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Molecular detection and identification of an enterovirus during an outbreak of aseptic meningitis
N Siafakas1, A Georgopoulou, P Markoulatos
1University of Essex, Department of Biological Sciences, John Tabor Laboratories, Essex, England.
Abstract:
Stool samples from sixteen cases of children with meningitis originating from four different and geographically isolated parts of Greece were investigated for enteroviruses. The conventional method of cell culture in four different cell lines was initially used for the isolation of enteroviruses. The results showed a cytopathic effect (CPE) in all cases after two, or even more successive passages in only one cell line (RD), although a less-than-satisfactory CPE was obtained in many cases. Seroneutralization with RIVM mixed hyperimmune antisera followed and the isolates were typed as Coxsackie B viruses. The method of RT-PCR with enterovirus-specific primers targeted to the highly conserved 5'-UTR of the genome was initially used for the detection of enteroviruses from the inoculated cell cultures. A positive RT-PCR result was obtained for all of the clinical samples rapidly and accurately and the isolates were further characterized with the aid of Restriction Fragment Length Polymorphism (RFLP) analysis and Single Strand Conformation Polymorphism analysis (SSCP) of the amplicons. The RFLP analysis showed first of all that the isolates had an identical restriction pattern with Coxsackie B5 Faulkner reference strain with 4 out of 5 restriction enzymes and secondly, both RFLP and SSCP analysis indicated the epidemiological association of the isolates. The speed of the molecular methodology that was used in comparison with the conventional methods and its possible significance for the description of virus evolution and circulation in the populations is discussed.
Insights
Molecular methods like RT-PCR rapidly detected enteroviruses, specifically Coxsackie B viruses, in children with meningitis. These techniques also confirmed epidemiological links between virus strains, offering insights into virus evolution and spread.
Area of Science:
- Virology
- Molecular Biology
- Epidemiology
Background:
- Enteroviruses are a common cause of viral meningitis in children.
- Geographically isolated regions can present unique challenges for epidemiological surveillance.
- Conventional cell culture methods for enterovirus detection can be slow and have variable sensitivity.
Purpose of the Study:
- To investigate enterovirus infections in pediatric meningitis cases from diverse Greek regions.
- To compare the efficacy of conventional cell culture with molecular methods for enterovirus detection and characterization.
- To analyze the epidemiological relationships between enterovirus isolates.
Main Methods:
- Enterovirus isolation using cell culture (RD cell line).
- Seroneutralization for virus typing.
- Reverse Transcription Polymerase Chain Reaction (RT-PCR) for rapid detection.
- Restriction Fragment Length Polymorphism (RFLP) and Single Strand Conformation Polymorphism (SSCP) for molecular typing and epidemiological analysis.
Main Results:
- Enteroviruses were detected in all sixteen pediatric meningitis cases.
- Coxsackie B viruses were identified as the causative agents.
- RT-PCR provided rapid and accurate detection of enteroviruses.
- RFLP and SSCP analyses confirmed the isolates as Coxsackie B5 and indicated epidemiological links.
Conclusions:
- Molecular methods, particularly RT-PCR, offer significant advantages in speed and accuracy over conventional cell culture for enterovirus detection in meningitis cases.
- RFLP and SSCP are valuable tools for characterizing enterovirus strains and understanding their epidemiological circulation.
- The study highlights the utility of molecular techniques for monitoring virus evolution and spread in populations.