Detection Of Legionella Pneumophila Research Articles

Naked-eye detection of Legionella pneumophila using smart fluorogenic polymers prepared as hydrophilic films, coatings, and electrospun nanofibers

Legionella pneumophila is a significant public health threat, responsible for severe diseases such as Legionnaires’ disease. Traditional detection methods are often labour-intensive, time-consuming, and require sophisticated equipment. This study introduces smart fluorogenic polymeric materials for the naked-eye detection of L. pneumophila via protease activity. These materials, prepared as hydrophilic films, cellulose-coated linear copolymers, and electrospun nanofibers, operate on an OFF/ON FRET system, emitting fluorescence under UV light upon interaction with L. pneumophila proteases. Characterisation confirmed the successful immobilisation of the peptide substrate and its response to proteases. The sensors showed moderate to high sensitivity and specificity, with detection limits of 2.91 × 105, 3.64 × 10⁵, and 4.04 × 105 CFUs/mL for the film, copolymer, and nanofiber formats, respectively. Cross-reactivity tests identified only Pseudomonas aeruginosa as an interferent. This novel approach offers rapid, simple, and cost-effective L. pneumophila detection with visible results under UV light, suitable for clinical and environmental samples. It highlights the potential for broader pathogen detection applications.

Validation of a Loop-Mediated Isothermal Amplification-Based Kit for the Detection of Legionella pneumophila in Environmental Samples According to ISO/TS 12869:2012.

Legionella pneumophila is a freshwater opportunistic pathogen and the leading cause of severe pneumonia known as Legionnaires' disease. It can be found in all water systems and survives in biofilms, free-living amoebae, and a wide variety of facilities, such as air conditioning and showers in hospitals, hotels and spas. The reference cultural method allows for the isolation and identification in many days, and in addition, it does not detect viable but rather non-culturable bacteria, increasing the risk of infection. In this context, a new LAMP-based (loop-mediated isothermal amplification) kit was developed, allowing for the rapid, sensitive, and labor-saving detection of L. pneumophila. The kit, "Legionella pneumophila Glow", was validated according to ISO/TS 12869:2012, testing sensitivity, inclusivity and exclusivity, and kit robustness. Sensitivity showed that the "Legionella pneumophila Glow" kit can detect up to 28 plasmid copies/µL. Robustness tests showed consistent results, with both contamination levels and the matrices used giving reproducible results. Furthermore, real samples were evaluated to compare the performance of the two methods. The LAMP kit "Legionella pneumophila Glow" proved a useful option for the rapid, efficient, and labor-saving screening of different typologies of water samples, offering significant advantages over the traditional method, as it is characterized by a high sensitivity, ease of use for laboratory testing, and a large reduction in analysis time, making it an asset to official controls.

A Diagnostic Chip for the Colorimetric Detection of Legionella pneumophila in Less than 3 h at the Point of Need.

Legionella pneumophila has been pinpointed by the World Health Organization as the highest health burden of all waterborne pathogens in the European Union and is responsible for many disease outbreaks around the globe. Today, standard analysis methods (based on bacteria culturing onto agar plates) need several days (~12) in specialized analytical laboratories to yield results, not allowing for timely actions to prevent outbreaks. Over the last decades, great efforts have been made to develop more efficient waterborne pathogen diagnostics and faster analysis methods, requiring further advancement of microfluidics and sensors for simple, rapid, accurate, inexpensive, real-time, and on-site methods. Herein, a lab-on-a-chip device integrating sample preparation by accommodating bacteria capture, lysis, and DNA isothermal amplification with fast (less than 3 h) and highly sensitive, colorimetric end-point detection of L. pneumophila in water samples is presented, for use at the point of need. The method is based on the selective capture of viable bacteria on on-chip-immobilized and -lyophilized antibodies, lysis, the loop-mediated amplification (LAMP) of DNA, and end-point detection by a color change, observable by the naked eye and semiquantified by computational image analysis. Competitive advantages are demonstrated, such as low reagent consumption, portability and disposability, color change, storage at RT, and compliance with current legislation.

Validation of quantitative real-time polymerase chain reaction for detection of Legionella pneumophila in hospital water networks

Rapid monitoring of Legionella pneumophila (Lp) is essential to reduce the risk of Legionnaires' disease in healthcare facilities. However, culture results take at least eight days, delaying the implementation of corrective measures. Here, we assessed the performance of a qPCR method and determined qPCR action thresholds for the detection of Lp in hospital hot water networks (HWNs). Hot water samples (N= 459) were collected from a hospital HWN. Lp were quantified using iQ-Check® Quanti real-time PCR Quantification kits (Bio-Rad) and the results were compared with those of culture. qPCR thresholds corresponding to the culture action thresholds of 10 and 1000cfu/L were determined on a training dataset and validated on an independent dataset. Lp concentrations measured by culture and qPCR were correlated for both the training dataset (Spearman's correlation coefficient ρ= 0.687, P<0.0001) and the validation dataset (ρ= 0.661, P<0.0001). Lp qPCR positivity thresholds corresponding to culture action thresholds of 10cfu/L was 91 genome units (gu) per litre (sensitivity, 86.4%; negative predictive value - NPV, 93.3%) and that corresponding to culture action thresholds of 1000cfu/L was 1048 gu/L (sensitivity, 100%; NPV, 100%). Detection of Lp by qPCR could be implemented with confidence in hospitals as a complement to culture in the monitoring strategy to speed up the implementation of corrective measures.

Surface acoustic wave-based lab-on-a-chip for the fast detection of Legionella pneumophila in water

Surface acoustic wave (SAW) -based immuno-biosensors are used for several applications, thanks to their versatility and faster response than conventional analytical methods. SAW immuno-biosensors can be usefully applied to promptly detect bacteria and prevent bacterial infections that can lead to severe diseases. Here, we present a SAW immuno-biosensor to detect Legionella pneumophila in water. Our device, working at ultra-high frequency (740 MHz), is functionalized with an anti-L. pneumophila antibody to maximize the specificity. We report the characteristic curve of the sensor, calculated measuring bacterial samples at known densities, and its related parameters. We also measure L. pneumophila samples contaminated with different Gram-positive and Gram-negative bacterial species (Escherichia coli and Enterococcus faecium) and samples diluted in mains waters. The proposed device is able to detect L. pneumophila in the range from 1·106 to 1·108 CFU/mL, with a limit of blank of 1.22·106 CFU/mL and a limit of detection of 2.01·106 CFU/mL. The nonspecific signal due to contaminant bacteria is very limited and measurements of L. pneumophila are not affected by contamination. We obtain a good detection also in mains water, representing a realistic matrix for L. pneumophila. Our results are encouraging and pave the way to the use of fast, easy-to-use, reliable and precise sensors to prevent bacterial infections in human activities.

Detection of Legionella pneumophila in the Water Samples of Food Industries and Hospitals in Bangladesh

Background: The human pathogen Legionella pneumophila causes a serious pneumonia-like respiratory disease called Legionnaires' disease, mainly in elderly and immunocompromised individuals. This pathogen can be found in the water distribution systems of large constructions with cooling towers which is a common phenomenon at present in Bangladesh due to its rapid economic growth. But there is a dearth of information on the incidence of Legionella in Bangladesh. Therefore, the current study aimed to investigate the presence of Legionella pneumophila in hospital and industrial water distribution systems in Dhaka, Bangladesh. Methods: A total of 114 water samples collected from two hospitals and five food industries were inoculated on the Legionella-specific medium Buffer Charcoal Yeast Extract (BCYE) agar medium before and after the treatment with acid, heat, or a combination of both. Samples producing Legionella-like colonies on BCYE agar medium were screened by Legionella Latex Test Kit, and the metagenomic DNAs obtained from these samples were analyzed by PCR using L. pneumophila-specific 16S rRNA primers. Results: Among 114 samples, Legionella-like colonies were observed in 30 water samples which demonstrated no agglutination in the Latex agglutination test. PCR analysis showed the presence of L. pneumophila in seven water samples, four in the potable water, chiller water, and cooling tower water of two different food industries, and three in ICU tap water, cooling tower water, and Fan Coil Units of two different hospitals. Sequence analysis of amplicons revealed that all seven sequences had 100% similarity with L. pneumophila. Conclusion: The presence of L. pneumophila in the water samples of local hospitals and food industries indicates that these habitats might serve as a potential site for Legionnaires' infection in Bangladesh. The results also showed that PCR, contrary to the conventional culture methods, could be more efficient and rapid in the identification of L. pneumophila.

CASE REPORT OF ICU ADMISSION DUE TO LEGIONELLA PNEUMOPHILA SEVERE PNEUMONIA IN VIET NAM

Legionnaires’ disease is a serious type of atypical pneumonia caused by Legionella bacteria. In this case study, the first case of ICU admission due to Legionella pneumophila severe infection in Vietnam is reported. A 40-year-old Japanese male patient with a history of hypertension for over 4 years was hospitalized with fever and frequent breathing difficulties. Diagnosis upon hospitalization was sepsis shock and ARDS pulmonary consolidation. The sputum specimen was then tested with multiplex PCR using Film array panel and the result was positive with Legionella pneumophila detection. The patient was medicated according to the results ofbacterial identification which gave good response and was discharged after 22 days of treatment.

Design of an optical nanobiosensor for detection of Legionella pneumophila in water samples.

Legionella spp. is a causative agent of Legionnaires' disease that creates public health problems. Isolation of these bacteria from water sources is essential to identify outbreak origins and prevent disease. Diagnostic biosensors for water quality control to protect consumers from water-borne infections can predict many outbreaks. Gold nanoparticles conjugated probes are a new generation of diagnostic tools. In this study, an optical nano biosensor was designed and characterized to detect Legionella pneumophila in water samples rapidly. Thiolated probes designed for the mip gene were attached to gold nanoparticles and then water samples containing Legionella pneumophila were examined. The limit of detection for PCR and biosensor was 104 and 103 copy numbers/μl, respectively. Biosensor sensitivity and PCR were reported to be 90% (18 out of 20) and 85% (17 out of 20), respectively. Specificity 100% has been reported for both methods. According to the obtained results, this method has the potential to diagnose L. pneumophila with high sensitivity and specificity. This system can be employed as a practical tool for rapid, accurate, high-sensitivity, and acceptable detection of Legionella pneumophila in contaminated water, which is cost-effective in terms of cost and time.

Influencing factors of Legionella reproduction in secondary water supply operation and management

Objective: To analyze the pollution status and influencing factors of Legionella pneumophila in a secondary water supply facility in a city. Methods: From June to August 2020, a survey on the level of Legionella pneumophila in secondary water supply unit was carried out in a city in northern China, and 304 sets of secondary water supply facilities were included in the study. A total of 760 water samples were collected from the inlet and outlet water of the secondary water supply facilities and some water samples in the water tank were collected for the detection of Legionella pneumophila, standard plate-count bacteria and related physical and chemical indicators. Through questionnaire survey, the basic information of secondary water supply facilities and daily management of water quality were collected. Multivariate logistic regression model was used to analyze the influencing factors of Legionella pneumophila contamination. Results: Among 304 sets of secondary water supply facilities, most of them were located in residential buildings [57.24% (174/304)]. High and low water tank water supply, low water tank variable frequency conversion water supply and non-negative pressure water supply accounted for 26.6% (81/304), 36.8% (112/304) and 36.5% (111/304), respectively. About 25.7% of facilities (78/304) were positive for Legionella pneumophila. Among them, the positive rates of Legionella pneumophila in high and low water tank water supply, low water tank variable frequency conversion water supply and non-negative pressure water supply facilities were 38.3% (31/81), 29.5% (33/112) and 12.6% (14/111), respectively. The results of multivariate logistic regression model analysis showed that the disinfectant residue could reduce the risk of Legionella pneumophila contamination in water samples, and the OR (95%CI) value was 0.083 (0.022-0.317). The increase of the standard plate-count bacteria and conductivity might increase the risk of Legionella pneumophila contamination in water samples. The OR (95%CI) values were 3.160 (1.667-5.99) and 1.004 (1.001-1.006), respectively. Compared with the non-negative pressure water supply, the risk of Legionella pneumophila contamination of secondary water supply facilities was increased by water supply from high and low water tanks and variable frequency conversion water supply from low water tanks, with OR (95%CI) values of 4.296 (2.096-8.803) and 2.894 (1.449-5.782), respectively. Conclusion: The positive rate of Legionella pneumophila in secondary water supply in the study city is high. Disinfectant residue, conductivity and method of water supply are associated with the positive rate of Legionella pneumophila.

Polymer Brushes on GaAs and GaAs/AlGaAs Nanoheterostructures: A Promising Platform for Attractive Detection of Legionella pneumophila.

This work reports on the potential of polymer brushes (PBs) grown on GaAs substrates (PB-GaAs) as a promising platform for the detection of Legionella pneumophila(Lp). Three functionalization approaches of the GaAs surface were used, and their compatibility with antibodies against Lp was evaluated using Fourier transform infrared spectroscopy and fluorescence microscopy. The incorporation of PBs on GaAs has allowed a significant improvement of the antibody immobilization by increased surface coverage. Bacterial capture experiments demonstrated the promising potential for enhanced immobilization of Lp in comparison with the conventional alkanethiol self-assembled monolayer-based biosensing architectures. Consistent with an eightfold improved capture of bacteria on the surface of a PB-functionalized GaAs/AlGaAs digital photocorrosion biosensor, we report the attractive detection of Lp at 500 CFU/mL.

Selective Detection of Legionella pneumophila Serogroup 1 and 5 with a Digital Photocorrosion Biosensor Using Antimicrobial Peptide-Antibody Sandwich Strategy.

Rapid detection of Legionella pneumophila (L. pneumophila) is important for monitoring the presence of these bacteria in water sources and preventing the transmission of the Legionnaires’ disease. We report improved biosensing of L. pneumophila with a digital photocorrosion (DIP) biosensor functionalized with an innovative structure of cysteine-modified warnericin antimicrobial peptides for capturing bacteria that are subsequently decorated with anti-L. pneumophila polyclonal antibodies (pAbs). The application of peptides for the operation of a biosensing device was enabled by the higher bacterial-capture efficiency of peptides compared to other traditional ligands, such as those based on antibodies or aptamers. At the same time, the significantly stronger affinity of pAbs decorating the L. pneumophila serogroup-1 (SG-1) compared to serogroup-5 (SG-5) allowed for the selective detection of L. pneumophila SG-1 at 50 CFU/mL. The results suggest that the attractive sensitivity of the investigated sandwich method is related to the flow of an extra electric charge between the pAb and a charge-sensing DIP biosensor. The method has the potential to offer highly specific and sensitive detection of L. pneumophila as well as other pathogenic bacteria and viruses.

Multiple Cross Displacement Amplification Coupled with Lateral Flow Biosensor (MCDA-LFB) for rapid detection of Legionella pneumophila

BackgroundLegionella pneumophila is an opportunistic waterborne pathogen of significant public health problems, which can cause serious human respiratory diseases (Legionnaires’ disease). Multiple cross displacement amplification (MCDA), a isothermal nucleic acid amplification technique, has been applied in the rapid detection of several bacterial agents. In this report, we developed a MCDA coupled with Nanoparticles-based Lateral Flow Biosensor (MCDA-LFB) for the rapid detection of L. pneumophila.ResultsA set of 10 primers based on the L. pneumophila specific mip gene to specifically identify 10 different target sequence regions of L. pneumophila was designed. The optimal time and temperature for amplification are 57 min and 65 °C. The limit of detection (LoD) is 10 fg in pure cultures of L. pneumophila. No cross-reaction was obtained and the specificity of MCDA-LFB assay was 100%. The whole process of the assay, including 20 min of DNA preparation, 35 min of L. pneumophila-MCDA reaction, and 2 min of sensor strip reaction, took a total of 57 min (less than 1 h). Among 88 specimens for clinical evaluation, 5 (5.68%) samples were L. pneumophila-positive by MCDA-LFB and traditional culture method, while 4(4.55%) samples were L. pneumophila-positive by PCR method targeting mip gene. Compared with culture method, the diagnostic accuracy of MCDA-LFB method was higher.ConclusionsIn summary, the L. pneumophila-MCDA-LFB method we successfully developed is a simple, fast, reliable and sensitive diagnostic tool, which can be widely used in basic and clinical laboratories.

Evaluation of an Environmental Transport Medium for Legionella pneumophila Recovery

The collection and storage of water-related matrices such as biofilm from collection to processing are critical for the detection of Legionella pneumophila by cultural and molecular tests. SRK™ is a liquid medium that acts both as an antimicrobial neutralizing agent and a transport medium for bacterial culture enumeration and is useful to maintain the stability of the sample from collection to analysis. The aims of this study were to evaluate Legionella pneumophila viability and bacterial nucleic acids’ stability in SRK™ medium over time at different storage conditions. Artificial bacterial inoculates with an approximate concentration of 104, 103 and 102 CFU/mL were made using Legionella pneumophila certified reference material suspended in SRK™ medium. Bacteria recovery was analyzed by cultural and molecular methods at time 0, 24 and 48 h at room temperature and at 0, 24, 48 and 72 h at 2–8 °C, respectively. SRK™ medium supported Legionella pneumophila culture viability with CFU counts within the expected range. The recovery after 72 h at 2–8 °C was 83–100% and 75–95% after 48 h at room temperature. Real-time PCR appropriately detected Legionella pneumophila DNA at each temperature condition, dilution and time point. Results demonstrated a good performance of SRK™ medium for the reliable recovery of environmental Legionella.

Early Detection of Legionella pneumophila and Aspergillus by mNGS in a Critically Ill Patient With Legionella Pneumonia After Extracorporeal Membrane Oxygenation Treatment: Case Report and Literature Review.

Legionella pneumophila can cause pneumonia, leading to severe acute respiratory distress syndrome (ARDS). Because of its harsh growth requirements, limited detection methods, and non-specific clinical manifestations, diagnosing Legionella pneumonia remains still challenging. Metagenomic next-generation sequencing (mNGS) technology has increased the rate of detection of Legionella. This study describes a patient who rapidly progressed to severe ARDS during the early stage of infection and was treated with extracorporeal membrane oxygenation (ECMO). Although his bronchoalveolar lavage fluid (BALF) was negative for infection and his serum was negative for anti-Legionella antibody, mNGS of his BALF and blood showed only the presence of Legionella pneumophila (blood mNGS reads 229, BALF reads 656). After antibiotic treatment and weaning from ECMO, however, he developed a secondary Aspergillus and Klebsiella pneumoniae infection as shown by mNGS. Mechanical ventilation and antibiotic treatment were effective. A search of PubMed showed few reports of secondary Aspergillus infections after Legionella infection. Severe pneumonia caused by any type of pathogenic bacteria may be followed by Aspergillus infection, sometimes during extremely early stages of infection. Patients with severe pneumonia caused by Legionella infection should undergo early screening for secondary infections using methods such as mNGS, enabling early and precise treatment, thereby simplifying the use of antibiotics and improving patient prognosis.

Atypical Pathogen Distribution in Chinese Hospitalized AECOPD Patients: A Multicenter Cross-Sectional Study.

PurposeThe proportion of atypical pathogens in patient with AECOPD within mainland China is unknown. The objectives of this study were to determine the distribution of atypical pathogens among Chinese patients with AECOPD, to evaluate the clinical characteristics of different atypical pathogen infections, and to compare different detection methods for atypical pathogens.Patients and MethodsSpecimens were collected from patients with AECOPD from March 2016 to November 2018 at eleven medical institutions in eight cities in China. Double serum, sputum, and urine samples were obtained from 145 patients. Serological and nucleic acid tests were used to assess for Mycoplasma pneumonia and Chlamydia pneumoniae; serological, urinary antigen, and nucleic acid tests were applied to detect Legionella pneumophila. The clinical characteristics of atypical pathogen-positive and -negative groups were also compared.ResultsThe overall positivity rate for Mycoplasma pneumoniae was 20.69% (30/145), with the highest rate being 20.00% (29/145) when determined by passive agglutination.The overall positive rates for Chlamydia pneumoniae and Legionella pneumophila were 29.66% (43/145) and 10.34% (15/145), respectively. The most common serotype of Legionella pneumophila was type 6. The maximum hospitalized body temperature, ratio of eosinophils, C-reactive protein (CRP) level, and procalcitonin (PCT) level of the Mycoplasma pneumoniae-positive group were significantly higher than those of the Mycoplasma pneumoniae-negative group. Patients in the Chlamydia pneumoniae-positive group smoked more, had higher proportions of comorbidities and frequent aggravations in the previous two years than those in the Chlamydia pneumoniae-negative group. Furthermore, the forced expiratory volume in one second to forced vital capacity (FEV1/FVC) ratio assessment of lung function was higher, and the concentration of arterial blood bicarbonate (HCO3−) was lower in the Legionella pneumophila-positive group than in the Legionella pneumophila-negative group.ConclusionOverall, atypical pathogens play an important role in AECOPD. Regarding the testing method, serological testing is a superior method to nucleic acid testing.

Efficacy of PCR Analysis of Mip, Doth and Gspd Genes with Culture in Detection of Legionella pneumophila.

The article's abstract is not available.

Evaluation of Legiolert™ for the Detection of Legionella pneumophila and Comparison with Spread-Plate Culture and qPCR Methods.

Legionella pneumophila, the organism responsible for Legionnaires' disease, a potentially lethal pneumonia, is an opportunistic bacterium spread via inhalation of contaminated, aerosolized water. The detection and control of L. pneumophila is crucial to reduce the risk it poses to human health. L. pneumophila is generally detected and quantified by the plating method, ISO 11731:2017 and by qPCR. ISO 11731 is based on the filtration of the water sample through a membrane, which is placed on selective agar medium, and after colony growth, presumptive Legionella are then confirmed by subculturing, serology, or PCR. Quantitative Polymerase Chain Reaction (qPCR) is based on the amplification of a DNA sequence specific to L. pneumophila, usually within the mip gene. The objective of this study was to compare these methods to a new, liquid culture method based on the Most Probable Number (MPN) technique, Legiolert™/Quanti-Tray® with data obtained with ISO 11731 and a viability quantitative qPCR (v-qPCR), for quantification of L. pneumophila in potable and non-potable waters. Data showed that the Legiolert method revealed concentrations of L. pneumophila greater than ISO 11731 and generally similar results to those of v-qPCR. The Legiolert method was highly specific and easy to use, representing a significant advancement in the quantification of L. pneumophila from potable and non-potable waters.

Biofilm Community Structures and Opportunistic Pathogen Gene Markers in Drinking Water Mains and the Role of Pipe Materials

Due to the difficulty of sampling, the characteristics of biofilm communities in drinking water mains are still poorly understood. In this study, 13 water mains were collected to investigate the occurrence of opportunistic pathogens, to explore the biofilm community structures, and to decipher the mechanisms of biofilm community assembly. The results showed high frequencies of detection of Legionella pneumophila, Acanthamoeba, and Vermamoeba vermiformis. The number of mycobacterial gene copies in ductile iron pipes (DIPs) was significantly higher than that in cast iron pipes (CIPs), indicating that pipe materials affected colonization by opportunistic pathogens. The biofilm samples were clearly clustered into two groups by pipe materials, highlighting the critical role of the pipe material in shaping biofilm communities. Furthermore, nitrifying bacteria were dominant in DIPs, while denitrifying bacteria were more abundant in CIPs than in DIPs. Overall, this work provides ecological insights into a poorly understood ecosystem.

Short Ligand, Cysteine-Modified Warnericin RK Antimicrobial Peptides Favor Highly Sensitive Detection of Legionella pneumophila.

Culture-based methods for the detection of Legionellapneumophila are prohibitively slow and frequently inadequate. The problem has been addressed with biosensing technology that employs a variety of ligands for the specific capture of bacteria. However, the limited success of the application of mammalian antibodies, aptamers, and nucleic acid-based probes for sensitive biosensing has generated growing interest in exploring alternative biosensing architectures, such as those based on antimicrobial peptides (AMP) that are known for their attractive therapeutic applications. We report on the successful employment of cysteine-modified warnericin RK AMP for the operation of a highly sensitive biosensor of L. pneumophila based on digital photocorrosion of GaAs/AlGaAs nanoheterostructures. The replacement of the relatively cumbersome procedure commonly applied for the attachment of antibodies to COOH-terminated mercaptohexadecanoic acid self-assembled monolayers has allowed for a significant reduction in the distance at which bacteria are immobilized above the biosensor surface. An important consequence of this approach is the attractive limit of detection of L. pneumophila estimated at 2 × 102 CFU/mL. The target bacteria were captured four times more efficiently than P. fluorescens, B. subtilis, and E. coli, which is highly promising for environmental monitoring.

Management of bacteriological specimens of patients suffering from coronavirus disease 2019 (COVID-19)

Management of bacteriological specimens of patients suffering from coronavirus disease 2019 (COVID-19)