Performance evaluation of direct fluorescent antibody, Focus Diagnostics Simplexa™ Flu A/B & RSV and multi-parameter customized respiratory Taqman® array card in immunocompromised patients

Background: Molecular assays for diagnosis of Flu A, Flu B, and RSV with short turn-around-time (TAT) are of considerable clinical importance. In addition, rapid and accurate diagnosis of a large panel of viral and atypical pathogens can be crucial in immunocompromised patients. Objectives: First, to evaluate the performance of the Simplexa (TM) Direct assay system in comparison with direct fluorescent antibody (DFA) arid customized Taqman (R) Array Card (TAC) testing for RSV, Flu A, and Flu B in immunocompromised patients. Second, to evaluate different algorithms for the detection of respiratory pathogens in terms of cost, turn-around-time (TAT) and diagnostic yield. Study design: We collected 125 nasopharyngeal swabs (NTS) and 25 BAL samples from symptomatic immunocompromised patients. Samples for which Simplexa (TM) and TAC results were discordant underwent verification testing. The TAC assay is based on singleplex RT-PCR, targeting 24 viruses, 8 bacteria and 2 fungi simultaneously. Results: The overall sensitivity was significantly lower for DFA testing than for the two molecular methods (p < 0.05). Performance characteristics of Simplexe(TM) testing were not significantly different compared to TAC testing (p > 0.1). For BAL samples only, the sensitivity and specificity of the Simplex (TM) assay was 100%. In total, 6.7, 16 and 18% of samples were positive for Flu A, Flu B or RSV by DFA, Simplexa (TM) and TAC testing, respectively. When considering not only these pathogens but also all results for TAC, the method identified 93 samples with one or more respiratory pathogens (62%). A co-infection rate of 15.3% was found by TAC. The estimated costs and TAT were 8.2(sic) and 2 h for DFA, 31.8(sic) and 1.5 h for Simplexa (TM) and 55 and 3 h for TAC testing. Conclusions: Performing the Simplexa (TM) test 24 h a day/7 days a week instead of DFA would considerably improve the overall sensitivity and time-to-result, albeit at a higher cost generated in the laboratory. Performing the TAC would increase the diagnostic yield and detection of co-infections significantly.