Development and evaluation of nucleic acid sequence based amplification (NASBA) for diagnosis of enterovirus infections using the NucliSens® Basic Kit

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Abstract

Background: Molecular methods based on RNA amplification are needed for sensitive detection of enteroviruses in clinical samples. Many ‘in house’ methods based on reverse-transcribed PCR (RT-PCR) could be difficult to use in the routine diagnostic laboratory since they tend to be time-consuming, use reagents from many different suppliers and include non-routine procedures. Objectives: The aim of this study was to develop and evaluate methods based on nucleic acid sequence based amplification (NASBA) for detection of enterovirus sequences. Study design: ‘In house’ prepared and commercially available reagents were utilised to develop enterovirus-specific NASBA assays. Optimised methods were evaluated using clinical samples (cerebrospinal fluid, respiratory and stool samples), titred virus controls and in vitro produced synthetic RNA. Results for NASBA were compared with RT-PCR and virus culture. Results: Kit-based reagents gave an equivalent sensitivity to the more laborious ‘in house’ molecular assays (NASBA and RT-PCR) on clinical material and controls. All molecular methods picked up enterovirus positive clinical samples that were not identified by culture. End point detection sensitivity for the NASBA assay based on the NucliSens® Basic Kit was ≤1 tissue culture infective dose 50% of a range of enteroviruses or <100 copies RNA input. The assay was specific for enteroviruses and did not pick up high titre rhinovirus preparations. Enterovirus Basic Kit NASBA results for clinical samples were easily obtained within a single working day. Conclusions: NASBA is a suitable alternative to RT-PCR for sensitive amplification and detection of enterovirus sequences in a range of clinical specimens. The use of kit-based reagents will enable a wide range of laboratories to undertake molecular-based diagnostic procedures for RNA viruses and provide results within a time frame relevant to patient management.

Introduction

Enteroviruses are important pathogens with a wide range of clinical manifestations. Non-polio enteroviruses account for most hospital admissions for viral meningitis. Enteroviruses can also cause severe neonatal infection (large hospital outbreaks have been described) pyrexias, rashes and respiratory symptoms (Melnick, 1996). The association of enteroviruses with diabetes and chronic fatigue syndrome has also been widely reported and warrants further study (Yousef et al., 1988, Clements et al., 1995a, Clements et al., 1995b, Galbraith et al., 1995, Vedhara et al., 1997). Isolation methods have been widely used for diagnosis of enterovirus infection but many of the viruses in this diverse group do not grow well in tissue culture (Grandien et al., 1995). There has been much discussion about the safety and ethics of using wild caught primary monkey cells for virus isolation. Many laboratories still rely on primary cells for isolation directly from clinical material despite the availability of continuous cell lines and, as such cells become less widely available, culture for a wide range of enterovirus types will become difficult in some centres.

Sensitive molecular-based amplification methods are required to detect enterovirus nucleic acid in cerebrospinal fluid (CSF). Many procedures based on reverse-transcribed polymerase chain reaction (RT-PCR) have been described (Rotbart, 1990, Zoll et al., 1992, Glimåker et al., 1993, Nicholson et al., 1994, Abzug et al., 1995, Clements et al., 1995b). Although these assays have been useful for diagnosis of enterovirus infections, laboratories often find ‘in house’ RT-PCR time consuming and difficult to control. Many protocols do not fit easily into a diagnostic laboratory set-up and utilise complicated mixes of reagents from a variety of sources.

NASBA is an isothermal, transcription-based amplification method first described by Guatelli et al. (1990) which has some advantages over RT-PCR for diagnostic detection of RNA targets. The method has been reviewed recently along with other isothermal RNA amplification formats (Chan and Fox, 1999). The aim of this study was to develop and evaluate methods based on NASBA for diagnosis of enterovirus infections as an alternative to RT-PCR. The availability of reagents for extraction, amplification and probe-specific detection of NASBA products allowed comparison of ‘in house’ and simple to use kit-based reagents for undertaking NASBA in a diagnostic setting.

Section snippets

Samples and controls

During the course of this study a range of different enteroviruses and rhinovirus cultures, spiked into negative stool, respiratory sample or CSF were utilised (n=30). Cultures included preparations of polioviruses 1, 2, 3, enterovirus 70, echoviruses 4, 6, 11, 30, coxsackievirus B1, 2, 3, 4, 5, coxsackievirus A9 and rhinoviruses 1B, 2, 7, 9, 14, 16, 41, 58 and 70. Enteroviruses and rhinoviruses were propagated and typed using well-validated methods (Johnston et al., 1995, Grandien et al., 1995

Extraction and sample preparation

Manual and automated extraction gave equivalent results for cultures and clinical samples evaluated during the course of the study (data not shown). Preparation of RNA using the NucliSens® extractor was less time consuming than the manual method (timings given in Fig. 1).

Optimisation of NASBA using the Basic Kit

Initial experiments utilised enterovirus positive and negative culture material (n=10) to assess the effect of KCl concentration on amplification efficiency. The standard protocol with 70 mM KCl (final concentration) gave good

Discussion

The enterovirus Basic Kit NASBA assay, in its optimised, final format, will be useful in a diagnostic setting. Many enterovirus types grow poorly in tissue culture and Basic Kit NASBA provides a time saving compared with most frequently used nucleic acid amplification procedures. Results can be made available in a time frame which is relevant to patient management. Rapid and sensitive diagnosis of enterovirus meningitis will avoid unnecessary and costly inappropriate antibiotic use. The

Acknowledgements

The authors would like to thank Dr Sally Corden and diagnostic staff in Virology, Cardiff Public Health Laboratory for provision of diagnostic support and quality control material. Keith Williams and Amanna Rahman provided excellent technical assistance for this work. Synthetic poliovirus RNA and ruthenium-labelled enterovirus-specific probe were prepared by Dr Peter Sillekens and Monique van der Meer (Organon Teknika Ltd). Stock viruses and quality control material were provided by Dr Geoff

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  • Cited by (0)

    1

    Present address: Department of Medical Genetics, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK.

    2

    Present address: Division of Clinical Virology, F68, Huddinge University Hospital, Stockholm, S-14186, Sweden.

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