Background
Malaysia is in the pre-elimination phase of the malaria elimination programme [
1]. Malaysia is fully committed to controlling malaria, providing good infrastructure for control which dates back to the programme’s implementation in 1961. The impact of the malaria eradication programme has resulted in a major reduction of malaria cases from 243,870 in 1961 to 44,226 in 1980 [
1,
2] and has provided the basis for the subsequent malaria control programme which began in 1982. In 2010, the National Malaria Control Programme was re-oriented from control to elimination, with the implementation of the “Malaria Elimination Programme”. This is in line with the global vision of malaria elimination and the aim of achieving malaria elimination status in Peninsular Malaysia by 2012 and Malaysian Borneo by 2020 [
3,
4].
The majority of the malaria cases reported in Malaysia in 2012 originated from two states in northern Borneo, Sabah (45%) and Sarawak (29%) (Vector Borne Disease Control 2012, unpublished data). This can largely be attributed to inaccessibility, climate and migration across the nearby border with Indonesia and results in a highly heterogeneous distribution of vector and parasite species as well as drug resistance patterns.
Drug resistance remains a major obstacle to malaria elimination efforts in the region [
5,
6]. Sulphadoxine-pyrimethamine (SP) has been in use for the treatment of uncomplicated falciparum malaria in Malaysia since the 1970s. It is used in remote areas for uncomplicated chloroquine-resistant
Plasmodium falciparum infections for outpatient as well as inpatient malaria cases. In spite of clinical resistance reported in some places, this combination is still widely employed [
7‐
10].
The genetic background of SP resistance is better documented than any other anti-malarial drug. Mutations in the dihydropteroate synthase (
dhps) and dihydrofolate reductase (
dhfr) genes, both coding for essential enzymes in the folate biosynthesis pathway, mediate drug resistance to SP [
11‐
13].
Pfdhfr codon ser108asn is likely to play a key role in pyrimethamine resistance with mutations at 51ile, 59arg and 164 leu modulating the level of resistance [
14]. The
pfdhfr triple mutation 51ile, 59arg and 108asn, has been shown to be associated with SP treatment failure, regardless of
pfdhps genotype. Study have shown that findings
in vitro may have indication or consistent with reports of failure treatment in the country [
15‐
17]. Sulphadoxine resistance in
P. falciparum is associated with mutations at five
pfdhps codons; 436ala/phe, 437gly, 540glu, 581gly and 613ser [
18‐
20]. A strong indicator for SP treatment failure is the quintuple mutations in three
pfdhfr codons (108asn + 51ile + 59arg) and two
pfdhps codons (437gly + 540glu) [
21,
22].
In spite of a massive reduction in malaria cases in Malaysia, the drug resistance situation remains poorly documented [
9]. Particularly in times of pre-elimination, a better understanding of the epidemiology of drug resistance has become vital for the region. The aim of the present study was therefore to investigate the distribution of
pfdhfr and
pfdhps gene polymorphism in
P. falciparum field isolates from Kalabakan, Sabah, in northern Borneo.
Methods
Study area and sample collection
The study was conducted between 2008 and 2009 in Kalabakan, 100 km from Tawau, in Sabah, bordering East Kalimantan. Samples were collected by active case detection in villages, logging and road construction camps within a radius of 50 to 80 km from Kalabakan. Kalabakan contributes most of the malaria cases in Sabah and was reported to have the highest number of malaria cases in 2008 and 2009 with 21.54% and 22.79%, respectively, of the total number of cases in Sabah. At the time this study was conducted, SP was the official first-line treatment for uncomplicated falciparum malaria and it remains widely used.
The study protocol was reviewed and approved by the institutional review board of the Institute for Medical Research, Kuala Lumpur and the Medical Review and Ethics Committee of the Ministry of Health, Malaysia. Individuals who consented to participate in the study were screened for malaria using rapid diagnostic tests (RDT) (Paramax- 3TM; Zephyr Biomedicals, India). In addition, blood films for malaria parasite (BFMP) were prepared to determine parasite density. All study participants diagnosed positive for malaria infection by RDT had 500 μl of whole blood collected by venepuncture. Blood was then spotted on 3MM Whatman filter paper. The filter paper was allowed to dry completely, transferred into individual plastic bags, labelled, and transported to the Institute for Medical Research in Kuala Lumpur where confirmation of species by PCR and genotype analysis was conducted. Speciation for
Plasmodium vivax,
P. falciparum, Plasmodium malariae and
Plasmodium knowlesi was undertaken using a modified version of published method [
23,
24].
DNA from filter paper was extracted using QIAampTM DNA mini kit (QIAmp; QIAGEN, Hilden, Germany), according to the manufacturer’s instructions (dried blood spots protocol) with the only modification being an adjustment of the elution buffer volume used to elute the DNA. DNA samples were then kept at −20°C until further processing.
Genotyping of pfdhfr and pfdhps by PCR-RFLP
PCR reaction and the restriction fragment polymorphism protocol (PCR-RFLP) were used for the detection of mutation on
pfdhfr as described elsewhere [
25] with some modification to DNA and primer concentrations. The first round PCR reaction mixture consisted of 50 ng of genomic DNA, 0.20 μM of each primer, 200 μM dNTPs, 1.5 mM MgCl
2, and 2.5 U of Taq polymerase in a final reaction volume of 50 μl. Two microlitres of the amplified product from the first PCR were subjected to two sets of secondary round PCR reaction mixtures containing 0.20 μM of each primer, 200 μM dNTPs, 2.0 mM MgCl2, and 2.5 U Taq polymerase. The PCR reaction used primers M4-F amplifying a 326 basepairs (bp) fragment containing cys59arg, ser108asn and ser108thr, and primers M3-F/amplifying a 522 bp fragment containing ala16val, asn51ile, ser108asn and ile164leu (Table
1).
Table 1
The primer pairs, the cycling temperature and restriction enzymes used in detection of gene polymorphism on
pfdhfr
F: 5′-GAA- | 94°C for 2 min; | 326 | cys |
XmnI
| 189. 163 | 26, 137, |
ATG-TAA-TTC- | 94°C for 1 min, | | 59 | | | 163, |
CCT-AGA-TAT- | 45°C for 1 min, | | arg | | | |
GGA-ATA-TT-3′ | 72°C for 1 min | | |
BstNI
| 326 | 145,181 |
| (5 cycles) | | ser | | | |
M4: 5′-TTA- | | | 108 | | | |
ATT-TCC-CAA- | continue with | | thr |
AluI
| 118, 180 | 299 |
GTA-AAA-CTA- | 40 cycles, | | | | | |
TTA-GAG- | 94°C for 1 min, | | ser | | | |
CTTC-3′ | 45°C for 1 min, | | 108 | | | |
| 72°C for 1 min Further extension 72°C for 10 min | | asp | | | |
M3: 5′-TTT- | Same cycle as | 522 | ala |
NlaIII
| 53, 93, 376 | 146, 245 |
ATG-ATG-GAA- | F-M4 PCR | | 16 | | | |
CAA-GTC-TGC-GAC-GTT- 3′ | | | val | | | |
| | | asn |
Tsp5091
| 55, 65, 120, | 55, 65, 120, |
F/: 5′-AAA-TTC- | | | 51 | | 153 | 218 |
TTG-ATA-AAC- | | | ile | | | |
AAC-GGA-ACC- | | | | | | |
TTT-TA-3′ | | | | | | |
| | | ser |
BsrI
| 522 | 190, 332 |
| | | 108 | | | |
| | | asn | | | |
| | | ile |
Dra I
| 107, 171, | 107, 143, |
| | | 164 | | 245 | 245 |
| | | leu | | | |
Similarly, nested PCR was conducted for the detection of mutation in the
pfdhps gene as described previously [
25] with modification on the cycling temperature for the second-round PCR, DNA and primers concentration. The first round PCR was performed using the primer sets R1-R2, followed by two sets of second-round PCR using K-K/and L-L/primers pair (Table
2). The PCR reaction used primers K-K/amplifying a 438 bp fragment containing ala437gly and lys540glu, and primers L-L/amplifying a 161 bp fragment containing ala581gly. The PCR and the nested PCR reactions used the same final concentrations as in the first round PCR carried out for
pfdhfr.
Table 2
The primer pairs, the cycling temperature and restriction enzymes used in detection of gene polymorphism on
pfdhps
K: 5′ TGC-TAG- | 94°C for 3 min | 438 | ala |
Ava II
| 438 | 404 |
TGT-TAT-AGA- | | | 437 | | | |
TAT-AGG-ATG- | 40 cycles of | | gly | | | |
AGC-ATC-3′ | 94°C for 1 min | | | | | |
| at 45°C for 45 | | lys |
FokI
| 438 | 85, 320 |
K/: 5′-CTA- | sec, 72°C for 1 | | 540 | | | |
TAA-CGA-GGT-ATT-GCA-TTT-AAT-GCA-AGA-A-3′ | min, further extension 72°C for 10 min | | glu | | | |
L: 5′-ATA- | 94°C for 2 min, | 161 | ala |
BstUI
| 105 | 138 |
GGA-TAC-TAT- | 45°C for 2 min, | | 581 | | | |
TTG-ATA-TTG- | 72°C for 1 min | | gly | | | |
GAC-CAG- | 30 sec (5 cycles) | | | | | |
GAT-TCG-3′ L/: 5′-TAT-TAC-AAC-ATT-TTG-ATC-ATT-CGC-GCA-ACC-GG-3′ | followed by 35 cycles 94°C for 1 min; 45°C for 1 min, 72°C for 1 min 30 sec. | | | | | |
| Further extension at 72°C for 10 min | | | | | |
The products of the secondary PCR containing the polymorphic region were subjected to enzyme digestion for the detection of mutations at the various sites. The enzyme digestions were conducted according to manufacturer’s instructions (New England Biolabs, Beverly, MA). The details of primer sequences, cycling temperatures, restriction enzyme digestion and fragment sizes for each codon are shown in Tables
1 and
2. DNA of laboratory strain
P. falciparum 3D7, K1, W2 and T9.96 were included in each reaction of PCR and RLFP and served as positive and negative controls. Water was used to replace the DNA template for the negative control.
Analysis of PCR-RFLP products using the Agilent 2100 Bioanalyzer
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 instructions (Agilent Technologies, Molecular Probes Inc, USA). The results were then viewed and analysed using the Agilent 2100 software.
Discussion
The study report the prevalence of mutations in the
pfdhfr and
pfdhps genes in
P. falciparum field isolates collected from individuals in Kalabakan, Sabah (Northern Borneo). This region remains the focus of
P. falciparum infection in a country that has reached pre-elimination and is striving for malaria elimination in the coming years. SP has been used extensively in the region for more than 30 years, as first-line drug until 2010 [
26]. This is surprising as SP treatment failures have been reported from Malaysia as early as 1982 [
7]. By the late 1990s, SP resistance had reached 47.4% in Peninsular Malaysia [
8] and 29.4% in Tawau, Sabah [
9] and first evidence of the molecular background of SP resistance in Malaysia came from a study conducted on mainland Malaysia and Borneo reported in 2001 [
27].
The findings of the study indicate that all samples collected harbour at least one of the markers known to be involved in SP resistance on both the
pfdhfr as well as the
pfdhps gene. Without exception all samples (100%) had the
pfdhps mutation 437gly, which is a common observation in areas where SP is widely used. Earlier studies suggest that its presence alone or in combination with 540glu is predictive of early SP treatment failure [
28,
29]. The 540glu is typically found together with 437gly, particularly in Africa [
30,
31]. However, the findings of the study showed that all the samples harboured the
pfdhps 437gly mutation together with 581 gly (74.19%) rather than 540glu. In fact, the studies indicate the complete absence of the 540glu mutation in the patient samples. Both combinations have been associated with sulphadoxine resistance [
32,
33]. The findings for 581gly also confirm earlier reports from the region [
27]. In spite of extensive use of the drug there were surprisingly few changes in the epidemiology of
pfdhps and
pfdhfr mutations within these seven years.
The other mutation that seems to affect virtually all
P. falciparum parasite samples in the region is 59arg (Table
3). This mutation is believed to modulate pyrimethamine resistance [
21,
34]. A study conducted in Burkino Faso, showed that
pfdhfr 59arg, with 51ile and 108asn is an important marker for SP treatment failure [
35]. Another study from Mozambique suggested that the two mutations at
pfdhfr 59arg and
pfdhps 437gly were enough to predict SP treatment failure [
36].
The study suggests that there are two predominant
pfdhfr mutation genotypes; 59arg + 164leu (22.58%) and 59arg + 108asn + 164leu (51.61%). There was not a single quadruple mutant (51ile + 59arg + 108asn + 164leu) in the samples, which is considered to be an indication of the highest levels of resistance to pyrimethamine [
37]. Similar to the 540glu mutation in
pfdhps, this study did not identify any sample with the 51ile mutation. However, Dokomajilar
et al. suggested that 59arg, even in the absence of 51ile, may be more important as a marker for pyrimethamine resistance [
35].
However, the results observed the presence of a sextuple mutation consisting of four pfdhfr (16val, 59arg, 108asn and 164leu) and two pfdhps (437gly, 581gly) mutations in a single individual from loggers’ camps near the border of Kalabakan to East Kalimantan. There were two sets of three pfdhfr and two pfdhps mutation (a quintuple) observed in the samples; a mutation genotype consisting of 59arg + 108asn + 164le + 437gly + 581gly, making up 41.94% of the samples and 16 val + 59arg + 164leu + 437gly + 581gly, which was found in a single sample only. The association of molecular findings with clinical treatment response was not possible due to the very low malaria prevalence and the active case detection used in this study covering a huge and largely inaccessible catchment area.
Acknowledgements
The authors would like to thank the Director General Of Health Malaysia for the permission to publish this paper. We thank the Director for the Institute for Medical Research (IMR), Kuala Lumpur for her critical review and support in publishing this paper. This study was supported by the National Institute of Health, Ministry of Health Malaysia. We also thank all staff of Tawau Health Department, Sabah for their contributions and all patients for their participation in this important study.
Competing interest
The authors declare that they have no competing interest.
Authors’ contributions
NRA prepared study proposal and protocol, study design, lead the study in Kalabakan, data analysis and interpretation and preparation of manuscript. NAA conducted the molecular genetic studies, analysis of data, data statistics and partly drafted the manuscript. HN participated in coordinating the study, reviewing the data, data analysis and English editing. LHS, ZI, HMS oversight the project. JJ participated in the study design in the field. All authors critically reviewed the manuscript and approved the final version before submission to the Journal. All authors read and approved the final manuscript.