Detection and quantification ofLegionella pneumophilain water samples using competitive PCR

2006 ◽  
Vol 52 (6) ◽  
pp. 584-590 ◽  
Author(s):  
Priscilla Declerck ◽  
Jonas Behets ◽  
Elke Lammertyn ◽  
Ilya Lebeau ◽  
Jozef Anné ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Water Samples ◽  
Cooling Tower ◽  
Tap Water ◽  
Cost Effective ◽  
Culture Method ◽  
Environmental Water ◽  
Competitive Pcr ◽  
Promising Alternative ◽  
Standard Culture

The presence of high levels of Legionella pneumophila in man-made aquatic systems correlates with the incidence of nosocomial Legionnaires' disease. This requires a rapid, reliable, and sensitive quantification of L. pneumophila concentrations in suspected water systems. In this research, a homologous competitor was developed and evaluated in a L. pneumophila competitive polymerase chain reaction (cPCR) to quantify this human pathogen in a quick, cost-effective, and reliable way. Accuracy of cPCR was evaluated by analyzing cooling tower and tap water samples spiked with known concentrations of L. pneumophila bacteria, in parallel with the standard culture method. Legionella pneumophila amounts detected and calculated from cPCR and culture correlated very well: r = 0.998, P = 0.002 for tap water and r = 0.990, P = 0.009 for cooling tower water. Nevertheless, for both kinds of water samples, mean numbers of L. pneumophila calculated from cPCR results were always higher than those obtained by culture. This study makes it clear that the rapid, sensitive, and cost-effective L. pneumophila cPCR is a promising alternative to the standard time-consuming culture method and expensive real-time PCR to enumerate L. pneumophila bacteria in environmental water samples.Key words: Legionella pneumophila, competitive PCR, cost-effective, cooling tower water, tap water, sensitive detection.

scholarly journals Comparison of Updated Methods for Legionella Detection in Environmental Water Samples

2021 ◽  
Vol 18 (10) ◽  
pp. 5436
Author(s):  
Daniela Toplitsch ◽  
Sabine Platzer ◽  
Romana Zehner ◽  
Stephanie Maitz ◽  
Franz Mascher ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Water Samples ◽  
Limit Of Detection ◽  
Culture Method ◽  
Environmental Water ◽  
Microbial Flora ◽  
Culture Methods ◽  
Outbreak Investigations ◽  
Standard Culture ◽  
Selection Of

The difficulty of cultivation of Legionella spp. from water samples remains a strenuous task even for experienced laboratories. The long incubation periods for Legionellae make isolation difficult. In addition, the water samples themselves are often contaminated with accompanying microbial flora, and therefore require complex cultivation methods from diagnostic laboratories. In addition to the recent update of the standard culture method ISO 11731:2017, new strategies such as quantitative PCR (qPCR) are often discussed as alternatives or additions to conventional Legionella culture approaches. In this study, we compared ISO 11731:2017 with qPCR assays targeting Legionella spp., Legionella pneumophila, and Legionella pneumophila serogroup 1. In samples with a high burden of accompanying microbial flora, qPCR shows an excellent negative predictive value for Legionella pneumophila, thus making qPCR an excellent tool for pre-selection of negative samples prior to work-intensive culture methods. This and its low limit of detection make qPCR a diagnostic asset in Legionellosis outbreak investigations, where quick-risk assessments are essential, and are a useful method for monitoring risk sites.

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 Rapid Detection and Enumeration of Legionella pneumophila in Hot Water Systems by Solid-Phase Cytometry

2004 ◽  
Vol 70 (3) ◽  
pp. 1651-1657 ◽  
Author(s):  
Helena Aurell ◽  
Philippe Catala ◽  
Pierre Farge ◽  
France Wallet ◽  
Matthieu Le Brun ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Natural Waters ◽  
Solid Phase ◽  
Tap Water ◽  
Culture Method ◽  
Water Systems ◽  
Cross Reactivity ◽  
Hot Water ◽  
Standard Culture ◽  
Direct Counts

ABSTRACT A new method for the rapid and sensitive detection of Legionella pneumophila in hot water systems has been developed. The method is based on an IF assay combined with detection by solid-phase cytometry. This method allowed the enumeration of L. pneumophila serogroup 1 and L. pneumophila serogroups 2 to 6, 8 to 10, and 12 to 15 in tap water samples within 3 to 4 h. The sensitivity of the method was between 10 and 100 bacteria per liter and was principally limited by the filtration capacity of membranes. The specificity of the antibody was evaluated against 15 non-Legionella strains, and no cross-reactivity was observed. When the method was applied to natural waters, direct counts of L. pneumophila were compared with the number of CFU obtained by the standard culture method. Direct counts were always higher than culturable counts, and the ratio between the two methods ranged from 1.4 to 325. Solid-phase cytometry offers a fast and sensitive alternative to the culture method for L. pneumophila screening in hot water systems.

Studies evaluating the applicability of utilising the same concentration techniques for the detection of protozoan parasites and viruses in water

1995 ◽  
Vol 31 (5-6) ◽  
pp. 417-423 ◽  
Author(s):  
R. Kfir ◽  
C. Hilner ◽  
M. du Preez ◽  
B. Bateman
Keyword(s):  
Water Samples ◽  
Tap Water ◽  
Cost Effective ◽  
Environmental Water ◽  
Similar Efficacy ◽  
Sample Volume ◽  
Water Recovery ◽  
Protozoan Parasites ◽  
Cartridge Filter ◽  
Giardia Muris

In this study concentration techniques regularly used for viral detection, i.e. flat-bed ultrafiltration and Filterite cartridge filtration, were evaluated for their efficacies in the recovery of protozoan parasites from water. Recovery of cysts was studied using tap water seeded with Giardia muris cysts and compared to methods designed for the detection of protozoan parasites. Recovery of cysts utilizing 1.2µm membrane filters was 11.1% (4.5-23%) compared to 11.6% (2.7-25.5%) with ultrafiltration (pore size 46-50 Å, with a molecular cut off of 50 000 daltons). Comparison of these methods for the isolation of Giardia cysts and Cryptosporidium oocysts from environmental water samples also indicated a similar efficacy. The recovery of cysts from 1001 of seeded samples using a Cuno wynd cartridge filter was 12.2% (1.6-46%) compared to 13.4% (5-24.2%) using a Filterite cartridge filter. Although the results indicated similar recovery efficacies for these two methods, use of Filterite resulted in a more consistent recovery rate. This study also indicated that the use of cartridge filters for the processing of large volume water samples (1001) showed a slightly better recovery efficacy than the flat-bed filtration technique which limits sample volume to about 101. This study shows that concentration techniques utilised for the isolation of enteric viruses can also be applied for the detection of protozoan parasites from water. This procedure allows for co-analysis of both viruses and protozoan parasites and provides a more rapid and cost-effective evaluation of water quality.

scholarly journals Assessment of Viability of Legionella Pneumophila in Environmental Samples: On Filter Application of Ethidium Monoazide Bromide

2021 ◽  
Author(s):  
Michela Consonni ◽  
Anna Grassi ◽  
Stefania Scuri ◽  
Maria Gori ◽  
Elisabetta Tanzi ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Water Samples ◽  
Environmental Samples ◽  
Dna Amplification ◽  
Culture Method ◽  
Environmental Water ◽  
Ethidium Monoazide ◽  
Cultivable Bacteria ◽  
Source Of Infection ◽  
Commercial Kit

Abstract Purpose: Culture method, Real-Time PCR (qPCR) and Ethidium Monoazide Bromide (EMA) qPCR have been compared in order to detect Legionella pneumophila (Lp) in water samples, to identify a method able to speed up the procedures, detect the “viable but not cultivable” bacteria (VBNC) and exclude dead bacteria using a commercial kit for extraction and amplification and modifying the protocol.Methods: Using these three methods, 34 environmental water samples and a series of samples artificially spiked with alive, dead and VBNC Lp ATCC 33152 were analysed. ISO 11731-2-2004 culture method was applied, whereas a commercial kit was selected for both qPCR and EMA qPCR pretreatment.Results: only 35% (12/34) of the environmental samples were positive in both culture and qPCR methods. With regard to EMA qPCR, results showed the absence of dye toxicity on viable and VBNC strains and an incomplete effectiveness on the dead ones. In both viable and VBNC strains a decrease of bacterial DNA amplification was recorded as a function of sample dilution but not of EMA concentration.Conclusions: Discrepancies between culture method and EMA-qPCR were observed and could be due to different causes as membrane-dye interactions, presence of interfering compounds and the relatively low sensitivity of the kit used.Significance and Impact of the Study: In presence of one or more suspected cases of nosocomial legionellosis, the application of a rapid molecular method able to identify only the viable and VBNC Lp would be useful in order to quickly identify the source of infection and to intervene with sanitation treatments. However, because in our experience EMA pretreatment on filter membrane has not given the expected results, it would be necessary to proceed with other experiments and different dyes.

Mobile-phone-based colourimetric analysis for determining nitrite content in water

2018 ◽  
Vol 15 (7) ◽  
pp. 403 ◽  
Author(s):  
Chanida Puangpila ◽  
Jaroon Jakmunee ◽  
Somkid Pencharee ◽  
Wipada Pensrisirikul
Keyword(s):  
Mobile Phone ◽  
Water Samples ◽  
Diazonium Salt ◽  
Field Measurements ◽  
Cost Effective ◽  
Environmental Water ◽  
Sulfanilic Acid ◽  
Calibration Graph ◽  
Nitrite Ion ◽  
Griess Reaction

Environmental contextA widespread pollutant in groundwater, rivers and lakes is nitrite, which is commonly determined batchwise by using colourimetry. The batchwise method, however, requires relatively large and expensive instrumentation, and hence is unsuitable for in-field measurements. This work introduces a simple and portable colourimetric analyser based on a mobile-phone camera for monitoring nitrite concentrations in environmental water samples. AbstractA cost-effective and portable colourimetric analyser installed on a mobile phone was used to measure nitrite in water samples in Chiang Mai City, Thailand. The colourimetric detection was based on the Griess reaction, in which nitrite ion reacts with sulfanilic acid under acidic conditions to produce a diazonium salt that further reacts with N-(1-naphthyl)-ethylenediamine dihydrochloride to form a red–violet azo dye. Under controlled conditions using a light-tight box with LED flash lights, images of the red–violet solution were captured using a built-in camera and further analysed by a program, Panalysis, on the mobile phone. The calibration graph was created by measuring the red colour intensity of a series of standard nitrite solutions from 0.09–1.8 mg N L−1. The calibration equation was then automatically stored for nitrite analysis. The results demonstrated good performance of the mobile phone analyser as an analytical instrument. The accuracy (RE <4%) and precision (RSD ≤ 1%, intra- and inter-day) were also obtained with a detection limit of 0.03 mg N L−1 and a sample throughput of 40 samples per hour. Our results establish this simple, inexpensive and portable device as a reliable in-field monitor of nitrite in environmental waters.

scholarly journals Performance of Legiolert Test vs. ISO 11731 to Confirm Legionella pneumophila Contamination in Potable Water Samples

Pathogens ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 690 ◽  
Author(s):  
Maria Scaturro ◽  
Matteo Buffoni ◽  
Antonietta Girolamo ◽  
Sandra Cristino ◽  
Luna Girolamini ◽  
...  
Keyword(s):  
Risk Assessment ◽  
Legionella Pneumophila ◽  
Water Samples ◽  
Gold Standard ◽  
Liquid Culture ◽  
Potable Water ◽  
Culture Method ◽  
Alternative Methods ◽  
Significant Difference

Detection and enumeration of Legionella in water samples is of great importance for risk assessment analysis. The plate culture method is the gold standard, but has received several well-known criticisms, which have induced researchers to develop alternative methods. The purpose of this study was to compare Legionella counts obtained by the analysis of potable water samples through the plate culture method and through the IDEXX liquid culture Legiolert method. Legionella plate culture, according to ISO 11731:1998, was performed using 1 L of water. Legiolert was performed using both the 10 mL and 100 mL Legiolert protocols. Overall, 123 potable water samples were analyzed. Thirty-seven (30%) of them, positive for L. pneumophila, serogroups 1 or 2–14 by plate culture, were used for comparison with the Legiolert results. The Legiolert 10 mL test detected 34 positive samples (27.6%) and the Legiolert 100 mL test detected 37 positive samples, 27.6% and 30% respectively, out of the total samples analyzed. No significant difference was found between either the Legiolert 10 mL and Legiolert 100 mL vs. the plate culture (p = 0.9 and p = 0.3, respectively) or between the Legiolert 10 mL and Legiolert 100 mL tests (p = 0.83). This study confirms the reliability of the IDEXX Legiolert test for Legionella pneumophila detection and enumeration, as already shown in similar studies. Like the plate culture method, the Legiolert assay is also suitable for obtaining isolates for typing purposes, relevant for epidemiological investigations.

On-Site Monitoring of Steroid Hormones in Environmental Waters with a Low-Cost, Integrated Polymer Lab-on-Chip Device

2013 ◽  
Vol 448-453 ◽  
pp. 396-401
Author(s):  
Nuno Miguel Matos Pires ◽  
Tao Dong
Keyword(s):  
Steroid Hormones ◽  
Optical Sensors ◽  
Water Samples ◽  
Low Cost ◽  
Cost Effective ◽  
Environmental Water ◽  
Lab On Chip ◽  
Gold Thin Film ◽  
Site Monitoring ◽  
On Chip

Routine analysis of steroid hormones in environmental water samples demands for cost-effective tools that can detect multiple targets simultaneously. This study reports a high-throughput polymer platform integrated to polymer optical sensors for on-site monitoring of hormones in water. This opto-microfluidic device concept is fully compatible to low-cost fabrication methods. A competitive chemiluminescence immunoassay was performed onto gold thin film coated chambers, and a detection resolution of roughly 0.2 ng/mL was obtained using 17β-estradiol as the model target. Furthermore, the integrated polymer platform showed good recovery for the estradiol target when spiked in surface water samples.

scholarly journals Detection of Vibrio cholerae O1 and O139 in Environmental Water Samples by an Immunofluorescent-Aggregation Assay

2010 ◽  
Vol 76 (16) ◽  
pp. 5520-5525 ◽  
Author(s):  
Duochun Wang ◽  
Xuebin Xu ◽  
Xiaoling Deng ◽  
Changyi Chen ◽  
Baisheng Li ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
High Specificity ◽  
Culture Method ◽  
Environmental Water ◽  
Estuarine Water ◽  
Conventional Culture ◽  
Conventional Culture Method

ABSTRACT Environmental waters are an important reservoir for Vibrio cholerae, and effective surveillance of the pathogen can help to warn of and prevent infection with this potentially fatal pathogen. An immunofluorescent-aggregation (IFAG) assay to detect V. cholerae O1 and O139 was established and evaluated with estuarine water samples. The practical application of this assay was compared with the conventional culture method and real-time PCR. The IFAG method had a sensitivity of 103 CFU/ml for detection of V. cholerae O1 and O139 strains in a suspension containing 10 different species of enterobacterial strains (total, 105 CFU/ml). Ten fluorescent bacterial aggregate colonies were randomly picked and tested positive in serum agglutination tests for the V. cholerae O1 and O139 strains, showing a high specificity. The enrichment broths of 146 samples of estuarine water were tested, and the percentage positive by the IFAG assay was 19.9% (29/146), which was significantly higher than that of the conventional culture method (10.3%, 15/146; P < 0.01) but lower than that of real-time PCR (29.5%, 43/146; P < 0.01). The coincidence rates of real-time PCR and IFAG detection were decreased with the reduction of the V. cholerae concentration. The IFAG method, with a high specificity and a relatively high sensitivity, may be used for detection and isolation of V. cholerae in environmental water samples.

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