High prevalence of mutation in the Plasmodium falciparum dhfr and dhps genes in field isolates from Sabah, Northern Borneo

Abstract

Background: Sulphadoxine-pyrimethamine (SP) has been in use for the treatment of uncomplicated falciparum malaria in Malaysia since the 1970s and is still widely employed in spite of widespread clinical resistance. Resistance to SP is known to be mediated by mutations in the pfdhfr and pfdhps genes. The aim of the present study was to investigate the distribution of pfdhfr and pfdhps gene polymorphism in Plasmodium falciparum field isolates from Kalabakan, Sabah, in northern Borneo.

Methods: A total number of 619 individuals were screened from 23 study sites of which 31 were positive for P. falciparum. Analysis of restriction fragment length polymorphisms (RFLP) was used to identify polymorphism in the pfdhfr and pfdhps genes at positions 16, 51, 59, 108, 164 and 437, 540, 581, respectively.

Results: All samples had at least one mutation in each of the genes associated with drug resistance. The prevalence of pfdhfr 59arg, 164leu and 108asn were 100%, 80.65% and 58.06%, respectively. Pfdhps mutants 437gly and 581gly accounted for 100% and 74.19% respectively. In pfdhfr, the most common mutant genotypes were combination 59arg + 164leu (22.58%) and 59arg + 108asn + 164leu (51.61%). In pfdhps the most common genotype was 437gly + 581gly (74.19%). One individual (3.22%) harboured parasites with four pfdhfr (16 val + 59arg + 108asn + 164leu) and two pfdhps (437gly + 581gly) mutations. The highest quintuple pfdhfr/pfdhps (41.94%) was three pfdhfr (59arg + 108asn + 164gly) and two pfdhps (437gly + 581gly).

Conclusion: The data suggest a high prevalence of genetic variations conferring resistance to SP which can predict treatment failure before becoming clinically evident. In areas like this, the use of SP may no longer be indicated.

Figure 1

PCR-RFLP of the pfdhfr gene, amplified region of M3-F/primers.BsrI cleaved the 522 bp fragment into 190 and 332 bp indication of 108asn mutation (A). DraI detect 164leu mutations producing 28, 107, 171 and 245 bp fragments for wild type and 107, 143 and 245 bp for mutant (B). Digestion with NlaIII produce 53, 93 and 376 bp for wild type and 146 and 245 bp for mutant at codon 16 (C) and digestion with Tsp5091 yielded 55, 65, 120, and 153 bp fragments for wild type and 55, 65, 120 and 218 bp fragments for mutant for detection on polymorphism at codon 51 (D). Lane L: DNA ladder of Agilent DNA 1000 Kit (Agilent Technologies, Molecular Probes Inc, USA), The controls are in Lane 1 and 2: A: Lane 1: Plasmodium falciparum KI strain (mutant); Lane 2: P. falciparum T9.96 strain (wild type); Lane 3–9: field samples from Kalabakan; well 10: PCR negative control (no DNA was added to the PCR reaction). B and C: Lane 1: Plasmodium falciparum KI strain (wild type); Lane 2: P. falciparum T9.96 strain (wild type); Lane 3–9: field samples from Kalabakan; well 10: PCR negative control (no DNA was added to the PCR reaction). D: Lane 1: Plasmodium falciparum KI strain (wild type); Lane 2: P. falciparum W2 strain (mutant) Lane 3–9: field samples from Kalabakan; well 10: PCR negative control (no DNA was added to the PCR reaction).

Figure 2

PCR-RFLP of the pfdhfr gene, amplified region of M4-F primers. The 326 bp fragment was cut by Alu I into 180 and 118 bp fragments, indications for wild type ser108 and 299 bp for mutant (A). BstNI digested only mutant allele into 145 and 181 bp indicates mutation 108thr. All the tested samples showed wild type (B). Reaction with XmnI produced fragment 163 and 189 bp for wild type; and 26, 137 and 163 bps for 59arg mutation (C). Lane L: DNA ladder of Agilent DNA 1000 Kit (Agilent Technologies, Molecular Probes Inc, USA), The controls are in Lane 1 and 2: A: Lane 1: Plasmodium falciparum KI strain (mutant); Lane 2: P. falciparum T9.96 strain (wild type); Lane 3–9: samples from Kalabakan; Lane 10: PCR negative control (no DNA was added to the PCR reaction). B: Lane 1: Plasmodium falciparum KI strain (wild type); Lane 2: P. falciparum T9.96 strain (wild type); Lane 3–9: samples from Kalabakan; Lane 10: PCR negative control (no DNA was added to the PCR reaction). C: Lane 1: Plasmodium falciparum KI strain (mutant); Lane 2: P. falciparum T9.96 strain (wild type); Lane 3–9: samples from Kalabakan; Lane 10: PCR negative control (no DNA was added to the PCR reaction).

Figure 3

PCR-RFLP of pfdhps gene. Amplified region of K-K/primers was targeted to ala437gly with AvaII and lys540glu with FokI, showed 404 bp (mutant) and 438 bps (wild type) respectively (A, B). Reaction of L-L/amplified region with BstUI showed presence of 581gly mutation with a 138 bp fragment and wild type ala581 producing a 105 bp (C). Lane L: DNA ladder of Agilent DNA 1000 Kit (Agilent Technologies, Molecular Probes Inc, USA). The controls are in Lane 1 and 2: A: Lane 1: Plasmodium falciparum 3D7 strain (mutant); Lane 2: P. falciparum K1 strain (mutant); B: Lane 1: Plasmodium falciparum K1 strain (wild type); Lane 2: P. falciparum T9.96 strain (wild type); C: Lane 1: Plasmodium falciparum K1 strain (mutant); Lane 2: P. falciparum W2 strain (wild type). Lane 3–10: samples from Kalabakan; Lane 11: PCR negative control (no DNA was added to the PCR reaction).