Mutations of pvdhfr and pvdhps genes in vivax endemic-malaria areas in Kota Marudu and Kalabakan, Sabah

Plasmodium vivax samples were collected from 23 villages in Kota Marudu, and 26 villages, road construction and loggers’ camps in Kalabakan, Sabah. Kalabakan is located 100 km from Tawau, towards the south of Sabah bordering Kalimantan, Indonesia while Kota Marudu is located in Kudat Division in the northern region of Sabah with 1083 km from Philippine (Fig. 1). The samples from Kalabakan were collected in 2008 and 2009 by active case detection. During those years, Kalabakan contributed to most of the malaria cases with 21.54 and 22.79 %, respectively, of the total cases in Sabah (Vector Borne Disease Control, 2008, 2009 unpublished data). Sample collections in Kota Marudu in 2011 and 2012 were by both active case detection and passive case detection. At the time the study was conducted, the number of malaria cases in Kota Marudu contributed 10 % of total cases in Sabah in 2011 (Vector Borne Disease Control, 2011 unpublished data).

The study protocol was reviewed and approved by the Research Review Committee of the Institute for Medical Research (IMR) and Medical Research Ethics Committee (MREC), Ministry of Health Malaysia. For active case detection, the team visited the villages and the study was announced and briefed to all the population. Study information sheets were given to all who came to the briefing. All the population received the study information sheet, informed consent form and they were assisted whenever required in understanding the study. Individuals who consented to participate in the study were screened for malaria infection by finger-prick blood diagnosis in the field using rapid diagnosis test (RDT) kit (Paramax-3™, Zephyr Biomedicals, India). Some finger-prick blood was also used to prepare blood film for malaria parasite (BFMP) for determination of parasite density.

Individuals diagnosed positive for malaria infection by RDT had approximately 500 µl of blood collected by venipuncture and transferred to EDTA tubes. BFMP and blood in anticoagulant EDTA tubes were brought to the field station to be processed. The EDTA blood was spotted onto filter paper 3MM^® Whatman (Brentford, UK). The filter paper was allowed to dry completely by hanging on a wire line in the field station. After drying, they were packed into a small plastic zipper bags containing silica gels, labelled appropriately and transported to Institute for Medical Research for further molecular study. The BFMP were stained with Giemsa and examined for presence of malaria parasites by microscopy to determine the density of the infection. Individuals diagnosed positive with malaria were treated with a standard anti-malaria treatment by health clinic staff. The study managed to collect 58 samples from Kalabakan and 52 samples from Kota Marudu.

Parasite DNA was extracted from blood spot filter paper by using QIAamp^® DNA Mini Kit (QIAmp; QIAGEN, Hilden, Germany) following the manufacturer’s instruction (Dried Blood Spot Protocol). The DNA was eluted with 150 µl AE Buffer (10 mM Tris–HCl pH 9.0 and 0.5 mM EDTA). The parasite DNA was used for molecular diagnosis of the malaria species and for genotyping studies. All samples underwent molecular speciation using Fuehrer et al. [23] for P. falciparum, P. vivax and Plasmodium malariae. All samples also underwent speciation for P. knowlesi using protocol from Imwong et al. [24]. Samples confirmed positive for P. vivax infection by the molecular diagnosis were included in the study.

The pvdhfr and pvdhps genes were amplified from genomic DNA by nested-PCR. The amplifications of the pvdhfr was carried out as described previously [11, 25] with some modification on the final volume of the PCR reaction mix. In the amplification of P. vivax dhfr-ts gene, size of 1876 bp, excluding the stop codon, the PCR reaction was amplified by using the oligonucleotide pair VDT OF and VDT OR. The final volume of the reaction mix was 50 µl, consisted of 50 ng of template DNA, 1X Buffer I-Taq (BioRad Laboratories Inc), 125 µM dNTPs (BioRad Laboratories Inc), 2.0 mM of Magnesium (Mg) (BioRad Laboratories Inc), 200 nM of each primer, and 2.5 U i-Taq DNA polymerase (BioRad Laboratories Inc). The cycling parameters for the amplification reactions were as follow: an initial denaturation step at 95 °C for 5 min was followed by 25 cycles of denaturation steps at 94 °C for 1 min, annealing steps at 68 °C for 2 min and extension at 72 °C for 2 min. PCR product were hold at 16 °C after a final extension at 72 °C for 5 min.

Two microlitre of the PCR reaction was then used to amplify the P. vivax dhfr (pvdhfr) domain of size 711 bp by using oligonucleotide pair VDT OF and VDF OR (first PCR). The PCR reaction mixtures were the same as used in the first PCR reaction except for the Mg concentration and the primer concentration. The cycling temperature was also similar to the first PCR amplification except for the annealing temperature and the number of cycles (see Table 1).

Nested PCR were carried out separately to amplify three regions on the pvdhfr genes. The PCR reaction used primer pairs VDF13NF and VDFNR58 amplifying 232 base pair (bp) fragment containing Ile13Leu, Pro33Leu, Ser58Arg, and Thr61Met, primer pair using VDNF58and VDF NR amplifying 472 bp fragment containing Phe58Ile/Leu and Ser117Thr/Asn, and primer pair VDTOF and VDFNR amplifying 608 bp fragment containing Phe58Ile/Leu and Ile173Leu. The PCR reaction were similar to the PCR reaction of the first PCR, except for the concentration of Mg and primer concentration. The cycling temperature was also similar to that of the first PCR except for the annealing temperature and the cycles (see Table 1).

Amplification of the P. vivax dhps (pvdhps) gene was carried out as described previously [19]. The first round PCR was performed by using the primer sets VDHPS OF and VDHPS OR, followed by two sets of second round PCR using primer sets VDHPS NF and VDHPS NR amplifying 703 bp fragment containing codon Ala383Gly, and primer sets VDHPS-553OF and VDHPS NR amplifying 170 bp fragment containing codon 553. The first and second rounds PCR were carried out in a final volume of 50 µl with similar reaction as carried out for pvdhfr, except for the primers and Mg concentration and the cycling temperature (see Table 2).

The PCR products were electrophoresed on 2 % agarose gel (Biorad Laboratories Inc) with GelTed™ Nucleic Acid Gel Stain and visualized on an ultraviolet transilluminator. The lack of cross-contamination was monitored by the inclusion of negative control samples (water was used to replace the DNA template) in each of amplifications carried out.

The presence of mutations on pvdhfr at codons 13Leu, 33Leu, 57Ile/Leu, 58Arg, 61Met, 117Asn/Thr, and 173Leu and mutation on pvdhps at codons 383Gly and 553Gly were analysed by RFLP following protocol as described previously [7]. The enzyme digestions were conducted according to manufacturer’s instruction (New England Biolabs, Beverly, MA, USA). Details of the restriction enzymes used and the digestion product sizes indicating wild type and mutant are described in Table 3. The detection of mutation at 13Leu, 33Leu, 58Arg, 61Met, and 173Leu was determined by HaeII, SacII, AluI, Tsp451, and StyI, respectively. The detection of mutation at 57Ile/Leu was determined by reaction of PCR (VDT-OF/VDFNR) with Xmn1, which did not cleave the 608 bp product. To confirm the mutation, either 57Ile or 57Leu, the PCR (VDNF57/VDF NR) was digested with BsrGI to digest the 472 bp products to 444 and 28 bp (mutation 57Ile) and no digestion (mutation 57Leu). The detection of mutation 117Thr/Asn was determined by reaction of PCR (VDNF57/VDF NR) with PvuII, which did not digest the 472 bp product. The codon Ser117Thr was determined by reaction with BsrI, which did not digest the 472 bp. The mutation at 117Thr was determined by reaction with BstNI, which cleaved the 472 bp product to 258 and 215 bp. The mutation at 117Asn was determined by reaction with BsrI which cleaved the 472 bp to 253 and 219 bp. The mutation at 117Asn was confirmed by reaction with BstNI, which did not cleave the 472 bp product. Amplification of pvdhps gene, region VDHPS NF/VDHPS NR, detected mutation at 383Gly. The mutation was detected by reaction of the PCR product with MspI, which cleaved the 703 bp product to 655 and 48 bp. The mutation at 553Gly was detected by reaction of PCR VDHPS553OF/VDHPSNR with MscI, which did not digest the 170 bp.

The PCR–RFLP products were analysed using the Agilent 2100 Bioanalyzer and the Agilent DNA 1000 Kit (Agilent Technologies, Molecular Probes Inc, USA). The procedures were conducted according to manufacturer’s instruction. The result was then viewed and analysed using Agilent 2100 software.

A total of 619 and 2119 individuals from 23 and 26 sites (villagers, logger camps, road construction workers) in Kalabakan and Kota Marudu, respectively, participated in the study. In Kalabakan, 58 individuals (9.37 %) (95 % [CI] 7.07–11.67) tested positive for malaria of which 26 (4.2 %) (95 % [CI] 2.6–5.8 %) were positive for P. vivax. While in Kota Marudu, 52 individuals (2.45 %) (95 % [CI]1.8–3.1 %) tested positive for malaria of which 11 (0.52 %) (95 % [CI] 0.2–0.8 %) were positive for P. vivax. Total number of P. vivax samples used in the study was 37. The pvdhfr and pvdhps genes were successfully amplified and RFLP analysis for detection of mutation on the genes was conducted.

Findings of the pvdhfr gene showed presence of mutations at 58Arg and 61Met (Fig. 2), at 57Leu and 117Thr (Fig. 3) and 57Leu (Fig. 4). Findings of pvdhps gene showed mutation at 383Gly (Fig. 5). Wild type was observed at Ile13, Pro33, Ile173 on pvdhfr gene and Ala553 on pvdhps gene.The prevalent of wild type and mutant on pvdhfr and pvdhps genes is summarized in Table 4. No mutation was observed at codon 13, 33 and 173 on pvdhfr and at codon 553 on pvdhps gene on samples from Kalabakan and Kota Marudu. Common mutations (100 %) on pvdhfr were at 57Leu and 117Thr (Table 4). Mutations at 58Arg and 61Met were observed to be higher in Kota Marudu, 72.73 % (95 % [CI] 43.44–90.26). Mutation at 383Gly on pvdhps was highest in Kalabakan with 80.77 % (95 % [CI] 62.12–91.49) (Table 4).

The findings showed that there are four distinct haplotypes of pvdhfr/pvdhps combination among the 37 samples (Table 5). The four haplotypes are: