The specificity is defined as the inability of the IMM to detect the target microorganism when it is not detected by the reference culture method, as follows: (d × 100) / (c + d). Finally, the efficiency is a general single parameter, which gives the agreement between the response obtained by the IMM and the reference culture method, as follows: (a + d) × 100 / n, where n is the total number of tests. The percentage of false positives is calculated as (c × 100) / (a + c), and the percentage of false negatives is calculated as (b × 100) SB431542 concentration / (b + d). A qualitative test can

be used as screening assay with confirmation. Only in this case, positive presumptive result confirmed as negative by the confirming culture method can be re-categorized as true negative. Performance characteristics were also calculated with this consideration, according to the guidelines of certification bodies [40]. Calculation BKM120 cost of detection limit Detection limit was established as the lowest number of cultivable L. pneumophila organisms that can be detected with a probability of 50%. This parameter

so-called LOD50 is estimated using a statistical model (Spearman-Kärber test) but not directly measured [37, 38, 40]. Collaborative trial A collaborative trial involving twelve independent laboratories was performed to evaluate the validity of the IMM by testing identical samples. The collaborative trial was designed and conducted according to internationally accepted guidelines [37, VAV2 41–49]. It has been shown that concentration methods can have highly variable recovery rates, making difficult to obtain identical samples especially for low concentrations of L. pneumophila[50]. Since the objective was the evaluation of the detection part of the IMM, the tested sample simulated the concentrated sample that is habitually obtained in the laboratory from an original sample, thus avoiding the concentration phase. In this collaborative

trial, a microbiological reference material in pill format was used (BaCuanti, Labaqua, Spain). According to the manufacturer´s instructions, water samples were obtained by diluting these pills. The twelve participating laboratories received pills of L. pneumophila at four levels: (i) pills P6 and P8 as negative control, (ii) pills P1 and P3, containing a medium level of L. pneumophila, (iii) pills P2, P5 and P9, containing a high level of L.pneumophila, and (iv) pills P4 and P7, containing a low level of L. pneumophila. To minimize any interlaboratory variability, all the required reagents were purchased from Biótica, Bioquímica Analítica S. L. Each participant received a detailed protocol describing the culture technique, the immunomagnetic run, and a reporting form to record the obtained results. Samples preparation The pills were supplied to the participating laboratories into individual sealed vials.