To compare Simplexa Flu A/B & RSV PCR with cytospin-immunofluorescence and laboratory-developed TaqMan PCR methods, 445 nasopharyngeal samples were tested. Of these, 199 were positive (46 for respiratory syncytial virus [RSV], 120 for influenza A, and 33 for influenza B) and 246 were negative. The direct fluorescent-antibody assay (DFA) detected 132 (66.3%) positive samples, Simplexa direct detected 162 (81.4%), Simplexa using extracts detected 177 (88.9%), and lab-developed TaqMan PCR reference methods detected 199 (100%). The specificities were 99.6%, 100%, 100%, and 100%, respectively. The two Simplexa methods were more sensitive than the DFA (P ؍ 0.0001) but less sensitive than the TaqMan reverse transcriptase PCR (P ؍ 0.0001).
Influenza virus and respiratory syncytial virus (RSV) cause a substantial disease burden in people of all ages. A rapid laboratory diagnosis can facilitate early antiviral therapy, discontinuation of antibiotics, and implementation of infection control measures (1). Our laboratory uses the cytospin-enhanced direct fluorescent-antibody assay (DFA) as an initial on-demand screen and laboratory-developed real-time TaqMan (LDT) PCR when greater sensitivity is needed (2). In this report, we evaluated Simplexa Flu A/B & RSV PCR as a potential replacement for DFA and LDT PCR.Nasopharyngeal swab specimens submitted in 3 ml M4 medium (Remel, Lenexa, KS) from February to April 2011 were tested by DFA and LDT PCR as requested by the clinician within 2 to 12 h of collection. The original samples and extracts were then frozen at Ϫ70°C, and a subset of the positive and negative samples were retested with the Simplexa PCR within 2 to 4 months.The cytospin-DFA was performed using SimulFluor reagents (Millipore, Billerica, MA) as previously described (3). For influenza detection and subtyping, the CDC real-time reverse transcriptase (RT) PCR primers and probes were used (2). For RSV, the protocol published by van Elden et al. (4) was used, but the subtypes were not differentiated. Total nucleic acid was extracted from 200 l of sample and eluted in 55 l on an EasyMag instrument (bioMérieux, Durham, NC), and 5 l of extract was added to 20 l AgPath-ID one-step RT-PCR master mix (Life Technologies, Carlsbad, CA) without an internal control (IC), and the manufacturers' cycling parameters (10 min at 45°C, 10 min at 95°C, 15 s at 95°C, 45 s at 60°C) were used. Amplification was performed for 45 cycles using an ABI 7500 instrument (Applied Biosystems, Foster City, CA). Positive samples with cycle threshold (C T ) values of Ͻ38 were accepted as positive. For C T values of Ն38, amplification was repeated in duplicate and accepted if one replicate was positive.For Simplexa Flu A/B & RSV PCR (Focus Diagnostics, Cypress, CA), frozen extracts were thawed, vortexed, and pulse spun. To each well of a 96-well disc was added 5 l of master mix containing primers, an IC, and 5 l of thawed extract or positive or negative control material. Discs were sealed and placed in a 3 M integrated cycler (Focus Diagnostics). Amp...