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Plasmodium vivax drug resistance markers: Genetic polymorphisms and mutation patterns in isolates from Malaysia
Acta Tropica 206 (2020) 105454
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Plasmodium vivax drug resistance markers: Genetic polymorphisms and mutation patterns in isolates from Malaysia
T
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Fei-Wen Cheonga, , Shairah Dzula,b, Mun-Yik Fonga, Yee-Ling Laua, Sasheela Ponnampalavanarc a
Department of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia Division of Management Services, Ministry of International Trade and Industry, 50480 Kuala Lumpur, Malaysia c Department of Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia b
A R T I C LE I N FO
A B S T R A C T
Keywords: Plasmodium vivax Drug resistance markers pvcrt-o pvmdr1 pvdhfr pvdhps
Transmission of Plasmodium vivax still persist in Malaysia despite the government's aim to eliminate malaria in 2020. High treatment failure rate of chloroquine monotherapy was reported recently. Hence, parasite drug susceptibility should be kept under close monitoring. Mutation analysis of the drug resistance markers is useful for reconnaissance of anti-malarial drug resistance. Hitherto, information on P. vivax drug resistance marker in Malaysia are limited. This study aims to evaluate the mutations in four P. vivax drug resistance markers pvcrt-o (putative), pvmdr1 (putative), pvdhfr and pvdhps in 44 isolates from Malaysia. Finding indicates that 27.3%, 100%, 47.7%, and 27.3% of the isolates were carrying mutant allele in pvcrt-o, pvmdr1, pvdhfr and pvdhps genes, respectively. Most of the mutant isolates had multiple point mutations rather than single point mutation in pvmdr1 (41/44) and pvdhfr (19/21). One novel point mutation V111I was detected in pvdhfr. Allelic combination analysis shows significant strong association between mutations in pvcrt-o and pvmdr1 (X2 = 9.521, P < 0.05). In the present study, 65.9% of the patients are non-Malaysians, with few of them arrived in Malaysia 1–2 weeks before the onset of clinical manifestations, or had previous history of malaria infection. Besides, few Malaysian patients had travel history to vivax-endemic countries, suggesting that these patients might have acquired the infections during their travel. All these possible imported cases could have placed Malaysia in a risk to have local transmission or outbreak of malaria. Six isolates were found to have mutations in all four drug resistance markers, suggesting that the multiple-drugs resistant P. vivax strains are circulating in Malaysia.
1. Introduction Malaria continues to place heavy public health and economy burden, causing 228 million cases and 405,000 deaths in 2018 with approximately US$ 2.7 billion spent in global malaria control and elimination (World Health Organization, 2019). Plasmodium vivax, which has the widest geographical distribution of the human malaria parasites, has the second highest prevalence rate in Malaysia after P. knowlesi. Although malaria control efforts have effectively reduced the incidence of vivax infection, transmission persists in rural areas due to asymptomatic or submicroscopic reservoir (Cheng et al., 2015). In November 2016, a P. vivax malaria outbreak has occurred in Pos Kemar Orang Asli settlement, Perak, with a total of 137 indigenous people, including 79 children (aged 14 and below), who were tested positive for P. vivax (The Star Online, 2017). However, some of the positively diagnosed villagers are reluctant to get treatment, which could be a
possible factor that contributed to the rapid spread of diseases in that area (Astro Awani Malaysia News, 2016). Besides, importation of a considerable fraction of vivax malaria by undocumented illegal workers from malaria endemic countries poses another major challenge towards local intervention (WHO, 2015). Anti-malarial drug chloroquine (CQ) resistance in P. vivax was first reported in Papua New Guinea (PNG) in 1989 (Rieckmann et al., 1989), and continued to emerge in Asia, Oceania and South America, with monotherapy failure rate exceeding 80% in PNG (Marques et al., 2014; Sumawinata et al., 2003; Waheed et al., 2015). High levels of CQ resistance had forced some countries to switch their treatment drug to sulfadoxine-pyrimethamine (SP). However, evidence of P. vivax resistance to pyrimethamine alone was documented in 1950s (Young and Burgess 1959), and its resistance against SP developed rapidly within several years after deployment of SP as monotherapy drug in many areas (Peters 1998; Pukrittayakamee et al., 2000). Atovaquone
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Corresponding author. E-mail addresses: [email protected] (F.-W. Cheong), [email protected] (S. Dzul), [email protected] (M.-Y. Fong), [email protected] (Y.-L. Lau), [email protected] (S. Ponnampalavanar). https://doi.org/10.1016/j.actatropica.2020.105454 Received 13 December 2019; Received in revised form 19 March 2020; Accepted 19 March 2020 Available online 20 March 2020 0001-706X/ © 2020 Elsevier B.V. All rights reserved.
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2.2. Study area and sample collection
resistance developed easily when it was use as a monotherapy drug. Although the combination of atovaquone-proguanil was effective in elimination of P. vivax erythrocytic forms, the parasitemia was found to be recurred in most of the patients due to the relapse of hypnozoites, indicating that this drug combination is ineffective against P. vivax hypnozoites (Looareesuwan et al., 1996). In P. falciparum, CQ resistance is linked to CQ resistance transporter (pfcrt) gene, which parasites with mutations in pfcrt could reduce the concentration of intravacuolar drug by increasing efflux of CQ from the digestive vacuole (Sidhu et al., 2002). Molecular mechanisms underlying CQ resistance in P. vivax remain unclear. Nonetheless, P. vivax multidrug resistance 1 gene (pvmdr1) and CQ resistance transporter gene (pvcrt-o), which are orthologous to pfmdr1 and pfcrt genes, respectively, have been identified as putative CQ drug resistance markers of P. vivax (Lu et al., 2011; Rungsihirunrat et al., 2015). Nonsynonymous point mutations or change of the gene copy numbers of pvmdr1 have been shown to alter the parasite's in vitro susceptibility to several anti-malarial drugs including CQ, mefloquine, amodiaquine, and artemisinin derivatives (Rungsihirunrat et al., 2015; Suwanarusk et al., 2008; Suwanarusk et al., 2007). Mutations in parasite's folate biosynthetic pathway genes, dihydrofolate reductase (pvdhfr) and dihydropteroate synthase (pvdhps) confer resistance to pyrimethamine and sulfadoxine, respectively, resulting decreased drug affinity and treatment failure in clinical settings (Imwong et al., 2001; Imwong et al., 2005; Korsinczky et al., 2004). CQ resistant-P. vivax has been documented in Sabah, Malaysia in 1996 (Ahlm et al., 1996), and a high treatment failure rate of CQ monotherapy by day 28 in vivax-infected patients has also been demonstrated in a recent randomized controlled trial (Grigg et al., 2016). Malaysian Ministry of Health malaria treatment guideline has been updated in 2016 to recommend artemisinin combination treatment (ACT) over CQ as first-line asexual treatment for all uncomplicated malaria including P. vivax (Barber et al., 2017). However, the implementation throughout the country remains unclear and CQ might still been prescribed in rural areas. Therefore, parasite drug susceptibility should be kept under close surveillance in Malaysia as our country is located in close geographical proximity to countries with anti-malarial drug resistant parasite strains such as Cambodia, Thailand, Vietnam, Indonesia, and is highly vulnerable to importation of these parasite strains via migrant workers. In order to assess the true drug resistance, ex vivo drug susceptibility assay should be performed. However, due to the difficulties in collecting fresh isolates and the tediousness in experiment procedures, the feasibility of this assay to be performed routinely is low. Hence mutation analysis of the drug resistance molecular markers could thus serve as an alternative method for the reconnaissance of anti-malarial drug resistance. Genotyping of these markers can serve as a surveillance tool to detect and monitor the prevalence and influx of resistant Plasmodium strains in a geographic region, which could provide useful information for implementation and intervention of government malaria control activities. Hitherto, studies on the prevalence, emergence or influx of P. vivax drug resistance marker genes in Malaysia are scarce. The present study studied the mutations in anti-malarial drug resistance markers crt, mdr1, dhfr and dhps in P. vivax, and the prevalence of wild type or mutant alleles in the P. vivax isolates in Malaysia.
A total of 44 P. vivax-infected patient blood samples was collected from 2011 to 2017, from state and district government hospitals in Malaysia: University Malaya Medical Center (UMMC), Kuala Lumpur Hospital (HKL), Kuala Lumpur; Pulau Pinang District Health Department, Penang; Permaisuri Ipoh Hospital, Perak; Kuala Kubu Bharu Hospital, Selangor; Tuanku Jaafar Hospital, Negeri Sembilan; Sultanah Nur Zahirah Hospital, Terengganu; General Health Laboratory Kota Bharu Perol, Kelantan; Kuala Lipis Hospital, Pahang; Jerantut Hospital, Pahang; and for East Malaysia, Sabah District Health Department, Sabah; and Sarawak District Health Department, Sarawak. Genomic DNA of P. vivax was extracted from these patient blood samples using DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Plasmodium species in each patient was confirmed by microscopic examination of Giemsastained blood smears and nested polymerase chain reaction (PCR) based on 18S rRNA gene (Singh et al., 1999; Singh et al., 2004). 2.3. Amplification of drug resistance markers pvcrt-o, pvmdr1, pvdhfr and pvdhps The extracted DNA which has been confirmed to be P. vivax was subjected to PCR targeting the pvcrt-o (K10 insertion), pvmdr1 (T958M, Y976F, and F1076L mutation points), pvdhfr (F57L/I, S58R, T61M, S117T/N, and I173F/L mutation points) and pvdhps (S382A/C, A383G, K512M/T, and A553G/C mutation points). The amino acid mutation positions and PCR regions of each genes were showed in Fig. 1. Amplifications were carried out according to previous described protocols by Lu et al. (2012) with modifications. Primer sets used in the study were listed in Table 1. DNA, 1 uL of was used for each reaction in a final volume of 20 uL reaction mixture, with 1× GoTaq® Flexi Buffer, 4 mM MgCl2, 0.2 μM of each primer, 0.2 μM dNTP and 1 U GoTaq® Flexi DNA Polymerase (Promega, Madison, USA). Single step PCR was carried out with the following thermal cycling conditions: initial denaturation at 94 C for 5 min; 35 cycles of denaturation at 94 C for 30 s, annealing of varying temperature for 1 min, extension at 72 C for 1 min; and final extension at 72 C for 8 min. Annealing temperature for pvcrt-o, pvmdr1, pvdhfr, and pvdhps were 61 C, 62 C, 58 C, and 56 C, respectively. 2.4. Determination of polymorphisms in gene markers by sequence analysis PCR amplicons were sequenced in both directions using the respective molecular marker forward and reverse primers by commercial company (First BASE Laboratories Sdn Bhd, Malaysia). The nucleotide sequences and deduced amino acid sequences were aligned and analyzed using BioEdit Sequence Alignment Editor Software. P. vivax strain Salvador 1 reference wild type sequences in National Center for Biotechnology Information (GenBank accession no. AF314649 for pvcrto; AY571984 for pvmdr1; X98123 for pvdhfr; AY186730 for pvdhps) were used to compare with the amino acid sequences of P. vivax isolates to identify the genetic polymorphisms and mutation patterns, where insertions and deletions were verified manually. Prevalence of the wild type or mutant alleles in the isolates was determined. Allelic combination analysis of drug resistance markers was performed by using statistical software SPSS V23. The association between mutation (with or without mutation) in pvcrt-o and pvmdr1 was analysed. On the other hand, association between mutation (with or without mutation) in pvdhfr and pvdhps was analysed.
2. Materials and methods 2.1. Ethical approval
3. Results
Use of human samples in this study was approved by University of Malaya Medical center Medical Ethics Committee (MEC Ref. No: 817.18) and Medical Research & Ethics Committee, Ministry of Health Malaysia [NMRR-15-67,223,975 (IIR)]. Informed consent from the donor or the next of kin was obtained for use of these samples in screening.
3.1. Descriptive characteristics of patients Twenty nine of the 44 (65.9%) vivax-infected patients are found to be non-Malaysian. Majority of these non-Malaysians were from 2
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Fig. 1. Schematic diagrams of amino acid point mutation positions and PCR regions of (A) pvcrt-o, (B) pvmdr1, (C) pvdhfr, and (D) pvdhps genes (Adapted from Lu et al. (2012)). Several previously reported point mutations are indicated in the diagrams, and targeted amino acid mutations in the present study are marked in red. In (A), (C) and (D), boxes indicate exons, and lines indicate introns. In (C), R indicates repeat region GGDN/TSGGDN/THGGDN. In (D), R indicates repeat region GEAKLTN-GEGKLTN-GEAKLTN-GEGKLTN-GEAKLTN-GEGKLTN-GDAKLTN-GDSKLTN-GEAKLTN.
resistance-conferring mutations region were selected for amplification, with expected amplicon sizes of 1186 bp, 604 bp, 716 bp and 1301 bp, respectively. A total of 44 vivax-single infection samples were used in this study. For each isolates, all of the four drug resistance markers were successfully amplified and sequenced. The polymorphisms and mutations for all 44 isolates in each of the four genes were tabulated in Supplementary Table 1.
Table 1 Primer sets for amplification of pvcrt-o, pvmdr1, pvdhfr and pvdhps genes. Genes
Primers
Amplicon size (bp)
pvcrt-o
F: 5′-AAGAGCCGTCTAGCCATCC-3′ R: 5′-AGTTTCCCTCTACACCCG-3′ F: 5′-GGATAGTCATGCCCCAGGATTG-3′ R: 5′-CATCAACTTCCCGGCGTAGC-3′ F: 5′-ATGGAGGACCTTTCAGATGTATT-3′ R: 5′-CCACCTTGCTGTAAACCAAAAAGTCCAGAG-3′ F: 5′-GGTTTATTTGTCGATCCTGTG-3′ R: 5′-GAGATTACCCTAAGGTTGATGTATC-3′
1186
pvmdr1 pvdhfr pvdhps
604 716
3.3. Analysis of mutation pattern in pvcrt-o gene
1301
Compared to Salvador 1 reference wild type sequence, nucleotide sequencing result of isolates showed that an insertion of lysine at amino acid position 10 (K10, nucleotide sequence AAG) was detected in 12 of 44 isolates (27.3%) (Table 2), where 32 isolates were carrying wild type allele without insertion (72.7%).
Pakistan (11/29, 37.9%). The other nationality including Indian (5/29, 17.2%), Indonesian (4/29, 13.8%), Nepali (4/29, 13.8%), Bangladeshi (2/29, 6.9%), Vietnamese (1/29, 3.4%), Myanmarese (1/29, 3.4%) and South African (1/29, 3.4%). Details of the patients including sex, age, parasitemia, and travel history were recorded in Supplementary Table 1.
3.4. Analysis of point mutation patterns in pvmdr1 gene Of the 44 isolates, no wild type alleles were detected. All isolates carried the point mutation T958M (100%), whereas point mutations Y976F and F1076L were found in five (11.4%) and 41 (93.2%) isolates, respectively (Table 2). All five isolates carrying the Y976F point mutation were combined with F1076L mutation, yet majority of the isolates carrying F1076L mutation is having wild type alleles at codon 976.
3.2. Amplification of drug resistance markers pvcrt-o, pvmdr1, pvdhfr and pvdhps Fragments of pvcrt-o, pvmdr1, pvdhfr and pvdhps covering respective 3
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mutations are showed in Table 3.
Table 2 Prevalence of each mutations in pvcrt-o, pvmdr1, pvdhfr, and pvdhps genes in isolates from Malaysia (n = 44). Mutations
3.6. Analysis of point mutation patterns in pvdhps gene
No. of isolates carrying mutation (%)
pvcrt-o K10 insertion pvmdr1 T958M Y976F F1076L pvdhfr F57L S58R T61M V111I S117T S117N I173F pvdhps S382A A383G A553G
The prevalence rate of point mutations S382A, A383G, A553G is 2.3% (1/44), 27.3% (12/44), 11.4% (5/44), respectively (Table 2). Point mutations at position 512 were not detected in all the isolates. The most common allele in the samples is wild type allele (32/44, 72.7%), while allele with single point mutation A383G accounted for 15.9% of total isolates (7/44). Four of the isolates (9.1%) were carrying two point mutations A383G + A553G, whereas only one isolate (2.3%) was carrying three point mutations S382A + A383G + A553G (Table 3).
12 (27.3) 44 (100.0) 5 (11.4) 41 (93.2) 11 (25.0) 18 (40.9) 9 (20.5) 1 (2.3) 8 (18.2) 10 (22.7) 2 (4.5)
3.7. Analysis of pvcrt-o and pvmdr1 allelic combinations Allelic combinations of pvcrt-o and pvmdr1 were analysed as these two genes are potentially undergoing same drug (CQ) pressure. Total of 12 isolates were carrying K10 insertion in pvcrt-o, combined with different point mutations in pvmdr1. Pearson's chi-square test showed that there is significant association between the mutations in pvcrt-o and pvmdr1 (X2 = 9.521, P < 0.05). The Cramer's V value is 0.465, indicating a strong association between the mutations in the two genes.
1 (2.3) 12 (27.3) 5 (11.4)
Table 3 Frequency of alleles with single or multiple mutations in pvcrt-o, pvmdr1, pvdhfr, and pvdhps genes in isolates from Malaysia (n = 44). Mutations
Pvcrt-o Wild type K10 insertion pvmdr1 Wild type T958M T958M + F1076L T958M + Y976F + F1076L pvdhfr Wild type S58R S117N F57L + S58R F57L + I173F S58R + S117N S58R + T61M + S117N F57L + S58R + T61M + S117T F57L + S58R + T61M + S117N F57L + V111I + S117T + I173F pvdhps Wild type A383G A383G + A553G S382A + A383G + A553G
Single/multiple mutations
No. of isolates carrying mutation(s) (%)
Nil Single
32 (72.7) 12 (27.3)
Nil Single Double Triple
0 (0.0) 3 (6.8) 36 (81.8) 5 (11.4)
Nil Single Single Double Double Double Triple Quadruple Quadruple Quadruple
23 (52.3) 1 (2.3) 1 (2.3) 1 (2.3) 1 (2.3) 7 (15.9) 1 (2.3) 7 (15.9) 1 (2.3) 1 (2.3)
Nil Single Double Triple
32 (72.7) 7 (15.9) 4 (9.1) 1 (2.3)
3.8. Analysis of pvdhfr and pvdhps allelic combinations Sulfadoxine and pyrimethamine are both antifolates that inhibit biosynthesis of folate in parasites. Hence, allelic combinations of pvdhfr and pvdhps were analysed as these two genes are responsible for biosynthesis of folate and are potentially undergoing similar drug group pressure. A total of 25 isolates are carrying point mutation(s) in either pvdhfr or pvdhps genes. Among them, 8 isolates are carrying point mutations in both pvdhfr and pvdhps genes. Pearson's chi-square test revealed no significant association between the point mutations in pvdhfr and pvdhps (X2 = 26.468, P > 0.05). The Cramer's V value is 0.448, indicating a strong association between the point mutations in the two genes. 4. Discussion The insertion of lysine (K) in the first exon (amino acid 10) of pvcrt-o has been associated with significant increase in CQ IC50 (Suwanarusk et al., 2007), whereas the isolates with Y976F mutation in pvmdr1 have also been found to have significantly increased CQ IC50 compared to isolates with wild type allele (Suwanarusk et al., 2008; Suwanarusk et al., 2007). In the present study, both the mutations were detected in the isolates in Malaysia (12 isolates for pvcrt-o K10 insertion and five isolates for pvmdr1 Y976F), while two of the isolates (4.5%) were carrying these two mutations in both the genes. Our result could be supported by study of Grigg et al., 2016 that recorded a 61.1% of in vivo CQ resistance, indicating the high prevalence of CQ-resistant P. vivax in Malaysia. We detected T958M mutation in pvdmr1 of all samples. Similar findings were reported from countries with both low and high levels of CQ resistance, suggesting that this point mutation could be an allelic variant of the wild type and might not be associated with the CQ resistance phenotypically (Joy et al., 2018; Schousboe et al., 2015). In majority of the isolates, the residue L was present at position 1076 in pvmdr1 as F1076L in the present study. However, this point mutation might not confer drug resistance in the parasite. Imwong et al. (2008) have identified this residue L at the correspond codon in the orthologous gene in P. falciparum and P. cynomolgi (pfmdr1 and pcmdr1, respectively), thus suggesting that this residue L could be a neutral allele that does not alter the fitness of the parasite. The resistance of P. vivax to antifolates has been linked to mutations in pvdhfr and pvdhps. With supportive results from in vitro drug assays and clinical assessments (Imwong et al., 2005; Rungsihirunrat et al.,
Of that, most of the isolates (36/44, 81.8%) were carrying two point mutations T958M + F1076L, followed by three point mutations T958M + Y976F + F1076L (5/44, 11.4%). Only three isolates were found to carry single point mutation T958M (6.8%) (Table 3). 3.5. Analysis of point mutation patterns in pvdhfr gene Seven point mutations were observed compared to the reference strain. Point mutation S58R was observed in most of the isolates (18/ 44, 40.9%), followed by F57L (11/44, 25.0%), S117N (10/44, 22.7%), T61M (9/44, 20.5%), S117T (8/44, 18.2%), and I173F (2/44, 4.5%). Novel mutation V111I was detected in one of the isolates (2.3%) (Table 2). More than half of the isolates (23/44, 52.3%) were carrying wild type allele. Two point mutations S58R + S117N and four point mutations F57L + S58R + T61M + S117T were common among the isolates, each accounting for 15.9% (7/44) of the isolates. The frequency distributions of other alleles with single or multiple point 4
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indigenous children have been reported in Pos Lenjang, Pahang in July 2019 (The Star Online, 2019), despite the government aims to achieve malaria elimination in 2020. Moreover, six of these isolates in the present study were found to have mutations in all four drug resistance markers pvcrt-o, pvmdr1, pvdhfr and pvdhps, suggesting that the multiple-drugs resistant P. vivax strains have been distributed in Malaysia and leading to a more worrying situation. With the aid of mutations analysis of drug resistance markers, surveillance and monitoring on efficacy of anti-malarial drugs could be performed in a more thorough way. The findings of the study serve as an update of the prevalence of wild type or mutant alleles in the P. vivax isolates in Malaysia, and could also provide fundamental data for future study to evaluate whether genotyping of these few drug resistance markers could reflect the occurrence of phenotypic drug resistance by performing in vitro or ex vivo drug susceptibility tests.
2007), the pvdhfr and pvdhps genotyping data could be useful to indicate the SP resistance status in P. vivax. Rungsihirunrat et al. (2007) demonstrated that the increase number of mutations (from double to quadruple point mutation) in pvdhfr is associated with the increase of antifolates IC50, suggesting the quadruple mutant alleles confer higher pyrimethamine resistance than double mutant alleles. Similar finding was shown by Imwong et al. (2001) that patients harbouring triple mutations in pvdhfr had a significant lower parasite reduction ratio compared to those patients with double mutations. Of that, point mutations F57L, S58R and S117N are found to be associated with SP resistance. In our findings, the three highest frequency of point mutations among the 21 mutant isolates are S58R, F57L and S117N, with the prevalence rate of 85.7% (18/21), 52.4% (11/21), and 47.6% (10/21), respectively. High number of isolates were having double mutations S58R + S117N. This finding is in concordance with a previous study which discovered that the initial mutations would occur at position 58 and 117 when drug pressure is applied (Imwong et al., 2003). On the other hand, seven isolates were carrying quadruple mutations F57L + S58R + T61M + S117T. Imwong et al. (2003) has demonstrated that in highly mutated isolates that carrying quadruple point mutation, the residue 117 would carry the S→T mutation rather than the S→N mutation, followed by mutations at position 57 and 61. Only two isolates were found to harbor single point mutation in the present study, indicating high percentage of highly mutated P. vivax strain is circulating in Malaysia. In pvdhps, majority of the isolates were carrying wild type allele. We detected three point mutations at residues 382, 383 and 553 in the isolates, in which these mutations have been reported previously in Thailand isolates (Imwong et al., 2005; Korsinczky et al., 2004). These mutations were hypothesized to be associated with reduced susceptibility to both sulfone and sulphonamide groups. Imwong et al. (2005) reported a slower parasite clearance in SP-treated patients carrying six or more combined point mutations in both pvdhfr and pvdhps genes, compared to patient with fewer mutations in these genes. Central Asia is accounted for more than 80% of global population at risk for Plasmodium vivax, particularly in India and Pakistan (Khan et al., 2016); while the high endemicity countries in Southeast Asia including Indonesia and Papua New Guinea (Battle et al., 2012). Population movement from malaria-endemic areas to non-endemic areas is one of the main causes for imported malaria cases, and the transmission of parasites can be continued with the presence of suitable mosquito vectors. Large proportion of the vivax-infected patients in the present study are found to be non-Malaysian. Few of these patients arrived in Malaysia one to two weeks before the onset of clinical manifestations. Besides, three of them (one Indian and two Indonesians) had previous history of malaria infection in their countries six months to three years ago. Although treatment was prescribed to the patients during the previous infection, the patients’ compliance for the treatment drugs is uncertain, especially for primaquine, the anti-hypnozoite drug that should be administered daily up to 14 days. Hence, the current infection in these few patients in the present study could be due to relapse of the P. vivax hypnozoites. On the other hand, three of the Malaysian patients had travel history to Pakistan and Papua New Guinea before the onset of symptoms. It is possible that these patients might have acquired the infections during their travel to those vivax-endemic countries. Besides, one of the Malaysian patients came back from Papua New Guinea three months ago, but this patient was diagnosed to have vivax infection after return and had relapse history. All these possible imported cases could have placed Malaysia in a risk to have local transmission or outbreak of malaria.
Author statement The authors declare that they have no competing interests. The material is original and has not been published. The material has not and will not be submitted for publication elsewhere so long as it is under consideration by Acta Tropica. If accepted, it will not be published elsewhere in the same form in English or in any other language. Fei-Wen Cheong, Shairah Dzul, Mun-Yik Fong, Yee-Ling Lau, and Sasheela Ponnampalavanar have participated in the study and concur with the submission and subsequent revisions submitted by the corresponding author. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This study was supported by BKP grant, University of Malaya, Malaysia (BK005-2017). Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.actatropica.2020.105454. References Ahlm, C., Wistrom, J., Carlsson, H., 1996. Chloroquine-Resistant Plasmodium vivax Malaria in Borneo. J. Travel Med. 3, 124. Astro Awani Malaysia News. 2016. Malaria cases likely to rise – Dr Mah. Retrieved from:http://english.astroawani.com/malaysia-news/malaria-cases-likely-rise-drmah-123972. Accessed Nov 21, 2019. Barber, B.E., Grigg, M.J., William, T., Yeo, T.W., Anstey, N.M., 2017. The treatment of Plasmodium knowlesi Malaria. Trends Parasitol. 33, 242–253. Battle, K.E., Gething, P.W., Elyazar, I.R., Moyes, C.L., Sinka, M.E., Howes, R.E., Guerra, C.A., Price, R.N., Baird, K.J., Hay, S.I., 2012. The global public health significance of Plasmodium vivax. Adv. Parasitol. 80, 1–111. Cheng, Q., Cunningham, J., Gatton, M.L., 2015. Systematic review of sub-microscopic P. vivax infections: prevalence and determining factors. PLoS Negl. Trop. Dis. 9, e3413. Grigg, M.J., William, T., Menon, J., Barber, B.E., Wilkes, C.S., Rajahram, G.S., Edstein, M.D., Auburn, S., Price, R.N., Yeo, T.W., Anstey, N.M., 2016. Efficacy of artesunatemefloquine for chloroquine-resistant Plasmodium vivax Malaria in Malaysia: an openlabel, randomized, controlled trial. Clin. Infect. Dis. 62, 1403–1411. Imwong, M., Pukrittakayamee, S., Looareesuwan, S., Pasvol, G., Poirreiz, J., White, N.J., Snounou, G., 2001. Association of genetic mutations in Plasmodium vivax dhfr with resistance to sulfadoxine-pyrimethamine: geographical and clinical correlates. Antimicrob. Agents Chemother. 45, 3122–3127. Imwong, M., Pukrittayakamee, S., Cheng, Q., Moore, C., Looareesuwan, S., Snounou, G., White, N.J., Day, N.P., 2005. Limited polymorphism in the dihydropteroate synthetase gene (dhps) of Plasmodium vivax isolates from Thailand. Antimicrob. Agents Chemother. 49, 4393–4395. Imwong, M., Pukrittayakamee, S., Pongtavornpinyo, W., Nakeesathit, S., Nair, S.,
5. Conclusion The incidence of vivax infection has declined markedly in Malaysia over the past decade. However, fifteen diagnosed vivax infections in 5
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Thailand. Am. J. Trop. Med. Hyg. 76, 1057–1065. Schousboe, M.L., Ranjitkar, S., Rajakaruna, R.S., Amerasinghe, P.H., Morales, F., Pearce, R., Ord, R., Leslie, T., Rowland, M., Gadalla, N.B., Konradsen, F., Bygbjerg, I.C., Roper, C., Alifrangis, M., 2015. Multiple origins of mutations in the mdr1 Gene–A putative marker of chloroquine resistance in P. vivax. PLoS Negl. Trop. Dis. 9, e0004196. Sidhu, A.B., Verdier-Pinard, D., Fidock, D.A., 2002. Chloroquine resistance in Plasmodium falciparum malaria parasites conferred by pfcrt mutations. Science 298, 210–213. Singh, B., Bobogare, A., Cox-Singh, J., Snounou, G., Abdullah, M.S., Rahman, H.A., 1999. A genus- and species-specific nested polymerase chain reaction malaria detection assay for epidemiologic studies. Am. J. Trop. Med. Hyg. 60, 687–692. Singh, B., Lee, K.S., Matusop, A., Radhakrishnan, A., Shamsul, S.S., Cox-Singh, J., Thomas, A., Conway, D.J., 2004. A large focus of naturally acquired Plasmodium knowlesi infections in human beings. Lancet 363, 1017–1024. Sumawinata, I.W., Bernadeta, Leksana, B., Sutamihardja, A., Purnomo, Subianto, B., Sekartuti, Fryauff, D.J., Baird, J.K., 2003. Very high risk of therapeutic failure with chloroquine for uncomplicated Plasmodium falciparum and P. vivax malaria in Indonesian Papua. Am. J. Trop. Med. Hyg. 68, 416–420. Suwanarusk, R., Chavchich, M., Russell, B., Jaidee, A., Chalfein, F., Barends, M., Prasetyorini, B., Kenangalem, E., Piera, K.A., Lek-Uthai, U., Anstey, N.M., Tjitra, E., Nosten, F., Cheng, Q., Price, R.N., 2008. Amplification of pvmdr1 associated with multidrug-resistant Plasmodium vivax. J. Infect. Dis. 198, 1558–1564. Suwanarusk, R., Russell, B., Chavchich, M., Chalfein, F., Kenangalem, E., Kosaisavee, V., Prasetyorini, B., Piera, K.A., Barends, M., Brockman, A., Lek-Uthai, U., Anstey, N.M., Tjitra, E., Nosten, F., Cheng, Q., Price, R.N., 2007. Chloroquine resistant Plasmodium vivax: in vitro characterisation and association with molecular polymorphisms. PLoS ONE 2, e1089. The Star Online. 2017. Malaria outbreak at pos kemar is over. Retrieved from:https:// www.thestar.com.my/news/nation/2017/01/03/dr-mah-malaria-outbreak-at-poskemar-is-over/. Accessed Nov 11, 2019. The Star Online. 2019. Mild malaria outbreak in kuala lipis. Retrieved from:https://www. thestar.com.my/news/nation/2019/07/16/mild-malaria-outbreak-in-kuala-lipis. Accessed November 14, 2019. Waheed, A.A., Ghanchi, N.K., Rehman, K.A., Raza, A., Mahmood, S.F., Beg, M.A., 2015. Vivax malaria and chloroquine resistance: a neglected disease as an emerging threat. Malar. J. 14, 146. World Health Organization. 2015. Eliminating malaria: case study 8. Progress towards elimination in Malaysia. Available at:http://www.who.int/malaria/publications/ atoz/9789241508346/en/. Accessed November 25, 2019. World Health Organization, 2019. World Malaria Report 2019. WHO. Available at: http://apps.who.int/iris/bitstream/handle/10665/275867/9789241565653-eng. pdf?ua=1. Accessed Nov 25, 2019 Young, M.D., Burgess, R.W., 1959. Pyrimethamine resistance in Plasmodium vivax malaria. Bull. World Health Organ. 20, 27–36.
Newton, P., Nosten, F., Anderson, T.J., Dondorp, A., Day, N.P., White, N.J., 2008. Gene amplification of the multidrug resistance 1 gene of Plasmodium vivax isolates from Thailand, Laos, and Myanmar. Antimicrob. Agents Chemother. 52, 2657–2659. Imwong, M., Pukrittayakamee, S., Renia, L., Letourneur, F., Charlieu, J.P., Leartsakulpanich, U., Looareesuwan, S., White, N.J., Snounou, G., 2003. Novel point mutations in the dihydrofolate reductase gene of Plasmodium vivax: evidence for sequential selection by drug pressure. Antimicrob. Agents Chemother 47, 1514–1521. Joy, S., Mukhi, B., Ghosh, S.K., Achur, R.N., Gowda, D.C., Surolia, N., 2018. Drug resistance genes: pvcrt-o and pvmdr-1 polymorphism in patients from malaria endemic South Western Coastal Region of India. Malar. J. 17, 40. Khan, A., Godil, F.J., Naseem, R., 2016. Chloroquine-resistant Plasmodium vivax in Pakistan: an emerging threat. Lancet Glob. Health 4, e790. Korsinczky, M., Fischer, K., Chen, N., Baker, J., Rieckmann, K., Cheng, Q., 2004. Sulfadoxine resistance in Plasmodium vivax is associated with a specific amino acid in dihydropteroate synthase at the putative sulfadoxine-binding site. Antimicrob. Agents Chemother 48, 2214–2222. Looareesuwan, S., Viravan, C., Webster, H.K., Kyle, D.E., Hutchinson, D.B., Canfield, C.J., 1996. Clinical studies of atovaquone, alone or in combination with other antimalarial drugs, for treatment of acute uncomplicated malaria in Thailand. Am. J. Trop. Med. Hyg. 54, 62–66. Lu, F., Lim, C.S., Nam, D.H., Kim, K., Lin, K., Kim, T.S., Lee, H.W., Chen, J.H., Wang, Y., Sattabongkot, J., Han, E.T., 2011. Genetic polymorphism in pvmdr1 and pvcrt-o genes in relation to in vitro drug susceptibility of Plasmodium vivax isolates from malariaendemic countries. Acta Trop 117, 69–75. Lu, F., Wang, B., Cao, J., Sattabongkot, J., Zhou, H., Zhu, G., Kim, K., Gao, Q., Han, E.T., 2012. Prevalence of drug resistance-associated gene mutations in Plasmodium vivax in Central China. Korean J. Parasitol. 50, 379–384. Marques, M.M., Costa, M.R., Santana Filho, F.S., Vieira, J.L., Nascimento, M.T., Brasil, L.W., Nogueira, F., Silveira, H., Reyes-Lecca, R.C., Monteiro, W.M., Lacerda, M.V., Alecrim, M.G., 2014. Plasmodium vivax chloroquine resistance and anemia in the western Brazilian Amazon. Antimicrob. Agents Chemother 58, 342–347. Peters, W., 1998. Drug resistance in malaria parasites of animals and man. Adv. Parasitol. 41, 1–62. Pukrittayakamee, S., Chantra, A., Simpson, J.A., Vanijanonta, S., Clemens, R., Looareesuwan, S., White, N.J., 2000. Therapeutic responses to different antimalarial drugs in vivax malaria. Antimicrob. Agents Chemother. 44, 1680–1685. Rieckmann, K.H., Davis, D.R., Hutton, D.C., 1989. Plasmodium vivax resistance to chloroquine? Lancet 2, 1183–1184. Rungsihirunrat, K., Muhamad, P., Chaijaroenkul, W., Kuesap, J., Na-Bangchang, K., 2015. Plasmodium vivax drug resistance genes; Pvmdr1 and Pvcrt-o polymorphisms in relation to chloroquine sensitivity from a malaria endemic area of Thailand. Korean J. Parasitol. 53, 43–49. Rungsihirunrat, K., Na-Bangchang, K., Hawkins, V.N., Mungthin, M., Sibley, C.H., 2007. Sensitivity to antifolates and genetic analysis of Plasmodium vivax isolates from
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Plasmodium vivax drug resistance markers: Genetic polymorphisms and mutation patterns in isolates from Malaysia
T
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Fei-Wen Cheonga, , Shairah Dzula,b, Mun-Yik Fonga, Yee-Ling Laua, Sasheela Ponnampalavanarc a
Department of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia Division of Management Services, Ministry of International Trade and Industry, 50480 Kuala Lumpur, Malaysia c Department of Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia b
A R T I C LE I N FO
A B S T R A C T
Keywords: Plasmodium vivax Drug resistance markers pvcrt-o pvmdr1 pvdhfr pvdhps
Transmission of Plasmodium vivax still persist in Malaysia despite the government's aim to eliminate malaria in 2020. High treatment failure rate of chloroquine monotherapy was reported recently. Hence, parasite drug susceptibility should be kept under close monitoring. Mutation analysis of the drug resistance markers is useful for reconnaissance of anti-malarial drug resistance. Hitherto, information on P. vivax drug resistance marker in Malaysia are limited. This study aims to evaluate the mutations in four P. vivax drug resistance markers pvcrt-o (putative), pvmdr1 (putative), pvdhfr and pvdhps in 44 isolates from Malaysia. Finding indicates that 27.3%, 100%, 47.7%, and 27.3% of the isolates were carrying mutant allele in pvcrt-o, pvmdr1, pvdhfr and pvdhps genes, respectively. Most of the mutant isolates had multiple point mutations rather than single point mutation in pvmdr1 (41/44) and pvdhfr (19/21). One novel point mutation V111I was detected in pvdhfr. Allelic combination analysis shows significant strong association between mutations in pvcrt-o and pvmdr1 (X2 = 9.521, P < 0.05). In the present study, 65.9% of the patients are non-Malaysians, with few of them arrived in Malaysia 1–2 weeks before the onset of clinical manifestations, or had previous history of malaria infection. Besides, few Malaysian patients had travel history to vivax-endemic countries, suggesting that these patients might have acquired the infections during their travel. All these possible imported cases could have placed Malaysia in a risk to have local transmission or outbreak of malaria. Six isolates were found to have mutations in all four drug resistance markers, suggesting that the multiple-drugs resistant P. vivax strains are circulating in Malaysia.
1. Introduction Malaria continues to place heavy public health and economy burden, causing 228 million cases and 405,000 deaths in 2018 with approximately US$ 2.7 billion spent in global malaria control and elimination (World Health Organization, 2019). Plasmodium vivax, which has the widest geographical distribution of the human malaria parasites, has the second highest prevalence rate in Malaysia after P. knowlesi. Although malaria control efforts have effectively reduced the incidence of vivax infection, transmission persists in rural areas due to asymptomatic or submicroscopic reservoir (Cheng et al., 2015). In November 2016, a P. vivax malaria outbreak has occurred in Pos Kemar Orang Asli settlement, Perak, with a total of 137 indigenous people, including 79 children (aged 14 and below), who were tested positive for P. vivax (The Star Online, 2017). However, some of the positively diagnosed villagers are reluctant to get treatment, which could be a
possible factor that contributed to the rapid spread of diseases in that area (Astro Awani Malaysia News, 2016). Besides, importation of a considerable fraction of vivax malaria by undocumented illegal workers from malaria endemic countries poses another major challenge towards local intervention (WHO, 2015). Anti-malarial drug chloroquine (CQ) resistance in P. vivax was first reported in Papua New Guinea (PNG) in 1989 (Rieckmann et al., 1989), and continued to emerge in Asia, Oceania and South America, with monotherapy failure rate exceeding 80% in PNG (Marques et al., 2014; Sumawinata et al., 2003; Waheed et al., 2015). High levels of CQ resistance had forced some countries to switch their treatment drug to sulfadoxine-pyrimethamine (SP). However, evidence of P. vivax resistance to pyrimethamine alone was documented in 1950s (Young and Burgess 1959), and its resistance against SP developed rapidly within several years after deployment of SP as monotherapy drug in many areas (Peters 1998; Pukrittayakamee et al., 2000). Atovaquone
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Corresponding author. E-mail addresses: [email protected] (F.-W. Cheong), [email protected] (S. Dzul), [email protected] (M.-Y. Fong), [email protected] (Y.-L. Lau), [email protected] (S. Ponnampalavanar). https://doi.org/10.1016/j.actatropica.2020.105454 Received 13 December 2019; Received in revised form 19 March 2020; Accepted 19 March 2020 Available online 20 March 2020 0001-706X/ © 2020 Elsevier B.V. All rights reserved.
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2.2. Study area and sample collection
resistance developed easily when it was use as a monotherapy drug. Although the combination of atovaquone-proguanil was effective in elimination of P. vivax erythrocytic forms, the parasitemia was found to be recurred in most of the patients due to the relapse of hypnozoites, indicating that this drug combination is ineffective against P. vivax hypnozoites (Looareesuwan et al., 1996). In P. falciparum, CQ resistance is linked to CQ resistance transporter (pfcrt) gene, which parasites with mutations in pfcrt could reduce the concentration of intravacuolar drug by increasing efflux of CQ from the digestive vacuole (Sidhu et al., 2002). Molecular mechanisms underlying CQ resistance in P. vivax remain unclear. Nonetheless, P. vivax multidrug resistance 1 gene (pvmdr1) and CQ resistance transporter gene (pvcrt-o), which are orthologous to pfmdr1 and pfcrt genes, respectively, have been identified as putative CQ drug resistance markers of P. vivax (Lu et al., 2011; Rungsihirunrat et al., 2015). Nonsynonymous point mutations or change of the gene copy numbers of pvmdr1 have been shown to alter the parasite's in vitro susceptibility to several anti-malarial drugs including CQ, mefloquine, amodiaquine, and artemisinin derivatives (Rungsihirunrat et al., 2015; Suwanarusk et al., 2008; Suwanarusk et al., 2007). Mutations in parasite's folate biosynthetic pathway genes, dihydrofolate reductase (pvdhfr) and dihydropteroate synthase (pvdhps) confer resistance to pyrimethamine and sulfadoxine, respectively, resulting decreased drug affinity and treatment failure in clinical settings (Imwong et al., 2001; Imwong et al., 2005; Korsinczky et al., 2004). CQ resistant-P. vivax has been documented in Sabah, Malaysia in 1996 (Ahlm et al., 1996), and a high treatment failure rate of CQ monotherapy by day 28 in vivax-infected patients has also been demonstrated in a recent randomized controlled trial (Grigg et al., 2016). Malaysian Ministry of Health malaria treatment guideline has been updated in 2016 to recommend artemisinin combination treatment (ACT) over CQ as first-line asexual treatment for all uncomplicated malaria including P. vivax (Barber et al., 2017). However, the implementation throughout the country remains unclear and CQ might still been prescribed in rural areas. Therefore, parasite drug susceptibility should be kept under close surveillance in Malaysia as our country is located in close geographical proximity to countries with anti-malarial drug resistant parasite strains such as Cambodia, Thailand, Vietnam, Indonesia, and is highly vulnerable to importation of these parasite strains via migrant workers. In order to assess the true drug resistance, ex vivo drug susceptibility assay should be performed. However, due to the difficulties in collecting fresh isolates and the tediousness in experiment procedures, the feasibility of this assay to be performed routinely is low. Hence mutation analysis of the drug resistance molecular markers could thus serve as an alternative method for the reconnaissance of anti-malarial drug resistance. Genotyping of these markers can serve as a surveillance tool to detect and monitor the prevalence and influx of resistant Plasmodium strains in a geographic region, which could provide useful information for implementation and intervention of government malaria control activities. Hitherto, studies on the prevalence, emergence or influx of P. vivax drug resistance marker genes in Malaysia are scarce. The present study studied the mutations in anti-malarial drug resistance markers crt, mdr1, dhfr and dhps in P. vivax, and the prevalence of wild type or mutant alleles in the P. vivax isolates in Malaysia.
A total of 44 P. vivax-infected patient blood samples was collected from 2011 to 2017, from state and district government hospitals in Malaysia: University Malaya Medical Center (UMMC), Kuala Lumpur Hospital (HKL), Kuala Lumpur; Pulau Pinang District Health Department, Penang; Permaisuri Ipoh Hospital, Perak; Kuala Kubu Bharu Hospital, Selangor; Tuanku Jaafar Hospital, Negeri Sembilan; Sultanah Nur Zahirah Hospital, Terengganu; General Health Laboratory Kota Bharu Perol, Kelantan; Kuala Lipis Hospital, Pahang; Jerantut Hospital, Pahang; and for East Malaysia, Sabah District Health Department, Sabah; and Sarawak District Health Department, Sarawak. Genomic DNA of P. vivax was extracted from these patient blood samples using DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Plasmodium species in each patient was confirmed by microscopic examination of Giemsastained blood smears and nested polymerase chain reaction (PCR) based on 18S rRNA gene (Singh et al., 1999; Singh et al., 2004). 2.3. Amplification of drug resistance markers pvcrt-o, pvmdr1, pvdhfr and pvdhps The extracted DNA which has been confirmed to be P. vivax was subjected to PCR targeting the pvcrt-o (K10 insertion), pvmdr1 (T958M, Y976F, and F1076L mutation points), pvdhfr (F57L/I, S58R, T61M, S117T/N, and I173F/L mutation points) and pvdhps (S382A/C, A383G, K512M/T, and A553G/C mutation points). The amino acid mutation positions and PCR regions of each genes were showed in Fig. 1. Amplifications were carried out according to previous described protocols by Lu et al. (2012) with modifications. Primer sets used in the study were listed in Table 1. DNA, 1 uL of was used for each reaction in a final volume of 20 uL reaction mixture, with 1× GoTaq® Flexi Buffer, 4 mM MgCl2, 0.2 μM of each primer, 0.2 μM dNTP and 1 U GoTaq® Flexi DNA Polymerase (Promega, Madison, USA). Single step PCR was carried out with the following thermal cycling conditions: initial denaturation at 94 C for 5 min; 35 cycles of denaturation at 94 C for 30 s, annealing of varying temperature for 1 min, extension at 72 C for 1 min; and final extension at 72 C for 8 min. Annealing temperature for pvcrt-o, pvmdr1, pvdhfr, and pvdhps were 61 C, 62 C, 58 C, and 56 C, respectively. 2.4. Determination of polymorphisms in gene markers by sequence analysis PCR amplicons were sequenced in both directions using the respective molecular marker forward and reverse primers by commercial company (First BASE Laboratories Sdn Bhd, Malaysia). The nucleotide sequences and deduced amino acid sequences were aligned and analyzed using BioEdit Sequence Alignment Editor Software. P. vivax strain Salvador 1 reference wild type sequences in National Center for Biotechnology Information (GenBank accession no. AF314649 for pvcrto; AY571984 for pvmdr1; X98123 for pvdhfr; AY186730 for pvdhps) were used to compare with the amino acid sequences of P. vivax isolates to identify the genetic polymorphisms and mutation patterns, where insertions and deletions were verified manually. Prevalence of the wild type or mutant alleles in the isolates was determined. Allelic combination analysis of drug resistance markers was performed by using statistical software SPSS V23. The association between mutation (with or without mutation) in pvcrt-o and pvmdr1 was analysed. On the other hand, association between mutation (with or without mutation) in pvdhfr and pvdhps was analysed.
2. Materials and methods 2.1. Ethical approval
3. Results
Use of human samples in this study was approved by University of Malaya Medical center Medical Ethics Committee (MEC Ref. No: 817.18) and Medical Research & Ethics Committee, Ministry of Health Malaysia [NMRR-15-67,223,975 (IIR)]. Informed consent from the donor or the next of kin was obtained for use of these samples in screening.
3.1. Descriptive characteristics of patients Twenty nine of the 44 (65.9%) vivax-infected patients are found to be non-Malaysian. Majority of these non-Malaysians were from 2
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Fig. 1. Schematic diagrams of amino acid point mutation positions and PCR regions of (A) pvcrt-o, (B) pvmdr1, (C) pvdhfr, and (D) pvdhps genes (Adapted from Lu et al. (2012)). Several previously reported point mutations are indicated in the diagrams, and targeted amino acid mutations in the present study are marked in red. In (A), (C) and (D), boxes indicate exons, and lines indicate introns. In (C), R indicates repeat region GGDN/TSGGDN/THGGDN. In (D), R indicates repeat region GEAKLTN-GEGKLTN-GEAKLTN-GEGKLTN-GEAKLTN-GEGKLTN-GDAKLTN-GDSKLTN-GEAKLTN.
resistance-conferring mutations region were selected for amplification, with expected amplicon sizes of 1186 bp, 604 bp, 716 bp and 1301 bp, respectively. A total of 44 vivax-single infection samples were used in this study. For each isolates, all of the four drug resistance markers were successfully amplified and sequenced. The polymorphisms and mutations for all 44 isolates in each of the four genes were tabulated in Supplementary Table 1.
Table 1 Primer sets for amplification of pvcrt-o, pvmdr1, pvdhfr and pvdhps genes. Genes
Primers
Amplicon size (bp)
pvcrt-o
F: 5′-AAGAGCCGTCTAGCCATCC-3′ R: 5′-AGTTTCCCTCTACACCCG-3′ F: 5′-GGATAGTCATGCCCCAGGATTG-3′ R: 5′-CATCAACTTCCCGGCGTAGC-3′ F: 5′-ATGGAGGACCTTTCAGATGTATT-3′ R: 5′-CCACCTTGCTGTAAACCAAAAAGTCCAGAG-3′ F: 5′-GGTTTATTTGTCGATCCTGTG-3′ R: 5′-GAGATTACCCTAAGGTTGATGTATC-3′
1186
pvmdr1 pvdhfr pvdhps
604 716
3.3. Analysis of mutation pattern in pvcrt-o gene
1301
Compared to Salvador 1 reference wild type sequence, nucleotide sequencing result of isolates showed that an insertion of lysine at amino acid position 10 (K10, nucleotide sequence AAG) was detected in 12 of 44 isolates (27.3%) (Table 2), where 32 isolates were carrying wild type allele without insertion (72.7%).
Pakistan (11/29, 37.9%). The other nationality including Indian (5/29, 17.2%), Indonesian (4/29, 13.8%), Nepali (4/29, 13.8%), Bangladeshi (2/29, 6.9%), Vietnamese (1/29, 3.4%), Myanmarese (1/29, 3.4%) and South African (1/29, 3.4%). Details of the patients including sex, age, parasitemia, and travel history were recorded in Supplementary Table 1.
3.4. Analysis of point mutation patterns in pvmdr1 gene Of the 44 isolates, no wild type alleles were detected. All isolates carried the point mutation T958M (100%), whereas point mutations Y976F and F1076L were found in five (11.4%) and 41 (93.2%) isolates, respectively (Table 2). All five isolates carrying the Y976F point mutation were combined with F1076L mutation, yet majority of the isolates carrying F1076L mutation is having wild type alleles at codon 976.
3.2. Amplification of drug resistance markers pvcrt-o, pvmdr1, pvdhfr and pvdhps Fragments of pvcrt-o, pvmdr1, pvdhfr and pvdhps covering respective 3
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mutations are showed in Table 3.
Table 2 Prevalence of each mutations in pvcrt-o, pvmdr1, pvdhfr, and pvdhps genes in isolates from Malaysia (n = 44). Mutations
3.6. Analysis of point mutation patterns in pvdhps gene
No. of isolates carrying mutation (%)
pvcrt-o K10 insertion pvmdr1 T958M Y976F F1076L pvdhfr F57L S58R T61M V111I S117T S117N I173F pvdhps S382A A383G A553G
The prevalence rate of point mutations S382A, A383G, A553G is 2.3% (1/44), 27.3% (12/44), 11.4% (5/44), respectively (Table 2). Point mutations at position 512 were not detected in all the isolates. The most common allele in the samples is wild type allele (32/44, 72.7%), while allele with single point mutation A383G accounted for 15.9% of total isolates (7/44). Four of the isolates (9.1%) were carrying two point mutations A383G + A553G, whereas only one isolate (2.3%) was carrying three point mutations S382A + A383G + A553G (Table 3).
12 (27.3) 44 (100.0) 5 (11.4) 41 (93.2) 11 (25.0) 18 (40.9) 9 (20.5) 1 (2.3) 8 (18.2) 10 (22.7) 2 (4.5)
3.7. Analysis of pvcrt-o and pvmdr1 allelic combinations Allelic combinations of pvcrt-o and pvmdr1 were analysed as these two genes are potentially undergoing same drug (CQ) pressure. Total of 12 isolates were carrying K10 insertion in pvcrt-o, combined with different point mutations in pvmdr1. Pearson's chi-square test showed that there is significant association between the mutations in pvcrt-o and pvmdr1 (X2 = 9.521, P < 0.05). The Cramer's V value is 0.465, indicating a strong association between the mutations in the two genes.
1 (2.3) 12 (27.3) 5 (11.4)
Table 3 Frequency of alleles with single or multiple mutations in pvcrt-o, pvmdr1, pvdhfr, and pvdhps genes in isolates from Malaysia (n = 44). Mutations
Pvcrt-o Wild type K10 insertion pvmdr1 Wild type T958M T958M + F1076L T958M + Y976F + F1076L pvdhfr Wild type S58R S117N F57L + S58R F57L + I173F S58R + S117N S58R + T61M + S117N F57L + S58R + T61M + S117T F57L + S58R + T61M + S117N F57L + V111I + S117T + I173F pvdhps Wild type A383G A383G + A553G S382A + A383G + A553G
Single/multiple mutations
No. of isolates carrying mutation(s) (%)
Nil Single
32 (72.7) 12 (27.3)
Nil Single Double Triple
0 (0.0) 3 (6.8) 36 (81.8) 5 (11.4)
Nil Single Single Double Double Double Triple Quadruple Quadruple Quadruple
23 (52.3) 1 (2.3) 1 (2.3) 1 (2.3) 1 (2.3) 7 (15.9) 1 (2.3) 7 (15.9) 1 (2.3) 1 (2.3)
Nil Single Double Triple
32 (72.7) 7 (15.9) 4 (9.1) 1 (2.3)
3.8. Analysis of pvdhfr and pvdhps allelic combinations Sulfadoxine and pyrimethamine are both antifolates that inhibit biosynthesis of folate in parasites. Hence, allelic combinations of pvdhfr and pvdhps were analysed as these two genes are responsible for biosynthesis of folate and are potentially undergoing similar drug group pressure. A total of 25 isolates are carrying point mutation(s) in either pvdhfr or pvdhps genes. Among them, 8 isolates are carrying point mutations in both pvdhfr and pvdhps genes. Pearson's chi-square test revealed no significant association between the point mutations in pvdhfr and pvdhps (X2 = 26.468, P > 0.05). The Cramer's V value is 0.448, indicating a strong association between the point mutations in the two genes. 4. Discussion The insertion of lysine (K) in the first exon (amino acid 10) of pvcrt-o has been associated with significant increase in CQ IC50 (Suwanarusk et al., 2007), whereas the isolates with Y976F mutation in pvmdr1 have also been found to have significantly increased CQ IC50 compared to isolates with wild type allele (Suwanarusk et al., 2008; Suwanarusk et al., 2007). In the present study, both the mutations were detected in the isolates in Malaysia (12 isolates for pvcrt-o K10 insertion and five isolates for pvmdr1 Y976F), while two of the isolates (4.5%) were carrying these two mutations in both the genes. Our result could be supported by study of Grigg et al., 2016 that recorded a 61.1% of in vivo CQ resistance, indicating the high prevalence of CQ-resistant P. vivax in Malaysia. We detected T958M mutation in pvdmr1 of all samples. Similar findings were reported from countries with both low and high levels of CQ resistance, suggesting that this point mutation could be an allelic variant of the wild type and might not be associated with the CQ resistance phenotypically (Joy et al., 2018; Schousboe et al., 2015). In majority of the isolates, the residue L was present at position 1076 in pvmdr1 as F1076L in the present study. However, this point mutation might not confer drug resistance in the parasite. Imwong et al. (2008) have identified this residue L at the correspond codon in the orthologous gene in P. falciparum and P. cynomolgi (pfmdr1 and pcmdr1, respectively), thus suggesting that this residue L could be a neutral allele that does not alter the fitness of the parasite. The resistance of P. vivax to antifolates has been linked to mutations in pvdhfr and pvdhps. With supportive results from in vitro drug assays and clinical assessments (Imwong et al., 2005; Rungsihirunrat et al.,
Of that, most of the isolates (36/44, 81.8%) were carrying two point mutations T958M + F1076L, followed by three point mutations T958M + Y976F + F1076L (5/44, 11.4%). Only three isolates were found to carry single point mutation T958M (6.8%) (Table 3). 3.5. Analysis of point mutation patterns in pvdhfr gene Seven point mutations were observed compared to the reference strain. Point mutation S58R was observed in most of the isolates (18/ 44, 40.9%), followed by F57L (11/44, 25.0%), S117N (10/44, 22.7%), T61M (9/44, 20.5%), S117T (8/44, 18.2%), and I173F (2/44, 4.5%). Novel mutation V111I was detected in one of the isolates (2.3%) (Table 2). More than half of the isolates (23/44, 52.3%) were carrying wild type allele. Two point mutations S58R + S117N and four point mutations F57L + S58R + T61M + S117T were common among the isolates, each accounting for 15.9% (7/44) of the isolates. The frequency distributions of other alleles with single or multiple point 4
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indigenous children have been reported in Pos Lenjang, Pahang in July 2019 (The Star Online, 2019), despite the government aims to achieve malaria elimination in 2020. Moreover, six of these isolates in the present study were found to have mutations in all four drug resistance markers pvcrt-o, pvmdr1, pvdhfr and pvdhps, suggesting that the multiple-drugs resistant P. vivax strains have been distributed in Malaysia and leading to a more worrying situation. With the aid of mutations analysis of drug resistance markers, surveillance and monitoring on efficacy of anti-malarial drugs could be performed in a more thorough way. The findings of the study serve as an update of the prevalence of wild type or mutant alleles in the P. vivax isolates in Malaysia, and could also provide fundamental data for future study to evaluate whether genotyping of these few drug resistance markers could reflect the occurrence of phenotypic drug resistance by performing in vitro or ex vivo drug susceptibility tests.
2007), the pvdhfr and pvdhps genotyping data could be useful to indicate the SP resistance status in P. vivax. Rungsihirunrat et al. (2007) demonstrated that the increase number of mutations (from double to quadruple point mutation) in pvdhfr is associated with the increase of antifolates IC50, suggesting the quadruple mutant alleles confer higher pyrimethamine resistance than double mutant alleles. Similar finding was shown by Imwong et al. (2001) that patients harbouring triple mutations in pvdhfr had a significant lower parasite reduction ratio compared to those patients with double mutations. Of that, point mutations F57L, S58R and S117N are found to be associated with SP resistance. In our findings, the three highest frequency of point mutations among the 21 mutant isolates are S58R, F57L and S117N, with the prevalence rate of 85.7% (18/21), 52.4% (11/21), and 47.6% (10/21), respectively. High number of isolates were having double mutations S58R + S117N. This finding is in concordance with a previous study which discovered that the initial mutations would occur at position 58 and 117 when drug pressure is applied (Imwong et al., 2003). On the other hand, seven isolates were carrying quadruple mutations F57L + S58R + T61M + S117T. Imwong et al. (2003) has demonstrated that in highly mutated isolates that carrying quadruple point mutation, the residue 117 would carry the S→T mutation rather than the S→N mutation, followed by mutations at position 57 and 61. Only two isolates were found to harbor single point mutation in the present study, indicating high percentage of highly mutated P. vivax strain is circulating in Malaysia. In pvdhps, majority of the isolates were carrying wild type allele. We detected three point mutations at residues 382, 383 and 553 in the isolates, in which these mutations have been reported previously in Thailand isolates (Imwong et al., 2005; Korsinczky et al., 2004). These mutations were hypothesized to be associated with reduced susceptibility to both sulfone and sulphonamide groups. Imwong et al. (2005) reported a slower parasite clearance in SP-treated patients carrying six or more combined point mutations in both pvdhfr and pvdhps genes, compared to patient with fewer mutations in these genes. Central Asia is accounted for more than 80% of global population at risk for Plasmodium vivax, particularly in India and Pakistan (Khan et al., 2016); while the high endemicity countries in Southeast Asia including Indonesia and Papua New Guinea (Battle et al., 2012). Population movement from malaria-endemic areas to non-endemic areas is one of the main causes for imported malaria cases, and the transmission of parasites can be continued with the presence of suitable mosquito vectors. Large proportion of the vivax-infected patients in the present study are found to be non-Malaysian. Few of these patients arrived in Malaysia one to two weeks before the onset of clinical manifestations. Besides, three of them (one Indian and two Indonesians) had previous history of malaria infection in their countries six months to three years ago. Although treatment was prescribed to the patients during the previous infection, the patients’ compliance for the treatment drugs is uncertain, especially for primaquine, the anti-hypnozoite drug that should be administered daily up to 14 days. Hence, the current infection in these few patients in the present study could be due to relapse of the P. vivax hypnozoites. On the other hand, three of the Malaysian patients had travel history to Pakistan and Papua New Guinea before the onset of symptoms. It is possible that these patients might have acquired the infections during their travel to those vivax-endemic countries. Besides, one of the Malaysian patients came back from Papua New Guinea three months ago, but this patient was diagnosed to have vivax infection after return and had relapse history. All these possible imported cases could have placed Malaysia in a risk to have local transmission or outbreak of malaria.
Author statement The authors declare that they have no competing interests. The material is original and has not been published. The material has not and will not be submitted for publication elsewhere so long as it is under consideration by Acta Tropica. If accepted, it will not be published elsewhere in the same form in English or in any other language. Fei-Wen Cheong, Shairah Dzul, Mun-Yik Fong, Yee-Ling Lau, and Sasheela Ponnampalavanar have participated in the study and concur with the submission and subsequent revisions submitted by the corresponding author. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This study was supported by BKP grant, University of Malaya, Malaysia (BK005-2017). Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.actatropica.2020.105454. References Ahlm, C., Wistrom, J., Carlsson, H., 1996. Chloroquine-Resistant Plasmodium vivax Malaria in Borneo. J. Travel Med. 3, 124. Astro Awani Malaysia News. 2016. Malaria cases likely to rise – Dr Mah. Retrieved from:http://english.astroawani.com/malaysia-news/malaria-cases-likely-rise-drmah-123972. Accessed Nov 21, 2019. Barber, B.E., Grigg, M.J., William, T., Yeo, T.W., Anstey, N.M., 2017. The treatment of Plasmodium knowlesi Malaria. Trends Parasitol. 33, 242–253. Battle, K.E., Gething, P.W., Elyazar, I.R., Moyes, C.L., Sinka, M.E., Howes, R.E., Guerra, C.A., Price, R.N., Baird, K.J., Hay, S.I., 2012. The global public health significance of Plasmodium vivax. Adv. Parasitol. 80, 1–111. Cheng, Q., Cunningham, J., Gatton, M.L., 2015. Systematic review of sub-microscopic P. vivax infections: prevalence and determining factors. PLoS Negl. Trop. Dis. 9, e3413. Grigg, M.J., William, T., Menon, J., Barber, B.E., Wilkes, C.S., Rajahram, G.S., Edstein, M.D., Auburn, S., Price, R.N., Yeo, T.W., Anstey, N.M., 2016. Efficacy of artesunatemefloquine for chloroquine-resistant Plasmodium vivax Malaria in Malaysia: an openlabel, randomized, controlled trial. Clin. Infect. Dis. 62, 1403–1411. Imwong, M., Pukrittakayamee, S., Looareesuwan, S., Pasvol, G., Poirreiz, J., White, N.J., Snounou, G., 2001. Association of genetic mutations in Plasmodium vivax dhfr with resistance to sulfadoxine-pyrimethamine: geographical and clinical correlates. Antimicrob. Agents Chemother. 45, 3122–3127. Imwong, M., Pukrittayakamee, S., Cheng, Q., Moore, C., Looareesuwan, S., Snounou, G., White, N.J., Day, N.P., 2005. Limited polymorphism in the dihydropteroate synthetase gene (dhps) of Plasmodium vivax isolates from Thailand. Antimicrob. Agents Chemother. 49, 4393–4395. Imwong, M., Pukrittayakamee, S., Pongtavornpinyo, W., Nakeesathit, S., Nair, S.,
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