Real-time reverse transcription-PCR for detection of rotavirus and adenovirus as causative agents of acute viral gastroenteritis in children

Abstract

Viral pathogens are the most common cause of gastroenteritis in developed countries. Human rotavirus and adenovirus infections are major causes of acute outbreaks and sporadic cases of gastroenteritis, occurring primarily among children less than 2 years of age. Patient hospitalization is often required, with enormous infection control implications. This work describes the development of real-time PCR assays for the detection of group F adenovirus, rotavirus A, and rotavirus C from stool specimens. Two hundred twenty stool samples from pediatric patients exhibiting symptoms of diarrhea and/or vomiting were examined. PCR results were compared with those of virus detection by electron microscopy and latex agglutination antigen detection. The incorporation of an internal-control RNA that was spiked into individual stool extracts functioned as an internal validation for the reporting of PCR-negative results. Rotavirus C was not detected by real-time PCR in the patient stool samples examined. Real-time reverse transcription-PCR resulted in 175% and 111% increases in the rates of detection of adenovirus F and rotavirus A, respectively, compared with latex agglutination testing. Molecular detection increased the number of stool specimens in which causative agents of gastroenteritis were identified by 155% compared to electron microscopy. Genotyping of a proportion of the rotavirus and adenovirus strains identified only genotype G1 rotavirus and both adenovirus genotypes 40 and 41 in circulation within the patient cohort examined. The results highlight the significance of rapid molecular methods for the routine screening of stool samples in hospital laboratories to provide rapid definitive diagnoses.

FIG. 1.

Consensus phylogenetic tree derived from nucleotide sequence alignment of cloned amplicons generated by rotavirus genotyping PCRs and sequences available in GenBank. Tree was generated using MEGA 3.1 as described in Materials and Methods. Bootstrap values (from 1,000 replicates) are expressed as percentages for each node. DNA sequences obtained from GenBank are listed as the accession number and serotype number. DNA sequences S1 to S4, corresponding to clinical specimens identified as negative for rotavirus by latex agglutination testing, are indicated with a white square. Stool specimens corresponding to sequences S5 to S9 were positive for rotavirus by latex agglutination testing, as indicated by the shaded square. All clone sequences clearly cluster with G1 serotype rotavirus sequences.