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Dr Richard W Gilpin’s Blog

Accurate VBNC Legionella Testing with mAb-DFA

The CDC and ISO culture methods are predictive when used with potable water samples, but not with cooling tower samples. The BCYE culture method for finding legionella in cooling tower water samples is inappropriate, based on our 36-years of commercial legionella testing experience.

Starved viable but non-culturable (VBNC) Legionella strains can infect and replicate in amoebae and human macrophages. Water Research online February 3, 2018
https://www.sciencedirect.com/science/article/pii/S0043135418300721
Surveillance of water systems is based on microbiological culture-based techniques; however, it has been shown that high percentages of the Legionella populations in water systems are not culturable. If cells remain in the VBNC state, the real number of active legionellae will be underestimated using culture-based standard techniques: VBNC legionellae are infective even after one year of starvation in ultrapure water;  VBNC legionellae can infect human macrophages without prior resuscitation: Longer co-culture times and high MOIs are required for infection by VBNC cells; Only after infecting macrophages did starved VBNC legionellae recover culturability.

Schrammel et al. BMC Microbiology (2018) 18:75  studied specific outer-membrane epitopes of L. pneumophila used in diagnostic applications to determine whether the outer-membrane epitopes are influenced by environmental stress conditions.
https://doi.org/10.1186/s12866-018-1220-x   With ELISA and single cell immuno-fluorescence analysis, Schrammel et al. show that for most of the investigated mAb-strain combinations, the total number of mAb-stained Legionella cells stayed constant for up to 400 days. The results demonstrate the continuous presence of outer membrane epitopes of L. pneumophila during short-term and long-term starvation. Thus, culture-independent mAb-based diagnostic and detection tools are applicable for both L. pneumophila in the culturable and the VBNC state.
This may explain why our mAb-DFA test method is more accurate than culture in some situations.

Our research with cooling tower samples in collaboration with the CDC Special Pathogens Laboratory in the early 1980's found that culture predictability was only 20% when compared to the direct fluorescent antibody (DFA) epifluorescence microscopy method performed by trained technicians. Legionella in cooling tower water plated on BCYE agar media are often overgrown or inhibited by competing microbial flora in the tower water that masks the presence of legionella, particularly with water samples taken from warm water-containing mechanical equipment exposed to the environment such as fountains, cooling towers, and saunas. Culture on BCYE agar with or without supplements, antimicrobial agents, water sample heat or acid treatment lacks the sensitivity of our quantitative DFA test.

Current DFA reagents use monoclonal antibodies specific for legionella cell wall surface antigens, yielding more accurate results than the earlier polyclonal antibody reagents first developed at CDC. We use these monoclonal antibody reagents to test for 14 serogroups of Legionella pneumophila and 15 other Legionella species associated with human disease. Our DFA method is a modification of the original DFA method published by the CDC in 1978 by William B. Cherry. Our DFA method was published in Water Research Vol. 19, No. 7, pp.839-848, 1985, except for a change to more specific monoclonal antibody DFA reagents in the 1990's.

Recent research corroborates our earlier findings:
Building and Environment. Online 13 June 2017. “Ten questions concerning the aerosolization and transmission of Legionella in the built environment.” Aaron J. Prussin II, David Otto Schwake, Linsey C. Marr. https://doi.org/10.1016/j.buildenv.2017.06.024
“Researchers have suggested that direct comparisons between Legionella detection by qPCR and culture are difficult to interpret, and thus it is important to simultaneously determine the total concentration of Legionella bacteria through qPCR (viable and non-viable), culturable Legionella by culturing techniques, and viable but non-culturable (VBNC) Legionella through DFA epifluorescence microscopy.” Quantitative polymerase chain reaction (qPCR) is a good method for potable water samples, but cooling tower water samples contain dirt, metals, and chemicals that interfere with the qPCR test..

“The two most common approaches to enumerating Legionella, quantitative polymerase chain reaction (qPCR) and cultivation, are inherently difficult to compare and can produce either similar or radically different results from identical samples. Concentration data have been collected at length for many environmental sources frequently contaminated with Legionella, although more research designed to assess the aggregated data will be needed to determine truly “typical” concentrations for various sources.”  ‘Early infectivity assays for Legionella demonstrated lethality in guinea pigs that were exposed to approximately 1000–10,000 aerosolized cells, while more recent quantitative microbial risk assessment predicts an approximately 1 in 10,000 risk from one cell deposited in the lungs for humans. These values, however, may not be reflective of real-world conditions and are highly dependent on many factors such as strain/species, cell state, aerosol size (which affects deposition efficiency in the respiratory tract), and host immunity.” “Cooling towers are considered another major source for legionellosis outbreaks; however, to our knowledge there are few studies correlating concentrations of Legionella in cooling tower water with those in air.”