Annals of Clinical Microbiology, The official Journal of the Korean Society of Clinical Microbiology

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Original article

Genetic polymorphisms in the pvdhfr, pvmdr1, and pvdhps genes of Plasmodium vivax in patients at a secondary hospital in South Korea

Jaewoong Lee1,2, Hyunjung Kim1,3, Yang Ree Kim4, Haekyung Lee1,3

1Department of Laboratory Medicine, College of medicine, The Catholic University of Korea, Seoul, 2Department of Laboratory Medicine, Incheon St. Mary’s Hospital, The Catholic University of Korea, Incheon, 3Department of Laboratory Medicine, Uijeongbu St. Mary’s Hospital, The Catholic University of Korea, Uijeongbu, 4Division of Infectious Diseases, Department of Internal Medicine, Uijeongbu St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Uijeongbu, Korea

Corresponding to Hae Kyung Lee, E-mail: hkl@catholic.ac.kr

Ann Clin Microbiol 2024;27(1):11-16. https://doi.org/10.5145/ACM.2022.25.1.2
Received on 8 June 2021, Revised on 5 March 2022, Accepted on 5 March 2022, Published on 20 March 2022.
Copyright © Korean Society of Clinical Microbiology.

Abstract

Background: Plasmodium vivax is a major pathogen that causes malaria in Korea. Several genetic polymorphisms in dihydrofolate reductase (pvdhfr), P. vivax multidrug resistance protein 1 (pvmdr1), and P. vivax hydroxymethylpterin pyrophosphokinase-dihydropteroate synthetase (pvdhps) genes are known to be associated with drug resistance in P. vivax. The objective of this study was to profile the known polymorphisms of P. vivax resistance genes in patients at a secondary hospital in Korea. 

Methods: A total of 12 patients with confirmed P. vivax infections were enrolled for this study. Sanger sequencing was performed for the pvdhfr, pvmdr1, and pvdhps genes to detect polymorphisms of these drug resistance genes. 

Results: Each specimen had single or double polymorphism in pvdhfr. One specimen had a polymorphism in pvdhps. However, no specimen had any polymorphisms in pvmdr1. There was no strain with multi-polymorphisms exceeding double polymorphisms, which reported the geographic location of treatment failure. 

Conclusion: No specimen showed chloroquine-resistance polymorphism in pvmdr1. Treatment with first-line therapy was successful. The prevalence of F57L in pvdhfr was higher than that reported previously. This change must be confirmed by further monitoring and surveillance of the strains with multi-polymorphisms.

Keywords

Drug resistance, Genetic polymorphism, Plasmodium vivax, Korea

Introduction

Most Plasmodium vivax infection in Korea occurs near the demilitarized zone (DMZ). From 2014, about 500 cases of P. vivax infections have occurred in Korea every year. Long-term military chemoprophylaxis since 2005 has contributed to a decrease in malaria cases. However, this might have facilitated the development of chloroquine (CQ)-resistant P. vivax strains [1].

CQ has been used for decades as a first-line treatment for uncomplicated P. vivax cases. Since the first report of CQ treatment failures in P. vivax malaria in Papua, Indonesia in 1989 [2,3], reports of antimalarial treatment failures related to genetic polymorphisms in P. vivax dihydrofolate reductase (pvdhfr), P. vivax multidrug resistance protein 1 (pvmdr1) and P. vivax hydroxymethylpterin pyrophosphokinasedihydropteroate synthetase (pvdhps) have increased, causing worldwide concerns about drug-resistant strain spread [49]. Especially, strains with multi-polymorphisms are thought to be the cause of antimalarial treatment failure. Despite the continuous occurrence of malaria infections in Korea, studies of CQ resistance polymorphisms were not reported. Here, we profiled known polymorphisms of P. vivax genes associated with drug resistance from clinical specimens in a secondary hospital in Korea.

Materials and methods

Patients with confirmed P. vivax infections in Uijeongbu St. Mary’s Hospital were enrolled from July 2014 to June 2017. A total of 12 P. vivax infection samples were collected. P. vivax infections were confirmed through SD Bioline Malaria Ag Pf/Pan™ rapid diagnosis test kits (Abbott, Chicago, IL, USA) and referral Plasmodium spp. PCR (polymerase chain reactin; SMLab, Seoul, Korea). Electronic medical records and complete blood count (CBC) findings were reviewed for all patients. In the 2015 World Health Organization (WHO) guidelines [10], antimalarial drug resistance is defined as the survival or increased number of parasites despite the use of an antimalarial drug at an usual recommended dose. According to the definition by the WHO, treatment failure in this study was defined as the persistence or an increase in parasite count despite the first-line therapy.

DNA extraction, PCR, and Sanger sequencing of pvdhfr, pvmdr1, and pvdhps genes were performed following methods reported in previous studies [6,1113]. The following primers were used for PCR and Sanger sequencing [13]: primer PV1F (sense) 5′-CAGTGAAGGGACAAAGAATGAACC-3′, primer PV1R (antisense) 5′-ACTCGGGGAAGAAGACGTCAC-3′ for pvdhfr (560 bps), primer PV7F (sense) 5′-GCCATGTTCATTTCTGAGACGCTG-3′, primer PV7R (antisense) 5′-TCGCTCTGATGGCAAACACTC-3′ for pvmdr1 (337 bps), primer PV9F (sense) 5′-GCGGTTTATTTGTCGATCCTGTG-3′ and primer PV9R (antisense) 5′-TTTTTCCTGGCATCACTTGCTG-3′ for pvdhps (244 bps). Amino acid sequences were compared with reference wild-type sequences, where insertions and deletions were manually verified. Identified single-nucleotide polymorphisms (SNPs), insertions, and deletions were compared with reported SNPs known to be associated with antimalarialresistance in previous studies [5,6,8,11,14,15].

Results

Ten of 12 patient were males. Initial hemoglobin level varied. CBCs on the first hospital visit of the twelve patients showed no specific pattern, although all cases showed cytopenia including various degrees of thrombocytopenia (Table 1). All 12 specimens had polymorphisms in pvdhfr. Only one out of 12 specimens had a polymorphism in pvdhps. No polymorphism was present in pvmdr1. Four previously reported nonsynonymous polymorphisms (F57L, S58R, H99S, S117N) and one previously reported in-frame deletion (c.292_309del, = T98A) were detected in pvdhfr. A nonsynonymous polymorphism (M399I) was detected in pvdhps. F57L in pvdhfr was only found as double polymorphisms, with c.292_309del found in 5 out of 12 cases. Two cases had C.292_309del and three cases had H99S in pvdhfr. One case had both S58R and S117N in pvdhfr. Only one case had M399I in pvdhps, with C.292_309del in pvdhfr (Table 2). All species with H99S did not have tandem repeat insertions or deletions (e.g., C.292_309del) in pvdhfr.

Table 1. CBC results of patients with confirmed Plasmodium vivax infections

Patient No.SexAge (yr)WBC (103/μL)Hb (g/dL)PLT (103/μL)Neu (%)Lym (%)Mono (%)Eos (%)Baso (%)
1M223.5515.14281.012.0 6.00.01.0
2M226.16 6.512266.121.310.61.80.2
3M642.5615.16378.012.0 8.002.0
4F502.4013.16581.212.3 5.70.50.3
5M575.39 9.42886.0 6.0 8.00.00.0
6M643.5113.85382.013.0 3.01.01.0
7M235.0011.76493.0 2.0 4.00.01.0
8M226.4813.610749.038.012.01.00.0
9F684.9611.83192.0 1.0 2.00.00.0
10M294.1515.64051.920.818.06.52.8
11M531.2813.81662.031.0 2.01.00.0
12M431.6715.52680.020.0 0.00.00.0

Abbreviations: CBC, complete blood count; WBC, white blood cells; Hb, hemoglobin; PLT, platelets; Neu, neutrophils; Lym, lymphocytes; Mono, monocytes; Eos, eosinophils; Baso, basophils; M, male; F,female.

Table 2. Identified polymorphisms in pvdhfr of Plasmodium vivax

VariantsNo. of cases
pvdhfr F57L, C.292_309del5
pvdhfr C.292_309del2
pvdhfr H99S3
pvdhfr S58R, S117N1
pvdhfr C.292_309del & pvdhps M399I1

Nine out of 12 cases were cured with first-line regimen of three days of hydroxycholoroquine (HCQ) followed by two weeks of primaquine (PQ). The patient No. 8 with the S58R/S117N double polymorphism in this study recovered after three days of atovaquone (AQ) followed by two weeks of PQ. Patient No. 10 who was transferred out after three days of HCQ was confirmed to be cured later (Table 3).

One of the three H99S patients was lost to follow-up after the first visit. However, the other two patients recovered after three days of HCQ followed by two weeks of PQ. All c.292_309del cases with or without F57L in pvdhfr recovered after three days of HCQ followed by two weeks of PQ. The patient with c.292_309del in pvdhfr and M399I in pvdhps was transferred out after three days of HCQ medication. It was confirmed that this patient recovered later.

Table 3. Clinical course of patients with confirmed Plasmodium vivax infections

Patient No. Medication Parasite count (/μL) [FU day*]
Initial 1st FU 2nd FU 3rd FU 4th FU 5th FU
1 3 days of HCQ followed by 2 weeks of PQ 1,704 305 [2] 298 [3] negative [8]
2 3 days of HCQ followed by 2 weeks of PQ 10,903 negative [4]
3 3 days of HCQ followed by 2 weeks of PQ 6,104 518 [2] 110 [3] 26 [4] negative [5]
4 3 days of HCQ followed by 2 weeks of PQ 1,344 60 [2] 30 [3] 29 [4] 32 [5] negative [9]
5 FU loss after 3 days of AQ 63,117 FU loss
6 3 days of HCQ followed by 2 weeks of PQ 6,809 271 [4] negative [11]
7 3 days of HCQ followed by 2 weeks of PQ 8,400 426 [3] negative [14]
8 3 days of AQ followed by 2 weeks of PQ 12,247 negative [5]
9 3 days of HCQ followed by 2 weeks of PQ 9,313 3,432 [2] 195 [3] 77 [4] negative [5]
10 Transferred out after 3 days of HCQ 428 transferred
11 3 days of HCQ followed by 2 weeks of PQ 36,632 1,432 [2] 164 [3] 152 [4] negative [6]
12 3 days of HCQ followed by 2 weeks of PQ 17,889 11,289 [2] 1,234 [3] 326 [4] 197 [5] negative [6]

*Days from initial medication. Abbreviations: FU, follow-up; HCQ, hydroxycholoroquine; PQ, primaquine; AQ, atovaquone.

Discussion

Among polymorphisms detected in pvdhfr (F57L, S58R, H99S, S117N, and c.292_309del), the S58R/ S117N double polymorphism has been previously reported in Indonesia, Thailand, Ethiopia, China, East Timor, Philippines, Vanuatu, Vietnam, Papua New Guinea, Madagascar, Iran [16], and French Guiana with in vitro resistance to drug combination of sulfadoxine and pyrimethamine (SP) [11]. Because the S58R/ S117N double polymorphism case in this study was treated with AQ and PQ, we could not assume that this polymorphism resulted in SP or CQ resistance in vivo.

It has been reported that the F57L polymorphism in the pvdhfr gene can increase resistance of P. vivax to antifolate agents such as pyrimethamine [4,9]. F57L strains are known to be less prevalent in the region where CQ is used as a first-line regimen [17], which means low selection pressure of antifolate agents. In a previous study [18], pvdhfr F57L was found in 23% (22/97) of P. vivax strains in Korea. In the present study, it was found in 42% (5/12) of P. vivax strains (Table 2). This might be due to the difference in sample size. Actual change within the period between the two studies is also possible.

M399I in the pvdhps gene was reported in a previous report [19], although its association with drug resistance was unknown. This polymorphism was unlikely to cause in vivo CQ resistance because the patient recovered after HCQ medication (Table 3).

Unlike previous reports from other geographic locations, multi-polymorphisms exceeding double polymorphisms were not found in the present study. These reports of multi-polymorphisms are from the region in which antimalarial treatment failures and are thought to be the cause of treatment failures [11,15,16]. This justifies further monitoring of genetic profile and drug resistance of P. vivax in Korea.

Although every specimen had single or double polymorphisms known to cause antimalarial drug resistance, there was no reported treatment failure with first-line therapy. Because all cases except two with AQ therapy were cured with first-line HCQ therapy and none of the 12 cases had polymorphism in pvmdr1, the current regimen of three days of HCQ followed by two weeks of PQ seems to be proper as a first-line therapy in Korea.

To the best of our knowledge, this is the first study analyzing polymorphisms in pvdhfr, pvdhps and pvmdr1 genes altogether in Korea. Although all cases were susceptible to first-line therapy, pvdhfr F57L proportion in this study was higher than that in a previous report. Whether this change was true without a selection pressure by antifolate drugs or it was just an error due to small sample size was unclear. This is a limitation of this study. To clarify this change and to surveil emergence of multi-polymorphism strain reported abroad, further monitoring of genetic profile and drug resistance of P. vivax is needed.

Ethics statement

This study protocol was approved by the Institutional Review Board of the Catholic University of Korea (IRB No. UC14TISI0006). This study was performed in accordance with the Declaration of Helsinki. Written consent was obtained from all patients in accordance with local regulations.

Conflicts of interest

No potential conflicts of interest relevant to this article were reported.

Funding

None.

References

1. Kim YK, Kim C, Park I, Kim HY, Choi JY, Kim JM. Therapeutic efficacy of chloroquine in Plasmodium vivax and the pvmdr1 polymorphisms in the Republic of Korea under mass chemoprophylaxis. Am J Trop Med Hyg 2011;84:532-4.

2. Rieckmann KH, Davis DR, Hutton DC. Plasmodium vivax resistance to chloroquine? Lancet 1989;2:1183-4.

3. Schwartz IK, Lackritz EM, Patchen LC. Chloroquine-resistant Plasmodium vivax from Indonesia. N Engl J Med 1991;324:927.

4. Tjitra E, Baker J, Suprianto S, Cheng Q, Anstey NM. Therapeutic efficacies of artesunatesulfadoxine-pyrimethamine and chloroquine-sulfadoxine-pyrimethamine in vivax malaria pilot studies: relationship to Plasmodium vivax dhfr mutations. Antimicrob Agents Chemother 2002;46:3947-53.

5. Barnadas C, Ratsimbasoa A, Tichit M, Bouchier C, Jahevitra M, Picot S, et al. Plasmodium vivax resistance to chloroquine in Madagascar: clinical efficacy and polymorphisms in pvmdr1 and pvcrt-o genes. Antimicrob Agents Chemother 2008;52:4233-40.

6. Orjuela-Sánchez P, de Santana Filho FS, Machado-Lima A, Chehuan YF, Costa MR, Alecrim M, et al. Analysis of single-nucleotide polymorphisms in the crt-o and mdr1 genes of Plasmodium vivax among chloroquine-resistant isolates from the Brazilian Amazon region. Antimicrob Agents Chemother 2009;53:3561-4.

7. Vargas-Rodríguez Rdel C, da Silva Bastos M, Menezes MJ, Orjuela-Sánchez P, Ferreira MU. Single-nucleotide polymorphism and copy number variation of the multidrug resistance-1 locus of Plasmodium vivax: local and global patterns. Am J Trop Med Hyg 2012;87:813-21.

8. Lu F, Wang B, Cao J, Sattabongkot J, Zhou H, Zhu G, et al. Prevalence of drug resistanceassociated gene mutations in Plasmodium vivax in Central China. Korean J Parasitol 2012;50:379-84.

9. Khattak AA, Venkatesan M, Khatoon L, Ouattara A, Kenefic LJ, Nadeem MF, et al. Prevalence and patterns of antifolate and chloroquine drug resistance markers in Plasmodium vivax across Pakistan. Malar J 2013;12:310.

10. WHO. WHO guidelines approved by the guidelines review committee. In: Guidelines for the treatment of malaria. Geneva; World Health Organization, 2015.

11. Barnadas C, Musset L, Legrand E, Tichit M, Briolant S, Fusai T, et al. High prevalence and fixation of Plasmodium vivax dhfr/dhps mutations related to sulfadoxine/pyrimethamine resistance in French Guiana. Am J Trop Med Hyg 2009;81:19-22.

12. Hawkins VN, Joshi H, Rungsihirunrat K, Na-Bangchang K, Sibley CH. Antifolates can have a role in the treatment of Plasmodium vivax. Trends Parasitol 2007;23:213-22.

13. Marques MM, Costa MR, Santana Filho FS, Vieira JL, Nascimento MT, Brasil LW, et al. Plasmodium vivax chloroquine resistance and anemia in the western Brazilian Amazon. Antimicrob Agents Chemother 2014;58:342-7.

14. Kuesap J, Rungsrihirunrat K, Thongdee P, Ruangweerayut R, Na-Bangchang K. Change in mutation patterns of Plasmodium vivax dihydrofolate reductase (Pvdhfr) and dihydropteroate synthase (Pvdhps) in P. vivax isolates from malaria endemic areas of Thailand. Mem Inst Oswaldo Cruz 2011;106 (S) 1:130-3.

15. Mint Lekweiry K, Ould Mohamed Salem Boukhary A, Gaillard T, Wurtz N, Bogreau H, Hafid JE, et al. Molecular surveillance of drug-resistant Plasmodium vivax using pvdhfr, pvdhps and pvmdr1 markers in Nouakchott, Mauritania. J Antimicrob Chemother 2012;67:367-74.

16. Gama BE, Oliveira NK, Souza JM, Daniel-Ribeiro CT, Ferreira-da-Cruz Mde F. Characterisation of pvmdr1 and pvdhfr genes associated with chemoresistance in Brazilian Plasmodium vivax isolates. Mem Inst Oswaldo Cruz 2009;104:1009-11.

17. Valecha N, Joshi H, Eapen A, Ravinderan J, Kumar A, Prajapati SK, et al. Therapeutic efficacy of chloroquine in Plasmodium vivax from areas with different epidemiological patterns in India and their Pvdhfr gene mutation pattern. Trans R Soc Trop Med Hyg 2006;100:831-7.

18. Lu F, Lim CS, Nam DH, Kim K, Lin K, Kim TS, et al. Mutations in the antifolate-resistanceassociated genes dihydrofolate reductase and dihydropteroate synthase in Plasmodium vivax isolates from malaria-endemic countries. Am J Trop Med Hyg 2010;83:474-9.

19. Choi YK. Genetic diversity and antimalarial drug resistance of Plasmodium vivax in Korea [dissertation]. Seoul: Kyung Hee University; 2010.

Table 1
Table 2
Table 3

1. Kim YK, Kim C, Park I, Kim HY, Choi JY, Kim JM. Therapeutic efficacy of chloroquine in Plasmodium vivax and the pvmdr1 polymorphisms in the Republic of Korea under mass chemoprophylaxis. Am J Trop Med Hyg 2011;84:532-4.

2. Rieckmann KH, Davis DR, Hutton DC. Plasmodium vivax resistance to chloroquine? Lancet 1989;2:1183-4.

3. Schwartz IK, Lackritz EM, Patchen LC. Chloroquine-resistant Plasmodium vivax from Indonesia. N Engl J Med 1991;324:927.

4. Tjitra E, Baker J, Suprianto S, Cheng Q, Anstey NM. Therapeutic efficacies of artesunatesulfadoxine-pyrimethamine and chloroquine-sulfadoxine-pyrimethamine in vivax malaria pilot studies: relationship to Plasmodium vivax dhfr mutations. Antimicrob Agents Chemother 2002;46:3947-53.

5. Barnadas C, Ratsimbasoa A, Tichit M, Bouchier C, Jahevitra M, Picot S, et al. Plasmodium vivax resistance to chloroquine in Madagascar: clinical efficacy and polymorphisms in pvmdr1 and pvcrt-o genes. Antimicrob Agents Chemother 2008;52:4233-40.

6. Orjuela-Sánchez P, de Santana Filho FS, Machado-Lima A, Chehuan YF, Costa MR, Alecrim M, et al. Analysis of single-nucleotide polymorphisms in the crt-o and mdr1 genes of Plasmodium vivax among chloroquine-resistant isolates from the Brazilian Amazon region. Antimicrob Agents Chemother 2009;53:3561-4.

7. Vargas-Rodríguez Rdel C, da Silva Bastos M, Menezes MJ, Orjuela-Sánchez P, Ferreira MU. Single-nucleotide polymorphism and copy number variation of the multidrug resistance-1 locus of Plasmodium vivax: local and global patterns. Am J Trop Med Hyg 2012;87:813-21.

8. Lu F, Wang B, Cao J, Sattabongkot J, Zhou H, Zhu G, et al. Prevalence of drug resistanceassociated gene mutations in Plasmodium vivax in Central China. Korean J Parasitol 2012;50:379-84.

9. Khattak AA, Venkatesan M, Khatoon L, Ouattara A, Kenefic LJ, Nadeem MF, et al. Prevalence and patterns of antifolate and chloroquine drug resistance markers in Plasmodium vivax across Pakistan. Malar J 2013;12:310.

10. WHO. WHO guidelines approved by the guidelines review committee. In: Guidelines for the treatment of malaria. Geneva; World Health Organization, 2015.

11. Barnadas C, Musset L, Legrand E, Tichit M, Briolant S, Fusai T, et al. High prevalence and fixation of Plasmodium vivax dhfr/dhps mutations related to sulfadoxine/pyrimethamine resistance in French Guiana. Am J Trop Med Hyg 2009;81:19-22.

12. Hawkins VN, Joshi H, Rungsihirunrat K, Na-Bangchang K, Sibley CH. Antifolates can have a role in the treatment of Plasmodium vivax. Trends Parasitol 2007;23:213-22.

13. Marques MM, Costa MR, Santana Filho FS, Vieira JL, Nascimento MT, Brasil LW, et al. Plasmodium vivax chloroquine resistance and anemia in the western Brazilian Amazon. Antimicrob Agents Chemother 2014;58:342-7.

14. Kuesap J, Rungsrihirunrat K, Thongdee P, Ruangweerayut R, Na-Bangchang K. Change in mutation patterns of Plasmodium vivax dihydrofolate reductase (Pvdhfr) and dihydropteroate synthase (Pvdhps) in P. vivax isolates from malaria endemic areas of Thailand. Mem Inst Oswaldo Cruz 2011;106 (S) 1:130-3.

15. Mint Lekweiry K, Ould Mohamed Salem Boukhary A, Gaillard T, Wurtz N, Bogreau H, Hafid JE, et al. Molecular surveillance of drug-resistant Plasmodium vivax using pvdhfr, pvdhps and pvmdr1 markers in Nouakchott, Mauritania. J Antimicrob Chemother 2012;67:367-74.

16. Gama BE, Oliveira NK, Souza JM, Daniel-Ribeiro CT, Ferreira-da-Cruz Mde F. Characterisation of pvmdr1 and pvdhfr genes associated with chemoresistance in Brazilian Plasmodium vivax isolates. Mem Inst Oswaldo Cruz 2009;104:1009-11.

17. Valecha N, Joshi H, Eapen A, Ravinderan J, Kumar A, Prajapati SK, et al. Therapeutic efficacy of chloroquine in Plasmodium vivax from areas with different epidemiological patterns in India and their Pvdhfr gene mutation pattern. Trans R Soc Trop Med Hyg 2006;100:831-7.

18. Lu F, Lim CS, Nam DH, Kim K, Lin K, Kim TS, et al. Mutations in the antifolate-resistanceassociated genes dihydrofolate reductase and dihydropteroate synthase in Plasmodium vivax isolates from malaria-endemic countries. Am J Trop Med Hyg 2010;83:474-9.

19. Choi YK. Genetic diversity and antimalarial drug resistance of Plasmodium vivax in Korea [dissertation]. Seoul: Kyung Hee University; 2010.