Abstract

Background: Sulphadoxine-pyrimethamine (SP) and chloroquine (CQ) have been used in treatment of falciparum and vivax malaria in Nepal. Recently, resistance to both drugs have necessitated a change towards artemisinin combination therapy (ACT) against Plasmodium falciparum in highly endemic areas. However, SP is still used against P. falciparum infections in low endemic areas while CQ is used in suspected cases in areas with lack of diagnostic facilities. This study examines the prevalence of molecular markers of CQ and SP resistance in P. falciparum and Plasmodium vivax to determine if high levels of in vivo resistance are reflected at molecular level as well.

Methods: Finger prick blood samples (n=189) were collected from malaria positive patients from two high endemic districts and analysed for single nucleotide polymorphisms (SNPs) in the resistance related genes of P. falciparum and P. vivax for CQ (Pfcrt, Pfmdr1, Pvmdr1) and SP (Pfdhfr, Pfdhps, Pvdhfr), using various PCR-based methods.

Results and discussion: Positive P. vivax and P. falciparum infections were identified by PCR in 92 and 41 samples respectively. However, some of these were negative in subsequent PCRs. Based on a few P. falciparum samples, the molecular level of CQ resistance in P. falciparum was high since nearly all parasites had the Pfcrt mutant haplotypes CVIET (55%) or SVMNT (42%), though frequency of the Pfmdr1 wild type haplotype was relatively low (35%). Molecular level of SP resistance in P. falciparum was found to be high. The most prevalent Pfdhfr haplotype was double mutant CNRNI (91%), while frequency of Pfdhps double mutant SGEAA and AGEAA were 38% and 33% respectively. Combined, the frequency of quadruple mutations (CNRNI-SGEAA/AGEAA) was 63%. Based on P. vivax samples, low CQ and SP resistance were most likely due to low prevalence of Pvmdr1 Y976F mutation (5%) and absence of triple/quadruple mutations in Pvdhfr.

Conclusions: Based on the limited number of samples, prevalence of CQ and SP resistance at molecular levels in the population in the study area were determined as high in P. falciparum and low in P. vivax. Therefore, CQ could still be used in the treatment of P. vivax infections, but this remains to be tested in vivo while the change to ACT for P. falciparum seems justified.

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Figures

Figure 1
Figure 1
Distribution of malaria in Nepal. Distribution of malaria in 75 districts of Nepal. Jhapa (1) and Banke (9) districts are the study sites. High and low malaria endemic districts are categorized on the basis of abundance of two principle vectors; Anopheles minimus and Anopheles fluviatilis, and malaria transmission rate: where annual malaria transmission rate are ≥ 1/1,000 and 0- 1/1,000 population respectively [5,53]. The figure has been modified from [54] to provide more detail.
Figure 2
Figure 2
Prevalence of haplotypes of Pfcrt in samples from Jhapa and Banke districts in Nepal. Prevalence of haplotypes at codons 72-76 of the Pfcrt gene related to P. falciparum chloroquine resistance, including the mixed haplotypes (n = 31).
Figure 3
Figure 3
Frequency of haplotypes of Pfmdr1 in samples from Jhapa and Banke districts in Nepal. Frequency of constructed haplotypes of SNPs in the Pfmdr1 gene at c86, c184 and c1246 associated with P. falciparum chloroquine resistance (n = 26).
Figure 4
Figure 4
Frequency of haplotypes of Pfdhfr in samples from Jhapa and Banke districts in Nepal. Frequency of constructed haplotypes of SNPs in the Pfdhfr gene at c50, c51, c59, c108 and c164 associated with P. falciparum pyrimethamine resistance (n = 32).
Figure 5
Figure 5
Frequency of haplotypes of Pfdhps in samples from Jhapa and Banke districts in Nepal. Frequency of constructed haplotypes of SNPs in the Pfdhps gene at c436, c437, c540, c581 and c613 linked with P. falciparum sulphadoxine resistance (n = 28).
Figure 6
Figure 6
Frequency of haplotypes of Pvmdr1 in samples from Jhapa and Banke districts in Nepal. Frequency of constructed haplotypes of SNPs in the Pvmdr1 gene at c958, c976 and c1076 associated with P. vivax chloroquine resistance (n = 39).
Figure 7
Figure 7
Frequency of haplotypes of Pvdhfr in samples from Jhapa and Banke districts in Nepal. Frequency of constructed haplotypes of SNPs in the Pvdhfr gene at c57, c58, c61 and c117 linked with P. vivax pyrimethamine resistance (n = 55).
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