Association of the G4 rotavirus genotype with gastroenteritis in adults.

Rotavirus is the most common etiological cause of acute viral gastroenteritis in infants and young children worldwide, yet its role in the adult population is less well understood. We have recently identified rotavirus as the causative agent of severe diarrhea in adults, specifically in two gastroenteritis outbreaks in separate care for the elderly homes. Strain typing has shown the continued presence of P[8]G1, the emergence of P[8]G9, and the reemergence of P[8]G4. A total of 26 community cases and 6 outbreak cases of rotavirus infection, positive via a molecular screening assay, were subsequently amplified using VP4 and VP7 specific primers (Con2/Con3 and 1A/1B primer sets, respectively). The age range of patients investigated was from <1 year to 89 years. The resulting PCR products were cloned into TOPO10 PCR IV vector and sequenced to give the P- and G-type accordingly. All sequence data were subjected to BLAST analysis. Three different rotavirus types P[8]G1, P[8]G4, and P[8]G9 were identified. Types P[8]G1 and P[8]G9 were identified as circulating within the community, whereas the third type P[8]G4 was identified only in an elderly care outbreak. The identification of G9 rotaviruses supports evidence of emergence of the genotype on a global scale.

A touchdown nucleic acid amplification protocol as an alternative to culture backup for immunofluorescence in the routine diagnosis of acute viral respiratory tract infections.

BACKGROUND: Immunofluorescence and virus culture are the main methods used to diagnose acute respiratory virus infections. Diagnosing these infections using nucleic acid amplification presents technical challenges, one of which is facilitating the different optimal annealing temperatures needed for each virus. To overcome this problem we developed a diagnostic molecular strip which combined a generic nested touchdown protocol with in-house primer master-mixes that could recognise 12 common respiratory viruses. RESULTS: Over an 18 month period a total of 222 specimens were tested by both immunofluorescence and the molecular strip. The specimens came from 103 males (median age 3.5 y), 80 females (median age 9 y) and 5 quality assurance scheme specimens. Viruses were recovered from a number of specimen types including broncho-alveolar lavage, nasopharyngeal secretions, sputa, post-mortem lung tissue and combined throat and nasal swabs. Viral detection by IF was poor in sputa and respiratory swabs. A total of 99 viruses were detected in the study from 79 patients and 4 quality control specimens: 31 by immunofluorescence and 99 using the molecular strip. The strip consistently out-performed immunofluorescence with no loss of diagnostic specificity. CONCLUSIONS: The touchdown protocol with pre-dispensed primer master-mixes was suitable for replacing virus culture for the diagnosis of respiratory viruses which were negative by immunofluorescence. Results by immunofluorescence were available after an average of 4-12 hours while molecular strip results were available within 24 hours, considerably faster than viral culture. The combined strip and touchdown protocol proved to be a convenient and reliable method of testing for multiple viruses in a routine setting.