Title: Validation of Legionella Viable-PCR Technology Using CDC-ELITE Proficiency Testing
Learner Objectives:
The objective of this presentation is to teach the audience about the concept of Legionella Polymerase Chain Reaction (PCR) testing using viable dye technology to discriminates between the DNA extracted from viable versus nonviable Legionella bacteria; and then to show data from CDC-ELITE proficiency testing samples to convince the audience of the validity of the viable Legionella vPCR technology.
Content/Topic Outline:
Presenter:
Richard D. Miller, Ph.D.
Presentation Description:
The detection of Legionella in building water systems is important in validation of Legionella control as specified in building Water Management Programs for risk management of legionellosis. Culture techniques are the Gold Standard for Legionella analyses because of their detection and quantitation of Colony-Forming Units (CFU), that are closely equivalent to Infectious Bacteria, despite the 7-10 day wait for the results. Polymerase chain reaction technology for detecting Legionella in water samples has been around for over 30 years. Current real-time qPCR has the advantages of quantitation, along with high specificity and speed (same day or next day results), but qPCR has limitations in its broad detection of multiple forms of Legionella DNA in water samples, including live, infectious Legionella, but also non-infectious, dead intact Legionella cells, as well as free Legionella DNA. Viable Legionella PCR techniques (called vPCR) using qPCR in combination with the viable dye, propidium monoazide (PMA) for distinguishing DNA from viable vs non-viable Legionella, have been published in recent years (Scaturro et al. Diagn. Microbiol. Infect. Dis. 85: 283, 2016; and Bonetta et al. Int. J. Environ. Res. Publ. Hlth 14: 467, 2017). In these studies, the vPCR technology results compared favorably to culture and illustrated the over-estimation of Legionella numbers by regular qPCR. Our current study was designed to follow-up on our presentation of Legionella vPCR at AWT 2020, where we compared vPCR to culture in actual building water samples. In the current study, we validated the viable Legionella vPCR technology using CDC-ELITE proficiency samples (two rounds of six samples, with each containing defined concentrations of different Legionella species and serogroups. The Legionella pneumophila vPCR test correctly identified the presence/absence of L. pneumophila serogroup 1 and L. pneumophila serogroups 2-15 correctly in every one of the ELITE samples. The quantitative numbers of L. pneumophila detected by vPCR versus culture (ELITE laboratories average and the CDC estimate), were within the correct orders of magnitude. These results confirm our previous results on actual building water samples; and support the use of vPCR technology for rapid validation of Legionella control measures in building water systems. The continued validation of this technology with CDC-ELITE and other proficiency testing samples is ongoing.
Presenter Bio:
Richard D. Miller, Ph.D. is a founder (1993), President, and Chief Scientific Officer at Environmental Safety Technologies, Inc. (EST); and also an Emeritus Professor of Microbiology & Immunology in the School of Medicine at the University of Louisville (since 1977). He served on the ASHRAE committees that developed the Legionella Guideline 12:2000 document, as well as the initial publication of ANSI/ASHRAE Standard 188-2015, Legionellosis: Risk Management for Building Water Systems. Dr. Miller has over 43 years of experience working with Legionella, through basic research and teaching, as well as through providing environmental testing (validation) and risk assessments for Legionella in cooling towers and potable water systems nationwide. He also has extensive knowledge of other important healthcare-associated waterborne pathogens, their risk for disease, the testing methodologies, results and interpretations. Dr. Miller may be reached at rmiller@estechlab.com.