Identification of Enteroviruses from Central Nervous System Infections by RT-PCR and Cell Culture Methods

Kilic I., Altuglu I. , Cicek C., Pullukcu H., Bayram N., Sirin H. , ...Daha Fazla

MIKROBIYOLOJI BULTENI, cilt.45, ss.468-477, 2011 (SCI İndekslerine Giren Dergi) identifier identifier identifier

  • Cilt numarası: 45 Konu: 3
  • Basım Tarihi: 2011
  • Sayfa Sayıları: ss.468-477


Viruses are the major causes of aseptic meningitis and encephalitis. Enteroviruses account for more than 80% of the aseptic meningitis cases for which an etiologic agent is identified. The aims of the present study were to identify agents of enteroviral meningitis by viral culture and reverse transcriptase polymerase chain reaction (RT-PCR) methods, to evaluate the appropriateness of a commercial RTPCR kit for its use in routine laboratory, and to obtain epidemiological data about enteroviral meningitis. Sixty six cerebrospinal fluid (CSF) samples from patients with suspected viral central nervous system (CNS) infection by clinical and CSF biochemical findings, sent to Ege University Faculty of Medicine, Department of Medical Microbiology were included in the study. The CSF samples were all negative for tested bacteria, mycobacteria, fungi, herpes simplex virus and cytomegalovirus. Thirty-four (51.5%) of the samples were from female and 32 (48.5%) were from male patients. Twenty-three (34.8%) patients were children (5 months-18 years) and 43 (65.2%) were adults (19-86 years). Shell vial rapid cell culture method by using Vero, HEp-2 and RD cell lines was performed for virus isolation and the results were evaluated on 48(th) hours after staining the cells with fluorescein labeled polyclonal antibodies (Pan-Enterovirus Blend, Light Diagnostics, USA). Enteroviral RNA in the samples was detected by a commercial RT-PCR kit (Enterovirus Consensus Kit, Argene, France). Sixty-one (92.4%) of 66 samples from patients with suspected viral CNS infection were found to be negative for enterovirus both with RT-PCR and shell vial cell culture methods. Three samples (4.5%) were positive by shell vial culture method. In one CSF sample that was culture positive, RT-PCR was also positive. However, the remaining two culture positive samples yielded negative result by RT-PCR. Intermediate results with RT-PCR were obtained in two samples (3%) that were identified as negative by cell culture. Two of the three positive samples in cell culture were identified as echovirus, however, the remaining sample could not be identified due to small sample amount. As a result, the commercial assay was found non-practical and labor intensive, giving indeterminant results in some cases and missing two culture positive samples. Since it didn't have an advantage over the cell culture method used, it was found inappropriate for routine diagnosis in our laboratory. On the other hand, it has been known that nucleic acid amplification tests (NAT) have markedly improved the diagnosis of enterovirus infections by increasing the sensitivity compared with cell culture methods. An alternative NAT method should be evaluated in parallel with cell culture method especially in CSF samples of children with suspected viral central nervous system infections.