Distinct Allelic Diversity of Plasmodium vivax Merozoite Surface Protein 3-Alpha (PvMSP-3α) Gene in Thailand Using PCR-RFLP.

Considering the importance of merozoite surface proteins (MSPs) as vaccine candidates, this study was conducted to investigate the polymorphism and genetic diversity of Plasmodium vivax merozoite surface protein 3-alpha (PvMSP-3α) in Thailand. To analyze genetic diversity, 118 blood samples containing P. vivax were collected from four malaria-endemic areas in western and southern Thailand. The DNA was extracted and amplified for the PvMSP-3α gene using nested PCR. The PCR products were genotyped by PCR-RFLP with Hha I and Alu I restriction enzymes. The combination patterns of Hha I and Alu I RFLP were used to identify allelic variants. Genetic evaluation and phylogenic analysis were performed on 13 sequences, including 10 sequences from our study and 3 sequences from GenBank. The results revealed three major types of PvMSP-3α, 91.5% allelic type A (∼1.8 kb), 5.1% allelic type B (∼1.5 kb), and 3.4% allelic type C (∼1.2 kb), were detected based on PCR product size with different frequencies. Among all PvMSP-3α, 19 allelic subtypes with Hha I RFLP patterns were distinguished and 6 allelic subtypes with Alu I RFLP patterns were identified. Of these samples, 73 (61%) and 42 (35.6%) samples were defined as monoallelic subtype infection by Hha I and Alu I PCR-RFLP, respectively, whereas 77 (65.3%) samples were determined to be mixed-allelic subtype infection by the combination patterns of Hha I and Alu I RFLP. These results strongly indicate that PvMSP-3α gene is highly polymorphic, particularly in blood samples collected from the Thai-Myanmar border area (the western part of Thailand). The combination patterns of Hha I and Alu I RFLP of the PvMSP-3α gene could be considered for use as molecular epidemiologic markers for genotyping P. vivax isolates in Thailand.

Development of Nested PCR-Heteroduplex Mobility Assay for Determination of Genetic Diversity in the Block 2 Region of the Plasmodium falciparum Merozoite Surface Protein 1 Gene.

Genetic diversity of Plasmodium parasite has significantly related to malaria control and vaccine development. The P. falciparum merozoite surface protein 1 (Pfmsp1) gene is a commonly used molecular marker to differentiate genetic diversity. This study is aimed at developing a nested PCR-Heteroduplex Mobility Assay (nPCR-HMA) for determination of the block 2 of the Pfmsp1 gene. The MAD20 family allele of P. falciparum was used as a control for optimization of the annealing and polyacrylamide gel electrophoresis conditions. In order to evaluate the developed nPCR-HMA, 8 clinical P. falciparum isolates were examined for allelic variants. The results revealed 9 allelic variants. Our study indicated that the successful nPCR-HMA with good precision and accuracy offers a more rapid, efficient, and cheap method for large-scale molecular epidemiological studies as compared to nucleotide sequencing.

Physical influence on larvicidal and pupicidal activity of the silicone-based monomolecular film.

Although silicone-based monomolecular film (MMF) has been accepted as larvicide in several countries, its mosquito control potential has never been investigated in Thailand. Laboratory assessment in this study was conducted to determine the MMF efficacy against Aedes aegypti. At the recommended dosage (1mL/m(2) of water surface), mortality of pupae (99.17±0.83%) was significantly greater than mortality of old and young larvae (73.33±9.13, 11.67±3.47%; respectively). Pupicidal activity was rapidly exhibited within hours while larvicidal activity took at least one day. Interestingly, among the survived mosquitoes after MMF exposure, larval length (3.6±0.18mm), pupation (0%) and adult emergence (0%) were significantly less than the control group. Gravid females also avoided laying eggs in MMF-treated oviposition cups. There was no influence of physical factors on MMF efficacy and no toxic effects on fish and plants. These results indicated the MMF is promising to provide not only larvicidal and pupicidal activity but also inhibition of larval development as indicated by both larval length and stage transformation.

Biosensor as a molecular malaria differential diagnosis.

BACKGROUND: In malaria diagnosis, specific gene identification is required in cases with subclinical infection or cases with mixed infection. This study applied the biosensor technology based on quartz crystal microbalance (QCM) to differentially diagnose the most common and severe malaria, Plasmodium falciparum and Plasmodium vivax. METHOD: The QCM surface was immobilized with malaria biotinylated probe. Specific DNA fragments of malaria-infected blood were amplified. Hybridization between the amplified products and the immobilized probe resulted in quartz frequency shifts which were measured by an in-house frequency counter. Diagnostic potency and clinical application of the malaria QCM were evaluated. RESULT: The malaria QCM could differentially diagnose blood infected with P. falciparum from that infected with P. vivax (p-value<0.05). No cross reaction with human DNA indicated the QCM specificity. Clinical application was evaluated using 30 suspected samples. Twenty-seven samples showed consistent diagnosis of the QCM with microscopy and rapid diagnosis tests (RDTs). Three samples reported "no malaria found" by microscopy showed P. falciparum infection by both QCM and the RDTs. CONCLUSION: The malaria QCM was developed with high accuracy, specificity, sensitivity, stability and cost-effectiveness. It is field applicable in malaria endemic area and might be a promising point of care testing.

Asymptomatic malaria infections among foreign migrant workers in Thailand.

OBJECTIVE: To determine the prevalence of malaria infections among foreign migrant workers in Thailand. METHODS: Giemsa-stained thin and thick blood films were prepared from blood samples of 294 foreign migrant workers recruited in the study. Microscopic examination of these blood films was performed for malaria detection. RESULTS: Blood film examination revealed 1.36% malaria infections in these 294 subjects. All positive cases were male Myanmar workers in which their blood films only ring stage of Plasmodium spp. was found at low parasite density (mean= 144 parasites/µ L of blood). The prevalence of malaria infections was not significantly different among foreign migrant workers classified by age, gender, and resident province (P>0.05). Thin blood films of these workers also showed 78.91% hypochromic erythrocytes and 61.9% relative Eosinophilia. CONCLUSIONS: These findings indicate a high risk of malaria transmission. Therefore active malaria surveillance by using molecular methods with more sensitive and specific than microscopy should be considered for malaria control in foreign migrant workers.

Diagnosis and genotyping of Plasmodium falciparum by a DNA biosensor based on quartz crystal microbalance (QCM).

BACKGROUND: Malaria infection with Plasmodium falciparum is an important basic health problem in the tropical and sub-tropical countries. The standard diagnostic method is blood film examination to visualize parasite morphology. However, in cases of low parasitemia or mixed infection with very low cryptic species, microscopy is not sensitive enough. Therefore, molecular techniques have been widely employed. METHODS: A label-free DNA biosensor based on quartz crystal microbalance (QCM) to diagnose and genotype P. falciparum was developed. Avidin-biotin interaction was used to coat the specific biotinylated probe on the gold surface of QCM. The gene encoding merozoite surface protein 2 (msp2) was amplified and the PCR products were then cut with restriction enzyme (MwoI). Enzymatic cutting made the PCR products suitable for QCM development. Hybridization between probe and enzymatic cutting DNA fragments resulted in frequency changes of the QCM. RESULTS: The newly developed QCM was tested for its diagnosis ability using both malaria laboratory strains and clinical isolates. The biosensor was sensitive at the sub-nanogram level, specific for only P. falciparum detection, no cross-reaction with P. vivax, and stable at room temperature for up to 6 months. Selection of msp2 as a target gene and a geno-typing marker made the QCM potentially useful for falciparum diagnosis simultaneously with genotyping. Potency was tested by genotyping two allelic families of P. falciparum, FC27 and IC1, using malaria laboratory strains, K1 and 3D7, respectively. CONCLUSIONS: The dual function QCM was successfully developed with high sensitivity and specificity, and was cost-effective, stable and field adaptable.

Molecular screening of Plasmodium infections among migrant workers in Thailand.

BACKGROUND & OBJECTIVE: A cross - sectional study was conducted to determine the prevalence of Plasmodium infections among migrant workers in Thailand. METHODS: A total of 241 migrants at Kanchanaburi, Pathumthani and Nakornpathom provinces of Thailand were recruited in our surveillance. Blood samples were examined for human malaria parasites by using microscopy and semi - nested multiplex PCR (SnM- PCR). RESULTS: Laboratory diagnosis revealed 6.2% total positive rate. As compared to microscopy (26.7%), SnM- PCR was more sensitive (93.3%) for malaria. Plasmodium falciparum was predominant than P. vivax (53% : 40%, respectively). The majority of positive cases were from Myanmar workers who had low parasitaemia and without symptoms. The highest prevalence (13.7%) was found among migrant workers from Kanchanaburi province in western Thailand. CONCLUSION: These findings indicate risk of malaria transmission from migrant workers. Malaria surveillance should be included in the health- screening program for migrants in Thailand to manage this health risk.

Infection of Blastocystis hominis in primary schoolchildren from Nakhon Pathom province, Thailand.

A study was conducted to evaluate the infection status of Blastocystis hominis in children from four public schools in Phuttamonthon district, Nakhon Pathom province, Thailand during November to December 2004. A total of 814 faecal specimens were used for B. hominis cultivation using Jones' medium. Mixed infections with other intestinal parasites were also examined by formalin ethyl acetate concentration method. It was found that 13.51% (110 of 814) of the children examined were infected with B. hominis. Mixed infections with other intestinal protozoa and helminths were observed in 10.91% (12 of 110) of B. hominis positive specimens. There were Giardia lamblia cysts (4.55%), Trichomonas hominis trophozoites (1.82%), Entamoeba histolytica cysts (0.91%), Endolimax nana cysts (0.91%), Strongyloides stercoralis larvae (0.91%), hookworm eggs (0.91%), and Trichuris trichiura eggs (0.91%). Of the children positive for B. hominis, there was no significant differences between sex (P > 0.05) and showed no correlation between age and the percentage of infection. The different infection rates among four schools indicated the involvement of hygienic factors which promoted the infection of this common intestinal protozoan. Variation in size of B. hominis was found in culture medium, which might indicate to the presence of different strains of B. hominis infection.

The exsheathment of Necator americanus infective larvae.

Infective 3rd-stage larvae of Necator americanus were treated with human sweat under various conditions, and compared with human serum, 1.5% saline solution, and distilled water. The infective larvae were observed under inverted microscopy. The highest percentage (14.0%) of the exsheathed larvae was found in human sweat after 2 hours' incubation at 37 degrees C. The proportion of exsheathed larvae in human sweat was significantly different from human serum (p<0.001), 1.5% saline solution (p<0.001), and distilled water (p<0.001). This may reflect the effect of human sweat on the process of skin penetration by Necator americanus larvae.

Measuring allelic heterogeneity in Plasmodium falciparum by a heteroduplex tracking assay.

We developed a novel Plasmodium falciparum genotyping strategy based on the heteroduplex tracking assay (HTA) method commonly used to genotype viruses. Because it can detect both sequence and size polymorphisms, we hypothesized that HTA is more sensitive than current methods. To test this hypothesis, we compared the ability of HTA and a nested polymerase chain reaction (PCR) to detect genetic diversity in 17 Thai samples. The HTA detected more MSP1 sequence variants in eight isolates (47%), less sequence variants in three isolates (18%), and an equal number of sequence variants in six isolates (35%), suggesting that HTA is equal to or more sensitive than the nested PCR. This study is a proof of concept that HTA is a sensitive allelic discrimination method able to determine genetic diversity in P. falciparum and warrants its use in studies of antimalarial drug efficacy.

Methylene blue staining method for identification of Opisthorchis viverrini egg.

Methylene blue staining method was used to distinguish O. viverrini eggs from Haplorchis taichui and Prosthodendrium molenkampi eggs. All eggs were obtained from dissected adult worms, fixed in 10% formalin, and stained with methylene blue prior to light microscopy observation. The distinct musk-melon-like texture of the O. viverini eggshell surface and the thread-like texture of H. taichui eggshell surface were recognized, while P. molenkampi eggs showed a smooth eggshell. We also evaluated the sensitivity and specificity of the method by training investigators to differentiate surface textures. After training, the investigators were randomly tested with 10 slides containing fluke eggs. The sensitivity and specificity were 95% and 95%, respectively.