Comparative study of Legiolert with ISO 11731-1998 standard method-conclusions from a Public Health Laboratory.

BACKGROUND: Legionella pneumophila (L. pneumophila) is responsible for 96% of Legionnaires' disease (LD) and 10% of all worldwide pneumonia cases. Legiolert™, a liquid culture method for most probable number (MPN) enumeration of L. pneumophila, was developed by IDEXX Laboratories. The method detects all serogroups of L. pneumophila in potable and non-potable water samples. OBJECTIVE: The goal of this study is to establish that Legiolert is a suitable alternative method to meet testing requirements in Spain for the enumeration of Legionella in water samples. METHODOLOGY: The laboratory analyzed 118 environmental water samples from the Barcelona region (56 potable and 62 non-potable) in parallel by the Standard method for detection and enumeration of Legionella (ISO 11731:1998) and by Legiolert. Comparison of the recovery of the alternative method (Legiolert) and the Standard was made using ISO 17994:2014 and McNemar's binomial test statistical methods. RESULTS: 44 samples were positive for Legionella (36 potable and 8 non-potable). Legiolert and the Standard method detected a similar percentage of positive samples, with Legiolert being slightly higher (31 vs 30%) and detecting higher concentrations of Legionella within the samples. ISO 17994:2014 analysis of the potable water samples found Legiolert was more sensitive than the Standard at detecting Legionella, even when complete Legionella species (L. spp.) results were considered for both methods. The two methods also demonstrated equivalent detection of L. spp. according to the McNemar's test. The comparison is significantly more in favor of Legiolert when only L. pneumophila results are considered. Each confirmation run with material extracted from positive Legiolert wells contained L. pneumophila, giving the method a specificity of 100%. Although statistical results for non-potable waters are not included because of the low number of samples, the two methods trended towards equivalence. CONCLUSIONS: Relative to the Standard method, Legiolert has a greater sensitivity and selectivity, and appears to have higher recovery for L. pneumophila, and equivalent recovery when L. spp. is included in the comparison. Legiolert also has high specificity. The procedural advantages of Legiolert allow laboratories to save on resources, costs, and time and consequently to test more frequently. In conclusion, the study finds IDEXX Legiolert a suitable alternative to ISO 11731:1998.

Comparison of the Legiolert™/Quanti-Tray® MPN test for the enumeration of Legionella pneumophila from potable water samples with the German regulatory requirements methods ISO 11731-2 and ISO 11731.

Due to the promising results of a previous study of the performance of the novel MPN method (Legiolert™/Quanti-Tray®) compared to ISO 11731-2, this study was performed to compare Legiolert for Legionella pneumophila with the German regulatory requirements methods ISO 11731-2 (100 ml membrane filtration) and ISO 11731 (1 ml direct plating) for the enumeration of L. pneumophila and Legionella spp. from potable water. Data from a multi-laboratory study according to ISO 17994 showed that Legiolert yielded on average higher counts of L. pneumophila than the ISO 11731-2 method, but the comparison with ISO 11731 was inconclusive due to the number of samples needing to be tested. Likewise, comparisons of the MPN method for 100 ml to the highest result of either ISO 11731 or ISO 11731-2 according to Federal Environmental Agency recommendation (2012) yielded no conclusive difference, regardless of whether non-pneumophila species of Legionella were included in the evaluation. The MPN method has a high specificity for L. pneumophila of 97.9% which compares favourably to the specificity of 95.3% quoted for ISO 11731. The new method represents a significant improvement in the enumeration of L. pneumophila from drinking water and related samples.

Legionella spp. isolation and quantification from greywater.

Legionella, an opportunistic human pathogen whose natural environment is water, is transmitted to humans through inhalation of contaminated aerosols. Legionella has been isolated from a high diversity of water types. Due its importance as a pathogen, two ISO protocols have been developed for its monitoring. However, these two protocols are not suitable for analyzing Legionella in greywater (GW). GW is domestic wastewater excluding the inputs from toilets and kitchen. It can serve as an alternative water source, mainly for toilet flushing and garden irrigation; both producing aerosols that can cause a risk for Legionella infection. Hence, before reuse, GW has to be treated and its quality needs to be monitored. The difficulty of Legionella isolation from GW strives in the very high load of contaminant bacteria. Here we describe a modification of the ISO protocol 11731:1998 that enables the isolation and quantification of Legionella from GW samples. The following modifications were made:•To enable isolation of Legionella from greywater, a pre-filtration step that removes coarse matter is recommended.•Legionella can be isolated after a combined acid-thermic treatment that eliminates the high load of contaminant bacteria in the sample.