Evaluation of a Commercially Available Reverse Transcription-PCR Assay for Diagnosis of Enteroviral Infection in Archival and Prospectively Collected Cerebrospinal Fluid Specimens

Abstract

A commercially available reverse transcription (RT)-PCR method (AMPLICOR EV; Roche Diagnostic Systems, Inc., Branchburg, N.J.) was evaluated for detection of enteroviruses in cerebrospinal fluid from patients with neurological disease. This assay was compared with virus isolation in cell culture and an in-house RT-PCR method designed with a nonoverlapping region of the enteroviral genome. A panel of 200 cerebrospinal fluid specimens prospectively collected from patients with a wide variety of neurological symptoms, including 50 patients involved in three different outbreaks of acute aseptic meningitis, was assayed. A second panel of 97 archived cerebrospinal fluid specimens, stored for 2 to 5 years, from patients with aseptic meningitis associated with several enterovirus outbreaks was also studied. From the first panel, enteroviruses were detected in 13 of 50 specimens by cell culture (26%), in 43 of 50 specimens by AMPLICOR EV (86%), and in 46 of 50 specimens by the in-house assay (92%) from patients with aseptic meningitis associated with outbreak and 1 of 29, 3 of 29, and 4 of 29 specimens, respectively, from sporadic cases of aseptic meningitis. The remaining 121 cerebrospinal fluid specimens from patients with other neurological syndromes were negative by all tests. From the second panel, enteroviral RNA was detected by the AMPLICOR test (31 of 97 specimens, 32%) and the in-house assay (39 of 97 specimens, 40%). According to our results, patients with aseptic meningitis should be analyzed for enteroviral infection in cerebrospinal fluid by RT-PCR methods, and the AMPLICOR EV test is a suitable tool for performing such studies. Archival cerebrospinal fluid specimens are less suitable for evaluation of the performance of RT-PCR methods designed for enterovirus detection.

Evaluation of a commercially available reverse transcription-PCR enzyme immunoassay (Enterovirus Consensus kit) for the diagnosis of enterovirus central nervous system infections

“…These figures were also similar to those reported for different amplification methods, such as the Roche Amplicor EV test [16], a real-time RT-PCR assay [21,35] and nucleic acid sequence-based amplification methodology [17,37]. As reported previously [16,30,36,38], there was a statistically significant improvement in the detection of enterovirus central nervous system disease with PCR compared to culture, and a very good correlation (80-85.9%) between the two assays [16,34]. Recently, Buck et al [39] compared a newly described shell vial assay, in which a mixture of human colon carcinoma and genetically engineered buffalo green monkey kidney cells (Super E-mix) was used, with two commercially available RT-PCR assays (one of which was the Enterovirus Consensus kit) and conventional cell culture for the diagnosis of enterovirus meningitis.…”

“…This was not a problem in the present study, since rhinovirus is found only in respiratory samples, and 60 clinical specimens analysed in parallel with the Enterovirus Consensus kit and PCR techniques described by Zoll et al [15] and Rotbart et al [18] yielded similar results, with a sensitivity for the Enterovirus Consensus kit that was equivalent to that obtained by other techniques described previously for clinical samples [24,25]. Moreover, Penter primers amplified all 64 serotypes of enteroviruses, including both prototype and field strains [25], while the primers described by Zoll et al [15] did not recognise coxsackieviruses A11, A17 and A24 and echovirus 16, and the Rotbart primers did not always recognise echoviruses 1 and 5 [30].…”

Combining Multiplex Reverse Transcription-PCR and a Diagnostic Microarray To Detect and Differentiate Enterovirus 71 and Coxsackievirus A16

“…Progress in molecular biology is making direct assaying of low-copy viral DNA or RNA sequences from clinical samples increasingly possible. In fact, reverse transcription-PCR (RT-PCR) has been used to diagnose enterovirus infections early (13,29), and DNA sequencing has been used for serotyping (7,14,35). However, genomic information for many enterovirus serotypes is limited, and there is a high genetic heterogeneity among different strains, so misdiagnosis might be frequent (27).…”

Application of Molecular Diagnostic Techniques for Viral Testing

“…Cell culture has a low sensitivity, so molecular techniques like RT-PCR have been developed for the diagnosis of this kind of viruses [80]. Moreover, RT-PCR has higher sensitivity than cell culture for detecting enteroviruses in the CSF [81].…”