Routine use of a highly automated and internally controlled real-time PCR assay for the diagnosis of herpes simplex and varicella-zoster virus infections.
BACKGROUND
Detection of herpes viruses can be significantly improved by PCR. The development of real-time PCR, which has overcome several limitations of conventional PCR, improved the prospects for implementation of PCR-based assays in diagnostic laboratory.


OBJECTIVE
To compare the diagnostic performance of an automated sample extraction procedure in combination with an internally controlled real-time PCR assay for detection of herpes simplex virus (HSV) and varicella-zoster virus (VZV) to conventional shell vial culture.


METHODS
One hundred eighty-two consecutive specimens from patients suspected of HSV or VZV infection were examined by internally controlled PCR and shell vial culture. An internal control consisting of phocine herpes virus was processed along with the specimens during the entire procedure and permitted to monitor extraction and amplification efficiency, including inhibition.


RESULTS
A total of 48 (26.4%) specimens were positive for HSV or VZV by culture, and 77 (42.3%) by real-time PCR. Thus, overall sensitivity increased by 60.4%. All culture-positive specimens were detected and typed correctly by PCR, except for a single specimen that contained PCR inhibitors. Specifically, the real-time PCR assay increased the detection rate for HSV-1 and HSV-2 by 43.9% and 62.5%, respectively. In PCR-positive specimens, lower levels of viral DNA were found in culture-negative than in culture-positive specimens. The increase of HSV detection rates by PCR varied with the origin of specimen and was particularly significant for skin specimens (7/14 versus 3/14 detected by culture) and bronchoalveolar lavages (8/8 versus 1/8). In addition, real-time PCR significantly increased the detection rate for VZV.


CONCLUSIONS
Compared to shell vial culture, our real-time PCR assay demonstrated a superior sensitivity and an added value of using internal control for checking the quality of examination of each specimen. These results provide a solid basis for implementation of real-time PCR in the routine diagnosis of HSV and VZV infections in various clinical specimens.
Stránská
Růzena
R
Department of Virology, Eijkman-Winkler Center, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. r.stranska@azu.nl
Schuurman
Rob
R
de Vos
Machiel
M
van Loon
Anton M
AM
D001331:Automation; D001992:Bronchoalveolar Lavage Fluid / Q000821:virology; D004279:DNA, Viral / Q000097:blood; D006561:Herpes Simplex / Q000175:diagnosis* / Q000821:virology; D006562:Herpes Zoster / Q000175:diagnosis* / Q000821:virology; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D018258:Herpesvirus 2, Human / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D014645:Herpesvirus 3, Human / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012867:Skin / Q000821:virology; D014776:Virus Cultivation; D014779:Virus Replication
D004279:DNA, Viral