Evaluation of quantitative and type-specific real-time RT-PCR assays for detection of respiratory syncytial virus in respiratory specimens from children.
BACKGROUND
Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract morbidity in young children and immunosuppressed patients.


OBJECTIVE
To rapidly and accurately quantify and subtype RSV in respiratory samples, we developed and evaluated two real-time RT-PCR assays.


METHODS
A quantitative assay was designed using primers for a consensus region of the matrix protein gene and a subtype-specific assay for RSV-A and RSV-B detection was designed using primers for the polymerase gene. Quantitative RSV RT-PCR results of pediatric nasal wash samples submitted to the University of Washington Virology Laboratory from December 2002, through May 2003, were compared to those of an indirect fluorescent antibody RSV antigen detection assay (FA).


RESULTS
Specificity of the RT-PCR assay was high, with no amplification of eleven common respiratory viruses and eight herpes viruses. Among 751 samples, RSV was detected in 267 (35.6%) by FA and in 286 (38.1%) by RT-PCR. Median RSV copy number in nasal wash samples that were positive by both FA and RT-PCR was 2.5 x 10(7) copies/mL versus a median of 3.0 x 10(4) copies/mL for samples positive by RT-PCR only (P < 0.001). The detection and quantity of RSV in respiratory specimens was associated with younger age, but not with gender or hospitalization. Among positive samples from this Seattle cohort, 52% were subtype A and 48% were subtype B. Both subtypes were detected with similar viral loads among all patient groups (stratified by age, gender, and hospitalization), and throughout the specimen collection period.


CONCLUSIONS
These real-time RT-PCR assays provide a rapid, specific, and highly sensitive alternative for detecting, quantifying, and subtyping RSV in clinical specimens.
Kuypers
Jane
J
Department of Laboratory Medicine, University of Washington, CHRMC, 4800 Sand Point Way N.E., Virology Office, G-815, 8G-3, Seattle, WA 98105, USA. jane.kuypers@seattlechildrens.org
Wright
Nancy
N
Morrow
Rhoda
R
D000293:Adolescent; D000328:Adult; D000367:Age Factors; D000956:Antigens, Viral / Q000032:analysis; D001483:Base Sequence; D002648:Child; D002675:Child, Preschool; D017931:DNA Primers / Q000235:genetics; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods / Q000706:statistics & numerical data; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D018357:Respiratory Syncytial Virus Infections / Q000175:diagnosis* / Q000821:virology*; D018113:Respiratory Syncytial Virus, Human / Q000145:classification / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity
D000956:Antigens, Viral; D017931:DNA Primers; D012367:RNA, Viral