Intensively water testing was made to compare the IMM to the ISO 11731 reference culture method. Tap, cooling tower and natural water samples were collected from different locations and different seasons during three years in the period 2008–2011. A total number of 459 water samples were tested. From these samples, 189 were naturally contaminated samples and 270 were artificially contaminated samples. Distribution of naturally contaminated samples was the following: 84 samples from cooling towers, 94 samples from tap water, 8 samples from water wells and 3 waste water samples. Distribution of artificially contaminated samples was the following: 104 samples from cooling towers, 166 samples from tap water. Both the collection L. pneumophila strain (ATCC 33152) and an environmental isolate of L. pneumophila sg 1 were used as inoculums to prepare artificially contaminated samples. Legionella pneumophila was grown for 3 days on BCYE agar (Buffered Charcoal Yeast Extract) supplemented with glycine, vancomycin, polymixine and cycloheximide (GVPC medium) to obtain exponential-phase cultures. These cultures were used to inoculate water samples. Each sample was tested for the level of background flora by standard plate count of dilutions series of each type of sample. The concentration of Legionella pneumophila ranged from 102 CFU to 107 CFU in the volume examined, between 0.1 L to 1.0 L (usually 1.0 L). Generally, the level of total bacterial counting was below 50 CFU/mL for the tap water samples, and this level was ranging from 102 to 105 CFU/mL for cooling tower water samples, most of them between 103 and 104 CFU/mL. Each of these examined volumes were concentrated by filtration through 0.4-μm-pore-size, 47-mm-diameter polycarbonate sterile membranes (Sartorius, Germany), following the instructions of the International Standard method ISO11731-Part 1. After filtration, each membrane was directly placed in a screw cap sterile container containing 10 mL of the reagent L0 (Biótica, Spain). Then L. pneumophila was eluted by vortex mixing for 2 min. An average of 47% of the seeded L. pneumophila organisms were recovered by filtration. This concentrate represented the prepared sample. The volume of this sample was divided into two portions: 9 mL for IMM test and 1 mL for the culture test. The positivity or negativity of the water samples by the IMM was visually recorded by the colorimetric end-point reaction.
The detection limit was determined considering validation protocols of international certification bodies [37, 38]. Both tap and cooling tower waters were collected and tested negative for the L. pneumophila before its use as matrices. Legionella pneumophila sg 1 (ATCC 33152, Laboratoire BioRéférence, ipl-Groupe, France) was resuspended into 20 mL of a sterile saline solution at room temperature under gently agitation. These 20 mL-suspensions were used to inoculate one liter of selected matrices. Five levels of target contamination were prepared to obtain fractional positive results by the IMM method. Level one corresponded to absence of the target organism, level two corresponded to a proportion of IMM positive results minor than 50%, level three corresponded to a proportion of IMM positive results around the 50%, level four corresponded to a proportion of IMM positive results higher than 50%, and finally level five corresponded to a proportion of IMM positive results of 100%.
These artificially contaminated 1.0 L-samples left to equilibrate for 15–16 hours at 4°C prior starting analysis, to stabilize the inoculated target organism. Each 1.0 L-sample was then divided into ten 100 mL-aliquots as replicates. A total of 66 100 mL-aliquots were examined. Each of these 100 mL-aliquots was concentrated by filtration following the instructions of the International Standard Method ISO11731-Part 1. The volume of each 10 mL-concentrated sample was divided into two portions: 9 mL for IMM test and 1 mL for the culture test. The positivity or negativity of the water samples by the IMM was visually recorded by the colorimetric end-point reaction. The proportion of positive results by the IMM was determined for each batch of ten 100 mL-replicates for each sample.
Reference culture method
For water testing and detection limit study, ISO11731-Part 1 was applied. Water samples were concentrated as described above. Briefly, after filtration of the volume examined, 0.1 mL-portion of the prepared sample was spread on the surface of BCYE agar (Buffered Charcoal Yeast Extract) medium supplemented with glycine, vancomycin, polymixine and cicloheximide (GVPC medium) (bioMérieux, Spain), while a 9 mL-portion of the prepared sample was tested by the IMM. The samples inoculated with high concentrations of L. pneumophila were first diluted with the same water matrix to ensure the count of colony forming units (CFU). The cultures were incubated for 10 days at 37± 1°C in humid atmosphere containing 5% of CO2.
The IMM test (Legipid® Legionella Fast Detection kit, Biótica, Spain), contained different reagents (L0, L1, L2, L3, L4, L5, and L6) and an easy to handle magnetic particle concentrator comprised by a magnet and two glass cuvettes. Unless otherwise stated, aall steps were conducted at room temperature in the magnetic particle concentrator. Nine milliliters portions of each prepared sample for water testing and detection limit studies were transferred to the kit glass cuvette, and 1 mL of L1 reagent containing Legionella pneumophila-binding magnetic beads (LPBM) suspension was added. The mixture was mildly rocked for 15 minutes. LPBM separation was performed by applying a magnet to the cuvette for 5 minutes, and the supernatant was discarded overturning the cuvettes. The LPBM was resuspended/washed with 5 ml of reagent L2 followed by magnetic separation as above. The LPBM were then incubated in 1 ml of reagent L3 for 10 minutes, were captured with the magnet (3 min), was resuspended/washed three times with 5 ml of reagent L2, and were magnetically captured again (3 min). Reagent L4 includes two powder co-substrates (1.3 mg each one) for the colorimetric reaction and it was dissolved in 1.3 mL of reagent L5. The LPBM were resuspended in this 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 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 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 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 . 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). The sensitivity was defined as the ability of IMM to detect the target microorganism compared to the reference culture method, as follows: (a × 100) / (a + b). 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) / (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 .
Calculation 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].
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, 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. 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.
The pills were supplied to the participating laboratories into individual sealed vials. For sample reconstitution, the safety seal was removed under aseptic conditions, the vial was opened, and 20 mL sterile distilled water were added, allowing to mix for 10 min at room temperature, gently shaking every 2 min. Sterile water was added up to a final volume of 100 mL. Then, three serial decimal dilutions (10-1, 10-2, and 10-3) of each sample were prepared.
Reference culture method
Determination of L. pneumophila by culture isolation was conducted in accordance with the ISO 11731-Part 2. Five milliliters of each sample, as well as its corresponding 10-fold serial dilutions were filtered through cellulose ester membranes (11406-47-ACN; Sartorius, Germany). The membranes were placed on the surface of the BCYE-α+GVPC medium (bioMérieux; Spain) and were incubated at 37°C, preferably in a 5% CO2 atmosphere for a period between 5 and 10 days.
Analysis using the IMM test kit was performed in accordance to the protocol described previously. Results were reported as presence/absence in 9 mL, and the aproximate concentrations of L. pneumophila were estimated by intercalation of the end-point colour developed in the analysed sample in the colour chart provided by the manufacturer. Accordingly, samples similar to the negative control one were labelled as <LOD (limit of detection), colour between the negative control and the first colour mark corresponded to 102–103 CFU/9 mL, colour similar to the first colour mark corresponded to 103 CFU/9 mL, colour between first and the second colour mark corresponded to 103–104 CFU/9 mL, colour similar to the second colour mark corresponded to 104 CFU/9 mL, and colour darker than the second colour mark was indicative of >104 CFU/9 mL.
Statistical data analysis
The results reported by eleven of the twelve participating laboratories were evaluated following statistical methods described in the ISO/DIS 13528. One laboratory was rejected due to incorrect application of the trial protocol.