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      Metodi diagnostici e di laboratorio (2020 - 2017)

        [ultimo aggiornamento 25/11/2020]



Evaluation of Legiolert for Quantification of Legionella pneumophila from Bath Water Samples

Inoue H, Baba M, Tayama S.

Tsukuba Research Laboratories, Aquas Corporation, Japan.

Biocontrol Sci 2020;25(3):179-182.

Abstract: Testing for Legionella spp. in public bath water samples is regulated in Japan. In this study, we used a total of 132 public bath water samples to compare the performance of Legiolert® and the conventional plate culture method for the enumeration of Legionella pneumophila. When Legiolert and plate culturing were performed at the same detection limit, L. pneumophila was detected in 26.5% of 132 samples by Legiolert, while 12.9% contained Legionella spp. (11.4% contained L. pneumophila) based on the plate culture method. Moreover, results of 83.3% of the total samples were consistent between the two methods, meaning that they were both positive or both negative. In this study, we demonstrated that Legiolert is a simpler and more effective method of monitoring for L. pneumophila in bath water samples.


Droplet digital PCR for the detection and monitoring of Legionella pneumophila

Falzone L, Gattuso G, Lombardo C, Lupo G, Grillo CM, Spandidos DA, Libra M, Salmeri M.

Department of Biomedical and Biotechnological Sciences, Section of General Pathology, University of Catania.

Int J Mol Med 2020 Nov;46(5):1777-1782.

Abstract: Legionella pneumophila (L. pneumophila) is a harmful pathogen often found in water systems. In hospitals, the absence of L. pneumophila in water systems is mandatory by law, therefore, frequent and effective monitoring of water is of fundamental importance. Molecular methods based on reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) have been proposed for the detection of L. pneumophila, however, the sensitivity and accuracy of these methods have not been validated yet. Therefore, it is important to evaluate other strategies able to overcome the limits of culture‑based and RT‑qPCR methods. On these bases, we compared the sensitivity and accuracy of droplet digital PCR (ddPCR) and RT‑qPCR in water samples with known concentrations of L. pneumophila and in an in vitro model of water heat treatments. ddPCR showed a higher sensitivity rate and accuracy compared to RT‑qPCR in detecting low bacterial load. In addition, ddPCR is not affected by the presence of fragmented DNA and showed higher accuracy than RT‑qPCR in monitoring the efficacy of heat shock treatments. In conclusion, ddPCR represents an innovative strategy to effectively detect L. pneumophila in water samples. Thanks to its high robustness, ddPCR could be applied also for the detection of L. pneumophila in patients with suspected legionellosis.


Evaluation of GVPC and BCYE Media for Legionella Detection and Enumeration in Water Samples by ISO 11731: Does Plating on BCYE Medium Really Improve Yield?

Scaturro M, Poznanski E, Mupo M, Blasior P, Seeber M, Prast AM, Romanin E, Girolamo A, Rota MC, Bella A, Ricci ML, Stenico A.

Department of Infectious Diseases, Istituto Superiore di Sanità, Rome, Italy.

Pathogens 2020 Sep 16;9(9):757.

Abstract: Legionella spp are the causative agents of Legionnaires' diseases, which is a pneumonia of important public health concern. Ubiquitous freshwater and soil inhabitants can reach man-made water systems and cause illness. Legionella enumeration and quantification in water systems is crucial for risk assessment and culture examination is the gold standard method. In this study, Legionella recovery from potable water samples, at presumably a low concentration of interfering microorganisms, was compared by plating on buffered charcoal yeast extract (BCYE) and glycine, vancomycin, polymyxin B, cycloheximide (GVPC) Legionella agar media, according to the International Standard Organization (ISO) 11731: 2017. Overall, 556 potable water samples were analyzed and 151 (27.1%) were positive for LegionellaLegionella grew on both BCYE and GVPC agar plates in 85/151 (56.3%) water samples, in 65/151 (43%) on only GVPC agar plates, and in 1/151 (0.7%) on only BCYE agar plates. In addition, GVPC medium identified Legionella species other than pneumophila in six more samples as compared with the culture on BCYE. Although the medians of colony forming units per liter (CFU/L) detected on the BCYE and GVPC agar plates were 2500 and 1350, respectively (p-value < 0.0001), the difference did not exceed one logarithm, and therefore is not relevant for Legionella risk assessment. These results make questionable the need to utilize BCYE agar plates to analyze potable water samples.


Performance of the ImmuView and BinaxNOW assays for the detection of urine and cerebrospinal fluid Streptococcus pneumoniae and Legionella pneumophila serogroup 1 antigen in patients with Legionnaires' disease or pneumococcal pneumonia and meningitis

Edelstein PH, Jørgensen CS, Wolf LA.

Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

PLoS One 2020 Aug 31;15(8):e0238479.

Abstract: The performances of the ImmuView Streptococcus pneumoniae (Sp) and Legionella pneumophila (Lp) urinary antigen test were compared to that of the BinaxNOW Sp and Lp assays, using frozen urine from 166 patients with Legionnaires' disease (LD) and 59 patients with pneumococcal pneumonia. Thirty Sp-positive or contrived cerebrospinal fluids (CSF) were also tested. Test specimens were collected and tested at different sites, with each site testing unique specimens by technologists blinded to expected results. No significant differences in test concordances were detected for the ImmuView and BinaxNOW assays for the Sp or Lp targets for urine from patients with pneumococcal pneumonia or LD when performance from both sites were combined. At one of two test sites the ImmuView Lp assay was more sensitive than the BinaxNOW assay, with no correlation between test performance and Lp serogroup 1 monoclonal type. Urines from six of seven patients with LD caused by Legionella spp. bacteria other than Lp serogroup 1 were negative in both assays. Both tests had equivalent performance for Sp-positive CSF. The clinical sensitivities for pneumococcal pneumonia were 88.1 and 94.4% for the ImmuView and Binax assays, and 87.6 and 84.2% for the Lp assays, respectively. Test specificities for pneumococcal pneumonia were 96.2 and 97.0% for the ImmuView and Binax assays, and 99.6 and 99.1% for the Lp assays. Both assays were highly specific for Sp in pediatric urines from children with nasopharyngeal colonization by the bacterium. ImmuView and BinaxNOW assay performance was equivalent in these studies.


Rapid Detection of Legionella pneumophila in Drinking Water, Based on Filter Immunoassay and Chronoamperometric Measurement

Ezenarro JJ, Párraga-Niño N, Sabrià M, Del Campo FJ, Muñoz-Pascual FX, Mas J, Uria N.

Departament de Genètica i Microbiologia, Universitat Autònoma de Barcelona, Cerdanyola, Spain.

Biosensors (Basel) 2020 Aug 20;10(9):102.

Abstract: Legionella is a pathogenic bacterium, ubiquitous in freshwater environments and able to colonise man-made water systems from which it can be transmitted to humans during outbreaks. The prevention of such outbreaks requires a fast, low cost, automated and often portable detection system. In this work, we present a combination of sample concentration, immunoassay detection, and measurement by chronoamperometry. A nitrocellulose microfiltration membrane is used as support for both the water sample concentration and the Legionella immunodetection. The horseradish peroxidase enzymatic label of the antibodies permits using the redox substrate 3,3',5,5'-Tetramethylbenzidine to generate current changes proportional to the bacterial concentration present in drinking water. Carbon screen-printed electrodes are employed in the chronoamperometric measurements. Our system reduces the detection time: from the 10 days required by the conventional culture-based methods, to 2-3 h, which could be crucial to avoid outbreaks. Additionally, the system shows a linear response (R2 value of 0.99), being able to detect a range of Legionella concentrations between 101 and 104 cfu·mL-1 with a detection limit (LoD) of 4 cfu·mL-1.


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

Scaturro M, Buffoni M, Girolamo A, Cristino S, Girolamini L, Mazzotta M, Bucci Sabattini MA, Zaccaro CM, Chetti L, Laboratory MAN, Bella A, Rota MC, Ricci ML.

Department of Infectious Diseases, Istituto Superiore di Sanità, Rome, Italy.

Pathogens 2020 Aug 23;9(9):690.

Abstract: 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.


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

Fricke C, Xu J, Jiang FL, Liu Y, Harms H, Maskow T.

Department of Environmental Microbiology, HelmholtzCentre for Environmental Research – UFZ, Leipzig Germany.

Microb Biotechnol 2020 Jul;13(4):1262-1272.

Abstract: 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 (CFU·ml-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.


Sensitivity and Selectivity of Two Commercially Available Media for Legionella spp. Recovery from Environmental Water Samples

Ditommaso S, Giacomuzzi M, Memoli G, Garlasco J, Zotti CM.

Department of Public Health and Pediatrics, University of Turin, Turin, Italy.

Pathogens 2020 Jun 29;9(7):523.

Abstract: The quality control of culture media used for Legionella spp. isolation and enumeration is paramount to achieve a satisfactory degree of comparability among water testing results from different laboratories. Here, we report on a comparative assessment of the sensitivity and selectivity of MWY and BCYEα media supplied by two different manufacturers (i.e., Xebios Diagnostics GmbH and Oxoid Ltd) for the detection of Legionella spp. from environmental water samples. Even though our analysis showed an excellent agreement between the recovery rates of the four media tested (90.5%), the quantitative recovery of Legionella spp. colonies using Xebios media was significantly greater than that achieved by Oxoid media (P = 0.0054). Furthermore, the sensitivity of detection was significantly higher when samples were plated on MWY Xebios agar (P = 0.0442), while the selectivity of MWY appeared to be the same regardless of the manufacturer. Furthermore, MWYXebios agar favored the growth of much larger colonies compared to those observed on MWYOxoid agar. Finally, MWYXebios medium enhanced the recovery of non-pneumophila Legionella species. Collectively, our findings demonstrate that quality control is crucial to ensure high selectivity and sensitivity of the culture media used for the detection and enumeration of Legionella spp. from environmental water resources. As water remediation measures strictly depend on Legionella spp. recovery, culture protocol standardization, as well as quality control of the culture media, is essential to achieve intra- and interlaboratory reproducibility and accuracy.



What Is the Risk of Missing Legionellosis Relying on Urinary Antigen Testing Solely? A Retrospective Belgian Multicenter Study

Muyldermans A, Descheemaeker P, Boel A, Desmet S, Van Gasse N, Reynders M; National Expert Committee on Infectious Serology.

Department of Medical Microbiology, AZ Sint-Jan Hospital, Bruges, Belgium.

Eur J Clin Microbiol Infect Dis 2020 Apr;39(4):729-734.

Abstract: Currently, diagnosis of legionellosis relies mainly on urinary antigen testing (UAT) for Legionella pneumophila serogroup 1 (Lp1). However, this test has several limitations, particularly missing non-Lp1 infections. The purpose of this large multicenter study was to investigate the risk of missing legionellosis relying on UAT solely. Molecular results of Legionella detection as part of a first line (syndromic) testing algorithm for severe respiratory tract infections were investigated retrospectively and compared with UAT results in 14 Belgian laboratories. Overall, 44.4% (20/45) UAT results appeared false negative and were reclassified as legionellosis based on PCR findings [Legionnaires' disease, 37.5% (15/40); Pontiac fever, 100% (5/5)]. A total of 39.4% (26/66) diagnosis probably would have been missed or delayed without a syndromic approach, as UAT or specific molecular testing for Legionella was not requested by the clinician. Furthermore, we confirmed the higher sensitivity of molecular Legionella detection in lower respiratory tract compared with upper respiratory tract specimens (p=0.010).


Serotyping and detection of pathogenecity loci of environmental isolates of Legionella pneumophila using MALDI-TOF MS

Kyritsi MA, Kristo I, Hadjichristodoulou C.

Laboratory of Hygiene and Epidemiology, School of Health Sciences, Department of Medicine, Larissa, Greece.

Int J Hyg Environ Health 2020 Mar;224:113441.

Abstract: BACKGROUND: The majority of Legionnaires' disease cases is attributed to Legionella pneumophila serogroup 1 (Lp1). Moreover, pathogenicity loci lvh and rtxA were associated with the ability of Lp strains to cause the disease. Consequently, except from serogroup assignment the detection of the aforementioned virulence genes during Legionella detection in water samples, could help environmental risk assessment and the implementation of targeted control measures. AIM: To establish and validate a rapid and robust MALDI-TOF MS-based method for the assignment of Lp isolates to serogroup and identify distinct peak biomarkers for the detection of lvh and rtxA loci during environmental investigations. METHOD: Fifteen reference strains and 150 Lp environmental isolates (70 Lp1 and 80 Lp2-15 strains) were used. All strains were PCR-tested for the presence of lvh and rtxA loci. Independent training and validation strain sets were constituted, and all strains were protein-extracted and submitted to MALDI-TOF MS analysis. The raw spectra of the training set strains obtained, were introduced into the Mass-Up software platform for biomarker detection, for both serogroup assignment and pathogenicity loci detection. Validation of the assigned biomarkers followed using the validation set strains. RESULTS: For serogroup assignment, the Mass-up analysis indicated five potential discriminating peaks and correctly classified 115 out of 132 validation set strains, displaying sensitivity of 87.5%, specificity of 86.7% and 87.1% accuracy. Concerning the pathogenicity loci detection, the Mass-up analysis indicated two ion peaks for rtxA locus discrimination and one peak for lvh locus discrimination. Concerning the lvh virulence gene, the algorithm correctly classified 113 out of 137 positive and all negative strains 14 in total-showing sensitivity of 82.5%, specificity of 100% and 84.1% accuracy. For rtxA locus, 134 out of 134 positive and 14 out of 17 negative strains were correctly classified with sensitivity of 100%, specificity of 76.5% and 97.4% accuracy. CONCLUSION: MALDI-TOF MS displayed good performance for Lp serogroup assignment and detection of the lvh and rtxA virulence genes. These findings could contribute to the rapid, inexpensive and comprehensive case investigation and risk assessment. Further studies are needed to standardize and evaluate the method using the direct target plate protein profiling instead of protein extraction in order to simplify the protocol.


Method Comparison of the ImmuView L. pneumophila and L. longbeachae Urinary Antigen Test with BinaxNOW Legionella Urinary Antigen Card for the Detection of L. pneumophila Serogroup 1 Antigen in Urine

Badoux P, Kracht-Kosten L, Herpers B, Euser S.

Regional Public Health Laboratory Kennemerland, Haarlem, the Netherlands.

J Clin Microbiol 2020 Feb;58(3):e01429-19.

Abstract: We compared the clinical performance of the ImmuView L. pneumophila and L. longbeachae Urinary Antigen test (SSI Diagnostica A/S, Hillerød, Denmark) to the BinaxNOW Legionella Urinary Antigen Card (Binax, Abbott, Lake Buff, IL) using urine specimens from patients suspected of having pneumonia. In total, 100 frozen urine samples (derived from 50 Legionella cases and 50 non-cases) were analyzed with both tests, as well as 200 non-frozen prospectively collected samples. For urine samples of five Legionella cases and two non-Legionella cases, the analytical sensitivity (limit of detection) and repeatability were examined. The urine samples of the five Legionella cases were diluted with urine samples that tested Legionella urinary antigen negative with both tests. The analyses of the 100 frozen samples resulted in a sensitivity and specificity of both the ImmuView and the BinaxNOW of 96.0% (48/50) and 100% (50/50). Of the 200 non-frozen samples, there were three samples that in both tests showed a positive result for L. pneumophila The analyses of reproducibility showed that for the 34 (diluted) samples that were tested at two consecutive times, 33 samples showed a consistent result for both the ImmuView and the BinaxNOW test (Cohen's kappa's: 0.916 and 0.928). In addition, the ImmuView test may have detected two L. longbeachae positive urine samples, although other diagnostic tests could not confirm this. Both ImmuView and BinaxNOW showed high sensitivity and specificity for the detection of L. pneumophila serogroup 1 antigen in urine samples from clinical patients with a suspected lower respiratory tract infection.


The Relevance of Molecular Genotyping to Allocate Cases in a Suspected Outbreak of Legionella Pneumonia in Patients with Prolonged Immunosuppressive Therapy

Borella P, Vecchi E, Incerti F, Marchesi I, Meacci M, Frezza G, Fregni Serpini G, Mansi A, Paduano S, Bargellini A.

Department of Biomedical, Metabolic and Neural Sciences, Section of Public Health, University of Modena and Reggio Emilia, Modena, Italy.

Int J Infect Dis 2020 Feb;91:174-176.

Abstract: Three cases of pneumonia caused by Legionella pneumophila serogroup 1 (Lp1) in immunosuppressed patients with repeated hospitalization were suspected as a healthcare-associated cluster. The environmental investigation did not reveal the presence of legionellae in the hospital patient rooms. Water samples collected from the homes of two patients were also negative for Legionella spp. In the absence of environmental strains potentially involved in the infections, we proceeded to genotype environmental Lp1 strains isolated in the hospital during routine water sampling during the decade 2009-2019 and recovered after long-term storage at -20°C. These 'historical' strains exhibited a high grade of similarity and stability over time, regardless of the disinfection systems. The different molecular profiles shown among the clinical and environmental strains excluded a nosocomial outbreak. The study suggests that the application of molecular typing may be a useful tool to discriminate hospital vs community-acquired cases, mostly for severely immunosuppressed patients in whom the symptomatology could be insidious and the incubation period could be prolonged. Moreover, the genotyping allowed us to exclude any link between the cases.


Molecular Detection of Opportunistic Premise Plumbing Pathogens in Rural Louisiana's Drinking Water Distribution System

Isaac TS, Sherchan SP.

Department of Global Environmental Health Sciences, School of Public Health and Tropical Medicine, Tulane University, New Orleans, LA, USA.

Environ Res Feb 2020;181:108847.

Abstract: Opportunistic premise plumbing pathogens (OPPPs) in drinking water distribution systems are responsible for causing numerous infections such as Legionnaires' disease and pneumonia through the consumption of contaminated drinking water. The incidence of opportunistic pathogens and the number of individuals at risk of contracting infections caused by these OPPPs in drinking water has risen drastically in the past decade. Pre-flush and post-flush water samples were collected from 64 houses in a rural town in northeast Louisiana to determine drinking water quality in terms of understanding abiotic and biotic factors on potential proliferation of OPPPs. Physical and chemical water quality parameters, such as pH, temperature, dissolved oxygen, salinity, and specific conductance were also measured. The quantitative polymerase chain reaction (qPCR) results indicated that Legionella spp. had the highest prevalence and was found in 46/64 samples (72%), followed by Mycobacterium spp. which was found in 43/64 samples (67%), E. coli in 31/64 samples (48%) and, Naegleria fowleri in 4/64 samples (6%) respectively. The results indicate the persistence of Legionella spp. DNA marker in these water samples.


Development of a DGGE Method to Explore Legionella Communities

Bayle S, Martinez-Arribas B, Jarraud S, Giannoni P, Garrelly L, Roig B, Cadière A.

EA7352 CHROME, University of Nîmes, Nîmes, France.

Heliyon 2020 Jan;6(1):e03149.

Abstract: Legionella risk assessment is nowadays based on the presence and concentration of either Legionella pneumophila or Legionella spp. Many species of Legionella can cause Legionnaires' disease, indeed about half of the known species have been associated with infection. The aim of this work was to develop a method to assess the composition of the Legionella species community in an environmental sample in order to have a better understanding of the contamination of the ecosystem by pathogenic strains. The method is based on the comparison of PCR-DGGE profile of DNA sample with a database consisting in DGGE profiles of Legionella species. Such a database includes all pathogenic Legionella strains. In order to homogenize and normalize the different DGGE fingerprint, a reference marker has been built and added during DGGE gel analysis. This study gives a valuable advance in the methods available for the understanding of Legionella contamination of water environments.


Loop-mediated Amplification as Promising On-Site Detection Approach for Legionella pneumophila and Legionella Spp

Reuter C, Slesiona N, Hentschel S, Aehlig O, Breitenstein A, Csáki A, Henkel T, Fritzsche W.

Leibniz Institute of Photonic Technology (Leibniz IPHT) Jena, Member of the Leibniz Research Alliance-Leibniz Health Technologies, Jena, Germany.

Appl Microbiol Biotechnol 2020 Jan;104(1):405-415.

Abstract: Recently Legionella pneumophila is the main causative waterborne organism of severe respiratory infections. Additionally, other Legionella species are documented as human pathogens. In our work, we describe a rapid detection method which combines two advantages for sensitive and specific detection of the genus Legionella: the fast isothermal amplification method "Loop-mediated isothermal AMPlification" (LAMP), and a colorimetric detection method using the metal indicator hydroxynaphtol blue (HBN) which allows to determine an optical signal with a simple readout (with the naked eye). Moreover, we present two approaches for minimizing the assay volume using a stationary microchip LAMP and droplet digital-based LAMP (ddLAMP) as promising highly sensitive setups.


Investigations on Contamination of Environmental Water Samples by Legionella Using Real-Time Quantitative PCR Combined with Amoebic Co-Culturing

Edagawa A, Kimura A, Miyamoto H.

Department of Environment Health, Osaka Prefectural Institute of Public Health, Osaka, Japan.

Biocontrol Sci 2019;24(4):213-220.

Abstract: We analyzed the contamination of environmental water samples with Legionella spp. using a conventional culture method, real-time quantitative PCR (qPCR), and real-time qPCR combined with an amoebic co-culture method. Samples (n=110) were collected from 19 cooling towers, 31 amenity water facilities, and 60 river water sources of tap water in Japan. Legionella was detected in only three samples (3/110, 2.7%) using the culture method. The rate of Legionella detection using amoebic co-culture followed by qPCR was 74.5%, while that using qPCR without amoebic co-culture was 75.5%. A higher than 10-fold bacterial count was observed in 19 samples (19/110, 17.3%) using real-time qPCR subsequent to amoebic co-culture, compared with identical samples analyzed without co-culture. Of these 19 samples, 13 were identified as Legionella spp., including L. pneumophila and L. anisa, and the non-culturable species were identified as L. lytica and L. rowbothamii. This study showed that the detection of Legionella spp., even in those samples where they were not detected by the culture method, was possible using real-time qPCR and an amoebic co-culture method. In addition, this analytical test combination is a useful tool to detect viable and virulent Legionella spp.


Culture of Clinical Specimens Reveals Extensive Diversity of Legionella pneumophila Strains in Arizona

Raphael BH, Huynh T, Brown E, Smith JC, Ruberto I, Getsinger L, White S, Winchell JM.

Respiratory Diseases Branch, CDC, Atlanta, Georgia, USA.

mSphere 2019;4(1):e00649-18.

Abstract: Between 2000 and 2017, a total of 236 Legionella species isolates from Arizona were submitted to the CDC for reference testing. Most of these isolates were recovered from bronchoalveolar lavage specimens. Although the incidence of legionellosis in Arizona is less than the overall U.S. incidence, Arizona submits the largest number of isolates to the CDC for testing compared to those from other states. In addition to a higher proportion of culture confirmation of legionellosis cases in Arizona than in other states, all Legionella pneumophila isolates are forwarded to the CDC for confirmatory testing. Compared to that from other states, a higher proportion of isolates from Arizona were identified as belonging to L. pneumophila serogroups 6 (28.2%) and 8 (8.9%). Genome sequencing was conducted on 113 L. pneumophila clinical isolates not known to be associated with outbreaks in order to understand the genomic diversity of strains causing legionellosis in Arizona. Whole genome multilocus sequence typing (wgMLST) revealed 17 clusters of isolates sharing at least 99% identical allele content. Only two of these clusters contained isolates from more than one individual with exposure at the same facility. Additionally, wgMLST analysis revealed a group of 31 isolates predominantly belonging to serogroup 6 and containing isolates from three separate clusters. Single nucleotide polymorphism (SNP) and pangenome analysis were used to further resolve genome sequences belonging to a subset of isolates. This study demonstrates that culture of clinical specimens for Legionella spp. reveals a highly diverse population of strains causing legionellosis in Arizona which could be underappreciated using other diagnostic approaches. IMPORTANCE: Culture of clinical specimens from patients with Legionnaires' disease is rarely performed, restricting our understanding of the diversity and ecology of Legionella Culture of Legionella from patient specimens in Arizona revealed a greater proportion of non-serogroup 1 Legionella pneumophila isolates than in other U.S. isolates examined. Disease caused by such isolates may go undetected using other diagnostic methods. Moreover, genome sequence analysis revealed that these isolates were genetically diverse and understanding these populations may help in future environmental source attribution studies.


Isolation of Legionella pneumophila by Co-culture With Local Ameba, Canada

Dey R, Mount H, Ensminger AW, Tyrrell GJ, Ward LP, Ashbolt NJ.

University of Alberta, Edmonton, Alberta, Canada.

Emerg Infect Dis 2019 Nov;25(11):2104-2107.

Abstract: Legionellosis was diagnosed in an immunocompromised 3-year-old girl in Canada. We traced the source of the bacterium through co-culture with an ameba collected from a hot tub in her home. We identified Legionella pneumophila serogroup 6, sequence type 185, and used whole-genome sequencing to confirm the environmental and clinical isolates were of common origin.


Comparison of Whole-Genome Sequences of Legionella pneumophila in Tap Water and in Clinical Strains, Flint, Michigan, USA, 2016

Brown CL, Garner E, Jospin G, Coil DA, Schwake DO, Eisen JA, Mukhopadhyay B, Pruden AJ.

Department of Civil and Environmental Engineering, Virginia Tech, Blacksburg, VA, USA.

Emerg Infect Dis 2019 Nov;25(11):2013-2020.

Abstract: During the water crisis in Flint, Michigan, USA (2014-2015), 2 outbreaks of Legionnaires' disease occurred in Genesee County, Michigan. We compared whole-genome sequences of 10 clinical Legionella pneumophila isolates submitted to a laboratory in Genesee County during the second outbreak with 103 water isolates collected the following year. We documented a genetically diverse range of L. pneumophila strains across clinical and water isolates. Isolates belonging to 1 clade (3 clinical isolates, 3 water isolates from a Flint hospital, 1 water isolate from a Flint residence, and the reference Paris strain) had a high degree of similarity (2-1,062 single-nucleotide polymorphisms), all L. pneumophila sequence type 1, serogroup 1. Serogroup 6 isolates belonging to sequence type 2518 were widespread in Flint hospital water samples but bore no resemblance to available clinical isolates. L. pneumophila strains in Flint tap water after the outbreaks were diverse and similar to some disease-causing strains.


Arthritis Caused by Legionella micdadei and Staphylococcus aureus: Metagenomic Next-Generation Sequencing Provides a Rapid and Accurate Access to Diagnosis and Surveillance

Huang Y, Ma Y, Miao Q, Pan J, Hu B, Gong Y, Lin Y.

Department of infectious diseases, Zhongshan Hospital, Fudan University, Shanghai, China.

Ann Transl Med 2019 Oct;7(20):589.

Abstract: Legionella spp. is an important pulmonary pathogen but rarely causes extra-pulmonary infections. We report a case of joint infection caused by Legionella micdadei and Staphylococcus aureus in a 54-year-old male with medication history of oral steroid for systemic lupus erythematosus (SLE). He developed arthritis in his right metacarpophalangeal (MCP) joints without precursor pneumonia. In the joint aspirate, S. aureus was detected through culture. The existence of L. micdadei and S. aureus were indicated by metagenomic next-generation sequencing (mNGS) and confirmed by 16S rRNA sequence analysis. After oral levofloxacin treatment for 54 days, the patient's symptoms ameliorated and blood test results improved, which were consistent with the dynamic trend of reads numbers in mNGS data. Our case included, arthritis caused by Legionella spp. have been reported in 11 patients. However, our case is the first to report septic arthritis caused by L. micdadei in native joints and monitored by mNGS. This case demonstrated an application of mNGS for etiological diagnosis and semi-quantification in joint aspirate. mNGS may serve as a promising tool for rapid and accurate etiological diagnosis and surveillance, contributing to appropriate antimicrobial drug applications and timely medication adjustments when necessary.


A Real-Time PCR for Specific Detection of the Legionella pneumophila Serogroup 1 ST1 Complex

Ginevra C, Chastang J, David S, Mentasti M, Yakunin E, Chalker VJ, Chalifa-Caspi V, Valinsky L, Jarraud S, Moran-Gilad J; ESCMID Study Group for Legionella Infections (ESGLI).

ESCMID Study Group for Legionella Infections (ESGLI), Basel, Switzerland; School of Public Health, Faculty of Health Sciences, Ben-Gurion University of the Negev. Beer-Sheva, Israel; Public Health Services, Ministry of Health, Jerusalem, Israel.

Clin Microbiol Infect 2019 Sep:S1198-743X(19)30487-2.

Abstract: Objective: Legionella pneumophila serogroup 1 (Lp1) sequence type (ST) 1 is globally widespread in the environment and accounts for a significant proportion of Legionella infections, including nosocomial Legionnaires' disease (LD). This study aimed to design a sensitive and specific detection method for Lp ST1 that will underpin epidemiological investigations and risk assessment. Methods: A total of 628 Lp genomes (126 ST1s) were analyzed by comparative genomics. Interrogation of more than 900 accessory genes revealed seven candidate targets for specific ST1 detection and specific primers and hydrolysis probes were designed and evaluated. The analytical sensitivity and specificity of the seven primer and probe sets were evaluated on serially diluted DNA extracted from the reference strain CIP107629 and via qPCR applied on 200 characterized isolates. The diagnostic performance of the assay was evaluated on 142 culture-proven clinical samples from LD cases and a real-life investigation of a case cluster. Results: Of seven qPCR assays that underwent analytical validation, one PCR target (lpp1868) showed higher sensitivity and specificity for ST1 and ST1-like strains. The diagnostic performance of the assay using respiratory samples corresponded to a sensitivity of 95% (19/20) (95% CI (75.1-99.9)) and specificity of 100% (122/122) (95% CI (97-100)). The ST1 PCR assay could link two out of three culture-negative hospitalized LD cases to ST1 during a real-time investigation. Conclusion: Using whole genome sequencing (WGS) data, we developed and validated a sensitive and specific qPCR assay for the detection of Lp1 belonging to the ST1 clonal complex by amplification of the lpp1868 gene. The ST1 qPCR is expected to deliver an added value for Lp control and prevention, in conjunction with other recently developed molecular assays.


DVC-FISH to Identify Potentially Pathogenic Legionella Inside Free-Living Amoebae from Water Sources

Moreno Y, Moreno-Mesonero L, García-Hernández J.

Research Institute of Water and Environmental Ingeneering (IIAMA), Universitat Politècnica de València, Valencia, Spain.

Environ Res 2019 Sep;176:108521.

Abstract: Despite all safety efforts, drinking and wastewater can still be contaminated by Legionella and free-living amoebae (FLA) since these microorganisms are capable of resisting disinfection treatments. An amoebae cyst harboring pathogenic Legionella spp. can be a transporter of this organism, protecting it and enhancing its infection abilities. Therefore, the aim of this work is to identify by DVC-FISH viable Legionella spp and Legionella pneumophila cells inside FLA from water sources in a specific and rapid way with the aim of assessing the real risk of these waters. A total of 55 water samples were processed, 30 reclaimed wastewater and 25 drinking water. FLA presence was detected in 52.7% of the total processed water samples. When DVC-FISH technique was applied, the presence of viable internalized Legionella spp. cells was identified in 69.0% of the total FLA-positive samples, concretely in 70.0% and 66.7% of wastewater and drinking water samples, respectively. L. pneumophila was simultaneously identified in 48.3% of the total FLA-positive samples, specifically in 50.0% and 44.4% of wastewater and drinking water samples, respectively. By culture, potentially pathogenic Legionella cells were recovered in 27.6% of the total FLA-positive bacteria, particularly in 35.0% and 11.1% of wastewater and drinking water samples, respectively. These findings demonstrate that FLA may promote resistance of bacteria to the performed disinfection treatments for drinking as well as for wastewater. So, in addition to the risk for the presence of pathogenic FLA in water it is necessary to take into account that these can be transporters of the pathogenic bacteria Legionella, which are able to survive inside them. The DVC-FISH method described here has been proved to be a rapid and specific tool to identify pathogenic Legionella spp. and L. pneumophila viable cells harboured by FLA in these water sources, posing particular public health concern.


Isolation and Identification of Legionella Spp. In Environmental Water Sources Based on Macrophage Infectivity Potentiator ( mip) Gene Sequencing in Southwest Iran

Moosavian M, Moradzadeh M, Ghadiri A, Saki M.

Infectious and Tropical Diseases Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.

AIMS Microbiol 2019 Aug;5(3):223-231.

Abstract: Legionella species are widespread in natural water sources and man-made aqueous environments, as well as freshwater. The present study was conducted owing to the lack of research regarding the prevalence of Legionella spp in the water sources of Ahvaz city in southwest Iran. In this study the macrophage infectivity potentiator (mip) gene sequencing was used for identification of various Legionella species isolated from different water sources. In this study, 144 water samples were collected and inoculated on the buffered charcoal-yeast extract (BCYE) agar and modified Wadowsky-Yee (MWY) medium. The DNA was extracted from positive cultures. The Legionella species were confirmed by amplifying a 654 bp fragment of the 16S rRNA gene. The mip gene of all isolates were amplified by PCR and purified for sequencing. The mip gene sequences were analyzed by jPHYDIT software version 1. The results showed a 13.9% (20/144) prevalence of Legionella spp. in water sources of Ahvaz city, southwest Iran. Analyzing of the mip gene sequences showed, out of 20 Legionella isolates, 13 isolates (54.1%) were positive for L. pneumophila, 5 isolates (20.8%) were positive for L. worsleinsis, one isolates for each one of L. dumoffi and L. fairfieldensis (4.1%). According to our research, the occurrence of Legionella spp in water sources could be a hazard for the health systems especially in the hospitals. The regular monitoring of these water sources by health planners may therefore be useful for decreasing the risk for Legionella spp. infections.


Potential False-Positive Urine Legionella Enzyme Immunoassay Test Results

Como J, Moffa MA, Bhanot N, Min Z, Cole KS, Kuzyck J, Walsh TL.

Department of Medicine and Division of Infectious Diseases, Allegheny General Hospital, Pittsburgh, PA, USA.

Eur J Clin Microbiol Infect Dis 2019 Jul;38(7):1377-1382.

Abstract: The objective of this study was to identify potential false-positive urine Legionella pneumophila (Legionella) enzyme immunoassay test results. A total of 107 consecutive patients with positive EIA tests were retrospectively analyzed over a 34-month period. Concurrent blood, urine, and sputum cultures, as well as chest radiographic findings, were reviewed in these patients. Twenty patients (19%) had no radiographic evidence of pulmonary disease despite a positive EIA test. In those 20 patients, 14 also had growth of non-Legionella bacteria. Of patients with an infiltrate or opacity on chest imaging, only 27 had Legionella sputum cultures obtained, with Legionella culture growth occurring in 7 (26%). Nine other patients had negative Legionella sputum cultures but the growth of another pathogenic organism in blood, sputum, and/or urine cultures. Pseudomonas aeruginosa was the most common organism isolated, found in 20% of patients in the entire cohort. Twenty-five patients (23%) were characterized as having probable false-positive Legionella urinary antigen EIA testing, and an additional 17 patients (16%) were characterized as having possible false-positive Legionella EIA tests. Our findings suggest that urine Legionella EIA tests may lead to a substantial number of cases being misdiagnosed as Legionnaires' disease in patients with non-Legionella bacterial colonization or infection.


Comparison of the Novel Immunocatch Legionella Test with Sofia Legionella FIA Assay and With BinaxNOW Legionella Card Assay for Detection of Legionella Pneumophila (Serogroup 1) Antigen in Urine Samples

Congestrì F, Morotti M, Vicari R, Pedna MF, Sparacino M, Torri A, Bertini S, Sambri V.

Unit of Microbiology, Greater Romagna Hub Laboratory, Pievesestina di Cesena, Italy.

J Clin Microbiol 2019 Jul;57(8):e00305-19.

Abstract: Legionnaires' disease (LD) refers to a serious form of acute pneumonia caused by Legionella species. LD can be difficult to diagnose because the signs and symptoms are nonspecific, and therefore a rapid laboratory diagnosis is of paramount importance. In this study, a recently introduced immunochromatographic test (Immunocatch Legionella; Eiken Chemical Co., Ltd.) for Legionella pneumophila (serogroup 1) urinary antigen detection was compared with the Sofia Legionella fluorescent immunoassay (FIA) (Quidel) (routinely used in our laboratory) and with the widely used BinaxNOW Legionella assay (Alere). A total of 248 urine samples (60 frozen and 188 fresh) were evaluated. All of the samples were collected from patients with high clinical suspicion of Legionnaires' disease. The three assays were performed simultaneously according to the manufacturers' instructions. A total of 180 concordant negative and 66 concordant positive results were obtained. Only 2 discrepant results were registered. The sensitivity and specificity of Immunocatch compared with Sofia were, respectively, 98.5% and 99.4%. Cohen's kappa coefficient and overall percent agreement between Immunocatch and Sofia were also calculated and resulted in, respectively, 0.97 and 99.2%. These performances suggest that the Immunocatch test is a useful tool for Legionella pneumophila (serogroup 1) urinary antigen detection.


Comparison of Legiolert and a Conventional Culture Method for Detection of Legionella pneumophila From Cooling Towers in Québec

Barrette I.

EnvironeX Group, 2325 Fernand-Lafontaine Blvd, Longueuil, QC, Canada.

J AOAC Int 2019 Jul;102(4):1235-1240.

Abstract: Background: Legionnaires' disease is a potentially lethal pneumonia contracted through inhalation of aerosolized water contaminated with Legionella bacteria. Detection and control of L. pneumophila, the primary species responsible for the disease, is critical to public health. In Québec, cooling towers and evaporative condensers are required to follow a maintenance and testing program to ensure L. pneumophila concentrations remain at acceptable levels. Objective: This study compared a new culture method based on the most probable number approach, Legiolert®, with the formal culture method used at EnvironeX for regulatory compliance testing to quantify L. pneumophila from cooling tower waters in Québec. Methods: A split-sample analysis was performed in which 401 samples from cooling towers in Québec were tested with both methods. Results: Results with 74 positive samples showed that Legiolert provided a significant increase in sensitivity for L. pneumophila compared with the agar plate method. Cooling tower samples often contain non-Legionella flora that necessitate multiple treatment and plating conditions to prevent interference with the test. Legiolert showed little to no impact from non-Legionella organisms in this study. Conclusions: Overall, Legiolert showed several advantages over the agar plate method, including increased sensitivity, reduced interference, a simplified test procedure, and an easy-to-read positive signal.


Validation of a Diagnostic Score Model for the Prediction of Legionella pneumophila Pneumonia

Miyashita N, Horita N, Higa F, Aoki Y, Kikuchi T, Seki M, Tateda K, Maki N, Uchino K, Ogasawara K, Kiyota H, Watanabe A.

First Department of Internal Medicine, Division of Respiratory Medicine, Infectious Disease and Allergology, Kansai Medical University, Japan.

J Infect Chemother 2019 Jun;25(6):407-412.

Abstract: Background: Community-acquired pneumonia (CAP) due to Legionella has a high mortality rate in patients who do not receive adequate antibiotic therapy. In a previous study, we developed a simple Legionella Score to distinguish patients with Legionella and non-Legionella pneumonia based on clinical information at diagnosis. In the present study, we validated this Legionella Score for the presumptive diagnosis of Legionella CAP. Methods: This validation cohort included 109 patients with Legionella CAP and 683 patients with non-Legionella CAP. The Legionella Score includes six parameters by assigning one point for each of the following items: being male, absence of cough, dyspnea, C-reactive protein (CRP) ≥18 mg/dL, lactate dehydrogenase (LDH) ≥260 U/L, and sodium <134 mmol/L. Results: When the Legionella CAP and non-Legionella CAP were compared by univariate analysis, most of the evaluated symptoms and laboratory test results differed substantially. The six parameters that were used for the Legionella Score also indicated clear differences between the Legionella and non-Legionella CAP. All Legionella patients had a score of 2 points or higher. The median Legionella Scores were 4 in the Legionella CAP cases and 2 in the non-Legionella CAP cases. A receiver operating characteristics curve showed that the area under the curve was 0.93. The proposed best cutoff, total score ≥3, had sensitivity of 93% and specificity of 75%. Conclusion: Our Legionella Score was shown to have good diagnostic ability with a positive likelihood of 3.7 and a negative likelihood of 0.10.


Urinary Antigene and PCR Can Both Be Used to Detect Legionella pneumophila in Children's Hospital-Acquired Pneumonia

Mojtahedi SY, Rahbarimanesh A, Noorbakhsh S, Shokri H, Jamali-Moghadam-Siyahkali S, Izadi A.

Department of Pediatric Infectious disease, Tehran University of Medical science, Tehran, Iran.

Eur J Transl Myol, 2019 May;29(2):8120.

Abstract: Legionella pneumophila is the causative agent of more than 95% cases of severe Legionella pneumonia. Nosocomial pneumonias in different hospital wards is an important medical and pharmaceutical concern. This study aimed to detect Legionella with two methods: polymerase chain reaction (PCR) and detection of urine antigenic test (UAT) in patients suffering from nosocomial pneumonia admitted to pediatric intensive care unit (PICU) of children hospitals. This study was conducted in PICU wards of Rasool Akram and Bahrami children hospitals, Tehran, Iran during 2013-2014. In patients diagnosed with hospital-acquired pneumonia, intratracheal secretion samples for PCR and urine sample for UAT were taken. Simultaneously, PCR and urinary antigen test were conducted using commercial kits. The results of urinary antigen test and PCR were analyzed by SPSS v.19 for statistical comparison. In this study, 96 patients aging 2.77 years on average with two age peaks of less than 1 year and 7-8 year were enrolled. More than half of the patients were under 1 year old. The most common underlying diseases were seizure, Acute Lymphoblastic Lymphoma, Down syndrome and metabolic syndromes. The positivity rate of Legionella urinary antigen test was 16.7% and positivity rate of PCR test was 19.8%. There were no significant associations between the results obtained by both assays with age, gender or underlying diseases. In conclusion, PCR is a better detection method for Legionella infection than urinary antigen test, but the difference between the two methods was not significant.


DNA-based Bioassay of Legionella Pneumonia Pathogen Using Gold Nanostructure: A New Platform for Diagnosis of Legionellosis

Mobed A, Hasanzadeh M, Babaie P, Agazadeh M, Mokhtarzadeh A, Rezaee MA.

Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.

Int J Biol Macromol 2019 May;128:692-699.

Abstract: The specific diagnosis of hard-growing bacteria is one of the most important concerns of medical bacteriology. Legionella pneumophila is one of the most important bacteria in hard growth. In spite remarkable trends in bacteriology, now day, culture is the gold standard for detection of L. pneumophila. This work is an attempt to quantification of L. pneumophila bacteria using a bioassay. The fabrication of a new electrochemical DNA-based bioassay using gold nano architecture combined with as a transducer substrate combined with toluidine blue (TB) as a redox marker was performed successful. Also, the mixture of beta‑cyclodextrin and dopamine as Poly (dopamine‑β‑Cyclodextrin) was used to proper a biointerface for stabilization of gold nanoparticles optimum immobilize of pDNA sequence (5-SH-TCGA TAC TCT CCC CGC CCC TT T TGTATCGACG-3). So, a specific thiolated pDNA was immobilized on the transducer substrate and DNA hybridization was followed by C-DNA sequence (5-ACA AAA GGG GCG GGG AGA GTA-3) using square wave voltammetry and differential pulse voltammetry. At the optimum conditions, linear range was 1 μM to 1 ZM and low limit of quantification (LLOQ) was 1 Zepto-molar. L. pneumophila were sensitively distinguished by the planned DNA sensor. Finally, the engineered DNA based bioassay could be used for identifying the L. pneumophila samples from patients or environments.


Validation and In-Field Testing of a New On-Site qPCR System for Quantification of Legionella pneumophila According to ISO/TS 12869:2012 in HVAC Cooling Towers

Ahmed S, Liwak-Muir U, Walker D, Zoldowski A, Mears A, Golovan S, Mohr S, Lem P, Harder C.

Spartan Bioscience Inc., 2934 Baseline Road, Suite 500, Ottawa, ON, Canada.

J Water Health 2019 Apr;17(2):237-253.

Abstract: Legionella pneumophila, found in engineered water systems such as HVAC cooling towers, poses a significant public health risk. Culture, though routinely used to quantify L. pneumophila, has several disadvantages including long turnaround time, low sensitivity, and inter-laboratory variability. In this study, we validated the performance of an on-site quantitative polymerase chain reaction (qPCR) detection system for L. pneumophila in accordance with International Standards Organization Technical Specification 12869:2012. We evaluated specificity, limit of detection and quantification, and calibration curve linearity. Additionally, we evaluated whole system recovery and robustness using samples taken from taps and evaporative cooling towers. We then compared the system's performance against laboratory culture and laboratory qPCR across 53 cooling towers in a 12-week in-field study. We found that concordance between on-site qPCR and culture was both laboratory- and site/sample-dependent. Comparison of laboratory qPCR with on-site qPCR revealed that laboratory results were highly variable and showed little concordance. Some discordance may be explained by time delay between sample collection and testing ('shipping effect') which may lead to inaccurate reporting. Overall, our study highlights the value of on-site qPCR detection of L. pneumophila, demonstrates that laboratories are prone to misreporting results due to shipping effects, and reveals significant discordance between laboratory qPCR and culture.


Loop-mediated Isothermal Amplification for Detection of Legionella pneumophila in Respiratory Specimens of Hospitalized Patients in Ahvaz, Southwest Iran

Moosavian M, Seyed-Mohammadi S, Saki M, Shahi F, Khoshkholgh Sima M, Afshar D, Barati S.

Department of Microbiology, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.

Infect Drug Resist 2019 Mar;12:529-534.

Abstract: Background: Legionnaires' disease is an important public health problem that can cause substantial mortality and morbidity. Legionnaires' disease-risk estimation may be compromised by uncertainties in Legionella-detection methods. The aim of this study was the detection of L. pneumophila in respiratory specimens of hospitalized patients with respiratory symptoms by culture, PCR, and loop-mediated isothermal amplification (LAMP) methods. Methods: Sputum and bronchoalveolar lavage samples were obtained from patients with pneumonia admitted to teaching hospitals in Ahvaz, Iran from June 2016 to March 2017. Isolation of Legionella spp. was done by culturing the samples directly onto buffered charcoal-yeast extract and modified Wadowsky-Yee agar medium. Then, PCR and LAMP assays were performed for detection of L. pneumophila via its mip gene in respiratory specimens. Results: A total of 100 respiratory specimens were collected. Our results showed that 1% of the samples were culture positive for Legionella spp., and 3% and 7% of samples were positive for L. pneumophila using the mip gene on PCR and LAMP assays, respectively.

Conclusion: Legionnaires' disease should be considered in the diagnosis of pulmonary infectious diseases. Also, the LAMP assay is a faster method with higher sensitivity and specificity than conventional methods, such as PCR and culture, for laboratory diagnosis of Legionnaires' disease.


Single Cell Analysis of Legionella and Legionella-Infected Acanthamoeba by Agarose Embedment

Personnic N, Striednig B, Hilbi H.

Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland.

Methods Mol Biol 2019;1921:191-204.

Abstract: Legionella pneumophila resides in multispecies biofilms, where it infects and replicates in environmental protozoa such as Acanthamoeba castellanii. Studies on L. pneumophila physiology and host-pathogen interactions are frequently conducted using clonal bacterial populations and population level analysis, overlooking the remarkable differences in single cell behavior. The fastidious nutrient requirements of extracellular L. pneumophila and the extraordinary motility of Acanthamoeba castellanii hamper an analysis at single cell resolution. In this chapter, we describe a method to study L. pneumophila and its natural host A. castellanii at single cell level by using an agarose embedment assay. Agarose-embedded bacteria and infected cells can be monitored over several hours up to several days. Using properly adapted flow chambers, agarose-embedded specimens can be subjected to a wide range of fluctuating conditions.


Migration of Acanthamoeba castellanii through Legionella Biofilms

Hochstrasser R, Hilbi H.

Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland.

Methods Mol Biol 2019;1921:79-89.

Abstract: The amoeba-resistant bacterium Legionella pneumophila infects humans through aerosols and thereby can cause a life-threatening pneumonia termed Legionnaires' disease. In the environment L. pneumophila forms and colonizes biofilms, which usually comprise complex multispecies communities. In these biofilms L. pneumophila persists and replicates intracellularly in protozoa, such as the amoeba Acanthamoeba castellanii. The interactions between sessile L. pneumophila in biofilms and their natural protozoan hosts are not understood on a molecular level. Here, we describe a method to visualize by confocal microscopy the formation and architecture of mono-species L. pneumophila biofilms. Furthermore, we describe and quantify the migration or "grazing" of A. castellanii in the biofilm. This allows investigating on a molecular and cellular level L. pneumophila biofilm formation and Legionella-amoeba interactions within biofilms.


Quantification of Viable but Non-Culturable Cells of Legionella pneumophila

Braun RS, Mendis N, Li L, Faucher SP.

Faculty of Agricultural and Environmental Sciences, Department of Natural Resource Sciences, McGill University, Ste-Anne-de-Bellevue, QC, Canada.

Methods Mol Biol 2019;1921:45-53. 

Abstract: Legionella pneumophila, among other bacteria, may enter a viable but non-culturable state as a means for survival in stressful conditions. Bacterial cells in the viable but non-culturable state cannot grow on standard medium; however, they continue to exhibit characteristics that are associated with live cells, such as respiration, transcription, and cell wall integrity. The present paper outlines a detailed protocol for the detection of viable but non-culturable L. pneumophila cells via Syto® 9 and propidium iodide staining coupled with flow cytometry.


Quantification of Legionella DNA Certified Reference Material by Digital Droplet PCR

Baume M, Cariou A, Leveau A, Fessy N, Pastori F, Jarraud S, Pierre S.

Hospices Civils de Lyon, Centre National de Référence des Légionelles, Lyon, France.

J Microbiol Methods 2019 Feb;157:50-53.

Abstract: A value was assigned in 2009 to the Legionella DNA Certified Reference Material, and the stability study conducted using quantitative PCR found a low level of degradation. Herein, the Digital Droplet PCR method for Legionella DNA was qualified and used to provide absolute quantification of the CRM.


Optimization of Viability qPCR for Selective Detection of Membrane-Intact Legionella pneumophila

Kontchou JA, Nocker A.

IWW Water Centre, Applied Microbiology, Mülheim an der Ruhr, Germany.

J Microbiol Methods 2019 Jan;156:68-76.

Abstract: Although a number of viability qPCR assays have been reported to selectively detect signals from membrane-intact Legionella pneumophila, the efficient suppression of amplification of DNA from dead membrane-compromised bacteria remains an ongoing challenge. This research aimed at establishing a new oligonucleotide combination that allows for a better exclusion of dead Legionella pneumophila on basis of the mip gene. Propidium monoazide (PMA) was chosen as viability dye. An oligonucleotide combination for the amplification of a 633 bp sequence was established with 100% specificity for different Legionella pneumophila strains compared with 17 other Legionella species tested. Apart from increasing amplicon length, the study aimed at optimizing dye incubation time and temperature. An incubation temperature of 45°C for 10 min was found optimal. Dye treatment of heat-killed bacteria in the presence of EDTA improved signal suppression, whereas deoxycholate also affected signals from live intact bacteria. Suppression of signals from heat-treated bacteria was found to be approx. twice as efficient compared to a commercial kit, although the detection sensitivity is superior when targeting short amplicons. With a limit of detection of 10 genome copies per PCR well and a 6-log signal reduction of bacteria killed at 80°C, the assay appears useful for applications where pathogen numbers are not limiting and where the priority is on the distinction between intact and damaged Legionella pneumophila for the evaluation of hygienic risk and of disinfection efficiency.


Legionella SBT Applied Directly to Respiratory Samples as a Rapid Molecular Epidemiological Tool

Quero S, Párraga-Niño N, Sabria M, Barrabeig I, Sala MR, Jané M, Mateu L, Sopena N, Pedro-Botet ML, Garcia-Nuñez M.

Infectious Diseases Unit, Hospital Universitari Germans Trias i Pujol, Badalona, Spain. e

Sci Rep 2019 Jan;9(1):623.

Abstract: Legionnaires' disease (LD) is an atypical pneumonia caused by the inhalation of Legionella. The methods used for the diagnosis of LD are direct culture of respiratory samples and urinary antigen detection. However, the sensitivity of culture is low, and the urinary antigen test is specific only for L. pneumophila sg1. Moreover, as no isolates are obtained, epidemiological studies cannot be performed. The implementation of Nested-sequence-based typing (Nested-SBT) makes it possible to carry out epidemiological studies while also confirming LD, especially in cases caused by non-sg 1. Sixty-two respiratory samples from patients with Legionella clinically confirmed by positive urinary antigen tests were cultured and tested by Nested-SBT, following the European Study Group for Legionella Infections (ESGLI) protocol. Only 2/62 (3.2%) respiratory samples were culture positive. Amplification and sequencing of Nested-SBT genes were successfully performed in 57/62 samples (91.9%). The seven target genes were characterised in 39/57 (68.4%) respiratory samples, and the complete sequence type (ST) was obtained. The mip gene was the most frequently amplified and sequenced. Nested-SBT is a useful method for epidemiological studies in culture-negative samples, achieving a 28.7-fold improvement over the results of culture studies and reducing the time needed to obtain molecular epidemiological results.


Bioassays: The Best Alternative for Conventional Methods in Detection of Legionella pneumophila

Mobed A, Hasanzadeh M, Agazadeh M, Mokhtarzadeh A, Rezaee MA, Sadeghi J.

Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.

Int J Biol Macromol 2019 Jan;121:1295-1307.

Abstract: Fastidious bacteria are group of bacteria that not only grow slowly but also have complex nutritional needs. In this review, recent progress made on development of biosensing strategies towards quantification of Legionella pneumophila as fastidious bacteria in microbiology was investigated. In coincidence with medical bacteriology, it is the most widely used biomonitoring, biosensors based on DNA and antibody. Also, all of Legionella pneumophila genosensors and immunosensors that developed in recent years were collected and analyzed. This review is meant to provide an overview of the various types of bioassays have been developed for determination of Legionella, along with significant advances over the last several years in related technologies. In addition, this review described: i) Most frequently applied principles in bioassay/biosensing of Legionella. ii) The aspects of fabrication in the perspective of bioassay/biosensing applications. iii) The potential of various electrochemical and optical bioassay/biosensing for the determination of Legionella and the circumvention of the most serious problem in immunosensing/immunoassay was discussed. iv) Some of bioassay/biosensing has been discussed with and without labels. v) We also summarize the latest developments in the applications of bioassay/biosensing methods for detection of Legionella. vi) The development trends of optical and electrochemical based bioassay/biosensing are also introduced.


Improvement of Legionnaires' Disease Diagnosis Using Real-Time PCR Assay: A Retrospective Analysis, Italy, 2010 to 2015

Ricci ML, Grottola A, Fregni Serpini G, Bella A, Rota MC, Frascaro F, Pegoraro E, Meacci M, Fabio A, Vecchi E, Girolamo A, Rumpianesi F, Pecorari M, Scaturro M.

Department of Infectious Diseases, Istituto Superiore di Sanità, Rome, Italy.

Euro Surveill 2018 Dec;23(50):1800032.

Abstract: Aim: To evaluate real-time PCR as a diagnostic method for Legionnaires' disease (LD). Detection of Legionella DNA is among the laboratory criteria of a probable LD case, according to the European Centre for Disease Prevention and Control, although the utility and advantages, as compared to culture, are widely recognised. Methods: Two independent laboratories, one using an in-house and the other a commercial real-time PCR assay, analysed 354 respiratory samples from 311 patients hospitalised with pneumonia between 2010-15. The real-time PCR reliability was compared with that of culture and urinary antigen tests (UAT). Concordance, specificity, sensitivity and positive and negative predictive values (PPV and NPV, respectively) were calculated. Results: Overall PCR detected eight additional LD cases, six of which were due to Legionella pneumophila (Lp) non-serogroup 1. The two real-time PCR assays were concordant in 99.4% of the samples. Considering in-house real-time PCR as the reference method, specificity of culture and UAT was 100% and 97.9% (95% CI: 96.2-99.6), while the sensitivity was 63.6% (95%CI: 58.6-68.6) and 77.8% (95% CI: 72.9-82.7). PPV and NPV for culture were 100% and 93.7% (95% CI: 91.2-96.3). PPV and NPV for UAT were 87.5% (95% CI: 83.6-91.4) and 95.8% (95% CI: 93.5-98.2). Conclusion: Regardless of the real-time PCR assay used, it was possible to diagnose LD cases with higher sensitivity than using culture or UAT. These data encourage the adoption of PCR as routine laboratory testing to diagnose LD and such methods should be eligible to define a confirmed LD case.


Insights into the Long-Term Persistence of Legionella in Facilities from Whole-Genome Sequencing

Wells M, Lasek-Nesselquist E, Schoonmaker-Bopp D, Baker D, Thompson L, Wroblewski D, Nazarian E, Lapierre P, Musser KA.

Wadsworth Center, New York State Department of Health, Albany, NY, USA.

Infect Genet Evol 2018 Nov;65:200-209.

Abstract: We investigated the value of whole-genome sequencing (WGS) and single nucleotide polymorphism (SNP) analyses in determining the relationships among and evolutionary rates of Legionella species with long-term persistence in three healthcare facilities. We examined retrospective clinical and environmental isolates of Legionella micdadei and Legionella pneumophila serogroup 1 isolates with identical PFGE DNA fingerprints sampled over the course of up to 18 years. WGS analyses demonstrated that heterogeneous populations of Legionella were present within each facility despite displaying the same PFGE profiles. Additionally, clustering of some clinical isolates with those from a separate but related institution exposed a source of infection not previously detected, underscoring the importance of considering phylogenetic relationships when assessing epidemiological links. The data supported an average substitution rate of 0.80 SNPs per genome per year for L. micdadei but a reliable estimate for L. pneumophila serogroup 1 could not be obtained due to complicating factors such as non-chronological links among isolates and inadequate sampling depths. While the substitution rate for L. micdadei is consistent with previous estimates for L. pneumophila, the lack of a temporal signal in our sequence data for L. pneuomphila serogroup 1 isolates suggests either insufficient change to provide an estimate or variable evolutionary rates, which could reflect the presence of both actively dividing and viable but non-culturable Legionella spp. in the built environment. This study highlights the increased discriminatory power of WGS SNP analysis as compared to PFGE, emphasizes the need for extended sampling, and provides insight into the evolution of Legionella from longitudinal investigations.


New System for the Detection of Legionella pneumophila in Water Samples

Párraga-Niño N, Quero S, Ventós-Alfonso A, Uria N, Castillo-Fernandez O, Ezenarro JJ, Muñoz FX, Garcia-Nuñez M, Sabrià M.

Unitat de Malalties Infeccioses, Fundació Institut d'Investigació Germans Trias i Pujol, Badalona, Spain. e

Talanta 2018 Nov;189:324-331.  

Abstract: Waterborne pathogens are a global concern for public health worldwide. Despite continuing efforts to maintain water safety, water quality is still affected by deterioration and pollution. Legionella pneumophila colonizes man-made water systems and can infect humans causing Legionnaire's disease (LD), pneumonia. The prevention of LD is a public health issue and requires specific systems to control and detect these microorganisms. Culture plate is the only technique currently approved but requires more than 10 days to obtain results. A rapid test that inform in hours about the presence of Legionella pneumophila in water samples will improve the control of this pathogen colonization. In order to control colonization by L. pneumophila we developed a membrane filter method to capture and immunodetect this microorganism in water samples. This membrane filter is used to retain the bacteria using a nitrocellulose disc inside a home-made cartridge. Subsequently we perform the immunodetection of the bacteria retained in the nitrocellulose (blocking, antibody incubation, washings and developing). On comparing our test with the gold-standard, the most important finding is the considerably reduction in time maintaining the same detection limit. This rapid test is easily automated for L. pneumophila detection allowing a comprehensive surveillance of L. pneumophila in water facilities and reducing the variability in the analyses due to the low need for manipulation. Moreover, corrective measures may be applied the same day of the analysis. This method considerably reduces the detection time compared with the conventional, gold-standard detection culture method that requires more than 10 days, being decisive to prevent outbreaks.


One Step for Legionella pneumophila Detection in Environmental Samples by DNA-gold Nanoparticle Probe

Nuthong B, Wilailuckana C, Tavichakorntrakool R, Boonsiri P, Daduang S, Bunyaraksyotin G, Suphan O, Daduang J.

Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand.

J Appl Microbiol  2018 Nov;125(5):1534-1540. 

Abstract: Aims: To develop and evaluate a DNA-gold nanoparticle (DNA-AuNP) probe assay to detect Legionella pneumophila, which causes Legionnaires' disease, compared with the gold standard culture method. Methods and results: Gold nanoparticles (AuNPs) were conjugated with DNA probes to detect the mip gene of L. pneumophila. The DNA-AuNP probe assay was evaluated for its specificity, sensitivity and stability. The results showed that only L. pneumophila mixed with this probe resulted in a red solution that was easily detected by the naked eye, and the colour was stable when 10 mmol l-1 MgSO4 was added. The 100 Legionella isolates and 10 other bacteria led to 100% specificity. Compared with the culture method, our method showed a 100% negative predictive value, 100% sensitivity (kappa=0.87), and a detection limit of 4.5 ng DNA μl-1 with a 6-min response time for the 124 colonies suspected of being Legionella. The DNA-AuNP probe reagents were stable for more than 6 months. Conclusions: The developed DNA-AuNP probe assay has good negative predictive value, sensitivity, rapidity and ease of use, which is helpful for ruling out negative samples. Significance and impact of the study: The DNA-AuNP probe assay can detect the mip gene of L. pneumophila. Therefore, it may be an alternative method for screening colonies suspected of being L. pneumophila.


Rapid Detection of Viable Legionella pneumophila in Tap Water by a qPCR and RT-PCR-based Method

Boss R, Baumgartner A, Kroos S, Blattner M, Fretz R, Moor D.

Federal Food Safety and Veterinary Office, Berne, Switzerland. e

Appl Microbiol 2018 Oct;125(4):1216-1225.

Abstract: Aims: A molecular method for a rapid detection of viable Legionella pneumophila of all serogroups in tap water samples was developed as an alternative to the reference method (ISO). Legionellae are responsible for Legionnaires' disease, a severe pneumonia in humans with high lethality. Methods and results: The developed method is based on a nutritional stimulation and detection of an increase in precursor 16S rRNA as an indicator for viability. For quantification, DNA was detected by qPCR. This method was compared to the ISO method using water samples obtained from public sports facilities in Switzerland. The sensitivity and specificity were 91 and 97%, respectively, when testing samples for compliance with a microbiological criterion of 1000 cell equivalents per l. Conclusion: The new method is sensitive and specific for L. pneumophila and allows results to be obtained within 8 h upon arrival, compared to one week or more by the ISO method. Significance and impact of the study: The method represents a useful tool for a rapid detection of viable L. pneumophila of all serogroups in water by molecular biology. It can be used as an alternative to the ISO method for official water analysis for legionellae and particularly when a short test time is required.


Legionella pneumophila Recurrently Isolated in a Spanish Hospital: Two Years of Antimicrobial Resistance Surveillance

Graells T, Hernández-García M, Pérez-Jové J, Guy L, Padilla E.

Departament de Microbiologia, CATLAB Centre Analítiques AIE, Viladecavalls, Barcelona, Spain.

Environ Res 2018 Oct:166:638-646.

Abstract: Objectives: The aim of this study was to monitor the spread, persistence and antibiotic resistance patterns of Legionella spp. strains found in a hospital water distribution system. These environmental studies are intended to help detect the presence of antibiotic resistant strains before they infect patients. Methods: Antimicrobial surveillance tests were performed at 27 different sampling points of the water network of a large Spanish hospital over two years. Water samples were screened for Legionella according to ISO 11731:2007. Legionella spp. isolates were identified by serotyping and by mass spectrometry (MALDI-ToF). Epidemiological molecular typing was done by Pulse-Field Gel Electrophoresis (PFGE) and by Sequence-Based Typing (SBT). Antibiotic susceptibility tests were performed using disk diffusion and ETEST®. Results: Legionella spp. were recurrently isolated for 2 years. All isolates belonged the same group, L. pneumophila serogroups 2-14. Isolates were all attributed by SBT to sequence type (ST) ST328, although PFGE revealed 5 different patterns. No significant change in antibiotic susceptibility could be observed for this study period, irrespectively of the method used. Conclusion: Colonization of water systems by Legionella spp. is still occurring, although all the prevention rules were strictly followed. Antibiotic resistance monitoring may help us to find resistance in bacteria with environmental reservoirs but difficult to isolate from patients. The knowledge of the antibiotic susceptibility in environmental strains may help us to predict changes in clinical strains. This study might also help reconsidering Legionnaires' disease (LD) diagnostic methods. L. pneumophila serogroups 2-14 present all along the time of the investigation in the water distribution system can cause LD. However, they may not be detected by routine urine tests run on patients, thereby missing an ongoing LD infection.


Evaluation of Legiolert for Quantification of Legionella pneumophila From Non-potable Water

Rech MM, Swalla BM, Dobranic JK.

EMSL Analytical, Inc., 1010 Yuma Street, Denver, CO, 80204, USA.

Curr Microbiol 2018 Oct;75(10):1282-1289.

Abstract: Legiolert® is a new culture method for quantification of Legionella pneumophila, which is the primary species associated with Legionnaires' disease. The test is based on a most probable number approach and differs significantly from traditional culture methods by providing results at 7 days, rapid sample preparation and analysis, and objective interpretation of test results. In this study, we compared the performance of Legiolert with the U.S. Centers for Disease Control and Prevention (CDC) method for detection of L. pneumophila from non-potable samples, primarily comprising cooling tower waters. Our results demonstrated no significant difference between Legiolert and the CDC method for quantification of L. pneumophila. However, Legiolert showed a significant increase in sensitivity when water samples containing higher L. pneumophila concentrations were examined. Cooling tower waters often contain non-Legionella organisms (NLO) that interfere with traditional Legionella test methods, and we observed varying degrees of NLO interference on many CDC method plates. In contrast, Legiolert was resistant to NLO interference and produced a very low rate of false-positive results. Collectively, Legiolert is a sensitive and specific method for quantification of L. pneumophila from non-potable water that provides advantages over the CDC method.


Low Genomic Diversity of Legionella pneumophila within Clinical Specimens

David S, Mentasti M, Parkhill J, Chalker VJ.

Centre for Genomic Pathogen Surveillance, Wellcome Genome Campus, Hinxton, Cambridge, United Kingdom.

Clin Microbiol Infect 2018 Sep;24(9):1020.e1-1020.e4.

Abstract: Objectives: Legionella pneumophila is the leading cause of Legionnaires' disease, a severe form of pneumonia acquired from environmental sources. Investigations of both sporadic cases and outbreaks rely mostly on analysis of a single to a few colony pick(s) isolated from each patient. However, because of the lack of data describing diversity within single patients, the optimal number of picks is unknown. Here, we investigated diversity within individual patients using sequence-based typing (SBT) and whole-genome sequencing (WGS). Methods: Ten isolates of L. pneumophila were obtained from each of ten epidemiologically unrelated patients. SBT and WGS were undertaken, and single-nucleotide polymorphisms (SNPs) were identified between isolates from the same patient. Results: The same sequence type (ST) was obtained for each set of ten isolates. Using genomic analysis, zero SNPs were identified between isolates from seven patients, a maximum of one SNP was found between isolates from two patients, and a maximum of two SNPs was found amongst isolates from one patient. Assuming that the full within-host diversity has been captured with ten isolates, statistical analyses showed that, on average, analysis of one isolate would yield a 70% chance of capturing all observed genotypes, and seven isolates would yield a 90% chance. Conclusions: SBT and WGS analyses of multiple colony picks obtained from ten patients showed no, or very low, within-host genomic diversity in L. pneumophila, suggesting that analysis of one colony pick per patient will often be sufficient to obtain reliable typing data to aid investigation of cases of Legionnaires' disease.


Assessment of Flow Cytometry for Microbial Water Quality Monitoring in Cooling Tower Water and Oxidizing Biocide Treatment Efficiency

Helmi K, David F, Di Martino P, Jaffrezic MP, Ingrand V.

Veolia Recherche et Innovation, Chemin de la Digue, Maisons-Laffitte, France.

J Microbiol Methods 2018 Sep;152:201-209.

Abstract: The control of Legionella proliferation in cooling tower water circuits requires regular monitoring of water contamination and effective disinfection procedures. In this study, flow cytometry was assessed to monitor water contamination and disinfection treatment efficiency on bacterial cells regarding nucleic acid injury (SYBR® Green II), cell integrity (SYBR® Green II and propidium iodide) and metabolism activity (ChemChrome V6). A total of 27 cooling tower water samples were analyzed in order to assess water contamination levels regarding viable populations: standard culture, ATP measurement and flow cytometry methods were compared. Flow cytometry and plate counts methods showed a significant correlation for changes in concentrations despite a 1 to 2-log difference regarding absolute quantification. Concerning intracellular activity, the use of two different flow cytometers (FACSCanto™ II and Accuri™ C6) showed no statistical difference while a difference was observed between flow cytometry and usual methods (culture and ATP measurement). The standard culture and flow cytometry methods were also compared for in vitro bacteria inactivation measurements in the presence of 3 different types of oxidizing biocides commonly used for cooling tower disinfection. Reductions observed ranged between 1 and 2 log depending on (1) the detection method, (2) the bacterial population origin and/or (3) the active biocide molecule used. In conclusion, flow cytometry represents an efficient, accurate and fast approach to monitor water contamination and biocide treatment efficiency in cooling towers.


Use of Serology and Polymerase Chain Reaction to Detect Atypical Respiratory Pathogens During Acute Exacerbation of Chronic Obstructive Pulmonary Disease

Jung CY, Choe YH, Lee SY, Kim WJ, Lee JD, Ra SW, Choi EG, Lee JS, Park MJ, Na JO.

Division of Pulmonology, Department of Internal Medicine, Soonchunhyang University College of Medicine, Cheonan, Korea.

Korean J Intern Med 2018 Sep;33(5):941-951.

Abstract: Background/aims: To use serological and multiplex polymerase chain reaction (PCR) assays to examine sputum samples from patients experiencing acute exacerbation of chronic obstructive pulmonary disease (AECOPD) for the presence of atypical pathogens, including Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella pneumophila. Methods: From September 2012 to February 2014, 341 patients with AECOPD attending outpatient clinics were enrolled as part of a randomized, double-blind, multicenter study. A commercial enzyme-linked immunosorbent assay was used to measure serum immunoglobulin M (IgM) and IgG antibody titers on the first day of the study and at 36 days post-enrollment. Multiplex PCR was used to test sputum samples for the presence of atypical pathogens. A urinary antigen test for L. pneumophila was performed on the first day. Results: Nineteen patients (5.6%) showed serological evidence of acute infection with M. pneumoniae. Also, one and seven patients (2%) showed serological evidence of acute infection with C. pneumoniae and L. pneumophila, respectively. All DNA samples were negative for M. pneumoniae, C. pneumoniae, and L. pneumophila according to PCR. Only one urine sample was positive for L. pneumophila antigen, but serologic evidence was lacking. Conclusion: Serological testing suggested that infection by atypical pathogens during AECOPD was relatively uncommon. In addition, PCR provided no direct evidence of infection by atypical pathogens. Thus, atypical pathogens may not be a major cause of AECOPD in South Korea.


Comparison of the Legiolert™/Quanti-Tray ® MPN Test for the Enumeration of Legionella pneumophila from Potable Water Samples with the German Regulatory Requirements Methods ISO 11731-2 and ISO 11731

Spies K, Pleischl S, Lange B, Langer B, Hübner I, Jurzik L, Luden K, Exner M.

Universitätsklinikum Bonn, Institut für Hygiene und Öffentliche Gesundheit, Bonn, Germany.

Int J Hyg Environ Health 2018 Aug;221(7):1047-1053.

Abstract: Due to the promising results of a previous study of the performance of the novel MPN method (Legiolert™/Quanti-Tray®) compared to ISO 11731-2, this study was performed to compare Legiolert for Legionella pneumophila with the German regulatory requirements methods ISO 11731-2 (100 ml membrane filtration) and ISO 11731 (1 ml direct plating) for the enumeration of L. pneumophila and Legionella spp. from potable water. Data from a multi-laboratory study according to ISO 17994 showed that Legiolert yielded on average higher counts of L. pneumophila than the ISO 11731-2 method, but the comparison with ISO 11731 was inconclusive due to the number of samples needing to be tested. Likewise, comparisons of the MPN method for 100 ml to the highest result of either ISO 11731 or ISO 11731-2 according to Federal Environmental Agency recommendation (2012) yielded no conclusive difference, regardless of whether non-pneumophila species of Legionella were included in the evaluation. The MPN method has a high specificity for L. pneumophila of 97.9% which compares favourably to the specificity of 95.3% quoted for ISO 11731. The new method represents a significant improvement in the enumeration of L. pneumophila from drinking water and related samples.


Detection of Legionella anisa in Water from Hospital Dental Chair Units and Molecular Characterization by Whole-Genome Sequencing

Fleres G, Couto N, Lokate M, van der Sluis LWM, Ginevra C, Jarraud S, Deurenberg RH, Rossen JW, García-Cobos S, Friedrich AW.

Department of Medical Microbiology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.

Microorganisms 2018 Jul;6(3):71.

Abstract: This study aims to assess contamination with Legionella spp. in water from dental chair units (DCUs) of a hospital dental ward and to perform its molecular characterization by whole-genome sequencing (WGS). We collect eight water samples (250 mL) from four DCUs (sink and water-syringe). Samples are tested for the presence of Legionella spp. (CFUs/mL) by culturing according to the Nederland Norm (NEN) 6265. Three DCUs are found positive for Legionella anisa, and four isolates are cultured (sink n=2, water-syringe n=1; two isolates from the same chair) with 1×10² CFU/mL. Whole-genome multi-locus sequence typing (wgMLST) results indicate that all strains belong to the same cluster with two to four allele differences. Classical culture combined with WGS allows the identification of a unique clone of L. anisa in several DCUs in the same hospital dental ward. This may indicate a common contamination source in the dental unit waterlines, which was fixed by replacing the chairs and main pipeline of the unit. Our results reveal tap water contamination in direct contact with patients and the usefulness of WGS to investigate bacterial molecular epidemiology.


Comparison of Database Search Methods for the Detection of Legionella pneumophila in Water Samples Using Metagenomic Analysis

Borthong J, Omori R, Sugimoto C, Suthienkul O, Nakao R, Ito K.

Division of Bioinformatics, Research Center for Zoonosis Control, Hokkaido University, Sapporo, Japan.

Front Microbiol 2018 Jun;9:1272.

Abstract: Metagenomic analysis has become a powerful tool to analyze bacterial communities in environmental samples. However, the detection of a specific bacterial species using metagenomic analysis remains difficult due to false positive detections of sequences shared between different bacterial species. In this study, 16S rRNA amplicon and shotgun metagenomic analyses were conducted on samples collected along a stream and ponds in the campus of Hokkaido University. We compared different database search methods for bacterial detection by focusing on Legionella pneumophila. In this study, we used L. pneumophila-specific nested PCR as a gold standard to evaluate the results of the metagenomic analysis. Comparison with the results from L. pneumophila-specific nested PCR indicated that a blastn search of shotgun reads against the NCBI-NT database led to false positive results and had problems with specificity. We also found that a blastn search of shotgun reads against a database of the catalase-peroxidase (katB) gene detected L. pneumophila with the highest area under the receiver operating characteristic curve among the tested search methods; indicating that a blastn search against the katB gene database had better diagnostic ability than searches against other databases. Our results suggest that sequence searches targeting long genes specifically associated with the bacterial species of interest is a prerequisite to detecting the bacterial species in environmental samples using metagenomic analyses.


Quantitative Imaging Flow Cytometry of Legionella-Infected Dictyostelium Amoebae Reveals the Impact of Retrograde Trafficking on Pathogen Vacuole Composition

Welin A, Weber S, Hilbi H.

Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland.

Appl Environ Microbiol 2018 May;84(11):e00158-18.

Abstract: The ubiquitous environmental bacterium Legionella pneumophila survives and replicates within amoebae and human macrophages by forming a Legionella-containing vacuole (LCV). In an intricate process governed by the bacterial Icm/Dot type IV secretion system and a plethora of effector proteins, the nascent LCV interferes with a number of intracellular trafficking pathways, including retrograde transport from endosomes to the Golgi apparatus. Conserved retrograde trafficking components, such as the retromer coat complex or the phosphoinositide (PI) 5-phosphatase D. discoideum 5-phosphatase 4 (Dd5P4)/oculocerebrorenal syndrome of Lowe (OCRL), restrict intracellular replication of L. pneumophila by an unknown mechanism. Here, we established an imaging flow cytometry (IFC) approach to assess in a rapid, unbiased, and large-scale quantitative manner the role of retrograde-linked PI metabolism and actin dynamics in the LCV composition. Exploiting Dictyostelium discoideum genetics, we found that Dd5P4 modulates the acquisition of fluorescently labeled LCV markers, such as calnexin, the small GTPase Rab1 (but not Rab7 and Rab8), and retrograde trafficking components (Vps5, Vps26, Vps35). The actin-nucleating protein and retromer interactor WASH (Wiskott-Aldrich syndrome protein [WASP] and suppressor of cAMP receptor [SCAR] homologue) promotes the accumulation of Rab1 and Rab8 on LCVs. Collectively, our findings validate IFC for the quantitative and unbiased analysis of the pathogen vacuole composition and reveal the impact of retrograde-linked PI metabolism and actin dynamics on the LCV composition. The IFC approach employed here can be adapted for a molecular analysis of the pathogen vacuole composition of other amoeba-resistant pathogens. IMPORTANCE: Legionella pneumophila is an amoeba-resistant environmental bacterium which can cause a life-threatening pneumonia termed Legionnaires' disease. In order to replicate intracellularly, the opportunistic pathogen forms a protective compartment, the Legionella-containing vacuole (LCV). An in-depth analysis of the LCV composition and the complex process of pathogen vacuole formation is crucial for understanding the virulence of L. pneumophila. Here, we established an imaging flow cytometry (IFC) approach to assess in a rapid, unbiased, and quantitative manner the accumulation of fluorescently labeled markers and probes on LCVs. Using IFC and L. pneumophila-infected Dictyostelium discoideum or defined mutant amoebae, a role for phosphoinositide (PI) metabolism, retrograde trafficking, and the actin cytoskeleton in the LCV composition was revealed. In principle, the powerful IFC approach can be used to analyze the molecular composition of any cellular compartment harboring bacterial pathogens.


Adaptation of Amoeba Plate Test to Recover Legionella Strains from Clinical Samples

Descours G, Hannetel H, Reynaud JV, Ranc AG, Beraud L, Kolenda C, Campese C, Lina G, Ginevra C, Jarraud S.

Hospices Civils de Lyon, Groupement Hospitalier Nord, National Reference Centre for Legionella, Institute for Infectious Agents, Lyon, France.

J Clin Microbiol 2018 Apr;56(5):e01361-17.

Abstract: The isolation of Legionella from respiratory samples is the gold standard for diagnosis of Legionnaires' disease (LD) and enables epidemiological studies and outbreak investigations. The purpose of this work was to adapt and to evaluate the performance of an amoebic coculture procedure (the amoeba plate test [APT]) for the recovery of Legionella strains from respiratory samples, in comparison with axenic culture and liquid-based amoebic coculture (LAC). Axenic culture, LAC, and APT were prospectively performed with 133 respiratory samples from patients with LD. The sensitivities and times to results for the three techniques were compared. Using the three techniques, Legionella strains were isolated in 46.6% (n=62) of the 133 respiratory samples. The sensitivity of axenic culture was 42.9% (n=57), that of LAC was 30.1% (n=40), and that of APT was 36.1% (n=48). Seven samples were positive by axenic culture only; for those samples, there were <10 colonies in total. Five samples, all sputum samples, were positive by an amoebic procedure only (5/5 samples by APT and 2/5 samples by LAC); all had overgrowth by oropharyngeal flora with axenic culture. The combination of axenic culture with APT yielded a maximal isolation rate (i.e., 46.6%). Overall, the APT significantly reduced the median time for Legionella identification to 4 days, compared with 7 days for LAC (P<0.0001). The results of this study support the substitution of LAC by APT, which could be implemented as a second-line technique for culture-negative samples and samples with microbial overgrowth, especially sputum samples. The findings provide a logical basis for further studies in both clinical and environmental settings.


Comparative Genome Analysis Reveals a Complex Population Structure of Legionella pneumophila Subspecies

Kozak-Muiznieks NA, Morrison SS, Mercante JW, Ishaq MK, Johnson T, Caravas J, Lucas CE, Brown E, Raphael BH, Winchell JM.

Division of Bacterial Diseases, National Center for Immunization and Respiratory Diseases, CDC, Atlanta, GA, USA.

Infect Genet Evol 2018 Apr;59:172-185.

Abstract: The majority of Legionnaires' disease (LD) cases are caused by Legionella pneumophila, a genetically heterogeneous species composed of at least 17 serogroups. Previously, it was demonstrated that L. pneumophila consists of three subspecies: pneumophila, fraseri and pascullei. During an LD outbreak investigation in 2012, we detected that representatives of both subspecies fraseri and pascullei colonized the same water system and that the outbreak-causing strain was a new member of the least represented subspecies pascullei. We used partial sequence-based typing consensus patterns to mine an international database for additional representatives of fraseri and pascullei subspecies. As a result, we identified 46 sequence types (STs) belonging to subspecies fraseri and two STs belonging to subspecies pascullei. Moreover, a recent retrospective whole genome sequencing analysis of isolates from New York State LD clusters revealed the presence of a fourth L. pneumophila subspecies that we have termed raphaeli. This subspecies consists of 15 STs. Comparative analysis was conducted using the genomes of multiple members of all four L. pneumophila subspecies. Whereas each subspecies forms a distinct phylogenetic clade within the L. pneumophila species, they share more average nucleotide identity with each other than with other Legionella species. Unique genes for each subspecies were identified and could be used for rapid subspecies detection. Improved taxonomic classification of L. pneumophila strains may help identify environmental niches and virulence attributes associated with these genetically distinct subspecies.


Detection of Respiratory Bacterial Pathogens Causing Atypical Pneumonia by Multiplex Lightmix ® RT-PCR

Wagner K, Springer B, Imkamp F, Opota O, Greub G, Keller PM.

Institute of Medical Microbiology, University of Zurich, Switzerland. pkeller@imm.uzh

Int J Med Microbiol 2018 Apr;308(3):317-323.

Abstract: Pneumonia is a severe infectious disease. In addition to common viruses and bacterial pathogens (e.g. Streptococcus pneumoniae), fastidious respiratory pathogens like Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella spp. can cause severe atypical pneumonia. They do not respond to penicillin derivatives, which may cause failure of antibiotic empirical therapy. The same applies for infections with B. pertussis and B. parapertussis, the cause of pertussis disease, that may present atypically and need to be treated with macrolides. Moreover, these fastidious bacteria are difficult to identify by culture or serology, and therefore often remain undetected. Thus, rapid and accurate identification of bacterial pathogens causing atypical pneumonia is crucial. We performed a retrospective method evaluation study to evaluate the diagnostic performance of the new, commercially available Lightmix® multiplex RT-PCR assay that detects these fastidious bacterial pathogens causing atypical pneumonia. In this retrospective study, 368 clinical respiratory specimens, obtained from patients suffering from atypical pneumonia that have been tested negative for the presence of common agents of pneumonia by culture and viral PCR, were investigated. These clinical specimens have been previously characterized by singleplex RT-PCR assays in our diagnostic laboratory and were used to evaluate the diagnostic performance of the respiratory multiplex Lightmix® RT-PCR. The multiplex RT-PCR displayed a limit of detection between 5 and 10 DNA copies for different in-panel organisms and showed identical performance characteristics with respect to specificity and sensitivity as in-house singleplex RT-PCRs for pathogen detection. The Lightmix® multiplex RT-PCR assay represents a low-cost, time-saving and accurate diagnostic tool with high throughput potential. The time-to-result using an automated DNA extraction device for respiratory specimens followed by multiplex RT-PCR detection was below 4 h, which is expected to significantly improve diagnostics for atypical pneumonia-associated bacterial pathogens.


The Diagnosis of Legionella pneumophila Serogroup 5 Bacteremic Pneumonia During Severe Neutropenia Using Loop-mediated Isothermal Amplification

Moriguchi S, Abe M, Kimura M, Yoshino C, Baba M, Okada C, Izutsu K, Taniguchi S, Araoka H, Yoneyama A.

Department of Hematology, Toranomon Hospital, Japan.

Intern Med 2018 Apr;57(7):1045-1048.

Abstract: A 60-year-old man developed pneumonia after undergoing autologous peripheral blood stem cell transplantation for diffuse large-B cell lymphoma. A urinary antigen test and sputum culture were both negative for Legionella pneumophila; however, a sputum sample that was examined by loop-mediated isothermal amplification (LAMP) was positive for Legionella spp. On admission, the results of blood culturing using a BACTEC system were negative for 7 days. However, L. pneumophila serogroup 5 was detected in a blood subculture using WYOα medium. The patient was successfully treated with a fluoroquinolone-based regimen. LAMP is useful for the diagnosis of Legionella spp.


Legionella Contamination in Warm Water Systems: A Species-Level Survey

Dilger T, Melzl H, Gessner A.

AGROLAB Labor GmbH, Eching am Ammersee, Germany.

Int J Hyg Environ Health 2018 Mar;221(2):199-210.

Abstract: Legionellae constitute a frequent contamination of warm water systems and can lead to serious infections. Therefore, in many countries it is mandatory to monitor warm water systems for their presence. The method of examination in Germany is regulated by guideline ISO 11731 and DIN EN ISO 11731-2, and the results are reported as concentration of Legionella spp. Only limited information is available on the presence of individual species of Legionellae in the examined systems, since most investigations and research focus solely on Legionella pneumophila as the most important human pathogen. In this study 76,220 samples obtained from 13,397 warm water systems originating from 24 different zip code districts covering an area of more than 71,000km2 in southern Germany were examined. This resulted in the identification of 47,924 Legionella isolates to the species level using a MALDI-TOF mass spectrometry-based method. Legionella species distribution was analyzed with respect to warm water system type, geographic region (defined as zip code district) and temperature during sample taking. Overall, 20.7% of the samples were found positive for Legionella species and 14 different species of Legionella were recovered. These were not equally present throughout the geographic area investigated, but instead an individual regional diversity of Legionella species was observed for the examined zip code districts. Although Legionella pneumophila represented 84% of all contaminations found, depending on the geographical region its proportion varied substantially between 57.5% and 91.2%. The occurrence of other species was also of importance since they accounted for up to 42% of contaminations regionally, with Legionella londiniensis being most prominent representing up to 38.8% of recovered colonies. In addition, the influence of temperature on the individual species was disparate, but the temperature range between 50°C and 59°C was identified as the optimal condition for facilitating emergence of the majority of recovered Legionella species. The identification of Legionella to the species level by MALDI-TOF allowed for a more concise depiction of the regional distribution and the ecology of this genus and may be of additional value when counter measures need to be initiated.


Evaluation of a Most Probable Number Method for the Enumeration of Legionella pneumophila From North American Potable and Nonpotable Water Samples

Petrisek R, Hall J.

H.P. Environmental, Inc., 104 Elden Street, Herndon, VA, USA.

J Water Health 2018 Feb;16(1):25-33.

Abstract: This study compares the performance of a novel most probable number (MPN) method (Legiolert™/Quanti-Tray®) with Standard Methods for the Examination of Water and Wastewater 9260 J for the enumeration of Legionella pneumophila from potable and nonpotable waters. Data from the study showed that Legiolert exhibited higher sensitivity for the detection of L. pneumophila for potable water and equivalent sensitivity for nonpotable water. The Legiolert medium had a high specificity with no false positive signals reported for either water type. The new method represents a significant improvement in usability and accuracy in the enumeration of L. pneumophila.


Molecular Typing of Legionella pneumophila Isolates from Environmental Water Samples and Clinical Samples Using a Five-Gene Sequence Typing and Standard Sequence-Based Typing

Zhan XY, Zhu QY.

Guangzhou KingMed Center for Clinical Laboratory, Guangzhou, China.

PLoS One 2018 Feb;13(2):e0190986. 

Abstract: Inadequate discriminatory power to distinguish between L. pneumophila isolates, especially those belonging to disease-related prevalent sequence types (STs) such as ST1, ST36 and ST47, is an issue of SBT scheme. In this study, we developed a multilocus sequence typing (MLST) scheme based on two non-virulence loci (trpA, cca) and three virulence loci (icmK, lspE, lssD), to genotype 110 L. pneumophila isolates from various natural and artificial water sources in Guangdong province of China and compared with the SBT. The isolates were assigned to 33 STs of the SBT and 91 new sequence types (nSTs) of the MLST. The indices of discrimination (IODs) of SBT and MLST were 0.920 and 0.985, respectively. Maximum likelihood trees of the concatenated SBT and MLST sequences both showed distinct phylogenetic relationships between the isolates from the two environments. More intragenic recombinations were detected in nSTs than in STs, and they were both more abundant in natural water isolates. We found out the MLST had a high discriminatory ability for the disease associated ST1 isolates: 22 ST1 isolates were assigned to 19 nSTs. Furthermore, we assayed the discrimination of the MLST for 29 reference strains (19 clinical and 10 environmental). The clinical strains were assigned to eight STs and ten nSTs. The MLST could also subtype the prevalent clinical ST36 or ST47 strains: eight ST36 strains were subtyped into three nSTs and two ST47 strains were subtyped into two nSTs. We found different distribution patterns of nSTs between the environmental and clinical ST36 isolates, and between the outbreak clinical ST36 isolates and the sporadic clinical ST36 isolates. These results together revealed the MLST scheme could be used as part of a typing scheme that increased discrimination when necessary.


Legionella Confirmation in Cooling Tower Water. Comparison of Culture, Real-Time PCR and Next Generation Sequencing

Farhat M, Shaheed RA, Al-Ali HH, Al-Ghamdi AS, Al-Hamaqi GM, Maan HS, Al-Mahfoodh ZA, Al-Seba HZ.

Department of Biochemistry, Imam Abdulrahman Bin Faisal University, Dammam, Kindgdom of Saudi Arabia.

Saudi Med J 2018 Feb;39(2):137-141.

Abstract: Objectives: To investigate the presence of Legionella spp in cooling tower water. Legionella proliferation in cooling tower water has serious public health implications as it can be transmitted to humans via aerosols and cause Legionnaires' disease. Methods: Samples of cooling tower water were collected from King Fahd Hospital of the University (KFHU) (Imam Abdulrahman Bin Faisal University, 2015/2016). The water samples were analyzed by a standard Legionella culture method, real-time polymerase chain reaction (RT-PCR), and 16S rRNA next-generation sequencing. In addition, the bacterial community composition was evaluated. Results: All samples were negative by conventional Legionella culture. In contrast, all water samples yielded positive results by real-time PCR (105 to 106 GU/L). The results of 16S rRNA next generation sequencing showed high similarity and reproducibility among the water samples. The majority of sequences were Alpha-, Beta-, and Gamma-proteobacteria, and Legionella was the predominant genus. The hydrogen-oxidizing gram-negative bacterium Hydrogenophaga was present at high abundance, indicating high metabolic activity. Sphingopyxis, which is known for its resistance to antimicrobials and as a pioneer in biofilm formation, was also detected. Conclusion: Our findings indicate that monitoring of Legionella in cooling tower water would be enhanced by use of both conventional culturing and molecular methods.


Antibody Test for Legionella pneumophila Detection

Párraga-Niño N, Quero S, Uria N, Castillo-Fernandez O, Jimenez-Ezenarro J, Muñoz FX, Sabrià M, Garcia-Nuñez M.

Unitat de Malalties Infeccioses, Fundació Institut d'Investigació Germans Trias I Pujol, Badalona, Spain.

Diagn Microbiol Infect Dis 2018 Feb;90(2):85-89.

Abstract: Legionella pneumophila is responsible for Legionnaires' disease (LD). Its detection in both environmental and clinical samples is mainly performed by culture plate method which requires up to 10days to obtain results. Nowadays, there are commercial antibodies against this bacterium, but they have not been tested against all subgroups of L. pneumophila sg 1 or serogroups 1-16 or their cross-reactions with other non-Legionella bacteria. Indeed, many of these antibodies became available when only 8 serogroups of L. pneumophila had been described. We tested 7 antibodies and found that 2 (Mab 8/5 and OBT) specifically detected all the subgroups of L. pneumophila sg 1, one without cross-reactions (Mab8/5). Moreover, the LP3IIG2 antibody detected almost all serogroups tested with lower rates of cross-reactivity, resulting in a specific sensitive antibody for the detection of L. pneumophila. LP3IIG2 presented higher rate of cross-reactivity against respiratory non-Legionella isolates, thereby contraindicating its clinical applicability.


Quantification of Viable and Non-Viable Legionella Spp. By Heterogeneous Asymmetric Recombinase Polymerase Amplification (haRPA) on a Flow-Based Chemiluminescence Microarray

Kober C, Niessner R, Seidel M.

Institute of Hydrochemistry, Chair of Analytical Chemistry and Water Chemistry, Technical University of Munich, München, Germany.

Biosens Bioelectron 2018 Feb;100:49-55.

Abstract: Increasing numbers of legionellosis outbreaks within the last years have shown that Legionella are a growing challenge for public health. Molecular biological detection methods capable of rapidly identifying viable Legionella are important for the control of engineered water systems. The current gold standard based on culture methods takes up to 10 days to show positive results. For this reason, a flow-based chemiluminescence (CL) DNA microarray was developed that is able to quantify viable and non-viable Legionella spp. as well as Legionella pneumophila in one hour. An isothermal heterogeneous asymmetric recombinase polymerase amplification (haRPA) was carried out on flow-based CL DNA microarrays. Detection limits of 87 genomic units (GU) µL-1 and 26GUµL-1 for Legionella spp. and Legionella pneumophila, respectively, were achieved. In this work, it was shown for the first time that the combination of a propidium monoazide (PMA) treatment with haRPA, the so-called viability haRPA, is able to identify viable Legionella on DNA microarrays. Different proportions of viable and non-viable Legionella, shown with the example of L. pneumophila, ranging in a total concentration between 101 to 105GUµL-1 were analyzed on the microarray analysis platform MCR 3. Recovery values for viable Legionella spp. were found between 81% and 133%. With the combination of these two methods, there is a chance to replace culture-based methods in the future for the monitoring of engineered water systems like condensation recooling plants.


Water Cultures Are More Sensitive Than Swab Cultures for the Detection of Environmental Legionella

Decker BK, Harris PL, Toy DL, Muder RR, Sonel AF, Clancy CJ.

Infectious Diseases Section, Veterans Affairs Pittsburgh Healthcare System, Pittsburgh, Pennsylvania.

Infect Control Hosp Epidemiol 2018 Jan;39(1):108-110.

Abstract: Water cultures were significantly more sensitive than concurrently collected swab cultures (n=2,147 each) in detecting Legionella pneumophila within a Veterans Affairs healthcare system. Sensitivity for water versus swab cultures was 90% versus 30% overall, 83% versus 48% during a nosocomial Legionnaires' disease outbreak, and 93% versus 22% post outbreak.


MALDI-TOF MS Analysis as a Useful Tool for an Identification of Legionella pneumophila, a Facultatively Pathogenic Bacterium Interacting with Free-Living Amoebae: A Case Study from Water Supply System of Hospitals in Bratislava (Slovakia)

Trnková K, Kotrbancová M, Špaleková M, Fulová M, Boledovičová J, Vesteg M.

Department of the Environment, Faculty of Natural Sciences, Matej Bel University, Tajovského 55, 974 01 Banská Bystrica, Slovakia.

Exp Parasitol 2018 Jan;184:97-102.

Abstract: Legionellae, i.e. Legionella pneumophila, are human bacterial hydrophilic facultative pathogens causing pneumonia (Legionnaires' disease). Free-living amoebae (FLA) can serve as natural hosts and thus as reservoirs of many amoebae-resistant bacteria. An encysted amoeba can contribute to the resistance of intracellular L. pneumophila to various chemical and physical treatments. Humans can be infected by droplets containing bacteria from an environmental source or human-made devices such as shower heads, bathtubs, air-conditioning units or whirlpools. In this study, we were investigating the presence of FLA and L. pneumophila in plumbing systems of healthcare facilities in Bratislava (Slovakia) by standard diagnostic methods, while the presence of L. pneumophila was verified also by MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) analysis. The results showed the occurrence of L. pneumophila and FLA in 62.26% and 66.4% of samples taken from four pediatric clinics, respectively. Both standard methods and MALDI-TOF MS showed comparable results and they can be successfully applied for the identification of L. pneumophila strains in environmental samples. Our approach could be useful for further monitoring, prevention and decreasing risk of Legionella infection also in other hospitals.


A gyrB Oligonucleotide Microarray for the Specific Detection of Pathogenic Legionella and Three Legionella pneumophila Subsp

Xi D, Dou Y, Ren W, Yang S, Feng L, Cao B, Wang L.

The Key Laboratory of Molecular Microbiology and Technology of the Ministry of Education, Nankai University, Tianjin, China.

Antonie Van Leeuwenhoek 2017 Dec;110(12):1515-1525.

Abstract: Among the 50 species and 70 serogroups of Legionella identified, Legionella pneumophila, comprising three subsp. (subsp. pneumophila, subsp. fraseri, and subsp. pasculleii), is recognized as the major cause of epidemic legionellosis. Rapid and reliable assays to identify pathogenic Legionella spp., and the three L. pneumophila subsp. in particular, are in great demand. In this study, we analyzed the gyrB genes of eleven Legionella spp. and subsp., comprising L. anisa, L. bozemanii, L. dumoffii, L. feeleii, L. gormanii, L. longbeachae, L. micdadei, L. waltersii, L. pneumophila subsp. pneumophila, L. pneumophila subsp. fraseri, and L. pneumophila subsp. pasculleii. We developed a rapid oligonucleotide microarray detection technique to identify accurately these common pathogenic Legionella spp. and L. pneumophila subsp. To detect multiple Legionella species with high specificity, 31 reproducible probes were designed in the array. Sixty-one strains were analyzed in total, including 37 target pathogens and 24 non-target bacterial species used to validate the microarray. The sensitivity of the detection was 1.0 ng using genomic DNA of three Legionella spp., L. anisa, L. dumoffii, and L. waltersii, or 13 CFU/100 mL using the cultured L. pneumophila subsp. pneumophila. Eight isolated strains were tested using the microarray with 100% accuracy. The data indicated that the technique is an efficient method to diagnose and detect Legionella spp. and subsp. in basic microbiology, clinical diagnosis, epidemiological surveillance, and food safety applications. In addition, a phylogenetic study based on the gyrB gene revealed the genetic relationship among the different Legionella spp. and subsp.


DNA Microarray for Rapid Detection and Identification of Food and Water Borne Bacteria: From Dry to Wet Lab

Ranjbar R, Behzadi P, Najafi A, Roudi R.

Department of Microbiology, College of Basic Sciences, Shahr-e-Qods Branch, Islamic Azad University, Tehran, Iran.

Open Microbiol J 2017 Nov;11:330-338.

Abstract: Background: A rapid, accurate, flexible and reliable diagnostic method may significantly decrease the costs of diagnosis and treatment. Designing an appropriate microarray chip reduces noises and probable biases in the final result. Objective: The aim of this study was to design and construct a DNA Microarray Chip for a rapid detection and identification of 10 important bacterial agents. Method: In the present survey, 10 unique genomic regions relating to 10 pathogenic bacterial agents including Escherichia coli (E.coli), Shigella boydii, Sh. dysenteriae, Sh. flexneri, Sh. sonnei, Salmonella typhi, S. typhimurium, Brucella sp., Legionella pneumophila, and Vibrio cholera were selected for designing specific long oligo microarray probes. For this reason, the in-silico operations including utilization of the NCBI RefSeq database, Servers of PanSeq and Gview, AlleleID 7.7 and Oligo Analyzer 3.1 was done. On the other hand, the in-vitro part of the study comprised stages of robotic microarray chip probe spotting, bacterial DNAs extraction and DNA labeling, hybridization and microarray chip scanning. In wet lab section, different tools and apparatus such as Nexterion® Slide E, Qarraymini spotter, NimbleGen kit, TrayMixTM S4, and Innoscan 710 were used. Results: A DNA microarray chip including 10 long oligo microarray probes was designed and constructed for detection and identification of 10 pathogenic bacteria. Conclusion: The DNA microarray chip was capable to identify all 10 bacterial agents tested simultaneously. The presence of a professional bioinformatician as a probe designer is needed to design appropriate multifunctional microarray probes to increase the accuracy of the outcomes.


Viability qPCR, a New Tool for Legionella Risk Management

Lizana X, López A, Benito S, Agustí G, Ríos M, Piqué N, Marqués AM, Codony F.

GenIUL, Carrer de la Ciutat d'Asunción, Barcelona, Spain.

Int J Hyg Environ Health 2017 Nov;220(8):1318-1324.

Abstract: Background: Viability quantitative Polymerase Chain Reaction (v-qPCR) is a recent analytical approach for only detecting live microorganisms by DNA amplification-based methods This approach is based on the use of a reagent that irreversibly fixes dead cell’s DNA. In this study, we evaluate the utility of v-qPCR versus culture method for Legionellosis risk management. Methods: The present study was performed using 116 real samples. Water samples were simultaneously analysed by culture, v-qPCR and qPCR methods. Results were compared by means of a non-parametric test. Results: In 11.6% of samples using both methods (culture method and v-qPCR) results were positive, in 50.0% of samples both methods gave rise to negative results. As expected, equivalence between methods was not observed in all cases, as in 32.1% of samples positive results were obtained by v-qPCR and all of them gave rise to negative results by culture. Only in 6.3% of samples, with very low Legionella levels, was culture positive and v-qPCR negative. In 3.5% of samples, overgrowth of other bacteria did not allow performing the culture. When comparing both methods, significant differences between culture and v-qPCR were in the samples belonging to the cooling towers-evaporative condensers group. The v-qPCR method detected greater presence and obtained higher concentrations of Legionella spp. (p<0.001). Otherwise, no significant differences between methods were found in the rest of the groups. Conclusions: The v-qPCR method can be used as a quick tool to evaluate Legionellosis risk, especially in cooling towers-evaporative condensers, where this technique can detect higher levels than culture. The combined interpretation of PCR results along with the ratio of live cells is proposed as a tool for understanding the sample context and estimating the Legionellosis risk potential according to 4 levels of hierarchy.


Legionella pneumophila LPS to Evaluate Urinary Antigen Tests

Ranc AG, Carpentier M, Beraud L, Descours G, Ginevra C, Maisonneuve E, Verdon J, Berjeaud JM, Lina G, Jarraud S.

Hospices Civils de Lyon, Centre National de Référence des Légionelles, France.

Diagn Microbiol Infect Dis 2017 Oct;89 (2):89-91.

Abstract: Three urinary antigen tests were compared using purified Legionella pneumophila (Lp) LPS. For Lp serogroup1, Sofia®FIA and Binax®EIA limits of detection (LOD) were similar; that of BinaxNOW® lower. For all tests the LOD was higher with LPS from non-Pontiac compared to Pontiac-strains. The LOD was variable for other Lp serogroups.


Comparison of the ImmuView and the BinaxNOW Antigen Tests in Detection of Streptococcus pneumoniae and Legionella pneumophila in Urine

Athlin S, Iversen A, Özenci V.

Department of Infectious Diseases, Faculty of Medicin and Health, Örebro University, Örebro, Sweden.

Eur J Clin Microbiol Infect Dis 2017 Oct;36(10):1933-1938.

Abstract: The use of urinary antigen tests (UATs) may provide early etiology in pneumonia and facilitates rapid and directed antibiotic treatment. In this study, we evaluated the novel lateral flow ImmuView Streptococcus pneumoniae and Legionella pneumophila UAT, which detects pneumococcal and L. pneumophila serogroup 1 antigens in a combined test. We compared the ImmuView UAT with the BinaxNOW S. pneumoniae UAT and the BinaxNOW L. pneumophila UAT in 147 patients with pneumococcal bacteremia (n=48), non-pneumococcal non-Legionella bacteremia (n=93) and Legionella infections in the lower airways (L. pneumophila, n=5; L. bozemanii, n=1). In three cases, the ImmuView test was invalid before and after boiling while the BinaxNOW tests were valid in all cases. In 144 cases, the three UATs demonstrated a very good inter-assay agreement for detection of pneumococcal antigen (κ=0.86) and L. pneumophila antigen (κ=1.00). The ImmuView and BinaxNOW S. pneumoniae tests had similar sensitivities (62% vs 60%; p=ns) in 48 cases with pneumococcal bacteremia and both tests had specificities of 97% in 96 cases with non-pneumococcal infections. Furthermore, the ImmuView and BinaxNOW L. pneumophila tests were positive for Legionella antigen in five patients with confirmed L. pneumophila serogroup 1 infections, and negative in all non-L. pneumophila cases. The ImmuView and BinaxNOW tests performed similarly when evaluated on urine samples from bacteremic and non-bacteremic patients with identified etiology.


Incubation of Premise Plumbing Water Samples on Buffered Charcoal Yeast Extract Agar at Elevated Temperature and pH Selects for Legionella pneumophila

Veenendaal HR, Brouwer-Hanzens AJ, van der Kooij D.

KWR Watercycle Research Institute, Nieuwegein, The Netherlands.

Water Res 2017 Oct;123:439-447.

Abstract: Worldwide, over 90% of the notified cases of Legionnaires' disease are caused by Legionella pneumophila. However, the standard culture medium for the detection of Legionella in environmental water samples, Buffered Charcoal Yeast Extract (BCYE) agar of pH 6.9±0.4 with or without antimicrobial agents incubated at 36±1°C, supports the growth of a large diversity of Legionella species. BCYE agar of elevated pH or/and incubation at elevated temperature gave strongly reduced recoveries of most of 26 L. non-pneumophila spp. tested, but not of L. pneumophila. BCYE agar of pH 7.3±0.1, incubated at 40±0.5°C (BCYE pH 7.3/40°C) was tested for selective enumeration of L. pneumophila. Of the L. non-pneumophila spp. tested, only L. adelaidensis and L. londiniensis multiplied under these conditions. The colony counts on BCYE pH 7.3/40°C of a L. pneumophila serogroup 1 strain cultured in tap water did not differ significantly from those on BCYE pH 6.9/36°C when directly plated and after membrane filtration and showed repeatability's of 13-14%. By using membrane filtration L. pneumophila was detected in 58 (54%) of 107 Legionella-positive water samples from premise plumbing systems under one or both of these culture conditions. The L. pneumophila colony counts (log-transformed) on BCYE pH 7.3/40°C were strongly related (r2=0.87) to those on BCYE pH 6.9/36°C but differed significantly (p<0.05) by a mean of -0.12±0.30 logs. L. non-pneumophila spp. were detected only on BCYE pH 6.9/36°C in 49 (46%) of the samples. Hence, BCYE pH 7.3/40°C can facilitate the enumeration of L. pneumophila and their isolation from premise plumbing systems with culturable L. non-pneumophila spp., some of which, e.g. L. anisa, can be present in high numbers.


Evaluation of Timing of Re-Appearance of VBNC Legionella for Risk Assessment in Hospital Water Distribution Systems

Marinelli L, Cottarelli A, Solimini AG, Del Cimmuto A, De Giusti M.

Department of Public Health and Infectious Diseases, Sapienza University of Rome, Italy.

Ann Ig 2017 Sep-Oct;29(5):431-439.

Abstract: Background: In this study we estimated the presence of Legionella species, viable but non-culturable (VBNC), in hospital water networks. We also evaluated the time and load of Legionella appearance in samples found negative using the standard culture method. Methods: A total of 42 samples was obtained from the tap water of five hospital buildings. The samples were tested for Legionella by the standard culture method and were monitored for up to 12 months for the appearance of VBNC Legionella. Results: All the 42 samples were negative at the time of collection. Seven of the 42 samples (17.0%) became positive for Legionella at different times of monitoring. The time to the appearance of VBNC Legionella was extremely variable, from 15 days to 9 months from sampling. The most frequent Legionella species observed were Legionella spp and L. anisa and only in one sample L. pneumophila srg.1. Conclusions: Our study confirms the presence of VBNC Legionella in samples resulting negative using the standard culture method and highlights the different time to its appearance that can occur several months after sampling. The results are important for risk assessment and risk management of engineered water systems.


On-filter Direct Amplification of Legionella pneumophila for Rapid Assessment of Its Abundance and Viability

Samhan FA, Stedtfeld TM, Waseem H, Williams MR, Stedtfeld RD, Hashsham SA.

Department of Civil and Environmental Engineering, Michigan State University, East Lansing, MI, USA.

Water Res 2017 Sep;121:162-170.

Abstract: Guidelines and regulations to control Legionella pneumophila in cooling water systems of large buildings are evolving due to the increasing number of outbreaks. Rapid, on-site, simple, and sensitive quantification methods that are also able to assess viability may be extremely useful in monitoring and control. Culture-based methods for measuring L. pneumophila may take 4-10 days and qPCR-based methods are also slow, requiring at least a day from sample to result, albeit mainly due to the need for sample transport to a centralized laboratory. This study reports a rapid isothermal amplification method for L. pneumophila concentration and detection with live/dead differentiation under field conditions. Using an on-filter direct amplification (i.e., amplification of cells without DNA extraction and purification) approach with propidium monoazide (PMA), and a real time isothermal amplification platform (Gene-Z), L. pneumophila could be detected in 1-2 h at 1 cfu/100 ml of tap water. Signature sequences from 16S rRNA and cadA genes were used as genetic markers for L. pneumophila and loop-mediated isothermal amplification (LAMP) primers were designed using Primer Explorer V4. Result were also compared with direct amplification of cells spiked into distilled, tap, and cooling water samples as well as extracted DNA by qPCR. This method may be useful to managers of cooling water systems in large buildings for rapid detection of L. pneumophila. The overall approach of on-site sample concentration, on-filter amplification, and live/dead differentiation may be extended to other organisms where analytical sensitivity and speed are equally important.


Multiplex Polymerase Chain Reaction of Genetic Markers for Detection of Potentially Pathogenic Environmental Legionella pneumophila Isolates

Valavane A, Chaudhry R, Malhotra P.

Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India.

Indian J Med Res 2017 Sep;146(3):392-400.

Abstract: Background & objectives: Genomic constitution of the bacterium Legionella pneumophila plays an important role in providing them a pathogenic potential. Here, we report the standardization and application of multiplex polymerase chain reaction (PCR) for the detection of molecular markers of pathogenic potential in L. pneumophila in hospital environment. Methods: Culture of the standard strains of L. pneumophila was performed in buffered charcoal-yeast extract agar with L-cysteine at pH 6.9. Primers were designed for multiplex PCR, and standardization for the detection of five markers annotated to L. pneumophila plasmid pLPP (11A2), lipopolysaccharide synthesis (19H4), CMP-N-acetylneuraminic acid synthetase (10B12), conjugative coupling factor (24B1) and hypothetical protein (8D6) was done. A total of 195 water samples and 200 swabs were collected from the hospital environment. The bacterium was isolated from the hospital environment by culture and confirmed by 16S rRNA gene PCR and restriction enzyme analysis. A total of 45 L. pneumophila isolates were studied using the standardized multiplex PCR. Results: The PCR was sensitive to detect 0.1 ng/μl DNA and specific for the two standard strains used in the study. Of the 45 hospital isolates tested, 11 isolates had four markers, 12 isolates had three markers, 10 isolates had two markers, nine isolates had one marker and three isolates had none of the markers. None of the isolates had all the five markers. Interpretation & conclusions: The findings of this study showed the presence of gene markers of pathogenic potential of the bacterium L. pneumophila. However, the genomic constitution of the environmental isolates should be correlated with clinical isolates to prove their pathogenic potential. Rapid diagnostic methods such as multiplex PCR reported here, for elucidating gene markers, could help in future epidemiological studies of bacterium L. pneumophila.


Diagnostic Testing for Legionnaires' Disease

Pierre DM, Baron J, Yu VL, Stout JE.

Department of Medicine, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA.

Ann Clin Microbiol Antimicrob 2017 Aug;16(1):59.

Abstract: Legionnaires' disease is commonly diagnosed clinically using a urinary antigen test. The urinary antigen test is highly accurate for L. pneumophila serogroup 1, however other diagnostic tests should also be utilized in conjunction with the urinary antigen as many other Legionella species and serogroups are pathogenic. Culturing of patient specimens remains the gold standard for diagnosis of Legionnaires' disease. Selective media, BYCE with the addition of antibiotics, allows for a high sensitivity and specificity. Culturing can identify all species and serogroups of Legionella. A major benefit of culturing is that it provides the recovery of a patient isolate, which can be used to find an environmental match. Other diagnostic tests, including DFA and molecular tests such as PCR and LAMP, are useful tests to supplement culturing. Molecular tests provide much more rapid results in comparison to culture; however, these tests should not be a primary diagnostic tool given their lower sensitivity and specificity in comparison to culturing. It is recommended that all laboratories develop the ability to culture patient specimens in-house with the selective media.


Digital PCR for Detection and Quantification of Fluoroquinolone Resistance in Legionella pneumophila

Hennebique A, Bidart M, Jarraud S, Beraud L, Schwebel C, Maurin M, Boisset S.

Institut de Biologie et de Pathologie, Centre Hospitalier Universitaire Grenoble Alpes, Grenoble, France. e

Antimicrob Agents Chemother 2017 Aug;61(9):e00628-17.

Abstract: The emergence of fluoroquinolone (FQ)-resistant mutants of Legionella pneumophila in infected humans was previously reported using a next-generation DNA sequencing (NGS) approach. This finding could explain part of the therapeutic failures observed in legionellosis patients treated with these antibiotics. The aim of this study was to develop digital PCR (dPCR) assays allowing rapid and accurate detection and quantification of these resistant mutants in respiratory samples, especially when the proportion of mutants in a wild-type background is low. We designed three dPCRgyrA assays to detect and differentiate the wild-type and one of the three gyrA mutations previously described as associated with FQ resistance in L. pneumophila: at positions 248CT (T83I), 259GA (D87N), and 259GC (D87H). To assess the performance of these assays, mixtures of FQ-resistant and -susceptible strains of L. pneumophila were analyzed, and the results were compared with those obtained with Sanger DNA sequencing and real-time quantitative PCR (qPCR) technologies. The dPCRgyrA assays were able to detect mutated gyrA sequences in the presence of wild-type sequences at up to 1:1,000 resistant/susceptible allele ratios. By comparison, Sanger DNA sequencing and qPCR were less sensitive, allowing the detection of gyrA mutants at up to 1:1 and 1:10 ratios, respectively. When testing 38 respiratory samples from 23 legionellosis patients (69.6% treated with an FQ), dPCRgyrA detected small amounts of gyrA mutants in four (10.5%) samples from three (13.0%) patients. These results demonstrate that dPCR is a highly sensitive alternative to quantify FQ resistance in L. pneumophila, and it could be used in clinical practice to detect patients that could be at higher risk of therapeutic failure.


Molecular Typing of Legionella pneumophila From Air-Conditioning Cooling Waters Using Mip Gene, SBT, and FAFLP Methods

Gong X, Li J, Zhang Y, Hou S, Qu P, Yang Z, Chen S.

Guangzhou Center for Disease Control and Prevention, Guangzhou, China.

J Microbiol Methods 2017 Aug;139:1-7.

Abstract: Legionella spp. are important waterborne pathogens. Molecular typing has become an important method for outbreaks investigations and source tracking of Legionnaires’ disease. In a survey program conducted by the Guangzhou Center for Disease Control and Prevention, multiple serotypes Legionella pneumophila (L. pneumophila) were isolated from waters in air-conditioning cooling towers in urban Guangzhou region, China between 2008 and 2011. Three genotyping methods, mip (macrophage infectivity potentiator) genotyping, SBT (sequence-based typing), and FAFLP (fluorescent amplified fragment length polymorphism analysis) were used to type these waterborne L. pneumophila isolates. The three methods were capable of typing all the 134 isolates and a reference strain of L. pneumophila (ATCC33153), with discriminatory indices of 0.7034, 0.9218, and 0.9376, for the mip, SBT, and FAFLP methods respectively. Among the 9 serotypes of the 134 isolates, 10, 50, and 34 molecular types were detected by the mip, SBT, and FAFLP methods respectively. The mip genotyping and SBT typing are more feasible for inter-laboratory results sharing and comparison of different types of L. pneumophila. The SBT and FAFLP typing methods were rapid with higher discriminatory abilities. Combinations of two or more of the typing methods enables more accurate typing of Legionella isolates for outbreak investigations and source tracking of Legionnaires’ disease.


Complete Genome Sequences of Two Geographically Distinct Legionella micdadei Clinical Isolates

Osborne AJ, Jose BR, Perry J, Smeele Z, Aitken J, Gardner PP, Slow S.

School of Biological Sciences, University of Canterbury, Christchurch, New Zealand.

Genome Announc 2017 Jun;5(22):e00436-17.

Abstract: Legionella is a highly diverse genus of intracellular bacterial pathogens that cause Legionnaire's disease (LD), an often-severe form of pneumonia. Two L. micdadei sp. clinical isolates, obtained from patients hospitalized with LD from geographically distinct areas, were sequenced using PacBio SMRT cell technology, identifying incomplete phage regions, which may impact virulence.


Complete Genome Sequence of a Legionella longbeachae Serogroup 1 Strain Isolated from a Patient with Legionnaires' Disease

Slow S, Anderson T, Miller J, Singh S, Murdoch D, Biggs PJ.

Department of Pathology, University of Otago, Christchurch, New Zealand.

Genome Announc 2017 Jun;5(24):e00564-17.

Abstract: Legionella longbeachae serogroup 1, predominantly found in soil and composted plant material, causes the majority of cases of Legionnaires' disease (LD) in New Zealand. Here, we report the complete genome sequence of an L. longbeachae serogroup 1 (sg1) isolate derived from a patient hospitalized with LD in Christchurch, New Zealand.


Evaluation of Legionella Air Contamination in Healthcare Facilities by Different Sampling Methods: An Italian Multicenter Study

Montagna MT, De Giglio O, Cristina ML, Napoli C, Pacifico C, Agodi A, Baldovin T, Casini B, Coniglio MA, D'Errico MM, Delia SA, Deriu MG, Guida M, Laganà P, Liguori G, Moro M, Mura I, Pennino F, Privitera G, Romano Spica V, Sembeni S, Spagnolo AM, Tardivo S, Torre I, Valeriani F, Albertini R, Pasquarella C.

Department of Biomedical Science and Human Oncology, University of Bari "Aldo Moro", Bari, Italy.

Int J Environ Res Public Health 2017 Jun;14(7):670. 

Abstract: Healthcare facilities (HF) represent an at-risk environment for legionellosis transmission occurring after inhalation of contaminated aerosols. In general, the control of water is preferred to that of air because, to date, there are no standardized sampling protocols. Legionella air contamination was investigated in the bathrooms of 11 HF by active sampling (Surface Air System and Coriolis®μ) and passive sampling using settling plates. During the 8-hour sampling, hot tap water was sampled three times. All air samples were evaluated using culture-based methods, whereas liquid samples collected using the Coriolis®μ were also analyzed by real-time PCR. Legionella presence in the air and water was then compared by sequence-based typing (SBT) methods. Air contamination was found in four HF (36.4%) by at least one of the culturable methods. The culturable investigation by Coriolis®μ did not yield Legionella in any enrolled HF. However, molecular investigation using Coriolis®μ resulted in eight HF testing positive for Legionella in the air. Comparison of Legionella air and water contamination indicated that Legionella water concentration could be predictive of its presence in the air. Furthermore, a molecular study of 12 L. pneumophila strains confirmed a match between the Legionella strains from air and water samples by SBT for three out of four HF that tested positive for Legionella by at least one of the culturable methods. Overall, our study shows that Legionella air detection cannot replace water sampling because the absence of microorganisms from the air does not necessarily represent their absence from water; nevertheless, air sampling may provide useful information for risk assessment. The liquid impingement technique appears to have the greatest capacity for collecting airborne Legionella if combined with molecular investigations.


Evaluation of Legionella Real-Time PCR Against Traditional Culture for Routine and Public Health Testing of Water Samples

Collins S, Stevenson D, Walker J, Bennett A.

Biosafety Air and Water Microbiology Group, Public Health England, Porton Down, Salisbury, UK.

J Appl Microbiol 2017 Jun;122(6):1692-1703.

Abstract: Aims: To evaluate the usefulness of Legionella qPCR alongside traditional culture for enumeration of Legionella from water samples as part of both routine and public health investigation testing. Methods and results: Routine water samples (n=2002) and samples from public health investigations (n=215) were analysed by culture and qPCR for Legionella spp., Legionella pneumophila and L. pneumophila sg 1. A negative qPCR result was highly predictive of a negative culture result for all water systems (negative predictive values, NPV from 97.4 to 100%). Positive predictive values (PPV) were lower (0-50%). Results for qPCR were generally larger than culture with average log10 differences of 1.1 for Legionella spp. and 1.2 for L. pneumophila. Alert and action levels of 1000 and 10 000 GU per litre, respectively, are proposed for Legionella qPCR for hot and cold water systems (HCWS). The use of qPCR significantly reduced the time to results for public health investigations by rapidly identifying potential sources and ruling out others, thus enabling a more rapid and efficient response. Conclusions: The high NPV of qPCR supports its use to rapidly screen out negative samples without culture. Alert and action levels for Legionella qPCR for HCWS are proposed. Quantitative PCR will be a valuable tool for both routine and public health testing. Significance and impact of the study: This study generated comparative data of >2000 water samples by qPCR and culture. Action and alert levels have been recommended that could enable duty holders to interpret qPCR results to facilitate timely Legionella control and public health protection.


Evaluation of the bioNexia Legionella Test, Including Impact of Incubation Time Extension, for Detection of Legionella pneumophila Serogroup 1 Antigen in Urine

Badoux P, Euser SM, Bruin JP, Mulder PPG, Yzerman EPF.

Regional Public Health Laboratory Kennemerland, Boerhaavelaan, Haarlem, the Netherlands.

J Clin Microbiol 2017 Jun;55(6):1733-1737.

Abstract: In this study, we compared the bioNexia test (bioMérieux, Marcy-l'Étoile, France), a new immunochromatographic assay for the detection of Legionella pneumophila serogroup 1 in urine, with the BinaxNOW urinary antigen test (Alere, Waltham, Massachusetts, USA). After 15 min of incubation (in accordance with the manufacturers' instructions), the sensitivities and specificities were, respectively, 76.5% and 97.2% for the bioNexia test and 87.1% and 100% for the BinaxNOW test. After a prolonged incubation time of 60 min, the sensitivities and specificities increased to, respectively, 89.4% and 97.2% for the bioNexia test and 91.8% and 100% for the BinaxNOW test. When the tests were read after 15 min, the concentration of discrepant urine samples increased the sensitivities to 94.1% for both tests. In conclusion, we found that although the bioNexia test showed lower sensitivity for the detection of L. pneumophila antigen in nonconcentrated urine compared to the BinaxNOW test, a prolonged incubation time as well as the use of concentrated samples showed comparable sensitivities for both tests.


Rapid On-Site Monitoring of Legionella pneumophila in Cooling Tower Water Using a Portable Microfluidic System

Yamaguchi N, Tokunaga Y, Goto S, Fujii Y, Banno F, Edagawa A.

Osaka Institute of Public Health, Osaka, Japan.

Sci Rep 2017 Jun;7(1):3092.

Abstract: Legionnaires' disease, predominantly caused by the bacterium Legionella pneumophila, has increased in prevalence worldwide. The most common mode of transmission of Legionella is inhalation of contaminated aerosols, such as those generated by cooling towers. Simple, rapid and accurate methods to enumerate L. pneumophila are required to prevent the spread of this organism. Here, we applied a microfluidic device for on-chip fluorescent staining and semi-automated counting of L. pneumophila in cooling tower water. We also constructed a portable system for rapid on-site monitoring and used it to enumerate target bacterial cells rapidly flowing in the microchannel. A fluorescently labelled polyclonal antibody was used for the selective detection of L. pneumophila serogroup 1 in the samples. The counts of L. pneumophila in cooling tower water obtained using the system and fluorescence microscopy were similar. The detection limit of the system was 104 cells/ml, but lower numbers of L. pneumophila cells (101 to 103 cells/ml) could be detected following concentration of 0.5-3 L of the water sample by filtration. Our technique is rapid to perform (1.5 h), semi-automated (on-chip staining and counting), and portable for on-site measurement, and it may therefore be effective in the initial screening of Legionella contamination in freshwater.


A Bioinformatics Tool for Ensuring the Backwards Compatibility of Legionella pneumophila Typing in the Genomic Era

Gordon M, Yakunin E, Valinsky L, Chalifa-Caspi V, Moran-Gilad J; ESCMID Study Group for Legionella Infections.

Ben-Gurion University of the Negev, Beer-Sheva, Israel.

Clin Microbiol Infect 2017 May;23(5):306-310.

Abstract: Objectives: Whole genome sequencing (WGS) has revolutionized the subtyping of Legionella pneumophila but calling the traditional sequence-based type from genomic data is hampered by multiple copies of the mompS locus. We propose a novel bioinformatics solution for rectifying that limitation, ensuring the feasibility of WGS for cluster investigation. Methods: We designed a novel approach based on the alignment of raw reads with a reference sequence. With WGS, reads originating from either of the two mompS copies cannot be differentiated. Therefore, when non-identical copies were present, we applied a read-filtering strategy based on read alignment to a reference sequence via unique 'anchors'. If minimal read coverage was achieved after filtration (≥3X), a consensus sequence was built based on mapped reads followed by calling the sequence-based typing allele. The entire procedure was implemented using a Perl script. Results: The method was validated using a diverse sample of 265 L. pneumophila genomes, consisting of 59 different sequence types (STs) and 23 mompS variants; 57 of the 265 (22%) had non-identical mompS copies. In 237 of the 265 samples (89.4%), mompS calling was successful and no erroneous calling occurred. A 98.1% success was recorded among 109 samples meeting quality requirements. The method was superior to alternative approaches. Conclusions: As WGS becomes more accessible, technical difficulties in routine clinical and surveillance work will arise. The case of mompS in L. pneumophila serves as an example for such limitations that necessitate the development of novel computational solutions that meet end-user demands.


Can We Truly Rely on the Urinary Antigen Test for the Diagnosis? Legionella Case Report

Miyata J, Huh JY, Ito Y, Kobuchi T, Kusukawa K, Hayashi H.

Division of Family Medicine University of Fukui Hospital Fukui Japan.

J Gen Fam Med 2017 Apr;18(3):139-143.

Abstract: It is critical to diagnose and treat Legionella pneumonia (LP) immediately after infection because of the associated high mortality. The urine antigen test (UAT) is often used for the diagnosis of LP; however, it cannot detect the serogroups of all Legionella species. A detained medical history and several clinical findings such as liver enzyme elevation and hyponatremia are useful in diagnosis. Some specific types of Legionella are found in compost. Herein, we report a case of LP in which the patient's medical history and several clinical findings were useful for diagnosis.


Accuracy of Diagnostic Tests for Legionnaires' Disease: A Systematic Review

Cristovam E, Almeida D, Caldeira D, Ferreira JJ, Marques T.

Laboratory of Microbiology and Molecular Biology, Centro Hospitalar de Lisboa Ocidental, Lisbon, Portugal.

J Med Microbiol 2017 Apr;66(4):485-489.

Abstract: Purpose: Rapid and effective diagnosis of Legionnaires' disease (LD) cases is extremely important so that timely and appropriate therapy can be provided, thereby lowering the morbidity and mortality rates and reducing the health and economic costs associated with this disease. Methodology: Diagnosis is established solely by microbiological tests. There are several methods available, each with different performance, sensitivity and specificity characteristics, and further understanding is required. Our objective was to assess the accuracy of urinary antigen detection, direct fluorescent antibody (DFA) staining, serological testing and the polymerase chain reaction (PCR) method versus culture analysis (the reference standard) in patients suspected of being infected with Legionella or patients with laboratory-confirmed LD. We performed a MEDLINE search in November 2014. Two authors independently assessed the trials and extracted data. Pooled analysis was performed through Meta-DiSc version 1.4. Result: The inclusion criteria were met by 11 studies. All the studies evaluated PCR and DFA tests to detect Legionella in clinical specimens, comparing them to culture techniques, and were included in the meta-analysis. The pooled sensitivity and specificity for PCR were 83 % [95% confidence interval (CI): 79-87%] and 90% (95% CI: 88-92%), respectively. DFA was evaluated in one study and the sensitivity and specificity of this test were 67% (95% CI: 30-93%) and 100% (95% CI: 91-100%), respectively. PCR had high sensitivity and specificity for early diagnosis of LD. Conclusion: Culture analysis is deemed necessary for epidemiological studies, molecular strain typing and antibiotic sensibility evaluations; however, the performance of PCR in recent studies calls for additional, well-designed studies in order to achieve the best standard test, which will enable optimization of the Legionella infection diagnostic.


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

Bonetta S, Pignata C, Bonetta S, Meucci L, Giacosa D, Marino E, Gilli G, Carraro E.

Department of Public Health and Pediatrics, University of Torino, Torino, Italy.

Int J Environ Res Public Health 2017 Apr;14(5): 467.

Abstract: 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.


Evaluation of a Most Probable Number Method for the Enumeration of Legionella pneumophila From Potable and Related Water Samples

Sartory DP, Spies K, Lange B, Schneider S, Langer B.

SWM Consulting, Little Ness, Shrewsbury, UK.

Lett Appl Microbiol 2017 Apr;64(4):271-275.

Abstract: This study compared the performance of a novel MPN method (Legiolert/Quanti-Tray) with the ISO 11731-2 membrane filtration method for the enumeration of Legionella pneumophila from 100 ml potable water and related samples. Data from a multi-laboratory study analysed according to ISO 17994 showed that Legiolert™/Quanti-Tray® yielded on average higher counts of L. pneumophila. The Legiolert medium had a high specificity of 96·4%. The new method represents a significant improvement in the enumeration of L. pneumophila from drinking water-related samples. Significance and impact of the study: Legionella pneumophila is an opportunistic pathogen of major concern. The current large volume quantitative method employs membrane filtration (MF) and selective culture on GVPC agar followed by confirmation of isolates by serology (ISO 11731-2). We present here the results of a multi-laboratory evaluation of a most probable number (MPN) in-situ confirmed method (Legiolert™/Quanti-Tray®). The results indicate that Legiolert/Quanti-Tray yielded on average higher counts of L. pneumophila than ISO 11731-2. This development significantly improves and simplifies the enumeration of L. pneumophila from potable water samples.


Development of a Genus-Specific Next Generation Sequencing Approach for Sensitive and Quantitative Determination of the Legionella Microbiome in Freshwater Systems

Pereira RP, Peplies J, Brettar I, Höfle MG.

Department of Vaccinology and Applied Microbiology, RG Microbial Diagnostics, Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany.

BMC Microbiol 2017 Mar;17(1):79.

Abstract: Background: Next Generation Sequencing (NGS) has revolutionized the analysis of natural and man-made microbial communities by using universal primers for bacteria in a PCR based approach targeting the 16S rRNA gene. In our study we narrowed primer specificity to a single, monophyletic genus because for many questions in microbiology only a specific part of the whole microbiome is of interest. We have chosen the genus Legionella, comprising more than 20 pathogenic species, due to its high relevance for water-based respiratory infections. Methods: A new NGS-based approach was designed by sequencing 16S rRNA gene amplicons specific for the genus Legionella using the Illumina MiSeq technology. This approach was validated and applied to a set of representative freshwater samples. Results: Our results revealed that the generated libraries presented a low average raw error rate per base (<0.5%); and substantiated the use of high-fidelity enzymes, such as KAPA HiFi, for increased sequence accuracy and quality. The approach also showed high in situ specificity (>95%) and very good repeatability. Only in samples in which the gamma bacterial clade SAR86 was present more than 1% non-Legionella sequences were observed. Next-generation sequencing read counts did not reveal considerable amplification/sequencing biases and showed a sensitive as well as precise quantification of L. pneumophila along a dilution range using a spiked-in, certified genome standard. The genome standard and a mock community consisting of six different Legionella species demonstrated that the developed NGS approach was quantitative and specific at the level of individual species, including L. pneumophila. The sensitivity of our genus-specific approach was at least one order of magnitude higher compared to the universal NGS approach. Comparison of quantification by real-time PCR showed consistency with the NGS data. Overall, our NGS approach can determine the quantitative abundances of Legionella species, i.e. the complete Legionella microbiome, without the need for species-specific primers. Conclusions: The developed NGS approach provides a new molecular surveillance tool to monitor all Legionella species in qualitative and quantitative terms if a spiked-in genome standard is used to calibrate the method. Overall, the genus specific NGS approach opens up a new avenue to massive parallel diagnostics in a quantitative, specific and sensitive way.


Laboratory Tests for Legionnaire's Disease

Dunne WM Jr, Picot N, van Belkum A.

Scientific Office, BioMérieux, 3, Route de Port Michaud, La Balme Les Grottes 38390, France.

Infect Dis Clin North Am 2017 Mar;31(1):167-178.

Abstract: Legionella pneumophila is one of the more recently discovered bacterial pathogens of humans. The last 2 decades have seen tremendous progress in the evolution of diagnostic tests, for detection and characterization of this pathogen and for defining the host response to infection. This has generated several diagnostic tools that span the range from simple immunologic assays to modern genome sequencing. This review describes the state of affairs of this continuously evolving field regarding the diagnosis of Legionnaire's disease and covers detection, assessment of antibiotic susceptibility, and epidemiologic characterization of isolates of L. pneumophila and other pathogenic species within the genus.


Discrimination Between Legionnaires' Disease and Pneumococcal Pneumonia Based on the Clinical and Laboratory Features: A Quantitative Approach Using the Modified Winthrop-University Hospital Weighted Point System

Yamakuchi H, Hamada Y, Urakami T, Aoki Y.

Infectious Disease and Hospital Epidemiology, Kagoshima Seikyou Hospital, Japan.

Intern Med 2017;56(5):487-491.

Abstract: Objective: Legionnaires' disease (LD) is a common form of lobar pneumonia, but the optimum diagnostic modality has long been a subject of debate due to incomplete sensitivity and specificity. A delay in the initiation of specific therapy for LD is associated with increased mortality. The decision to treat a patient for Legionella must be made quickly. The purpose of this study was to evaluate the ability of the modified Winthrop-University Hospital WUH system to identify LD while discriminating against pneumococcal pneumonia at the time of hospitalization for community-acquired pneumonia. Methods: Five patients with LD and 13 patients with pneumococcal pneumonia were retrospectively analyzed. Results: The WUH system identified 4 of 5 patients with LD (sensitivity, 80%) while excluding legionellosis in 12 of 13 patients with pneumococcal pneumonia (specificity, 92%). The positive and negative likelihood ratios were 10.4 and 0.2. The area under the receiver operating characteristic curve was 0.969. Conclusion: The WUH system is useful for obtaining a rapid presumptive clinical diagnosis of LD. Further investigation with a larger number of patients is strongly recommended.


A Case of Legionella pneumophila Evaluated with CT and Ultrasound

D'Angelo A, De Simone C, Pagnottella M, Rossi S, Pepe R, Ruggieri G, Cocco G, Schiavone C.

UOSD Ecografia Internistica, Università degli studi G. d'Annunzio Chieti-Pescara, Chieti, Italy.

J Ultrasound 2017 Feb;20(3):243-245.

Abstract: A 36-year-old man was admitted to the emergency department of "SS Annunziata" hospital in Chieti complaining of a sharp chest pain arisen some hours before admission. On examination, the patient looked sweaty; his vital signs showed tachycardia and augmented breath rate; sinus tachycardia and normal ventricular repolarization were observed on ECG, and no abnormalities were observed in the echoscan of the hearth. According to the clinical and electrocardiographic findings, and to previous episode of DVT in anamnesis, a thorax CT scan was performed in order to rule out pulmonary embolism. It showed an "area of parenchymal consolidation involving almost all the left lower lobe with patent bronchial structures"; given the patient's CURB 65 score, he was then admitted to the pneumology ward where empiric treatment with levofloxacin (750 mg PO once daily) was initiated. Thoracic ultrasound was performed using a multifrequency convex transducer, and the posterior left area was examined through intercostal approach, placing the patient in a sitting position. A subpleural patchy hypoechoic lesion with irregular boundaries was detected; the maximum diameter was 11 cm, and the multiple hyperechoic spots inside it (elsewhere defined as "air bronchogram") showed no Doppler signal. Given the positive result of the Legionella urinary antigen test, antibiotic treatment was switched to Levofloxacin 1000 mg PO once daily and Claritromicin 500 mg PO twice daily. After 3 days, his clinical conditions improved dramatically. Ultrasound performed after 5 days from the diagnosis showed decreased dimensions of the lesion previously identified (maximum diameter 8.25 cm) and a marked reduction of the hyperechoic spots in it. The patient was discharged in good clinical conditions, and both thorax CT scan obtained after 1 and 4 months from the diagnosis showed radiological resolution of the parenchymal consolidation. The key to ultrasound visualization of pneumonia is its contact with the pleural surface (86-98% in cases of CAP) and the relative loss of aeration of the portion involved by the infection and a concomitant increase in the fluid content. A paradigmatic US image for parenchymal inflammatory infiltrate has not been established yet; anyway, some typical findings, when combined with the clinical features, can confirm the diagnostic hypothesis.


Approach to Determine the Diversity of Legionella Species by Nested PCR-DGGE in Aquatic Environments

Huang WC, Tsai HC, Tao CW, Chen JS, Shih YJ, Kao PM, Huang TY, Hsu BM.

Department of Earth and Environmental Sciences, National Chung Cheng University, Chiayi, Taiwan, ROC.

PLoS One 2017 Feb;12(2):e0170992. 

Abstract: In this study, we describe a nested PCR-DGGE strategy to detect Legionella communities from river water samples. The nearly full-length 16S rRNA gene was amplified using bacterial primer in the first step. After, the amplicons were employed as DNA templates in the second PCR using Legionella specific primer. The third round of gene amplification was conducted to gain PCR fragments apposite for DGGE analysis. Then the total numbers of amplified genes were observed in DGGE bands of products gained with primers specific for the diversity of Legionella species. The DGGE patterns are thus potential for a high-throughput preliminary determination of aquatic environmental Legionella species before sequencing. Comparative DNA sequence analysis of excised DGGE unique band patterns showed the identity of the Legionella community members, including a reference profile with two pathogenic species of Legionella strains. In addition, only members of Legionella pneumophila and uncultured Legionella sp. were detected. Development of three step nested PCR-DGGE tactic is seen as a useful method for studying the diversity of Legionella community. The method is rapid and provided sequence information for phylogenetic analysis.


Detection of Legionella pneumophila in Urine and Serum Specimens of Neutropenic Febrile Patients with Haematological Malignancies

Farzi N, Abrehdari-Tafreshi Z, Zarei O, Chamani-Tabriz L.

Reproductive Biotechnology Research Center, Avicenna Research Institute (ACECR), Tehran, Iran.

Int J Hematol Oncol Stem Cell Res 2017 Jan;11(1):49-53.

Abstract: Background: Legionella pneumophila (L. pneumophila) is a gram-negative bacterium which causes Legionnaires' disease as well as Pontiac fever. The Legionella infections in patients suffering from neutropenia- as a common complication of cancer chemotherapy- can distribute rapidly. We aimed to detect of L. pneumophila in hematological malignancy suffering patients with neutropenic fever by targeting the (macrophage infectivity potentiator) mip gene. Subjects and Methods: Serum and urine specimens were obtained from 80 patients and presence of mip gene of L. pneumophila in specimens was investigated by PCR. Results: The L. pneumophila infection was detected in 21 (26.2%) and 38 ‎‎(47.5%) of urine and serum specimens, respectively. Conclusion: Our findings indicated that the relative high prevalence of L. pneumophila in the studied patient’s group which show the necessity of considering this microorganism in future studies from detection and treatment point of view in cancer patients.