Aree Seugorn

Molecular characterisation of drug-resistant Plasmodium falciparum from Thailand

BackgroundThe increasing levels of Plasmodium falciparum resistance to chloroquine (CQ) in Thailand have led to the use of alternative antimalarials, which are at present also becoming ineffective. In this context, any strategies that help improve the surveillance of drug resistance, become crucial in overcoming the problem.MethodsIn the present study, we have established the in vitro sensitivity to CQ, mefloquine (MF), quinine (QUIN) and amodiaquine (AMQ) of 52 P. falciparum isolates collected in Thailand, and assessed the prevalence of four putative genetic polymorphisms of drug resistance, pfcrt K76T, pfmdr1 N86Y, pfmdr1 D1042N and pfmdr1 Y1246D, by PCR-RFLP.ResultsThe percentage of isolates resistant to CQ, MF, and AMQ was 96% (50/52), 62% (32/52), and 58% (18/31), respectively, while all parasites were found to be sensitive to QUIN. In addition, 41 (79%) of the isolates assayed were resistant simultaneously to more than one drug; 25 to CQ and MF, 9 to CQ and AMQ, and 7 to all three drugs, CQ, MF and AMQ. There were two significant associations between drug sensitivity and presence of particular molecular markers, i) CQ resistance / pfcrt 76T (P = 0.001), and ii) MF resistance / pfmdr1 86N (P < 0.001)Conclusionsi) In Thailand, the high levels of CQ pressure have led to strong selection of the pfcrt 76T polymorphism and ii) pfmdr1 86N appears to be a good predictor of in vitro MF resistance.

Genotyping of polymorphic marker (MSP3α and MSP3β) genes of Plasmodium vivax field isolates from malaria endemic of Thailand

Two polymorphic marker genes, merozoite surface protein 3α (PvMSP3α) and merozoite surface protein 3β (PvMSP3β), from 100 Plasmodium vivax field isolates, were investigated using polymerase chain reaction and restriction fragment length polymorphism (PCR/RFLP). Genotyping of PvMSP3α and PvMSP3β revealed marked polymorphisms in length and sequence. Three major types of PvMSP3α (Type A, B and C) and two major types of PvMSP3β (Type A and B) were detected based on the length of PCR products. Fourteen alleles of both genes with difference frequencies were distinguished by restriction fragment length polymorphism, and these results strongly support that P. vivax isolates in Thailand are markedly diverse. PvMSP3α and PvMSP3β are reliable polymorphic markers for population genetic analysis of P. vivax, and PCR/RFLP provides a powerful method for genotyping and identification of mixed parasite infections without requirement of gene sequencing.

The patterns of mutation and amplification of Plasmodium falciparum pfcrt and pfmdr1 genes in Thailand during the year 1988 to 2003

The study investigated the patterns of pfmdr1 and pfcrt genetic polymorphisms in Plasmodium falciaprum isolates collected from Thailand during the periods 1988-1993 (35 isolates), and 2003 (21 isolates). Pfcrt polymorphisms were almost universal for the mutations at codons K76T, A220S, Q271E, N326S, and R371I. All parasites displayed the chloroquine (CQ)-resistant phenotypes. This data suggested that pfcrt gene was sufficient to CQ resistance but did not mediate level of resistance. The prevalence [number of isolates (%)] of pfmdr1 polymorphisms at codons N86Y, Y184F, S1034C, N1042D and D1246Y were five (9%), 48 (86%), ten (18%), and 15 (27%), respectively. All isolates carried the wild-type nucleotide at position 1246. Results support the role of pfmdr1 in modulating susceptibilities of the P. falciparum to CQ, QN, and MQ. The frequencies of the S1034C and N1042D pfmdr1 polymorphisms and number of gene copy were significantly different in isolates collected during the two periods, with a trend of increasing prevalence of wild-type genotypes and number of gene copy from 1988 to 2003. The prominent pattern of pfmdr1 at codons 86/184/1034/1042/1246 was NFSND, with prevalence increasing from 40% to 95% during the 10-year period.

Association between chloroquine resistance phenotypes and point mutations in pfcrt and pfmdr1 in Plasmodium falciparum isolates from Thailand.

The relationship between the in vitro susceptibility of Plasmodium falciparum isolates to the quinoline antimalarials chloroquine (CQ), mefloquine (MQ), and quinine (QN), and pfcrt and pfmdr1 gene polymorphisms were investigated. Field isolates (110 samples) were collected from various endemic areas of Thailand throughout 2002-2004. The pfcrt 76T allele was identified in 109 isolates (99.1%) while pfcrt 76K was found in a single (0.9%) isolate. The pfmdr 86N, 86Y, and the combination (86N+86Y) alleles were identified in 83 (75.5%), 22 (20%), and 5 (4.5%) isolates, respectively. The pfmdr1 1042N, 1042D alleles and a mixture (1042N+1042D) of the alleles were found in 94 (85.5%), 12 (10.9%) and 4 (3.6%) isolates, respectively. The pfmdr1 1246Y allele was detected in a single (0.9%) isolate. The pfmdr1 gene polymorphisms (86-1042-1246) was grouped into seven haplotypes as follows: N-N-D (68 isolates; 61.2%), Y-N-D (22 isolates; 19.8%), N-D-D (11 isolates; 9.9%), N-D-Y (1 isolate; 0.9%), N/Y-N-D (4 isolates; 3.6%), N-N/D-D (3 isolates; 2.7%), and N/Y-N/D-D (1 isolate; 0.9%). Eight different combinations of pfcrt-pfmdr1 genotypes were observed. Only one CQ-, MQ- and QN-sensitive isolate was found at the Thai-Laos border and no cases of QN resistance were found in this study.

Diversity and population structure of Plasmodium falciparum in Thailand based on the spatial and temporal haplotype patterns of the C-terminal 19-kDa domain of merozoite surface protein-1

BackgroundThe 19-kDa C-terminal region of the merozoite surface protein-1 of the human malaria parasite Plasmodium falciparum (Pf MSP-119) constitutes the major component on the surface of merozoites and is considered as one of the leading candidates for asexual blood stage vaccines. Because the protein exhibits a level of sequence variation that may compromise the effectiveness of a vaccine, the global sequence diversity of Pf MSP-119 has been subjected to extensive research, especially in malaria endemic areas. In Thailand, Pf MSP-119 sequences have been derived from a single parasite population in Tak province, located along the Thailand-Myanmar border, since 1995. However, the extent of sequence variation and the spatiotemporal patterns of the MSP-119 haplotypes along the Thai borders with Laos and Cambodia are unknown.MethodsSixty-three isolates of P. falciparum from five geographically isolated populations along the Thai borders with Myanmar, Laos and Cambodia in three transmission seasons between 2002 and 2008 were collected and culture-adapted. The msp-1 gene block 17 was sequenced and analysed for the allelic diversity, frequency and distribution patterns of Pf MSP-119 haplotypes in individual populations. The Pf MSP-119 haplotype patterns were then compared between parasite populations to infer the population structure and genetic differentiation of the malaria parasite.ResultsFive conserved polymorphic positions, which accounted for five distinct haplotypes, of Pf MSP-119 were identified. Differences in the prevalence of Pf MSP-119 haplotypes were detected in different geographical regions, with the highest levels of genetic diversity being found in the Kanchanaburi and Ranong provinces along the Thailand-Myanmar border and Trat province located at the Thailand-Cambodia border. Despite this variability, the distribution patterns of individual Pf MSP-119 haplotypes seemed to be very similar across the country and over the three malarial transmission seasons, suggesting that gene flow may operate between parasite populations circulating in Thailand and the three neighboring countries.ConclusionThe major MSP-119 haplotypes of P. falciparum populations in all endemic populations during three transmission seasons in Thailand were identified, providing basic information on the common haplotypes of MSP-119 that is of use for malaria vaccine development and inferring the population structure of P. falciparum populations in Thailand.

Comparison of protein patterns between Plasmodium falciparum mutant clone T9/94-M1-1(b3) induced by pyrimethamine and the original parent clone T9/94.

OBJECTIVE To compare the protein patterns from the extracts of the mutant clone T9/94-M1-1(b3) induced by pyrimethamine, and the original parent clone T9/94 following separation of parasite extracts by two-dimensional electrophoresis (2-DE). METHODS Proteins were solubilized and separated according to their charges and sizes. The separated protein spots were then detected by silver staining and analyzed for protein density by the powerful image analysis software. RESULTS Differentially expressed protein patterns (up- or down-regulation) were separated from the extracts from the two clones. A total of 223 and 134 protein spots were detected from the extracts of T9/94 and T9/94-M1-1(b3) clones, respectively. Marked reduction in density of protein expression was observed with the extract from the mutant (resistant) clone compared with the parent (sensitive) clone. A total of 25 protein spots showed at least two-fold difference in density, some of which exhibited as high as ten-fold difference. CONCLUSIONS These proteins may be the molecular targets of resistance of Plasmodium falciparum to pyrimethamine. Further study to identify the chemical structures of these proteins by mass spectrometry is required.