Evaluation of two (semi-)nested VP1 based-PCRs for typing enteroviruses directly from cerebral spinal fluid samples.
Human enteroviruses (EVs) are the leading cause of CNS-associated disease in childhood. Identification of the EV types that patients are infected with is essential for monitoring outbreaks, the emergence of new types or variants, epidemiological surveillance and contributes to patient management. Rapid and sensitive molecular detection methods are frequently used to detect EVs/HPeVs directly from CSF. This requires that sensitive EV typing methods from CSF material need to be developed. In the present study two nested PCR-based typing assays were evaluated. The performance of the EV-A and -B specific nested PCR protocol and the Codehop-based PCR protocol were analyzed with several TCID(50)-titrated EV-A to D strains and 22 EV positive CSF samples. The EV-A and -B protocol was found to be more sensitive than the Codehop protocol. The Codehop protocol showed a high degree of aspecific amplification products when run on a gel, and required additional gel purification. The detection limit of the two protocols varied between the types, ranging from 0.1TCID(50)/mL sample to 10(6)TCID(50)/mL sample. From the 22 EV positive CSF samples, 15 (68%) samples were typed using either protocol. All samples were characterized as members of species B (E30 (9), CAV9 (2), E6 (1), E11 (1), E21 (1), E25 (1)). Three samples (E30 (2) and E25 (1)) could only be typed using the EV-B protocol. In this study, selected EV strains could be typed using both assays at low virus concentrations, typically found in CSF. However, the EV-A and -B protocol was more sensitive than the Codehop protocol for primary typing of CSF samples.
Laboratory of Clinical Virology, Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
R P
RP
Minnaar
G
G
Koen
K
K
de Haan
K C
KC
Wolthers
K S M
KS
Benschop
D020121:5' Untranslated Regions; D036022:Capsid Proteins / Q000032:analysis* / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D029821:Enterovirus A, Human / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D029822:Enterovirus B, Human / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D004769:Enterovirus Infections / Q000134:cerebrospinal fluid* / Q000821:virology; D060005:Genotyping Techniques / Q000379:methods; D006801:Humans; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D012680:Sensitivity and Specificity; D019562:Viral Load
D020121:5' Untranslated Regions; D036022:Capsid Proteins; D012367:RNA, Viral; C116597:echovirus 9 capsid protein vp1