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Background: While malaria incidence in Indonesia has decreased threefold in the last decade, more than 200,000 cases were reported in 2016. Different endemicity of Plasmodium falciparum malaria among several islands in Indonesia has been recognized and two unique mutations of P. falciparum dihydropteroate synthase (pfdhps) affecting sulfadoxine-pyrimethamine (SP) resistance were detected from the research of SP efficiency and genotype analysis in South Kalimantan. In this study, geographical distribution and origin of these pfdhps K540T and I588F mutations were analysed., Methods: Malaria parasites DNA from several endemic areas in Indonesia; Sumatera, Java, Kalimantan, Lombok, Sumbawa, Timor, Sulawesi, and Papua islands; in two periods, 2004-2006 and 2009-2012 were subjected for pfdhfr and pfdhps sequence analysis., Results: Different genotype polymorphisms of pfdhfr and pfdhps were observed in the parasites from various regions in Indonesia and relatively more divergent genotypes were determined from Kalimantan isolates in both 2004-2006 and 2009-2012. The parasites containing K540T mutation were identified in 2004-2006 isolates from East Kalimantan, East Java and Sumbawa as an SGTGA haplotype. The other I588F mutation was also determined in 2004-2006 parasites, isolated from Lombok and Sumbawa islands as an SGEAA(588F) haplotype. The parasites with pfdhfr/pfdhps quintuple or sextuple mutation, a genotype marker of SP resistance, were determined mostly in Kalimantan in both 2004-2006 and 2009-2012., Conclusion: Analysis of the prevalence and pfdhfr/pfdhps combined genotypes of K540T or I588F mutations suggested that K540T might be origin in Kalimantan Island and I588F in Sumbawa Island and then these were spread to other areas along with people movement. This research indicates regular monitoring of drug efficacy and parasite genotype analysis is important to keep efficiency and prevent the spread of resistance. It is also essential for the latest anti-malarial drug artemisinin-based combination therapy.

Background: Mutations in pfdhfr and pfdhps genes have been shown to associate with sulphadoxine-pyrimethamine (SP) resistance of Plasmodium falciparum parasites. However, pfdhfr, pfdhps genotypes and the correlations to SP treatment outcome in Indonesia has not yet been well analysed., Methods: After obtaining informed consent, 61 uncomplicated falciparum malaria patients were recruited in Banjar district, South Kalimantan Province, Indonesia, from October 2009 to August 2010. They were treated by a single oral dose of SP and its effects on clinical and parasitological status were followed until day 28 after treatment. Occasionally, a thick smear blood film for microscopy observation and blood spot on a filter paper for pfdhfr and pfdhps genotype analysis were collected., Results: Pfdhfr and pfdhps genotypes from 24 P. falciparum-infected patients consisting of adequate clinical parasitological response (ACPR) (n = 6; 25.0%) and early treatment failure (ETF) (n = 10; 41.7%) or late parasitological failure (LPF) (n = 8; 33.3%) were obtained by sequencing. Two novel mutations of pfdhps gene, K540T and I588F, were determined in ten and five isolates, respectively. These mutations were present in the pfdhfr/pfdhps combined haplotypes of ANRNI/SGTGA (n = 6), ANRNL/SGTGA (n = 4), and ANRNI/SGEAA(588F) (n = 5), (mutation codons are bold typed); these haplotypes were mostly belonging to parasitological failure (ETF or LPF). The parasites acquiring five mutations in pfdhfr/pfdhps haplotypes and four mutations with additional I588F did not respond adequately to SP treatment., Conclusion: Many of Plasmodium falciparum infected patients in Banjar district, South Kalimantan, Indonesia did not respond adequately to SP treatment and these low ineffectiveness of SP in this area was associated with two novel mutations of pfdhps, K540T and I588F.

The amino-terminal region of the serine repeat antigen (SERA) of Plasmodium falciparum is a major malaria-vaccine candidate. Variation in this molecule is essentially dimorphic and alleles may be grouped into the types FCR3, K1 and Honduras1. The Honduras1-type is thought to be the product of homologous recombination between FCR3 and K1 alleles. Here we have examined patterns of sequence diversity in exon II of SERA gene, which encodes most of the amino-terminal region of the antigen, in wild P. falciparum isolates from Indonesia (n=60), Myanmar (n=10) and Thailand (n=14). Among the Indonesian isolates the FCR-3 type predominated (56/60), twenty of which we characterized as novel alleles. A new K1-type allele was also found. In Myanmar, however, all isolates displayed K1-type SERA sequences, which included one new allele. The Honduras1-type was not detected in both countries. In contrast, the 14 isolates from Thailand displayed all three allelic types, with one new Honduras1-type and three new K1-type alleles. On examining the global distribution of SERA alleles by combining previously published sequence data with our results, the FCR3-type alleles predominated in Indonesia, Brazil, and Solomon Islands, but were not found in wild isolates from Myanmar and Africa. Brazil was the only area where K1-type alleles were not found. The distribution of Honduras1-type alleles seems to be mostly restricted to parasite populations from Vietnam, Thailand and Africa. In the allelic families FCR3 and K1, most diversity resulted from variation in sequence and number of octamer repeat units and of allotypes encoding the stretch of serine residues. Sequence analysis indicated that both insertions and deletions of repetitive motifs (creating variation within dimorphic allelic families) and homologous recombination between alleles belonging to different allelic families (creating Honduras1-type alleles) play a role in generating new SERA alleles. Since repeat motifs in the amino-terminal region of SERA contain epitopes recognized by parasite-inhibitory antibodies, sequence variation in exon II may represent one of the parasite's immune-evasion strategies.

In vitro growth of Plasmodium falciparum is restricted in glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes (RBC), as a result of oxidative stress. Bathocuproine disulphonate (BCS), a copper chelator, as well as cysteine have been shown to synergistically stimulate the in vitro growth of various mammalian cells and Trypanosoma under oxygenated conditions. We examined the effects of these two chemicals on the in vitro growth of P. falciparum in G6PD-deficient RBC, and found that addition of BCS and cysteine synergistically enhanced the growth of the P. falciparum FCR-3 strain in these RBC to the same level as in normal RBC. However, BCS or cysteine alone had no stimulatory effect. To explain this synergistic enhancement, changes in thiol, NADPH and glutathione contents were investigated. After addition of cysteine alone, thiol content in the medium decreased rapidly, but when BCS was added, it was maintained at about 35% at 24 hours after incubation, suggesting that BCS stimulates parasite growth in G6PD-deficient RBC by inhibiting copper-mediated oxidation of cysteine in the medium. In these RBC, no increase in NADPH level, but a slight increase in glutathione, was observed in the presence of both BCS and cysteine. The slight increase of glutathione, was probably due to incorporation of cysteine from the medium, although this could not fully explain the synergistic growth enhancement. These findings taken together suggest that cysteine incorporated into G6PD-deficient RBC may help maintain the thiol groups in many proteins, such as membrane proteins, hemoglobin and enzymes, and plays an important role in maintaining an appropriate culture state necessary for parasite growth. We also examined the effects of BCS and cysteine on adaptation of wild isolates of P. falciparum to in vitro cultivation using the candle jar method. Although there was no drastic effect on growth enhancement, the presence of BCS and cysteine accelerated the appearance of schizonts in many isolates.