Evaluation and Verification of the Seeplex Diarrhea-V ACE Assay for Simultaneous Detection of Adenovirus, Rotavirus, and Norovirus Genogroups I and II in Clinical Stool Specimens

Abstract

Acute viral gastroenteritis is an intestinal infection that can be caused by several different viruses. Here we describe the evaluation and verification of Seeplex Diarrhea-V ACE (Seeplex DV), a novel commercial multiplex reverse transcription-PCR (RT-PCR) assay that detects 5 diarrheal pathogens, including adenovirus, rotavirus, norovirus genogroup I (GI) and GII, and astrovirus. We describe a retrospective study of 200 clinical specimens of which 177 were stool specimens previously tested for the presence of gastrointestinal viruses by electron microscopy (EM) and/or real-time RT-PCR (rRT-PCR). The remaining 23 specimens comprised other human pathogens of viral or bacterial origin. Discordant norovirus GI and GII results were resolved using a commercial kit; discordant adenovirus and rotavirus results were resolved using a home brew multiplex rRT-PCR assay. Diagnostic sensitivities and specificities were calculated before and after discordant analysis. After discordant analysis, estimated diagnostic sensitivities were 100% for adenovirus, rotavirus, and norovirus GI and 97% for norovirus GII. Diagnostic specificities after discordant analysis were 100% for adenovirus, rotavirus, and norovirus GI and 99.4% for norovirus GII. The 95% limits of detection were 31, 10, 2, and 1 genome equivalent per reaction for adenovirus, rotavirus, and norovirus GI and GII, respectively. The results demonstrate that the Seeplex DV assay is sensitive, specific, convenient, and reliable for the simultaneous detection of several viral pathogens directly in specimens from patients with gastroenteritis. Importantly, this novel multiplex PCR assay enabled the identification of viral coinfections in 12 (6.8%) stool specimens.Acute viral gastroenteritis is the second most common infectious disease worldwide (20, 5), affecting humans of all age groups (23, 17) but mostly the young, the elderly, and people in enclosed communities, such as hospitals, nursing homes, military bases, and cruise ships. Known enteric viral pathogens include adenovirus group F serotypes 40 and 41, rotavirus, norovirus, and astrovirus, with rotavirus and norovirus being the predominant causative agents of gastroenteritis (3,7,30). In recent years the number of reported gastroenteritis outbreaks of suspected viral etiology has increased, hence the need for fast, sensitive, and reliable diagnostic assays.The established method for detection of enteric viruses at the Ontario Agency for Health Protection and Promotion (OAHPP) Public Health Laboratories (PHL) relies on the conventional and labor-intensive electron microscopy (EM). In recent years, home brew real-time reverse transcription-PCR (rRT-PCR) assays were implemented for the detection of norovirus GI and GII (11). Similar home brew rRT-PCR assays for the detection of adenovirus (6) and rotavirus (35) have been described. These rRT-PCR assays enable the detection of a specific virus by the amplification of a unique genomic sequence within its RNA or DNA. However, the simultaneous detection of several viruses c...

Acute diarrhea in adults consulting a general practitioner in France during winter: incidence, clinical characteristics, management and risk factors

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Microbiological Diagnosis of Severe Diarrhea in Kidney Transplant Recipients by Use of Multiplex PCR Assays

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A 3-year-old girl with vomiting and diarrhea

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