Detection of Respiratory Viruses in Sputum from Adults Using Automated Multiplex Polymerase Chain Reaction (PCR)
Background: Respiratory tract infections (RTI) frequently cause hospital admissions among adults. Diagnostic viral RT-PCR of nose and throat swabs (NTS) is useful for patient care by allowing better antibiotic management, timely antiviral use and appropriate patient isolation. Previously, we found that the addition of sputum RT-PCR testing improves the diagnostic yield for respiratory viruses. However, automated RT-PCR systems are not amenable to utilizing sputum due to its viscous nature. Thus, we evaluated a simple method of processing sputum for use in the fully automated FilmArray Respiratory Viral Panel RT-PCR assay (FilmArray).
Methods: Archived sputum and NTS samples collected in the winters of 2008-2012 from hospitalized adults with RTI were used. From this collection, a random subset of sputum samples previously positive for viruses (Flu A, Flu B, RSV A, coronaviruses OC43 & 229E, human metapneumovirus (HMPV), parainfluenza viruses 1-3 and rhinovirus) by manual RNA extraction and uniplex real-time RT-PCR were selected. A sterile cotton tip swab was dunked in the sputum followed by swirling in 700 uL of sterile water (dunk method) and tested by FilmArray. In addition, quantitative RT-PCR was performed on dunked sputum and NTS samples for Flu A, RSV, OC43 and HMPV.
Results: Virus was identified in 31% of 965 illnesses using uniplex RT-PCR. The addition of sputum RT-PCR significantly increased the diagnostic yield compared to NTS alone (302/965 [31%] vs. 197/965 [20%]; p=.0001). Sputum was the only sample positive for 105 subjects, including 35% (22/64) of influenza cases. Of 108 randomly selected sputa previously positive by uniplex PCR, 99 (92%) were also positive by FilmArray using the dunk method. Quantitative RT-PCR revealed significantly higher mean viral loads in dunked sputum compared to NTS samples for Flu A, RSV and HMPV (p=0.0001, p=0.006, p=0.011).
Conclusion: The dunk method is a simple and practical method for processing sputum for use in a fully automated PCR system with a yield of 92% compared to manual extraction and uniplex RT-PCR. The higher viral load in sputum may allow detection when the NTS viral load is below the limits of detection. Testing of sputum may be particularly important in patients with lower respiratory tract disease during influenza season.
Hologic: Consultant, Consulting fee
Sanofi Pasteur: Research Contractor, Research grant
AstraZeneca: Research Contractor, Research grant
ADMA Biologic Inc: Research Contractor, Research grant
E. Walsh, Clearpath Incorporated: Consultant, Consulting fee
Alios Biopharma: Consultant, Consulting fee