Detection Of Legionella Research Articles

Detection of Legionella pneumophila in a Biological Treatment Plant by Co- Cultivation with Acanthamoeba castellanii

Legionella pneumophila was identified in the aeration ponds of a biological wastewater treatment plant at the pulp and paper industry Borregaard, Sarpsborg, Norway. After 3 outbreaks of Legionaires’ disease reported in this area in 2005 and 2008, the aeration ponds were shut down by the Norwegian authorities in September 2008. During the shutdown of these ponds, September to December 2008, the viable counts of L. pneumophila decreased from 107 to < 10 CFU/mL measured using the International Standard growth (ISO11731) method. The aim of this work was to use amoebal coculture with Achantamoebae castellanii to recover and detect L. pneumophila from the complex microbial community in the pond during the shutdown period. This work shows that the viable counts of the environmental L. pneumophila ST 462 outbreak strain present in the pond samples during shutdown, was increased from 0-10 CFU/mL (no amoebae added) to 107 -108 CFU/mL in co-culture with A. castellanii. This indicates that pathogenic L. pneumophila isolates present in the environment may not be detected using standard culture methods. As a consequence, methodological improvements are needed to ensure more reliable detection and isolation of Legionella. By using amoebal co-culture, the concentration of L. pneumophila increased by 5-7 log units, allowing low concentrations and bacteria not detected using standard growth methods (according to the ISO11731), to be detected. Cells in the viable but non-culturable (VBNC) form will not be detected using the ISO 11731 standard culture method, and growth on agar media may be inhibited by other organisms and inhibitors present in complex environmental samples. The methodological procedure described in this paper may assist in providing a general more robust and sensitive approach to detect L. pneumophila in more complex environmental samples and may assist in providing improved hazard assessments.

Seasonal variation of Legionella in Taiwan's reservoir and its relationships with environmental factors.

In this study, the presence of Legionella in major water reservoirs of Taiwan was examined with respect to seasonal variation, geographical variation, and water quality parameters using TaqMan real-time qPCR. Water samples were collected quarterly at 19 reservoirs in Taiwan between November 2012 and August 2013. The detection rate for Legionella was 35.5% (27/76), and Legionella was detected in all seasons. The Legionella concentration was relatively high in spring and summer, reaching 3.86 × 10(8) and 7.35 × 10(8) cells/L, respectively. By sampling the area, Legionella was detected at a higher proportion in reservoirs in the northern and southern areas, and the difference was consistent in all seasons. Significant association was found between detection of Legionella and various water quality parameters, including conductivity, chlorophyll a, and dissolved oxygen (Mann-Whitney U test, P < 0.05). Results of Spearman rank test showed negative correlation for Legionella detection with pH (P = 0.030, R = -0.497) and dissolved oxygen (P = 0.007, R = -0.596) in fall and positive correlation with Carlson's trophic state index (P = 0.049, R = 0.457) in spring. The identified species included Legionella pneumophila and Legionella drancourtii. The detection of Legionella in reservoirs was indicative of a potential public health risk and should be further evaluated.

Vectorial role of Acanthamoeba in Legionella propagation in water for human use

Legionella spp. is the causative agent of Legionnaires' disease and is transmitted through aerosols emanating from man-made water systems. Legionella resistance to water treatments has been related to its association with environmental amoebae such as Acanthamoeba. Due to the high presence of this protozoon in Spain and the high rate of notification of Legionnaires' disease of this country, the aims of this work were to study the coexistence of these bacteria and protozoa in water as well as their interaction. The usefulness of Acanthamoeba co-culture for the isolation of environmental Legionella was also studied. For this purpose, 70 water samples were collected in 2011 from three Drinking Water Treatment Plants, three Wastewater Treatment Plants and five Natural Pools in Spain. Acanthamoeba was found by PCR in 87.1% (61/70) samples and, by culture in 85.7% (60/70) samples. Legionella was detected by PCR in 58.6% (41/70) of water samples, in 5.7% (4/70) by agar culture and 75.7% (53/70) by Acanthamoeba co-culture. From the 54 Acanthamoeba water isolates, Legionella was detected in 43 of them independently of Acanthamoeba's genotype (T3, T4 and T11). Legionella feeleii, Legionella birminghamiensis, Legionella gresilensis/berliardensis, Legionella fairfieldensis, Legionella drozanski and Legionella falloni were identified. In conclusion, our results showed that environmental Acanthamoeba is infected by Legionella to a high percentage, and due to its ubiquity, high resistance and its pathogenic potential per se, new methods for its elimination should be studied. Also, the high effectivity of Acanthamoeba co-culture for Legionella detection has been shown.

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

Quantitative polymerase chain reaction (qPCR) offers rapid, sensitive, and specific detection of Legionella in environmental water samples. In this study, qPCR and qPCR combined with propidium monoazide (PMA-qPCR) were both applied to hot-water system samples and compared to traditional culture techniques. In addition, we evaluated the ability of PMA-qPCR to monitor the efficacy of different disinfection strategies. Comparison between the quantification obtained by culture and by qPCR or PMA-qPCR on environmental water samples confirms that the concentration of Legionella estimated by GU/L is generally higher than that estimated in CFU/L. Our results on 57 hot-water-system samples collected from 3 different sites show that: i) qPCR results were on average 178-fold higher than the culture results (Δ log10=2.25), ii) PMA-qPCR results were on average 27-fold higher than the culture results (Δ log10=1.43), iii) propidium monoazide–induced signal reduction in qPCR were nearly 10-fold (Δ log10=0.95), and that iv) different degrees of correlations between the 3 methods might be explained by different matrix properties, but also by different disinfection methods affecting cultivability of Legionella. In our study, we calculated the logarithmic differences between the results obtained by PMA-qPCR and those obtained by culture, and we suggested an algorithm for the interpretation of PMA-qPCR results for the routine monitoring of healthcare water systems using a commercial qPCR system (iQ-check real-time PCR kit; Bio-Rad, Marnes-la-Coquette, France).

Legionella in water samples: How can you interpret the results obtained by quantitative PCR?

Evaluation of the potential risk associated with Legionella has traditionally been determined from culture-based methods. Quantitative polymerase chain reaction (qPCR) is an alternative tool that offers rapid, sensitive and specific detection of Legionella in environmental water samples.In this study we compare the results obtained by conventional qPCR (iQ-Check™ Quanti Legionella spp.; Bio-Rad) and by culture method on artificial samples prepared in Page's saline by addiction of Legionella pneumophila serogroup 1 (ATCC 33152) and we analyse the selective quantification of viable Legionella cells by the qPCR-PMA method.The amount of Legionella DNA (GU) determined by qPCR was 28-fold higher than the load detected by culture (CFU).Applying the qPCR combined with PMA treatment we obtained a reduction of 98.5% of the qPCR signal from dead cells. We observed a dissimilarity in the ability of PMA to suppress the PCR signal in samples with different amounts of bacteria: the effective elimination of detection signals by PMA depended on the concentration of GU and increasing amounts of cells resulted in higher values of reduction.Using the results from this study we created an algorithm to facilitate the interpretation of viable cell level estimation with qPCR-PMA.

Method modification of the Legipid® Legionella fast detection test kit.

Legipid(®) Legionella Fast Detection is a test based on combined magnetic immunocapture and enzyme-immunoassay (CEIA) for the detection of Legionella in water. The test is based on the use of anti-Legionella antibodies immobilized on magnetic microspheres. Target microorganism is preconcentrated by filtration. Immunomagnetic analysis is applied on these preconcentrated water samples in a final test portion of 9 mL. The test kit was certified by the AOAC Research Institute as Performance Tested Method(SM) (PTM) No. 111101 in a PTM validation which certifies the performance claims of the test method in comparison to the ISO reference method 11731-1998 and the revision 11731-2004 "Water Quality: Detection and Enumeration of Legionella pneumophila" in potable water, industrial water, and waste water. The modification of this test kit has been approved. The modification includes increasing the target analyte from L. pneumophila to Legionella species and adding an optical reader to the test method. In this study, 71 strains of Legionella spp. other than L. pneumophila were tested to determine its reactivity with the kit based on CEIA. All the strains of Legionella spp. tested by the CEIA test were confirmed positive by reference standard method ISO 11731. This test (PTM 111101) has been modified to include a final optical reading. A methods comparison study was conducted to demonstrate the equivalence of this modification to the reference culture method. Two water matrixes were analyzed. Results show no statistically detectable difference between the test method and the reference culture method for the enumeration of Legionella spp. The relative level of detection was 93 CFU/volume examined (LOD50). For optical reading, the LOD was 40 CFU/volume examined and the LOQ was 60 CFU/volume examined. Results showed that the test Legipid Legionella Fast Detection is equivalent to the reference culture method for the enumeration of Legionella spp.

Detection of Legionella, L. pneumophila and Mycobacterium avium complex (MAC) along potable water distribution pipelines.

Inhalation of potable water presents a potential route of exposure to opportunistic pathogens and hence warrants significant public health concern. This study used qPCR to detect opportunistic pathogens Legionella spp., L. pneumophila and MAC at multiple points along two potable water distribution pipelines. One used chlorine disinfection and the other chloramine disinfection. Samples were collected four times over the year to provide seasonal variation and the chlorine or chloramine residual was measured during collection. Legionella spp., L. pneumophila and MAC were detected in both distribution systems throughout the year and were all detected at a maximum concentration of 103 copies/mL in the chlorine disinfected system and 106, 103 and 104 copies/mL respectively in the chloramine disinfected system. The concentrations of these opportunistic pathogens were primarily controlled throughout the distribution network through the maintenance of disinfection residuals. At a dead-end and when the disinfection residual was not maintained significant (p < 0.05) increases in concentration were observed when compared to the concentration measured closest to the processing plant in the same pipeline and sampling period. Total coliforms were not present in any water sample collected. This study demonstrates the ability of Legionella spp., L. pneumophila and MAC to survive the potable water disinfection process and highlights the need for greater measures to control these organisms along the distribution pipeline and at point of use.

Detection of Legionella by cultivation and quantitative real-time polymerase chain reaction in biological waste water treatment plants in Norway.

Cases of Legionnaires' disease associated with biological treatment plants (BTPs) have been reported in six countries between 1997 and 2010. However, knowledge about the occurrence of Legionella in BTPs is scarce. Hence, we undertook a qualitative and quantitative screening for Legionella in BTPs treating waste water from municipalities and industries in Norway, to assess the transmission potential of Legionella from these installations. Thirty-three plants from different industries were sampled four times within 1 year. By cultivation, 21 (16%) of 130 analyses were positive for Legionella species and 12 (9%) of 130 analyses were positive for Legionella pneumophila. By quantitative real-time polymerase chain reaction (PCR), 433 (99%) of 437 analyses were positive for Legionella species and 218 (46%) of 470 analyses were positive for L. pneumophila. This survey indicates that PCR could be the preferable method for detection of Legionella in samples from BTPs. Sequence types of L. pneumophila associated with outbreaks in Norway were not identified from the BTPs. We showed that a waste water treatment plant with an aeration basin can produce high concentrations of Legionella. Therefore, these plants should be considered as a possible source of community-acquired Legionella infections.

Uncertainties associated with assessing the public health risk from Legionella.

Legionella is an opportunistic pathogen of public health concern. Current regulatory and management guidelines for the control of this organism are informed by risk assessments. However, there are many unanswered questions and uncertainties regarding Legionella epidemiology, strain infectivity, infectious dose, and detection methods. This review follows the EnHealth Risk Assessment Framework, to examine the current information available regarding Legionella risk and discuss the uncertainties and assumptions. This review can be used as a tool for understanding the uncertainties associated with Legionella risk assessment. It also serves to highlight the areas of Legionella research that require future focus. Improvement of these uncertainties will provide information to enhance risk management practices for Legionella, potentially improving public health protection and reducing the economic costs by streamlining current management practices.

A case of nosocomial Legionella pneumonia associated with a contaminated hospital cooling tower

We report the epidemiological investigation of a nosocomial pneumonia case due to Legionella pneumophila linked to a contaminated hospital cooling tower in an immune-compromised patient. A 73-year-old female patient was diagnosed with nosocomial Legionella pneumonia proven by a culture of L.pneumophila serogroup 1 from bronchoalveolar lavage fluid. Two strains isolated from the patient and two strains isolated from two cooling towers were found to be identical using repetitive-sequence-based-PCR with a 95% probability. This Legionella pneumonia case might be caused by aerosol from cooling towers on the roof of the hospital building which was contaminated by L.pneumophila. We increased up the temperature of hot water supply appropriately for prevention of Legionella breeding in an environment of patients' living. On the other hand, as the maintenance of cooling tower, we increased the frequency of Legionella culture tests from twice a year to three times a year. In addition, we introduced an automated disinfectants insertion machine and added one antiseptic reagent (BALSTER ST-40 N, Tohzai Chemical Industry Co., Ltd., Kawasaki, Japan) after this Legionella disease, and thereafter, we have no additional cases of Legionella disease or detection of Legionella spp. from the cooling tower or hot water supply. This case demonstrates the importance of detecting the infection source and carrying out environmental maintenance in cooperation with the infection control team.

Detection of Legionella spp. and Legionella pneumophila in water samples of Spain by specific real-time PCR

Legionella pneumophila is the primary cause of the legionellosis diseases (90%) (Yu et al. in J Infect Dis 186:127-128, 2002; Doleans et al. in J Clin Microbiol 42:458-460, 2004; Den Boer et al. in Clin Microbiol Infect 14:459-466, 2008). In this study, methodologies based on molecular biology were developed in order to provide a quick diagnosis of the bacterial presence in water samples of Spain. Multiplex real-time polymerase chain reaction assays were realized to target the 16S rRNA and macrophage infectivity potentiator (mip) genes of, respectively, Legionella spp. and L. pneumophila including in the design of an internal control. The results obtained by the culture and the gene amplification methods agreed in 94.44% for the 16S rRNA gene, and a concordance of 66.67% of the cases was obtained for the mip gene.

Evaluation of Legionella presence in the water system of a public building by semi-nested polymerase chain reaction as a rapid screening method complementary to plate count

The aim of this study was to apply a fast and efficient protocol for monitoring levels of Legionella contamination in high-risk water systems using molecular techniques and culture methods. Forty-nine water samples from a public building were analyzed by a culture method (BCYE agar) and a specific semi-nested polymerase chain reaction (PCR). The first 30 analyses using plate counts were positive in 28% of hot water and 8% of cold water samples. The analysis by PCR, obtained within 24 h of collection, revealed the presence of DNA in 100 and 54% respectively. Only four PCR results coincided with the cultures. A second survey (19 samples) was performed 1 year later. Semi-nested PCR revealed that 53% of the samples were positive; however, plate counts yielded no positive results. The 16S rRNA sequence comparison between the first and second samplings showed 100% homology. In conclusion, the study of the design of the building's water system, the use of a fast screening semi-nested PCR and a culture method for the detection of Legionella allowed accurate assessment of the contamination, thus contributing to the early implementation of measures to eliminate the presence of the bacteria in water systems and consequently reduce a latent public health risk.

Diagnostic, therapeutic and economic consequences of a positive urinary antigen test for Legionella spp. in patients admitted with community-acquired pneumonia: a 7-year retrospective evaluation

AimsA positive urinary antigen test for Legionella spp. (Legionella urinary antigen test; LUAT) allows an early switch from empiric to targeted treatment (TT) in hospitalised, community-acquired pneumonia (CAP) patients. We...

Seasonal distribution of Legionella spp. and L. pneumophila in a river in Taiwan evaluated with culture-confirmed and direct DNA extraction methods

Summary In this study, we evaluated the presence and amount of Legionella in along a river in Taiwan, and the relations between seasonal distribution of Legionella spp. and geographic characteristics in the watershed were also evaluated. Water samples were pre-treated and analyzed with culture-confirmed and direct DNA extraction methods. For culture-confirmed method, water samples were cultivated through a series of selective media, and candidate colonies were confirmed by PCR. For direct DNA extraction method, direct DNA extraction was performed from pre-treated water samples. The DNA extracts were analyzed with PCR and DNA sequence analysis for species determination, quantitative PCR (qPCR) was performed to quantify Legionella concentration in the water sample. In all, 150 water samples were included in this study, with 73 (48.6%) water samples detected with Legionella spp., and 17 with L. pneumophila . Over 80% Legionella spp. detections were through direct DNA extraction method, but more than 80% L. pneumophila detections were through culture-confirmed method. While detection of Legionella spp. was done with two methods, positive results were found through only one method. Legionella spp. was detected in all seasons with detection rate ranging between 34.3–58.8% and seasonal average concentration from 1.9 × 10 2 to 7.1 × 10 3 CFU/L. Most of the L. pneumophila detections were from samples collected in fall (38.2%) and summer (6.0%), which also coincided with increased cases of Legionellosis reported through Center of Disease Control in Taiwan. The high prevalence and concentration of Legionella spp. and L. pneumophila in the surface waters should be further evaluated for potential health risks.

Identification and quantitative detection of Legionella spp. in various aquatic environments by real-time PCR assay

In this study, a SYBR green quantitative real-time PCR was developed to quantify and detect the Legionella spp. in various environmental water samples. The water samples were taken from watershed, water treatment plant, and thermal spring area in Taiwan. Legionella was detected in 13.6% (24/176), and the detection rate for river water, raw drinking water, and thermal spring water was 10, 21.4, and 16.6%, respectively. Using real-time PCR, concentration of Legionella spp. in detected samples ranged between 9.75 × 10(4) and 3.47 × 10(5) cells/L in river water, 6.92 × 10(4) and 4.29 × 10(5) cells/L in raw drinking water, and 5.71 × 10(4) and 2.12 × 10(6) cells/L for thermal spring water samples. The identified species included Legionella pneumophila (20.8%), Legionella jordanis (4.2%), Legionella nautarum (4.2%), Legionella sp. (4.2%), and uncultured Legionella sp. (66.6%). The presence of L. pneumophila in aquatic environments suggested a potential public health threat that must be further examined.

SPR based immunosensor for detection of Legionella pneumophila in water samples

Detection of legionellae by water sampling is an important factor in epidemiological investigations of Legionnaires' disease and its prevention. To avoid labor-intensive problems with conventional methods, an alternative, highly sensitive and simple method is proposed for detecting L. pneumophila in aqueous samples. A compact Surface Plasmon Resonance (SPR) instrumentation prototype, provided with proper microfluidics tools, is built. The developed immunosensor is capable of dynamically following the binding between antigens and the corresponding antibody molecules immobilized on the SPR sensor surface. A proper immobilization strategy is used in this work that makes use of an important efficient step aimed at the orientation of antibodies onto the sensor surface. The feasibility of the integration of SPR-based biosensing setups with microfluidic technologies, resulting in a low-cost and portable biosensor is demonstrated.

Susceptibility of &lt;i&gt;Legionella&lt;/i&gt; Strains to the Chlorinated Biocide, Monochloramine

Members of the Legionella genus find suitable conditions for their growth and survival in nuclear power plant cooling circuits. To limit the proliferation of Legionella pathogenic bacteria in nuclear power plant cooling circuits, and ensure that levels remain below regulatory thresholds, monochloramine treatment can be used. Although the treatment is highly effective, i.e. it reduces Legionella numbers by over 99%, Legionella bacteria can still be detected at low concentrations and rapid re-colonisation of circuits can occur after the treatment has ceased. The aim of this study was to develop an in vitro methodology for determining the intrinsic susceptibility of L. pneumophila strains, collected from various nuclear power plant cooling circuits subjected to different treatment conditions. The methodology was developed by using an original approach based on response surface methodology (RSM) combined with a multifactorial experimental design. The susceptibility was evaluated by the Ct factor. The susceptibility of environmental strains varies widely and is, for some strains, greater than that of known tolerant species; however, strain susceptibility was not related to treatment conditions. Selection pressure induced by monochloramine use did not result in the selection of more tolerant Legionella strains and did not explain the detection of Legionella during treatment or the rapid re-colonisation of cooling circuits after disinfection has ceased.

Quality of air and water in dental healthcare settings during professional toothcleaning

Introduction. Professional toothcleaning with ultrasonic scaler produces microbial aerosols. These microorganisms come from dental unit waterlines (DUWLs) -thus potentially including opportunistic pathogens, or from patients -thus potentially including human pathogens. Aim. To investigate the association between levels and quality of contamination of air samples and DUWLs during professional toothcleaning, thus providing information regarding the nature of air contamination produced by ultrasonic scaler use. Material and methods. Before treating the first patient of the day, 100 mL of water was aseptically collected from the DUWL designated for the ultrasonic scaler, water was not disinfected or flushed. Aliquots were plated on Plate Count Agar to determine total viable flora (TVF) and Charcoal-Yeast Extract Agar supplemented with α Growth Supplement to determine Legionella. Two sets of settle plates were placed on the tray in front of the patient, one before and another during patient treatment to determine TVF and Legionella. The association between TVF and Legionella levels and prevalence in DUWLs and in air samples was assessed using correlation coefficients. Results. 82 testing occasions were performed. The mean TVF levels in DUWLs and air were 21.2 (95% confidence interval, 95CI, 13.8-32.6) CFU/mL and 12.4 (95CI, 9.7-15.8) CFU/plate/h, respectively. The mean Legionella detection rates were 1.2% (DUWLs) and 0% (air). Correlations between air and water TVF and Legionella were not significant. Conclusion. Air contamination during ultrasonic scaler use was frequent and high, but it was not associated with DUWL contamination, suggesting that airborne microorganisms could come from patients and be potentially pathogens for humans.

Predictors of Legionella occurrence in dental unit waterlines of a highly colonized dental hospital

Introduction. Legionella is frequently detected in Dental Unit Waterlines (DUWLs). Although such a high occurrence is not necessarily associated with high risk for Legionnaire's disease among patients and staff, it is prudent to monitor DUWLs for Legionella periodically. Since this procedure is long and expensive, surrogate markers are frequently used. Aim. To investigate whether surrogate markers are predictive of Legionella detection in DUWLs in a highly colonized dental hospital. Material and methods. DUWLs from a dental hospital where legionellae were detected intermittently throughout a period of ten years was considered. The investigated predictors were total viable flora (TVF) at 37°C and at 22°C, Pseudomonas (legionellae competitor) occurrence and season. Multivariate analysis was made and, using the best fitting logistic regression model, the probability to detect legionellae in water from DUWLs was estimated. Results. Legionellae were detected in 52% water samples collected in summertime and never detected in wintertime at levels ranging between 0 and 200 colony forming units(CFU)/L. The odds ratio of legionellae occurrence were 25.0 for Pseudomonas undetected vs. detected, 108.3 for summertime vs. wintertime, 2.2-2.3 for TVF levels at 37°C and 22°C >200 CFU/mL vs. ≤200 CFU/mL. A 29% probability to detect legionellae from DUWLs, where Pseudomonas was undetected, TVF levels were >200 CFU/mL and in summertime, was estimated. Conclusion. Despite legionellae were ubiquitous in the dental hospital during the study period, in the most favourable conditions for Legionella growth (lack of competitor, high biofilm and hot weather), legionellae were detected in almost one third of DUWLs.

Photonic crystals on copolymer film for bacteria detection

The development of two-dimensional photonic crystals (PCs) on a copolymer film is described in connection with Fresnel reflection spectroscopy and fluorescence microscopy. Label-free detection of Legionella pneumophila was performed using a PC platform with a detection limit of 200cells/mL. L. pneumophila is well known as the cause of Legionnaires' disease and a lesser form called Pontiac fever. Since death by L. pneumophila infection depends on the early anti-microbial treatment, rapid diagnosis of this disease is critical for efficient treatment and patient survival. Conventional assays have turn-around times measurable in several hours to days, and are limited in their detection of various serogroups. Due to the recent introduction of regulatory guidelines for routine testing of water cooling towers and treatment facilities, biosensors for the on-field detection of Legionella spp. are highly in demand. The versatile and economical immunochips described here can be easily adapted for the monitoring of L. pneumophila serogroups in clinical and environmental samples in a few minutes.