Verification and validation of diagnostic laboratory tests in clinical virology

Rabenau, Holger F.; Kessler, Harald H.; Kortenbusch, Marhild; Steinhorst, Andreas; Raggam, Reinhard B.; Berger, Annemarie

doi:10.1016/j.jcv.2007.07.009

Cited by 97 publications

(90 citation statements)

References 3 publications

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“…These tests are considerably advanced and require additional funds and staff for up-front research and development, quality assurance, and laboratory accreditation (8,13,15,28). In recent years, commercial products such as those identified in this communication have allowed for the implementation of molecular testing without the added costs of research and development and additional quality assurance costs.…”

Section: Discussionmentioning

confidence: 99%

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

Higgins¹,

Beniprashad²,

Cardona³

et al. 2011

J Clin Microbiol

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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...

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Section: Discussionmentioning

confidence: 99%

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

Higgins¹,

Beniprashad²,

Cardona³

et al. 2011

J Clin Microbiol

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“…To confirm the reproducibility of this method, intra-assay (each sample tested three times within an experiment) and interassay (each sample tested one time in three different experiments) precision was evaluated (28). For each reference virus, 10 5 -copy dilutions were mixed.…”

Section: Resultsmentioning

confidence: 99%

Simultaneous Detection of Seven Enteric Viruses Associated with Acute Gastroenteritis by a Multiplexed Luminex-Based Assay

Liu

Zhang

et al. 2012

J Clin Microbiol

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Rapid and broad diagnostic methods are needed for the identification of viral agents of gastroenteritis. In this study, we used Luminex xMAP technology to develop a multiplexed assay for the simultaneous identification of major enteric viral pathogens, including rotavirus A (RVA), noroviruses (NoVs) (including genogroups GI and GII), sapoviruses (SaV), human astrovirus (HAstV), enteric adenoviruses (EAds), and human bocavirus 2 (HBoV2). The analytical sensitivity allowed detection of 10 3 (EAds, HBoV2, and RVA) and 10 4 (NoV GI and GII, SaV, and HAstV) copies per reaction mixture. Compared to conventional PCR, the Luminex-based assay yielded greater than 75% sensitivity and 97% specificity for each virus, and the kappa correlation for detection of all viruses ranged from 0.75 to 1.00. In conclusion, this multiplexed Luminex-based assay provides a potentially rapid, high-throughput, and maneuverable diagnostic tool for major viral pathogens associated with gastroenteritis.

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“…Verification should be performed for a new test or test system for viral antibodies, antigens and nucleic acid. If these tests are home brew tests or test systems, they must be validated (Rabenau et al, 2007). As verification Afr.…”

Section: Verification and Validation Proceduresmentioning

confidence: 99%

ISO 15189 accreditation experience at the Microbiology Laboratory of a Turkish University Hospital

Gürol¹,

Kurt²,

Karaltı³

et al. 2011

Afr. J. Microbiol. Res.

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We aimed to share the experience of the ISO/IEC 15189:2007 accreditation process in a University Hospital's Medical Microbiology Laboratory before and after the assessment, and tried to seek answers to some questions in applying the standard. Our ISO 15189 accreditation process began in 2008 after having acquired JCI accreditation in 2007. The aim was to be the first accreditated university hospital laboratory in our country. The applied tests in bacteriology, mycology, parasitology, virology, immunology and molecular serology were accreditated. We completed the validation and verification procedures mostly applied to quantitative analyses. Regarding the external quality controls and the internal controls, the corrective actions provided us great control and self correction. We increased the rate of success in the quality controls. We encouraged our laboratory personnel to fill out a laboratory discrepancy form to control our process and to arrange training. During the course of 2009, 60 of the 85 forms were completed in the microbiology laboratory among the central laboratories. Statistical analyses were performed throughout a year and this information helped us in making improvements. The number of training hours in one year was found as 78 h and 30 min during the routine laboratory process. We should provide support in bringing quality to laboratories in our country. As a matter of fact, having our own external quality controls is believed to bring cost effectiveness in acquiring an accreditation process.

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Verification and validation of diagnostic laboratory tests in clinical virology

Cited by 97 publications

References 3 publications

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

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

Simultaneous Detection of Seven Enteric Viruses Associated with Acute Gastroenteritis by a Multiplexed Luminex-Based Assay

ISO 15189 accreditation experience at the Microbiology Laboratory of a Turkish University Hospital

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