Background
Malaria incidence in Indonesia has decreased threefold in the last decade [
1], and there is no evidence for the presence of the parasites resistant to artemisinin-based combination therapy (ACT) [
2]. However, about 25% of Indonesia’s population (total population: 261 million) is at risk of malaria, and more than 200,000 positive cases were reported in 2016 [
3]. Detection of drug resistant malaria parasites and prevention of these spreading are critical to keep efficacy of the malaria treatment and to obtain elimination of malaria. In this research, origin and distribution of unique mutations of
Plasmodium falciparum sulfadoxine–pyrimethamine (SP) resistance in Indonesia were analysed.
SP has been widely used as an anti-malarial drug for treatment of uncomplicated malaria, and for intermittent preventive treatment in vulnerable populations, pregnant women (IPTp) and infants (IPTi) in high malaria transmission areas in Africa [
4,
5]. However, emergence and spread of SP resistant
P. falciparum has been reported worldwide [
6‐
9]. In Indonesia, SP was recommended as a second line anti-malarial drug after chloroquine resistance had been determined in 1973, and
P. falciparum resistance to SP was reported for the first time in Jayapura (Papua Province) in 1979 [
10,
11]. Chloroquine and SP had been used in Indonesia until 2008, when the malaria treatment policy was changed. ACT, using a combination of an artemisinin derivative with another anti-malarial, such as piperaquine, lumefantrine or amodiaquine, is provided as the first line anti-malarial drug for treatment of uncomplicated malaria, and SP is not administered for malaria treatment. However, people, especially in local areas, use SP when they are suffering from malaria, or for chemoprophylaxis [
12], because of some effectiveness, low cost, simple administration as a single oral dose, and fewer side effects. It is important to obtain information about SP resistance in malaria endemic areas in Indonesia.
The mutations in
P. falciparum dihydrofolate reductase (
pfdhfr) and dihydropteroate synthase (
pfdhps) are responsible for pyrimethamine and sulfadoxine resistance, respectively [
13‐
16]. Stepwise accumulation of point mutations in
pfdhfr and
pfdhps genes is associated with higher level of resistance to SP in vitro and in vivo [
17,
18]. The amino acid substitution at position 108 serine to asparagine or threonine (S108
N/
T) in
pfdhfr is essential for subsequent A16
V, N51
I, C59
R and I164
L mutations (underlined bold type indicates the mutant allele), leading to high-level of resistance to cycloguanil or pyrimethamine [
19]. Similarly, a single mutation in the
pfdhps converting alanine to glycine at amino acid position 437 (A437
G), which is normally the first mutation under the sulfadoxine drug pressure, conferred on the parasite a fivefold higher level of drug resistance [
16]. Additional mutations K540
E and A581
G, then S436
A/
F and A613
S/
T are associated with increasing resistance to sulfadoxine. A combination of
pfdhfr triple (N51
I, C59
R and S108
N) and
pfdhps double (A437
G and K540
E) mutations collectively form the quintuple mutations, which is strongly associated with in vitro and in vivo SP resistance [
17,
20‐
24].
There are several reports of mutation analysis of
pfdhfr and
pfdhps genes from different malaria endemic areas in Indonesia. Different ratio of mutation level was reported from sample analysis of several island and district parasites. From the West Papua sample analysis obtained in 1996 to 1999 by Nagesha et al. [
25], C59
R and S108
N mutations in
pfdhfr and A437
G in
pfdhps were commonly determined in SP resistant parasites. In addition, they reported an additional K540
E mutation in
pfdhps was observed in more resistant level parasites. Extensive analysis of the parasite genotypes from eight malaria endemic areas were reported by Syafruddin et al. in 2005, representing a broad region of the western and eastern Indonesian archipelago [
26]. Polymorphisms in
pfdhfr gene at S108
N/
T, A16
V and C59
R were frequently identified, in which A16
V were observed in association with S108
T and these were differently distributed, more common among samples from eastern regions. The polymorphism in
pfdhps was less frequent in this report; about 15% of A437
G and less than 5% of K540
E were detected. Among the Sumba island samples of 2007, less frequent mutations were reported by Asih et al. in 2009 [
27]; about 25% parasites presented S108
N and C59
R mutations in
pfdhfr and only few % of the isolates presented A437
G mutant allele of
pfdhps. More prevalence of mutations in
pfdhfr and
pfdhps was observed from a study of SP efficacy and genotype analysis in South Kalimantan in 2009–2010 [
28]. More than 90% of the isolates exhibited S108
N and C59
R mutations in
pfdhfr gene, in addition I164
L substitution was detected in 30% of the parasites. The alterations in
pfdhps were detected at the amino acid positions of A437, K540, A581 and I588 to glycine (97%), glutamine or threonine (36%, 36%), glycine (45%) and phenylalanine (23%), respectively. This result of
pfdhfr and
pfdhps genotypes is quite unique compared with the previous Indonesia parasites; more prevalence of mutation ratio and detection of novel mutations of
pfdhps K540
T and I588
F.
In this study, pfdhfr and pfdhps sequences of P. falciparum from different malaria endemic regions, mostly eastern part of Indonesia were analysed to investigate polymorphisms of pfdhfr and pfdhps genotypes and predict susceptibility for sulfadoxine–pyrimethamine in malaria parasites in Indonesia, and to obtain information about distribution of the previously identified pfdhps K540T and I588F novel mutations.
Methods
Study sites and malaria patients
Malaria parasites collected from the following several endemic areas in Indonesia were analysed in this study: Sumatera (Indragiri Hilir, Merangin), Kalimantan (Paser, Seruyan, Banjar), Java (Pacitan), Lombok (West Lombok), Sumbawa (Sumbawa), Timor (Timor Tengah Selatan), Sulawesi (Gorontalo), and Papua (Jayapura) islands in 2004–2006 and in 2009–2012. Malaria patients were recruited at primary health centers, district hospitals or local field areas and written informed consent were obtained from each participant, or from caretakers if participations were under 12 years old, after explanation of the purpose of the study in local language. Finger prick blood samples were collected on a glass slide for microscopical observation and on a filter paper (Advantec, Toyo Roshi Kaisha, Ltd., Japan) for parasite DNA analysis. Thick smear blood films were stained with Giemsa (Merck, Germany) and examined microscopically for the presence of malaria parasites. Dried blood spot on a filter paper was kept in a small plastic clips prior to parasite DNA extraction. In some field studies, BinaxNOW Malaria diagnosis kit (Binax Inc, Portland, ME, USA) were used to identify P. falciparum malaria patients. Patients with positive malaria diagnosis results were treated with an anti-malarial drug according to national policy. The study protocol was reviewed and approved by the Ethical Committee, Faculty of Medicine, Universitas Airlangga, Surabaya, Indonesia and Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan.
Parasite DNA analysis
Parasite DNA was extracted from the dried blood spots on filter paper by using QIAamp DNA blood mini kit (Qiagen, Netherlands) and kept at − 30 °C.
Plasmodium falciparum samples were selected by nested PCR methodology using species specific primer sets of 18S rRNA genes described in Snounou et al. [
29] and Kimura et al. [
30].
Pfdhfr and
pfdhps genotypes were determined by sequencing as previously described by Isozumi et al. [
31]. using amplified PCR product as a template directly for sequence analysis. Alleles corresponding to amino acid positions at 16, 51, 59, 108 and 164 of the
pfdhfr gene and at 436, 437, 540, 581, 588 and 613 of the
pfdhps gene were read more carefully, and at least two independent PCR products were prepared for sequence analysis in the case of rare mutations. Previously reported results from South Kalimantan Province [
28] were included in this analysis.
Statistical analysis
Data were entered in Microsoft Excel and exported to SPSS version 17.0 for analysis. Chi square and Fisher’s exact tests were used, where applicable, to assess the relationship of mutations between two periods of studies. Allele proportions were calculated as the number carrying a certain allele divided by the number of samples with positive PCR outcome.
Discussion
In this report, polymorphisms of
pfdhfr and
pfdhps genes in several malaria endemic areas in Indonesia are analysed in two periods of time, 2004–2006 and 2009–2012. Previously reported amino acid substitutions, important for sulfadoxine–pyrimethamine (SP) resistance in Indonesian parasites, were determined in this analysis. The C59
R, S108
N and I164
L mutations in
pfdhfr gene and A437
G, K540
E/
T, A581
G and I588
F in
pfdhps were observed from both 2004–2006 and 2009–2012 samples, but
pfdhfr A16
V and S108
T mutations for cycloguanil resistance were not detected (Fig.
1). In
pfdhfr gene, wild type ANCSI and three mutant ANC
NI, AN
RNI and AN
RNL alleles were observed (Table
1). This simple accumulation pattern of mutations in the
pfdhfr genotype supports the stepwise selection hypothesis of resistant gene evolution [
19]. Wild type SAKAA and six different mutant alleles were determined in
pfdhps gene (Table
2). Comparison of wild type and each mutant alleles suggests stepwise accumulation of mutations in the
pfdhps genotypes.
The unique K540
T and I588
F mutations of
pfdhps, both of which were detected previously from 2009 to 2010 South Kalimantan sample analysis [
28] and the former was also reported by Lau et al. [
32] from 2010 Sabah, Malaysia parasites, were identified in 2004–2006 parasites. The evidence presenting here that K540
T was detected in 2004–2006 as an S
GTGA haplotype from several parasites in East Kalimantan, East Java and Sumbawa Island suggests a possibility of Kalimantan Island origin for this mutation. In East Kalimantan, the S
GTGA was found from many patients as a combination genotype of either AN
RNI/S
GTGA or AN
RNL/S
GTGA, additionally as a mixed infection of both genotypes (Table
3). Meanwhile, the S
GTGA existed as the combination genotype of AN
RNI/S
GTGA only in East Java and Sumbawa parasites.
Another novel mutation I588F was also detected in 2004–2006 samples from Lombok and Sumbawa islands; the parasites of SGEAA both with and without I588F mutation were detected in Sumbawa, whereas SGEAA(588F) and wild type SAKAA were observed in Lombok. It suggests a possibility that the initial mutation of I588F had occurred in SGEAA type parasites in Sumbawa Island, then introduced into its neighbor Lombok Island. Further analysis and comparison of microsatellite loci around the pfdhps haplotypes will provide additional information for understanding the origin and spreading of these K540T and I588F mutant alleles.
In Riau and Jambi Provinces (Sumatera Island), only small number of malaria patients had been detected and these were mostly infected with P. vivax and only a few P. falciparum were observed in both 2004–2006 and 2009–2012 periods. Timor Tengah Selatan (Timor Island) and Gorontalo (Sulawesi Island) were similar situation in 2009–2012. Remarkably pfdhps I588F mutation was observed in Gorontalo as the same combined genotype of ANRNI/SGEAA(588F).
Genotype comparison of 2004–2006 and 2009–2012 parasites in each malaria areas provides additional information (Table
3). In Lombok, parasites of the major combined genotype AN
RNI/SAKAA and a few number of minor type were detected in both 2004–2006 and 2009–2012 periods. The parasites might have well responded to SP or other malaria therapies and new SP resistant type parasites had not settled in Lombok. On the other hand, increased number of
pfdhfr/
pfdhps combined genotype was determined in 2009–2012 parasites from Papua Island compared to 2004–2006 parasites. The genotypes were AN
RNI/S
GEAA(588
F) and new combinations with either
pfdhfr or
pfdhps wild type. This suggests some parasites with wild type
pfdhfr ANCSI and
pfdhps SAKAA were introduced as the both wild type combination or either haplotype individually between two periods. The AN
RNI/S
GEAA had been selected under SP pressure before 2004–2006, and the new parasites with
pfdhfr and/or
pfdhps wild type alleles and AN
RNI/S
GEAA(588
F) were maintained under reduced SP pressure after DHP was commonly used. ACT was introduced in Papua Province in 2005; first artesunate-amodiaquine (AA) was used experimentally, then changed to dihydroartemisinin–piperaquine (DHP) in 2008 [
33]. Wild type
pfdhfr and
pfdhps genotype detection was reported in the parasites from West Papua and several islands by Syafruddin et al. in 2005 [
26]. It suggests the parasites with wild type allele were introduced from a neighbour districts or it might exist in Papua in 2004–2006, however it could not detected because the number of samples was not sufficient.
Parasite populations from different Provinces in Kalimantan Island presented diverse polymorphisms. Analysis of Paser (East Kalimantan) 2004–2006 parasites revealed dominance of the
pfdhps K540
T mutation (S
GTGA haplotype, Fig.
2b) and many cases of mixed genotype infections (Table
3). This suggests high
P. falciparum infection rate under strong pressure of SP. The mixed genotype infection was not common in 2009–2012 samples from Middle and South Kalimantan, and the parasites from these Provinces presented characteristic genotype polymorphisms. Most of the parasites from Seruyan (Middle Kalimantan) acquired
pfdhfr I164
L mutation (AN
RNL haplotype, Fig.
2a), and several
pfdhps genotypes involving unique
pfdhps K540
T or I588
F mutations were detected in Banjar (South Kalimantan) parasites (Fig.
2b).
High heterogeneity of malaria epidemiology and divergent genetic polymorphisms across islands, districts, and even close neighbour sub-districts in one island are not uncommon in Indonesia [
26]. Divergent polymorphisms in
pfdhfr/
pfdhps genotype of the parasite populations from different Kalimantan provinces were observed in this study from analysis of the parasite samples before introduction of ACT at each research site in these Provinces. AA was implemented in 2006 in East Kalimantan and then DHP treatment was started in 2009. DHP has been first-line treatment therapy against malaria in Middle Kalimantan and South Kalimantan since 2010. However in fact, ACT was introduced gradually form one site to the other, and the parasite samples from Kalimantan analysed in this research were collected before ACT was applied in the research areas. Current situation and comparison of genetic divergence among Kalimantan provinces are important subject to provide information how application of ACT influences malaria parasite populations on genotypes and polymorphisms.
Different prevalence of quintuple or sextuple mutant parasites in
pfdhfr/
pfdhps combined genotypes were observed (Fig.
3). While the parasites from Kalimantan and Pacitan (East Java) belonged to SP resistant quintuple or sextuple mutant genotype, parasites from the other areas in Indonesia presented four or less mutations in combined genotype that tend to adequate response for SP treatment. In addition, efficacy of SP treatment for
P. vivax malaria infection in Indonesia was reported by Asih et al. [
34] recently. These suggest SP could be considered for prevention or treatment of malaria as a single prescription or in combination with artemisinin in Indonesia except in Kalimantan Island. In such a case, regular monitoring of the efficacy and regular genotype analysis are essential to prevent spread of resistance.
Mobility of people among thousands of islands is one of the important factors for malaria control in Indonesia. Some of outbreaks, resurgences of malaria had occurred under the unique circumstances of people migration. Marwoto et al. reported immigrant workers who worked as transmigrants or seasonal workers in malaria endemic areas outside Java Island returned to their home villages brought imported malaria cases [
35,
36]. Many migrants and temporal workers have moved from Pacitan (East Java) to several islands historically. Similar
pfdhfr/
pfdhps genotype polymorphisms in 2004–2006 parasites from Pacitan (East Java) and 2009–2012 Saruyan (Middle Kalimantan) in Table
3 suggests the malaria parasites in these districts could have been transferred along with human migrations.
The Indonesia National Malaria Control Programme desires to eliminate malaria in the whole country by 2030 [
37]. Most districts in Java and Bali islands, also several districts in other islands fall under the World Health Organization criteria of elimination and malaria cases have gradually decreased over the last several years in Indonesia [
38].
Considering presence of malaria vector mosquitoes in most of the places used to be malaria endemic, and current situation of frequent human migrations within the country, continuous maintenance of early malaria diagnosis and treatment system is essential for malaria elimination programme. Parasite genotype analysis is helpful to follow malaria transmission and drug treatment efficiencies.
Authors’ contributions
SB and HU prepared the protocol, summarized and interpreted data, wrote the paper. SB, F and HU analysed the samples. F, PMR, K, PA, DS, SR, I and BA coordinated field studies and collected data at the field locations. B developed protocol. YPD and HK reviewed and discussed the protocol, results, interpretation and the manuscript. All authors read and approved the final manuscript.