Development and evaluation of a real-time RT-PCR assay on the LightCycler for the rapid detection of enterovirus in cerebrospinal fluid specimens.
BACKGROUND
Detection of enteroviral nucleic acid in cerebrospinal fluid (CSF) specimens has been demonstrated to improve the management of patients with aseptic meningitis.


OBJECTIVE
To develop on the LightCycler (LC) instrument a real-time RT-PCR assay based on TaqMan technology for the detection of enteroviruses (EV) in cerebrospinal fluid (CSF) specimens.


METHODS
After evaluation of the analytical performances, seventy-four CSF samples collected prospectively from patients who have been suspected for a clinical diagnosis of meningitis were evaluated by two LC real-time RT-PCR assays and one conventional RT-PCR assay.


RESULTS
Our assay detected all 30 different EV species tested, whereas no reactivity was observed with other neurotropic viruses. The analytical sensitivity of both LC RT-PCR real-time assays was 1 TCID50 for LC one-step and two-step RT-PCR assays. Results for LC one-step and LC two-step RT-PCR were compared to results of the conventional RT-PCR: of the 74 CSF specimens tested, 11 were positive and 56 were negative by all methods. Four other specimens were positive for EV by at least two of the methods (including the LC two-step RT-PCR and the conventional RT-PCR), two other CSF specimens were positive by the LC two-step RT-PCR assay only, and another one CSF specimen was positive by the LC one-step RT-PCR assay only. No CSF specimens were negative by the LC two-step RT-PCR assay and positive by the conventional RT-PCR assay. The sensitivity, specificity, positive and negative predictive values of both LC RT-PCR assays by using conventional RT-PCR as the "gold standard" were, respectively, 73.3, 98.3, 91.7, 93.5% for the LC one-step RT-PCR and 100, 96.6, 88.2, 100% for the LC two-step RT-PCR. There was substantial agreement between the three assays (k=0.80).


CONCLUSIONS
The LC two-step RT-PCR assay is a rapid, sensitive and reliable method which can be routinely performed with CSF samples for diagnosis of EV infection and is an important improvement for optimal patient management.
Petitjean
J
J
Laboratory of Human and Molecular Virology, University Hospital, Avenue G. Clemenceau, 14033 Caen Cedex, France. petitijean-j@chu-caen.fr
Vabret
A
A
Dina
J
J
Gouarin
S
S
Freymuth
F
F
D002555:Cerebrospinal Fluid / Q000821:virology*; D004770:Enterovirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D004769:Enterovirus Infections / Q000134:cerebrospinal fluid / Q000175:diagnosis* / Q000821:virology; D006801:Humans; D008587:Meningitis, Viral / Q000134:cerebrospinal fluid / Q000175:diagnosis* / Q000821:virology; D011237:Predictive Value of Tests; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D012367:RNA, Viral