Evaluation of quantitative and type-specific real-time RT-PCR assays for detection of respiratory syncytial virus in respiratory specimens from children

Abstract

Background: Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract morbidity in young children and immunosuppressed patients.

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

Study design: 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.

Fig. 1

The median number of RSV, expressed as log 10 copies/ml, quantified by real time RT-PCR, was determined for FA positive and FA negative respiratory specimens from children. Among 286 specimens positive by RT-PCR, 265 were FA positive and 21 were negative. Among 280 RSV positive specimens that were subtyped, 133 subtype A and 126 subtype B were FA positive; 13 subtype A and 8 subtype B were FA negative. The number of RSV detected in specimens that were FA positive was significantly higher than the number detected in FA negative specimens (P<0.001) for all groups.

Fig. 2

The number of RSV, expressed as log 10 copies/ml, quantified in 280 samples by real-time RT-PCR is plotted against the patient’s age in months. Specimens are sorted by the RSV subtype and the FA result. Six RSV RT-PCR positive specimens were not tested with the type-specific assay.