Cross‐reactivity of the IDEXX Legiolert method with other Gram‐negative bacteria and waterborne pathogens leads to false‐positive assay results

Abstract

Legionella species are the causative agent of Legionnaires’ disease, a potentially fatal bacterial pneumonia. New regulations and standards have prioritized the development of water safety plans to minimize the growth and spread of Legionella species in buildings. To determine the presence and type of Legionella in a water system, microbiological culturing is the gold standard method. However, recently new methodologies have been developed that claim to be sensitive and specific for Legionella at the genus or L. pneumophila at the species level. Published and anecdotal reports suggest that one of these newer culture‐based, enzyme‐substrate methods, the IDEXX Legiolert test, may exhibit false positivity with other microbes common to water sources. We experimentally evaluated the IDEXX Legiolert method using these other waterborne bacteria including Elizabethkingia meningoseptica, Pseudomonas aeruginosa, Proteus mirabilis and Serratia marcescens at real‐world environmental concentrations. We saw false‐positive results for the Legiolert test with several of these organisms, at sample concentrations as low as 60 CFU per ml. False‐positive Legionella results can trigger costly remediation and water‐use restrictions, that may be implemented while waiting for additional, confirmatory microbiological testing that could, in this case, yield no L. pneumophila.

Is flushing necessary during building closures? A study of water quality and bacterial communities during extended reductions in building occupancy

“…The resulting concentration was 10 MPN/L, which is at the limit of detection for the Legiolert test kit (10 cells/L; Legiolert: Detection of Legionella pneumophila -IDEXX US [WWW Document], 2018). Although a false positive rate of 3-4% has previously been reported for this method (Hirsh et al, 2021;Lytle et al, 2021, p. 20;Monteiro et al, 2021), the corresponding detection of mip via qPCR (<LoD; Figure 7) supports that L. pneumophila was present in this sample.…”

Legionella pneumophilaoccurrence in reduced-occupancy buildings in 11 cities during the COVID-19 pandemic

“…The samples that were only positive by Legiolert exhibited low concentrations (median: 32 MPN/L), and all but one (n=6 of 7) were collected from PA-1 and analyzed using ddPCR, indicating that the ddPCR LOD and/or DNA extraction recovery efficiency may have impeded detection. However, false positives using Legiolert have also been reported (3% to 4%), [111][112][113] which could have contributed to this discrepancy. The majority of samples that were positive only by qPCR/ddPCR (n=9 of 11) were quantifiable (above the LLOQ), spanning the full range of L. pneumophila concentrations (10 2 to 10 6 gc/L, Figure 3A), indicating the presence of dead or non-culturable L. pneumophila.…”

Water quality during extended stagnation and flushing in a college residential hall

“…60 Other Gram-negative bacteria, including P. aeruginosa , P. mirabilis , and S. marcescens , can produce false positive results with Legiolert. 61 Disagreements between Legiolert and DNA-based methods have been reported, particularly for samples with low concentrations of L. pneumophila as measured by Legiolert. 8 The lower limit of detection for Legiolert as compared to our mip qPCR assay might explain the discrepancy in the results.…”