Improved diagnosis on a daily basis of enterovirus meningitis using a one‐step real‐time RT‐PCR assay

Abstract

The detection of the enterovirus genome in cerebrospinal fluid (CSF) by PCR techniques has proved to be more sensitive than traditional cell culture for the diagnosis of enterovirus meningitis. However, PCR assays are time consuming and labor intensive, particularly if separate hybridization steps are used to confirm the specificity of positive findings. The aim of this study was to develop a one-step real-time RT-PCR assay with LightCycler (LC) technology that was sensitive, rapid, and easy to perform in routine practice. The enterovirus detection limit was determined by testing 10-fold limiting dilution series of cell culture stocks with the echovirus 25 (E-25) prototype strain and with the third European Union Quality Control Concerted Action (EU-QCCA) enterovirus proficiency panel. A total of 100 CSF specimens were investigated in a comparative study. With the E-25 strain, the detection limit of the real-time assay was 286 TCID50/ml (50% tissue culture infective dose). When samples of the EU-QCCA panel were tested, our assay gave identical results (detection limit down to 3.6 TCID50/ml) to those of the reference laboratory, which used one-step RT-PCR assay. When CSF specimens were tested, there was a correlation between the real-time assay and the conventional in-house assay in 96 of 100 CSFs tested. This one-step real-time assay allows rapid enterovirus detection in CSF since results are obtained in 3 hr as against 36 hr with the "in-house" RT-PCR assay. This new assay is now being used in routine practice, and allows diagnosis on a daily basis.

Impact of rapid enterovirus molecular diagnosis on the management of infants, children, and adults with aseptic meningitis

“…The recent guidelines for the management of bacterial meningitis of the Infectious Diseases Society of America support this practice [Tunkel et al, 2004;Graham and Murdoch, 2005]. In our study, the real-time EV-PCR results were obtained within 3 hr [Archimbaud et al, 2004], and were available on the same day from Monday to Friday in 44% of patients, and within the first 24 hr in 70%. This assay gave a shorter TAT than the classic PCR assay used before 2003, 1 day versus 3 days, respectively [Peigue-Lafeuille et al, 2002].…”

“…The time of assay and analysis was about 36 hr. In 2003, this assay was replaced by a more rapid one-step real-time RT-PCR assay [Archimbaud et al, 2004]. This molecular assay was used daily in the virology laboratory for infants, children and adults.…”

“…After extraction of the viral RNAs with the QIAamp viral RNA mini kit (Qiagen, Courtaboeuf, France) from 140 ml of CSF, the one-step real-time enterovirus PCR assay was performed as previously reported [Archimbaud et al, 2004] with the following modifications. The nucleotide at the 5 0 terminus region of the reverse primer EV2-LC was deleted (5 0 -ATT GTC ACC ATA AGC AGC C-3 0 ), the T4 gene 32 protein was not used, and the annealing temperature for each cycle was 518C.…”

“…For diagnosis, the ''gold standard'' for enterovirus meningitis is the detection of enterovirus genome by reverse transcriptase-polymerase chain reaction (RT-PCR) from cerebrospinal fluid (CSF) specimens. This assay is an effective alternative to viral culture, since it can provide rapid results within 5-24 hr of receipt of the sample [Ramers et al, 2000;Stellrecht et al, 2002;Archimbaud et al, 2004]. Previous studies of children suggested that the use of the enterovirus PCR assay has a clinical benefit by reducing antibiotic or antiviral therapy, and avoiding ancillary tests, and also it reduces hospital related costs allowing earlier discharge [Hamilton et al, 1999;Ramers et al, 2000;Robinson et al, 2002;Stellrecht et al, 2002].…”

Applications of Real-time Reverse Transcription Polymerase Chain Reaction in Clinical Virology Laboratories for the Diagnosis of Human Diseases

“…The detection of the enterovirus genome in csf has proved to be more sensitive than traditional cell culture. Hence, a one-step rRT-PCR assay developed with LightCycler (LC) technology allowed the detection of enterovirus in csf in 3 hrs as opposed to 36 hrs of regular RT-PCR assay [24] . In another study, a single-tube, rRT-PCR/hybridization assay using TaqMan with LC technology was optimized for enterovirus screening that was carried out in 2 hrs [25] .…”