scholarly journals Confirming the Presence of Legionella pneumophila in Your Water System: A Review of Current Legionella Testing Methods

2021 ◽  
Author(s):  
James T Walker ◽  
Paul McDermott
Keyword(s):  
Legionella Pneumophila ◽  
Water System ◽  
Culture Method ◽  
Immunomagnetic Separation ◽  
Most Probable Number ◽  
Water Safety ◽  
Probable Number ◽  
Culture Independent ◽  
Multiple Species ◽  
Risk Managers

Abstract Legionnaires’ disease has been recognized since 1976 and Legionella pneumophila, still accounts for more than 95% of cases. Approaches in countries, including France, suggest that focusing risk reduction specifically on L. pneumophila is an effective strategy, as detecting L. pneumophila has advantages over targeting multiple species of Legionella. In terms of assays, the historically accepted plate culture method takes 10 days for confirmed Legionella spp. results, has variabilities which affect trending and comparisons, requires highly trained personnel to identify colonies on a plate in specialist laboratories and does not recover viable-but-non-culturable (VBNC) bacteria. Polymerase chain reaction is sensitive, specific and provides results in less than 24 h and determines the presence/absence of Legionella spp. and/or L. pneumophila DNA. Whilst specialist personnel and laboratories are generally required, there are now on-site PCR options but there is no agreement on comparing genomic units to colony forming units and action limits. Immunomagnetic separation assays are culture-independent, detect multiple Legionella species and results are available in 24 h, with automated processing options. Field-use lateral flow devices provide presence/absence determination of L. pneumophila serogroup 1 where sufficient cells are present, but testing potable waters is problematic. Liquid culture most probable number (MPN) assays provide confirmed L. pneumophila results in 7 days that are equivalent to or exceed plate culture, are robust and reproducible and can be performed in a variety of laboratory settings. MPN isolates can be obtained for epidemiological investigations. This accessible, non-technical review will be of particular interest to building owners, operators, risk managers and water safety groups to make informed decisions to reduce the risk of L. pneumophila.

Comparison of Legiolert and a Conventional Culture Method for Detection of Legionella pneumophila from Cooling Towers in Québec

2019 ◽  
Vol 102 (4) ◽  
pp. 1235-1240 ◽  
Author(s):  
Isabelle Barrette
Keyword(s):  
Legionella Pneumophila ◽  
Cooling Tower ◽  
Test Procedure ◽  
Agar Plate ◽  
Culture Method ◽  
Most Probable Number ◽  
Cooling Towers ◽  
Plate Method ◽  
Probable Number ◽  
Compliance Testing

Abstract Background: Legionnaires’ disease is a potentially lethal pneumonia contracted through inhalation of aerosolized water contaminated with Legionella bacteria. Detection and control of L. pneumophila, the primary species responsible for the disease, is critical to public health. In Québec, cooling towers and evaporative condensers are required to follow a maintenance and testing program to ensure L. pneumophila concentrations remain at acceptable levels. Objective: This study compared a new culture method based on the most probable number approach, Legiolert®, with the formal culture method used at EnvironeX for regulatory compliance testing to quantify L. pneumophila from cooling tower waters in Québec. Methods: A split-sample analysis was performed in which 401 samples from cooling towers in Québec were tested with both methods. Results: Results with 74 positive samples showed that Legiolert provided a significant increase in sensitivity for L. pneumophila compared with the agar plate method. Cooling tower samples often contain non-Legionella flora that necessitate multiple treatment and plating conditions to prevent interference with the test. Legiolert showed little to no impact from non-Legionella organisms in this study. Conclusions: Overall, Legiolert showed several advantages over the agar plate method, including increased sensitivity, reduced interference, a simplified test procedure, and an easy-to-read positive signal.

scholarly journals Integrated Real-Time PCR for Detection and Monitoring of Legionella pneumophila in Water Systems

2006 ◽  
Vol 73 (5) ◽  
pp. 1452-1456 ◽  
Author(s):  
Diaraf Farba Yaradou ◽  
Sylvie Hallier-Soulier ◽  
Sophie Moreau ◽  
Florence Poty ◽  
Yves Hillion ◽  
...  
Keyword(s):  
Real Time ◽  
Legionella Pneumophila ◽  
Cooling Tower ◽  
Water System ◽  
Culture Method ◽  
Hot Water ◽  
Pcr Assay ◽  
Standard Culture ◽  
Serial Samples ◽  
Over Time

ABSTRACT We evaluated a ready-to-use real-time quantitative Legionella pneumophila PCR assay system by testing 136 hot-water-system samples collected from 55 sites as well as 49 cooling tower samples collected from 20 different sites, in parallel with the standard culture method. The PCR assay was reproducible and suitable for routine quantification of L. pneumophila. An acceptable correlation between PCR and culture results was obtained for sanitary hot-water samples but not for cooling tower samples. We also monitored the same L. pneumophila-contaminated cooling tower for 13 months by analyzing 104 serial samples. The culture and PCR results were extremely variable over time, but the curves were similar. The differences between the PCR and culture results did not change over time and were not affected by regular biocide treatment. This ready-to-use PCR assay for L. pneumophila quantification could permit more timely disinfection of cooling towers.

scholarly journals Evaluation of Culture Methods To Identify Bovine Feces with High Concentrations of Escherichia coli O157

2007 ◽  
Vol 73 (16) ◽  
pp. 5253-5260 ◽  
Author(s):  
J. Trent Fox ◽  
David G. Renter ◽  
Michael W. Sanderson ◽  
Daniel U. Thomson ◽  
Kelly F. Lechtenberg ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Sensitivity And Specificity ◽  
Immunomagnetic Separation ◽  
Most Probable Number ◽  
Culture Methods ◽  
Potassium Tellurite ◽  
Probable Number ◽  
E Coli ◽  
Concentration Sensitivity ◽  
High Concentrations

ABSTRACT Our objective was to evaluate methods for identifying cattle with high concentrations of Escherichia coli O157 in their feces. In two experiments, feces were collected from cattle orally inoculated with nalidixic acid (Nal)-resistant E. coli O157, and direct plating of diluted feces on sorbitol MacConkey agar with cefixime and potassium tellurite (CT-SMAC) containing Nal was considered the gold standard (GS) method. In experiment 1, methods evaluated were preenrichment direct streak, immunomagnetic separation with most probable number (MPN), and postenrichment direct streak with MPN, all using CT-SMAC. The mean concentration of Nal-resistant E. coli O157 in samples (n = 59) by use of the GS was 3.6 log10 CFU/g. The preenrichment streak detected >3.0 log10 CFU/g samples with a 74.4% sensitivity and 68.8% specificity. Postenrichment direct streak-MPN and immunomagnetic separation-MPN concentrations were correlated significantly with GS concentrations (r = 0.53 and r = 0.39, respectively). In experiment 2 (480 samples), pre- and postenrichment direct streaking performed in triplicate and spiral plating on CT-SMAC were evaluated. For preenrichment streaks, sensitivity was 79.7% and specificity was 96.7% for detecting >3.0 log10 CFU/g when the criterion was positive cultures on at least two plates. For spiral plating at that concentration, sensitivity and specificity were 83.9% and 56.3%, respectively. Postenrichment streaking performed relatively poorly. Triplicate preenrichment streaks of 1:10-diluted feces on CT-SMAC may be useful for identifying cattle shedding high concentrations of E. coli O157. Estimates of sensitivity and specificity enable appropriate application of methods and interpretation of results and may enhance applied research, surveillance, and risk assessments.

Validation of the Legionella pneumophila SG1 DETECT Kit for Quantification of Legionella pneumophila Serogroup 1 Bacteria in Potable Waters, Process Waters and Surface Waters: AOAC Performance Tested MethodSM 052002

2020 ◽  
Author(s):  
Hans-Anton Keserue ◽  
Nathalie Cornillie ◽  
Anna-Katharina Ehlert ◽  
Dominic C Mills ◽  
Damien Morger ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Magnetic Particles ◽  
Limit Of Detection ◽  
Industrial Process ◽  
Reference Method ◽  
Immunomagnetic Separation ◽  
Environmental Matrices ◽  
Limit Of Quantitation ◽  
The Matrix ◽  
Culture Independent

Abstract The L.p.SG1 DETECT Kit is a rapid, quantitative method for the detection and enumeration of Legionella pneumophila serogroup 1 (L.p. SG1) bacteria from different water matrixes. The method is based on a combination of immunomagnetic separation (IMS) and flow cytometric (FCM) quantification. To this end, the method employs magnetic particles conjugated to anti-L.p. SG1 antibodies for the IMS of the target bacteria from environmental matrices and fluorescently labelled anti-L.p. SG1 antibodies for subsequent quantification by FCM. The IMS can be performed either manually with a magnetic rack (rqmicro.MIMS) or automated with the rqmicro.STREAM sample preparation instrument. Compared to the reference method ISO 11731:2017, which is based on culturing and enumeration of colony forming units (CFU) on agar plates, and can take up to 10 days until results are available, analysis with the L.p. SG1 DETECT Kit is culture-independent and delivers results within 2 h. This Performance Tested Method validation study demonstrates a robust method with recoveries exceeding 69%, inclusivity of 100%, exclusivity of 97.2% and a shelf life of at least 6 months at 4 °C or 40 days at 25 °C. The Limit of Detection (LoD) was determined at 21 CFU/L and the Limit of Quantitation (LoQ) at 80 CFU/L for potable water using the rqmicro.STREAM. The matrix study across 3 different types of water matrixes (potable, surface and industrial process water), demonstrates superior repeatability and reproducibility, as well as equivalent or even superior detection of L.p. SG1 bacteria compared to the standard ISO 11731 method.

An Investigation of Escherichia coli O157 Contamination of Cattle during Slaughter at an Abattoir

2005 ◽  
Vol 68 (3) ◽  
pp. 451-457 ◽  
Author(s):  
NARELLE FEGAN ◽  
GLEN HIGGS ◽  
PAUL VANDERLINDE ◽  
PATRICIA DESMARCHELIER
Keyword(s):  
Escherichia Coli ◽  
Oral Cavity ◽  
Immunomagnetic Separation ◽  
Most Probable Number ◽  
Escherichia Coli O157 ◽  
Probable Number ◽  
Fecal Samples ◽  
E Coli ◽  
Cell Counts ◽  
Carcass Contamination

The extent of contamination with Escherichia coli O157 was determined for 100 cattle during slaughter. Samples from 25 consecutively slaughtered cattle from four unrelated groups were collected from the oral cavity, hide, rumen, feces after evisceration, and pre- and postchill carcass. Ten random fecal samples were collected from the pen where each group of animals was held at the abattoir. E. coli O157 was detected using automated immunomagnetic separation (AIMS), and cell counts were determined using a combination of most probable number (MPN) and AIMS. E. coli O157 was isolated from 87 (14%) of the 606 samples collected, including 24% of 99 oral cavity samples, 44% of 100 hides, 10% of 68 fecal samples collected postevisceration, 6% of 100 prechill carcass swabs, and 15% of 40 fecal samples collected from holding pens. E. coli O157 was not isolated from rumen or postchill carcass samples. E. coli O157 was isolated from at least one sample from each group of cattle tested, and the prevalence in different groups ranged from less than 1 to 41%. The numbers of E. coli O157 differed among the animals groups. The group which contained the highest fecal (7.5 × 105 MPN/g) and hide (22 MPN/cm2) counts in any individual animal was the only group in which E. coli O157 was isolated from carcasses, suggesting a link between the numbers of E. coli O157 present and the risk of carcass contamination. Processing practices at this abattoir were adequate for minimizing contamination of carcasses, even when animals were heavily contaminated with E. coli O157.

Effect of Trisodium Phosphate on Campylobacter Attached to Post-Chill Chicken Carcasses

1994 ◽  
Vol 57 (4) ◽  
pp. 324-326 ◽  
Author(s):  
MICHAEL F. SLAVIK ◽  
JEONG-WEON KIM ◽  
MICHAEL D. PHARR ◽  
DENNIS P. RABEN ◽  
SONIA TSAI ◽  
...  
Keyword(s):  
Culture Method ◽  
Trisodium Phosphate ◽  
Most Probable Number ◽  
Log P ◽  
Probable Number ◽  
Conventional Culture ◽  
And Control ◽  
Conventional Culture Method

Trisodium phosphate (TSP) was evaluated as a means to reduce Campylobacter on chicken carcasses. Post-chill chicken carcasses were dipped into a 10% TSP solution at 50°C for 15 s. After storing the TSP-treated carcasses for 0, 1 or 6 days at 4°C, the carcasses were subjected to the recovery of Campylobacter. The incidence and reduction of Campylobacter attached to the carcasses were measured using a nitrocellulose (NC) membrane lift, conventional culture method, and a most probable number (MPN) technique. In trials 1 and 2, the incidence of Campylobacter was measured. For 1 day-stored groups, Campylobacter was present on 96 and 100% of control carcasses and present on 24 and 28% of TSP-treated carcasses as measured by NC membrane lift method. The reduction was less (4 to 36%) when measured by culture method. For carcasses immediately subjected for the recovery of cells after treatment, there was no difference between TSP-treated and control carcasses by either NC membrane or culture method. In trial 3, the reduction levels of Campylobacter were quantified by using a MPN method. The levels of Campylobacter on carcasses were decreased by 1.5 and 1.2 logs in 1- and 6-day stored, TSP-treated carcasses, respectively (p < 0.05). However, TSP treatment at 10°C reduced the level of Campylobacter only by 0.16 log (p > 0.10).

Prevalence, Concentrations, and Antibiotic Sensitivities of Salmonella Serovars in Poultry from Retail Establishments in Seattle, Washington

2014 ◽  
Vol 77 (6) ◽  
pp. 885-893 ◽  
Author(s):  
E. MAZENGIA ◽  
M. SAMADPOUR ◽  
H. W. HILL ◽  
K. GREESON ◽  
K. TENNEY ◽  
...  
Keyword(s):  
Significant Proportion ◽  
Performance Standards ◽  
Production Method ◽  
Immunomagnetic Separation ◽  
Product Type ◽  
Most Probable Number ◽  
Probable Number ◽  
Critical Data ◽  
Data Gaps ◽  
Processed Products

Poultry have been identified as one of the major sources of salmonellosis, with estimates ranging from 10 to 22% of total cases. Despite several advances in the industry and new performance standards, the incidence of salmonellosis in the population has not declined over the last 15 years. Salmonella is pervasive in a wide variety of foods, and thus, estimating its burden resulting from specific food categories has been challenging and plagued with uncertainty due to critical data gaps. The objective of this study was to conduct a year-long market survey (1,322 samples) to help bridge the data gaps on the contamination rates and levels of Salmonella on raw poultry by product type (i.e., breast, thighs, drums, wings, and split breast) and production method (conventional versus organic). The isolates recovered were serotyped and tested for antibiotic sensitivities. A PCR method was utilized for initial screening of samples after an overnight enrichment in tryptic soy broth. Three-tube most-probable-number (MPN) assays and anti-Salmonella immunomagnetic separation methods were utilized to determine the levels of Salmonella and aid with the recovery of Salmonella species, respectively. Eleven percent of the samples were positive for Salmonella. Significant differences in percent positive rates by product type included up to a 4-fold difference in percent positive rates between establishments, ranging from 7 to 31%. Of the samples positive for Salmonella species, 94% had <30 MPN/100 g. Production methods identified as organic or as not using antibiotics had significantly higher rates of recovery of Salmonella. On the other hand, all of the Salmonella isolates that were resistant to two or more antibiotics originated from conventional processing establishments where antibiotics were utilized. In addition, a significant proportion of isolates from conventionally processed products were serotypes clinically relevant to humans.

Service Water Life Cycle Management

2008 ◽  
Author(s):  
Eric J. Houston ◽  
Arlene S. Rahn ◽  
George J. Licina
Keyword(s):  
Nuclear Power ◽  
Deterministic Model ◽  
Operating Time ◽  
Water System ◽  
Water Systems ◽  
Most Probable Number ◽  
Probable Number ◽  
System Integrity ◽  
Service Water ◽  
Water Piping

Nuclear plant service water systems are a critical part of the facility’s infrastructure. System integrity and performance are vital for plant reliability and essential to achieving a plant life of 40 years and beyond. Corrosion, fouling (macrofouling, microfouling and sedimentation) and other effects that are detrimental to the reliability of the service water system led to the issue of NRC Generic Letter 89-13 “Service Water System Problems Affecting Safety-Related Equipment.” This generic letter continues to be a fundamental guideline for safety related service water systems at all U.S. nuclear plants. The low temperature and pressure service water piping systems are primarily degraded by corrosion. Because of the complexity and random nature of corrosion processes, it is nearly impossible to develop a mathematically deterministic model that accurately predicts pipe wall loss. However, if statistical distributions are used to describe the various corrosion processes, mathematical algorithms that incorporate all of the distributions, iterated a statistically significant number of times, can be used to forecast the most probable number of leaks. This paper predicts the condition of service water piping at Kewaunee Nuclear Power Plant using the described model and includes the expected number of through-wall leaks as a function of operating time.

scholarly journals Evaluation of a most probable number method for the enumeration of Legionella pneumophila from North American potable and nonpotable water samples

2017 ◽  
Vol 16 (1) ◽  
pp. 25-33 ◽  
Author(s):  
Ray Petrisek ◽  
Jonathon Hall
Keyword(s):  
Legionella Pneumophila ◽  
Water Samples ◽  
Potable Water ◽  
High Specificity ◽  
Most Probable Number ◽  
Water Type ◽  
Probable Number ◽  
Standard Methods ◽  
Water And Wastewater ◽  
Higher Sensitivity

Abstract This study compares the performance of a novel most probable number (MPN) method (Legiolert™/Quanti-Tray®) with Standard Methods for the Examination of Water and Wastewater 9260 J for the enumeration of Legionella pneumophila from potable and nonpotable waters. Data from the study showed that Legiolert exhibited higher sensitivity for the detection of L. pneumophila for potable water and equivalent sensitivity for nonpotable water. The Legiolert medium had a high specificity with no false positive signals reported for either water type. The new method represents a significant improvement in usability and accuracy in the enumeration of L. pneumophila.

Prevalence of Salmonella in Cashews, Hazelnuts, Macadamia Nuts, Pecans, Pine Nuts, and Walnuts in the United States

2017 ◽  
Vol 80 (3) ◽  
pp. 459-466 ◽  
Author(s):  
Guodong Zhang ◽  
Lijun Hu ◽  
David Melka ◽  
Hua Wang ◽  
Anna Laasri ◽  
...  
Keyword(s):  
United States ◽  
Culture Method ◽  
The United States ◽  
Most Probable Number ◽  
Contamination Level ◽  
Probable Number ◽  
Tree Nuts ◽  
Prevalence Estimates ◽  
Macadamia Nuts ◽  
Small Chain

ABSTRACT Nuts have been identified as a vector for salmonellosis. The objective of this project was to estimate the prevalence and contamination level of Salmonella in raw tree nuts (cashews, pecans, hazelnuts, macadamia nuts, pine nuts, and walnuts) at retail markets in the United States. A total of 3,656 samples of six types of tree nuts were collected from different types of retail stores and markets nationwide between October 2014 and October 2015. These samples were analyzed using a modified version of the Salmonella culture method from the U.S. Food and Drug Administration's Bacteriological Analytical Manual. Of the 3,656 samples collected and tested, 32 were culturally confirmed as containing Salmonella. These isolates represented 25 serotypes. Salmonella was not detected in pecans and in-shell hazelnuts. Salmonella prevalence estimates (and 95% confidence intervals) in cashews, shelled hazelnuts, pine nuts, walnuts, and macadamia nuts were 0.55% [0.15, 1.40], 0.35% [0.04, 1.20], 0.48% [0.10, 1.40], 1.20% [0.53, 2.40], and 4.20% [2.40, 6.90], respectively. The rates of Salmonella isolation from major or big chain supermarkets, small chain supermarkets, discount, variety, or drug stores, and online were 0.64% [0.38, 1.00], 1.60% [0.80, 2.90], 0.00% [0.00, 2.40], and 13.64% [2.90, 35.00], respectively (Cochran-Mantel-Haenszel test: P = 0.02). The rates of Salmonella isolation for conventional and organic nuts were not significantly different. Of the samples containing Salmonella, 60.7% had levels less than 0.003 most probable number (MPN)/g. The highest contamination level observed was 0.092 MPN/g. The prevalence and levels of Salmonella in these tree nut samples were comparable to those previously reported for similar foods.

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