Peeters B., Micalessi MI., Van Meensel B., Smismans A., Frans J.

1. Acknowledgements Acknowledgements STRATEGIES TO OVERCOME INVALID RESULTS WITH THE RESPIFINDER® SMART 22 ASSAY USED FOR DIAGNOSIS OF RESPIRATORY TRACT INFECTIONS Extraction in 100 µl onNuclisensEasyMAG(bioMérieux) NasopharyngealeSwab (Copan) 200 µl ReverseTranscription+ Pre-amplification Hybridizationwithspecific probes foreverypathogen Ligation/ Amplification/ Melting curve FluorescencedetectionduringmeltingonRotorGene Q (Qiagen) Timeframe 40 min 130 min 70 min 145 min Peeters B., Micalessi MI., Van Meensel B., Smismans A., Frans J. Laboratory of Clinical Microbiology, Imelda Hospital, Bonheiden, Belgium. Correspondence: bart.peeters@imelda.be OBJECTIVES The RespiFinder® SMART 22 assay (PathoFinder) is a multiplex PCR, which enables the detection and differentiation of 18 respiratory viruses as well as 4 bacteria in 6 h. An internal control (IC) is included in the assay to assess amplification inhibition. A result is reported as invalid when the internal control is not amplified and a negative result is obtained for the 22 respiratory pathogens. This assay, which is performed in our daily routine, exhibits an inhibition rate of 5.9% (55/949). To circumvent false negative results due to inhibition, the manufacturer recommends sample pretreatment with dithiothreitol and/or dilution of the sample extract. This study investigates how inhibition can be resolved without repeating the test, using a second RespiFinder SMART 22 reagent panel and delaying patient diagnosis. METHODS AND RESULTS Figure 1: RespiFinder SMART 22 workflow The complete workflowfromextractionuntillanalysis of the results is demonstrated in Figure 1. An IC is added to every sample beforeextraction to check forinhibition and extraction efficiency and a negative sample is added to every run to check forcontamination and to allow a correct threshold setting. Afteranalysis of the results, positive samples show a peak at a certaintemperature in the ROX or Cy5 channel. Eachpeak is specificfor a certainrespiratorypathogen. The experimental set-up and resultsare given in Table 1. Table 1: Experimental set-up and results We assume that the inhibitor present in the sample is either a protease which exerts its activity on the PCR enzymes or is a non-protease allosteric PCR-inhibitor. Allosteric inhibitors can result in competitive binding of reagents when the relative concentration of the inhibitor is extremely high compared to the target. CONCLUSION Invalidresults for the RespiFinder SMART 22 assay may be avoided by subjecting all samples to a one-time freeze thaw cycle with a freezing step (-70°C) of at least 30 min. Freezing samples prior to analysis does not affect sensitivity of the respifinder test. This sample pretreatment provides a fast, low-cost and easy to apply solution to prevent amplification inhibition and to obtain higher turnaround times. The inhibition mechanism occurs in both positive and negative samples. Acknowledgements The authorwouldlike to thankPathoFinder BV forproviding all reagents and the RespiFinder SMART 22 kits to conductthisstudy.