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Am. J. Trop. Med. Hyg., 69(6), 2003, pp. 614-620
Copyright © 2003 by The American Society of Tropical Medicine and Hygiene

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FREQUENCY DISTRIBUTION OF ANTIMALARIAL DRUG-RESISTANT ALLELES AMONG ISOLATES OF PLASMODIUM FALCIPARUM IN PURWOREJO DISTRICT, CENTRAL JAVA PROVINCE, INDONESIA

D. SYAFRUDDIN, PUJI B. S. ASIH, SONA L. AGGARWAL, AND ANURAJ H. SHANKAR
Eijkman Institute for Molecular Biology, Jakarta, Indonesia; Department of Parasitology, Faculty of Medicine, Hasanuddin University, Makassar, Indonesia; Department of International Health, Johns Hopkins School of Public Health; Johns Hopkins School of Medicine, Baltimore, Maryland; Helen Keller International, Jakarta, Indonesia


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Treatment failures to the first- and second-lines antimalarial drugs chloroquine and sulfadoxine-pyrimethamine have increased in the Purworejo district on the island of Java, Indonesia. A molecular epidemiologic study was conducted to determine the frequency distribution of mutant alleles of the genes associated with the resistance among the isolates of Plasmodium falciparum from the area. Analyses using a polymerase chain reaction and restriction fragment length polymorphism showed that nearly all of the 111 samples carried mutant alleles in genes associated with chloroquine resistance: P. falciparum multi-drug resistance 1 (pfmdr1) 86Y (92%), 1042D (4.5%), and P. falciparum chloroquine resistance transporter (pfcrt) 76T (99.1%). Mutant alleles of the in the dihydrofolate reductase (dhfr) gene were also high (84.7%), either as 108N and 108T or paired with 59R, and 16V, respectively. Mutant alleles in the dihydropteroate synthase gene were the least common, either as a single 437G mutation (35.3%) or paired with 540E (26.5%). These results are consistent with the antimalarial drug resistance situation in the area and emphasize the need for a proper treatment strategy.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Malaria incidence on much of the Indonesian islands of Java and Bali, Indonesia had been successfully eliminated or reduced to hypoendemic status by the Government-initiated malaria control program in 1952, although in some foci persistent transmission still occurs.1 The prolonged political and socioeconomic crisis within the last few years, however, has changed the malaria situation in many parts of Indonesia, including Java. Several outbreaks have been documented in areas previously declared malaria free. Malaria incidence has increased in the Menoreh Hills, a malaria-endemic focus in central Java that includes the Magelang, Purworejo, and Kulon Progo districts, as shown by the persistent increase of annual parasite incidence since 1995 (Ministry of Health, unpublished data).

Surveys conducted within the last few years indicated that chloroquine resistance has spread almost homogeneously in the entire archipelago.2–8 Resistance of Plasmodium falciparum to chloroquine, quinine, and mefloquine had been also documented in Purworejo district by either in vivo or in vitro drug resistance tests. In the subdistricts of Kemiri, Loano, and Bener, the prevalence of resistance to chloroquine was 73% R-I level, 22.4% R-II, and 4.5% R-III in 1995,9 while in the Pituruh subdistrict, chloroquine resistance was reported as R-I (52%), R-II (3.6%) and R-III (0.4%) (Ministry of Health, unpublished data). In vitro analyses conducted between 1981 and 1985 and 1986 and 1990 reported sulfadoxine-pyrimethamine resistance in 71% and 22% of cases, respectively, whereas the in vivo test found 12% resistance.9

As in other part of Indonesia, slide proven and/or clinically diagnosed malaria is usually treated with a standard curative regimen of chloroquine (25 mg base/kg of body weight in three doses over a 72-hour period), followed by 15 mg of primaquine base per day for two weeks for P. vivax or a single dose of 45 mg of primaquine base for P. falciparum. In cases of treatment failure, a standard formulation of sulfadoxine and pyrimethamine (supplied by the Ministry of Health) is given in accordance with the Ministry of Health guidelines.Physicians or clinic staff generally give therapy to patients mostly without supervision after the first dose.

Although treatment failure to the mainline antimalarial drugs is currently increasing, the use of other alternative drugs has not yet been recommended due to economic reasons. During the latest malaria epidemic in Purworejo in October 2000, it was recommended to intensify the control program through rational deployment of the available drugs.

Studies on the molecular basis of antimalarial drug resistance over the last few decades revealed various mutant alleles of the P. falciparum chlorquine resistance transporter (pfcrt) and the P. falciparum multi-drug resistance 1 (pfmdr 1) genes that were associated with resistance to chloroquine.10 Similarly, mutant alleles of the dhfr and dhps genes had also been implicated in the Plasmodium spp. resistance to antifolates and sulfa drugs, respectively.11 Many epidemiologic studies have been conducted on the frequency distribution of the alleles among the P. falciparum isolates from different geographic regions. These studies have proven to be an invaluable tool in evaluating the spread of drug resistance in many countries.12–22

The aim of the present study was to determine the frequency distribution of the mutant alleles of genes associated with resistance to chloroquine and sulfadoxine-pyrimethamine among the P. falciparum isolates from Purworejo district, Central Java Province, Indonesia.


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Study site. The location of the Purworejo district within the Central Java Province of Indonesia is shown in Figure 1Go. The Purworejo district is located along the western slope of Menoreh Hill and shares a border with the Kulon Progo District of the Yogjakarta special Province. Purworejo district has an area of 1,034 km2 and is divided into 16 subdistricts, and includes rugged terrain, valleys used for rice fields and teak or bamboo forest-covered hillsides. Most of the cultivated lands are used for rice fields, but coconut, salak, papaya, jackfruit, and pineapple are also cultivated. The majority of the 733,840 residents work as farmers. The survey was conducted in Sokoagung village in the Bagelen subdistrict. This village has a population of approximately 2,200 people and has been one of the malaria foci in the Purworejo district with mesoendemic status for infection by P. vivax and P. falciparum. The climate is typically tropical with a monsoon occurring between November and March. The village is characterized by rough terrain and was subject to heavy rains leading to landslides and floods during the surveys.



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  		    FIGURE 1. Map of the Central Java Province of Indonesia showing the location of Sokoagung village, the Bagelen Sub-district, the Purworejo District (gray area), the Menoreh Hill area (triangle), and Sokoagung village (oval). The inset shows the entire Indonesian archipelago.

 
Sample collection. A malariometric survey was conducted during October and November 2000, and 182 blood samples from malaria-infected individuals were collected as blots on filter paper (3MM; Whatman, Hillsboro, OR). Plasmodium falciparum-infected samples, as revealed by microscopic examination of a blood smear, were used for DNA isolation. This study was carried out with the approval by the Ethics Committees for Protection of Human Subjects at the Eijkman Institute for Molecular Biology (Jakarta, Indonesia).

Extraction of DNA. Parasite DNA was extracted from the blood samples using Chelex-100 (Bio-Rad Laboratories, Hercules, CA) ion exchanger, essentially according to the procedure previously described.23 The DNA was used immediately for a polymerase chain reaction (PCR) or stored at -20°C for subsequent use.

Amplification by PCR and detection of mutant alleles. A PCR) and restriction fragment length polymorphism (RFLP) were performed for four genes (dihydrofolate reductase [dhfr], dihydropteroate synthase [dhps], pfmdr1, and pfcrt) to identify the presence of mutant alleles. All reactions were carried out as described previously for dhfr, dhps, and pfmdr1.20,21,24,25


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
A total of 182 blood samples from P. falciparum-infected individuals were examined for the presence of mutations in the pfcrt, pfmdr1, dhfr, and dhps genes. Our results show that among the blood samples examined, only 111 gave PCR results. There were 35 samples (31.5%) that indicate mixed infections of both wild-type and mutant alleles, as shown by the pattern of RFLP in at least one of the gene PCR products (Table 1Go).


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TABLE 1
Genotypic profiles of Plasmodium falciparum isolates from Purworejo District, Indonesia*
 
Mutant alleles of the pfmdr1 and pfcrt genes. Analysis of pfmdr1 gene PCR products indicated that mutant alleles of this gene have spread to all samples examined in Purworejo District (Table 1Go). The most common allele of the pfmdr1 gene among the field isolates of P. falciparum was 86Y. The 1042 allele was also observed at a lower frequency (4.5%), and was paired with allele 86Y, except in one isolate. Mutant allele 76T in the pfcrt gene was found in most samples (99.1%), but it was mixed with wild-type allele 76K in one case. Except in four samples, all P. falciparum isolates carried mutant allele 76T of the pfcrt gene paired with the either 86Y or 1042D alleles of the pfmdr1 gene. Alleles 1034C and 1246Y were not found in any of the samples examined.

Mutant alleles of the dhfr and dhps genes. Mutant alleles in the dhfr gene were found in 94 samples (84.7%) either as a single 108N or 108T, or else paired with alleles 16V and 59R (Figure 2Go). In four samples, the 59R mutation was found as a single mutation. There were 42 isolates (30.2%) that carry alleles 108N and 59R simultaneously. The 108T allele was detected in 34 isolates (27.4%), and among these, 32 were paired with 16V. Mutant alleles at codons 50, 51, and 164 were not observed in any of the samples examined in Indonesia. Mutant alleles of the dhps gene were the least common among the four genes examined in this study (Figure 2Go). The most common allele was 437G (35.3%), and was found either as single mutation or paired with the 540E allele (26.5%). Mutant alleles at codons 436, 581, and 613 were not observed in any of the samples examined in this study. With the exception of two samples, mutations in the dhps gene was also paired with one or more mutations in dhfr gene.



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  		    FIGURE 2. Histogram of the frequency distribution of mutant alleles of Plasmodium falciparum associated with resistance to sulfadoxine-pyrimethamine. Mutant alleles 50R, 51I, and 164L of the dihydrofolate reductase (dhfr) gene were not found in any of the samples examined. Mutant alleles 436F/A, 581G, and 613S/T of the dihyropteroate synthase (dhps) gene were not observed in any of the isolates examined.

 

DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results showed that all of the field isolates of P. falciparum from the Purworejo district carried a mutant allele of at least one the genes that are associated with resistance to the front-line antimalarial drugs chloroquine and sulfadoxine-pyrimethamine. There were also high proportions of P. falciparum isolates that carry more than one mutant allele in all genes analyzed.

Mutant alleles associated with chloroquine resistance, 76T of the pfcrt gene paired with either 86Y or 1042D of the pfmdr1 gene, predominated the genotypic patterns of P. falciparum isolates in the Purworejo District. This may reflect the increasing cases of treatment failure with the standard dose of chloroquine within the last few years in the area. The antimalarial drug resistance situation in Purworejo has not been extensively studied until recently. In vivo and in vitro drug sensitivity tests performed between 1981 and 1995 reported resistance to chloroquine in 24–60% of the cases and resistance to sulfadoxine-pyrimethamine in 12–70% of the cases. However, this result has not altered the antimalarial drug policy in the area because only a small number of samples were examined.9 Our recent in vitro tests found three P. falciparum isolates collected from the nearby Pituruh sub-district that were highly resistant to chloroquine with similar genotypic patterns to the PCR samples. These data, if taken together, indicate that the current standard chloroquine treatment dose may no longer effectively eliminate the malaria infection.8,26

The molecular basis for the malaria parasite resistance to quinoline antimalarials has long been a central issue in malaria research because chloroquine is the most widely used antimalarial drug. Evidence to date indicates that the resistance may involve more than one gene. Various sets of polymorphism in pfcrt gene have been associated with chloroquine resistance. Mutant allele 76T of the pfcrt gene has been shown to play a determining role in chloroquine resistance, although it was also present to a lesser frequency in a chloroquine-sensitive strain.21,22 This evidence suggests that the existence of other mutant alleles is required, or that several genes may be involved in chloroquine resistance. Our findings that most P. falciparum isolates in the area carry both mutant alleles in the pfcrt and pfmdr1 genes simultaneously gives support for this idea. Linkage disequilibrium between pfcrt 76T and pfmdr1 86Y was noted in Africa.27,28 The 1042D allele of the pfmdr1 gene was also observed in few isolates of P. falciparum in Purworejo. This allele was mainly found in the eastern part of Indonesia, and was exclusively predominant in isolates from Flores. It is of interest to note the presence of isolates carrying both 86Y and 1042D alleles of the pfmdr1 gene. At this stage, it is unclear whether multiple mutations in this gene confer a higher degree of resistance to chloroquine and its derivatives. Allelic exchange experiments indicated that 1034C, 1042D, and 1246Y alleles of the pfmdr1 gene reduced the chloroquine 50% inhibitory concentration (IC50).29

Given the rapid spread of chloroquine resistance to many parts of Indonesia over the last two decades, it is not surprising to find that the P. falciparum isolates in Indonesia have indeed carried mutant alleles in genes that are associated with resistance to this drug.

Of the 111 P. falciparum isolates examined in this study, we found that only 19 carried wild-type allele of the dhfr gene. The remainder carried alleles 108N or 108T, either as a single mutation or in combination with the 59R and 16V mutations. Additionally, we also found the double mutant alleles 108N+59R in 42 isolates and 108T+16V in 32 isolates. These findings indicate that these isolates were resistant to anti-folate antimalarial drugs. Two antifolate drugs are currently used, pyrimethamine and proguanil (cycloguanil). Pyrimethamine has always been used in combination with sulfadoxine, whereas proguanil is available as a single drug, paludrine, and more recently also in combination with atovaquone (Malarone®; GlaxoSmithKline, Middlesex, United Kingdom), The molecular mechanism that underlies resistance to anti-folate antimalaria drugs involves the S108N/T mutation in the dhfr gene. Resistance to pyrimethamine is mainly linked to the 108N mutation, whereas cycloguanil resistance is exclusively linked to the 16V and 108T mutations. The 108N mutation alone would confer an approximately 100-fold increase in resistance to pyrimethamine compared with the wild-type allele 108S.30,31 The presence of other mutations (51I, 59R, and 164L) not only enhances resistance but also confers cross-resistance to both antifolate drugs.32–34

The results are also of particular interest in terms of resistance to cycloguanil. There were 32 P. falciparum isolates that genotypically indicate an exclusive resistance to this drug, the active form of proguanil, although this drug has never been officially used in Indonesia. There are several explanations that may explain this result. First, proguanil-containing anti-malarial drugs may have been used in the area or the parasite isolates may have been imported from an area where the drug has been used. Second, there may be other drugs with action similar to cycloguanil, such as trimethoprim, a weak inhibitor of dihydrofolate reductase, which are commonly used in combination with sulfamethoxazole for treatment of bacterial diseases.35

In this study, mutant alleles of the dhps gene were found less frequently in P. falciparum isolates compared with the other genes analyzed. However, this prevalence was much higher than the 3% previously observed in the adjacent sub-district of Kokap in 1999. This indicates that the wider use of the second-line antimalarial sulfadoxine-pyrimethamine within the last few years in the area has significantly increased the prevalence of P. falciparum isolates carrying the resistant genotypes. The role of dhps mutations in the mechanism of resistance to sulfa drugs has been well described. The 437G mutation confers resistance to sulfadoxine in P. falciparum, and when coupled with the additional mutant allele 540E, the level of resistance is increased.36 The finding that almost 25% of the P. falciparum isolates from Purworejo carry double mutations in the dhps gene emphasizes the need for careful prescription of sulfadoxine-pyrimethamine because it may be the last affordable antimalarial drug in this area.

The combination of pyrimethamine-sulfadoxine, also known as Fansidar® (F. Hoffmann-LaRoche, Basel, Switzerland), has been used mainly as a second-line antimalarial drug in Indonesia. Resistance to this drug has also consistently spread to many malaria-endemic areas in this country. It is not clear whether mutations in both the dhfr and dhps are required to confer resistance to Fansidar. Previous studies in Africa indicated that resistance to sulfadoxine-pyrimethamine in vivo was associated with the presence of triple mutant alleles 108N, 51I, and 59R in the dhfr gene with or without the mutant alleles 437G and 540E in the dhps gene.15–18 Although this study did not find any isolates that had quintuple mutations in the dhfr and dhps genes, it may be of particular importance to note that the current practice of unsupervised use of Fansidar will certainly facilitate the development of parasite resistance to this drug. Our previous in vivo test in western Papua New Guinea indicated that mutations in both the dhfr and dhps genes are required for resistance to Fansidar.20

In conclusion, molecular analyses of antimalarial drug resistant alleles of P. falcifarum isolates in the Purworejo district strongly indicate the widespread distribution of chloroquine-resistance, and to a lesser extent, sulfadoxine-pyrimethamine-resistant isolates in this area. These results emphasize the need for proper use of antimalarial drugs in this area and in Indonesia in general because most other alternative antimalarial drugs are currently unaffordable. With the current increase in malaria morbidity and mortality in Indonesia, a national malaria control strategy that permits malaria treatment without a prior laboratory diagnosis should be revisited.

Received March 26, 2003. Accepted for publication September 13, 2003.

Acknowledgments: We thank officials of the Ministry of Health in Purworejo District and at the selected subdistrict and villages for providing us with technical support during the malariometric surveys, Dr. F. Laihad (Malaria Section, Center for Communicable Diseases Control, The Ministry of Health) and Dr. M. Duffy (Australia-Indonesia Medical Research Initiative) for critically reading the manuscript. We also thank Professor Sangkot Marzuki (Director of the Eijkman Institute, Jakarta) for his invaluable support and input.

Financial support: This work was supported in part by The Indonesian Government through Bappenas, the Australia-Indonesia Medical Research Initiative project, a fellowship from the American Society of Tropical Medicine and Hygiene awarded to Sona L. Aggarwal, and by a Prebluda Fellowship grant awarded to Anuraj H. Shankar.

This work also received support from the Nutrition and Health Surveillance System Cooperative Agreement No. 497-a-00-99-00033-00 between USAID-Indosia and Helen Keller International.

Authors’ addresses: D. 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. Sona L. Aggarwal, Johns Hopkins School of Medicine, Baltimore, MD 21205. Anuraj H. Shankar, Helen Keller International, Jl. Bungur Dalam 23 A, Kemang, Jakarta 12730, Indonesia.

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


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 MATERIALS AND METHODS
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 DISCUSSION
 REFERENCES
 

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