Comparison of Viral Isolation and Multiplex Real-Time Reverse Transcription-PCR for Confirmation of Respiratory Syncytial Virus and Influenza Virus Detection by Antigen Immunoassays

Abstract

We evaluated the Prodesse ProFlu-1 real-time reverse transcription-PCR multiplex assay with the SmartCycler instrument for the detection of human respiratory syncytial virus (RSV) and influenza A and B viruses in comparison to conventional cell culture and antigen immunoassays with the BD Directigen A؉B and Binax NOW RSV assays over two successive respiratory virus seasons. Ninety-two percent of the 361 specimens tested were nasopharyngeal aspirates obtained from individual patients, of which 119 were positive for RSV and 59 were positive for influenza virus. The median age of the patients whose specimens were positive for RSV and influenza virus were 6.3 months and 42.4 years, respectively. The specificity of all of the methods tested was >99%, and the individual sensitivities of NOW RSV, RSV culture, Directigen A؉B, influenza virus culture, and the Proflu-1 PCR for influenza/RSV were 82% (95% confidence interval [CI], 73 to 88), 57% (95% CI, 44 to 69), 59% (95% CI, 44 to 72), 54% (95% CI, 38 to 69), and 98% (95% CI, 93 to 100)/95% (95% CI, 85 to 99), respectively. In a clinical setting where viral isolation is performed to confirm rapid antigen immunoassay results for these common respiratory viruses, one-step real-time reverse transcriptase PCR testing can be a more sensitive and timely confirmatory method.Human respiratory syncytial virus (RSV) and influenza A and B viruses are respiratory pathogens associated with substantial morbidity and mortality annually (43). Virtually all children become infected with RSV within 2 years after birth, and 1% require hospitalization (15). Although the importance of RSV as a cause of pneumonia and brochiolitis in young children is well recognized (21), the most serious morbidity and highest mortality associated with both RSV and influenza virus circulation occurs disproportionately among elderly persons (43). The first-line tests used to detect these virus infections in many hospitals are antigen-based immunoassays. It has been demonstrated that antigen immunoassays have exceedingly poor sensitivity in detecting RSV and influenza virus infections in the elderly, seriously limiting their utility for detecting and confirming institutional or community outbreaks (7,13,38). This study was intended to evaluate the performance of viral isolation in cell culture, one-step real-time multiplex reverse transcription-PCR (RT-PCR), and antigen immunoassays for the detection of influenza virus and RSV in respiratory specimens from adults and children during two respiratory virus seasons. MATERIALS AND METHODS Specimens.Upper respiratory tract specimens were collected from 353 individual symptomatic patients during two successive winter respiratory virus seasons encompassing

Current Best Practices for Respiratory Virus Testing

“…The specificities of RADTs are generally reported to be high (6,11,15,16,24,30,45), but a recent report suggests that this might be incorrect, and this warrants further investigation (44). Although the specificities of DFA and rapid cell culture are high, the sensitivities of the tests vary by virus (and sometimes by viral strain) from a low of 50% (adenovirus DFA and RSV culture) to a high of Ͼ80% (RSV DFA and influenza A virus culture) compared to NAATs (26,30,31,35,52). In addition, although the 8 viruses commonly detected are responsible for a large portion of viral respiratory tract infections, select coronaviruses (229E, OC43, NL63, and HKU-1), parainfluenza virus type 4, rhinovirus, and potentially bocavirus are also significant causes of respiratory disease and are generally only detected using NAATs (4 (8 viruses), 35% for rapid cell culture (7 viruses), and 63% for a comprehensive NAAT (15 viruses).…”

Comparison of Becton Dickinson Directigen EZ Flu A+B Test against the CDC Real-Time PCR Assay for Detection of 2009 Pandemic Influenza A/H1N1 Virus

“…Much of this attention has focused on RIDTs, which have been demonstrated previously to have lower sensitivity than RT-PCR, viral culture, and other methods in the detection of seasonal influenza (6). In published reports regarding the use of RIDT for detecting pandemic influenza A/H1N1 2009 virus, the performance characteristics of several commercial RIDTs have been reported to be low.…”

Comparison of the Simplexa FluA/B & RSV Direct Assay and Laboratory-Developed Real-Time PCR Assays for Detection of Respiratory Virus

“…T raditionally, rapid point-of-care detection of influenza A virus (FluA) and influenza B virus (FluB) and respiratory syncytial viruses (RSV) has been performed using antigen immunoassays, which have low sensitivity (1,2,3). In comparison, nucleic acid amplification tests (NAAT) have improved sensitivity but increased time to result (4).…”