Population Genetics of Plasmodium vivax in Four High Malaria Endemic Areas in Thailand

Recent trends of malaria in Thailand illustrate an increasing proportion of Plasmodium vivax, indicating the importance of P. vivax as a major causative agent of malaria. P. vivax malaria is usually considered a benign disease so the knowledge of this parasite has been limited, especially the genetic diversity and genetic structure of isolates from different endemic areas. The aim of this study was to examine the population genetics and structure of P. vivax isolates from 4 provinces with different malaria endemic settings in Thailand using 6 microsatellite markers. Total 234 blood samples from P. vivax mono-infected patients were collected. Strong genetic diversity was observed across all study sites; the expected heterozygosity values ranged from 0.5871 to 0.9033. Genetic variability in this study divided P. vivax population into 3 clusters; first was P. vivax isolates from Mae Hong Son and Kanchanaburi Provinces located on the western part of Thailand; second, Yala isolates from the south; and third, Chanthaburi isolates from the east. P. vivax isolates from patients having parasite clearance time (PCT) longer than 24 hr after the first dose of chloroquine treatment had higher diversity when compared with those having PCT within 24 hr. This study revealed a clear evidence of different population structure of P. vivax from different malaria endemic areas of Thailand. The findings provide beneficial information to malaria control programme as it is a useful tool to track the source of infections and current malaria control efforts.

Genetic diversity of the msp-1, msp-2, and glurp genes of Plasmodium falciparum isolates along the Thai-Myanmar borders

Objective:To study the genetic diversity at the msp-1, msp-2, and glurp genes of Plasmodium falciparum (P. falciparum) isolates from 3 endemic areas in Thailand: Tak, Kanchanaburi and Ranong provinces. Methods:A total of 144 P. falciparum isolates collected prior to treatment during January, 2012 to June, 2013 were genotyped. DNA was extracted;allele frequency and diversity of msp-1, msp-2, and glurp genes were investigated by nested polymerase chain reaction. Results:P. falciparum isolates in this study had high rate of multiple genotypes infection (96.5%) with an overall mean multiplicity of infection of 3.21. The distribution of allelic families of msp-1 was significantly different among isolates from Tak, Kanchanaburi, and Ranong but not for the msp-2. K1 and MAD20 were the predominant allelic families at the msp-1 gene, whereas alleles belonging to 3D7 were more frequent at the msp-2 gene. The glurp gene had the least diverse alleles. Population structure of P. falciparum isolates from Tak and Ranong was quite similar as revealed by the presence of similar proportions of MAD20 and K1 alleles at msp-1 loci, 3D7 and FC27 alleles at msp-2 loci as well as comparable mean MOI. Isolates from Kanchanaburi had different structures;the most prevalent alleles were K1 and RO33. Conclusions: The present study shows that P. falciparum isolates from Tak and Ranong provinces had similar allelic pattern of msp-1 and msp-2 and diversity but different from Kanchanaburi isolates. These allelic variant profiles are valuable baseline data for future epidemiological study of malaria transmission and for continued monitoring of polymorphisms associated with antimalarial drug resistance in these areas.

Genetic diversity of the msp-1, msp-2, and glurp genes of Plasmodium falciparum isolates along the Thai-Myanmar borders

<p><b>OBJECTIVE</b>To study the genetic diversity at the msp-1, msp-2, and glurp genes of Plasmodium falciparum (P. falciparum) isolates from 3 endemic areas in Thailand: Tak, Kanchanaburi and Ranong provinces.</p><p><b>METHODS</b>A total of 144 P. falciparum isolates collected prior to treatment during January, 2012 to June, 2013 were genotyped. DNA was extracted; allele frequency and diversity of msp-1, msp-2, and glurp genes were investigated by nested polymerase chain reaction.</p><p><b>RESULTS</b>P. falciparum isolates in this study had high rate of multiple genotypes infection (96.5%) with an overall mean multiplicity of infection of 3.21. The distribution of allelic families of msp-1 was significantly different among isolates from Tak, Kanchanaburi, and Ranong but not for the msp-2. K1 and MAD20 were the predominant allelic families at the msp-1 gene, whereas alleles belonging to 3D7 were more frequent at the msp-2 gene. The glurp gene had the least diverse alleles. Population structure of P. falciparum isolates from Tak and Ranong was quite similar as revealed by the presence of similar proportions of MAD20 and K1 alleles at msp-1 loci, 3D7 and FC27 alleles at msp-2 loci as well as comparable mean MOI. Isolates from Kanchanaburi had different structures; the most prevalent alleles were K1 and RO33.</p><p><b>CONCLUSIONS</b>The present study shows that P. falciparum isolates from Tak and Ranong provinces had similar allelic pattern of msp-1 and msp-2 and diversity but different from Kanchanaburi isolates. These allelic variant profiles are valuable baseline data for future epidemiological study of malaria transmission and for continued monitoring of polymorphisms associated with antimalarial drug resistance in these areas.</p>

Plasmodium falciparum Genotype Diversity in Artemisinin Derivatives Treatment Failure Patients along the Thai-Myanmar Border

Genetic characteristics of Plasmodium falciparum may play a role in the treatment outcome of malaria infection. We have studied the association between diversity at the merozoite surface protein-1 (msp-1), msp-2, and glutamate-rich protein (glurp) loci and the treatment outcome of uncomplicated falciparum malaria patients along the Thai-Myanmar border who were treated with artemisinin derivatives combination therapy. P. falciparum isolates were collected prior to treatment from 3 groups of patients; 50 cases of treatment failures, 50 recrudescences, and 56 successful treatments. Genotyping of the 3 polymorphic markers was analyzed by nested PCR. The distribution of msp-1 alleles was significantly different among the 3 groups of patients but not the msp-2 and glurp alleles. The allelic frequencies of K1 and MAD20 alleles of msp1 gene were higher while RO33 allele was significantly lower in the successful treatment group. Treatment failure samples had a higher median number of alleles as compared to the successful treatment group. Specific genotypes of msp-1, msp-2, and glurp were significantly associated with the treatment outcomes. Three allelic size variants were significantly higher among the isolates from the treatment failure groups, i.e., K1270-290, 3D7610-630, G650-690, while 2 variants, K1150-170, and 3D7670-690 were significantly lower. In conclusion, the present study reports the differences in multiplicity of infection and distribution of specific alleles of msp-1, msp-2, and glurp genes in P. falciparum isolates obtained from treatment failure and successful treatment patients following artemisinin derivatives combination therapy.

Genetic diversity of the msp-1, msp-2, and glurp genes of Plasmodium falciparum isolates along the Thai-Myanmar borders

Objective: To study the genetic diversity at the msp-1, msp-2, and glurp genes of Plasmodium falciparum (P. falciparum) isolates from 3 endemic areas in Thailand: Tak, Kanchanaburi and Ranong provinces. Methods: A total of 144 P. falciparum isolates collected prior to treatment during January, 2012 to June, 2013 were genotyped. DNA was extracted; allele frequency and diversity of msp-1, msp-2, and glurp genes were investigated by nested polymerase chain reaction. Results: P. falciparum isolates in this study had high rate of multiple genotypes infection (96.5%) with an overall mean multiplicity of infection of 3.21. The distribution of allelic families of msp-1 was significantly different among isolates from Tak, Kanchanaburi, and Ranong but not for the msp-2. K1 and MAD20 were the predominant allelic families at the msp-1 gene, whereas alleles belonging to 3D7 were more frequent at the msp-2 gene. The glurp gene had the least diverse alleles. Population structure of P. falciparum isolates from Tak and Ranong was quite similar as revealed by the presence of similar proportions of MAD20 and K1 alleles at msp-1 loci, 3D7 and FC27 alleles at msp-2 loci as well as comparable mean MOI. Isolates from Kanchanaburi had different structures; the most prevalent alleles were K1 and RO33. Conclusions: The present study shows that P. falciparum isolates from Tak and Ranong provinces had similar allelic pattern of msp-1 and msp-2 and diversity but different from Kanchanaburi isolates. These allelic variant profiles are valuable baseline data for future epidemiological study of malaria transmission and for continued monitoring of polymorphisms associated with antimalarial drug resistance in these areas.

Mechanism of action of drug-resistant malaria and the development of new antimalarial drugs.

Abstract not available

Assessment of a Real-Time PCR for the Efficacy Monitoring of Antimalarial Treatment

The objective of this study was to assess a Real-Time PCR for the efficacy monitoringof antimalarial treatment. The assay compared microscopy with Real-Time PCR forassessing the efficacy of artesunate-mefloquine combination in the treatment of falciparum malaria patients. Thirty six patients with uncomplicated falciparum were selected from fifty patients. Thick peripheral blood smears were taken on day 0, 3, 7, 14 and 28. The ratio of male and female was 4:1. The ratio of Thai and Burmist was 2:7.The results showed that all thick blood films of 36 patients were positive while theReal-Time PCR showed 33 positive for falciparum (Tm73-74๐C) and 3 positive for vivax(Tm76-77๐C). The thick blood film of 165 samples from 33 patients was negative. 16.33 % of these samples were positive when testing with Real-Time PCR. The thick blood film sample of 6 patients from 36 patients was negative on day 3, 7, 14 and 28 after treatment while the results of Real-Time PCR were P. falciparum on day 3 and 28 in 3 and 1 patients respectively. The real-time PCR showed positive P.vivax on day 7 in 2 patients. The results showed that thick blood film had a lower sensitivity than the Real-Time PCR method. However, microscopic detection remains the most reliable standard. In this result we classified every negative thick smear corresponding to a positive Real-Time PCR results as a false negative. Furthermore, the important finding in this study is that 16.67 % of treatment failure were detected by the Real-Time PCR method while, 5.56 % of treatment failure were detected by thick blood film. The Real-Time PCR method is useful for in vivo drug efficacy studies. It is important to detect resistant malaria parasites before they reach high resistance level. \ KEY Words : Real-Time PCR, Efficacy Monitoring, Antimalarial Treatment\ 

Appropriate laboratory tests for the diagnosis of dengue infection at a general hospital in Southern Thailand

Background: Laboratory diagnosis of dengue infection is complicated by the variability of either virus, antigen or antibody appearance in blood circulation. Patients came to the hospital at different time intervals after the onset of fever. This resulted in problems in the selection of appropriate laboratory tests. Objective: To determine the appropriate laboratory assays for the diagnosis of dengue infection at a general hospital in Southern Thailand. Methods: Serum samples were obtained from 175 dengue-suspected patients attending Nakhon Si Thammarat Provincial Hospital, in Southern Thailand during the period January-September 2006. Dengue infected patients were classified as having clinical symptoms according to the WHO criteria for dengue infection combined with positive ELISA test results for IgM and/or IgG antibodies for dengue virus. All were assayed for dengue viral infection by real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR), Platelia Dengue NS1 Ag test (NS1-Ag), Dengue IgM Capture ELISA (ELISA-IgM), Dengue Indirect IgG ELISA (ELISA-IgG) and Dengue Duo IgM and IgG rapid strip test (RST). Results: One hundred of the 175 dengue suspected cases were diagnosed as having dengue viral infection. Thirty-eight cases (22%) were positive by NS1-Ag test, 36 (21%) by real-time RT-PCR, 41 (23%) by RST, 74 (42%) by ELISA-IgM and 88 (50%) by ELISA-IgG. Among the 100 positive cases, 40 cases (40%) were determined as primary infection and 60 cases (60%) as secondary infection on the basis of the variability of specific IgG and IgM levels in patient serum. The NS1-Ag test kit, real-time RT-PCR and RST detected more positive cases in primary infected patients compared to secondary infected patients (p \< 0.0001). With ELISA-IgG when combined with NS1-Ag, RT-PCR or ELISA-IgM, 88, 89 and 96% of the cases could be detected. Three combinations of ELISA-IgG, ELISA-IgM with NS1-Ag or RT-PCR resulted in 99 and 100 % positive cases. Conclusion: A combination of ELISA-IgG, ELISA-IgM with NS1 Ag test was appropriate for laboratory diagnosis of dengue infection at the hospital where equipment for running ELISA tests was available.

Mixed-species malaria infection in high transmission areas of Thailand

Background: Mixed-species malaria infections examined by microscopy of Giemsa-stained thick blood films (GS-TBF) during 1996-2007 in Thailand were 0.3-0.5. However, there were reports of higher mixed-species infection rates detected by polymerase chain reaction (PCR) method. This study was conducted in order to ascertain the relative frequency of mixed-species malaria infection and possible determinants in top 10 malaria high transmission provinces of Thailand. Methods: This study was a survey of mixed Plasmodium species incidence in the top 10 malaria transmission provinces of Thailand. A total of 836 malaria patients were examined. The number of samples in each province was proportionate to the number of malaria patients in that province. A real-time PCR based on SYBR Green I detection system was used to detect and differentiate Plasmodium species. Results: Preliminary results from GS-TBF examined by the microscopists at the malaria clinics in the selected areas showed that 380 (45.5%) of 836 patients were infected by Plasmodium falciparum; 450 (53.8%), 2 (0.2%) and 4 (0.5%) were infected by P. vivax, P. malariae and mixed P. falciparum and P. vivax, respectively. Real-time PCR results of the corresponding samples from filter papers showed that 353 (42.2%) were infected by Plasmodium falciparum; 446 (53.4%), 1 (0.1%), 2 (0.2%), 32 (3.8%), 1 (0.1%) and 1 (0.1%) were infected by P. vivax, P. malariae, P. ovale, mixed P. falciparum and P. vivax, mixed P. falciparum and P. malariae and mixed P. vivax and P. ovale, respectively. Conclusion: Mixed-infection rates detected by real-time PCR were 8.48 (4.07/0.48) times higher than those detected by GS-TBS. Demographic factors including age, sex, occupation, place where contracted the disease and recurrence of the infection were not different between the groups with mono-species infection and mixed-species infection.

Drug Sensitivity of Plasmodium falciparum and Plasmodium vivax in the area of Mae Sot, Tak Province, northwestern Thailand

In vitro studies on the drug response of <I>Plasmodium falciparum</I> were conducted as from 1998 in the district of Mae Sot in northwestern Thailand near the border to Myanmar. In vitro studies on the drug response of <I>Plasmodium vivax</I> started in 2001 in the same area. For <I>P. falciparum</I> the investigations showed a very high degree of resistance. The sensitivity to mefloquine declined significantly between 198 and 2005, whereas the sensitivity to artemisinin and to quinine increased during the same period. Sensitivity to lumefantrine and atovaquone is still in the therapeutically feasible range. The sensitivity of <I>P. vivax</I> to chloroquine has declined between 2000 and 2004. Mefloquine and lumefantrine have been identified as potential replacement drugs. The activity of monodesbutyl-benflumetol proved to be higher than that of structurally related lumefantrine.