New Reverse Transcription-PCR Assay for Rapid and Sensitive Detection of Enterovirus Genomes in Cerebrospinal Fluid Specimens of Patients with Aseptic Meningitis

Abstract

Enterovirus (EV) detection by a new commercially available reverse transcription (RT)-PCR assay (Penter RT-PCR test) was compared with EV isolation from cell cultures and with EV detection by an in-houseEnteroviruses (EVs) of the Picornaviridae family (64 distinct serotypes) are etiological causes of neurological syndromes in infants and adults (9,14). Aseptic meningitis is the most common EV-related central nervous system syndrome and may be difficult to distinguish from meningitis caused by herpesviruses or bacteria, particularly in young infants and children (9, 10). When classical cell culture techniques are used, attempts to isolate EVs from cerebrospinal fluid (CSF) samples are frequently unsuccessful because of the low viral titers in clinical specimens and because some serotypes grow poorly in cell culture (3, 4). Therefore, in-house PCR techniques for the detection of the EV genome have been introduced, allowing sensitive and rapid identification of EV-infected CSF samples (1,11,12,17) and improving the management of young infants with meningitis (8). However, the application of in-house EV PCR assays to the clinical diagnosis of EV-related neurological syndromes is actually limited by the time-consuming methods and the lack of standardization of procedures (6, 13). In this study, a new commercially available and well-standardized reverse transcription (RT)-PCR test (Penter RT-PCR) (2) was compared with cell culture and with an inhouse reference RT-PCR assay for the detection of EV in CSF of patients with aseptic meningitis.Fifty-four archived CSF specimens taken from 49 children (mean age, 6 years; range, 10 days to 12 years) and 5 adults (mean age, 29 years; range, 19 to 33 years) hospitalized at the university hospital of Reims (Reims, France) for meningitis syndrome during a summer outbreak of aseptic meningitis (May to November 2001) were retrospectively selected. CSF samples were included in this study providing they revealed a typical profile of aseptic meningitis with elevated levels of protein (Ͼ0.45 g/liter) and/or nuclear cells (Ͼ6 cells/mm 3 ), normal glucose levels, negative Gram stain results, and negative bacterial and fungi cultures (15). Virus isolation had been prospectively performed for each CSF sample on human diploid fibroblasts (MRC-5) and rhesus monkey kidney cells (MA-104) as previously described (7). EV isolates were typed by the standard method of virus neutralization (Lim-Banyesch-Melnick immune serum pools) (4). After testing of CSF by viral culture, aliquots of CSF were stored at Ϫ80°C for later use in detecting enteroviral RNA by RT-PCR.The new EV Penter RT-PCR test (new-generation EV consensus kit; Argène Biosoft, Varhiles, France) utilized several pairs of stair primers selected for annealing in the 5Ј untranslated region, which is highly conserved in the EV group (2), and was used according to the manufacturer's instructions. Briefly, viral RNA was extracted from 140 l of CSF sample by a rapidextraction protocol (viral RNA minikit; Qiagen, Courtaboeuf, France) and elute...

Development, Technical Performance, and Clinical Evaluation of a NucliSens Basic Kit Application for Detection of Enterovirus RNA in Cerebrospinal Fluid

“…Our laboratory currently uses the NucliSens EV assay to aid in the differentiation of rhinovirus from enterovirus culture isolates. The assay did not detect parechovirus 1 (formerly echovirus 22) (8), a result consistent with reports by other investigators using the NucliSens EV primer set (5, 9) or other RT-PCR assays that use primers designed from the same 5Ј-NTR sequence region (2,7,10,13,15,18,19,(23)(24)(25).…”

GeneXpert Enterovirus Assay: One-Year Experience in a Routine Laboratory Setting and Evaluation on Three Proficiency Panels

“…Since the conventional means of diagnosis of enteroviral meningitis by cell culture from cerebrospinal fluid (CSF) is timeconsuming and expensive and lacks sensitivity, some commercial and several in-house PCR protocols as well as nucleic acid sequence-based amplification assays have been developed during the last 15 years in order to improve the ability to detect enterovirus from CSF (2-4, 6, 7, 11, 13, 16, 20-22, 24-26). The most widely used PCR-based assays for the routine diagnosis of enteroviral meningitis are those that detect PCR products in microtiter wells (6,21,22) and those that use the real-time PCR technology (2,3,13,(24)(25)(26). However, all of these approaches require specially trained laboratory staff, thus limiting the ability of the "real-time" technology to deliver patient results that would be most useful for making patient management decisions STAT.…”

“…Thus, each CSF sample was tested for the presence of enterovirus RNA by using GXEA and an in-house real-time PCR on the same day. Since the in-house real-time PCR assay had been implemented in the daily routine only 7 months before the start of the GXEA evaluation and because of a lack of an internal control for the in-house real-time PCR, all CSF samples were additionally tested once weekly by using the enterovirus consensus kit (Argene SA, Varilhes, France), following the manufacturer's instructions, as described previously (6). Briefly, 10 l of RNA previously extracted for the in-house real-time PCR and stored for a maximum of 1 week at Ϫ70°C was amplified by using a one-step reverse-transcription PCR and primers targeting a 425-bp region of the 5Ј noncoding region, followed by detection of PCR products in a microtiter plate by use of a biotinylated probe.…”

Fatal Echovirus 18 Leukoencephalitis in a Child

“…Moreover, serological results and vac- The use of a standardized PCR assay for EV RNA, followed by a hybridization assay, allowed us to determine the presence of EV genomic RNA in a CSF sample from the infant studied (3). This positive EV molecular detection associated with high levels of IFN-␣ was compatible with viral CSF infection in this study (3,7). Moreover, RT-PCR amplification of the VP1 gene and amplicon sequencing have been used to identify genetically identical EV-18 strains in paired CSF and throat samples (Fig.…”

“…The use of a standardized reverse transcription (RT)-PCR assay for EV, followed by microwell hybridization, assessed the presence of EV genomic RNA in throat and CSF samples (3). Genotyping of these EV strains by partial amplification and sequencing of the VP1 gene revealed 100% sequence homology in both throat and CSF samples from this infant.…”