Utility of Saliva for Detecting Influenza A Virus by Cost-Effective Extraction-Free SYBR Green-Based PCR

Respiratory specimens collected via nasopharyngeal and throat swabs are the recommended method of choice for the molecular detection of the Influenza A virus. However, they often cause discomfort to patients and require trained healthcare workers. The aim of this study is to validate a cost-effective nucleic acid extraction-free PCR method using SYBR Green chemistry with saliva samples to diagnose respiratory illnesses caused by the Influenza A virus. For this, we enrolled symptomatic pediatric and adult patients with influenza-like illness. A SYBR Green-based nucleic acid amplification test was used with CDC-recommended primers specific for detecting Influenza A virus. Results from saliva PCR tests were compared with those of standard TaqMan chemistry-based PCR detection from nasopharyngeal swabs. The sensitivities of saliva PCR tests with and without nucleic acid extraction were 93.6% and 87.1%, respectively. Both methods showed specificity of 96% and 98.4%, respectively. Overall, the sensitivity and specificity of saliva as a sample using the developed SYBR Green-based PCR for Influenza A virus detection were 90.3% and 97.7%, respectively. Given the high sensitivity, specificity, positive and negative predictive values, likelihood ratios, agreement percentage, and κ statistics, this study concluded that saliva is a potential specimen for diagnosing Influenza A virus infection by extraction-free nucleic acid amplification testing. Despite promising results, further validation through large, multicenter studies using fresh samples and subtype-specific analysis is warranted.