Comparing methods of determining Legionella spp. in complex water matrices

Waterborne diseases are a serious threat because of their ability to infect a high number of individuals in a short time span, such as during outbreaks of Legionellosis. This significantly highlights the need for the rapid detection and quantification of bacteria in environmental water samples. The aim of this study was to investigate the feasibility of quantitative Polymerase Chain Reaction (qPCR) for the detection of Legionellapneumophila (L. pneumophila) in environmental water samples and comparison of standard culture methods for Legionella detection with qPCR. Our study reached a negative predictive value (NPV) for L. pneumophila of 80.7% and for L. pneumophila serogroup 1 (sg1) the calculated NPV was 87.0%. The positive predictive value (PPV) for L. pneumophila was 53.9% and for L. pneumophila sg1 PPV was 21.4%. Results showed a correlation between qPCR and culture with an R2 value of 0.8973 for L. pneumophila, whereas no correlation was observed for the detection of L. pneumophila sg1. In our study, qPCR proved useful for the identification of L. pneumophila negative samples. However, despite the obvious benefits (sample handling, rapid generation of results), qPCR needs to be improved regarding the PPV before it can replace culture in water quality assessment.

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“…Of the 83 tested samples, 24.1% (20) were negative for Legionella spp. investigated with both methods.…”

Section: Pneumophila Detection Via Iso 11731 and Qpcrmentioning

confidence: 98%

Legionella Detection in Environmental Samples as an Example for Successful Implementation of qPCR

Toplitsch¹,

Platzer²,

Pfeifer³

et al. 2018

Water

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“…Since the first recognition of L. pneumophila in 1976, several methods were developed for the detection of this pathogen (Villari et al , 1998; Díaz‐Flores et al , 2015; Mobed et al , 2019). One of the earliest diagnostic tools was based on immunofluorescence labelling (Cherry et al , 1978; Berdal et al , 1979).…”

Section: Introductionmentioning

confidence: 99%

Rapid culture‐based detection of Legionella pneumophila using isothermal microcalorimetry with an improved evaluation method

Fricke

Jiang

et al. 2020

Microbial Biotechnology

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The detection and quantification of Legionella pneumophila (responsible for legionnaire's disease) in water samples can be achieved by various methods. However, the culture-based ISO 11731:2017, which is based on counts of colony-forming units per ml (CFUÁml -1 ) is still the gold standard for quantification of Legionella species (spp.). As a powerful alternative, we propose real-time monitoring of the growth of L. pneumophila using an isothermal microcalorimeter (IMC). Our results demonstrate that, depending on the initial concentration of L. pneumophila, detection times of 24-48 h can be reliably achieved. IMC may, therefore, be used as an early warning system for L. pneumophila contamination. By replacing only visual detection of growth by a thermal sensor, but otherwise maintaining the standardized protocol of the ISO 11731:2017, the new procedure could easily be incorporated into existing standards. The exact determination of the beginning of metabolic heat is often very difficult because at the beginning of the calorimetric signal the thermal stabilization and the metabolic heat development overlap. Here, we propose a new data evaluation based on the first derivation of the heat flow signal. The improved evaluation method can further reduce detection times and significantly increase the reliability of the IMC approach.

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“…When used in conjunction with the culture method, qPCR can serve as a powerful tool. There are, however, several drawbacks: qPCR typically overestimates Lp burden because it detects dead cells 19,20 and the presence of PCR inhibitors may prevent the use of this method. In addition, multiple processing steps are required which increases the overall cost of the qPCR method 21 .…”

Section: Introductionmentioning

confidence: 99%

Identification of two aptamers binding toLegionella pneumophilawith high affinity and specificity

Saad

Chinerman

Tabrizian

et al. 2019

Preprint

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19Legionella pneumophila (Lp) is a water borne bacterium causing Legionnaires' Disease (LD) in 20 humans. Rapid detection of Lp in water system is essential to reduce the risk of LD outbreaks. 21 The methods currently available require expert skills and are time intensive, thus delaying 22 intervention. In situ detection of Lp by biosensor would allow rapid implementation of control 23 strategies. To this end, a biorecognition element is required. Aptamers are considered promising 24 biorecognition molecules for biosensing. Aptamers are short oligonucleotide sequence folding 25 into a specific structure and are able to bind to specific molecules. Currently no aptamer and thus 26 no aptamer-based technology exists for the detection of Lp. In this study, Systemic Evolution of 27 Ligands through EXponential enrichment (SELEX) was used to identify aptamers binding 28 specifically to Lp. Ten rounds of positive selection and two rounds of counter-selection against 29 two Pseudomonas species were performed. Two aptamers binding strongly to Lp were identified 30 with KD of 116 and 135 nM. Binding specificity of these two aptamers to Lp was confirmed by 31 flow cytometry and fluorescence microscopy. Therefore, these two aptamers are promising 32 biorecognition molecules for the detection of Lp in water systems. 33 34 Key words: Legionella pneumophila, aptamers, SELEX, flow cytometry, fluorescence 35 microscopy, Pseudomonas 36 37 38 Legionella pneumophila (Lp) is a pathogenic Gram-negative bacterium responsible for two types 39 of respiratory diseases, namely the severe pneumonia Legionnaires' Disease (LD) and the milder 40 flu-like Pontiac fever 1 . Lp occurs in both natural and engineered water systems and is one of the 41 most prevalent pathogens in man-made, engineered water systems 2 . Infections occur when the 42 bacteria are aerosolized, and the contaminated aerosols are inhaled; Lp can then infect and replicate 43 inside alveolar macrophages 3 . Modern water systems provide optimal transmission conditions for 44 Lp by generating aerosols 4 . Leading sources of infection are cooling towers, hot water distribution 45 systems, humidifiers, misters, showers, fountains, spa pools and evaporative condensers 5 . 46 47 Outbreaks of LD consistently occur globally and have increased in recent years. The average 48 incidence rate is about 10-15 cases per million people 6 . According to the Centre for Disease 49 Control, incidences of legionellosis have increased by four and a half times between 2000 and 50 2016 7 . The rise in LD outbreaks can be attributed to several factors such as aging infrastructures 51and an aging population who is more vulnerable to such infections, as well as increases in diagnosis 52 and reporting 8,9 . Nevertheless, most LD outbreaks are the consequences of management failure of 53 man-made water systems 10 . Examples of these failures for water distribution systems include 54 keeping the temperature of hot water distribution system below 50°C and allowing water to 55 stagnate 10 . For coolin...

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Comparing methods of determining Legionella spp. in complex water matrices

Cited by 32 publications

References 40 publications

Legionella Detection in Environmental Samples as an Example for Successful Implementation of qPCR

Legionella Detection in Environmental Samples as an Example for Successful Implementation of qPCR

Rapid culture‐based detection of Legionella pneumophila using isothermal microcalorimetry with an improved evaluation method

Identification of two aptamers binding toLegionella pneumophilawith high affinity and specificity

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