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The mechanism of chloroquine (CQ) resistance in Plasmodium falciparum has been investigated and mutations in the P. falciparum CQ resistance transporter gene (pfcrt) located on chromosome 7, and the P. falciparum multidrug resistant gene 1 (pfmdr1), located on chromosome 5, have been implicated. The substitution of threonine for lysine in codon 76, K76T in the pfcrt gene, was shown in vitro to be associated with CQ resistance in isolates from Asia, Africa, South America, and Papua New Guinea.1,2 Sequence polymorphisms at position 72–76 of this gene have been associated with the geographic origin of parasite samples, with the CVIET pattern in resistant isolates from Asia and Africa, and with SVMNT in resistant isolates from Papua New Guinea and South America.1,3 The multidrug resistant gene pfmdr1 with a mutation of asparagine to tyrosine at position 86 (N86Y) has been associated with in vitro resistant strains.4 Although its participation is not clear, it has been suggested that the pfmdr1 mutation may confer some advantage to the parasite in the presence of CQ, thus increasing the level of CQ resistance.2,5 Furthermore, a recent study that included samples from four countries of Southeast Asia described the mutations N86Y in pfmdr1 and K76T in pfcrt genes as molecular markers for predicting clinical outcome of CQ treatment.6
In Indonesia, the first cases of resistance were reported in the early 1970s from Kalimantan and Irian Jaya. Although resistance has been reported on several islands in Indonesia,7 with resistance as high as 95% for P. falciparum and 84% for P. vivax,8 CQ continues to be the first-line treatment of P. falciparum and P. vivax malaria because of its safety and availability at very low cost. Here we examined the prevalence of polymorphisms in the pfmdr1 and pfcrt genes in 48 P. falciparum isolates from the West Lombok District of Indonesia. In addition, the possible origin of CQ resistance in Indonesia is discussed.
Blood samples were collected from 48 patients with uncomplicated P. falciparum malaria in sub-district Batulayar in West Lombok in the West Nusa Tenggara Province of Indonesia (Figure 1
) from June to September 2002. Sub-district Batulayar has a population of approximately 35,658. The climate is tropical and malaria transmission occurs more frequently during dry season between April and October, although low-level transmission occurs throughout the year, especially in the hilly-forested ranges of Sidemen to Pusuk. Cases of P. falciparum and P. vivax malaria and a few cases of P. malariae malaria have been reported in the area. The recommended first-line treatment is CQ, 25 mg/kg given over a three-day period. When a treatment failure occurs, the combination of sulfadoxine/pyrimethamine is prescribed. Inclusion criteria were a fever 
37.5°C during the last 48 hours and a positive result in the NOW ICT® (Binax, Portland, ME) rapid malaria test. Blood samples were collected on filter paper and transported to the Muninting district health center laboratory for malaria testing. Samples positive for P. falciparum malaria were processed thereafter in Japan at the Department of Protozoology of Nagasaki University. Informed consent was obtained from each individual. The study was reviewed and approved by the ethical committee of the Institute of Tropical Medicine of Nagasaki University and the executive committee of the Malaria Control Project in Lombok and Sumbawa under the Japanese International Cooperation Agency partnership program.
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To determine the presence of tyrosine at position 86 in pfmdr1, a nested polymerase chain reaction (PCR)-restriction fragment length polymorphism protocol was used as previously described.9 Digestion with the restriction endonuclease Apo I (New England Biolabs, Inc., Beverly, MA) detects tyrosine at position 86 after resolution of the products by electrophoresis on 1–3% agarose gels (Nusieve 3:1; BioWhittaker Molecular Applications, Rockand, ME). For pfcrt gene analysis, a first amplification was carried out with previously designed primers,9 and the products obtained were used as a template in a nested PCR encompassing the polymorphic codons 72–76 and 97 in exon 2 as previously reported.3 The PCR amplification products were purified using the QIAquick PCR purification kit (Qiagen) and directly sequenced on an ABI310 automated sequencer using ABI PRISM Big Dye Terminator Cycle kit (Applied Biosystems, Foster City, CA) following the manufacturer’s instructions.
The glurp gene, located on chromosome 11, which has a high degree of polymorphism, was assessed for evaluation of diversity of the P. falciparum isolates population in the region.10 The amplification products were resolved by electrophoresis on a 1% agarose gel and stained with ethidium bromide. The glurp amplification product sizes were estimated using DNAfrag version 3.03 Software (John Nash, Institute for Biologic Sciences, National Research Council of Canada, Ottawa, Ontario, Canada). For comparisons, Fisher’s exact test was used.
Seventeen isolates (35.4%) had the 86Y mutation in the pfmdr1 gene, 26 had wild type N86, and 5 carried both alleles (Table 1). In previous studies in Irian Jaya and West Papua, N86Y was found to show a correlation with CQ-resistant P. falciparum parasites.11,12 The mutation K76T in the pfcrt gene was found in all but one (47 of 48, 97.9%) of the isolates studied. A previous report showed that the K76T mutation showed a correlation with clinical resistance to CQ and as a molecular marker had a sensitivity of 93% and a specificity of 82%.13 Both point mutations in the pfmdr1 and pfcrt genes have been proposed to indicate a tendency toward reduced susceptibility to CQ. Thus, our results suggest potential CQ resistance in the region, although other factors may influence the final treatment outcome. The combination of geographic remoteness to health facilities and lack of interest in seeking medical attention driven by both financial reasons and lack of knowledge resulted in an overall follow-up rate of 23% (11 of 48). After 14 days of CQ treatment, 5 of 11 patients were not able to clear the parasites, and had tendency to harbor 86Y in the pfmdr1 gene (Table 1). However, a larger sample size is required to obtain conclusive results.
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The sequence analysis of codons 72–76 in the pfcrt gene (Table 1
) allowed identification of previously reported haplotypes SagtVMNT (42 of 48, 87.5%) and CVIET (5 of 48, 10.4%). The pfcrt SVMNT haplotype with serine coded by AGT has been found in Bougainville, Papua New Guinea,16 the main island of Papua New Guinea,3 and East Timor.17 The haplotype CVIET has been reported in countries of Southeast Asia.1,18 Since Lombok, Indonesia is located near Papua New Guinea (Figure 1), it is not unexpected that both the SVMNT and CVIET haplotypes were detected.
Furthermore, in our attempt to evaluate the diversity among the isolates studied, we assessed the glurp gene and 11 glurp genotypes were found in West Lombok, ranging from 450 to 1,100 basepairs. The West Lombok District, despite its small area, shows a high degree of diversity in the P. falciparum population that might be a product of high rate of transmission of malaria or human transmigration.
Upon examination for any linkage among the alleles studied in the pfmdr1, pfcrt, and glurp genes, significant associations were found between the pfcrt CVIET haplotype and pfmdr1 86Y (P = 0.0193), the CVIET haplotype and glurp 450 (P < 0.001), and pfmdr1 86Y and glurp 450 (P = 0.0057). Our findings showed that the majority of isolates have pfcrt haplotype SVMNT, pfmdr1 86N, and glurp with molecular masses greater than 450 homogeneously distributed in all the villages from West Lombok, indicating that these might be indigenous in the area. A few isolates harboring pfcrt haplotype CVIET, pfmdr1 86Y, and glurp 450, found mainly in Kedondong Atas village, were most likely introduced recently. Therefore, CQ resistance in Lombok might have the same origin as the Papua New Guinea strains, and the Southeast Asian pfcrt haplotype CVIET might have been introduced only recently in a particular region. Further studies are being carried out in isolates from Lombok and other Indonesian islands to determine the prevalence of the novel K76N mutation and its association with clinical outcome/in vitro susceptibility to CQ.
Received December 16, 2003. Accepted for publication February 4, 2004.
Acknowledgments: We are grateful to the Case Detection and Treatment Team at the Meninting Health Center (West Lombok, Indonesia) for their collaboration in this study.
Financial support: This study was partially supported by the Japanese International Cooperation Agency partnership program, and by grant for International Health Cooperation Research (13-C-5) from the Ministry of Health, Labor and Welfare. Maria Cecilia Huaman is the recipient of the Monbusho scholarship awarded by the Ministry of Education, Science, Sports and Culture of the Government of Japan.
Authors’ addresses: Maria Cecilia Huaman, Institute of Tropical Medicine, Nagasaki University, Sakamoto 1-12-4, Nagasaki 852-8523, Japan and Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Peru, E-mail: ceci_ huaman{at}yahoo.com. Kazumi Yoshinaga and Hiroji Kanbara, Institute of Tropical Medicine, Nagasaki University, Sakamoto 1-12-4, Nagasaki 852-8523, Japan, Telephone: 81-95-849-7838, Fax: 81-95-849-7805. E-mail: f0512{at}cc.nagasaki-u.ac.jp. Aan Suryanatha and Nyoman Suarsana, Meninting Health Center, West Lombok, Indonesia.
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