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    Map of the Indonesian archipelago and the sampling sites (boxes) of the isolates of Plasmodium falciparum.

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    Comparison of polymorphisms identified in west and east Indonesia. The 1042D polymorphism of the Plasmodium falciparum multidrug resistance 1 (pfmdr1) gene was only found in eastern Indonesia. pfcrt = P. falciparum chloroquine resistance transporter; dhfr = dihydrofolate reductase; dhps = dihydropteroate synthase.

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MOLECULAR EPIDEMIOLOGY OF PLASMODIUM FALCIPARUM RESISTANCE TO ANTIMALARIAL DRUGS IN INDONESIA

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  • 1 Eijkman Institute for Molecular Biology, Jakarta, Indonesia; Department of Parasitology, Faculty of Medicine, Hasanuddin University, Makassar, Indonesia; United States Naval Medical Research Unit No. 2, Jakarta, Indonesia; The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia, Papua New Guinea Institute of Medical Research, Goroka, Papua New Guinea

The extent of gene polymorphisms associated with resistance to chloroquine and sulfadoxine-pyrimethamine was examined in field isolates of Plasmodium falciparum from Indonesia. Eight malaria-endemic areas, representing a broad region of the western and eastern Indonesian Archipelago were surveyed. Blood from 20–50 patients was collected at each site, DNA was isolated, and the sequences of four different genes (dihydrofolate reductase [dhfr], dihydropteroate synthase [dhps], P. falciparum multidrug resistance 1 [pfmdr1], and P. falciparum chloroquine resistance transporter [pfcrt]) were analyzed using polymerase chain reaction and restriction fragment length polymorphisms to detect polymorphisms previously shown to be associated with resistance. This analysis identified polymorphisms in dhfr at 108-Asn/Thr, 16-Val, and 59-Arg. Polymorphisms in dhps were found less frequently, either 437-Gly alone or paired with 540-Glu. The pfcrt 76-Thr polymorphism was fixed in all parasite populations and pfmdr1 86-Tyr polymorphisms in all populations except in the most eastern regions. The pfmdr1 1042-Asp polymorphism occurred less frequently. These findings indicate that polymorphisms in genes associated with drug resistance in P. falciparum are found across a broad region of Indonesia.

INTRODUCTION

Since the first reports of chloroquine resistance in East Kalimantan and Indonesian Papua in 1975,1 chloroquine resistance has been observed in all areas of the archipelago where in vivo/in vitro studies have been conducted.2–12 As a consequence of this widespread resistance, a combination of the antifolates sulfadoxine and pyrimethamine was introduced as a second-line treatment of simple malaria. The synergistic combination of the two, which inhibit dihydropteroate synthase and dihydrofolate reductase, respectively, in the folate biosynthetic pathway, was believed to enhance their antimalarial potency and reduce the risk of drug resistance.13,14 However, resistance to this drug combination had already been observed in Indonesia,1 and has now spread across the archipelago.3,8,10,15–17 The resistance of malaria parasites to Fansidar® (Fansidar Hoffmann La Roche, Basel, Switzerland), the most commonly used combination of sulfadoxine-pyrimethamine, is now widespread in southeast Asia.18–22 Despite the spread of resistance to these antimalarials in Indonesia, chloroquine and sulfadoxine-pyrimethamine are still used as first-line and second-line antimalarial drugs, respectively.

Molecular studies over the last few decades have identified several mutations associated with chloroquine and sulfadoxine-pyrimethamine resistance in a number of Plasmodium falciparum genes. Polymorphisms in the P. falciparum chloroquine resistance transporter (pfcrt) gene, located on chromosome 7, were proposed to be important in chloroquine resistance and transfection experiments have shown that the polymorphism 76-Ser to Thr is tightly linked to the resistance phenotype.23,24 Additionally, polymorphisms in the P. falciparum multidrug resistance 1 (pfmdr1) gene have been shown by transfection to modulate higher levels of chloroquine resistance and also to affect mefloquine, halofantrine, and quinine resistance.25,26

The molecular basis of resistance to pyrimethamine and sulfadoxine has been more clearly defined. Polymorphisms in the dihydrofolate reductase (dfhr) gene that alter 108-Ser to Asn/Thr in the enzyme have been shown to confer resistance to pyrimethamine.27 Additional polymorphisms at amino acid positions 50, 51, 59, and 164 combined with 108-Asn confer increasing levels of pyrimethamine resistance.28 The combination of 16-Ala to 16-Val and 108-Ser to 108-Thr confers resistance to cycloguanil but retains sensitivity to pyrimethamine.29,30 Similarly, polymorphisms in the dihydropteroate synthase (dhps) gene confer resistance to sulfadoxine.31 The polymorphism 437-Gly in dhps appears to be the first to be selected by drug pressure and it encodes lower level resistance to sulfadoxine. Subsequent polymorphisms at positions 436, 540, 581, and 613 confer increasing levels of resistance to this drug.32

Epidemiologic studies have been conducted in all malaria endemic areas of the world looking at polymorphisms in the aforementioned genes and their relationship with treatment failure or resistant P. falciparum.7,17,33–37 The aim of the present study was to complement existing knowledge of in vivo and in vitro antimalarial drug responses in Indonesia by determining the extent of associated gene polymorphisms in P. falciparum isolates from different malaria-endemic areas. The information obtained will contribute to the development of strategies for therapeutic intervention of malaria in Indonesia.

MATERIALS AND METHODS

Study sites.

Eight malaria-endemic areas that represent the entire Indonesia archipelago (Figure 1) were selected for sample collection. Four areas (Nias, Lampung, Kokap, and Kutai) are located in western Indonesia and four (Minahasa Mamuju, Flores, and Armopa) in eastern Indonesia. In each area, 20–50 blood samples infected with P. falciparum isolates were collected from people either with malaria fever or apparently healthy individuals. In these selected areas, chloroquine is used as the first-line antimalarial drug and sulfadoxine-pyrimethamine is used as the second-line antimalarial drug. Primaquine, in combination with either of the above, is used for radical cure. Parenteral quinine is exclusively used as a life-saving antimalarial drug in severe and complicated malaria. This study was carried out with the approval of the Ethics Committees for Protection of Human Subjects at the Eijkman Institute for Molecular Biology (Jakarta, Indonesia) and The Walter and Eliza Hall Institute (Melbourne, Victoria, Australia).

Sample collection.

A malariometric survey was conducted in a selected village in each area, and blood samples were collected via one or more of the following methods: in anticoagulant tubes, smears on a glass slide, or as a blot on filter paper (3MM; Whatman, Hillsboro, OR). Plasmodium falciparum-infected samples, as revealed by microscopic examination of a slide smear, were used for DNA isolation.

Extraction of DNA.

Parasite DNA was extracted from the blood samples using Chelex-100 ion exchanger (Bio-Rad Laboratories, Hercules, CA) essentially according to the procedure described previously.38 The DNA was either used immediately for a polymerase chain reaction (PCR) or stored at −20°C for later analysis.

Polymerase chain reaction amplification.

Nested PCRs were performed for four genes: dhfr, dhps, pfmdr1, and pfcrt. All reactions were carried out in 50-μL reaction mixtures containing 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2, 200 mM dNTP, 1 unit of Taq polymerase (Sigma, St. Louis, MO), and a pair of primers (20 pM each). One to five microliters of DNA was used as template in the first reaction and 1–2 μL of the first-round PCR product was used as template for the secondary PCR. Positive (D10 DNA) and negative (water) controls were used in all PCRs. The PCR primers and conditions were as previously described for dhfr, dhps, and pfmdr1,39,40 and pfcrt.23,24 Secondary PCR products were resolved by electrophoresis on 1–2% agarose gels and visualized by staining with ethidium bromide.

Restriction fragment length polymorphism (RFLP).

Restriction enzyme digestion of either the native or introduced sites of the PCR products was performed to determine the presence of polymorphisms in dhfr, dhps, pfmdr1,39–42 and pfcrt.23–25 A number of restriction enzymes were used for RFLP of PCR products. For dhfr, the PCR products were digested with Nla III, Tai I, Tsp 509I, Xmn I, and Dra I to determine the polymorphisms at codons 16, 50, 51, 59, and 164, respectively. Three enzymes, Alu I, Bsr I, and Scr FI, were used to identify codon 108. For dhps, the PCR products were digested with Msp A1I, Ava II, and Fok I to determine the polymorphisms at codon 436, 437, and 540, respectively. In addition, restriction enzymes Bst UI and Bsl I were used to identify polymorphisms at codon 581, while Mwo I, Bsa WI, and Age I were used for codon 613. Similarly, pfmdr1 PCR products were digested with Afl III, Dde I, Ase I, and Eco RV to determine the presence of polymorphisms at codons 86, 1034, 1042, and 1246, respectively. For pfcrt PCR products, the polymorphism at codon 76 was examined by digestion with Apo I. To determine complete digestion, introduced or natural restriction sites in the DNA fragment served as controls. If no such restriction sites were present, a known PCR product carrying the required restriction sites was used. As an additional control in every RFLP experiment, 0.5 μg of purified lambda DNA (Promega, Madison, WI) was digested to monitor the enzyme activity. Digested products were subjected to electrophoresis on 1.5–3% agarose gels (Progen, Australia) or 1.5–3% ultrapure agarose gels (Progen, Australia) when a higher resolution was required. Loss of restriction sites in the DNA fragment for the enzymes Nla III, Tsp 509I, Dde I, and Ase I or a gain in the restriction sites for Tai I, Xmn I, Bsr I, Scr FI, Dra I, Msp A1I, Ava II, Fok I, Bsl I, Bsa WI, Age I, Afl III, and Eco RV indicated that the DNA fragments carried polymorphisms. If there was an indication of incomplete digestion in a sample, the digestion were repeated with overnight incubation to confidently accept the presence of a mixed allelic population. These cases were considered to be mixed infections.

RESULTS

A total of 213 P. falciparum isolates from eight malaria-endemic areas in Indonesia were analyzed. Some samples, particularly those collected on filter paper, failed to amplify using certain primers; thus, the gene polymorphism information obtained was incomplete. Based on gene analysis, there were 83 samples (38.9%) that gave PCR results indicating mixed isolates, either as polymorphic and wild-type or two polymorphic types.

Genotypic profiles of P. falciparum isolates from western Indonesia.

Polymorphisms in the pfmdr1 and pfcrt genes.

Analysis of 117 isolates from Nias Island, Hanura, Kokap, and Kutai showed that polymorphisms in the pfmdr1 and pfcrt genes have spread to all sample collection sites (Table 1). Except in one sample from Nias Island that failed to be amplified, all (116 of 116) isolates carried the 86Y polymorphism in pfmdr1. No polymorphisms at codons 1034, 1042, and 1246 of the pfmdr1 gene were observed in any of the isolates examined. Likewise, for pfcrt, 100% (85 of 85) of the amplified samples carried the 76T polymorphism.

Polymorphisms in the dhfr and dhps genes.

Amplification of dhfr 108 was successful with 92 isolates (Table 1). Of these, 54% (50 of 92) had either a single polymorphism, 108N, or the mixed S108N and 19% (17 of 92) had either 108T or 108T mixed with wild-type. An additional 13% (12 of 92) carried the mixture 108N/T. Except for one isolate from Kutai, the threonine polymorphism was found only at Hanura and Kokap. Only 14% (13 of 92) of the successfully amplified isolates for dhfr 108 carried a single wild-type haplotype. Analysis of codon 59 was successful in 90 of the 92 amplified isolates. This identified 54% (49 of 90) as being wild-type C59 while the other 46% (41 of 90) were either 59R or 59R mixed with wild-type. The C59R polymorphism was in all cases associated with one or other of the 108 resistance polymorphisms. At Kutai, the 59R polymorphism was not observed. At dhfr codon 16, the wild-type A16 was observed in 62% (16 of 26) of isolates from Kokap and the single polymorphism 16V or the mixed A16V was observed in 38% (10 of 26) of the isolates. This polymorphism was only observed in association with 108T as either a single or mixed population. Codon 16 was only observed as wild-type at all other locations in the western part of Indonesia. No polymorphisms were observed at codons 50, 51, or 164 in any of the samples examined.

Polymorphisms in the dhps gene were the least common among the four genes tested in this study (Table 1). The most common polymorphism was 437G, which was observed in Hanura, Kokap, and Kutai; however, only in 14% (16 of 117) of all isolates. The double polymorphism of codons 437G and 540E was observed in one sample from Kokap, in association with dhfr 59R and 108N/T. No polymorphisms were observed at codons 436, 581, or 613 in any of the samples examined in this study.

Genotypic profiles of P. falciparum isolates from eastern Indonesia.

Analysis of pfmdr1 and pfcrt genes.

Analysis of 71 isolates that successfully amplified pfcrt codon 76 identified 91% (64 of 70) with the polymorphism 76T and one isolate from each of Flores and Armopa were mixed K76T (Table 2). For pfmdr1, the results were more complex, with the 86Y polymorphism ranging from 95% (19 of 20) at Minahasa to 16% (3 of 19) at Flores. However, the 84% (16 of 19) of isolates from Flores that carried the wild-type N86 were observed to have the polymorphism 1042D. At Armopa, 27% (4 of 15) of the isolates carried the 86Y polymorphism and all isolates were wild-type at codon 1042. The 1042D polymorphism was also observed in 15% (3 of 20) of isolates from Minahasa.

Point mutations in the dhfr and dhps genes.

Eighty-three samples were successfully amplified at dhfr 108, of which only 10% (8 of 83) were wild-type S108. The rest were either 108T (34%, 28 of 83), S108T (20%, 17 of 83), 108N (19%, 16 of 83), S108N (12%, 10 of 83) or 108N/T (5%, 4 of 83). Eighty isolates were successfully analyzed for polymorphisms at codon 59. A total of 68% (54 of 80) were wild-type C59 and 32% (26 of 80) were either 59R or the mixed 59R with wild-type (Table 2). At codon 16, the polymorphism 16V was observed twice at Mamuju, once in association with 108T and once with 59R and 108T. In Flores, 16V was observed once in association with 108T and once as the mixed A16V in association with S108T. No polymorphisms were observed at codons 50, 51, and 164 in any of the samples examined.

Analysis of dhps showed that 13% (13 of 96) of the isolates were either 437G or A437G. One isolate from Minahasa carried the 540E polymorphism associated with 437G and dhfr S108T. A single isolate from Armopa carried dhps K540G in association with A437G and dhfr C59 and S108. No mutation was observed at codons 436, 581, and 613 in any of the samples examined in this study.

DISCUSSION

As might have been expected from the previous extensive studies on in vivo and in vitro antimalarial drug responses in Indonesia, the isolates of P. falciparum examined in this study were found to carry multiple genetic polymorphisms associated with resistance to chloroquine and sulfadoxine-pyrimethamine.

Although the molecular basis for the P. falciparum resistance to quinoline antimalarials is still being investigated, evidence indicates that resistance is multigenic. Initially, the role of mutations in pfmdr1 in the modulation of chloroquine resistance was shown.27 Allelic exchange experiments have indicated that the 1246 Tyr polymorphism in pfmdr1 increases the chloroquine 50% inhibitory concentration (IC50) and, both alone and in combination with the polymorphisms 1034 Cys and 1042 Asp, also modulates sensitivity to other anti-malarials drugs.28 However, a detailed linkage analysis and chromosomal mapping of progeny identified a second gene involved in chloroquine resistance, pfcrt, in which mutation at codon 76, K76T is strongly associated with the chloroquine resistance phenotypes in field and clinical studies.23–25 Furthermore, conclusive evidence that various mutant pfcrt haplotypes confer chloroquine resistance with characteristic verapamil reversibility and reduced chloroquine accumulation was demonstrated.26 Nevertheless, the role of other modifying factor such as pfmdr1 in this regard could not be excluded.

Previous field-based studies in Indonesia have associated the 86Y allele of the pfmdr1 gene to chloroquine resistance, both in vivo and in vitro.8,41 Other studies in Indonesia have reported that the 76T polymorphism of pfcrt is also associated with chloroquine resistance in vivo and in vitro, and the allele has the potential to be used as a predictor for chloroquine therapeutic treatment failure.7,42 Our present results show that the isolates of P. falciparum from Indonesia carry polymorphisms in both the pfcrt and pfmdr1 genes. In western Indonesia, all asymptomatic and mildly symptomatic malaria patients were carrying parasites with both pfcrt 76T and pfmdr1 86Y polymorphisms. The situation in eastern Indonesia was more complex (Figure 2). Northern Sulawesi had a resistant profile at these two codons, whereas southern Sulawesi had a lower frequency of pfmdr1 86Y polymorphism than may have been expected, but pfcrt was fixed in the parasite population. In Flores, all isolates carried 76T and all isolates carried a polymorphism in pfmdr1. However, most of these were the 1042D polymorphism, without the expected 86Y polymorphism. Recent findings have shown that Papua New Guinea and Papua Indonesia have a range of variants of chloroquine-resistant P. falciparum parasites at pfcrt codons 72–76.42,43 These variants encompass African, southeast Asian, and South American haplotypes. While work on this area of parasite evolution is still in its early stages, future studies may conceivably show an association between regional pfmdr1 and pfcrt haplotypes. Given that the pfmdr1 1042D polymorphism is far more common in South America, further work on this aspect could show interesting results. In isolated Papua, pfcrt 76T appears to be fixed in the parasite population; however, there is only a low rate of polymorphism in pfmdr1. In our previous study in Armopa, Papua Province involving Javanese migrants who have easier access to the Health center than the indigenous Papuans, a high prevalence of 86Y polymorphism was found in the P. falciparum isolates examined.8 A closer examination of the social and chemotherapeutic history in this particular area may shed more light on this apparent contradiction. It is of interest to note the occurrence of two isolates in Minahasa carrying both 86Y and 1042D polymorphisms of pfmdr1. A recent study also reported this allele from Purworejo district in the island of Java.12 This finding may be associated with the civil strife in the eastern parts of Indonesia that has resulted in migration to Java.

Given the spread of chloroquine treatment failure to many parts of Indonesia over the last two decades,5,7,8,9,11,17,44 it was not surprising to find that P. falciparum isolates in Indonesia carry the polymorphisms in pfmdr1 and pfcrt that have been associated with resistance to this drug.

The P. falciparum isolates examined carried polymorphisms in dhfr as either 108N or 108T as well as the 59R and 16V polymorphisms. There are two antifolate drugs that have been used in Indonesia, pyrimethamine and proguanil (cycloguanil). Pyrimethamine has always been used in combination with sulfadoxine, whereas proguanil is available as a single drug (Paludrine®; AstraZeneca Pty., North Ryde, New South Wales, Australia). Evidence to date indicates that the molecular mechanism that underlies resistance to antifolate antimalarials involves the S108N/T polymorphism of dhfr. There is a differential target in the dhfr enzyme between pyrimethamine and cycloguanil in which resistance to pyrimethamine is mainly linked to the 108N polymorphism, whereas cycloguanil resistance is linked to the 16V plus 108T polymorphism. However, additional polymorphisms at 50A, 51I, 59R, and 164L will confer cross-resistance to both anti-folate drugs.32 In our, study there was a relatively common distribution of the 108T dhfr mutation, either as a single polymorphism or else paired with 16V among the field isolates of P. falciparum from Indonesia. This single polymorphism has been reported in laboratory clones45 and enzyme kinetic analysis indicated that this mutant did not confer resistance to cyloguanil. Conversely, the enzyme harboring mutant 16V alone showed a high resistance to cycloguanil, but exhibited a very low kinetic parameter, indicating that this mutation may interfere with the fitness of the isolates and that the parasite carrying this polymorphism may not survive in the natural population of the parasite. The findings that no P. falciparum isolates in Indonesia carried the 16V as a single mutation are in line with this suggestion. However, as this previous work reports, the poor dhfr activity of the A16V polymorphism was restored in the presence of another mutation, 108T. Therefore, it is strongly suspected that the 108T mutation in Indonesia may serve as a precursor for the double mutant 16V plus 108T, which is highly resistant to cycloguanil.

The reported distribution of the single 108T polymorphism among field isolates of P. falciparum has been very limited and so far it has only been seen in isolates from Indonesia and Papua New Guinea.46,47 In this regard, its relatively more common distribution among the P. falciparum isolates from the eastern part of Indonesia suggests that the isolates carrying this polymorphism may share the same origin with the isolate found in Papua New Guinea.

In this study, polymorphisms in the dhps gene were found less frequently than polymorphisms in other genes. The highest rate of mutation (23%) was found in northern Sulawesi. The most common mutation was 437G and in one sample each from Kokap, northern Sulawesi, and Armopa; this was paired with the 540E polymorphism. The role of dhps polymorphisms in the mechanism of resistance to sulfa drugs has been well described.33,34 The presence of the 437G polymorphism confers resistance to sulfadoxine in P. falciparum, and when coupled with the additional polymorphism at 540E the level of resistance is increased.27 Our results indicate that the P. falciparum isolates from Indonesia are still predominantly wild-type dhps, a finding that reflects the results of recent studies in the region in Papua New Guinea.48

There has been a disturbing report from Nias Island on the high rate of P. falciparum treatment failure following sulfadoxine-pyrimethamine chemotherapy.17 In this study, no dhps polymorphisms were found in samples from Nias Island, and only very low rates of mutation at the other study sites. Not withstanding the recent encouraging report from Malawi on long-term sulfadoxine-pyrimethamine efficacy,49 this is a worrying indicator for Indonesia. The combination of sulfadoxine-pyrimethamine is used as the second-line antimalarial drug in Indonesia. While there is a lack of broad epidemiologic data available, what is published suggests that resistance to this drug combination has spread to malaria-endemic areas in Indonesia.8,15,17,44 Given this, serious consideration must now be given to the use of artemisinin combination therapy, which has already been shown to be effective against chloroquine-resistant strains of P. falciparum in Indonesia.6

In conclusion, molecular analysis of P. falciparum parasites from eight malaria-endemic areas in Indonesia indicated the widespread presence of isolates carry resistance polymorphisms to both the first-line antimalarial drug chloroquine and to the second-line treatment sulfadoxine-pyrimethamine. With widespread reports of treatment failure and the current increase of malaria morbidity and mortality in Indonesia, serious consideration should be given to the revision of the guidelines for the chemotherapeutic treatment of malaria in Indonesia.

Table 1

Genotypic pattern of Plasmodium falciparum isolates from western Indonesia*

DHFRDHPSPfmdr1
LocationsNo. of genotypes16V59R108N/T437G540E86Y1042DPfCRT 76TNo. of isolates
* DHFR = dihydrofolate reductase; DHPS = dihydropteroate synthase; Pfmdr1 = P. falciparum multidrug resistance 1; PfCRT = P. falciparum chloroquine resistance transporter.
Nias, North Sumatra1.ACSAKYNT1
2.ACNAKYNT2
3.ARNAKYNT9
4.AC/RS/NAKYNT3
5.ANAKYNT1
6.AS/NAKYNT1
7.AKYNT2
8.AKT1
Hanura, Lampung1.ARNAKYNT3
2.ARS/NAKYNT2
3.ARNA/GKYNT2
4.ACS/NAKYNT4
5.ARNA/GKYNT3
6.ACS/NA/GKYNT1
7.ARN/TAKYNT1
8.AC/RN/TAKYNT2
9.AC/RN/TGKYNT2
10.AC/RN/TA/GKYNT3
11.AC/RS/NA/GKYNT1
12.ACSAKYN3
13.ACTAKYN2
14.ARNAKYN1
15.AKYNT1
16.AKYN10
Kokap, Central Java1.ACSAKYNT2
2.ARNAKYNT3
3.ACTAKYNT2
4.VCTAKYNT2
5.AC/RNAKYNT1
6.VCN/TAKYNT1
7.ARN/TAKYNT1
8.ARN/TGEYNT1
9.AC/RS/NAKYNT1
10.A/VCS/TAKYNT1
11.A/VC/RN/TAKYNT1
12.VCTAKYNT3
13.ARNAKYNT1
14.ACS/TAKYN1
15.A/VCS/TAKYN1
16.ACNAKYN1
17.ACTAKYN2
18.A/VCTAKYN1
19.CTAKYN1
20.AKYNT1
Kutai, East Kalimantan1.ACSAKYNT5
2.ACNAKYNT5
3.ACS/TAKYNT1
4.ACS/NAKYNT3
5.ACSA/GKYNT1
6.ACNA/GKYNT2
7.ACSAKYN1
8.AKYNT2
9.AKYN8
Table 2

Genotypic pattern of Plasmodium falciparum isolates from eastern Indonesia*

DHFRDHPSPfmdr1
LocationsNo. of genotypes16V59R108N/T437G540E86Y1042DPfCRT 76TNo. of isolates
* For definition of abbreviations, see Table 1.
Minahasa, North Sulawesi1.ACTAKN/YNK1
2.ACNA/GKYNT1
3.ACS/NAKYNT2
4.ACS/NGKYNT1
5.AC/RN/TAKYNT1
6.ACS/NAKN/YN1
7.ACS/NGKYN1
8.ACS/TAKYD1
9.ACS/TGKYD1
10.ACS/TGEND1
11.ACN/TAKYT1
12.ACNAKNT1
13.ACS/TAKT2
14.ACN/TAKT1
15.AC/RN/TAKT1
16.ACS/TAKN2
17.ACS/TAK3
18.AKYNT3
19.AKYN1
20.A/GKYN1
21.AKNT1
22.AKYT2
23.A/GKN1
24.AKN2
25.A/GKY1
Mamuju, South Sulawesi1.ACTAKYNT2
2.VRTAKYNT1
3.ACNAKNNT4
4.ACNAKYNT1
5.ACS/NGKNNT1
6.ACTAKYN5
7.AC/RTAKYN1
8.ACTAKT2
9.AC/RTAKT1
10.VCTAKT1
11.ARTAKT2
12.ACTAK1
13.AKNNT1
Flores, East Nusa Tenggara1.ARTAKNDT2
2.ACSAKYNT1
3.VCTAKNDT1
4.ACTAKNDT5
5.ACTAKYNT1
6.ACSAKNDT1
7.ACS/TAKNDT1
8.ARS/TAKNDT3
9.ACTAKNDK/T1
10.AC/RS/TAKN/YN/DT1
11.AS/TAKNDT1
12.A/VS/TAKNDT1
13.ATAKT1
Armopa, West Papua1.ACSA/GK/ENNT1
2.AC/RS/NAKNNT3
3.ARNAKNNT2
4.ARNGKNNT1
5.ACSAKNNT2
6.ARNAKN/YNK/T1
7.ACSAKNNK1
8.ARNGKN/YNT1
9.ARNAKNNK1
10.ACSAKYNT2
11.ARNAK2
12.ARNGK1
13.AC/RS/NAK1
Figure 1.
Figure 1.

Map of the Indonesian archipelago and the sampling sites (boxes) of the isolates of Plasmodium falciparum.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 2; 10.4269/ajtmh.2005.72.174

Figure 2.
Figure 2.

Comparison of polymorphisms identified in west and east Indonesia. The 1042D polymorphism of the Plasmodium falciparum multidrug resistance 1 (pfmdr1) gene was only found in eastern Indonesia. pfcrt = P. falciparum chloroquine resistance transporter; dhfr = dihydrofolate reductase; dhps = dihydropteroate synthase.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 2; 10.4269/ajtmh.2005.72.174

Authors’ addresses: Din Syafruddin, Eijkman Institute for Molecular Biology, Jl. Diponegoro 69, Jakarta 10430, Indonesia and Department of Parasitology, Faculty of Medicine, Hasanuddin University, Jl. Perintis Kemerdekaan Km 10, Makassar 90245, Indonesia. Puji B. S. Asih, Eijkman Institute for Molecular Biology, Jl. Diponegoro 69, Jakarta 10430, Indonesia. Gerard J. Casey, Hadya S. Nagesha, and Alan F. Cowman, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia. Jason Maguire and J. Kevin Baird, United States Naval Medical Research Unit No. 2, Jakarta, Indonesia. John C. Reeder, Papua New Guinea Institute of Medical Research, Goroka EHP441, PO Box 60, Papua New Guinea.

Acknowledgments: We thank the officials of the Ministry of Health at the selected areas for providing us with technical support during the malariometric surveys in a the given areas, and Dr. Supargiyono (Gajah Mada University, Yogyakarta, Indonesia) and Dr. P. Hariyanto (Sam Ratulangi University, Manado, Indonesia) for helping us with sample collection. We also thank Professor Sangkot Marzuki (Director of the Eijkman Institute, Jakarta, Indonesia) for his input.

Financial support: This work was supported by the Indonesian and Australian Governments through Bappenas and the Australian Agency for International Development through the Australia Indonesia Medical Research Initiative program. Hadya S. Nagesha was supported by the Wellcome Trust (United Kingdom).

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Author Notes

Reprint requests: Din Syafruddin, Eijkman Institute for Molecular Biology, Jalan Diponegoro 69, Jakarta 10430, Indonesia, Telephone: 62-21-3917131, Fax: +62-21-3147982, E-mail: din@eijkman.go.id.
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