Abstract
A rapid, low cost, accurate point-of-care (POC) device to detect influenza virus is needed for effective treatment and control of both seasonal and pandemic strains. We developed a single-use microfluidic chip that integrates solid phase extraction (SPE) and molecular amplification via a reverse transcription polymerase chain reaction (RT-PCR) to amplify influenza virus type A RNA. We demonstrated the ability of the chip to amplify influenza A RNA in human nasopharyngeal aspirate (NPA) and nasopharyngeal swab (NPS) specimens collected at two clinical sites from 2008–2010. The microfluidic test was dramatically more sensitive than two currently used rapid immunoassays and had high specificity that was essentially equivalent to the rapid assays and direct fluorescent antigen (DFA) testing. We report 96% (CI 89%,99%) sensitivity and 100% (CI 95%,100%) specificity compared to conventional (bench top) RT-PCR based on the testing of n = 146 specimens (positive predictive value = 100%(CI 94%,100%) and negative predictive value = 96%(CI 88%,98%)). These results compare well with DFA performed on samples taken during the same time period (98% (CI 91%,100%) sensitivity and 96%(CI 86%,99%) specificity compared to our gold standard testing). Rapid immunoassay tests on samples taken during the enrollment period were less reliable (49%(CI 38%,61%) sensitivity and 98%(CI 98%,100%) specificity). The microfluidic test extracted and amplified influenza A RNA directly from clinical specimens with viral loads down to 103 copies/ml in 3 h or less. The new test represents a major improvement over viral culture in terms of turn around time, over rapid immunoassay tests in terms of sensitivity, and over bench top RT-PCR and DFA in terms of ease of use and portability.
Highlights
In a normal year, the influenza virus infects millions of individuals [1] causing approximately 350,000 hospitalizations and 50,000 deaths in the United States [2,3]
Nasopharyngeal aspirate (NPA) and nasopharyngeal swab (NPS) specimens that were collected with Institutional Review Board approval from patients presenting with symptoms consistent with influenza to two Boston hospitals, as described in the methods, were tested by bench top reverse transcription polymerase chain reaction (RT-polymerase chain reaction (PCR))
Results of direct fluorescent antigen testing (DFA) (n = 106) and rapid immunoassay (n = 119) testing that had been performed on specimens as part of routine care at each of the two hospitals were compared to results obtained from testing of those specimens by our bench top PCR reference standard
Summary
The influenza virus infects millions of individuals [1] causing approximately 350,000 hospitalizations and 50,000 deaths in the United States [2,3]. The most recent pandemic highlighted weaknesses in the methods widely used to diagnose influenza: rapid immunoassays (antigen tests), direct fluorescent antigen testing (DFA), and viral culture methods (culture followed by immunofluorescence or hemagglutination assay for virus identification) [7]. Rapid influenza tests on the market were widely used and found to be dramatically lacking in sensitivity [8,9,10,11] such that the Centers for Disease Control and Prevention (CDC, Atlanta, GA) recommended that a negative test result be ignored for clinical decision-making [12]. Viral culture remains the ‘‘gold standard’’ test; it requires 2–7 days to complete and lacks the ability to distinguish between different respiratory viruses and between influenza virus types without follow up testing [7]. The vast amount of PCR data generated during the 2009 H1N1 pandemic supported the hypothesis that PCR performs as well or better than culture, including the ability to verify and differentiate influenza types and subtypes [8,9,15]
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