Detection of respiratory viruses in sputum from adults by use of automated multiplex PCR

Abstract

Respiratory tract infections (RTI) frequently cause hospital admissions among adults. Diagnostic viral reverse transcriptase PCR (RT-PCR) of nose and throat swabs (NTS) is useful for patient care by informing antiviral use and appropriate isolation. However, automated RT-PCR systems are not amenable to utilizing sputum due to its viscosity. We evaluated a simple method of processing sputum samples in a fully automated respiratory viral panel RT-PCR assay (FilmArray). Archived sputum and NTS samples collected in 2008-2012 from hospitalized adults with RTI were evaluated. A subset of sputum samples positive for 10 common viruses by a uniplex RT-PCR was selected. A sterile cotton-tip swab was dunked in sputum, swirled in 700 μL of sterile water (dunk and swirl method) and tested by the FilmArray assay. Quantitative RT-PCR was performed on "dunked" sputum and NTS samples for influenza A (Flu A), respiratory syncytial virus (RSV), coronavirus OC43 (OC43), and human metapneumovirus (HMPV). Viruses were identified in 31% of 965 illnesses using a uniplex RT-PCR. The sputum sample was the only sample positive for 105 subjects, including 35% (22/64) of influenza cases and significantly increased the diagnostic yield of NTS alone (302/965 [31%] versus 197/965 [20%]; P = 0.0001). Of 108 sputum samples evaluated by the FilmArray assay using the dunk and swirl method, 99 (92%) were positive. Quantitative RT-PCR revealed higher mean viral loads in dunked sputum samples compared to NTS samples for Flu A, RSV, and HMPV (P = 0.0001, P = 0.006, and P = 0.011, respectively). The dunk and swirl method is a simple and practical method for reliably processing sputum samples in a fully automated PCR system. The higher viral loads in sputa may increase detection over NTS testing alone.

FIG 1

Quantitative PCR viral load of straight sputum, dunked sputum and NTS samples. Quantitative PCR viral loads in TCID₅₀/ml of RSV, FLU A, OC43, and HMPV samples. Each solid circle represents a sample with detectable viral RNA. The interval bars indicate the 95% confidence interval of the mean viral load for each virus, which is represented by an open circle with midline bar.

FIG 2

Comparison of quantitative viral loads of dunked sputum and NTS samples. Comparison of the quantitative PCR viral loads in TCID₅₀/ml of NTS and dunked sputum RSV, FLU A, OC43, and HMPV samples. Light gray bars represent NTS samples, and dark gray bars represent dunked sputum samples.

FIG 3

Influenza A quantitative PCR of straight sputum samples, dunked sputum samples, and NTS samples collected with flocked swabs. Quantitative PCR viral loads in TCID₅₀/ml of Flu A NTS samples obtained with flocked swabs. Each solid circle represents a sample with detectable viral RNA. The interval bars indicate the 95% confidence interval of each mean viral load, which is represented by an open circle with midline bar.