This was a cross-sectional study that involved the examination of 1,140
stool samples of outpatients and inpatients with acute gastroenteritis referred to Selleckchem ATM inhibitor HC–UFPR. The patients were admitted to pediatric wards or to the hematopoietic stem cells transplantation (HSCT) unit. The stool samples were collected from April of 2001 to December of 2008, and were sent to the virology laboratory for RVA detection and posterior genotyping studies. Medical records of infected patients were reviewed, and the clinical data were collected using specific forms. This study was approved by the Ethics of Research on Human Beings Committee of the HC-UFPR, under registration No. 4441.023/2002-04. Dehydration was classified as mild, moderate, or severe, and evaluated on a clinical dehydration scale for children as previously reported.11 Fecal samples were initially tested for group A rotavirus antigen by screening tests – LA, (Virotect Rota kit–Omega Diagnostics or Rotascreen kit–Microgen Bioproducts) and EIA (EIARA kit–Biomanguinhos or Rotascreen II kit–Microgen
Bioproducts), according to the manufacturer’s instructions. The performance of these Dabrafenib methods was analyzed and their results were compared to those previously reported.12 Positive samples were sequentially analyzed by molecular methods. Genomic RNA was extracted using aliquots of 200 μL of fecal suspension (10% wt/vol) and silica filter, in accordance with the process previously described.13 The RNA obtained was analyzed by a multiplex hemi-nested SDHB real time polymerase chain reaction (RT-PCR) to define the viral genotype, using previously described methods.13 Briefly, the RNA was reverse transcribed and amplified by using specific primers corresponding to a conserved nucleotide sequence of the VP4 and VP7 genes, fragments of 876 bp and 904 bp, respectively.13 The amplified fragment was used as a template to a second PCR, using a combined typing scheme of the pool of primers to
identify VP7: pool A (G1, G2, G3, G4, and G5), pool B (G8, G9, and G10), and pool C (G6 and G11); and to identify VP4: pool A [P4], [P6], [P8], [P9], and pNCDV [P1] genotypes. The results obtained were confirmed with individual primers for the identified genotype. Samples that were positive in the first-step PCR, and which could not be genotyped by multiplex nested PCR were analyzed by nucleotide sequencing. The PCR products were purified using Invisorb® Spin PCRapid kit (Invitek Inc–USA), after both DNA strands were directly sequenced as described in the Thermo Sequenase kit (USB Inc – Ohio, USA) manual. BigDye® Terminator method was used on an ABI 3100 (Applied Biosystems Inc – USA). Specific primers from the first and second PCR were used to detect the RVA. The BioEdit Sequence Alignment Editor was used to assemble the fragments into the most likely sequence.