Comparison of Updated Methods for Legionella Detection in Environmental Water Samples

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.

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Rapid Method for Enumeration of Viable Legionella pneumophila and Other Legionella spp. in Water

Applied and Environmental Microbiology ◽

10.1128/aem.71.7.4086-4096.2005 ◽

2005 ◽

Vol 71 (7) ◽

pp. 4086-4096 ◽

Cited By ~ 50

Author(s):

Pilar Delgado-Viscogliosi ◽

Tristan Simonart ◽

Virginie Parent ◽

Grégory Marchand ◽

Marie Dobbelaere ◽

...

Keyword(s):

Legionella Pneumophila ◽

Limit Of Detection ◽

Culture Method ◽

Epifluorescence Microscopy ◽

Specific Method ◽

Double Staining ◽

Culture Methods ◽

Experimental Conditions ◽

Short Period ◽

Standard Culture

ABSTRACT A sensitive and specific method has been developed to enumerate viable L. pneumophila and other Legionella spp. in water by epifluorescence microscopy in a short period of time (a few hours). This method allows the quantification of L. pneumophila or other Legionella spp. as well as the discrimination between viable and nonviable Legionella. It simultaneously combines the specific detection of Legionella cells using antibodies and a bacterial viability marker (ChemChrome V6), the enumeration being achieved by epifluorescence microscopy. The performance of this immunological double-staining (IDS) method was investigated in 38 natural filterable water samples from different aquatic sources, and the viable Legionella counts were compared with those obtained by the standard culture method. The recovery rate of the IDS method is similar to, or higher than, that of the conventional culture method. Under our experimental conditions, the limit of detection of the IDS method was <176 Legionella cells per liter. The examination of several samples in duplicates for the presence of L. pneumophila and other Legionella spp. indicated that the IDS method exhibits an excellent intralaboratory reproducibility, better than that of the standard culture method. This immunological approach allows rapid measurements in emergency situations, such as monitoring the efficacy of disinfection shock treatments. Although its field of application is as yet limited to filterable waters, the double-staining method may be an interesting alternative (not equivalent) to the conventional standard culture methods for enumerating viable Legionella when rapid detection is required.

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Detection and quantification ofLegionella pneumophilain water samples using competitive PCR

Canadian Journal of Microbiology ◽

10.1139/w05-156 ◽

2006 ◽

Vol 52 (6) ◽

pp. 584-590 ◽

Cited By ~ 10

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.

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Targeting Species-Specific Low-Affinity 16S rRNA Binding Sites by Using Peptide Nucleic Acids for Detection of Legionellae in Biofilms

Applied and Environmental Microbiology ◽

10.1128/aem.02918-05 ◽

2006 ◽

Vol 72 (8) ◽

pp. 5453-5462 ◽

Cited By ~ 23

Author(s):

Sandra A. Wilks ◽

C. William Keevil

Keyword(s):

16S Rrna ◽

Legionella Pneumophila ◽

Environmental Samples ◽

Culture Method ◽

Dna Probes ◽

Culture Methods ◽

Variable Regions ◽

Standard Culture ◽

Species Specific

ABSTRACT Using fluorescence in situ hybridization to detect bacterial groups has several inherent limitations. DNA probes are generally used, targeting sites on the 16S rRNA. However, much of the 16S rRNA is highly conserved, with variable regions often located in inaccessible areas where secondary structures can restrict probe access. Here, we describe the use of peptide nucleic acid (PNA) probes as a superior alternative to DNA probes, especially when used for environmental samples. A complex bacterial genus (Legionella) was studied, and two probes were designed, one to detect all species and one targeted to Legionella pneumophila. These probes were developed from existing sequences and are targeted to low-binding-affinity sites on the 16S rRNA. In total, 47 strains of Legionella were tested. In all cases, the Legionella spp. PNA probe labeled cells strongly but did not bind to any non-Legionella species. Likewise, the specific L. pneumophila PNA probe labeled only strains of L. pneumophila. By contrast, the equivalent DNA probes performed poorly. To assess the applicability of this method for use on environmental samples, drinking-water biofilms were spiked with a known concentration of L. pneumophila bacteria. Quantifications of the L. pneumophila bacteria were compared using PNA hybridization and standard culture methods. The culture method quantified only 10% of the number of L. pneumophila bacteria found by PNA hybridization. This illustrates the value of this method for use on complex environmental samples, especially where cells may be in a viable but noncultivable state.

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

10.21203/rs.3.rs-590583/v1 ◽

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.

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Viability PCR, a Culture-Independent Method for Rapid and Selective Quantification of Viable Legionella pneumophila Cells in Environmental Water Samples

Applied and Environmental Microbiology ◽

10.1128/aem.02878-08 ◽

2009 ◽

Vol 75 (11) ◽

pp. 3502-3512 ◽

Cited By ~ 100

Author(s):

Pilar Delgado-Viscogliosi ◽

Lydie Solignac ◽

Jean-Marie Delattre

Keyword(s):

Legionella Pneumophila ◽

Water Samples ◽

Environmental Water ◽

Hot Water ◽

Culture Methods ◽

Culture Techniques ◽

Viability Assay ◽

Viable Cells ◽

Wide Range

ABSTRACT PCR-based methods have been developed to rapidly screen for Legionella pneumophila in water as an alternative to time-consuming culture techniques. However, these methods fail to discriminate between live and dead bacteria. Here, we report a viability assay (viability PCR [v-PCR]) for L. pneumophila that combines ethidium monoazide bromide with quantitative real-time PCR (qPCR). The ability of v-PCR to differentiate viable from nonviable L. pneumophila cells was confirmed with permeabilizing agents, toluene, or isopropanol. v-PCR suppressed more than 99.9% of the L. pneumophila PCR signal in nonviable cultures and was able to discriminate viable cells in mixed samples. A wide range of physiological states, from culturable to dead cells, was observed with 64 domestic hot-water samples after simultaneous quantification of L. pneumophila cells by v-PCR, conventional qPCR, and culture methods. v-PCR counts were equal to or higher than those obtained by culture and lower than or equal to conventional qPCR counts. v-PCR was used to successfully monitor in vitro the disinfection efficacy of heating to 70°C and glutaraldehyde and chlorine curative treatments. The v-PCR method appears to be a promising and rapid technique for enumerating L. pneumophila bacteria in water and, in comparison with conventional qPCR techniques used to monitor Legionella, has the advantage of selectively amplifying only viable cells.

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Modern nanobiotechnologies for efficient detection and remediation of mercury

Sensor Review ◽

10.1108/sr-12-2020-0290 ◽

2021 ◽

Vol ahead-of-print (ahead-of-print) ◽

Author(s):

Mulayam Singh Gaur ◽

Rajni Yadav ◽

Mamta Kushwah ◽

Anna Nikolaevna Berlina

Keyword(s):

Heavy Metals ◽

Water Samples ◽

Low Cost ◽

High Sensitivity ◽

Environmental Water ◽

Mercury Ions ◽

Content Type ◽

Efficient Detection ◽

Sensitivity And Selectivity ◽

Selection Of

Purpose This information will be useful in the selection of materials and technology for the detection and removal of mercury ions at a low cost and with high sensitivity and selectivity. The purpose of this study is to provide the useful information for selection of materials and technology to detect and remove the mercury ions from water with high sensitivity and selectivity. The purpose of this study is to provide the useful information for selection of materials and technology to detect and remove the mercury ions from water with high sensitivity and selectivity. Design/methodology/approach Different nano- and bio-materials allowed for the development of a variety of biosensors – colorimetric, chemiluminescent, electrochemical, whole-cell and aptasensors – are described. The materials used for their development also make it possible to use them in removing heavy metals, which are toxic contaminants, from environmental water samples. Findings This review focuses on different technologies, tools and materials for mercury (heavy metals) detection and remediation to environmental samples. Originality/value This review gives up-to-date and systemic information on modern nanotechnology methods for heavy metal detection. Different recognition molecules and nanomaterials have been discussed for remediation to water samples. The present review may provide valuable information to researchers regarding novel mercury ions detection sensors and encourage them for further research/development.

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

Pathogens ◽

10.3390/pathogens9090690 ◽

2020 ◽

Vol 9 (9) ◽

pp. 690 ◽

Cited By ~ 1

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.

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Direct detection of Mycobacterium avium in environmental water and scale samples by loop-mediated isothermal amplification

Journal of Water and Health ◽

10.2166/wh.2013.007 ◽

2013 ◽

Vol 12 (2) ◽

pp. 211-219 ◽

Cited By ~ 4

Author(s):

Yukiko Nishiuchi ◽

Aki Tamaru ◽

Yasuhiko Suzuki ◽

Seigo Kitada ◽

Ryoji Maekura ◽

...

Keyword(s):

Mycobacterium Avium ◽

Environmental Samples ◽

Direct Detection ◽

Culture Method ◽

Environmental Water ◽

Isothermal Amplification ◽

Lamp Method ◽

Culture Methods ◽

Loop Mediated Isothermal Amplification ◽

Conventional Culture

We previously demonstrated the colonization of Mycobacterium avium complex in bathrooms by the conventional culture method. In the present study, we aimed to directly detect M. avium organisms in the environment using loop-mediated isothermal amplification (LAMP), and to demonstrate the efficacy of LAMP by comparing the results with those obtained by culture. Our data showed that LAMP analysis has detection limits of 100 fg DNA/reaction for M. avium. Using an FTA® elute card, DNA templates were extracted from environmental samples from bathrooms in the residences of 29 patients with pulmonary M. avium disease. Of the 162 environmental samples examined, 143 (88%) showed identical results by both methods; 20 (12%) and 123 (76%) samples were positive and negative, respectively, for M. avium. Of the remaining 19 samples (12%), seven (5%) and 12 (7%) samples were positive by the LAMP and culture methods, respectively. All samples that contained over 20 colony forming units/primary isolation plate, as measured by the culture method, were also positive by the LAMP method. Our data demonstrate that the combination of the FTA elute card and LAMP can facilitate prompt detection of M. avium in the environment.

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VIDAS® Enzyme-Linked Immunofluorescent Assay for Detection of Listeria in Foods: Collaborative Study

Journal of AOAC International ◽

10.1093/jaoac/83.4.903 ◽

2000 ◽

Vol 83 (4) ◽

pp. 903-918 ◽

Cited By ~ 15

Author(s):

Vidhya Gangar ◽

Michael S Curiale ◽

Armando D’Onorio ◽

Ann Schultz ◽

Ronald L Johnson ◽

...

Keyword(s):

False Positive Rate ◽

Collaborative Study ◽

False Negative ◽

Traditional Culture ◽

Culture Method ◽

Contamination Level ◽

Culture Methods ◽

Positive Rate ◽

Standard Culture ◽

Lis Method

Abstract The VIDAS LIS method and the traditional culture methods for detection of Listeria species in food were evaluated in a multilaboratory comparative study. The 6 foods tested were either naturally contaminated or inoculated with 3 different concentrations of Listeria. Results for each food and each contamination level with the VIDAS LIS method were as good as or better than those obtained with the traditional culture method. Of 1558 samples tested, 935 were positive: 839 by the VIDAS method and 809 by standard culture methods. Overall false negative rates were 10.3 and 13.5% for the VIDAS LIS and culture methods, respectively. The false positive rate for the VIDAS LIS assay was 1.4% based on 9 VIDAS LIS positive assays that did not confirm positive by isolation of Listeria. The agreement between the VIDAS LIS and culture methods for all samples tested was 86%.

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Quantitative Detection of Legionella pneumophila in Water Samples by Immunomagnetic Purification and Real-Time PCR Amplification of the dotA Gene

Applied and Environmental Microbiology ◽

10.1128/aem.71.7.3433-3441.2005 ◽

2005 ◽

Vol 71 (7) ◽

pp. 3433-3441 ◽

Cited By ~ 79

Author(s):

M. A. Yáñez ◽

C. Carrasco-Serrano ◽

V. M. Barberá ◽

V. Catalán

Keyword(s):

Cell Culture ◽

Real Time ◽

Legionella Pneumophila ◽

Water Samples ◽

Real Time Pcr ◽

Limit Of Detection ◽

Immunomagnetic Separation ◽

Pcr Analysis ◽

Quantitative Detection ◽

Specific Sequence

ABSTRACT A new real-time PCR assay was developed and validated in combination with an immunomagnetic separation system for the quantitative determination of Legionella pneumophila in water samples. Primers that amplify simultaneously an 80-bp fragment of the dotA gene from L. pneumophila and a recombinant fragment including a specific sequence of the gyrB gene from Aeromonas hydrophila, added as an internal positive control, were used. The specificity, limit of detection, limit of quantification, repetitivity, reproducibility, and accuracy of the method were calculated, and the values obtained confirmed the applicability of the method for the quantitative detection of L. pneumophila. Moreover, the efficiency of immunomagnetic separation in the recovery of L. pneumophila from different kinds of water was evaluated. The recovery rates decreased as the water contamination increased (ranging from 59.9% for distilled water to 36% for cooling tower water), and the reproducibility also decreased in parallel to water complexity. The feasibility of the method was evaluated by cell culture and real-time PCR analysis of 60 samples in parallel. All the samples found to be positive by cell culture were also positive by real-time PCR, while only eight samples were found to be positive only by PCR. Finally, the correlation of both methods showed that the number of cells calculated by PCR was 20-fold higher than the culture values. In conclusion, the real-time PCR method combined with immunomagnetic separation provides a sensitive, specific, and accurate method for the rapid quantification of L. pneumophila in water samples. However, the recovery efficiency of immunomagnetic separation should be considered in complex samples.

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