Comparative evaluation of Taqman real-time PCR and semi-nested VP1 PCR for detection of enteroviruses in clinical specimens.
ABSTRACT Molecular diagnostic tests to detect enterovirus in clinical specimens usually target highly conserved sites in the 5'-non-translated region, allowing detection of all members of the genus. The sequences in the 5'-NTR do not correlate with serotype, but PCR and sequencing of the VP1 region can be used for typing; this system has largely replaced traditional antigenic typing.
To investigate the relative performance of two common enterovirus assays.
We compared the relative sensitivities of Taqman real-time RT-PCR (rRT-PCR) and a VP1 semi-nested PCR (RT-snPCR) assay in which sequencing the VP1 amplicon also provides typing information.
There was 89% concordance between the two methods among the 371 clinical specimens tested (74 positive in both assays and 257 negative in both assays). Twenty-seven rRT-PCR-negative/VP1-positive specimens were confirmed positive by sequencing; 13 specimens were rRT-PCR-positive/VP1-negative.
The results suggest that either assay can produce satisfactory results, but that using both assays in parallel should provide the highest sensitivity for clinical diagnostic testing.
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ABSTRACT: Typing of human enterovirus (EV) remains a major goal for diagnosis and epidemiological purposes. Whereas sequencing the viral capsid (VP) 1 coding region is the gold standard for EV typing, a method relying on the sequencing of the VP2 coding region has been proposed as an alternative but has been validated only on cell culture supernatants. To avoid the selection of cultivable strains and to fasten the identification step, a new semi-nested PCR method targeting the VP2 region was developed by using the CODEHOP strategy. After validation of the method on reference and clinical strains, a total of 352 clinical specimens found positive for EV RNA (138 by the Xpert(®) EV kit and 214 by the Enterovirus R-gene(™) kit) during a three-year period (2010-2012) were analyzed prospectively for VP2 genotyping. Overall, 204 (58%) specimens were typeable. A higher proportion of throat swab/stool specimens was found typeable by comparison to cerebrospinal fluids (CSF) (94 out of 142 (66.2%) vs 83 out of 169 (49.1%), P<0.01 by chi square test). Moreover, the median Ct value obtained was lower for typeable specimens compared to untypeable ones (32.20 vs 33.01, P<0.05 and 25.96 vs 31.74, P<0.001, for GeneXpert and R-gene assays, respectively, by Mann-Whitney-Wilcoxon test). These results suggest that, in case of EV meningitis, a peripheral specimen (i.e. throat swab or stool) susceptible to exhibit a higher viral load should be used preferentially to CSF for identifying the causative EV genotype by using the VP2 typing method without cell culture isolation. This article is protected by copyright. All rights reserved.Clinical Microbiology and Infection 12/2013; · 4.58 Impact Factor
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ABSTRACT: The discovery of new or divergent viruses using metagenomics and high-throughput sequencing has become more commonplace. The preparation of a sample is known to have an effect on the representation of virus sequences within the metagenomic dataset yet comparatively little attention has been given to this. Physical enrichment techniques are often applied to samples to increase the number of viral sequences and therefore enhance the probability of detection. With the exception of virus ecology studies, there is a paucity of information available to researchers on the type of sample preparation required for a viral metagenomic study that seeks to identify an aetiological virus in an animal or human diagnostic sample. A review of published virus discovery studies revealed the most commonly used enrichment methods, that were usually quick and simple to implement, namely low-speed centrifugation, filtration, nuclease-treatment (or combinations of these) which have been routinely used but often without justification. These were applied to a simple and well-characterised artificial sample composed of bacterial and human cells, as well as DNA (adenovirus) and RNA viruses (influenza A and human enterovirus), being either non-enveloped capsid or enveloped viruses. The effect of the enrichment method was assessed by quantitative real-time PCR and metagenomic analysis that incorporated an amplification step. Reductions in the absolute quantities of bacteria and human cells were observed for each method as determined by qPCR, but the relative abundance of viral sequences in the metagenomic dataset remained largely unchanged. A 3-step method of centrifugation, filtration and nuclease-treatment showed the greatest increase in the proportion of viral sequences. This study provides a starting point for the selection of a purification method in future virus discovery studies, and highlights the need for more data to validate the effect of enrichment methods on different sample types, amplification, bioinformatics approaches and sequencing platforms. This study also highlights the potential risks that may attend selection of a virus enrichment method without any consideration for the sample type being investigated.Journal of virological methods 09/2013; · 2.13 Impact Factor
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ABSTRACT: Background Hand, foot, and mouth disease (HFMD) is generally considered a rare illness in adults. Classically, HFMD has been strongly associated with coxsackievirus strain A16 and enterovirus 71. The coxsackievirus A6 (CVA6) strain has been linked to severe worldwide outbreaks since 2008. CVA6 is associated with a more severe and profound course of disease, affecting both children and adults. Objectives To present a series of five adult patients diagnosed with HFMD due to CVA6. We investigate method of diagnosis and compare clinical presentation of adult cases to those in children. Study Design Each patient underwent a full-body skin exam as well as inspection of the oral cavity. Rapid plasma reagin (RPR) and serologic assays by complement fixation against coxsackievirus B (1-6) and A (2,4,7,9,10,16) were performed as indicated. As standard serological testing does not detect CVA6, real-time reverse transcription-polymerase chain reaction (qRT-PCR) of serum, buccal swabs, and skin scrapings were performed by the Centers for Disease Control and Prevention (CDC). Results: Each patient had clinical findings consistent with various stages of HFMD. One patient presented with delayed onychomadesis and desquamation of the palms and soles. RPR and serologic assays by complement fixation against CVB (1-6) and CVA (2,4,7,9,10,16) were mostly negative, although elevated in two patients due to cross-reactivity. qRT-PCR identified CVA6 genetic material in samples from all patients. Conclusion This series demonstrates that there is a wide array of disease presentation of CVA6 associated HFMD in adults.Journal of Clinical Virology 08/2014; · 3.47 Impact Factor