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Genetic polymorphisms in genes associated with drug resistance in Plasmodium vivax parasites from northeastern Myanmar

Genetic polymorphisms in genes associated with drug resistance in Plasmodium vivax parasites from... Background: Anti-malarial drug resistance is still a major threat to malaria elimination in the Great Mekong Sub- region. Plasmodium vivax parasites resistant to anti-malarial drugs are now found in Myanmar. Molecular surveillance on drug resistance genes in P. vivax parasites from northeastern Myanmar was aimed at estimating the underlying drug resistance in this region. Methods: Blood samples from patients with vivax malaria were collected from Laiza city in northeastern Myanmar in 2020. Drug resistance genes including Pvcrt-o, Pvmdr1, Pvdhfr and Pvdhps were amplified and sequenced. Genetic polymorphisms and haplotypes were analysed to evaluate the prevalence of mutant alleles associated with drug resistance. Results: A total of 149 blood samples from P. vivax patients were collected. The prevalence of Pvmdr1 mutations at codons 958 and 1076 was 100.0% and 52.0%, respectively, whereas no single nucleotide polymorphism was present at codon 976. The proportions of single and double mutant types were 48.0% and 52.0%, respectively. A K10 “AAG” insertion in the Pvcrt-o gene was not detected. Mutations in Pvdhfr at codons 57, 58, 61, 99 and 117 were detected in 29.9%, 54.3%, 27.6%, 44.9% and 55.1% of the samples, respectively. Wild type was predominant (46.3%), followed by quadruple and double mutant haplotypes. Of three types of tandem repeat variations of Pvdhfr, Type B, with three copies of GGDN repeats, was the most common. Pvdhps mutations were only detected at codons 383 and 553 and the wild type Pvdhps was dominant (78.0%). Eleven haplotypes were identified when combining the mutations of Pvdhfr and Pvdhps, among which the predominant one was the wild type (33.9%), followed by double mutant alleles S58R/S117N /WT (24.6%). Conclusions: This study demonstrated resistant P. vivax phenotypes exists in northeastern Myanmar. Continued surveillance of drug resistance markers is needed to update treatment guidelines in this region. Keywords: Genetic polymorphisms, Drug resistance, Plasmodium vivax, Northeastern Myanmar Background Plasmodium vivax, the most geographically widespread human malaria parasite, causes significant morbid - ity in Southeast Asia, the Western Pacific, Central and South America, and parts of Africa [1]. There were an *Correspondence: huangfang@nipd.chinacdc.cn estimated 2.5  billion people at risk of P. vivax infec- WHO Collaborating Centre for Tropical Diseases; National Center for International Research on Tropical Diseases, Shanghai, China tion worldwide and 6.4  million clinical vivax malaria Full list of author information is available at the end of the article © The Author(s) 2022. 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The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Huang et al. Malaria Journal (2022) 21:66 Page 2 of 9 cases in 2019, mainly distributed in Southeast Asia Sequencing of Pvmdr1 genes from several regions of [2]. In the past 20 years, the burden of P. vivax malaria the world has revealed more than fifty polymorphisms, has decreased dramatically, with many countries in as well as copy number variants (CNVs). Six SNPs have the Asia-Pacific and the Americas reporting reduc - been reported at high frequency in multiple studies in tions of 90% in the number of clinical cases [2]. As a regions with reported drug resistance: S513R, G698S, consequence, 34 countries are actively attempting to M908L, T958M, Y976F and F1076L. Among these, the eliminate malaria, and countries in Central America two most common mutations were Y976F and F1076L. and East Asia have declared their intention to eliminate The essential enzymes dihydropteroate synthase (DHPS) malaria from their regions by 2025 and 2030, respec- and dihydrofolate reductase-thymidylate synthase tively [3, 4]. (DHFR-TS) are involved in folate synthesis and are tar- Myanmar, formerly Burma, has the heaviest malaria gets of sulfadoxine and pyrimethamine, respectively [14, burden in the Greater Mekong Sub-region (GMS), with 25]. SP has been used to treat P. falciparum for a long more cases and deaths than the rest of the region com- time, but it is primarily used for intermittent preventive bined [2]. In recent years, Myanmar has made significant treatment in pregnancy (IPTp) and IPT for infants (IPTi), progress in reducing malaria morbidity and mortality [5– and seasonal malaria chemoprevention combining with 7]. Driven by the emergence and spread of artemisinin amodiaquine in some regions of Africa owing to its wide- resistance in the GMS, the World Health Organization spread resistance [26–28]. (WHO) declared their goal to eliminate malaria in the CQ is also used to treat vivax malaria in Southeast Asia, GMS by 2030 [8]. The National Plan for Malaria Elimina - the prevalence of high CQ-resistant falciparum malaria tion in Myanmar 2016–2030 was developed with the goal resulted for a time in the widespread use of SP, which was of decreasing the annual parasite index (API) to < 1 in available widely in areas where malaria was endemic and all states/regions by 2020, interrupting the transmission was still a first-line treatment in the adjacent countries of falciparum malaria in all states/regions by 2025 and Laos and Myanmar [29]. However, CQ and SP resistant eliminating malaria nationwide by 2030 [5]. However, P. falciparum and P. vivax were reported in Myanmar conflict-affected settings and regions with high popula - several decades ago, especially in southern Myanmar and tion mobility have posed challenges to malaria elimina- at the border between Myanmar and Thailand [29–35]. tion [9]. Recently, increasing clinical failures after CQ treatment Plasmodium vivax resistance to different anti-malarial have been reported in multiple regions of Myanmar drugs, including chloroquine (CQ), mefloquine, sulf - [36, 37]. Currently, the first-line treatment for P. vivax adoxine-pyrimethamine (SP), has been reported in many in Myanmar is CQ combined with primaquine (PQ) [5]. countries or regions [10–15]. Molecular surveillance of In northeastern Myanmar, the treatment efficacy of CQ drug resistance markers, as one of the tools in malaria was found to be relatively high, with a cumulative rate of surveillance, has been widely performed throughout the parasite recurrence less than 3% [35]. The northernmost world [16]. Several candidate drug resistance genes in P. state in Myanmar, with a long border of almost 2000 km vivax parasites have been identified [17]. Plasmodium with Tibet and Yunnan Province in China, mainly in vivax chloroquine resistance transporter (Pvcrt) and Kachin State, also called Jinghpaw Mung, where malaria the P. vivax multidrug resistance transporter (Pvmdr1) has a relatively high rate of transmission [6], resulting have been confirmed to be the Plasmodium falciparum from relatively low access to health services and difficul - orthologs involving CQ resistance genes [18, 19]. The ties in deploying interventions to hard-to-reach commu- Pvcrt-o gene was characterized nearly two decades ago nities [38, 39]. Limited data have defined the molecular [19]; it is similar to its P. falciparum orthologue and has epidemiology of P. vivax resistance markers in north- an intron-rich gene structure with 14 exons that encodes eastern Myanmar. In this study, genetic polymorphisms a protein with 451 amino acids and 10 membrane in genes potentially associated with drug resistance in P. domains [20]. Sequence polymorphism is relatively lim- vivax parasites from northeastern Myanmar were ana- ited in the Pvcrt-o locus but a lysine (AAG) insertion at lysed to estimate the underlying drug resistance, with the amino acid position 10 (K10), which was initially discov- aim of implementing an appropriate drug policy in this ered in Southeast Asian strains, has been suggested to be region. associated with CQ resistance [21]. Since then, this pol- ymorphism has been observed in both Southeast Asian Methods and South American parasites [17, 22, 23]. The Pvmdr1 Study site gene encodes a transmembrane protein of the parasite’s Samples were collected from central hospitals and com- digestive vacuole with 12 transmembrane domains and munity clinics in Laiza (24° 45′ 36″ N, 97° 33′ 48″ E), a 1464 amino acids, and was characterized in 2005 [24]. remote city in Kachin State in northeastern Myanmar, Huang  et al. Malaria Journal (2022) 21:66 Page 3 of 9 along the China-Myanmar border. This region is moun - filter paper (Whatman 903, GE Healthcare, USA), air tainous and its climate is defined as subtropical, with dried, and then stored at − 20 °C before DNA extraction. a dry season from October to April and a rainy season Parasite DNA was extracted from the dried blood spots from May to September. In recent years, malaria cases using a QIAamp DNA Mini Kit (Valencia, CA, USA) fol- have declined dramatically owing to cooperation between lowing the protocol. A polymerase chain reaction (PCR) China and Myanmar towards malaria elimination [38, amplifying the small-subunit rRNA gene of Plasmodium 40]. Local malaria transmission displayed a distinct sea- spp. [43] was performed to confirm the positive sam - sonality with two peaks, a large peak in April-August ples and identify the species before the four genes were and a small peak in November, except 2020 with a mall sequenced. peak in September (Fig. 1). Four human malaria parasites including P. falciparum, P. vivax, Plasmodium ovale and PCR amplification and sequencing Plasmodium malariae have been detected, and P. vivax is The Pvcrt-o gene was amplified by a single-round PCR the predominant species, accounting for more than 90% and Pvmdr1, Pvdhps and Pvdhfr were amplified by nested [41]. On the other side of Laiza city is Nabang township, PCR, as previously described [21, 34, 44–46]. The prim - Yunnan, China, and there is no barrier along the border. ers, cycling conditions and sizes of the PCR products A large number of immigrants from both countries cross are shown in Additional file  1. PCR reactions were per- the border frequently through Nabang port, one of the formed in a 25 µL reaction mixture that contained 1 µL provincial ports along the border. Anopheles minimus of genomic DNA, 12.5 µL 2×Taq Master Mix (TIAN- is reported to be the dominant mosquito species in this GEN, Beijing, China), 9.5 µL ddH O and 10 µM prim- area and most malaria infection occurred in population ers. The amplification products were analysed by 1.5% with subsistence activities associated with forest areas, agarose gel electrophoresis and directly sequenced. The such as logging, banana or rubber planting, and living in PCR products were purified using filter plates (Edge Bio - planting areas during the farming season or entire year systems, Gaithersburg, MD, USA) and sequenced on an [42]. ABI 3730XL automatic sequencer. Bidirectional sequenc- ing was performed, and all the products were sequenced Sample collection and DNA extraction twice using independently amplified PCR products. Blood samples were collected from P. vivax-infected patients prior to anti-malarial drug treatment in the cen- Data analysis tral hospitals and community clinics in Laiza city from The output sequence data were assembled, edited and July to October 2020. Blood samples were spotted on aligned using Geneious (version 2021.0.3) software, Fig. 1 Monthly reported cases of malaria from sentinel sites in Laiza in Kachin State, Myanmar, 2016–2020 Huang et al. Malaria Journal (2022) 21:66 Page 4 of 9 Prevalence of Pvmdr1 mutations and K10‑insertion comparing the samples with the reference sequences in Pvcrt‑o of P vmdr1 (accession number: AY618622), P vdhps Among the 149 samples, 82.6% (123/149) samples showed (accession number: XM001617159) and P vdhfr (acces- successful amplification of Pvmdr1. Mutations in codons sion number: XM001615032) from GenBank. The 958 and 1076 were identified, with a prevalence of 100% known polymorphisms relating to drug resistance at and 52.0% (Fig. 2), respectively. No single nucleotide poly- codons 958, 976, 1076 of the P vmdr1 gene and codons morphism (SNP) was present in codon 976. Analysis of 57, 58, 61, 117, 173 of the P vdhfr gene, and codons the Pvmdr1 haplotype prevalence showed that all the iso- 382, 383, 553, 580 and 558 of P vdhps gene, were lates were of the mutant type. Appropriately half of them evaluated for haplotype. The wild-type haplotypes of carried a single T958M mutation or a double T958M/ P vmdr1, P vdhfr and P vdhps genes were T958/Y976/ F1076L mutation, with a prevalence of 48.0% and 52.0%, F1076, F57/S58/T61/R76/S117/I173 and S382/A383/ respectively (Table  1). The percentage of 88.6% (132/149) A553/R580/V585, respectively. The mixed alleles were samples were successfully sequenced in Pvcrt-o gene, and determined according to the emergence of two chro- K10 “AAG” insertion was  not detected in any of the 132 matogram peaks at one nucleotide site through Muta- successfully sequenced samples (Table 1). tion Surveyor (Soft Genetics LLC., version 5.1, State College, PA, USA). The K10 “AAG” insertion in P vcrt- Polymorphisms in the Pvdhfr gene o gene was determined by comparing with the refer- The Pvdhfr amplicon was successfully sequenced for ence strain of Sal-1 retrieved from Plasmodium data 85.2% (127/149) P. vivax isolates. Amino acid changes in base [47]. The nucleotide sequences were submitted Pvdhfr due to mutations in codons 57, 58, 61, 99 and 117 to GenBank under accession numbers MZ819186- were detected in 29.9%, 54.3%, 27.6%, 44.9% and 55.1% MZ819695. SAS software (SAS Institute Inc, Version of the samples, respectively. In addition, the I13L and 9.2, Cary, NC, USA) was used for data processing and I173L was not detected in present study. The wild type statistical analysis. The chi-squared test and Fisher’s was predominant (46.3%, 57/127). The two most com - exact test were used to evaluate differences among the mon point mutations were S58R (54.3%) and S117T/N different subgroups. P value < 0.05 were used to iden- (55.1%), followed by H99S (44.9%), F57L/I (29.9%), and tify significant differences. T61M (27.6%). Among 70 isolates that carried a point mutation in codon 117, 38 carried 117T whereas 32 car- rying 117 N. Moreover, 38 samples with a point mutation Results in codon 57 included 34 isolates carrying 57I and 4 car- Demographics of patients rying 57 L. A single mutation in codon 117 was identified A total of 149 blood samples from patients with P. in only one isolate. In addition, one synonymous muta- vivax infections were collected before treatment tion in codon 69, TAT to TAC (Y), was detected in two from hospitals and clinics in Laiza city in 2020. All isolates. the patients were confirmed to be infected with P. The haplotype analysis revealed seven distinct allelic vivax by microscopic examination of Giemsa-stained forms of the Pvdhfr gene (Table 1); among them, the wild thick smears through passive case detection. They type was predominant. A quadruple-mutant haplotype, were treated for 3 days with CQ plus 14 days with PQ F57I/S58R/T61M/S117T (26.8%) and a double-mutant according to the anti-malarial drug policy in Myan- haplotype S58R/S117N (24.4%) were the two most com- mar. The majority of the patients were male (70.5%, mon mutant types (Fig.  2). The single-mutant haplo - 105/149) and aged 21–30 years (61.7%, 92/149). The type S117T, triple mutant types S58R/T61M/S117N and median (range) age of the 149 patients was 22 years S58R/T61M/S117T and quadruple-mutant haplotype (ranges: 6–79) (Additional file 2). Fig. 2 Tandem repeat variation between amino acid positions 88 and 103 in the Pvdhfr gene Huang  et al. Malaria Journal (2022) 21:66 Page 5 of 9 Table 1 Frequency of alleles with single or multiple mutations in the Pvmdr1, Pvdhfr, Pvdhps and Pvcrt-o genes from P. vivax isolates Mutations Single/multiple mutations Numbers (%) P value Pvmdr1 < 0.0001 Wild type ( T958/Y976/F1076) 0 Y976F Single mutation 0 T958M Single mutation 59 (48.0) T958M + F1076L Double mutations 64 (52.0) Pvdhfr* < 0.0001 Wild type (F57/S58/T61/S117/I173) 57 (46.3) F57I/L Single mutation 0 S117T Single mutation 1 (0.8) S58R + S117N Double mutations 31 (24.4) S58R + T61M + S117T Triple mutations 2 (1.6) S58R + T61M + S117N Triple mutations 1 (0.8) F57I + S58R + T61M + S117T Quadruple mutations 34 (26.8) F57L + S58R + T61M + S117N Quadruple mutations 1 (0.8) Pvdhps < 0.0001 Wild type (A383/A553) 103 (78.0) A383G Single mutation 0 V585A Single mutation 0 A553G Single mutation 2 (1.5) A383G + A553G Double mutations 27 (20.5) Pvcrt-o Wild type ( Without K10 insertion) 132 (100.0) * Among 32 samples, those with deletion of GGDN repeats from position 95 to 99 were not included. The I13L and I173L mutant alleles were not detected A total of 39 samples with mutations in codon 57 including 34 samples carrying 57I and 4 carrying 57L, but F57I/L only existed in quadruple mutations The chi-squared test or Fisher’s exact test was used to evaluate differences among the frequency of alleles of each gene F57I/S58R/T61M/S117N were rare, being identified in (Table  1). Point mutations in codons 382, 580 and 585 only one, two, one, and one isolate, respectively. were not observed in this study. Variations were identified in the central tandem repeat region between amino acid positions 88 and 103 of Pvd- Analysis of allelic combinations of Pvdhfr and Pvdhps hfr (Fig.  2). Three types of tandem repeat variation were Allelic combinations of the Pvdhfr and Pvdhps enzymes observed: Type A contained three copies of GGDN are responsible for the biosynthesis of folate and are repeats, which was the same as the reference strain potentially under similar drug pressure. A total of 118 (accession number:  X98123.1) designated as the wild isolates were sequenced successfully for both genes. type [48]; Type B also had three copies of GGDN repeats The combinations of point mutations in the Pvdhfr and but showed a mutant allele in codon 99; Type C lacked Pvdhps genes were analyzed in codons 57, 58, 61, and six amino acids from positions 98 to 103. 117, and codons 383 and 553, respectively (Table  2). Eleven haplotypes were identified, and 33.9% (40/118) Polymorphisms in the Pvdhps gene harboured the wild type, as the predominant allele. A percentage of 88.6% (132/149) samples were success- The double Pvdhfr mutant S58R/S117N and wild-type fully assessed for the Pvdhps gene. Pvdhps displayed lim- Pvdhps was the most common mutant haplotype, with ited polymorphisms and a relatively low prevalence of a frequency of 24.6% (29/118). The quadruple-mutant mutations. Nonsynonymous mutations were detected F57I/S58R/T61M/S117T/WT, quadruple Pvdhfr mutant only in codons 383 and 553, with a prevalence of 20.5% F57I/S58R/T61M/S117T and wild-type Pvdhps alleles (27/132) and 22.0% (29/132), respectively. The wild-type or double Pvdhps mutant A383G/A553G were present Pvdhps was dominant (78.0%) (Table  1; Fig.  2). Among at intermediate frequencies of 16.1%, 11.0% and 9.3%, the mutant types, the single-mutant A553G was rare and respectively. Rare haplotypes including single-, triple-, only detected in two isolates, whereas the double-mutant quadruple-, and quintuple-mutant alleles, were detected A383G/A553G was more frequent (20.5%, 27/132) in one isolate each (Table 2). Huang et al. Malaria Journal (2022) 21:66 Page 6 of 9 Table 2 Combination of Pvdhfr and Pvdhps mutations in P. vivax isolates from northeastern Myanmar Haplotypes Pvdhfr/Pvdhps Pvdhfr Pvdhps Number (%) F57I/L S58R T61M S117T/N A383G A553G WT/WT F S T S A A 40 (33.9) WT/A553G F S T S A G 1 (0.8) WT/A383G + A553G F S T S G G 11 (9.3) S58R + S117N/W T F R T N A A 29 (24.6) F57L + S58R + S117N/W T L R T N A A 1 (0.8) F57I + S58R + T61M + S117T/W T I R M T A A 19 (16.1) F57L + S58R + T61M + S117T/W T L R M T A A 1 (0.8) F57L + S58R + S117T/A553G L R T T A G 1 (0.8) S58R + S117N/A383G + A553G F R T N G G 1 (0.8) F57L + S58R + S117T/A383G + A553G L R T T G G 1 (0.8) F57I + S58R + T61M + S117T/A383G + A553G I R M T G G 13 (11.0) Total 118 WT: wild type Pvmdr1 T958M mutation is present in isolates from Discussion different countries having low to high levels of CQ resist - Myanmar has the heaviest malaria burden in the GMS ance, thus T958M appears to be an allelic variant of [49], and its geographical location between Southeast the wild type and is most likely not associated with CQ Asia and South Asia makes it a strategically important resistance [24, 56]. In this study, Pvmdr1 T958M was pre- point for the potential spread of resistant parasites. sent in all the tested samples, whereas F1076L occurred In the present study, genetic polymorphisms in the at an intermediate frequency, with a prevalence of 52.0%. Pvdhps and Pvdhfr genes associated with SP resistance However, Y976F was not detected in this study, although and Pvcrt-o and Pvmdr1, two putative molecular mark- it has been frequently reported in other regions with a ers of resistance to CQ, were analysed to evaluate the high prevalence [18, 21–23, 53, 57]. Another study also level of drug resistance in northeastern Myanmar. found that Y976F was rare in Myanmar [34], which sup- Previous studies have shown that 48.3–72.7% of ports the findings of this study. In addition, the associa - isolates carried Pvcrt-o  K10 insertions in Myanmar tion of the Pvmdr1 substitution Y976F with CQ-resistant between 2009 and 2016 [21, 34], and approximately 19% P. vivax in vitro was confirmed in Papua, Indonesia [53], of the samples from Yunnan Province bordering Myan- while other studies did not find such an association [24]. mar harbored this insertion [50]. In the present study, Pvdhfr and Pvdhps, the targets of SP drugs, disrupt among all the tested samples, the K10 “AAG” insertion folate synthesis in P. vivax [12, 14, 25]. Compared with in the Pvcrt-o gene was not identified, in contrast to Pfdhfr and Pfdhps in P. falciparum, Pvdhfr and Pvdhps the results of previous studies. However, these results are highly conserved in P. vivax [58]. The prevalence of are validated to some extent with the high CQ cure the Pvdhfr mutation (53.7%) was lower in this study rate for P. vivax along the China-Myanmar border [37, than in other studies from the China-Myanmar border, 51]. Interestingly, the K10 insertion is rarely observed southern Thailand and western Myanmar [23, 44, 50, 52]. in Thailand, the border region between Thailand and Moreover, amino acid changes in PvDHFR were detected Myanmar, and the border region between Thailand in 27.6–55.1% of the isolates, which was also lower than and Cambodia [44, 52]. Other studies have reported no the values in other studies [34, 44, 52]. With respect to association between the K10 insertion and in  vitro or Pvdhfr haplotypes, the double-mutant S58R/S117N and ex  vivo P. vivax CQ resistance [22, 53, 54]. Given the quadruple-mutant F57I/S58R/T61M/S117T were domi- geographical genetic differences among parasite popu - nant in northeastern Myanmar and were also the most lations, the prevalence of the Pvcrt-o K10 insertion common types in Myanmar, Thailand and southern shows significant temporal and spatial heterogeneity Yunnan Province, China [50, 52]. A previous study on [55] and the discrepancy may have resulted from dif- the expression of mutant PvDHFR-TS in a yeast system ferences in geography, population movement or sample demonstrated that these mutations confer high levels of size issues. Moreover, the role of the Pvcrt-o K10 inser- pyrimethamine resistance, and the quadruple mutation tion in CQ resistance in the parasite remains unknown. Huang  et al. Malaria Journal (2022) 21:66 Page 7 of 9 resulted in a thousand-fold increase in resistance to variations in drug resistance genes may suggest differ - pyrimethamine [17]. Interestingly, some researchers have ent drug selection pressures imposed on local P. vivax hypothesized that the T61M mutation may be a compen- populations. The factors that influence the evaluation satory mutation to offset possible fitness costs resulting of drug resistance are not completely known, but drug from the carriage of other resistance mutations, because pressure is probably a key element for the selection of a triple Pvdhfr mutant (58R/61M/117T) resulted in sus- resistant parasite mutants. ceptibilities similar to those of the wild-type to all drugs This study had limitations. First, Pvcrt-o and Pvmdr1 with the exception of pyrimethamine, for which this are only putative markers of CQ resistance and the haplotype conferred a 58-fold increase in resistance. In Pvmdr1 copy number was not evaluated in this study. this study, single mutations at residues 58 and 117 were There was limited evidence for CQ resistance without rare, as was the triple mutation, but the double muta- in vitro susceptibility test of P. vivax isolates and in vivo tion S58R/S117N was more common. These results sug - therapeutic efficacy study to highlight therapeutic fail - gest that vivax parasites in northeastern Myanmar may ure. Second, samples were collected only from Laiza city be under low to medium drug pressure. In addition, the through passive case detection, which may not describe majority (74.8%) had three copies of tandem repeats the whole picture of drug resistance in this region. in Pvdhfr gene and Type B was predominant (44.9%, 57/127), followed by Type A (29.9%) and Type C (25.2%). This pattern was different from that reported from south - Conclusions ern China [50], which showed Type C as the main type GMS has become a focus in the war against malaria (40.5%, 118/291), followed by Type B and Type A. How- after the emergence of artemisinin-resistant falciparum ever, the mechanism of action of the tandem repeat vari- malaria, especially in remote areas along the Myanmar ants in pyrimethamine resistance remains unclear [44], border. This study identified genetic polymorphisms in as these variants are not present in the Pfdhfr gene in P. genes associated with drug resistance in P. vivax parasites falciparum [59, 60]. in northeastern Myanmar, which indicated the risks asso- Similar to SP resistance in P. falciparum, mutations ciated with drug resistant P. vivax phenotypes, especially in Pvdhps alone are thought to be insufficient to con - antifolate resistance. Therefore, continued surveillance fer resistance to SP. Strains containing both the Pvdhps of anti-malarial drug resistance markers is needed in A383G and A553G mutations and mutant Pvdhfr alleles northeastern Myanmar to inform case management and have been implicated in SP treatment failure [61]. The update treatment guidelines. Pvdhps mutations exhibited much less diversity and lower prevalence in this study than in previous studies, Supplementary Information which reported a high mutation prevalence (80–90%) The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s12936- 022- 04084-y. in Pvdhps in Malaysia, Thailand, India, Indonesia, and China and high failure rates of SP treatment [14, 52, 62– Additional file 1: Table S1. Primers and cycling conditions for amplifica- 64]. Additionally, the prevalence of Pvdhfr mutations in tion of Pvcrt-o, Pvmdr1, Pvdhps and Pvdhfr by a PCR assay. regions where P. vivax is the dominant parasite is lower Additional file 2: Table S2. Demographics of patients included in this than that in regions where P. falciparum and P. vivax are study. co-endemic; the reason for this may be that the pressure of SP treatment of P. falciparum can lead to co-selection Acknowledgements of Pvdhfr mutant alleles, which could also occur for other We thank all the participants who provided blood samples for this study. drugs, including artemisinin partner drugs [63]. Recently, high treatment efficacy of CQ was found in Authors’ contributions FH conceived and designed the study. FH and YC conducted the laboratory Bangladesh, Bhutan, India and Nepal, but high failure work and data analysis. SL, PT, LJSP and LY collected the blood samples. HL rates of treatment with CQ were confirmed in some and DYB provided technique support for the data collection and analysis. FH sites in Myanmar and Timor-Leste [65]. However, this drafted the manuscript. All authors read and approved the final manuscript. study demonstrated that the frequency of mutant hap- Funding lotypes in northeastern Myanmar was relatively low, This study was supported by the Natural Science Foundation of Shanghai whereas the frequency of the wild type was increasing. (No. 18ZR1443400), the National Important Scientific & Technological Project (2018ZX10101002–002), and the Fifth Round of Three-Year Public Health In addition, genetic polymorphisms were rarer than Action Plan of Shanghai (No. GWV-10.1-XK13). in other regions of Myanmar, as shown in previous reports as well. This result is consistent with the treat - Availability of data and materials The nucleotide sequences were submitted to GenBank under accession num- ment efficacy of CQ reported from northeast Myan - bers MZ819186-MZ819695. The datasets analysed in this study are available mar [35] and the China-Myanmar border [51]. These from the corresponding author on reasonable request. Huang et al. Malaria Journal (2022) 21:66 Page 8 of 9 13. Korsinczky M, Fischer K, Chen N, Baker J, Rieckmann K, Cheng Q. Sulfadox- Declarations ine resistance in Plasmodium vivax is associated with a specific amino acid in dihydropteroate synthase at the putative sulfadoxine-binding site. Ethics approval and consent to participate Antimicrob Agents Chemother. 2004;48:2214–22. This study was approved by the Ethical Review Committee of National Insti- 14. Hastings MD, Maguire JD, Bangs MJ, Zimmerman PA, Reeder JC, Baird tute of Parasitic Diseases, Chinese Center for Disease Control and Prevention JK, et al. Novel Plasmodium vivax dhfr alleles from the Indonesian and the Ethics Committee of Laiza Central Ministry of Public Health. Archipelago and Papua New Guinea: association with pyrimethamine resistance determined by a Saccharomyces cerevisiae expression system. Consent for publication Antimicrob Agents Chemother. 2005;49:733–40. Not applicable. 15. Price RN, von Seidlein L, Valecha N, Nosten F, Baird JK, White NJ. Global extent of chloroquine-resistant Plasmodium vivax: a systematic review Competing interests and meta-analysis. Lancet Infect Dis. 2014;14:982–91. The authors declare that they have no competing interests. 16. WHO. Methods for surveillance of antimalarial drug efficacy. Geneva: World Health Organization; 2009. Author details 17. Buyon LE, Elsworth B, Duraisingh MT. The molecular basis of antimalarial National Institute of Parasitic Diseases, Chinese Center for Disease Control drug resistance in Plasmodium vivax. Int J Parasitol Drugs Drug Resist. and Prevention, Shanghai, China. Chinese Center for Tropical Diseases 2021;16:23–37. Research, Shanghai, China. NHC Key Laboratory of Parasite and Vector 18. Brega S, Meslin B, de Monbrison F, Severini C, Gradoni L, Udomsangpetch Biology, Shanghai, China. WHO Collaborating Centre for Tropical Diseases; R, et al. 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Genetic polymorphisms in genes associated with drug resistance in Plasmodium vivax parasites from northeastern Myanmar

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10.1186/s12936-022-04084-y
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Abstract

Background: Anti-malarial drug resistance is still a major threat to malaria elimination in the Great Mekong Sub- region. Plasmodium vivax parasites resistant to anti-malarial drugs are now found in Myanmar. Molecular surveillance on drug resistance genes in P. vivax parasites from northeastern Myanmar was aimed at estimating the underlying drug resistance in this region. Methods: Blood samples from patients with vivax malaria were collected from Laiza city in northeastern Myanmar in 2020. Drug resistance genes including Pvcrt-o, Pvmdr1, Pvdhfr and Pvdhps were amplified and sequenced. Genetic polymorphisms and haplotypes were analysed to evaluate the prevalence of mutant alleles associated with drug resistance. Results: A total of 149 blood samples from P. vivax patients were collected. The prevalence of Pvmdr1 mutations at codons 958 and 1076 was 100.0% and 52.0%, respectively, whereas no single nucleotide polymorphism was present at codon 976. The proportions of single and double mutant types were 48.0% and 52.0%, respectively. A K10 “AAG” insertion in the Pvcrt-o gene was not detected. Mutations in Pvdhfr at codons 57, 58, 61, 99 and 117 were detected in 29.9%, 54.3%, 27.6%, 44.9% and 55.1% of the samples, respectively. Wild type was predominant (46.3%), followed by quadruple and double mutant haplotypes. Of three types of tandem repeat variations of Pvdhfr, Type B, with three copies of GGDN repeats, was the most common. Pvdhps mutations were only detected at codons 383 and 553 and the wild type Pvdhps was dominant (78.0%). Eleven haplotypes were identified when combining the mutations of Pvdhfr and Pvdhps, among which the predominant one was the wild type (33.9%), followed by double mutant alleles S58R/S117N /WT (24.6%). Conclusions: This study demonstrated resistant P. vivax phenotypes exists in northeastern Myanmar. Continued surveillance of drug resistance markers is needed to update treatment guidelines in this region. Keywords: Genetic polymorphisms, Drug resistance, Plasmodium vivax, Northeastern Myanmar Background Plasmodium vivax, the most geographically widespread human malaria parasite, causes significant morbid - ity in Southeast Asia, the Western Pacific, Central and South America, and parts of Africa [1]. There were an *Correspondence: huangfang@nipd.chinacdc.cn estimated 2.5  billion people at risk of P. vivax infec- WHO Collaborating Centre for Tropical Diseases; National Center for International Research on Tropical Diseases, Shanghai, China tion worldwide and 6.4  million clinical vivax malaria Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Huang et al. Malaria Journal (2022) 21:66 Page 2 of 9 cases in 2019, mainly distributed in Southeast Asia Sequencing of Pvmdr1 genes from several regions of [2]. In the past 20 years, the burden of P. vivax malaria the world has revealed more than fifty polymorphisms, has decreased dramatically, with many countries in as well as copy number variants (CNVs). Six SNPs have the Asia-Pacific and the Americas reporting reduc - been reported at high frequency in multiple studies in tions of 90% in the number of clinical cases [2]. As a regions with reported drug resistance: S513R, G698S, consequence, 34 countries are actively attempting to M908L, T958M, Y976F and F1076L. Among these, the eliminate malaria, and countries in Central America two most common mutations were Y976F and F1076L. and East Asia have declared their intention to eliminate The essential enzymes dihydropteroate synthase (DHPS) malaria from their regions by 2025 and 2030, respec- and dihydrofolate reductase-thymidylate synthase tively [3, 4]. (DHFR-TS) are involved in folate synthesis and are tar- Myanmar, formerly Burma, has the heaviest malaria gets of sulfadoxine and pyrimethamine, respectively [14, burden in the Greater Mekong Sub-region (GMS), with 25]. SP has been used to treat P. falciparum for a long more cases and deaths than the rest of the region com- time, but it is primarily used for intermittent preventive bined [2]. In recent years, Myanmar has made significant treatment in pregnancy (IPTp) and IPT for infants (IPTi), progress in reducing malaria morbidity and mortality [5– and seasonal malaria chemoprevention combining with 7]. Driven by the emergence and spread of artemisinin amodiaquine in some regions of Africa owing to its wide- resistance in the GMS, the World Health Organization spread resistance [26–28]. (WHO) declared their goal to eliminate malaria in the CQ is also used to treat vivax malaria in Southeast Asia, GMS by 2030 [8]. The National Plan for Malaria Elimina - the prevalence of high CQ-resistant falciparum malaria tion in Myanmar 2016–2030 was developed with the goal resulted for a time in the widespread use of SP, which was of decreasing the annual parasite index (API) to < 1 in available widely in areas where malaria was endemic and all states/regions by 2020, interrupting the transmission was still a first-line treatment in the adjacent countries of falciparum malaria in all states/regions by 2025 and Laos and Myanmar [29]. However, CQ and SP resistant eliminating malaria nationwide by 2030 [5]. However, P. falciparum and P. vivax were reported in Myanmar conflict-affected settings and regions with high popula - several decades ago, especially in southern Myanmar and tion mobility have posed challenges to malaria elimina- at the border between Myanmar and Thailand [29–35]. tion [9]. Recently, increasing clinical failures after CQ treatment Plasmodium vivax resistance to different anti-malarial have been reported in multiple regions of Myanmar drugs, including chloroquine (CQ), mefloquine, sulf - [36, 37]. Currently, the first-line treatment for P. vivax adoxine-pyrimethamine (SP), has been reported in many in Myanmar is CQ combined with primaquine (PQ) [5]. countries or regions [10–15]. Molecular surveillance of In northeastern Myanmar, the treatment efficacy of CQ drug resistance markers, as one of the tools in malaria was found to be relatively high, with a cumulative rate of surveillance, has been widely performed throughout the parasite recurrence less than 3% [35]. The northernmost world [16]. Several candidate drug resistance genes in P. state in Myanmar, with a long border of almost 2000 km vivax parasites have been identified [17]. Plasmodium with Tibet and Yunnan Province in China, mainly in vivax chloroquine resistance transporter (Pvcrt) and Kachin State, also called Jinghpaw Mung, where malaria the P. vivax multidrug resistance transporter (Pvmdr1) has a relatively high rate of transmission [6], resulting have been confirmed to be the Plasmodium falciparum from relatively low access to health services and difficul - orthologs involving CQ resistance genes [18, 19]. The ties in deploying interventions to hard-to-reach commu- Pvcrt-o gene was characterized nearly two decades ago nities [38, 39]. Limited data have defined the molecular [19]; it is similar to its P. falciparum orthologue and has epidemiology of P. vivax resistance markers in north- an intron-rich gene structure with 14 exons that encodes eastern Myanmar. In this study, genetic polymorphisms a protein with 451 amino acids and 10 membrane in genes potentially associated with drug resistance in P. domains [20]. Sequence polymorphism is relatively lim- vivax parasites from northeastern Myanmar were ana- ited in the Pvcrt-o locus but a lysine (AAG) insertion at lysed to estimate the underlying drug resistance, with the amino acid position 10 (K10), which was initially discov- aim of implementing an appropriate drug policy in this ered in Southeast Asian strains, has been suggested to be region. associated with CQ resistance [21]. Since then, this pol- ymorphism has been observed in both Southeast Asian Methods and South American parasites [17, 22, 23]. The Pvmdr1 Study site gene encodes a transmembrane protein of the parasite’s Samples were collected from central hospitals and com- digestive vacuole with 12 transmembrane domains and munity clinics in Laiza (24° 45′ 36″ N, 97° 33′ 48″ E), a 1464 amino acids, and was characterized in 2005 [24]. remote city in Kachin State in northeastern Myanmar, Huang  et al. Malaria Journal (2022) 21:66 Page 3 of 9 along the China-Myanmar border. This region is moun - filter paper (Whatman 903, GE Healthcare, USA), air tainous and its climate is defined as subtropical, with dried, and then stored at − 20 °C before DNA extraction. a dry season from October to April and a rainy season Parasite DNA was extracted from the dried blood spots from May to September. In recent years, malaria cases using a QIAamp DNA Mini Kit (Valencia, CA, USA) fol- have declined dramatically owing to cooperation between lowing the protocol. A polymerase chain reaction (PCR) China and Myanmar towards malaria elimination [38, amplifying the small-subunit rRNA gene of Plasmodium 40]. Local malaria transmission displayed a distinct sea- spp. [43] was performed to confirm the positive sam - sonality with two peaks, a large peak in April-August ples and identify the species before the four genes were and a small peak in November, except 2020 with a mall sequenced. peak in September (Fig. 1). Four human malaria parasites including P. falciparum, P. vivax, Plasmodium ovale and PCR amplification and sequencing Plasmodium malariae have been detected, and P. vivax is The Pvcrt-o gene was amplified by a single-round PCR the predominant species, accounting for more than 90% and Pvmdr1, Pvdhps and Pvdhfr were amplified by nested [41]. On the other side of Laiza city is Nabang township, PCR, as previously described [21, 34, 44–46]. The prim - Yunnan, China, and there is no barrier along the border. ers, cycling conditions and sizes of the PCR products A large number of immigrants from both countries cross are shown in Additional file  1. PCR reactions were per- the border frequently through Nabang port, one of the formed in a 25 µL reaction mixture that contained 1 µL provincial ports along the border. Anopheles minimus of genomic DNA, 12.5 µL 2×Taq Master Mix (TIAN- is reported to be the dominant mosquito species in this GEN, Beijing, China), 9.5 µL ddH O and 10 µM prim- area and most malaria infection occurred in population ers. The amplification products were analysed by 1.5% with subsistence activities associated with forest areas, agarose gel electrophoresis and directly sequenced. The such as logging, banana or rubber planting, and living in PCR products were purified using filter plates (Edge Bio - planting areas during the farming season or entire year systems, Gaithersburg, MD, USA) and sequenced on an [42]. ABI 3730XL automatic sequencer. Bidirectional sequenc- ing was performed, and all the products were sequenced Sample collection and DNA extraction twice using independently amplified PCR products. Blood samples were collected from P. vivax-infected patients prior to anti-malarial drug treatment in the cen- Data analysis tral hospitals and community clinics in Laiza city from The output sequence data were assembled, edited and July to October 2020. Blood samples were spotted on aligned using Geneious (version 2021.0.3) software, Fig. 1 Monthly reported cases of malaria from sentinel sites in Laiza in Kachin State, Myanmar, 2016–2020 Huang et al. Malaria Journal (2022) 21:66 Page 4 of 9 Prevalence of Pvmdr1 mutations and K10‑insertion comparing the samples with the reference sequences in Pvcrt‑o of P vmdr1 (accession number: AY618622), P vdhps Among the 149 samples, 82.6% (123/149) samples showed (accession number: XM001617159) and P vdhfr (acces- successful amplification of Pvmdr1. Mutations in codons sion number: XM001615032) from GenBank. The 958 and 1076 were identified, with a prevalence of 100% known polymorphisms relating to drug resistance at and 52.0% (Fig. 2), respectively. No single nucleotide poly- codons 958, 976, 1076 of the P vmdr1 gene and codons morphism (SNP) was present in codon 976. Analysis of 57, 58, 61, 117, 173 of the P vdhfr gene, and codons the Pvmdr1 haplotype prevalence showed that all the iso- 382, 383, 553, 580 and 558 of P vdhps gene, were lates were of the mutant type. Appropriately half of them evaluated for haplotype. The wild-type haplotypes of carried a single T958M mutation or a double T958M/ P vmdr1, P vdhfr and P vdhps genes were T958/Y976/ F1076L mutation, with a prevalence of 48.0% and 52.0%, F1076, F57/S58/T61/R76/S117/I173 and S382/A383/ respectively (Table  1). The percentage of 88.6% (132/149) A553/R580/V585, respectively. The mixed alleles were samples were successfully sequenced in Pvcrt-o gene, and determined according to the emergence of two chro- K10 “AAG” insertion was  not detected in any of the 132 matogram peaks at one nucleotide site through Muta- successfully sequenced samples (Table 1). tion Surveyor (Soft Genetics LLC., version 5.1, State College, PA, USA). The K10 “AAG” insertion in P vcrt- Polymorphisms in the Pvdhfr gene o gene was determined by comparing with the refer- The Pvdhfr amplicon was successfully sequenced for ence strain of Sal-1 retrieved from Plasmodium data 85.2% (127/149) P. vivax isolates. Amino acid changes in base [47]. The nucleotide sequences were submitted Pvdhfr due to mutations in codons 57, 58, 61, 99 and 117 to GenBank under accession numbers MZ819186- were detected in 29.9%, 54.3%, 27.6%, 44.9% and 55.1% MZ819695. SAS software (SAS Institute Inc, Version of the samples, respectively. In addition, the I13L and 9.2, Cary, NC, USA) was used for data processing and I173L was not detected in present study. The wild type statistical analysis. The chi-squared test and Fisher’s was predominant (46.3%, 57/127). The two most com - exact test were used to evaluate differences among the mon point mutations were S58R (54.3%) and S117T/N different subgroups. P value < 0.05 were used to iden- (55.1%), followed by H99S (44.9%), F57L/I (29.9%), and tify significant differences. T61M (27.6%). Among 70 isolates that carried a point mutation in codon 117, 38 carried 117T whereas 32 car- rying 117 N. Moreover, 38 samples with a point mutation Results in codon 57 included 34 isolates carrying 57I and 4 car- Demographics of patients rying 57 L. A single mutation in codon 117 was identified A total of 149 blood samples from patients with P. in only one isolate. In addition, one synonymous muta- vivax infections were collected before treatment tion in codon 69, TAT to TAC (Y), was detected in two from hospitals and clinics in Laiza city in 2020. All isolates. the patients were confirmed to be infected with P. The haplotype analysis revealed seven distinct allelic vivax by microscopic examination of Giemsa-stained forms of the Pvdhfr gene (Table 1); among them, the wild thick smears through passive case detection. They type was predominant. A quadruple-mutant haplotype, were treated for 3 days with CQ plus 14 days with PQ F57I/S58R/T61M/S117T (26.8%) and a double-mutant according to the anti-malarial drug policy in Myan- haplotype S58R/S117N (24.4%) were the two most com- mar. The majority of the patients were male (70.5%, mon mutant types (Fig.  2). The single-mutant haplo - 105/149) and aged 21–30 years (61.7%, 92/149). The type S117T, triple mutant types S58R/T61M/S117N and median (range) age of the 149 patients was 22 years S58R/T61M/S117T and quadruple-mutant haplotype (ranges: 6–79) (Additional file 2). Fig. 2 Tandem repeat variation between amino acid positions 88 and 103 in the Pvdhfr gene Huang  et al. Malaria Journal (2022) 21:66 Page 5 of 9 Table 1 Frequency of alleles with single or multiple mutations in the Pvmdr1, Pvdhfr, Pvdhps and Pvcrt-o genes from P. vivax isolates Mutations Single/multiple mutations Numbers (%) P value Pvmdr1 < 0.0001 Wild type ( T958/Y976/F1076) 0 Y976F Single mutation 0 T958M Single mutation 59 (48.0) T958M + F1076L Double mutations 64 (52.0) Pvdhfr* < 0.0001 Wild type (F57/S58/T61/S117/I173) 57 (46.3) F57I/L Single mutation 0 S117T Single mutation 1 (0.8) S58R + S117N Double mutations 31 (24.4) S58R + T61M + S117T Triple mutations 2 (1.6) S58R + T61M + S117N Triple mutations 1 (0.8) F57I + S58R + T61M + S117T Quadruple mutations 34 (26.8) F57L + S58R + T61M + S117N Quadruple mutations 1 (0.8) Pvdhps < 0.0001 Wild type (A383/A553) 103 (78.0) A383G Single mutation 0 V585A Single mutation 0 A553G Single mutation 2 (1.5) A383G + A553G Double mutations 27 (20.5) Pvcrt-o Wild type ( Without K10 insertion) 132 (100.0) * Among 32 samples, those with deletion of GGDN repeats from position 95 to 99 were not included. The I13L and I173L mutant alleles were not detected A total of 39 samples with mutations in codon 57 including 34 samples carrying 57I and 4 carrying 57L, but F57I/L only existed in quadruple mutations The chi-squared test or Fisher’s exact test was used to evaluate differences among the frequency of alleles of each gene F57I/S58R/T61M/S117N were rare, being identified in (Table  1). Point mutations in codons 382, 580 and 585 only one, two, one, and one isolate, respectively. were not observed in this study. Variations were identified in the central tandem repeat region between amino acid positions 88 and 103 of Pvd- Analysis of allelic combinations of Pvdhfr and Pvdhps hfr (Fig.  2). Three types of tandem repeat variation were Allelic combinations of the Pvdhfr and Pvdhps enzymes observed: Type A contained three copies of GGDN are responsible for the biosynthesis of folate and are repeats, which was the same as the reference strain potentially under similar drug pressure. A total of 118 (accession number:  X98123.1) designated as the wild isolates were sequenced successfully for both genes. type [48]; Type B also had three copies of GGDN repeats The combinations of point mutations in the Pvdhfr and but showed a mutant allele in codon 99; Type C lacked Pvdhps genes were analyzed in codons 57, 58, 61, and six amino acids from positions 98 to 103. 117, and codons 383 and 553, respectively (Table  2). Eleven haplotypes were identified, and 33.9% (40/118) Polymorphisms in the Pvdhps gene harboured the wild type, as the predominant allele. A percentage of 88.6% (132/149) samples were success- The double Pvdhfr mutant S58R/S117N and wild-type fully assessed for the Pvdhps gene. Pvdhps displayed lim- Pvdhps was the most common mutant haplotype, with ited polymorphisms and a relatively low prevalence of a frequency of 24.6% (29/118). The quadruple-mutant mutations. Nonsynonymous mutations were detected F57I/S58R/T61M/S117T/WT, quadruple Pvdhfr mutant only in codons 383 and 553, with a prevalence of 20.5% F57I/S58R/T61M/S117T and wild-type Pvdhps alleles (27/132) and 22.0% (29/132), respectively. The wild-type or double Pvdhps mutant A383G/A553G were present Pvdhps was dominant (78.0%) (Table  1; Fig.  2). Among at intermediate frequencies of 16.1%, 11.0% and 9.3%, the mutant types, the single-mutant A553G was rare and respectively. Rare haplotypes including single-, triple-, only detected in two isolates, whereas the double-mutant quadruple-, and quintuple-mutant alleles, were detected A383G/A553G was more frequent (20.5%, 27/132) in one isolate each (Table 2). Huang et al. Malaria Journal (2022) 21:66 Page 6 of 9 Table 2 Combination of Pvdhfr and Pvdhps mutations in P. vivax isolates from northeastern Myanmar Haplotypes Pvdhfr/Pvdhps Pvdhfr Pvdhps Number (%) F57I/L S58R T61M S117T/N A383G A553G WT/WT F S T S A A 40 (33.9) WT/A553G F S T S A G 1 (0.8) WT/A383G + A553G F S T S G G 11 (9.3) S58R + S117N/W T F R T N A A 29 (24.6) F57L + S58R + S117N/W T L R T N A A 1 (0.8) F57I + S58R + T61M + S117T/W T I R M T A A 19 (16.1) F57L + S58R + T61M + S117T/W T L R M T A A 1 (0.8) F57L + S58R + S117T/A553G L R T T A G 1 (0.8) S58R + S117N/A383G + A553G F R T N G G 1 (0.8) F57L + S58R + S117T/A383G + A553G L R T T G G 1 (0.8) F57I + S58R + T61M + S117T/A383G + A553G I R M T G G 13 (11.0) Total 118 WT: wild type Pvmdr1 T958M mutation is present in isolates from Discussion different countries having low to high levels of CQ resist - Myanmar has the heaviest malaria burden in the GMS ance, thus T958M appears to be an allelic variant of [49], and its geographical location between Southeast the wild type and is most likely not associated with CQ Asia and South Asia makes it a strategically important resistance [24, 56]. In this study, Pvmdr1 T958M was pre- point for the potential spread of resistant parasites. sent in all the tested samples, whereas F1076L occurred In the present study, genetic polymorphisms in the at an intermediate frequency, with a prevalence of 52.0%. Pvdhps and Pvdhfr genes associated with SP resistance However, Y976F was not detected in this study, although and Pvcrt-o and Pvmdr1, two putative molecular mark- it has been frequently reported in other regions with a ers of resistance to CQ, were analysed to evaluate the high prevalence [18, 21–23, 53, 57]. Another study also level of drug resistance in northeastern Myanmar. found that Y976F was rare in Myanmar [34], which sup- Previous studies have shown that 48.3–72.7% of ports the findings of this study. In addition, the associa - isolates carried Pvcrt-o  K10 insertions in Myanmar tion of the Pvmdr1 substitution Y976F with CQ-resistant between 2009 and 2016 [21, 34], and approximately 19% P. vivax in vitro was confirmed in Papua, Indonesia [53], of the samples from Yunnan Province bordering Myan- while other studies did not find such an association [24]. mar harbored this insertion [50]. In the present study, Pvdhfr and Pvdhps, the targets of SP drugs, disrupt among all the tested samples, the K10 “AAG” insertion folate synthesis in P. vivax [12, 14, 25]. Compared with in the Pvcrt-o gene was not identified, in contrast to Pfdhfr and Pfdhps in P. falciparum, Pvdhfr and Pvdhps the results of previous studies. However, these results are highly conserved in P. vivax [58]. The prevalence of are validated to some extent with the high CQ cure the Pvdhfr mutation (53.7%) was lower in this study rate for P. vivax along the China-Myanmar border [37, than in other studies from the China-Myanmar border, 51]. Interestingly, the K10 insertion is rarely observed southern Thailand and western Myanmar [23, 44, 50, 52]. in Thailand, the border region between Thailand and Moreover, amino acid changes in PvDHFR were detected Myanmar, and the border region between Thailand in 27.6–55.1% of the isolates, which was also lower than and Cambodia [44, 52]. Other studies have reported no the values in other studies [34, 44, 52]. With respect to association between the K10 insertion and in  vitro or Pvdhfr haplotypes, the double-mutant S58R/S117N and ex  vivo P. vivax CQ resistance [22, 53, 54]. Given the quadruple-mutant F57I/S58R/T61M/S117T were domi- geographical genetic differences among parasite popu - nant in northeastern Myanmar and were also the most lations, the prevalence of the Pvcrt-o K10 insertion common types in Myanmar, Thailand and southern shows significant temporal and spatial heterogeneity Yunnan Province, China [50, 52]. A previous study on [55] and the discrepancy may have resulted from dif- the expression of mutant PvDHFR-TS in a yeast system ferences in geography, population movement or sample demonstrated that these mutations confer high levels of size issues. Moreover, the role of the Pvcrt-o K10 inser- pyrimethamine resistance, and the quadruple mutation tion in CQ resistance in the parasite remains unknown. Huang  et al. Malaria Journal (2022) 21:66 Page 7 of 9 resulted in a thousand-fold increase in resistance to variations in drug resistance genes may suggest differ - pyrimethamine [17]. Interestingly, some researchers have ent drug selection pressures imposed on local P. vivax hypothesized that the T61M mutation may be a compen- populations. The factors that influence the evaluation satory mutation to offset possible fitness costs resulting of drug resistance are not completely known, but drug from the carriage of other resistance mutations, because pressure is probably a key element for the selection of a triple Pvdhfr mutant (58R/61M/117T) resulted in sus- resistant parasite mutants. ceptibilities similar to those of the wild-type to all drugs This study had limitations. First, Pvcrt-o and Pvmdr1 with the exception of pyrimethamine, for which this are only putative markers of CQ resistance and the haplotype conferred a 58-fold increase in resistance. In Pvmdr1 copy number was not evaluated in this study. this study, single mutations at residues 58 and 117 were There was limited evidence for CQ resistance without rare, as was the triple mutation, but the double muta- in vitro susceptibility test of P. vivax isolates and in vivo tion S58R/S117N was more common. These results sug - therapeutic efficacy study to highlight therapeutic fail - gest that vivax parasites in northeastern Myanmar may ure. Second, samples were collected only from Laiza city be under low to medium drug pressure. In addition, the through passive case detection, which may not describe majority (74.8%) had three copies of tandem repeats the whole picture of drug resistance in this region. in Pvdhfr gene and Type B was predominant (44.9%, 57/127), followed by Type A (29.9%) and Type C (25.2%). This pattern was different from that reported from south - Conclusions ern China [50], which showed Type C as the main type GMS has become a focus in the war against malaria (40.5%, 118/291), followed by Type B and Type A. How- after the emergence of artemisinin-resistant falciparum ever, the mechanism of action of the tandem repeat vari- malaria, especially in remote areas along the Myanmar ants in pyrimethamine resistance remains unclear [44], border. This study identified genetic polymorphisms in as these variants are not present in the Pfdhfr gene in P. genes associated with drug resistance in P. vivax parasites falciparum [59, 60]. in northeastern Myanmar, which indicated the risks asso- Similar to SP resistance in P. falciparum, mutations ciated with drug resistant P. vivax phenotypes, especially in Pvdhps alone are thought to be insufficient to con - antifolate resistance. Therefore, continued surveillance fer resistance to SP. Strains containing both the Pvdhps of anti-malarial drug resistance markers is needed in A383G and A553G mutations and mutant Pvdhfr alleles northeastern Myanmar to inform case management and have been implicated in SP treatment failure [61]. The update treatment guidelines. Pvdhps mutations exhibited much less diversity and lower prevalence in this study than in previous studies, Supplementary Information which reported a high mutation prevalence (80–90%) The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s12936- 022- 04084-y. in Pvdhps in Malaysia, Thailand, India, Indonesia, and China and high failure rates of SP treatment [14, 52, 62– Additional file 1: Table S1. Primers and cycling conditions for amplifica- 64]. Additionally, the prevalence of Pvdhfr mutations in tion of Pvcrt-o, Pvmdr1, Pvdhps and Pvdhfr by a PCR assay. regions where P. vivax is the dominant parasite is lower Additional file 2: Table S2. Demographics of patients included in this than that in regions where P. falciparum and P. vivax are study. co-endemic; the reason for this may be that the pressure of SP treatment of P. falciparum can lead to co-selection Acknowledgements of Pvdhfr mutant alleles, which could also occur for other We thank all the participants who provided blood samples for this study. drugs, including artemisinin partner drugs [63]. Recently, high treatment efficacy of CQ was found in Authors’ contributions FH conceived and designed the study. FH and YC conducted the laboratory Bangladesh, Bhutan, India and Nepal, but high failure work and data analysis. SL, PT, LJSP and LY collected the blood samples. HL rates of treatment with CQ were confirmed in some and DYB provided technique support for the data collection and analysis. FH sites in Myanmar and Timor-Leste [65]. However, this drafted the manuscript. All authors read and approved the final manuscript. study demonstrated that the frequency of mutant hap- Funding lotypes in northeastern Myanmar was relatively low, This study was supported by the Natural Science Foundation of Shanghai whereas the frequency of the wild type was increasing. (No. 18ZR1443400), the National Important Scientific & Technological Project (2018ZX10101002–002), and the Fifth Round of Three-Year Public Health In addition, genetic polymorphisms were rarer than Action Plan of Shanghai (No. GWV-10.1-XK13). in other regions of Myanmar, as shown in previous reports as well. This result is consistent with the treat - Availability of data and materials The nucleotide sequences were submitted to GenBank under accession num- ment efficacy of CQ reported from northeast Myan - bers MZ819186-MZ819695. The datasets analysed in this study are available mar [35] and the China-Myanmar border [51]. These from the corresponding author on reasonable request. Huang et al. 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Journal

Malaria JournalSpringer Journals

Published: Mar 3, 2022

Keywords: Genetic polymorphisms; Drug resistance; Plasmodium vivax; Northeastern Myanmar

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