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.

TT virus infection in acute non-A to E hepatitis in northern Thailand.

TT virus is a novel DNA virus widely distributed in the general population. We examined the prevalence of TTV infection in a population with acute non-A to E hepatitis and in comparison groups located in Northern Thailand. The prevalence of TTV in subjects with non-A-E hepatitis was 19% (21/112), 6% (4/72) in healthy volunteers, 17% (12/72) in those with hepatitis A or B, and 17% (8/48) in hospitalized patients with non-hepatitis illnesses. A significant association with TTV infection and non-A-E hepatitis was seen in all groups (OR 3.9, p = 0.02) and in children (OR 25.8, p = 0.001). Among subjects with non-A-E hepatitis, TTV was associated with higher alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels (significant for AST, p = 0.02). Our observations suggest that TTV in our study population may be associated with non-A-E hepatitis and that children in particular may be at risk of hepatocellular injury as a result of TTV infection.

Perinatal dengue infection.

We report a case of vertical transmission of dengue infection in an infant. The mother's was a term pregnancy with a history of chronic hypertension. She presented with high fever of 3 days duration 5 days prior to delivery. Her initial complete blood count showed platelet count of 64,000/mm3. Dengue hemorrhagic fever was diagnosed 2 days later and symptomatic treatment was given. During labor her platelets dropped to 11,000/mm3 and platelet concentrate was given. Cesarean section was performed due to prolonged second stage of labor. Her infant was normal at birth except for petechiae on the left thigh. The child's platelet count was 34,000/mm3 and low grade fever was detected on the first day. Clinical sepsis was suspected and antibiotic treatment was started and continued for 4 days until all the cultures came back as negative. Both mother and her baby made an uneventful recovery and were discharged 6 days after delivery with normal platelet counts. Maternal blood was positive for IgM antibody to dengue virus. Both cord blood and the baby's blood were positive for dengue virus serotype 2 by PCR.