• 1.

    World Health Organization, 1981. Synopsis of the world malaria situation in 1979. Wkly Epidemiol Rec 56: 145149.

  • 2.

    Chai IH, Lim GI, Yoon SN, Oh WI, Kim SJ, Chai JY, 1994. Occurrence of tertian malaria in a male patient who has never been abroad. Korean J Parasitol 32: 195200.

    • Search Google Scholar
    • Export Citation
  • 3.

    Park JW, Klein TA, Lee HC, Pacha LA, Ryu SH, Yeom JS, Moon SH, Kim TS, Chai JY, Oh MD, Choe KW, 2003. Vivax malaria: a continuing health threat to the Republic of Korea. Am J Trop Med Hyg 69: 159167.

    • Search Google Scholar
    • Export Citation
  • 4.

    Yeom JS, Ryu SH, Oh S, Lee WJ, Kim TS, Kim KH, Kim YA, Ahn SY, Cha JE, Park JW, 2005. Status of Plasmodium vivax malaria in the Republic of Korea during 2001–2003. Am J Trop Med Hyg 73: 604608.

    • Search Google Scholar
    • Export Citation
  • 5.

    Brega S, Meslin B, de Monbrison F, Severini C, Gradoni L, Udomsangpetch R, Sutanto I, Peyron F, Picot S, 2005. Identification of the Plasmodium vivax mdr-like gene (pvmdr1) and analysis of single-nucleotide polymorphisms among isolates from different areas of endemicity. J Infect Dis 191: 272277.

    • Search Google Scholar
    • Export Citation
  • 6.

    Suwanarusk R, Russell B, Chavchich M, Chalfein F, Kenangalem E, Kosaisavee V, Prasetyorini B, Piera KA, Barends M, Brockman A, Lek-Uthai U, Anstey NM, Tjitra E, Nosten F, Cheng Q, Price RN, 2007. Chloroquine resistant Plasmodium vivax: in vitro characterisation and association with molecular polymorphisms. PLoS One 31: e1089.

    • Search Google Scholar
    • Export Citation
  • 7.

    World Health Organization, 2003. Monitoring Antimalarial Drug Resistance: A Report of a Consultation. Geneva, Switzerland: World Health Organization.

    • Search Google Scholar
    • Export Citation
  • 8.

    Contacos PG, Collins WE, Jeffery GM, Krotoski WA, Howard WA, 1972. Studies on the characterization of Plasmodium vivax strains from Central America. Am J Trop Med Hyg 21: 707712.

    • Search Google Scholar
    • Export Citation
  • 9.

    Yeom JS, Ryu SH, Oh S, Choi DH, Song KJ, Oh YH, Lee JH, Kim YA, Ahn SY, Yang HY, Cha JE, Park JW, 2005. Evaluation of anti-malarial effects of mass chemoprophylaxis in the Republic of Korea army. J Korean Med Sci 20: 707712.

    • Search Google Scholar
    • Export Citation
  • 10.

    Park K, Kang JK, 2003. Effectiveness of primaquine terminal prophylaxis against late primary attacks of Plasmodium vivax malaria: a case-control study among troops of the Republic of Korea army. Trans R Soc Trop Med Hyg 97: 441442.

    • Search Google Scholar
    • Export Citation
  • 11.

    Rombo L, Berqvist Y, Hellgren U, 1987. Chloroquine and desethychloroquine concentrations during regular long-term malaria prophylaxis. Bull World Health Organ 65: 879883.

    • Search Google Scholar
    • Export Citation
  • 12.

    Baird JK, 2004. Chloroquine resistance in Plasmodium vivax. Antimicrob Agents Chemother 48: 40744083.

  • 13.

    Lee SW, Lee M, Lee DD, Kim C, Kim YJ, Kim JY, Green MD, Klein TA, Kim HC, Nettey H, Ko DH, Kim H, Park I, 2009. Biological resistance of hydroxychloroquine for Plasmodium vivax malaria in the Republic of Korea. Am J Trop Med Hyg 81: 600604.

    • Search Google Scholar
    • Export Citation
  • 14.

    Peel SA, 2001. The ABC transporter genes of Plasmodium falciparum and drug resistance. Drug Resist Updat 4: 6674.

  • 15.

    Picot S, Brega S, Gérôme P, Velut G, de Monbrison F, Cheminel V, Peyron F, 2005. Absence of nucleotide polymorphism in a Plasmodium vivax multidrug resistance gene after failure of mefloquine prophylaxis in French Guyana. Trans R Soc Trop Med Hyg 99: 234237.

    • Search Google Scholar
    • Export Citation
  • 16.

    JM, Nomura T, Neves J, Baird JK, Wellems TE, del Portillo HA, 2005. Plasmodium vivax: allele variants of the mdr1 gene do not associate with chloroquine resistance among isolates from Brazil, Papua, and monkey-adapted strains. Exp Parasitol 109: 256259.

    • Search Google Scholar
    • Export Citation
  • 17.

    Suwanarusk R, Chavchich M, Russell B, Jaidee A, Chalfein F, Barends M, Prasetyorini B, Kenangalem E, Piera KA, Lek-Uthai U, Anstey NM, Tjitra E, Nosten F, Cheng Q, Price RN, 2008. Amplification of pvmdr1 associated with multidrug-resistant Plasmodium vivax. J Infect Dis 198: 15581564.

    • Search Google Scholar
    • Export Citation
  • 18.

    Baird JK, Hoffman SL, 2004. Primaquine therapy for malaria. Clin Infect Dis 39: 13361345.

 

 

 

 

Therapeutic Efficacy of Chloroquine in Plasmodium vivax and the pvmdr1 Polymorphisms in the Republic of Korea Under Mass Chemoprophylaxis

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  • Department of Internal Medicine, Yonsei University, Wonju College of Medicine, Wonju, Korea; Konyang University, Myunggok Medical Research Institute, Daejeon, Korea; Department of Preventive Medicine, Yonsei University College of Medicine, Seoul, Korea; Department of Preventive Medicine, The Armed Forces Medical Command, Sungnam, Korea; Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea

Chemoprophylaxis with hydroxychloroquine (HCQ) and primaquine has been used in the Republic of Korea (ROK) Army since 1997. It may facilitate the development of chloroquine (CQ)-resistant strains of Plasmodium vivax. We investigated the therapeutic efficacy of HCQ and the pvmdr1 gene polymorphisms in P. vivax. From June to September 2006, 102 soldiers with vivax malaria near the demilitarized zone in Gyeonggi-do, ROK, were enrolled in the study. We determined the status of compliance of chemoprophylaxis. In 85 patients, therapeutic efficacy was monitored 28 days after standard HCQ treatment; 66 (64.7%) of 102 malaria patients had taken all chemoprolaxis with HCQ. In all patients enrolled in the therapeutic efficacy monitoring, parasitemia had not been observed since 3 days after standard HCQ treatment. However, the ubiquitous presence of the F1076L mutation of the pvmdr1 was observed. There was no evidence that the F1076L mutation of pvmdr1 could contribute to failure of HCQ treatment.

Introduction

By the end of 1970s, the World Health Organization (WHO) declared that Plasmodium vivax had been eradicated in the Republic of Korea (ROK).1 In 1993, one case of malaria reemerged near the demilitarized zone (DMZ) that separates ROK (south) from the Democratic People's Republic of Korea (north).2 Since 1993, the number of malaria cases has increased exponentially, not only among soldiers but also among civilians living near the DMZ.3,4 Although malaria did initially increase exponentially, the increase has since been tempered by control measures. Since 1997, the number of soldiers who received chemoprophylaxis with hydroxychloroquine (HCQ) and primaquine has continuously increased to over 200,000 in 2005. Nonetheless, the number of cases of malaria has increased again since 2005 in the ROK Army. Military antimalarial chemoprophylaxis has contributed, in part, to the decrease in the number of malaria cases among military personnel. However, long-term chemoprophylaxis may facilitate the development of chloroquine (CQ)-resistant strains of P. vivax.

Although the mechanism of P. vivax CQ resistance is unknown, it has been suggested that the mutation of pvmdr1 that is the orthologue of the pfmdr1 of P. falciparum is related to the CQ resistance.5,6

We investigated the breakthrough malaria infection and the therapeutic efficacy of CQ in P. vivax in ROK. Additionally, the pvmdr1 gene polymorphisms and their relevance to CQ resistance in P. vivax were evaluated.

Materials and Methods

Study subjects.

From June 1 to September 30, 2006, we investigated the malaria patients who were admitted to the three referral Armed Forces Hospitals, Byeokje, Yangju, and Ildong Hospitals, located in the Northern Kyunggi province near the DMZ. The patients were diagnosed when they had any febrile illness and P. vivax in peripheral blood films. Blood samples were obtained from all patients who had given the informed consent. The protocol was approved by the Institutional Ethic Committee at the Armed Forces Medical Command (April 13, 2006).

Compliance of chemoprophylaxis.

The chemoprophylaxis with HCQ sulfate (400 mg, one time per week) was started on May 8, 2006, and it continued throughout the transmission season (May to September). Therefore, the chemoprophylaxis was provided throughout the study period. Fourteen-day prophylaxis with primaquine (15 mg base, one time per day) was started on the first day of the last week of CQ administration.

The status of chemoprophylaxis was determined by the military record, which was a malaria case report form submitted by the military doctor after the interview with the patient. According to the status of chemoprophylaxis, the patients were classified as complete compliance when they took all doses before malaria infection, poor compliance when they did not take the dose more than one time before malaria infection, and no prophylaxis when they did not take the dose at all.

Monitoring of therapeutic efficacy.

All patients diagnosed with vivax malaria received standard treatment: 2 g HCQ sulfate (day 1 dose, 1,200 mg; day 2 dose, 400 mg; day 3 dose, 400 mg) and 15 mg primaquine one time daily for 14 days. The patients were followed-up 28 days after the start of antimalarial treatment; the protocol used was a slightly modified version of that antimalarial drug resistance monitoring that was originally suggested by WHO (Table 1).7

Table 1

Modified protocol of the WHO for antimalarial drug monitoring

Day01237142128
Clinical examination and temperature××××××××
Treatment with CQ×××
Parasitemia××××××
Molecular markers×

Identification of pvmdr1 polymorphism.

Parasite genomic DNA from all blood samples collected into ethylenediaminetetraacetic acid (EDTA) tubes was extracted with a QIAamp DNA blood kit (QIAGEN Inc., Valencia, CA) following the manufacturer's instructions with minor modifications: the incubation time with proteinase K was increased to 1 hour at 56°C to improve the yield of the extraction, and DNA was eluted from the column by use of 100 mL polymerase chain reaction (PCR)-grade H2O. Genomic DNA from P. vivax was amplified in a total volume of 20 μL containing 0.20 mmol/L 2'-deoxynucleoside 5'-triphosphate (dNTP), 1 μmol/L each primer, 1 U Platinum Taq DNA Polymerase (Invitrogen), 1.5 mmol/L MgCl2, and 5 μL 10× PCR buffer. The sequences of PCR primers were as follows: pvmdr1-F (5′-CACCTTGCCCTTCTTCGT-3′), pvmdr1-R (5′-TTCACCTTCTTGTTGCAAATAAC-3′), pvmdr1-S (5′-ATAGTCATGCCCCAGGATTG-3′), and pvmdr1-AS (5′-ACGTTTGGTCTGGACAAGTATC-3′). PCR was performed under the following conditions: 37 cycles of denaturation at 94°C (5 min in the first cycle and 30 s in subsequent cycles), annealing for 30 s at 55°C, extension for 30 s at 72°C, and a final primer extension for 7 min. PCR products were sequenced using the ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). The sequences were compared with the pvmdr1 (GenBank accession number AY618622) of Sal-1, a CQ-susceptible strain from Salvador,8 using a basic local alignment search tool (BLAST) search of the National Center for Biotechnology Information databases (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Results

During the study period, a total of 149 patients were diagnosed as P. vivax malaria in the study hospitals. The status of chemoprophylaxis was determined for 102 patients whose military records were available, and the therapeutic efficacy was monitored for 28 days after standard HCQ treatment in the 85 patients who had given the informed consent (Figure 1); 66 (64.7%) patients were classified as complete compliance, 11 (10.8%) patients had poor compliance, and 25 (24.5%) patients had no prophylaxis (Table 2). All peripheral blood smears needed for monitoring had been done in all 85 patients, and all enrolled patients cleared their parasites within 3 days.

Figure 1.
Figure 1.

Study enrollment flowchart.

Citation: The American Society of Tropical Medicine and Hygiene 84, 4; 10.4269/ajtmh.2011.10-0486

Table 2

Baseline characteristics of patients with vivax malaria

Characteristics* (N = 102)Measurement
Mean age (in years) ± standard deviation (range)22.5 ± 3.3 (17–36)
Residency
Northwestern, Kyeonggi (Byeokje)44 (43.1%)
Mid-north, Kyeonggi (Yangju)36 (35.3%)
Northeastern, Kyeonggi (Ildong)23 (22.6%)
Compliance
Complete66 (64.7%)
Poor11 (10.8%)
No25 (24.5%)
Median parasitemia count per microliter (IQR)2,250 (490–8050)

All patients were male.

IQR = interquartile range.

Complete compliance group (2,057/1 μL) vs. poor and no prophylaxis groups (4,558/1 μL).

The PCR was performed from the blood samples that were obtained from the 85 patients who enrolled in therapeutic efficacy monitoring. After PCR, the 78 isolates were obtained successfully from the 85 patients. After sequencing the PCR products, the single-nucleotide polymorphisms of pvmdr1 were analyzed, and the phenylalanine (F) of codon 1076 in pvmdr1 was found to be substituted by leucine (L) in all isolates, including 63 patients with complete compliance. The other mutations, including Y976F, were not found.

Discussion

The mass chemoprophylaxis for the soldiers based in the risk area reduced the number of malaria cases, but long-term chemoprophylaxis could facilitate the development of drug resistance/tolerance or prophylaxis failure.9,10 In this study, 66 patients were defined as complete compliance by the military record. The drug level in whole blood of the patients with chemoprophylaxis was not measured at the malaria infection; it is highly likely that the drug concentration in patients with complete compliance could be higher than 100 ng/mL, because it is usually known that the drug concentration is maintained higher than 100 ng/mL by the long-term CQ prophylaxis given one time per week.11 P. vivax may be classified as biologically resistant if malaria infection that persists in the CQ level in whole blood is greater than 100 ng/mL, and it should completely cure blood-stage CQ-sensitive P. vivax.12 However, Lee and others13 showed that the HCQ and its metabolite could not be found in the blood of 46 of 74 soldiers who had taken HCQ chemoprophylaxis weekly, although the blood concentration of the HCQ and its metabolites was above 100 ng/mL in eight patients. Therefore, it is not truly breakthrough malaria in the 66 patients regarded as complete compliance group.

The pfmdr1 gene of P. falciparum is classified as the adenosine 5′-triphosphate (ATP) binding cassette (ABC) transporter, and it has been known to relate with the resistance against CQ, artemisinin, quinine, mefloquine, and halofantrine.14 The pvmdr1 gene of P. vivax is the orthologue of the pfmdr1 of P. falciparum, and there is controversy regarding whether the pvmdr1 gene relates with the CQ resistance.5,6,15,16 A recent study showed that the amplification of pvmdr1 and single-nucleotide polymorphisms was correlated with susceptibility of P. vivax to multiple antimalarial drugs and that the pvmdr1 Y976F allele had reduced susceptibility to CQ.17 In this study, the F → L change at codon 1076 of the pvmdr1, which was caused by a single mutation (TTT → CTT), was observed in all 78 isolates. Therefore, this ubiquitous presence of the F1076L mutation of the pvmdr1 might be correlated with geographical characteristics in Korean vivax malaria rather than drug resistance.

In this study, no treatment failure was observed after the standard treatment of CQ/primaquine in 85 patients for the 28 days of monitoring. Considering the blood schizonticidal effect of primaqine,18 it could not be determined that there was absolutely no CQ treatment failure, but it could be determined that there was no standard treatment failure.

In this study, there were limitations. For example, the blood levels of CQ and its metabolites, desethylchloroquine and desethylhydroxychloroquine, were not measured after the vivax malaria was developed. In conclusion, there was no treatment failure of HCQ and primaquine in Korea, and there was no evidence that that the F1076L mutation of pvmdr1 could contribute to HCQ resistance in P. vivax.

ACKNOWLEDGMENTS:

The authors thank Kyeongman Jeon and Tae Sung Park in the Armed Forces Capital Hospital for assistance in collecting the data, and we also thank many medical officers in Byeokje, Yangju, and Ildong Hospitals for collecting the blood samples.

  • 1.

    World Health Organization, 1981. Synopsis of the world malaria situation in 1979. Wkly Epidemiol Rec 56: 145149.

  • 2.

    Chai IH, Lim GI, Yoon SN, Oh WI, Kim SJ, Chai JY, 1994. Occurrence of tertian malaria in a male patient who has never been abroad. Korean J Parasitol 32: 195200.

    • Search Google Scholar
    • Export Citation
  • 3.

    Park JW, Klein TA, Lee HC, Pacha LA, Ryu SH, Yeom JS, Moon SH, Kim TS, Chai JY, Oh MD, Choe KW, 2003. Vivax malaria: a continuing health threat to the Republic of Korea. Am J Trop Med Hyg 69: 159167.

    • Search Google Scholar
    • Export Citation
  • 4.

    Yeom JS, Ryu SH, Oh S, Lee WJ, Kim TS, Kim KH, Kim YA, Ahn SY, Cha JE, Park JW, 2005. Status of Plasmodium vivax malaria in the Republic of Korea during 2001–2003. Am J Trop Med Hyg 73: 604608.

    • Search Google Scholar
    • Export Citation
  • 5.

    Brega S, Meslin B, de Monbrison F, Severini C, Gradoni L, Udomsangpetch R, Sutanto I, Peyron F, Picot S, 2005. Identification of the Plasmodium vivax mdr-like gene (pvmdr1) and analysis of single-nucleotide polymorphisms among isolates from different areas of endemicity. J Infect Dis 191: 272277.

    • Search Google Scholar
    • Export Citation
  • 6.

    Suwanarusk R, Russell B, Chavchich M, Chalfein F, Kenangalem E, Kosaisavee V, Prasetyorini B, Piera KA, Barends M, Brockman A, Lek-Uthai U, Anstey NM, Tjitra E, Nosten F, Cheng Q, Price RN, 2007. Chloroquine resistant Plasmodium vivax: in vitro characterisation and association with molecular polymorphisms. PLoS One 31: e1089.

    • Search Google Scholar
    • Export Citation
  • 7.

    World Health Organization, 2003. Monitoring Antimalarial Drug Resistance: A Report of a Consultation. Geneva, Switzerland: World Health Organization.

    • Search Google Scholar
    • Export Citation
  • 8.

    Contacos PG, Collins WE, Jeffery GM, Krotoski WA, Howard WA, 1972. Studies on the characterization of Plasmodium vivax strains from Central America. Am J Trop Med Hyg 21: 707712.

    • Search Google Scholar
    • Export Citation
  • 9.

    Yeom JS, Ryu SH, Oh S, Choi DH, Song KJ, Oh YH, Lee JH, Kim YA, Ahn SY, Yang HY, Cha JE, Park JW, 2005. Evaluation of anti-malarial effects of mass chemoprophylaxis in the Republic of Korea army. J Korean Med Sci 20: 707712.

    • Search Google Scholar
    • Export Citation
  • 10.

    Park K, Kang JK, 2003. Effectiveness of primaquine terminal prophylaxis against late primary attacks of Plasmodium vivax malaria: a case-control study among troops of the Republic of Korea army. Trans R Soc Trop Med Hyg 97: 441442.

    • Search Google Scholar
    • Export Citation
  • 11.

    Rombo L, Berqvist Y, Hellgren U, 1987. Chloroquine and desethychloroquine concentrations during regular long-term malaria prophylaxis. Bull World Health Organ 65: 879883.

    • Search Google Scholar
    • Export Citation
  • 12.

    Baird JK, 2004. Chloroquine resistance in Plasmodium vivax. Antimicrob Agents Chemother 48: 40744083.

  • 13.

    Lee SW, Lee M, Lee DD, Kim C, Kim YJ, Kim JY, Green MD, Klein TA, Kim HC, Nettey H, Ko DH, Kim H, Park I, 2009. Biological resistance of hydroxychloroquine for Plasmodium vivax malaria in the Republic of Korea. Am J Trop Med Hyg 81: 600604.

    • Search Google Scholar
    • Export Citation
  • 14.

    Peel SA, 2001. The ABC transporter genes of Plasmodium falciparum and drug resistance. Drug Resist Updat 4: 6674.

  • 15.

    Picot S, Brega S, Gérôme P, Velut G, de Monbrison F, Cheminel V, Peyron F, 2005. Absence of nucleotide polymorphism in a Plasmodium vivax multidrug resistance gene after failure of mefloquine prophylaxis in French Guyana. Trans R Soc Trop Med Hyg 99: 234237.

    • Search Google Scholar
    • Export Citation
  • 16.

    JM, Nomura T, Neves J, Baird JK, Wellems TE, del Portillo HA, 2005. Plasmodium vivax: allele variants of the mdr1 gene do not associate with chloroquine resistance among isolates from Brazil, Papua, and monkey-adapted strains. Exp Parasitol 109: 256259.

    • Search Google Scholar
    • Export Citation
  • 17.

    Suwanarusk R, Chavchich M, Russell B, Jaidee A, Chalfein F, Barends M, Prasetyorini B, Kenangalem E, Piera KA, Lek-Uthai U, Anstey NM, Tjitra E, Nosten F, Cheng Q, Price RN, 2008. Amplification of pvmdr1 associated with multidrug-resistant Plasmodium vivax. J Infect Dis 198: 15581564.

    • Search Google Scholar
    • Export Citation
  • 18.

    Baird JK, Hoffman SL, 2004. Primaquine therapy for malaria. Clin Infect Dis 39: 13361345.

Author Notes

*Address correspondence to June Myung Kim, Division of Infectious Diseases, Department of Internal Medicine, Yonsei University College of Medicine, 134 Shinchon-Dong, Seodaemun-Gu, Seoul 120-752, Republic of Korea. E-mail: jmkim@yuhs.ac

Financial support: This work was supported in part by Konyang University Myung Gok Research Fund of 2007.

Authors' addresses: Young Keun Kim, Department of Internal Medicine, Yonsei University, Wonju College of Medicine, Kangwon-do, Republic of Korea, E-mail: amoxj@yonsei.ac.kr. Changsoo Kim, Department of Preventive Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea, E-mail: preman@yuhs.ac. Inho Park, Chief of Professional Service, Armed Forces Seoul Hospital, Seoul, Republic of Korea, E-mail: park_in_ho@yahoo.co.kr. Hyo Youl Kim, Yonsei University, Wonju College of Medicine, Kangwon-do, Republic of Korea, E-mail: hyksos@yonsei.ac.kr. Jun Yong Choi and June Myung Kim, Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea, E-mails: seran@yuhs.ac and jmkim@yuhs.ac.

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