Diagnosis of human metapneumovirus and rhinovirus in patients with respiratory tract infections by an internally controlled multiplex real-time RNA PCR.
BACKGROUND
Adequate laboratory diagnosis of human rhinoviruses (hRV) and human metapneumoviruses (h MPV) requires molecular methods as viral culture lacks sensitivity. However, setting up individual PCRs for all respiratory viruses is not practical so preferentially multiplex PCRs are used.


OBJECTIVE
To develop for routine diagnosis a rapid real-time PCR assay for detection of hRV and h MPV including an internal control in a single tube multiplex reaction using probes carrying different fluorophores to discriminate targets.


METHODS
The multiplex real-time RNA PCR was optimized to include the internal control virus and a total of 358 respiratory samples from 239 patients taken over a one-year period were analyzed by the multiplex assay.


RESULTS
The multiplex assay with co-amplification of the internal control was as sensitive and specific as the individual assays. Application of this assay on clinical samples from 239 patients in a one-year period resulted in an incidence of hRV and h MPV of 41/239 (17.1%) and 6/239 (2.5%), respectively. Inhibition, defined as poor internal control amplification, was detected in 8 (2.2%) samples. Culture was performed on these samples and only four hRV were detected.


CONCLUSIONS
This real-time PCR method enables sensitive diagnosis of these two respiratory pathogens with the potential to expand the assay as part of a full molecular respiratory viral screen.
Department of Medical Microbiology, Center Infectious Diseases, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands.
S A
SA
Scheltinga
K E
KE
Templeton
M F C
MF
Beersma
E C J
EC
Claas
D000293:Adolescent; D000328:Adult; D000818:Animals; D002460:Cell Line; D002648:Child; D002675:Child, Preschool; D006801:Humans; D029121:Metapneumovirus / Q000235:genetics / Q000302:isolation & purification*; D018184:Paramyxoviridae Infections / Q000175:diagnosis / Q000821:virology; D010850:Picornaviridae Infections / Q000175:diagnosis / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis* / Q000302:isolation & purification; D012015:Reference Standards; D012137:Respiratory System / Q000382:microbiology; D012141:Respiratory Tract Infections / Q000175:diagnosis* / Q000821:virology; D012229:Rhinovirus / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013997:Time Factors
D012367:RNA, Viral