Overestimation of the Legionella spp. load in environmental samples by quantitative real-time PCR: pretreatment with propidium monoazide as a tool for the assessment of an association between Legionella concentration and sanitary risk

Ditommaso, Savina; Ricciardi, Elisa; Giacomuzzi, Monica; Rivera, Susan R. Arauco; Ceccarelli, Adriano; Zotti, Carla Maria

doi:10.1016/j.diagmicrobio.2014.09.010

Cited by 27 publications

(19 citation statements)

References 30 publications

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“…A similar mean reduction was reported in the studies of Ditommaso et al [7,14,15], but they observed a dissimilarity in the ability of PMA (50 µM) to suppress the PCR signal in samples with different amounts of bacteria: increasing cell amounts resulted in higher reduction values [14]. In our study, a total reduction (3.4 log units) was reached when an initial inoculum concentration of ≤10 3 CFU was used.…”

Section: Resultssupporting

confidence: 90%

“…This trend has been reported in other studies [6,7] and it could be related to the PCR detection method, which also includes the quantification of Legionella doublets or chains as individual cells, while they are counted as only 1 CFU by the culture method. Moreover, a slight reduction of genomic units with qPCR (heat-killed cells) compared to qPCR (viable cells) was observed (mean Δ log 0.52).…”

Section: Resultssupporting

confidence: 78%

“…Quantitative polymerase chain reaction (qPCR) has been proposed as an alternative tool for rapid, sensitive, and specific detection of Legionella in water samples. This method may overcome many disadvantages of traditional culture methods; however, qPCR does not allow viable cells to be distinguished from dead cells [7]. A promising approach for detecting viable cells involves the use of qPCR along with photoactivatable DNA intercalators, either ethidium monoazide (EMA) or propidium monoazide (PMA), which can penetrate the membrane of compromised cells and block PCR amplification [8,9].…”

Section: Introductionmentioning

confidence: 99%

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Viability of Legionella pneumophila in Water Samples: A Comparison of Propidium Monoazide (PMA) Treatment on Membrane Filters and in Liquid

Bonetta¹,

Pignata²,

Bonetta³

et al. 2017

IJERPH

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Legionella pneumophila is a ubiquitous microorganism widely distributed in aquatic environments and can cause Legionellosis in humans. A promising approach to detect viable cells in water samples involves the use of quantitative polymerase chain reaction (qPCR) in combination with photoactivatable DNA intercalator propidium monoazide (PMA). However, the PMA efficiency could be different depending on the experimental conditions used. The aim of this study was to compare two PMA exposure protocols: (A) directly on the membrane filter or (B) in liquid after filter washing. The overall PMA-induced qPCR means reductions in heat-killed L. pneumophila cells were 2.42 and 1.91 log units for exposure protocols A and B, respectively. A comparison between the results obtained reveals that filter exposure allows a higher PMA-qPCR signal reduction to be reached, mainly at low concentrations (p < 0.05). This confirms the potential use of this method to quantify L. pneumophila in water with low contamination.

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Section: Resultssupporting

confidence: 90%

Section: Resultssupporting

confidence: 78%

Section: Introductionmentioning

confidence: 99%

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Viability of Legionella pneumophila in Water Samples: A Comparison of Propidium Monoazide (PMA) Treatment on Membrane Filters and in Liquid

Bonetta¹,

Pignata²,

Bonetta³

et al. 2017

IJERPH

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“…Both reagents contain a photo-inducible azide group that covalently binds to nucleic acids after exposure to light with a specific wavelength which results in a significantly decreased signal in a subsequent qPCR due to the inhibition of the polymerase [13]. The usage of PMA and EMA has been proposed for the selective detection of a broad spectrum of organisms including bacteria [14–18], fungi [19, 20], various protozoa including incorporated bacteria [21–23] and nematode eggs [24]. The application of the method for the distinction between infectious and non-infectious viruses has been investigated thoroughly in lab scale [25–29].…”

Section: Introductionmentioning

confidence: 99%

From Lab to Lake – Evaluation of Current Molecular Methods for the Detection of Infectious Enteric Viruses in Complex Water Matrices in an Urban Area

et al. 2016

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Quantitative PCR methods are commonly used to monitor enteric viruses in the aquatic environment because of their high sensitivity, short reaction times and relatively low operational cost. However, conclusions for public health drawn from results of such molecular techniques are limited due to their inability to determine viral infectivity. Ethidium monoazide (EMA) and propidium monoazide (PMA) are capable to penetrate the damaged or compromised capsid of the inactivated viruses and bind to the viral nucleic acids. We assessed whether dye treatment is a suitable approach to improve the ability of qPCR to distinguish between infectious and non-infectious human adenovirus, enterovirus and rotavirus A in surface water of an urban river and sewage before and after UV disinfection. Like the gold standard of cell culture assays, pretreatment EMA-/PMA-qPCR succeeded in removing false positive results which would lead to an overestimation of the viral load if only qPCR of the environmental samples was considered. A dye pretreatment could therefore provide a rapid and relatively inexpensive tool to improve the efficacy of molecular quantification methods in regards to viral infectivity.

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“…Ditommaso S. et al reported that the L. pneumophilia detection rate was at 67% with realtime PCR compared with 46% with the culture method. Furthermore, 21% of samples tested positive by real time PCR were negative for SCT [16]. In our study, L. pneumophilia was not detected in any of the water samples by the SCT (0%), whereas 4.6% was found to be positive with real-time PCR.…”

Section: Discussion Of Resultsmentioning

confidence: 73%

Comparing Efficiency Between Conventional and Molecular Methods for Detecting Legionella pneumophilia in Dental Unit Waterline Systems

İslam¹,

Güvenir²,

Süer³

et al. 2017

Pol. J. Environ. Stud.

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The aim of our study was to detect the prevalence of Legionella pneumophilia (L. pneumophilia) in DUWL S using standard culture technique (SCT) and the real-time polymerase chain reaction (PCR) method in order to assess the risk of L. pneumophilia contamination within a dental setting. A total of 65 water samples were collected from 16 dental units and one cold water supply system from all clinical departments. L. pneumophilia could not be detected in any of the water samples using the standard SCT (0%), whereas L. pneumophilia was detected using real time PCR in three (4.6%) water samples collected from the tap system. Following the detection of L. pneumophilia, the tap systems were disinfected with surface disinfectant and water samples were recollected. The recollected water samples following disinfection were negative for L. pneumophilia once analyzed using culture and real time PCR technique. Although the culture method using BCYE media is the 'gold standard' for the detection of L. pneumophilia; Real Time PCR analysis may also be a quick, useful, and sensitive method for the detection of L. pneumophilia in order to control and prevent possible infections that may arise in the dental setting.

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Overestimation of the Legionella spp. load in environmental samples by quantitative real-time PCR: pretreatment with propidium monoazide as a tool for the assessment of an association between Legionella concentration and sanitary risk

Cited by 27 publications

References 30 publications

Viability of Legionella pneumophila in Water Samples: A Comparison of Propidium Monoazide (PMA) Treatment on Membrane Filters and in Liquid

Viability of Legionella pneumophila in Water Samples: A Comparison of Propidium Monoazide (PMA) Treatment on Membrane Filters and in Liquid

From Lab to Lake – Evaluation of Current Molecular Methods for the Detection of Infectious Enteric Viruses in Complex Water Matrices in an Urban Area

Comparing Efficiency Between Conventional and Molecular Methods for Detecting Legionella pneumophilia in Dental Unit Waterline Systems

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