Diagnosis of viral gastroenteritis by simultaneous detection of Adenovirus group F, Astrovirus, Rotavirus group A, Norovirus genogroups I and II, and Sapovirus in two internally controlled multiplex real-time PCR assays.
BACKGROUND
Norovirus, Rotavirus group A, Astrovirus, Sapovirus and Adenovirus serotypes 40 and 41, are common causes of gastroenteritis. Conventional diagnosis of these causative agents is based on antigen detection and electron microscopy.


OBJECTIVE
To improve the diagnostic possibilities for viral gastroenteritis, two internally controlled multiplex real-time PCRs have been developed.


METHODS
Individual real-time PCRs were developed and optimized for the specific detection of Norovirus genogroup I, Norovirus genogroup II, Rotavirus group A, Astrovirus, Adenovirus group F and Sapovirus. Subsequently, the PCRs were combined to two multiplex PCR reactions. The multiplex assays were clinically evaluated using 239 fecal samples submitted to our laboratory over a 1-year period for the routine detection of Rotavirus and/or Adenovirus antigens using the Vikia(®) Rota/Adeno test (bioMérieux, Boxtel, The Netherlands).


RESULTS
In general, the multiplex real-time PCR assays showed comparable sensitivity and specificity to the individual assays. A retrospective clinical evaluation showed increased pathogen detection in samples from 14% using conventional methods to 45% using PCR. Subsequently, the assay was implemented as a routine diagnostic tool. From September 2007 up to December 2009, 486 positive results were obtained in 1570 samples (31%) analyzed. Norovirus genogroup II was found the most frequently (61.1%), followed by Adenovirus (9.9%), Rotavirus (9.3%), Astrovirus (6.0%), Norovirus genogroup I (3.3%) and Sapovirus (0.4%).


CONCLUSIONS
Two internally controlled multiplex real-time PCR assays for the simultaneous detection of Astrovirus, Adenovirus group F, Rotavirus, Norovirus genogroups I and II and Sapovirus have shown significant improvement in the diagnosis of viral gastroenteritis.
van Maarseveen
Noortje M
NM
Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands.
Wessels
Els
E
de Brouwer
Caroline S
CS
Vossen
Ann C T M
AC
Claas
Eric C J
EC
D000256:Adenoviridae / Q000302:isolation & purification; D002675:Child, Preschool; D005243:Feces / Q000821:virology; D005759:Gastroenteritis / Q000821:virology*; D006801:Humans; D007223:Infant; D001257:Mamastrovirus; D029322:Norovirus / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D012401:Rotavirus / Q000302:isolation & purification; D029341:Sapovirus / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods*; D014777:Virus Diseases / Q000175:diagnosis* / Q000821:virology