Assessment of a swab collection method without virus transport medium for PCR diagnosis of coxsackievirus infections.

Classically, detection of human enterovirus (EV) infections is based on virus isolation in tissue culture, proper sample collection and handling that optimizes virus viability. Samples are collected in virus transport medium (VTM) to ensure virus stability. High sensitivity and rapid results have made polymerase chain reaction (PCR) analysis increasingly popular for routine diagnosis. The PCR method enables simple sample collection and storage for EV diagnostics, which may eventually allow self-sampling at home. Our aim was to test a modification of the conventional clinical swab sample collection method for molecular diagnosis of EV infection. We compared swabs (cotton or synthetic) without VTM and the classical standard synthetic swabs with VTM. Effects of storage temperature (+4 °C or -80 °C) and duration were studied. EV-RNA could be detected by reverse transcriptase and nested PCR in both swab types without VTM. Differences depended on the storage duration and temperature. Optimum conditions were immediate processing or storage at -80 °C. Storage without VTM at +4 °C for longer periods is not advisable. We conclude that swabs without VTM can be considered for clinical EV-diagnostics based on PCR, and ultimately for epidemiological sample collection.

Development of a new method for diagnosis of Group B Coxsackie genome by reverse transcription loop-mediated isothermal amplification.

BACKGROUND: Coxsackie B viruses (genus, Enterovirus; family, Picornaviridae) can cause aseptic meningitis, encephalitis, pleurodynia, and fatal myocarditis, and are implicated in the pathogenesis of dilated cardiomyopathy. The differentiation of the group B Coxsackieviruses into their subtypes has potential clinical and epidemiological implications. OBJECTIVE: In this study, we developed a one-step, single-tube genogroup-specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of group B Coxsackie genomes targeting 5' UTR region. MATERIALS AND METHODS: The amplification can be obtained in less than 1 hour by incubating all the reagents in a single tube with reverse transcriptase and Bst DNA polymerase at 63°C. Detection of gene amplification could be accomplished by agarose gel electrophoresis and the monitoring of gene amplification can also be visualised with the naked eye by using SYBR green I fluorescent dye. RESULTS: A total of 40 samples comprising 31 positive samples and 9 negative samples were used in this study for comparative evaluation. The results were compared with those from Real-Time Polymerase Chain Reaction (RT-PCR). None of the RT-PCR-positive samples were missed by RT-LAMP, thereby indicating a higher sensitivity of the RT-LAMP assay. CONCLUSION: Thus, due to easy operation without a requirement of sophisticated equipment and skilled personnel, the RT-LAMP assay reported here is extremely rapid, cost-effective, highly sensitive, and specific and has potential usefulness for rapid detection of non-polio enterovirus (NPEV) not only by well-equipped laboratories but also by peripheral diagnostic laboratories with limited financial resources in developing countries.

Visual detection of human enterovirus 71 subgenotype C4 and Coxsackievirus A16 by reverse transcription loop-mediated isothermal amplification with the hydroxynaphthol blue dye.

A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of human enterovirus 71 subgenotype C4 (EV71-C4) and Coxsackievirus A16 (CVA16) infection, respectively. The reaction was performed in one step in a single tube at 65°C for 60 min with the addition of the hydroxynaphthol blue (HNB) dye prior to amplification. The detection limits of the RT-LAMP assay were 0.33 and 1.58 of a 50% tissue culture infective dose (TCID(50)) per reaction based on 10-fold dilutions of a titrated EV71 or CVA16 strain, respectively. No cross-reaction was observed with Coxsackievirus A (CVA) viruses (CVA2, 4, 5, 7, 9, 10, 14, and 24), Coxsackievirus B (CVB) viruses (CVB1,2,3,4, and 5) or ECHO viruses (ECHO3, 6, 11, and 19). The assay was further evaluated with 47 clinical stool specimens diagnosed previously with EV71, CVA16 or other human enterovirus infections. Virus isolates from stool samples were confirmed by virus neutralization testing and sequencing. RT-LAMP with HNB dye was demonstrated to be a sensitive and cost-effective assay for rapid visual detection of human EV71-C4 and CVA16.