Genetic Polymorphisms in the pvdhfr, pvmdr1, and pvdhps Genes of Plasmodium vivax in Patients at a Secondary Hospital in South Korea
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
ABSTRACT
Background: Plasmodium vivax is a major pathogen that causes malaria in South 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 South 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, South Korea
INTRODUCTION
Most Plasmodium vivax infection in South Korea occurs near the demilitarized zone (DMZ). From 2014, about 500 cases of P. vivax infections have occurred in South 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 pyrophosphokinase-dihydropteroate synthetase (pvdhps) have increased, causing worldwide concerns about drug-resistant strain spread [4-9]. Especially, strains with multi-polymorphisms are thought to be the cause of antimalarial treatment failure. Despite the continuous occurrence of malaria infections in South 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 South Korea.
MATERIALS AND METHODS
Patients with confirmed P. vivax infections in Uijeongbu St. Mary’s Hospital were enrolled from July 2014 to June 2017. 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. 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 [6,11-13]. 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′-GCGGTTT-ATTTGTCGATCCTGTG-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 antimalarial-resistance 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 confirmedP lasmodium vivax infections
|
Patient No. |
Sex |
Age (yr) |
WBC (103/μL) |
Hb (g/dL) |
PLT (103/μL) |
Neu (%) |
Lym (%) |
Mono (%) |
Eos (%) |
Baso (%) |
|
1 |
M |
22 |
3.55 |
15.1 |
42 |
81.0 |
12.0 |
6.0 |
0.0 |
1.0 |
|
2 |
M |
22 |
6.16 |
6.5 |
122 |
66.1 |
21.3 |
10.6 |
1.8 |
0.2 |
|
3 |
M |
64 |
2.56 |
15.1 |
63 |
78.0 |
12.0 |
8.0 |
0 |
2.0 |
|
4 |
F |
50 |
2.40 |
13.1 |
65 |
81.2 |
12.3 |
5.7 |
0.5 |
0.3 |
|
5 |
M |
57 |
5.39 |
9.4 |
28 |
86.0 |
6.0 |
8.0 |
0.0 |
0.0 |
|
6 |
M |
64 |
3.51 |
13.8 |
53 |
82.0 |
13.0 |
3.0 |
1.0 |
1.0 |
|
7 |
M |
23 |
5.00 |
11.7 |
64 |
93.0 |
2.0 |
4.0 |
0.0 |
1.0 |
|
8 |
M |
22 |
6.48 |
13.6 |
107 |
49.0 |
38.0 |
12.0 |
1.0 |
0.0 |
|
9 |
F |
68 |
4.96 |
11.8 |
31 |
92.0 |
1.0 |
2.0 |
0.0 |
0.0 |
|
10 |
M |
29 |
4.15 |
15.6 |
40 |
51.9 |
20.8 |
18.0 |
6.5 |
2.8 |
|
11 |
M |
53 |
1.28 |
13.8 |
16 |
62.0 |
31.0 |
2.0 |
1.0 |
0.0 |
|
12 |
M |
43 |
1.67 |
15.5 |
26 |
80.0 |
20.0 |
0.0 |
0.0 |
0.0 |
|
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
|
Variants |
No. of cases |
|
pvdhfr F57L, C.292_309del |
5 |
|
pvdhfr C.292_309del |
2 |
|
pvdhfr H99S |
3 |
|
pvdhfr S58R, S117N |
1 |
|
pvdhfr C.292_309del & pvdhps M399I |
1 |
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).
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. |
|||||||
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.
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 South 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 South 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 South Korea.
To the best of our knowledge, this is the first study analyzing polymorphisms in pvdhfr, pvdhps and pvmdr1 genes altogether in South 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.
요약
배경: Plasmodium vivax (P. vivax)는 국내 말라리아 감염증의 주 감염원이다. 기존 연구에서 P. vivax의 유전자 다형성 중 약제내성에 관련된 것으로 보고된 것으로는 p. vivax dihydrofolate reductase (pvdhfr), p. vivax multidrug resistance protein 1 (pvmdr1), p. vivax hydroxymethylpterin pyrophosphokinase-dihydropteroate synthetase (pvdhps) 유전자의 다형성이 있다. 본 연구에서는 한국의 2차 병원에서 진단된 감염 증례에서 P. vivax 유전자의 약제내성 관련 다형성 프로필을 분석하였다.
방법: P. vivax 감염이 확인된 12명의 환자가 연구에 포함되었다. 각 증례에서 P. vivax의 pvdhfr, pvmdr1, pvdhps 유전자의 약제내성 관련 다형성 확인을 위해 Sanger sequencing을 수행하였다.
결과: 12건의 증례 모두에서 1개 또는 2개의 pvdhfr 다형성이 관찰되었고, 한 개 증례에서 pvdhps 다형성이 관찰되었다. pvmdr1의 다형성은 관찰되지 않았다. 치료실패가 보고된 지역에서 관찰된 3개 이상의 다중 다형성을 보유한 원충 주는 관찰되지 않았다.
결론: 클로로퀸 내성과 관련된 pvmdr1의 변이는 관찰되지 않았고 모든 증례는 1차 치료에서 치료되었다. pvdhfr의 F57L변이 빈도는 이전의 보고에 비해 높게 관찰되었다. 이 변화를 좀 더 확실히 확인하고 다중 다형성 원충 주가 출현하는지 감시하기 위해서는 추가적인 모니터링이 필요하다.
CONFLICTS OF INTEREST
No potential con.icts of interest relevant to this article were reported.
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