Validation of the MYChrOme™ Culture Plate for Detection and Differentiation of Rapid-Growing Nontuberculous Mycobacteria in Potable and Non-Potable Water: AOAC Performance Tested MethodSM 062101.

BACKGROUND: The MYChrOme™ Culture Plate is a chromogenic media for the detection and differentiation of rapid-growing nontuberculous mycobacteria (NTM) in water, aided by MYCOn™ decontamination to reduce background microbiota. OBJECTIVE: Evaluate the MYChrOme Culture Plates for the detection of rapid-growing NTM in potable and non-potable water as part of the AOAC Performance Tested Method(s)SM program. METHODS: Inclusivity and exclusivity of MYChrOme were evaluated with 50 target and 30 non-target organisms. Method robustness and lot stability of MYChrOme were analyzed. The candidate method was compared to a modified US Food and Drug Administration (FDA) Method: U.S. FDA-Isolation and Identification of Nontuberculous Mycobacteria in Tattoo Inks using an equivalency test. The matrix study consisted of artificially contaminated potable water and naturally contaminated non-potable water. Independent laboratory testing was conducted to verify method performance in non-potable water. RESULTS: The inclusivity of MYChrOme was 94% within one week, and 98% within two weeks. The exclusivity was 96% for untreated samples and 100% for treated samples. The candidate method remained statistically equivalent for robustness and a three-month shelf-life was confirmed. For both matrixes, the candidate and reference methods were not equivalent, with more colonies enumerated on the candidate method except for one contamination level of the potable matrix. CONCLUSION: The MYChrOme culture method can successfully detect and differentiate rapid-growing NTM in the matrixes tested, with sensitivity equivalent or higher than the reference method. HIGHLIGHTS: The MYChrOme culture plate offers differentiation of rapid-growing NTM colonies, improved detection in non-potable samples with MYCOn decontamination, and results within 7 days.

Inaccuracies of the ISO 11731 Method for Environmental Validation of Legionella in Building Water Systems: Opportunities to Improve Sensitivity and Detect Viable but Non-Culturable Legionella.

Current environmental diagnostics for the detection of Legionella fail to detect viable but non-culturable Legionella, have sensitivity limitations and are time-consuming (10-14 days to results). The objective of this study was to compare Legionella detection results between the standard ISO 11731 and an innovative Legionella detection method that utilizes a hybrid methodology of traditional microbiology and molecular detection. In this study, four hundred and seventy-six (476) potable building water samples were analyzed with ISO 11731 and the novel method in parallel. Of the 476 total samples that were tested, a discrepancy of 21% was observed when comparing the ISO 11731 method to the novel method. Separating the samples based on hazard control methods yielded a 15.4% discrepancy for chlorinated systems (n = 284) and a 29% discrepancy for monochloraminated systems (n = 192). The data presented here conclusively show inaccuracies in environmental validation for building water systems based on results returned by the standard ISO 11731 method. This is especially evident in systems primarily disinfected with monochloramines. Overall, these data highlight the need for new and innovative methods to overcome the inaccuracies of the traditional ISO 11731 spread plates to prevent disease and injury caused by Legionella.

Next Day Legionella PCR: a highly reliable negative screen for Legionella in the built environment.

The opportunistic, waterborne pathogen Legionella caused 9,933 cases of Legionnaires' disease in 2018 in the United States (CDC.gov). The incidence of Legionnaires' disease can be reduced by maintaining clean building water systems through water management programs (WMPs). WMPs often include validation testing to confirm the control of bacteria, but the traditional culture method for enumerating Legionella requires 10-14 days to obtain results. A rapid DNA extraction developed by Phigenics and a real-time PCR negative screen for the genus Legionella provided results the day after sampling. This study evaluated the Next Day Legionella PCR (Phigenics, LLC) compared with the traditional culture method (ISO 11731) on 11,125 building water samples for approximately 1 year. Two DNA extraction methods (Methods 1 and 2) were compared. The negative predictive value (NPV) of the Next Day Legionella PCR in comparison to traditional culture for Method 1 was 99.95%, 99.92%, 99.85%, and 99.17% at >10, >2, >1, and >0.1 CFU/ml limits of detection, respectively. The improved DNA extraction (Method 2) increased the NPV to 100% and 99.88% at >1 and >0.1 CFU/ml, respectively. These results demonstrate the reliability of the genus-level Legionella PCR negative screen to predict culture-negative water samples.

Water Management for Construction: Evidence for Risk Characterization in Community and Healthcare Settings: A Systematic Review.

Construction activities are a known risk contributing to the growth and spread of waterborne pathogens in building water systems. The purpose of the study is to integrate evidence for categorizing construction activity risk factors contributing to waterborne disease in community and healthcare settings, establish severity of such risk factors and identify knowledge gaps. Using a systematic review, the inclusion criteria were: 1) studies with disease cases suspected to be associated with construction activities and waterborne pathogens, and 2) active construction work described in a community or healthcare setting. Each construction activity risk factor was correlated across all studies with the number of disease cases and deaths to establish risk severity. The eligibility review and quantitative synthesis yielded 31 studies for inclusion (community, n = 7 and healthcare, n = 24). From 1965 to 2016, a total of 894 disease cases inclusive of 112 deaths were associated with nine construction activity risk factors and waterborne pathogens. The present study findings support the need for building owners, water management teams and public health professionals to address construction activity risk factors and the analysis of current knowledge deficiencies within the scope of an ongoing water management program. The impact of construction activities on waterborne disease is preventable and should no longer be considered incidental nor accidental.

Environmental Validation of Legionella Control in a VHA Facility Water System.

OBJECTIVES We conducted this study to determine what sample volume, concentration, and limit of detection (LOD) are adequate for environmental validation of Legionella control. We also sought to determine whether time required to obtain culture results can be reduced compared to spread-plate culture method. We also assessed whether polymerase chain reaction (PCR) and in-field total heterotrophic aerobic bacteria (THAB) counts are reliable indicators of Legionella in water samples from buildings. DESIGN Comparative Legionella screening and diagnostics study for environmental validation of a healthcare building water system. SETTING Veterans Health Administration (VHA) facility water system in central Texas. METHODS We analyzed 50 water samples (26 hot, 24 cold) from 40 sinks and 10 showers using spread-plate cultures (International Standards Organization [ISO] 11731) on samples shipped overnight to the analytical lab. In-field, on-site cultures were obtained using the PVT (Phigenics Validation Test) culture dipslide-format sampler. A PCR assay for genus-level Legionella was performed on every sample. RESULTS No practical differences regardless of sample volume filtered were observed. Larger sample volumes yielded more detections of Legionella. No statistically significant differences at the 1 colony-forming unit (CFU)/mL or 10 CFU/mL LOD were observed. Approximately 75% less time was required when cultures were started in the field. The PCR results provided an early warning, which was confirmed by spread-plate cultures. The THAB results did not correlate with Legionella status. CONCLUSIONS For environmental validation at this facility, we confirmed that (1) 100 mL sample volumes were adequate, (2) 10× concentrations were adequate, (3) 10 CFU/mL LOD was adequate, (4) in-field cultures reliably reduced time to get results by 75%, (5) PCR provided a reliable early warning, and (6) THAB was not predictive of Legionella results. Infect Control Hosp Epidemiol 2018;39:259-266.

HACCP-Based Programs for Preventing Disease and Injury from Premise Plumbing: A Building Consensus.

Thousands of preventable injuries and deaths are annually caused by microbial, chemical and physical hazards from building water systems. Water is processed in buildings before use; this can degrade the quality of the water. Processing steps undertaken on-site in buildings often include conditioning, filtering, storing, heating, cooling, pressure regulation and distribution through fixtures that restrict flow and temperature. Therefore, prevention of disease and injury requires process management. A process management framework for buildings is the hazard analysis and critical control point (HACCP) adaptation of failure mode effects analysis (FMEA). It has been proven effective for building water system management. Validation is proof that hazards have been controlled under operating conditions and may include many kinds of evidence including cultures of building water samples to detect and enumerate potentially pathogenic microorganisms. However, results from culture tests are often inappropriately used because the accuracy and precision are not sufficient to support specifications for control limit or action triggers. A reliable negative screen is based on genus-level Polymerase Chain Reaction (PCR) for Legionella in building water systems; however, building water samples with positive results from this test require further analysis by culture methods.

A comprehensive water management program for multicampus healthcare facilities.

OBJECTIVE: Develop and implement an effective program for hazard analysis and control of waterborne pathogens at a multicampus hospital with clinics. DESIGN: A longitudinal study. Several-year study including analysis of results from monitoring and tests of 26 building water systems. SETTING: Outpatient and inpatient healthcare facilities network. METHODS: The hazard analysis and critical control point (HACCP) process was used to develop a water management program (WMP) for the hospital campuses. The HACCP method systematically addressed 3 questions: (1) What are the potential waterborne hazards in the building water systems of these facilities? (2) How are the hazards being controlled? (3) How do we know that the hazards have been controlled? Microbiological and chemical tests of building water samples were used to validate the performance of the WMP; disease surveillance data further validated effective hazard control. RESULTS: Hazard analysis showed that waterborne pathogens were generally in good control and that the water quality was good in all facilities. The hospital network has had several legionellosis cases that were identified as presumptive hospital acquired, but none was confirmed or substantiated by water testing in follow-up investigations. Building water system studies unrelated to these cases showed that pressure tanks and electronic automatic faucets required additional hazard control. CONCLUSIONS: Application of the HACCP process for long-term building water systems management was practical and effective. The need for critical control point management of temperature, flow, and oxidant (chlorine) residual concentration was emphasized. The process resulted in discovery of water system components requiring additional hazard control.

Inaccuracy in Legionella tests of building water systems due to sample holding time.

Most Legionella culture tests are performed on building water samples that have been shipped to analytical laboratories for analysis. Significant (≥ 1 log10 unit) changes in results were observed in 52% of held samples (6 h or longer, ambient temperature) drawn from building water systems in a 42-sample initial survey. It was not practical to use the spread plate protocol for on-site "t = 0" cultures in a larger, more diverse survey of thousands of building water systems. Two thousand four hundred twenty-one (2421) building water samples were split for on-site analysis using a field culture protocol and then also cultured after overnight shipment to the lab for analysis with the standardized spread plate method. Legionella test results from building water system samples are usually interpreted as ≥ a numerical detection or action limit. Therefore, binary statistical analyses were calculated by setting t = 0 culture results to "true". Overall in this survey, 10.4% of water samples sent to the laboratory for analysis returned either false-positive or false-negative results. The overall positive predictive value of results was poor (36%). Most (83%) false-positive results were returned from utility water systems. Most (74%) false-negative results were returned from potable water systems. These inaccuracies have serious implications in regard to interpretation and use of Legionella test results. The overall negative predictive value of results was excellent (99%) and also it was good (92%) for results from a polymerase chain reaction (PCR) assay that can be therefore used as a negative screening method.