Genetic polymorphisms of Plasmodium vivax ookinete (sexual stage) surface proteins (Pvs25 and Pvs28) from Thailand.

Plasmodium vivax is the most geographically widespread malaria parasite in human presently. The ookinete surface proteins of sexual stage of malaria parasites, Pvs25 and Pvs28, are candidates for the transmission blocking vaccine. The antigenic variation in population might be barrier for vaccine development. The objective of this study was to investigate the genetic diversity of Pvs25 and Pvs28 in endemic areas of Thailand. P. vivax clinical isolates collected from Thai-neighboring border areas were analyzed using polymerase chain reaction and sequencing method. Three and 14 amino acid substitutions were observed in 43 Pvs25 and 48 Pvs28 sequences, respectively. Three haplotypes in Pvs25 and 14 haplotypes with 5-7 GSGGE/D tandem repeats in Pvs28 were identified. The nucleotide diversity of pvs25 (π = 0.00059) had lower level than pvs28 (π = 0.00517). Tajima's D value for both pvs25 and pvs28 genes were negative while no significant difference was found (P > 0.10). Low genetic diversity was found in pvs25 and pvs28 genes in Thailand. The finding of the most frequent amino acid substitutions was consistent with global isolates. Therefore, the data could be helpful in developing of effective transmission blocking vaccine in malaria endemic areas.

Surveillance of drug resistance molecular markers in Plasmodium vivax before and after introduction of dihydroartemisinin and piperaquine in Thailand: 2009-2019.

Resistance to antimalarial drugs is a serious issue around the world. Widespread Plasmodium vivax and P. falciparum coinfections are commonly found in Thailand. Dihydroartemisinin and piperaquine (DHA-PPQ) have been used as first-line treatments for P. falciparum since 2015, and chloroquine (CQ) and primaquine (PQ) have remained first-line drugs for P. vivax for more than 60 years. Coinfections may lead parasites to evolve with regard to genetics under selective drug pressure. This study is aimed at investigating genes linked to antimalarial resistance in P. vivax before and after introduction of DHA-PPQ as a new drug regimen in Thailand. A total of 400 P. vivax isolates were collected from samples along the Thai-Myanmar and Thai-Malaysian borders before (2009-2015) and after (2016-2019) introduction of DHA-PPQ. Genomic DNA of P. vivax was obtained and subjected to analysis of five drug resistance-associated genes (Pvdhfr, Pvdhps, Pvmdr1, Pvcrt-o, and PvK12) by nested polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), and nucleotide sequencing. A high prevalence of Pvdhfr was found in both endemic areas over the period. The quadruple (57I/58R/61M/117T) Pvdhfr haplotype was predominant in both periods in both endemic areas. Although the wild-type haplotype of Pvdhps was predominant in Thai-Malaysian isolates in both periods, a single mutant haplotype (383G) was dominant in Thai-Myanmar isolates during both periods. A low prevalence of the Pvmdr1 976F mutation was found in both periods among Thai-Myanmar isolates. A significant decrease in Pvmdr1 976F was identified in Thai-Malaysian isolates from the second period (p < 0.01). Only one nonsynonymous mutation of Pvcrt-o (193E) and one synonymous mutation of PvK12 (R584) were detected in four isolates (4.7%) and one isolate (0.5%) in the first period among Thai-Myanmar isolates, respectively. Thus, with limited clinical efficacy data, the low prevalence of drug-resistance markers may suggest that there is a low prevalence of P. vivax-resistant strains and that the current drug regimen for P. vivax is still effective for treating this P. vivax parasite population. Continued surveillance of antimalarial drug resistance markers and monitoring of clinical drug efficacy should be conducted for epidemiological and policy implications.

Assessment of pharmacognostic specification of Cannabis sativa leaves in Thailand.

Lack of quality control can affect the safety, efficacy, and acceptability of herbal products that may lead to health problems. Cannabis sativa L. (Cannabaceae) has been widely used as an ethnomedicinal practice for its medicinal values. This study aims to establish pharmacognostic specifications of C. sativa as per standard procedures. Macroscopic-microscopic characteristics, physicochemical parameters, thin-layer chromatography (TLC) fingerprinting, and phytochemical screening of C. sativa leaves collected from various locations throughout Thailand were investigated. Leaves are palmate consists of seven leaflets with green color, margin is serrate with acuminate apex. Anomocytic stomata were found in the upper epidermis while unicellular and glandular trichomes with cystolith were found in the lower epidermis and the epidermis layer covered with cuticle. The physicochemical analysis revealed that the loss on drying (4.068 ± 0.084 %w/w) was within acceptable limits, total ash (14.360 ± 0.084%w/w), acid insoluble ash (2.726 ± 0.080%w/w), ethanol-soluble extractive (11.101 ± 0.223%w/w), water-soluble extractive (23.038 ± 0.306%w/w), and water content (7.523 ± 0.524%w/w). TLC fingerprint showed nine spots with Rf value 0.14, 019, 0.23, 0.29, 0.32, 0.45, 0.58, 0.70, and 0.76. Phytochemical screening of Cannabis leaves indicated the presence of phenolic compounds, flavonoids, alkaloids, diterpenes, triterpenes, and steroids. This study provided referential data for the accurate plant identity, and establishment of cannabis leaves monograph in Thailand.

Genetic Diversity of Plasmodium vivax Merozoite Surface Protein-3 Alpha and Beta from Diverse Geographic Areas of Thailand.

Malaria is parasitic disease cause by Plasmodium infection. In Thailand, coinfections of Plasmodium vivax and Plasmodium falciparum are commonly found. P. vivax infection has been increasing in the past decade. The objective of this study was to investigate the genetic diversity patterns of P. vivax merozoite surface protein 3 (PvMSP-3) genes in a total of 450 isolates collected from Thai-neighboring border areas during two different periods (2009-2014 and 2015-2016) using polymerase chain reaction (PCR)-restriction fragment length polymorphism method. Three major types of PvMSP-3α (A, B, and C) and PvMSP-3ß (A, B, and C) were detected based on PCR product size. In total, 45 and 23 alleles of PvMSP-3α and 41 and 30 alleles of PvMSP-3ß with different frequencies of samples were distinguished from the first and second period, respectively. Our results strongly indicate genetic diversity patterns of PvMSP-3 in isolates from the second period, especially in samples from the Thai-Myanmar border area. These two polymorphic genes could be used as molecular epidemiologic markers for genotyping P. vivax isolate in Thailand.

Phytochemical screening, antimalarial activities, and genetic relationship of 16 indigenous Thai Asteraceae medicinal plants: A combinatorial approach using phylogeny and ethnobotanical bioprospecting in antimalarial drug discovery.

Emergence of artemisinin resistance leads the people to discover the new candidate for antimalarial drug. Combinatorial phylogeny and ethnobotanical approach may be useful to minimize the expenditure and time in laboratory testing. Seven hundred and thirty-three ethnomedicinal plants were listed from literature search. Obtained 340 internal transcribed spacer (ITS) sequences of plant list which met criteria were retrieved from GenBank NCBI and analyzed by MUSCLE and maximum likelihood phylogenetic test to generate the phylogenetic tree. Interactive phylogenetic tree was generated by Interactive Tree of Life (ITOL, https://itol.embl.de) and showed strong clustered pattern on Asteraceae. Afterward, 16 species of Asteraceae were selected to investigate the antimalarial activity, phytochemical, and genetic diversity. The presence of phytochemical was determined by standard method. DNA fluorescence-based assay was performed to determine the antimalarial activity against 3D7 Plasmodium falciparum. IC50µg/mL was used to categorize antimalarial activity. On the other hand, ITS universal primer was used to amplify and sequence the obtained extracted DNA of tested plant by cetyltrimethylammonium bromide method. Phylogenetic analyses were performed by MAFFT and RAxML with automatic bootstrapping. ITOL and Adobe Illustrator were used to generate interactive phylogenetic tree. All species tested showed the presence of phenolics and flavonoids, whereas alkaloids and terpenoids were shown vary among tested extracts. Among 16 species tested, 1 species exhibited good-moderate (Sphaeranthus indicus, IC506.59 µg/mL), 4 weak (Artemisia chinensis, Artemisia vulgaris, Tridax procumbens, and Blumea balsamifera), and 3 very weak (Eupatorium capillifolium, Wedelia trilobata, and Vernonia cinerea). Generated phylogenetic tree by ITS data was able to separate the tested species into their tribal classification. In addition, new medicinal properties of A. chinensis were discovered. Combining phylogeny approach with ethnobotanical data is useful to narrow down the selection of antimalarial plants candidate.

Assessment of phylogenetic relationship among twenty Curcuma species in Thailand using amplified fragment length polymorphism marker.

Plants in the genus Curcuma are a rhizomatous perennial herb which is widely distributed in Thailand. It has long been known for their uses as folk medicines, foods, spices, and cosmetics. However, the identification of plants in the genus Curcuma is very difficult due to morphological similarity in the early flowering stage. Recently, the molecular technique is one of the reliable and powerful tools for plant identification. In this study, the genetic relationship among twenty Curcuma species from Thailand was accessed by the amplified fragment length polymorphism (AFLP) method. AFLP fingerprint showed 98.54% highly polymorphisms with the number of bands (617 bands) ranging between 48 and 80 bands. The dendrogram generated from the unweighted pair group method of the arithmetic average could separate these Curcuma species into three major clusters. Cluster I can be subdivided into IA, which composed of Curcuma parviflora, Curcuma sparganiifolia, Curcuma alismatifolia, Curcuma larsenii, Curcuma Gracillima, and Curcuma rhabdota with similarity index (SI) 0.7926-0.9358 and IB composed of Curcuma petiolata and Curcuma rubrobracteata with the SI 0.9240. Cluster II can be subdivided into IIA being composed of Curcuma longa, Curcuma Zedoaria, and Curcuma aromatica with the SI 0.8989-0.9071, whereas Cluster IIB was composed of Curcuma leucorrhiza, Curcuma aeruginosa, Curcuma comosa, Curcuma mangga, Curcuma angustifolia, Curcuma amada, Curcuma sessilis, and Curcuma albicoma with the SI 0.8236-0.9500. Cluster III belongs to Curcuma singularis and Alpinia galanga (outgroup plant), which clearly separated into different clusters from twenty Curcuma species. In summary, the ten successful AFLP primer combinations could be used to determine the genetic relationship among closely related twenty Curcuma species in Thailand.

Evaluation of Plasmodium vivax isolates in Thailand using polymorphic markers Plasmodium merozoite surface protein (PvMSP) 1 and PvMSP3.

Malaria is a significant public health problem in several tropical countries including Thailand. The prevalence of Plasmodium vivax infection has been increasing in the past decades. Plasmodium vivax merozoite surface protein (PvMSP) gene encodes a malaria vaccine candidate antigen. Its polymorphic nature leads to antigenic variation, the barrier for vaccine development, drug resistance, and potential for multiple-clone infections within the malaria patients. The objective of this study was to investigate the genetic diversity of PvMSP1 and PvMSP3 gene in P. vivax populations in Thailand. A total of 100 P. vivax isolates collected from the western (Kanchanaburi and Tak Provinces) and southern (Ranong Provinces) regions along the Thai-Myanmar border were analyzed using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Analysis of the F1, F2, and F3 regions of PvMSP1 revealed 5, 2, and 3 allelic variants, respectively. Three major types of PvMSP3-α and two major types of PvMSP3-ß were identified based on the PCR product sizes. After digestion with restriction enzymes, 29, 25, 26, and 18 patterns were distinguished by RFLP for PvMSP1 (F2, Alu I), PvMSP1 (F2, Mnl I), PvMSP3-α, and PvMSP3-ß, respectively. Combination of each family variant (PvMSP1 and PvMSP3) resulted in high genetic polymorphism of P. vivax population. Additionally, using PvMSP1 polymorphic marker revealed a significant association between multiple-genotype infections and P. vivax parasitemia. The results strongly supported that P. vivax populations in the endemic areas along the Thai-Myanmar border are highly diverse.

Characterization of Mangifera indica cultivars in Thailand based on macroscopic, microscopic, and genetic characters.

Thai mango cultivars are classified into six groups plus one miscellaneous group according to germplasm database for mango. Characterization is important for conservation and the development of Thai mango cultivars. This study investigated macroscopic, microscopic leaf characteristics, and genetic relationship among 17 cultivars selected from six groups of mango in Thailand. Selected mango samples were obtained from three different locations in Thailand (n = 57). They were observed for their leaf and fruit macroscopic characteristics. Leaf measurement for the stomatal number, veinlet termination number, and palisade ratio was evaluated under a microscope attached with digital camera. DNA fingerprint was performed using CTAB extraction of DNA and inter-simple sequence repeat (ISSR) amplification. Forty-five primers were screened; then, seven primers that amplified the reproducible band patterns were selected to amplified and generate dendrogram by Unweighted Pair-Group Method with Arithmetic Average. These selected 17 Thai mango cultivars had individually macroscopic characteristics based on fruits and leaves. For microscopic characteristics, the stomatal number, veinlet termination number, and palisade ratio were slightly differentiable. For genetic identification, 78 bands of 190-2660 bps were amplified, of which 82.05% were polymorphic. The genetic relationship among these cultivars was demonstrated and categorized into two main clusters. It was shown that ISSR markers could be useful for Thai mango cultivar identification.

Patients' adherence and clinical effectiveness of a 14-day course of primaquine when given with a 3-day chloroquine in patients with Plasmodium vivax at the Thai-Myanmar border.

Primaquine is the only antimalarial drug available for eradicating the hypnozoite stage of Plasmodium vivax to prevent the disease from recurring. However, one limitation of its clinical use is the long treatment course of 14 days, which may result in poor patients' adherence and low treatment efficacy. The aim of the current study was to assess patients' adherence and the clinical effectiveness of the unsupervised standard 14-day primaquine regimen (daily dose of 15mg base/kg body weight daily for 14 days) when given together with 3-day chloroquine (25mg base/kg body weight over 3 days). The study was conducted in 85 patients with P. vivax malaria in a malaria endemic area along the Thai-Myanmar border. Patients' adherence to primaquine therapy was assessed based on primaquine concentrations in finger-prick dried blood spot (DBS) samples alongside patients' self-reporting on drug administration and pill counting methods. Results suggest high rate of patients' adherence to this 14-day primaquine regimen (95-98% based on primaquine concentrations in DBS on days 3, 7, and 14 of treatment, and 100% based on patients' self-reporting and pill counting methods. Clinical effectiveness was 100% during the 42-day follow-up.

Genetic Polymorphisms in Plasmodium vivax Dihydrofolate Reductase and Dihydropteroate Synthase in Isolates from the Philippines, Bangladesh, and Nepal.

Genetic polymorphisms of pvdhfr and pvdhps genes of Plasmodium vivax were investigated in 83 blood samples collected from patients in the Philippines, Bangladesh, and Nepal. The SNP-haplotypes of the pvdhfr gene at the amino acid positions 13, 33, 57, 58, 61, 117, and 173, and that of the pvdhps gene at the positions 383 and 553 were analyzed by nested PCR-RFLP. Results suggest diverse polymorphic patterns of pvdhfr alone as well as the combination patterns with pvdhps mutant alleles in P. vivax isolates collected from the 3 endemic countries in Asia. All samples carried mutant combination alleles of pvdhfr and pvdhps. The most prevalent combination alleles found in samples from the Philippines and Bangladesh were triple mutant pvdhfr combined with single mutant pvdhps allele and triple mutant pvdhfr combined with double wild-type pvdhps alleles, respectively. Those collected from Nepal were quadruple mutant pvdhfr combined with double wild-type pvdhps alleles. New alternative antifolate drugs which are effective against sulfadoxine-pyrimethamine (SP)-resistant P. vivax are required.

Plasmodium vivax drug resistance genes; Pvmdr1 and Pvcrt-o polymorphisms in relation to chloroquine sensitivity from a malaria endemic area of Thailand.

The aim of the study was to explore the possible molecular markers of chloroquine resistance in Plasmodium vivax isolates in Thailand. A total of 30 P. vivax isolates were collected from a malaria endemic area along the Thai-Myanmar border in Mae Sot district of Thailand. Dried blood spot samples were collected for analysis of Pvmdr1 and Pvcrt-o polymorphisms. Blood samples (100 µl) were collected by finger-prick for in vitro chloroquine susceptibility testing by schizont maturation inhibition assay. Based on the cut-off IC50 of 100 nM, 19 (63.3%) isolates were classified as chloroquine resistant P. vivax isolates. Seven non-synonymous mutations and 2 synonymous were identified in Pvmdr1 gene. Y976F and F1076L mutations were detected in 7 (23.3%) and 16 isolates (53.3%), respectively. Analysis of Pvcrt-o gene revealed that all isolates were wild-type. Our results suggest that chloroquine resistance gene is now spreading in this area. Monitoring of chloroquine resistant molecular markers provide a useful tool for future control of P. vivax malaria.

Proteomics analysis of antimalarial targets of Garcinia mangostana Linn.

OBJECTIVE: To investigate possible protein targets for antimalarial activity of Garcinia mangostana Linn. (G. mangostana) (pericarp) in 3D7 Plasmodium falciparum clone using 2-dimensional electrophoresis and liquid chromatography mass-spectrometry (LC/MS/MS). METHODS: 3D7 Plasmodium falciparum was exposed to the crude ethanolic extract of G. mangostana Linn. (pericarp) at the concentrations of 12µg/mL (IC50 level: concentration that inhibits parasite growth by 50%) and 30 µg/mL (IC90 level: concentration that inhibits parasite growth by 90%) for 12 h. Parasite proteins were separated by 2-dimensional electrophoresis and identified by LC/MS/MS. RESULTS: At the IC50 concentration, about 82% of the expressed parasite proteins were matched with the control (non-exposed), while at the IC90 concentration, only 15% matched proteins were found. The selected protein spots from parasite exposed to the plant extract at the concentration of 12 µg/mL were identified as enzymes that play role in glycolysis pathway, i.e., phosphoglycerate mutase putative, L-lactate dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase, and fructose-bisphosphate aldolase/phosphoglycerate kinase. The proteosome was found in parasite exposed to 30 µg/mL of the extract. CONCLUSIONS: Results suggest that proteins involved in the glycolysis pathway may be the targets for antimalarial activity of G. mangostana Linn. (pericarp).

Proteomics analysis of antimalarial targets of Garcinia mangostana Linn

Objective: To investigate possible protein targets for antimalarial activity of Garcinia mangostana Linn. (G. mangostana) (pericarp) in 3D7 Plasmodium falciparum clone using 2-dimensional electrophoresis and liquid chromatography mass-spectrometry (LC/MS/MS). Methods: 3D7 Plasmodium falciparum was exposed to the crude ethanolic extract of G.mangostana Linn. (pericarp) at the concentrations of 12μg/mL (IC50 level: concentration that inhibits parasite growth by 50%) and 30 μg/mL (IC90 level: concentration that inhibits parasite growth by 90%) for 12 h. Parasite proteins were separated by 2-dimensional electrophoresis and identified by LC/MS/MS.Results:At the IC50 concentration, about 82% of the expressed parasite proteins were matched with the control (non-exposed), while at the IC90 concentration, only 15% matched proteins were found. The selected protein spots from parasite exposed to the plant extract at the concentration of 12 μg/mL were identified as enzymes that play role in glycolysis pathway, i.e., phosphoglycerate mutase putative, L-lactate dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase, and fructose-bisphosphate aldolase/phosphoglycerate kinase. The proteosome was found in parasite exposed to 30 μg/mL of the extract.Conclusions:Results suggest that proteins involved in the glycolysis pathway may be the targets for antimalarial activity of G. mangostana Linn. (pericarp).

Distribution of dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) mutant alleles in Plasmodium vivax isolates from Thailand.

The analysis of prevalence and distribution of pvdhfr and pvdhps mutations were performed in 169 samples collected from patients with Plasmodium vivax infection who attended the malaria clinics in the provinces along the three international borders of Thailand (Thai-Myanmar, Thai-Cambodian, and Thai-Malaysian borders). SNP-haplotypes of the pvdhfr at amino acid positions 13, 33, 57, 58, 61, 117, and 173 and of the pvdhps at positions 383 and 553 were examined by nested PCR-RFLP. Significant differences in the prevalence and distribution of pvdhfr and pvdhps combination alleles were observed in P. vivax isolates collected from all the three border areas. The most prevalent combination alleles were triple mutant pvdhfr 57L/58R/117T alleles/double wild-type pvdhps alleles (n=18), double mutant pvdhfr 58R/117N alleles/double wild-type pvdhps alleles (n=10), and triple mutant pvdhfr 58R/61M/117N alleles/double wild-type pvdhps alleles (n=52) or with single mutant pvdhps 383G allele (n=28), respectively. These information on prevalence and patterns of pvdhfr and pvdhps polymorphisms obtained from the present study suggest the presence of SP pressure on P. vivax isolates in Thailand which could be linked to the introduction of malaria from neighboring countries. Results did not support the application of SP for P. vivax control program in Thailand as well as the neighboring countries.

The identities and anti-herpes simplex virus activity of Clinacanthus nutans and Clinacanthus siamensis.

OBJECTIVE: To distinguish the difference among the Clinacanthus nutans (Burm. f.) Lindau (C. nutans) and Clinacanthus siamensis Bremek (C. siamensis) by assessing pharmacognosy characteristics, molecular aspect and also to evaluate their anti-herpes simplex virus (HSV) type 1 and type 2 activities. METHODS: Macroscopic and microscopic evaluation were performed according to WHO Geneva guideline. Stomatal number, stomatal index and palisade ratio of leaves were evaluated. Genomic DNA was extracted by modified CTAB method and ITS region was amplified using PCR and then sequenced. Dry leaves were subsequently extracted with n-hexane, dichloromethane and methanol and antiviral activity was performed using plaque reduction assay and the cytotoxicity of the extracts on Vero cells was determined by MTT assay. RESULTS: Cross section of midrib and stem showed similar major components. Leaf measurement index of stomatal number, stomatal index and palisade ratio of C. nutans were 168.32±29.49, 13.83±0.86 and 6.84±0.66, respectively, while C. siamensis were 161.60±18.04, 11.93±0.81 and 3.37±0.31, respectively. The PCR amplification of ITS region generated the PCR product approximately 700 bp in size. There were 34 polymorphisms within the ITS region which consisted of 11 Indels and 23 nucleotide substitutions. The IC50 values of C. nutans extracted with n-hexane, dichloromethane and methanol against HSV-1 were (32.05±3.63) µg/mL, (44.50±2.66) µg/mL, (64.93±7.00) µg/mL, respectively where as those of C. siamensis were (60.00±11.61) µg/mL, (55.69±4.41) µg/mL, (37.39±5.85) µg/mL, respectively. Anti HSV-2 activity of n-hexane, dichloromethane and methanol C. nutans leaves extracts were (72.62±12.60) µg/mL, (65.19±21.45) µg/mL, (65.13±2.22) µg/mL, respectively where as those of C. siamensis were (46.52±4.08) µg/mL, (49.63±2.59) µg/mL, (72.64±6.52) µg/mL, respectively. CONCLUSIONS: The combination of macroscopic, microscopic and biomolecular method are able to authenticate these closely related plants and both of them have a potency to be an anti-HSV agent.

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.

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.

Genotyping of polymorphic marker (MSP3α and MSP3ß) genes of Plasmodium vivax field isolates from malaria endemic of Thailand.

Two polymorphic marker genes, merozoite surface protein 3α (PvMSP3α) and merozoite surface protein 3ß (PvMSP3ß), from 100 Plasmodium vivax field isolates, were investigated using polymerase chain reaction and restriction fragment length polymorphism (PCR/RFLP). Genotyping of PvMSP3α and PvMSP3ß revealed marked polymorphisms in length and sequence. Three major types of PvMSP3α (Type A, B and C) and two major types of PvMSP3ß (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 PvMSP3ß 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.

Monitoring of clinical efficacy and in vitro sensitivity of Plasmodium vivax to chloroquine in area along Thai Myanmar border during 2009-2010.

BACKGROUND: In Thailand, the proportion of Plasmodium vivax infection has become equal to Plasmodium falciparum. Reports of a trend of gradual decline of in vitro sensitivity of P. vivax to chloroquine in some areas of the country, together with accumulating evidences of chloroquine resistance P. vivax in other parts of the world, emphasize the need for closely and continuously monitoring clinical efficacy in conjunction with in vitro sensitivity of P. vivax isolates. METHODS: The study was conducted at Mae Tao clinic for migrant workers, Tak Province during March 2008-August 2009. A total of 130 patients (17 Thais and 113 Burmeses; 64 males and 66 females) with mono-infection of P. vivax malaria, aged between 15-60 years and weighing more than 40 kg, were included in the study. Patients received treatment with chloroquine (2,000 mg chloroquine phosphate over three days) and the anti-relapse drug primaquine (15 mg for 14 days). In vitro sensitivity of P. vivax isolates was evaluated by schizont maturation inhibition assay. RESULTS: All patients showed satisfactory response to treatment. The cure rate was virtually 100% within the follow-up period of 42 days. Neither recurrence of P. vivax parasitaemia nor appearance of P. falciparum occurred during the investigation period. In vitro data showed a stable sensitivity of chloroquine in this area since 2006. Geometric mean and median (95% CI) values of IC50 for chloroquine were 100.1 and 134.7 (1.1-264.9) nM, respectively. CONCLUSION: In vivo results suggest that the standard regimen of chloroquine was still very effective for the treatment of blood infections with P. vivax in the Thai-Myanmar border area. In vitro sensitivity data however, raise the possibility of potential advent of resistance in the future. Regular monitoring of the chloroquine sensitivity of P. vivax is essential to facilitate the early recognition of treatment failures and to expedite the formulation of appropriate changes to the drug policy.

Assessment of in vitro sensitivity of Plasmodium vivax fresh isolates.

OBJECTIVE: To compare the applicability of the SYBR Green-I assay with the standard schizont maturation assay, for determination of sensitivity of Plasmodium vivax (P. vivax) to chloroquine and a new antifolate WR 99210. METHODS: The study was conducted at Mae Tao Clinic for migrant workers, Tak Province during April 2009 to July 2010. A total of 64 blood samples (1 mL blood collected into sodium heparinized plastic tube) were collected from patients with mono-infection with P. vivax malaria prior to treatment with standard regimen of a 3-day chloroquine. In vitro sensitivity of P. vivax isolates was evaluated by schizont maturation inhibition and SYBR Green-I assays. RESULTS: A total of 30 out of 64 blood samples collected from patients with P. vivax malaria were successfully analyzed using both the microscopic schizont maturation inhibition and SYBR Green-I assays. The failure rates of the schizont maturation inhibition assay (50%) and the SYBR Green-I assay (54%) were similar (P=0.51). The median IC10s, IC50s and IC90s of both chloroquine and WR99210 were not significantly different from the clinical isolates of P. vivax tested. Based on the cut-off of 100 nM, the prevalences of chloroquine resistance determined by schizont maturation inhibition and SYBR Green-I assays were 19 and 11 isolates, respectively. The strength of agreement between the two methods was very poor for both chloroquine and WR99210. CONCLUSIONS: On the basis of this condition and its superior sensitivity, the microscopic method appears better than the SYBR Green-I Green assay for assessing in vitro sensitivity of fresh P. vivax isolates to antimalarial drugs.