3 mL of reagent L5. The LPBM were resuspended in this SHP099 concentration solution under gentle agitation for 2 minutes to generate the signal. Then 100 μL of L6 reagent was added to stop the reaction. The mixture
was rocked for 1 minute. The LPBM were captured again as described above, and after 5 minutes, the color was compared with a negative control (without L. pneumophila). The kit is intended to provide a semi-quantitative measure of L. pneumopila concentration, by interpolation of the color developed by the tested sample in the supplied color chart. If the colorimetric reaction showed no difference between sample and negative control Selleckchem GDC-0449 after two minutes, then the reaction was allowed to proceed for 10 minutes before stopping to trap low positives which correspond to an estimate level around the LOD50 of the IMM test. A test is considered positive if at 2 minutes or before 10 minutes color difference appears with the control. A positive L. pneumophila test must have a color higher than the color control at 2 minutes from starting colorimetric reaction. Then reaction was stopped following the protocol instructions. General estimation of the level of L. pneumophila in the sample was obtained comparing the test color with the color chart. If there was no color difference at 2 minutes, the reaction was allowed continue up to 10 minutes and then it was stopped. A positive L. pneumophila test must have a color higher
than the color control
IWP-2 molecular weight at 10 minutes from starting colorimetric reaction. In this case, the estimated level of L. pneumophila was low, up to two orders of magnitude (102 CFU/volume examined). A negative L. pneumophila test was considered if there was no color difference with the control after 10 minutes. Calculation of performance characteristics The test performance characteristics (specificity, sensitivity, false positives, false negatives, and efficiency) of the IMM were Phospholipase D1 determined. Available ISO guides are designed to validate methods based on the microbial growth and the key issue is the “growth unit” capable to growth in a nutrient media. Although the qualitative IMM kit is not based on the growth unit, a first categorization of the presumptive results was obtained by using a two-by-two contingency table, following the scheme provided by the norm ISO/TR13843 [39]. IMM presumptive results were compared with the ones obtained with the reference method (ISO11731). These results were divided into four categories: (a) number of presumptive positives by the IMM found positive by the reference culture method (true positives), (b) number of presumptive negatives by the IMM found positive by the reference culture method (false negatives), (c) number of presumptive positives by the IMM found negative by the reference culture method (false positives), and (d) number of presumptive negatives by the IMM found negative by the reference culture method (true negatives).