Development and evaluation of a real-time RT-PCR assay on the LightCycler for the rapid detection of enterovirus in cerebrospinal fluid specimens

doi:10.1016/j.jcv.2005.09.006

Journal of Clinical Virology

Volume 35, Issue 3, March 2006, Pages 278-284

https://doi.org/10.1016/j.jcv.2005.09.006 Get rights and content

Abstract

Background

Detection of enteroviral nucleic acid in cerebrospinal fluid (CSF) specimens has been demonstrated to improve the management of patients with aseptic meningitis.

Objective

To develop on the LightCycler (LC) instrument a real-time RT-PCR assay based on TaqMan technology for the detection of enteroviruses (EV) in cerebrospinal fluid (CSF) specimens.

Study design

After evaluation of the analytical performances, seventy-four CSF samples collected prospectively from patients who have been suspected for a clinical diagnosis of meningitis were evaluated by two LC real-time RT-PCR assays and one conventional RT-PCR assay.

Results

Our assay detected all 30 different EV species tested, whereas no reactivity was observed with other neurotropic viruses. The analytical sensitivity of both LC RT-PCR real-time assays was 1 TCID₅₀ for LC one-step and two-step RT-PCR assays. Results for LC one-step and LC two-step RT-PCR were compared to results of the conventional RT-PCR: of the 74 CSF specimens tested, 11 were positive and 56 were negative by all methods. Four other specimens were positive for EV by at least two of the methods (including the LC two-step RT-PCR and the conventional RT-PCR), two other CSF specimens were positive by the LC two-step RT-PCR assay only, and another one CSF specimen was positive by the LC one-step RT-PCR assay only. No CSF specimens were negative by the LC two-step RT-PCR assay and positive by the conventional RT-PCR assay. The sensitivity, specificity, positive and negative predictive values of both LC RT-PCR assays by using conventional RT-PCR as the “gold standard” were, respectively, 73.3, 98.3, 91.7, 93.5% for the LC one-step RT-PCR and 100, 96.6, 88.2, 100% for the LC two-step RT-PCR. There was substantial agreement between the three assays (k = 0.80).

Conclusions

The LC two-step RT-PCR assay is a rapid, sensitive and reliable method which can be routinely performed with CSF samples for diagnosis of EV infection and is an important improvement for optimal patient management.

Introduction

Enteroviruses are a common and important human pathogen associated with several clinical manifestations ranging from asymptomatic or mild febrile illness to severe and potentially fatal syndromes including paralysis, aseptic meningitis, encephalitis, myocarditis, neonatal systemic infection. They are the most common cause of aseptic meningitis in both children and adults, accounting for 70–90% of all cases in which a causative agent is identified (Berlin et al., 1993, Rotbart, 1995, Wilfert et al., 1983). Clinical criteria and CSF analysis are not enough to distinguish between enteroviral meningitis and other central nervous system infections caused by other neurotropic viruses and some bacteria. Thus, the availability of a rapid and reliable enteroviral diagnostic test in the early course of meningitis may both shorten hospitalizations and eliminates unecessary treatment, leading to improvements in patient management and reduces costs (Nigrovic and Chiang, 2000, Romero, 1999, Schlesinger et al., 1994).

Conventional diagnosis of EV by cell culture from CSF is time-consuming and lacks sensitivity. In recent years, molecular assays have been demonstrated to improve enteroviral detection from CSF specimens and are now well established in the routine clinical laboratory (Kammerer et al., 1994, Petitjean et al., 1994, Rotbart, 1990b, Sawyer et al., 1994, Stellrecht et al., 2000). However, the use of conventional PCR assays for the routine diagnosis is limited by the lack of standardisation (RNA extraction and nucleic acid amplification methods) and by time-consuming procedures. The simplified and standardized commercial Amplicor^® EV test have been developed and may present a good alternative to obtain rapid and reliable results in a diagnostic laboratory (Carroll et al., 2000, Lina et al., 1996, Muir et al., 1999, Pozo et al., 1998, Van Vliet et al., 1998, Yerly et al., 1996). This assay, however, has recently been taken from the market. Recently, a new reliable RT-PCR assay (EV Penter RT-PCR test, Argene biosoft, Varhiles, France) has been commercialized (Bourlet et al., 2003, Jacques et al., 2003).

The recently-introduced real-time PCR-based fluorescence technologies have many advantages: high sensitivity and specificity, usefulness as quantitative assays, operation in a closed system avoiding contamination and ability to yield results rapidly. Several groups have described real-time PCR methods for the detection of EV in CSF (Corless et al., 2002, Nijhuis et al., 2002, Verstrepen et al., 2001, Verstrepen et al., 2002, Watkins-Riedel et al., 2002).

The aim of this study was to develop and validate a rapid, sensitive and reliable real-time PCR assay using the LightCycler system (Roche Molecular Biochemicals, Lewes, United Kingdom) for routine diagnosis of EV meningitis in a clinical laboratory setting. After determination of the analytical sensitivity, specificity and reproducibility of the assay, CSF specimens from patients admitted for suspected meningitis were tested and the results were compared with those obtained in conventional RT-PCR assay.

Section snippets

Virus stocks and CSF samples

Stock cultures of prototype viral strains and clinical isolates of EV stored at −70 °C were used to optimize PCR conditions and evaluate analytical performances. Fifteen prototype strains were obtained from the French National Reference Center for Enteroviruses, Lyon, France: echovirus type 6 (E-6), E-7, E-11 and E-30, coxsackievirus A type 9 (CV-A9), CV-A16 and CV-A21, coxsackievirus B type 1 (CV-B1), CV-B2, CV-B3, CV-B4 and CV-B5, poliovirus type 1 (PV-1), PV-2 and PV-3. Clinical strains (E-3,

Analytical sensitivity and specificity

The results obtained for each RT-PCR assay with the E-30 reference strain RNA dilution series are shown in the Table 2. Analysis of the 10-fold dilution series of E-30 RNA extract standards demonstrated a five-log dynamic range (from 10⁻¹ to 10⁻⁶) by the LC one- and two-step RT-PCR assay. Both assays identified E-30 RNA down to a level of 1 TCID₅₀. The limit of detection is the same as that of our conventional RT-PCR. The mean of C_T values was determined on four assays and ranged from 17 to

Discussion

In children and young adults, EV infections cause annually a substantial number of aseptic meningitis requiring unnecessary hospitalisation and inappropriate treatment. So, the need for an accurate and rapid diagnostic test for EV infections is widely appreciated. Several studies have demonstrated the high sensitivity, specificity, and utility of the molecular methods for the diagnosis of EV central nervous system infections in comparison with conventional cell culture methods (Carroll et al.,

Conclusion

In conclusion, we have developed a rapid, sensitive and reliable real-time RT-PCR assay on LightCycler instrument for the routine diagnosis of EV meningitis. This real-time PCR method compares favourably to previously published real-time PCR methods for detection of EV and are an attractive alternative to conventional PCR techniques in the routine laboratory setting. Further investigation and prospective studies on large-scale CSF specimens involving one or heterologous population of EV

References (36)

K.C. Carroll et al.
Evaluation of the Roche AMPLICOR enterovirus PCR assay in the diagnosis of enteroviral central nervous system infections
J Clin Virol
(2000)
J. Petitjean et al.
Detection of enteroviruses in cerebrospinal fluids: enzymatic amplification and hybridization with a biotinylated riboprobe
Mol Cell Probes
(1994)
H.F. Rabenau et al.
Rapid detection of enterovirus infection by automated RNA extraction and real-time fluorescence PCR
J Clin Virol
(2002)
H.A. Rotbart
Diagnosis of enteroviral meningitis with the polymerase chain reaction
J Pediatr
(1990)
K.A. Stellrecht et al.
A one step RT-PCR assay using an enzyme-linked detection system for the diagnosis of enterovirus meningitis
J Clin Virol
(2000)
W.A. Verstrepen et al.
Evaluation of a rapid real-time RT-PCR assay for detection of enterovirus RNA in cerebrospinal fluid specimens
J Clin Virol
(2002)
T. Watkins-Riedel et al.
Rapid diagnosis of enterovirus infections by real-time PCR on the LightCycler using the TaqMan format
Diagn Microbiol Infect Dis
(2002)
L.E. Berlin et al.
Aseptic meningitis in infants <2 years of age: diagnosis and etiology
J Infect Dis
(1993)
T. Bourlet et al.
New PCR test that recognizes all human prototypes of enterovirus: application for clinical diagnosis
J Clin Microbiol
(2003)
C.E. Corless et al.
Development and evaluation of a ≪ real-time ≫ RT-PCR for the detection of enterovirus and parechovirus RNA in CSF and throat swab samples
J Med Virol
(2002)

M. Gorgievski-Hrisoho et al.

Detection by PCR of enteroviruses in cerebrospinal fluid during a summer outbreak of aseptic meningitidis in Switzerland

J Clin Microbiol

(1998)

J. Jacques et al.

New reverse transcription-PCR assay for rapid and sensitive detection of enterovirus genomes in cerebrospinal fluid specimens of patients with aseptic meningitis

J Clin Microbiol

(2003)

U. Kammerer et al.

Nested PCR for specific detection and rapid identification of human picornaviruses

J Clin Microbiol

(1994)

J.H. Knepp et al.

Comparaison of automated and manual nucleic acid extraction methods for detection of enterovirus RNA

J Clin Microbiol

(2003)

K.K. Lai et al.

Evaluation of real-time PCR versus PCR with liquid-phase hybridization for detection of enterovirus RNA in cerebrospinal fluid

J Clin Microbiol

(2003)

K.A. Lim et al.

Typing of viruses by combinaisons of antiserum pools. Application to typing of enteroviruses (Coxsackie and Echo)

J Immunol

(1960)

B. Lina et al.

Multicenter evaluation of a commercially available PCR assay for diagnosing enterovirus infection in a panel of cerebrospinal fluid specimens

J Clin Microbiol

(1996)

P. Muir et al.

Multicenter quality assessment of PCR methods for detection of enteroviruses

J Clin Microbiol

(1999)

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Development and evaluation of a real-time RT-PCR assay on the LightCycler for the rapid detection of enterovirus in cerebrospinal fluid specimens

Abstract

Background

Objective

Study design

Results

Conclusions

Introduction

Section snippets

Virus stocks and CSF samples

Analytical sensitivity and specificity

Discussion

Conclusion

J Clin Virol

Mol Cell Probes

J Clin Virol

J Pediatr

J Clin Virol

J Clin Virol

Diagn Microbiol Infect Dis

Aseptic meningitis in infants <2 years of age: diagnosis and etiology

J Infect Dis

New PCR test that recognizes all human prototypes of enterovirus: application for clinical diagnosis

J Clin Microbiol

Development and evaluation of a ≪ real-time ≫ RT-PCR for the detection of enterovirus and parechovirus RNA in CSF and throat swab samples

J Med Virol

Detection by PCR of enteroviruses in cerebrospinal fluid during a summer outbreak of aseptic meningitidis in Switzerland

J Clin Microbiol

New reverse transcription-PCR assay for rapid and sensitive detection of enterovirus genomes in cerebrospinal fluid specimens of patients with aseptic meningitis

J Clin Microbiol

Nested PCR for specific detection and rapid identification of human picornaviruses

J Clin Microbiol

Comparaison of automated and manual nucleic acid extraction methods for detection of enterovirus RNA

J Clin Microbiol

Evaluation of real-time PCR versus PCR with liquid-phase hybridization for detection of enterovirus RNA in cerebrospinal fluid

J Clin Microbiol

Typing of viruses by combinaisons of antiserum pools. Application to typing of enteroviruses (Coxsackie and Echo)

J Immunol

Multicenter evaluation of a commercially available PCR assay for diagnosing enterovirus infection in a panel of cerebrospinal fluid specimens

J Clin Microbiol

Multicenter quality assessment of PCR methods for detection of enteroviruses

J Clin Microbiol