Evaluation of <i>Legionella pneumophila</i> confirmation methods following IDEXX Legiolert analysis of groundwater

ABSTRACT The IDEXX Legiolert test has been implemented for the quantification of Legionella pneumophila in a variety of potable and non-potable water matrices. For example, through coordination with the state regulatory agency, the Southern Nevada Water Authority (SNWA) has implemented treatment strategies and corresponding L. pneumophila monitoring (via Legiolert) to ensure groundwater quality and public health protection. Because of its specificity to L. pneumophila , confirmation of a positive Legiolert test is often assumed to be unnecessary, although high false positive rates have previously been reported for Legiolert testing of groundwater systems. This study evaluated four confirmation methodologies on liquid media harvested from Legiolert-positive trays: latex agglutination, MALDI-MS, rapid qPCR for the mip and srkA genes, and BCYE plating (±L-cysteine). With the exception of BCYE, these confirmation methods provide results in &lt;24 h, allowing for rapid regulatory and/or public health response. Of 108 presumptive positive analyses of groundwater samples collected in Southern Nevada, only two were ultimately determined to be false positive Legiolert results, although one prompted the shutdown of the corresponding groundwater well. qPCR of the mip gene had the highest L. pneumophila confirmation rate (96%), followed by MALDI-MS (92%), latex agglutination (88%), and BCYE (±L-cysteine) (88%). Although Legiolert alone proved to be effective and relatively accurate in detecting L. pneumophila , confirmation testing yielded valuable supporting information, including serogroup determination and false positive identification ( Stenotrophomonas maltophilia ). Collectively, these data highlight the benefits and drawbacks of various L. pneumophila confirmation methodologies for reporting and decision-making by SNWA and other drinking water utilities. IMPORTANCE In the United States, Legionella pneumophila is the leading cause of drinking water-associated illnesses, hospitalization, and deaths. It is the causative agent of Legionnaires’ disease and the less severe Pontiac Fever. As awareness of L. pneumophila risks increases and monitoring plans are implemented, it is imperative that laboratory analysts, practitioners, and decision-makers understand the limitations of available methods and the value of confirmation in increasing data confidence and informing appropriate actions. Groundwater is commonly used as a drinking water source for public and private systems and generally has less stringent treatment requirements than other sources. Studies have described groundwater as an environmental reservoir for L. pneumophila , but there have also been reports of high false positive rates when monitoring groundwater, particularly when using IDEXX Legiolert. Therefore, the results from this study provide critical knowledge to those monitoring L. pneumophila and using the data for regulatory compliance and/or operational decision-making.