Molecular serotyping of dengue viruses in field-caught Aedes mosquitos by in-house RNA extraction/RT-PCR reagent kits.

We developed in-house RNA extraction and RT-PCR reagent kits for the molecular serotyping of dengue viruses in field-caught Aedes mosquitos. Mosquitos that showed positive results by ELISA or IFA were selected for the identification of dengue viruses in order to predict the distribution of the four dengue serotypes. Total RNA was extracted from one whole mosquito as well as from one dissected mosquito by our in-house RNA extraction reagents using the modified method of guanidinium thiocyanate denaturation and isopropanol precipitation. The extracted RNA was amplified by our in-house RT-PCR reagents specific for each dengue serotype under optimized conditions. Dengue viral RNA extracted from a single mosquito as well as from the head and thorax of one dissected mosquito could be detected successfully; it could not be found in the abdomen, however. These results indicated that most of the dengue viruses were located in the head and thorax rather than in the abdomen. The results of dengue serotyping showed a pure specific PCR product for each dengue serotype at 490, 230, 320 and 398 bp for DEN-1, DEN-2, DEN-3, and DEN-4 respectively. In addition, the detection sensitivity was very high: an amount of RNA template equivalent to approximately 1/80 of a single mosquito could be detected by agarose gel electrophoresis and ethidium bromide staining. The coupling of RT-PCR-based surveillance of dengue viral infection with disease mapping data (Geograpical Information System, GIS) could serve as an alternative epidemiological means of providing an early warning of dengue fever/dengue hemorrhagic fever epidemics.

Field trial of the ICT filariasis for diagnosis of Wuchereria bancrofti infections in an endemic population of Thailand.

The ICT Filariasis, a rapid card test format, which is based on qualitative detection by monoclonal antibody of the circulating antigen of Wuchereria bancrofti adult worm, is a new diagnostic test of choice for determining the infections under field conditions. By using clinical and recall techniques and microscopy (thick smear and capillary tube technique) as reference, we assessed the efficiency of the ICT card test in sera from 225 subjects living in W. bancrofti-endemic villages of Tak Province, Thailand, who were recruited during a cross-sectional community survey. The ICT card test gave a 20% antigen positive rate, while other tests gave lower positive rates of the same 5.8% by clinical and recall techniques and thick smear, and 5.3% by capillary tube technique, respectively. The ICT card test had a specificity of 100% when sera from microfilaremic subjects were positive, as when sera from W. bancrofti non-endemic subjects either with Brugia malayi microfilaremia or with other parasites, and those from normal controls were all negative by the test. When done in W. bancrofti microfilaremia sera, the ICT card test had a sensitivity of 100% using a microscopy as reference, and 84.6% when using clinical and recall techniques. However, the ICT card test was more sensitive than the others when done in endemic normal sera (14% positive). Such findings compared well with findings in endemic area of South America, suggested its usefulness to detect W. bancrofti infections in endemic area, especially on the Thai-Myanmar border.

Epidemiology of hookworm infection in Soong nern district, Korat province, Thailand.

Stool surveys in Soong Nern district, Korat Province, were done by the MF concentration technique. The intensity of hookworm infection, egg counts, results of treatment by anthelminthic drugs, observations on new infections and reinfections following treatment throughout the year and the density of nematode larvae in soil in the study area were investigated. Hookworm infection rates were high in Na-Glang village, both in adult villagers and school-aged children. The intensity of hookworm infections was considered mild and after treatment the numbers of eggs in the stools diminished or disappeared. New cases and reinfection increased during the rainy month, due to socio-environmental factors in the village that favoured the development of hookworm larvai in the soil. Although in this study it was impossible to distinguish hookworm larvae among the nematode larvae collected form soil it was highly probable that hookworm larvae were present. Control of hookworm infection in the study area was proposed by improvement of environmental sanitation and health education.