Metodi diagnostici e di laboratorio (aprile 2003 - aprile 2012)

Plasma-activated carbon nanotube-based high sensitivity immunosensors for monitoring Legionella pneumophila by direct detection of maltose binding protein peptidoglycan-associated lipoprotein (MBP-PAL)

Lee JY, Jin JH, Kim JH, Kim MJ, Lee CJ, Min NK.

Department of Biomicrosystem Technology, Korea University, Seoul 136-701, Republic of Korea. nkmin@korea.ac.kr

Biotechnol Bioeng. 2012 Jun;109(6):1471-8.

ABSTRACT: Transferred multi-walled carbon nanotube (MWCNT)-modified platinum thin-film immunosensing electrode material was engineered on a glass substrate and fabricated a fully-integrated electrochemical three-electrode system for monitoring Legionella pneumophila. The transferred MWCNT film was treated with oxygen plasma to improve its electrochemical response and electrical conductivity. We voltammetrically characterized and optimized the electrochemical performance of the fabricated electrode for direct detection of Legionella pneumophila-specific peptidoglycan-associated lipoprotein (PAL) and maltose binding protein (MBP) peptidoglycan-associated lipoprotein (MBP-PAL) fusion. The latter, as an intermediate product to yield the former, has important roles in the growth and purification of PAL, which commercial enzyme-linked immunosorbent assay (ELISA) kits require as a target substrate. Consequently, direct electrochemical detection of MBP-PAL compared to PAL by square-wave voltammetry showed a greater than 50% increase in sensitivity with a lower detection limit of 5pgmL(-1) . We also investigated the affinity properties by determining kinetic parameters of the PAL and the MBP-PAL in relation to polyclonal antibodies immobilized on transferred MWCNT substrates using Michaelis-Menten assumptions and a Hanes-Woolf plot. This new method presented herein could save the time and effort for the separation and purification of PAL form MBP-PAL fusions that are required for performing ELISA-based immunoassay. Biotechnol. Bioeng. 2012; 109:1471-1478. © 2011 Wiley Periodicals, Inc.

 

Contribution of Amoebic Coculture to Recovery of Legionella Isolates from Respiratory Samples: Prospective Analysis over a Period of 32 Months

Descours G, Suet A, Ginevra C, Campese C, Slimani S, Ader F, Che D, Lina G, Jarraud S.

ghislaine.descours@univ-lyon1.fr.

J Clin Microbiol. 2012 May;50(5):1725-6.

ABSTRACT: We evaluated the contribution of amoebic coculture to the recovery of Legionella spp. from 379 respiratory samples. The sensitivity of axenic culture was 42.1%. The combination of axenic culture with amoebic coculture increased the Legionella isolation rate to 47.1%. Amoebic coculture was particularly efficient in isolating Legionella spp. from respiratory samples contaminated with oropharyngeal flora.

 

Identification of Legionella pneumophila serogroups and other Legionella species by mip gene sequencing

Haroon A, Koide M, Higa F, Tateyama M, Fujita J.

Department of Infectious, Respiratory, and Digestive Medicine, Control and Prevention of Infectious Diseases (First Department of Internal Medicine), Faculty of Medicine, University of the Ryukyus, 207 Uehara, Nishihara-cho, Okinawa, 903-0215, Japan. koide-mi@med.u-ryukyu.ac.jp

J Infect Chemother. 2012 Apr;18(2):276-81.

ABSTRACT: The virulence factor known as the macrophage infectivity potentiator (mip) is responsible for the intracellular survival of Legionella species. In this study, we investigated the potential of the mip gene sequence to differentiate isolates of different species of Legionella and different serogroups of Legionella pneumophila. We used 35 clinical L. pneumophila isolates and one clinical isolate each of Legionella micdadei, Legionella longbeachae, and Legionella dumoffii (collected from hospitals all over Japan between 1980 and 2007). We used 19 environmental Legionella anisa isolates (collected in the Okinawa, Nara, Osaka, and Hyogo prefectures between 1987 and 2007) and two Legionella type strains. We extracted bacterial genomic DNA and amplified out the mip gene by PCR. PCR products were purified by agarose gel electrophoresis and the mip gene was then sequenced. The L. pneumophila isolates could be divided into two groups: one group was very similar to the type strain and was composed of serogroup (SG) 1 isolates only; the second group had more sequence variations and was composed of SG1 isolates as well as SG2, SG3, SG5, and SG10 isolates. Phylogenetic analysis displayed one cluster for L. anisa isolates, while other Legionella species were present at discrete levels. Our findings show that mip gene sequencing is an effective technique for differentiating L. pneumophila strains from other Legionella species.

 

Disposable Immunochips for the Detection of Legionella pneumophila Using Electrochemical Impedance Spectroscopy

Li N, Brahmendra A, Veloso AJ, Prashar A, Cheng XR, Hung VW, Guyard C, Terebiznik M, Kerman K.

Department of Physical and Environmental Sciences, ‡Department of Biological Sciences, University of Toronto Scarborough , 1265 Military Trail, Toronto, ON, M1C 1A4, Canada. kagan.kerman@utoronto.ca.

Anal Chem. 2012 Apr 17;84(8):3485-8.

ABSTRACT: The rapid diagnosis of Legionellosis is crucial for the effective treatment of this disease. Currently, most clinical laboratories utilize rapid immunoassays that are sufficient for the detection of Legionella serogroup 1, but not other clinically relevant serogroups. In this report, the development of a disposable immunochip system is described in connection with electrochemical impedance spectroscopy and fluorescence microscopy. The immunochips were prepared by covalently immobilizing fluorophore-conjugated L. pneumophilaantibodies on Au chips. The analytical performance of the immunochips was optimized as a prescreening tool for L. pneumophila. The versatile immunochips described here can be easily adapted for the monitoring of all Legionella serogroups in clinical and environmental samples.

 

Comparison of sputum and nasopharyngeal swab specimens for molecular diagnosis of Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella pneumophila

Cho MC, Kim H, An D, Lee M, Noh SA, Kim MN, Chong YP, Woo JH.

Department of Laboratory Medicine, University of Ulsan College of Medicine and Asan Medical Center, Seoul, Korea. mnkim@amc.seoul.kr

Ann Lab Med. 2012 Mar;32(2):133-8.

ABSTRACT: Background: Differentiation of atypical pathogens is important for community-acquired pneumonia (CAP). In this study, we compared sputum and nasopharyngeal swabs (NPS) for use in detection of Mycoplasma pneumoniae (MP), Chlamydophila pneumoniae (CP), and Legionella pneumophila (LP), using Seeplex PneumoBacter ACE Detection Assay (PneumoBacter; Seegene). Methods: Sputum and NPS specimens were collected from patients in 15 hospitals. DNA was extracted from sputum using QIAamp DNA Stool Mini Kit (Qiagen) and from NPS using easyMAG (bioMérieux). Both types of specimens were evaluated by multiplex PCR using PneumoBacter. To determine the diagnostic performance of this assay, sputum samples were also tested using BD ProbeTec ET Atypical Pneumonia Assay (APA; Becton Dickinson). Results: Among 217 sputum and NPS, 20 (9.2%), 2 (0.9%), and 0 sputum were positive for MP, LP, and CP, respectively, whereas 8 (3.7%) NPS were positive for MP. The sputum APA test yielded 186, 206, and 204 interpretable results for MP, LP, and CP, respectively. Of these, 21 (11.3%) were positive for MP, 2 (1.0%) were positive for LP, and 0 samples were positive for CP. Compared to APA, the sensitivity and specificity of the sputum assay for MP were 95.2% and 100.0%, respectively, whereas for the NPS assay, these were 38.1% and 93.9%. Sputum testing was more sensitive than NPS testing (P=0.002). For LP and CP diagnosis, PneumoBacter and APA tests agreed 100%. Conclusions: Specimen type is crucial and sputum is preferred over NPS for simultaneous detection of MP, LP, and CP using multiplex PCR in CAP.

 

Legionella pneumophila sequence type 1/Paris pulsotype subtyping by spoligotyping

Ginevra C, Jacotin N, Diancourt L, Guigon G, Arquilliere R, Meugnier H, Descours G, Vandenesch F, Etienne J, Lina G, Caro V, Jarraud S.

christophe.ginevra@univ-lyon1.fr.

J Clin Microbiol. 2012 Mar;50(3):696-701.

ABSTRACT: Endemic strains of Legionella pneumophila sequence type 1 (ST1), in particular the ST1/Paris pulsotype, are dispersed worldwide and represent about 10% of culture-proven clinical cases of Legionnaires' disease in France. The high rate of isolation of this strain from both clinical and environmental samples makes identification of the source of infection difficult during epidemiological investigations. The full-length genome sequence of this strain was recently determined, and it revealed the presence of a CRISPR/cas complex. The aim of this study was to develop and evaluate a spoligotyping tool based on the diversity of this CRISPR locus that would allow the accurate subtyping of the L. pneumophila serogroup 1 ST1/Paris pulsotype. The CRISPR loci of 28 L. pneumophila ST1/Paris pulsotype isolates were sequenced, and 42 different spacers regions were characterized. A membrane-based spoligotyping method was developed and used to determine the subtypes of 406 L. pneumophila isolates, including 233 with the ST1/Paris pulsotype profile that were collected in France from 2000 to 2011. A total of 46 different spoligotypes were detected, and 41 of these were specifically identified in the ST1/Paris pulsotype isolates. In 27 of 33 epidemiological investigations, the environmental source of contamination was confirmed by comparing spoligotypes of clinical isolates with those of environmental isolates. With an index of discrimination of 79.72% (95% confidence interval, 75.82 to 83.63), spoligotyping of the L. pneumophila ST1/Paris pulsotype has the potential to be a useful complementary genotyping tool for discriminating isolates with undistinguishable pulsed-field gel electrophoresis (PFGE) and ST genotypes, which could help to identify environmental sources of infection.

 

Counting Legionella cells within single amoeba host cells

Buse HY, Ashbolt NJ.

National Exposure Research Laboratory, Office of Research and Development, US Environmental Protection Agency, Cincinnati, Ohio, USA. buse.helen@epa.gov

Appl Environ Microbiol. 2012 Mar;78(6):2070-2.

ABSTRACT: Here we present the first attempt to quantify Legionella pneumophila cell numbers within individual amoeba hosts that may be released into engineered water systems. The maximum numbers of culturable L. pneumophila cells grown within Acanthamoeba polyphaga and Naegleria fowleri were 1,348 (mean, 329) and 385 (mean, 44) CFU trophozoite(-1), respectively.

 

Microbial environmental contamination in Italian dental clinics: A multicenter study yielding recommendations for standardized sampling methods and threshold values

Pasquarella C, Veronesi L, Napoli C, Castiglia P, Liguori G, Rizzetto R, Torre I, Righi E, Farruggia P, Tesauro M, Torregrossa MV, Montagna MT, Colucci ME, Gallè F, Masia MD, Strohmenger L, Bergomi M, Tinteri C, Panico M, Pennino F, Cannova L, Tanzi M; SItI Working Group Hygiene in Dentistry.

Dipartimento di Sanità Pubblica, Università degli Studi di Parma, Italy. ira.pasquarella@unipr.it

Sci Total Environ. 2012 Mar 15;420:289-99.

ABSTRACT: A microbiological environmental investigation was carried out in ten dental clinics in Italy. Microbial contamination of water, air and surfaces was assessed in each clinic during the five working days, for one week per month, for a three-month period. Water and surfaces were sampled before and after clinical activity; air was sampled before, after, and during clinical activity. A wide variation was found in microbial environmental contamination, both within the participating clinics and for the different sampling times. Before clinical activity, microbial water contamination in tap water reached 51,200cfu/mL (colony forming units per milliliter), and that in Dental Unit Water Systems (DUWSs) reached 872,000cfu/mL. After clinical activity, there was a significant decrease in the Total Viable Count (TVC) in tap water and in DUWSs. Pseudomonas aeruginosa was found in 2.38% (7/294) of tap water samples and in 20.06% (59/294) of DUWS samples; Legionella spp. was found in 29.96% (89/297) of tap water samples and 15.82% (47/297) of DUWS samples, with no significant difference between pre- and post-clinical activity. Microbial air contamination was highest during dental treatments, and decreased significantly at the end of the working activity (p<0.05). The microbial buildup on surfaces increased significantly during the working hours. This study provides data for the establishment of standardized sampling methods, and threshold values for contamination monitoring in dentistry. Some very critical situations have been observed which require urgent intervention. Furthermore, the study emphasizes the need for research aimed at defining effective managing strategies for dental clinics.

 

Application of multilocus sequence analysis (MLSA) for accurate identification of Legionella spp. Isolated from municipal fountains in Chengdu, China, based on 16S rRNA, mip, and rpoB genes

Guan W, Xu Y, Chen DL, Xu JN, Tian Y, Chen JP.

Department of Parasitology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu, 610041, Sichuan, P. R. China. jpchen007@163.com

J Microbiol. 2012 Feb;50(1):127-36.

ABSTRACT: Legionellosis (Legionnaires' disease; LD) is a form of severe pneumonia caused by species of Legionella bacteria. Because inhalation of Legionella-contaminated aerosol is considered the major infection route, routine assessments of potential infection sources such as hot water systems, air-conditioner cooling water, and municipal fountains are of great importance. In this study, we utilized in vitro culture and multilocus sequence analysis (MLSA) targeting 16S rRNA, mip, rpoB, and mip-rpoB concatenation to isolate and identify Legionella spp. from 5 municipal fountains in Chengdu City, Sichuan Province, China. Our results demonstrated that 16S rRNA was useful for initial identification, as it could recognize isolates robustly at the genus level, while the genes mip, rpoB, and mip-rpoB concatenation could confidently discriminate Legionella species. Notably, the three subspecies of L. pneumophila could be distinguished by the analysis based on rpoB. The serotyping result of strain CD-1 was consistent with genetic analysis based on the concatenation of mip and rpoB. Despite regular maintenance and sanitizing methods, 4 of the 5 municipal fountains investigated in this study were positive for Legionella contamination. Thus, regularly scheduled monitoring of municipal fountains is urgently needed as well as vigilant disinfection. Although the application of MLSA for inspection of potential sites of infection in public areas is not standard procedure, further investigations may prove its usefulness.

 

Evaluation of propidium monoazide (PMA) treatment directly on membrane filter for the enumeration of viable but non cultivable Legionella by qPCR

Slimani S, Robyns A, Jarraud S, Molmeret M, Dusserre E, Mazure C, Facon JP, Lina G, Etienne J, Ginevra C.

Hospices Civils de Lyon, Lyon, France. christophe.ginevra@univ-lyon1.fr

J Microbiol Methods. 2012 Feb;88(2):319-21.

ABSTRACT: A PMA (propidium monoazide) pretreatment protocol, in which PMA is applied directly to membrane filters, was developed for the PCR-based quantification (PMA-qPCR) of viable Legionella pneumophila. Using this method, the amplification of DNA from membrane-damaged L. pneumophila was strongly inhibited for samples containing a small number of dead bacteria.

 

Immunochromatic kits Xpect Legionella and BinaxNOW Legionella for detection of Legionella pneumophila urinary antigen have low sensitivities for the diagnosis of Legionnaires' disease

Svarrer CW, Lück C, Elverdal PL, Uldum SA.

Unit of Atypical Pneumonia, Department of Microbiological Surveillance and Research (AMOF), Statens Serum Institut, Ørestads Boulevard 5, DK 2300 Copenhagen S, Denmark. cwo@ssi.dk

J Med Microbiol. 2012 Feb;61(Pt 2):213-7.

ABSTRACT:Urinary antigen tests are the most widely used methods for diagnosing Legionnaires' disease (LD). However, all available urinary antigen tests have the disadvantage that they have low or no sensitivity for serogroups (sgs) other than Legionella pneumophila sg 1. Recently, Oxoid introduced the Xpect Legionella test for detection of L. pneumophila sg 1 and sg 6. In this study, we have evaluated the Xpect kit together with the BinaxNOW kit and compared them with the BinaxEIA kit. One hundred and fifteen urine samples from 91 patients with laboratory-confirmed LD were examined. Ninety-three samples were from 69 culture-proven cases of which 27 samples were from 23 non-sg 1 cases. At the patient level, the overall sensitivities for the three Legionella urinary antigen kits were 79 % for the BinaxEIA, 47 % for the BinaxNOW and 32 % for the Xpect kit. None of the urine samples from the 10 L. pneumophila sg 6 cases were positive by the Xpect kit whereas samples from four of the patients were positive by the BinaxEIA. Overall, the sensitivities for both immunochromatic assays were poor and they should not be used as the sole method for the diagnosis of LD.

 

Electrochemically amplified molecular beacon biosensor for ultrasensitive DNA sequence-specific detection of Legionella sp

Rai V, Nyine YT, Hapuarachchi HC, Yap HM, Ng LC, Toh CS.

Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, 21 Nanyang Link, Nanyang Technological University, Singapore 637171, Singapore. cstoh@ntu.edu.sg

Biosens Bioelectron. 2012 Feb 15;32(1):133-40.

ABSTRACT: An electrochemically amplified molecular beacon (EAMB) biosensor is constructed using thiolated hairpin DNA-ferrocene probes on gold electrode. The switching from "on" to "off" states of individual probes in the presence of complementary DNA target influences the electrode potential, besides the current, owing to changes in surface density of the electroactive hairpin DNA-ferrocene probes. The EAMB biosensor demonstrates linear range over 8 orders of magnitude with ultrasensitive detection limit of 2.3 × 10(-14)M for the quantification of a 21-mer DNA sequence. Its applicability is tested against PCR amplicons derived from genomic DNA of live Legionella pneumophila. Excellent specificity down to one and three nucleotides mismatches in another strain of L. pneumophila and a different bacterium species, respectively, is demonstrated.

 

Quantification of Legionella pneumophila by real-time quantitative PCR from samples with humic acid and ferric ion

Chen NT, Chang CW.

Institute of Environmental Health, College of Public Health, National Taiwan University, Taipei City 100, Taiwan. chingwenchang@ntu.edu.tw

Sci Total Environ. 2012 Jan 1;414:608-13.

ABSTRACT: This study determined and overcame the influences of humic acid (HA) and/or ferric ion (Fe) on quantification of Legionella pneumophila by real-time quantitative PCR (qPCR). Four commonly used DNA isolation methods, QiAamp DNA Mini Kit (Q), Q with Sepharose 4B gel column (Q/G), freeze-thaw/phenol-chloroform lysis (FT-PC), and FT-PC/G, were adopted to isolate L. pneumophila DNA from samples containing Fe alone (0-30 mg l(-1)) or Fe/HA (0/0-3/100 mg l(-1)). Among the four DNA isolation methods, Q removed HA effectively and obtained the greatest DNA yield regardless of Fe and HA concentration (P<0.05). For samples containing Fe (0.3-3 mg l(-1)) or Fe/HA (0.3/10-3/100 mg l(-1)), qPCR inhibition was found in all isolated DNA, especially in those obtained by Q/G and FT-PC/G. DNA dilution at either 10 or 100 folds reduced qPCR inhibition and increased cell recovery (P<0.05). Under 10-fold dilution, Q acquired the highest concentrations of L. pneumophila determined by qPCR. Consequently, Q with post 10-fold dilution is suggested prior to qPCR for quantifying L. pneumophila from water containing Fe (≤ 3 mg l(-1)) or Fe/HA (≤ 3/100 mg l(-1)).

 

Wild-type MIC distribution and epidemiological cut-off values in clinical Legionella pneumophila serogroup 1 isolates

Bruin JP, Ijzerman EP, den Boer JW, Mouton JW, Diederen BM.

Regional Laboratory of Public Health, Haarlem 2035 RC, The Netherlands. j.bruin@streeklabhaarlem.nl

Diagn Microbiol Infect Dis. 2012 Jan;72(1):103-8.

ABSTRACT: Objectives: The purpose of this study was to establish wild-type (WT) distributions and determine the epidemiological cut-off values (ECOFF) in clinical L. pneumophila serogroup 1 isolates for 10 antimicrobials commonly used for the treatment of Legionella infections using a method feasible in a routine clinical laboratory. Methods: MICs of 183 clinical L. pneumophila serogroup 1 isolates, collected as part of an outbreak detection program, were tested using E-test methodology on buffered charcoal yeast extract agar supplemented with α-ketoglutarate (BCYE-α). The MICs were read after 2 days of incubation at 35 °C with increased humidity and without CO(2). ECOFFs were determined according to EUCAST methodology and expressed as WT ≤ X mg/L. Results: All antimicrobials showed a WT distribution, although the width varied from 2 two-fold dilutions to 8 dilutions, depending on antibiotic class. The ECOFFs determined were 1.0 mg/L for ciprofloxacin, 0.50 mg/L for levofloxacin, 1.0 mg/L for moxifloxacin, 1.0 mg/L for erythromycin, 1.0 mg/L for azithromycin, 0.50 mg/L for clarithromycin, 1.0 mg/L for cefotaxime, 0.032 mg/L for rifampicin, 16 mg/L for tigecycline, and 8 mg/L for doxycycline. Conclusions: All isolates were inhibited by low concentrations of the fluoroquinolones and macrolides tested, with somewhat higher MICs for the fluoroquinolones. Rifampicin was found to be the most active against L. pneumophila isolates in vitro. These data can be used as a reference for the detection of resistance in clinical L. pneumophila isolates and as a setting of clinical breakpoints.

 

Evaluation of the SD Bioline test, a new assay for detecting Legionella pneumophila serogroup 1 antigen in urine

Bruin JP, Diederen BM.

Regional Laboratory of Public Health, Boerhaavelaan 26, 2035 RC Haarlem, The Netherlands. j.bruin@streeklabhaarlem.nl

J Infect. 2012 Jan;64(1):113-4.

Letter to the editor.

 

Evaluation of DNA isolation procedures for detecting and quantifying environmental Legionella by real-time quantitative PCR

Chen NT, Chang CW, Wu JD.

Institute of Environmental Health, College of Public Health, National Taiwan University, Taipei, 100, Taiwan. chingwenchang@ntu.edu.tw

Water Sci Technol. 2012;65(6):989-97.

ABSTRACT: Six methods, QiAamp DNA Mini Kit (Q), Q with Sepharose 4B gel column (Q/G), Q with low melting point agarose (Q/L), freeze-thaw/phenol-chloroform lysis (FT-PC), FT-PC/G, and FT-PC/L, were evaluated for their ability to isolate DNA of sufficient quality to quantify Legionella using qPCR. Samples of mixing Legionella pneumophila (ATCC33152) and humic acid (HA, 0-126.8 mg/l) were treated by the six methods. Q, Q/G, Q/L, FT-PC/G, and FT-PC/L removed HA from 1.9-126.8 to <1 mg/l determined by A260 with a spectrophotometer. Q obtained the highest DNA yield, followed by Q/G. Dilution (10- to 100-fold) of DNA arising from extraction using Q, Q/G, FT-PC, or FT-PC/G prevented qPCR inhibition. The highest recovery of cells was found in DNA extracted by Q and diluted 100-fold, and followed by Q/G. The applicability of Q and Q/G with dilution was further validated with cooling tower waters. Q or Q/G with 10-fold dilution increased L. pneumophila detection, whereas 100-fold dilution obtained the highest cell concentrations. Similar results were found for Legionella spp. except that both 10- and 100-fold dilutions increased cell concentrations. Thus, Q with 10-fold dilution is suggested to detect and quantify Legionella spp. and detect L. pneumophila. For L. pneumophila-positive samples, 100-fold diluted DNA must be re-analyzed to accurately quantify L. pneumophila.

 

Legionella wadsworthii pneumonia: gram stain usefulness

Lecsö-Bornet M, Brossier F, Lerat I, Gabarre J, Morelot-Panzini C, Similowski T, Jarlier V.

Department of Bacteriology, Pitié-Salpêtrière Hospital, AP–HP, 75013 Paris, France. florence.brossier@psl.aphp.fr

Med Mal Infect. 2011 Dec;41(12):669-70.

Letter to the Editor.

 

Development of a miniaturized DNA microarray for identification of 66 virulence genes of Legionella pneumophila

Żak M, Zaborowski P, Baczewska-Rej M, Zasada AA, Matuszewska R, Krogulska B.

Department of Zoonoses and Tropical Diseases, Medical University of Warsaw, Żwirki i Wigury 61 Str., 02-091 Warsaw, Poland. mariuszzakster@gmail.com

Postepy Hig Med Dosw (Online). 2011 Dec 20;65:838-48.

ABSTRACT: Introduction: For the last five years, Legionella sp. infections and legionnaire's disease in Poland have been receiving a lot of attention, because of the new regulations concerning microbiological quality of drinking water. This was the inspiration to search for and develop a new assay to identify many virulence genes of Legionella pneumophila to better understand their distribution in environmental and clinical strains. The method might be an invaluable help in infection risk assessment and in epidemiological investigations. Material/methods: The microarray is based on Array Tube technology. It contains 3 positive and 1 negative control. Target genes encode structural elements of T4SS, effector proteins and factors not related to T4SS. Probes were designed using OligoWiz software and data analyzed using IconoClust software. To isolate environmental and clinical strains, BAL samples and samples of hot water from different and independent hot water distribution systems of public utility buildings were collected. Results: We have developed a miniaturized DNA microarray for identification of 66 virulence genes of L. pneumophila. The assay is specific to L. pneumophila sg 1 with sensitivity sufficient to perform the assay using DNA isolated from a single L. pneumophila colony. Seven environmental strains were analyzed. Two exhibited a hybridization pattern distinct from the reference strain. Discussion: The method is time- and cost-effective. Initial studies have shown that genes encoding effector proteins may vary among environmental strains. Further studies might help to identify set of genes increasing the risk of clinical disease and to determine the pathogenic potential of environmental strains.

 

Detection of Legionella by quantitative-polymerase chain reaction (qPCR) for monitoring and risk assessment

Krøjgaard LH, Krogfelt KA, Albrechtsen HJ, Uldum SA.

Dept. of Microbiological Surveillance and Research, Statens Serum Institut, Ørestads Boulevard 5, Copenhagen S, Denmark. lkd@ssi.dk

BMC Microbiol. 2011 Nov 21;11:254.

ABSTRACT: Background: Culture and quantitative polymerase chain reaction (qPCR) assays for the detection of Legionella were compared on samples from a residential area before and after two interventions. A total of 84 samples were collected from shower hoses and taps as first flush samples and at constant temperature. Samples were grouped according to the origin of the sample, a) circulation water b) water from empty apartments c) water from shower hoses. The aims were to investigate the usefulness of qPCR compared to culture for monitoring remedial actions for elimination of Legionella bacteria and as a tool for risk assessment. Results: In water collected from the apartments Legionella spp were detected by qPCR in the concentration range from LOQ to 9.6*105GU/L while L. pneumophila were detected in a range from LOQ to 6.8*105 GU/L. By culturing, the legionellae were detected in the range from below detection limit (> 10 CFU/L) to 1.6*106 CFU/L. In circulating water and in first flush water from shower hoses, culture and qPCR showed the same tendencies. The overall correlation between the bacteria number detected by culture and the two developed qPCR assays (L. spp and L. pneumophila) was relatively poor (r2 = 0.31 for culture and Legionella spp. assay, r2 = 0.20 for culture and L. pneumophila assay).

Conclusions: Detection by qPCR was suitable for monitoring changes in the concentration of Legionella but the precise determination of bacteria is difficult. Risk assessment by qPCR only on samples without any background information regarding treatment, timing, etc is dubious. However, the rapid detection by qPCR of high concentrations of Legionella - especially Legionella pneumophila - is valuable as an indicator of risk, although it may be false positive compared to culture results. On the other hand, the detection of a low number of bacteria by qPCR is a strong indication for the absence of risk.

 

 

High-Throughput Typing Method To Identify a Non-Outbreak-Involved Legionella pneumophila Strain Colonizing the Entire Water Supply System in the Town of Rennes, France

Sobral D, Le Cann P, Gerard A, Jarraud S, Lebeau B, Loisy-Hamon F, Vergnaud G, Pourcel C.

Université Paris-Sud, Institut de Génétique et Microbiologie, 91405 Orsay, France. christine.pourcel@u-psud.fr

Appl Environ Microbiol. 2011 Oct;77(19):6899-907.

ABSTRACT: Two legionellosis outbreaks occurred in the city of Rennes, France, during the past decade, requiring in-depth monitoring of Legionella pneumophila in the water network and the cooling towers in the city. In order to characterize the resulting large collection of isolates, an automated low-cost typing method was developed. The multiplex capillary-based variable-number tandem repeat (VNTR) (multiple-locus VNTR analysis [MLVA]) assay requiring only one PCR amplification per isolate ensures a high level of discrimination and reduces hands-on and time requirements. In less than 2 days and using one 4-capillary apparatus, 217 environmental isolates collected between 2000 and 2009 and 5 clinical isolates obtained during outbreaks in 2000 and 2006 in Rennes were analyzed, and 15 different genotypes were identified. A large cluster of isolates with closely related genotypes and representing 77% of the population was composed exclusively of environmental isolates extracted from hot water supply systems. It was not responsible for the known Rennes epidemic cases, although strains showing a similar MLVA profile have regularly been involved in European outbreaks. The clinical isolates in Rennes had the same genotype as isolates contaminating a mall's cooling tower. This study further demonstrates that unknown environmental or genetic factors contribute to the pathogenicity of some strains. This work illustrates the potential of the high-throughput MLVA typing method to investigate the origin of legionellosis cases by allowing the systematic typing of any new isolate and inclusion of data in shared databases.

 

LAMP-based method for a rapid identification of Legionella spp. and Legionella pneumophila

Lu X, Mo ZY, Zhao HB, Yan H, Shi L.

College of Light Industry and Food Sciences, South China University of Technology, 510640, Guangzhou, China. ziyaomo@gmail.com

Appl Microbiol Biotechnol. 2011 Oct;92(1):179-87.

ABSTRACT: Legionella pneumophila is accounted for more than 80% of Legionella infection. However it is difficult to discriminate between the L. pneumophila and non-L. pneumophila species rapidly. In order to detect the Legionella spp. and distinguish L. pneumophila from Legionella spp., a real-time loop-mediated isothermal amplification (LAMP) platform that targets a specific sequence of the 16S rRNA gene was developed. LS-LAMP amplifies the fragment of the 16S rRNA gene to detect all species of Legionella genus. A specific sequence appears at the 16S rRNA gene of L. pneumophila, while non-L. pneumophila strains have a variable sequence in this site, which can be recognized by the primer of LP-LAMP. In the present study, 61 reference strains were used for the method verification. We found that the specificity was 100% for both LS-LAMP and LP-LAMP, and the sensitivity of LAMP assay for L. pneumophila detection was between 52 and 5.2 copies per reaction. In the environmental water samples detection, a total of 107 water samples were identified by the method. The culture and serological test were used as reference methods. The specificity of LS-LAMP and LP-LAMP for the samples detection were 91.59% (98/107) and 93.33% (56/60), respectively. The sensitivity of LS-LAMP and LP-LAMP were 100% (51/51) and 100% (18/18). The results suggest that real-time LAMP, as a new assay, provides a specific and sensitive method for rapid detection and differentiation of Legionella spp. and L. pneumophila and should be utilized to test environmental water samples for increased rates of detection.

 

Enhanced isolation of Legionella species from composted material

McCabe S, Brown A, Edwards GF, Lindsay D.

Scottish Haemophilus Legionella Meningococcus Pneumococcus Reference Laboratory (SHLMPRL), House on the Hill, Stobhill Hospital, Glasgow, UK. Diane.Lindsay@ggc.scot.nhs.uk

Clin Microbiol Infect. 2011 Oct;17(10):1517-20.

ABSTRACT: Legionella pneumophila and Legionella species were isolated from composted material when freshly prepared buffered charcoal yeast extract (BCYE) was supplemented with glycine (1.5 g/L), polymyxin B sulfate (40000 IU/L), vancomycin hydrochloride (0.5 mg/L) and cycloheximide (40 mg/L) (GVPC medium) and Modified Wadowsky-Yee (MWY) (Oxoid, Cambridge, UK) plates were used for cultivation, but not with commercially sourced pre-poured GVPC and MWY plates (Oxoid). Legionella cincinnatiensis and pathogenic L.pneumophila serogroup (Sg) 1 Benidorm and France/Allentown were identified, as well as a non-typeable (NT) strain of L. pneumophila. As most laboratories no longer produce their own media, this may contribute to the lack of positive cultures from composted material. The antigenicity of the NT strain is discussed.

 

Accuracy and precision of Legionella isolation by US laboratories in the ELITE program pilot study

Lucas CE, Taylor TH Jr, Fields BS.

Division of Bacterial Diseases, Centers for Disease Control and Prevention, 1600 Clifton Rd NE MS G03, Atlanta, GA 30333, USA. chl9@cdc.gov

Water Res. 2011 Oct 1;45(15):4428-36.

ABSTRACT: A pilot study for the Environmental Legionella Isolation Techniques Evaluation (ELITE) Program, a proficiency testing scheme for US laboratories that culture Legionella from environmental samples, was conducted September 1, 2008 through March 31, 2009. Participants (n=20) processed panels consisting of six sample types: pure and mixed positive, pure and mixed negative, pure and mixed variable. The majority (93%) of all samples (n=286) were correctly characterized, with 88.5% of samples positive for Legionella and 100% of negative samples identified correctly. Variable samples were incorrectly identified as negative in 36.9% of reports. For all samples reported positive (n=128), participants underestimated the cfu/ml by a mean of 1.25 logs with standard deviation of 0.78 logs, standard error of 0.07 logs, and a range of 3.57 logs compared to the CDC re-test value. Centering results around the interlaboratory mean yielded a standard deviation of 0.65 logs, standard error of 0.06 logs, and a range of 3.22 logs. Sampling protocol, treatment regimen, culture procedure, and laboratory experience did not significantly affect the accuracy or precision of reported concentrations. Qualitative and quantitative results from the ELITE pilot study were similar to reports from a corresponding proficiency testing scheme available in the European Union, indicating these results are probably valid for most environmental laboratories worldwide. The large enumeration error observed suggests that the need for remediation of a water system should not be determined solely by the concentration of Legionella observed in a sample since that value is likely to underestimate the true level of contamination.

 

Bacteria misagglutination in legionella surveillance programmes

Orsini M, Cristino S, Grottola A, Romano-Spica V.

Center for Advanced Studies, Research and Development in Sardinia, Bioinformatics Group, Pula, Italy. orsini@crs4.it

J Hosp Infect. 2011 Oct;79(2):179-80.

Letter to the Editor.

 

Total and viable Legionella pneumophila cells in hot and natural waters as measured by immunofluorescence-based assays and solid-phase cytometry

Parthuisot N, Binet M, Touron-Bodilis A, Pougnard C, Lebaron P, Baudart J.

UPMC Université Paris 06, UMR 7621, LOMIC, Observatoire Océanologique, F-66650 Banyuls-sur-Mer, France. baudart@obs-banyuls.fr

Appl Environ Microbiol. 2011 Sep;77(17):6225-32.

ABSTRACT: A new method was developed for the rapid and sensitive detection of viable Legionella pneumophila. The method combines specific immunofluorescence (IF) staining using monoclonal antibodies with a bacterial viability marker (ChemChrome V6 cellular esterase activity marker) by means of solid-phase cytometry (SPC). IF methods were applied to the detection and enumeration of both the total and viable L. pneumophila cells in water samples. The sensitivity of the IF methods coupled to SPC was 34 cells liter(-1), and the reproducibility was good, with the coefficient of variation generally falling below 30%. IF methods were applied to the enumeration of total and viable L. pneumophila cells in 46 domestic hot water samples as well as in cooling tower water and natural water samples, such as thermal spring water and freshwater samples. Comparison with standard plate counts showed that (i) the total direct counts were always higher than the plate counts and (ii) the viable counts were higher than or close to the plate counts. With domestic hot waters, when the IF assay was combined with the viability test, SPC detected up to 3.4 × 10(3) viable but nonculturable L. pneumophila cells per liter. These direct IF methods could be a powerful tool for high-frequency monitoring of domestic hot waters or for investigating the occurrence of viable L. pneumophila in both man-made water systems and environmental water samples.

 

Detection of Legionella species in potting mixes using fluorescent in situ hybridisation (FISH)

Whiley H, Taylor M, Bentham R.

School of the Environment, Environmental Health, Flinders University, GPO Box 2100, 5001 Adelaide, Australia. harriet.whiley@flinders.edu.au

J Microbiol Methods. 2011 Sep;86(3):304-9.

ABSTRACT: This study used Fluorescent in situ Hybridisation (FISH) with rRNA targeted oligonucleotide probes combined with scanning confocal laser microscopy to successfully detect Legionella spp. in commercially available potting mix. A range of techniques were explored to optimise the FISH method by reducing background fluorescence and preventing non-specific binding of probes. These techniques included the use of a blocking agent, UV light treatment, image subtraction of a nonsense probe and spectral unmixing of specific probes fluorescence and autofluorescence dependent on the specific emission spectra of probe fluorophores. Spectral unmixing was the best microscopy technique for reducing background fluorescence and non-specific binding of probes was not observed. The rapid turnaround time and increased sensitivity of the FISH provides as an alternative to traditional culture methods, which are tedious and often give varied results. FISH is also advantageous compared to PCR methods as it provides information on the structure of the microbial community the bacteria is situated in. This study demonstrates that FISH could provide an alternative method for Legionella detection and enumeration in environmental samples.

 

Development and validation of ELISA for detection of antibodies to Legionella pneumophila serogroup 1, 3 and 6 in human sera

Elverdal PL, Svarrer CW, Jørgensen CS, Skovsted IC, Uldum SA.

Unit of Atypical Pneumonia, Department of Microbiological Surveillance and Research, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark. pel@ssi.dk

J Microbiol Methods. 2011 Sep;86(3):298-303.

ABSTRACT: The aim of this study was to determine whether separate measurement of immunoglobulin (Ig) M and G antibodies to Legionella (L.) pneumophila serogroups (sg) 1, 3 and 6 as single antigens can facilitate an early diagnosis of Legionnaires' disease. The developed ELISA was evaluated and compared with an in-house indirect Legionella immunofluorescence antibody test (IFAT) measuring Total Ig. A total of 193 sera from 128 patients with confirmed L. pneumophila infections were used to assess the sensitivity of the developed ELISA. The sensitivity was assessed in different time-periods after onset of symptoms. It was found that the sensitivity of the test increased during the first month of infection, IgM being the most sensitive; ranging from 13% in the first week after onset of symptoms, 45% in the second week to 84% in the third week; in the fourth (and beyond) week a drop to 67% was observed. The IFAT detecting L. pneumophila sg 1-6 had a sensitivity of 11%, 27%, 80% and 59%, respectively, during these time-periods. The test with the lowest sensitivity was the IgG ELISA (0%, 21%, 36% and 52%), but by combining the IgG results with the IgM results, the overall sensitivity of the assay was improved (13%, 48%, 88% and 70%). This study confirms that detection of IgG and IgM antibodies by ELISA is an important diagnostic tool especially during the initial phase of the disease, when supported by other tests like the urinary antigen test, PCR or culture. Furthermore, we showed that the ELISA is suitable for the detection of significant changes in antibody levels in paired serum samples. It was found that the sensitivity was higher for the ELISA assays than for the IFAT. Both the in-house IgM ELISA and the IFAT had a low false positive rate, while a 14% false positive rate was found for the IgG ELISA among serum samples from patients with other infections.

 

Investigation of the population structure of Legionella pneumophila by analysis of tandem repeat copy number and internal sequence variation

Visca P, D'Arezzo S, Ramisse F, Gelfand Y, Benson G, Vergnaud G, Fry NK, Pourcel C.

Dipartimento di Biologia, Università Roma Tre, Rome, Italy. christine.pourcel@u-psud.fr

Microbiology. 2011 Sep;157(Pt 9):2582-94.

ABSTRACT: The population structure of the species Legionella pneumophila was investigated by multilocus variable number of tandem repeats (VNTR) analysis (MLVA) and sequencing of three VNTRs (Lpms01, Lpms04 and Lpms13) in selected strains. Of 150 isolates of diverse origins, 136 (86%) were distributed into eight large MLVA clonal complexes (VACCs) and the rest were either unique or formed small clusters of up to two MLVA genotypes. In spite of the lower degree of genome-wide linkage disequilibrium of the MLVA loci compared with sequence-based typing, the clustering achieved by the two methods was highly congruent. The detailed analysis of VNTR Lpms04 alleles showed a very complex organization, with five different repeat unit lengths and a high level of internal variation. Within each MLVA-defined VACC, Lpms04 was endowed with a common recognizable pattern with some interesting exceptions. Evidence of recombination events was suggested by analysis of internal repeat variations at the two additional VNTR loci, Lpms01 and Lpms13. Sequence analysis of L. pneumophila VNTR locus Lpms04 alone provides a first-line assay for allocation of a new isolate within the L. pneumophila population structure and for epidemiological studies.

 

Faster Legionella testing on horizon

Pearson S.

susan@wordways.co.uk

Health Estate. 2011 Aug;65(7):44-6.

ABSTRACT: While the "traditional" way to measure Legionella quantitatively in water is based on a complex culture method where results can take up to 14 days, the last few years have seen the availability of very rapid real-time monitoring of the bacterium in water systems, with the development of quantitative polymerase chain reaction (qPCR), a process which gives results "within hours". To date, however, a lack of consensus on how to interpret such results in relation to those from culture has been a stumbling block, although, as Susan Pearson, a freelance journalist and public relations consultant specialising in medicine and the environment, reports, the positive results of a recent multi-centre European study mean this could soon all change.

 

PCR methods for the rapid detection and identification of four pathogenic Legionella spp. and two Legionella pneumophila subspecies based on the gene amplification of gyrB

Zhou G, Cao B, Dou Y, Liu Y, Feng L, Wang L.

Key Laboratory of Molecular Microbiology and Technology of the Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071, China. wanglei@nankai.edu.cn

Appl Microbiol Biotechnol. 2011 Aug;91(3):777-87.

ABSTRACT: A total of 25 gyrB gene sequences from 20 Legionella pneumophila subsp. pneumophila strains and five L. pneumophila subsp. fraseri strains were obtained and analyzed, and a multiplex PCR for the simultaneous detection of Legionella bozemanae, Legionella longbeachae, Legionella micdadei and Legioenella pneumophila, and two single PCRs for the differentiation of L. pneumophila subsp. pneumophila and L. pneumophila subsp. fraseri were established. The multiplex PCR method was shown to be highly specific and reproducible when tested against 41 target strains and 17 strains of other bacteria species. The sensitivity of the multiplex PCR was also analyzed and was shown to detect levels as low as 1 ng of genomic DNA or 10 colony-forming units (CFUs) per milliliter in mock water samples. Sixty-three air conditioner condensed water samples from Shanghai City were examined, and the result was validated using 16S rRNA sequencing. The data reported here demonstrate that the multiplex PCR method described is efficient and convenient for the detection of Legionella species in water samples. Twenty L. pneumophila subsp. pneumophila strains and five L. pneumophila subsp. fraseri strains were used for the validation of the two L. pneumophila subspecies-specific PCR methods, and the results indicated that the two PCR methods were both highly specific and convenient for the identification of L. pneumophila at the subspecies level.

  

Faster Legionella testing on horizon

Pearson S.

susan@wordways.co.uk

Health Estate. 2011 Aug;65(7):44-6.

ABSTRACT: While the "traditional" way to measure Legionella quantitatively in water is based on a complex culture method where results can take up to 14 days, the last few years have seen the availability of very rapid real-time monitoring of the bacterium in water systems, with the development of quantitative polymerase chain reaction (qPCR), a process which gives results "within hours". To date, however, a lack of consensus on how to interpret such results in relation to those from culture has been a stumbling block, although, as Susan Pearson, a freelance journalist and public relations consultant specialising in medicine and the environment, reports, the positive results of a recent multi-centre European study mean this could soon all change.

 

Methodologies for quantifying culturable, viable, and total Legionella pneumophila in indoor air

Chang CW, Chou FC.

Institute of Environmental Health, College of Public Health, National Taiwan University, Taipei, Republic of China. chingwenchang@ntu.edu.tw

Indoor Air. 2011 Aug;21(4):291-9.

ABSTRACT: Legionella pneumophila, aerosolized from numerous indoor facilities (e.g., shower heads, hot tubs, spas), may cause Pontiac fever (PF) and lethal pneumonia named Legionnaires' disease (LD) in humans. Reliable methods on quantitative exposure assessment of this bioaerosol are essential for the prevention of PF and LD. Coupled with culture, ethidium monoazide with qPCR, and qPCR assays, the collection efficiency for culturable, viable, and total L. pneumophila was assessed by means of filtration sampling (IOM with gelatin filter and cassette with polycarbonate filter) and liquid-based sampling methods (BioSampler, AGI-30, MAS-100 sampler with Tween mixture and deionized water (DW)). Results show IOM/gelatin filter was comparable to cassette/polycarbonate filter (P = 0.33) and performed greater than all of tested liquid-based methods for total cell collection. On the other hand, IOM/gelatin filter obtained greater efficiencies than cassette/polycarbonate filter by a factor of 3.8-8.6 for viable cells (P = 0.0006) and two orders of magnitude for culturable cells (P = 0.00002). Further comparison between liquid impingement and filtration methods indicates the sampling by IOM/gelatin filter, AGI-30, and BioSampler with DW were the most appropriate for viable cells, while culturable cells were collected most efficiently by BioSampler/DW with periodical replenishment during the sampling. PRACTICAL IMPLICATIONS: This study recommends the most suitable methodologies for quantifying culturable, viable, and total Legionella pneumophila in indoor air. By using appropriate sampling and analytical methods, the residents and building owners are able to obtain the reliable data and further characterize the exposure risk and/or intervention efficacy against L. pneumophila. Moreover, the adoption of suitable monitoring methods also assists the investigators to explore the sources linked to PF and LD during the outbreaks. Considering reliable microbial monitoring is fundamental for epidemiological survey and risk assessment, the present information should be taken into account in assessing L. pneumophila indoors.

 

Usefulness of real-time PCR as a complementary tool to the monitoring of Legionella spp. and Legionella pneumophila by culture in industrial cooling systems

Touron-Bodilis A, Pougnard C, Frenkiel-Lebossé H, Hallier-Soulier S.

EDF Research and Development, Laboratoire National d'Hydraulique et d'Environnement, Chatou Cedex, France. aurelie.touron-bodilis@edf.fr

J Appl Microbiol. 2011 Aug;111(2):499-510.

ABSTRACT: AIMS: This study was designed to evaluate the usefulness of quantification by real-time PCR as a management tool to monitor concentrations of Legionella spp. and Legionella pneumophila in industrial cooling systems and its ability to anticipate culture trends by the French standard method (AFNOR T90-431).

METHODS AND RESULTS: Quantifications of Legionella bacteria were achieved by both methods on samples from nine cooling systems with different water qualities. Proportion of positive samples for L. pneumophila quantified by PCR was clearly lower in deionized or river waters submitted to a biocide treatment than in raw river waters, while positive samples for Legionella spp. were quantified for almost all the samples. For some samples containing PCR inhibitors, high quantification limits (up to 4·80 × 10(5) GU l(-1) ) did not allow us to quantify L. pneumophila, when they were quantified by culture. Finally, the monitoring of concentrations of L. pneumophila by both methods showed similar trends for 57-100% of the samples.

CONCLUSIONS: These results suggest that, if some methodological steps designed to reduce inhibitory problems and thus decrease the quantification limits, could be developed to quantify Legionella in complex waters, the real-time PCR could be a valuable complementary tool to monitor the evolution of L. pneumophila concentrations.

SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the possibility of using real-time PCR to monitor L. pneumophila proliferations in cooling systems and the importance to adapt nucleic acid extraction and purification protocols to raw waters.

 

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and database for identification of Legionella species

He Y, Chang TC, Li H, Shi G, Tang YW.

Department of Pathology and Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA. yiwei.tang@vanderbilt.edu

Can J Microbiol. 2011 Jul;57(7):533-8.

ABSTRACT: More than 20 species of Legionella have been identified in relation to human infections. Rapid detection and identification of Legionella isolates is clinically useful to differentiate between infection and contamination and to determine treatment regimens. We explored the use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) Biotyper system (Bruker Daltonik GmbH, Bremen, Germany) for the identification of Legionella species. The MALDI MS spectra were generated and compared with the Biotyper database, which includes 25 Legionella strains covering 22 species and four Legionella pneumophila serogroups. A total of 83 blind-coded Legionella strains, consisting of 54 reference and 29 clinical strains, were analyzed in the study. Overall, the Biotyper system correctly identified 51 (61.4%) of all strains and isolates to the species level. For species included in the Biotyper database, the method identified 51 (86.4%) strains out of 59 Legionella strains to the correct species level, including 24 (100%) L. pneumophila and 27 (77.1%) non-L. pneumophila strains. The remaining 24 Legionella strains, belonging to species not covered by the Biotyper database, were either identified to the Legionella genus level or had no reliable identification. The Biotyper system produces constant and reproducible MALDI MS spectra for Legionella strains and can be used for rapid and accurate Legionella identification. More Legionella strains, especially the non-L. pneumophila strains, need to be included in the current Biotyper database to cover varieties of Legionella species and to increase identification accuracy.

 

A rapid detection method using flow cytometry to monitor the risk of Legionella in bath water

Taguri T, Oda Y, Sugiyama K, Nishikawa T, Endo T, Izumiyama S, Yamazaki M, Kura F.

Nagasaki Prefectural Institute for Environmental Research and Public Health, 2-1306-11 Ikeda, Omura, Nagasaki 856-0026, Japan. tagurit@pref.nagasaki.lg.jp

J Microbiol Methods. 2011 Jul;86(1):25-32.

ABSTRACT: Legionella species are the causative agents of human legionellosis, and bathing facilities have been identified as the sources of infection in several outbreaks in Japan. Researchers in Japan have recently reported evidence of significant associations between bacterial counts and the occurrence of Legionella in bathing facilities and in a hot tub model. A convenient and quantitative bacterial enumeration method is therefore required as an indicator of Legionella contamination or disinfection to replace existing methods such as time-consuming Legionella culture and expensive Legionella-DNA amplification. In this study, we developed a rapid detection method (RDM) to monitor the risk of Legionella using an automated microbial analyzing device based on flow cytometry techniques to measure the total number of bacteria in water samples within two minutes, by detecting typical patterns of scattered light and fluorescence. We first compared the results of our RDM with plate counting results for five filtered hot spring water samples spiked with three species of bacteria, including Legionella. Inactivation of these samples by chlorine was also assessed by the RDM, a live/dead bacterial fluorescence assay and plate counting. Using the RDM, the lower limit of quantitative bacterial counts in the spiked samples was determined as 3.0×10(3)(3.48log)counts mL(-1). We then used a laboratory model of a hot tub and found that the RDM could monitor the growth curve of naturally occurring heterotrophic bacteria with 1 and 2 days' delayed growth of amoeba and Legionella, respectively, and could also determine the killing curve of these bacteria by chlorination. Finally, samples with ≥3.48 or <3.48log total bacterial counts mL(-1) were tested using the RDM from 149 different hot tubs, and were found to be significantly associated with the positive or negative detection of Legionella with 95% sensitivity and 84% specificity. These findings indicated that the RDM can be used for Legionella control at bathing facilities, especially those where the effectiveness of chlorine is reduced by the presence of Fe(2+), Mn(2+), NH(4)(+), skin debris, and/or biofilms in the water.

 

Recovery of Legionella species from water samples using an internal method based on ISO 11731: suggestions for revision and implementation

Ditommaso S, Gentile M, Giacomuzzi M, Zotti CM.

Dipartimento di Sanità Pubblica e di Microbiologia, Università degli Studi di Torino, Via Santena, 5 bis, 10126 Turin, Italy. savina.ditommaso@unito.it

Diagn Microbiol Infect Dis. 2011 Jun;70(2):200-6.

ABSTRACT: The study aim was to determine retrospectively whether the parallel use of 2 media [buffered charcoal yeast extract (BCYE) and medium of Wadowsky and Yee (MWY)] to isolate Legionella spp. from water samples taken from hospital water supply systems increased the sensitivity of the culture method as compared with methods/protocols in which only seeding on a selective medium is used. We analyzed the results obtained from 931 positive water samples. In 484 of the 931 positive water samples, Legionella spp. was isolated in the presence of other microorganisms; in 83% (400/484), we used MWY to count suspected colonies, which gave a lower number of unreadable plates. In the 447 samples containing only Legionella spp., the highest frequency of positive samples (93%, 418/447) was obtained with BCYE, whereas seeding on MWY yielded 78% (348/447) (P < 0.001). Evaluation of the influence of the media on the Legionella spp. counts obtained by the 2 media showed that BCYE agar produced significantly higher counts than MWY (P < 0.001). The major conclusions that may be drawn from our data are as follows: 1) BCYE gives a high recovery rate of positive samples (93%) and a much greater yield of Legionella spp. than MWY; 2) BCYE was necessary for the detection of non-L. pneumophila spp. which grew poorly on selective media; 3) selective media [MWY or GVPC (glycine, vancomycin, polymyxin B, and cycloheximide)] were necessary for the recovery of Legionella spp. when the non-selective medium (BCYE) was difficult to interpret because of contaminating background flora. The use of different media is recommended for routine water tests in hospitals.

 

Detection of Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella spp. in clinical specimens using a single-tube multiplex real-time PCR assay

Thurman KA, Warner AK, Cowart KC, Benitez AJ, Winchell JM.

Respiratory Diseases Branch, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA. jwinchell@cdc.gov

Diagn Microbiol Infect Dis. 2011 May;70(1):1-9.

ABSTRACT: A multiplex real-time PCR assay for the detection of Mycoplasma pneumoniae (MP181), Chlamydia (Chlamydophila) pneumoniae (CP-Arg), Legionella spp. (Pan-Leg), and the human RNase P (RNase P) gene was developed for rapid testing of atypical bacterial respiratory pathogens in clinical specimens. This method uses 4 distinct hydrolysis probes to detect 3 leading causes of community-acquired pneumonia. The assay was evaluated for specificity and sensitivity by testing against 35 related organisms, a dilution series of each specific target and 197 clinical specimens. Specificity testing demonstrated no cross-reactivity. A comparison to previously validated singleplex real-time PCR assays for each agent was also performed. The analytical sensitivity for specific pathogen targets in both the singleplex and multiplex was identical (50 fg), while efficiencies ranged from 82% to 97% for the singleplex assays and from 90% to 100% for the multiplex assay. The clinical sensitivity of the multiplex assay was improved for the Pan-Leg and CP-Arg targets when compared to the singleplex. The MP181 assay displayed equivalent performance. This multiplex assay provides an overall improvement in the diagnostic capability for these agents by demonstrating a sensitive, high-throughput and rapid method. This procedure may allow for a practical and efficient means to test respiratory clinical specimens for atypical pneumonia agents in health care settings and facilitate an appropriate public health response to outbreaks.

 

Use of nested polymerase chain reaction based on sequence-based typing of clinical samples to determine the source of infection for hospital-acquired Legionnaires' disease

Scaturro M, Fontana S, Ricci ML.

Department of Infectious, Parasitic, and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy. maria.scaturro@iss.it

Infect Control Hosp Epidemiol. 2011 May;32(5):510-2.

ABSTRACT: The source of infection of a hospital-acquired Legionnaires' disease case was determined for the first time by nested polymerase chain reaction based on sequence-based typing. The typing was performed directly on DNA extracted from tissue samples, allowing a rapid epidemiological correlation with environmental isolates.

 

Quantification of viable Legionella pneumophila cells using propidium monoazide combined with quantitative PCR

Yáñez MA, Nocker A, Soria-Soria E, Múrtula R, Martínez L, Catalán V.

LABAQUA, SA, Alicante, Spain. vicente.catalan@labaqua.com

J Microbiol Methods. 2011 May;85(2):124-30.

ABSTRACT: One of the greatest challenges of implementing fast molecular detection methods as part of Legionella surveillance systems is to limit detection to live cells. In this work, a protocol for sample treatment with propidium monoazide (PMA) in combination with quantitative PCR (qPCR) has been optimized and validated for L. pneumophila as an alternative of the currently used time-consuming culture method. Results from PMA-qPCR were compared with culture isolation and traditional qPCR. Under the conditions used, sample treatment with 50 μM PMA followed by 5 min of light exposure were assumed optimal resulting in an average reduction of 4.45 log units of the qPCR signal from heat-killed cells. When applied to environmental samples (including water from cooling water towers, hospitals, spas, hot water systems in hotels, and tap water), different degrees of correlations between the three methods were obtained which might be explained by different matrix properties, but also varying degrees of non-culturable cells. It was furthermore shown that PMA displayed substantially lower cytotoxicity with Legionella than the alternative dye ethidium monoazide (EMA) when exposing live cells to the dye followed by plate counting. This result confirmed the findings with other species that PMA is less membrane-permeant and more selective for the intact cells. In conclusion, PMA-qPCR is a promising technique for limiting detection to intact cells and makes Legionella surveillance data substantially more relevant in comparison with qPCR alone. For future research it would be desirable to increase the method's capacity to exclude signals from dead cells in difficult matrices or samples containing high numbers of dead cells.

 

Automated immunomagnetic processing and separation of Legionella pneumophila with manual detection by sandwich ELISA and PCR amplification of the ompS gene

Reidt U, Geisberger B, Heller C, Friedberger A.

Department IW-SI-Sensors, Electronics & Systems Integration, EADS Innovation Works, Munich, Germany. ulrich.reidt.external@eads.net

J Lab Autom. 2011 Apr;16(2):157-64.

ABSTRACT: The culture-independent and automated detection of bacteria in the environment is a scientific and technological challenge. For detection alone, a number of sensitive methods are known (e.g., PCR, enzyme-linked immunosorbent assay [ELISA], fluorescent in situ hybridization) but a major problem remaining is the enrichment and separation of the bacteria that usually occur at low concentrations. Here, we present an automated capturing and separation system, which can easily be combined with one of the sensitive detection techniques. We have developed a method for enrichment and detection of Legionella pneumophila in liquid media. Concentrated microorganisms were either detected by PCR or by sandwich ELISA. The limit of detection with the immunological assay was about 750 bacteria. Using PCR, the equivalent of about 2000 genomes could be detected. The assays were then transferred to a laboratory prototype for automated processing. It was possible to automatically enrich L. pneumophila by immunomagnetic separation (IMS), and again, the bacteria were detected by sandwich ELISA and PCR amplification of the ompS gene. As a novel aspect, ompS gene was used for the first time as a target for the detection of L. pneumophila on magnetic beads. The aim of this work was to develop an automated procedure and a device for IMS of bacteria. With Legionella as a model organism, we could show that such a novel fully automated system can be an alternative to time-consuming conventional cultivation methods for detecting bacteria or other microorganisms.

 

Comparison of commercial systems for extraction of nucleic acids from DNA/RNA respiratory pathogens

Yang G, Erdman DE, Kodani M, Kools J, Bowen MD, Fields BS.

Division of Blood Disorders, National Center for Birth Defects and Developmental Disabilities, Centers for Disease Control and Prevention, Atlanta, GA 30329, USA. gyang@cdc.gov

J Virol Methods. 2011 Jan;171(1):195-9.

ABSTRACT: This study compared six automated nucleic acid extraction systems and one manual kit for their ability to recover nucleic acids from human nasal wash specimens spiked with five respiratory pathogens, representing Gram-positive bacteria (Streptococcus pyogenes), Gram-negative bacteria (Legionella pneumophila), DNA viruses (adenovirus), segmented RNA viruses (human influenza virus A), and non-segmented RNA viruses (respiratory syncytial virus). The robots and kit evaluated represent major commercially available methods that are capable of simultaneous extraction of DNA and RNA from respiratory specimens, and included platforms based on magnetic-bead technology (KingFisher mL, Biorobot EZ1, easyMAG, KingFisher Flex, and MagNA Pure Compact) or glass fiber filter technology (Biorobot MDX and the manual kit Allprep). All methods yielded extracts free of cross-contamination and RT-PCR inhibition. All automated systems recovered L. pneumophila and adenovirus DNA equivalently. However, the MagNA Pure protocol demonstrated more than 4-fold higher DNA recovery from the S. pyogenes than other methods. The KingFisher mL and easyMAG protocols provided 1- to 3-log wider linearity and extracted 3- to 4-fold more RNA from the human influenza virus and respiratory syncytial virus. These findings suggest that systems differed in nucleic acid recovery, reproducibility, and linearity in a pathogen specific manner.

 

Diagnosis of fulminant pneumonia caused by Legionella pneumophila serogroup 8 with the sequence analysis of the 16S rRNA gene

Kawanami T, Yatera K, Fukuda K, Yamasaki K, Kunimoto M, Nagata S, Nishida C, Ishimoto H, Ogawa M, Taniguchi H, Mukae H.

Department of Respiratory Medicine, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan. namihei@med.uoeh-u.ac.jp

Tohoku J Exp Med. 2011;225(1):65-9.

ABSTRACT: Pneumonia is the fourth leading cause of death in Japan. Accurate and rapid detection of the causative pathogen(s) is necessary and important for appropriate antimicrobial treatment, especially in patients with rapidly progressive pneumonia or immunocompromised patients. Conventional methods, such as cultivations, detection of urinary antigens or PCR amplification of specific genes, inevitably require the precise presumption of potential pathogens in each case, and pneumonia caused by unanticipated microorganisms might lead to inadequate antimicrobial treatments and unfortunate consequences. We herein report an immunocompromised female patient (69 years old) with fulminant pneumonia caused by Legionella (L.) pneumophila serogroup 8. Ordinary cultivation methods and urinary antigen detection failed to identify the causative organisms. Accordingly, DNA was extracted from the bronchoalveolar lavage fluid and used for the PCR-based cloning of the bacterial 16S rRNA gene. Sequencing analysis of the isolated clones revealed the predominance of L. pneumophila. Based on this information, the patient received an appropriate and successful antimicrobial treatment. In addition, L. pneumophila serogroup 8 was identified with culturing the bronchoalveolar lavage fluid and serotyping with L. pneumophila antisera. The 16S rRNA gene sequencing analysis can reveal the potential pathogens without any presumption about the organism, and can evaluate the kinds and ratio of bacterial species in each specimen. In conclusion, this cultivation-independent method is a potential diagnostic modality for pneumonia, especially in patients with rapidly progressive pneumonia or those who are immunocompromised.

 

Assessment of real-time PCR for quantification of Legionella spp. in spa water

Guillemet TA, Lévesque B, Gauvin D, Brousseau N, Giroux JP, Cantin P.

Institut national de santé publique du Québec, Québec, QC, Canada. philippe.cantin@mddep.gouv.qc.ca

Lett Appl Microbiol. 2010 Dec;51(6):639-44.

ABSTRACT: AIMS: Legionella bacteria ubiquitously colonize natural freshwater and are responsible for legionellosis in humans. Several cases of legionellosis have been associated in particular with the use of whirlpool spas. The objective of this study was to verify whether real-time PCR is applicable for the quantification of Legionella spp. in spa water.

METHODS AND RESULTS: The study compared concentrations obtained by real-time PCR vs that obtained by conventional culture for 101 spa water samples. For the culture method, Legionella spp. were detected and quantified in 14 of 101 samples with measured concentrations ranging from 250 to 3.5 × 10(5) CFU l(-1). With the real-time PCR method, Legionella spp. were detected and quantified in 42 of 101 samples with concentrations ranging from 1000 to 6.1 × 10(7) GU l(-1). Results revealed a significant but weak correlation (r(2) = 0.1867) between the two methods. The positive predictive value (35%) of the PCR method compared to conventional culture herein was low. In contrast, the negative predictive value was excellent, reaching 93%.

CONCLUSIONS: Real-time PCR could be used as a screening tool to rapidly ascertain the absence of Legionella spp. in spa water. However, a positive result involves the need to resort to conventional culture.

SIGNIFICANCE AND IMPACT OF THE STUDY: Data of this study highlighted the pros and cons of quantification of Legionella spp. in spa water with real-time PCR using a commercial quantitative PCR kit in a routine laboratory, when compared to conventional culture.

 

Sessile Legionella pneumophila is able to grow on surfaces and generate structured monospecies biofilms

Pécastaings S, Bergé M, Dubourg KM, Roques C.

LU 49, Adhesion bacterienne et formation de biofilms, UPS, Universite de Toulouse, Toulouse, France. sophie@pecastaings.net

Biofouling. 2010 Oct;26(7):809-19.

ABSTRACT: Currently, models for studying Legionella pneumophila biofilm formation rely on multi-species biofilms with low reproducibility or on growth in rich medium, where planktonic growth is unavoidable. The present study describes a new medium adapted to the growth of L. pneumophila monospecies biofilms in vitro. A microplate model was used to test several media. After incubation for 6 days in a specific biofilm broth not supporting planktonic growth, biofilms consisted of 5.36 ± 0.40 log (cfu cm(-2)) or 5.34 ± 0.33 log (gu cm(-2)). The adhered population remained stable for up to 3 weeks after initial inoculation. In situ confocal microscope observations revealed a typical biofilm structure, comprising cell clusters ranging up to approximately 300 μm in height. This model is adapted to growing monospecies L. pneumophila biofilms that are structurally different from biofilms formed in a rich medium. High reproducibility and the absence of other microbial species make this model useful for studying genes involved in biofilm formation.

 

Inter-laboratory validation of a rapid assay for the detection and quantification of Legionella spp. in water samples

Bargellini A, Marchesi I, Leoni E, Mansi A, Cristino S, Marcelloni AM, Borella P.

Department of Public Health Sciences, University of Modena and Reggio Emilia, Modena, Italy. annalisa.bargellini@unimore.it

Lett Appl Microbiol. 2010 Oct;51(4):421-7.

ABSTRACT:AIMS: To compare the standard culture method with a new, rapid test (ScanVIT-Legionella™) using fluorescently labelled gene probes for the detection and enumeration of Legionella spp. The new technique was validated through experiments conducted on both artificially and naturally contaminated water and through an inter-laboratory comparison.

METHODS AND RESULTS: All samples were processed by the ScanVIT test according to the manufacturer's instructions and by a culture method (ISO 11731). ScanVIT detected significantly more positive samples, although concentrations were similar and a strong positive correlation between the two methods was observed (r = 0.888, P < 0.001). The new test was more accurate in identifying the co-presence of Legionella pneumophila and Leg. non-pneumophila. ScanVIT showed a slightly higher Legionella recovery from water samples artificially contaminated with Leg. pneumophila alone or together with Pseudomonas aeruginosa. Lastly, the inter-laboratory comparison revealed that the ScanVIT test exhibits a lower variability than the traditional culture test (mean coefficient of variation 8.7 vs 16.1%).

CONCLUSIONS: The results confirmed that the ScanVIT largely overlaps the reference method and offers advantages in terms of sensitivity, quantitative reliability and reduced assay time.

SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed method may represent a useful validated alternative to traditional culture for the rapid detection and quantification of Legionella spp. in water.

 

High-resolution in situ genotyping of Legionella pneumophila populations in drinking water by multiple-locus variable-number tandem-repeat analysis using environmental DNA

Kahlisch L, Henne K, Draheim J, Brettar I, Höfle MG.

Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research (HZI), 38124 Braunschweig, Germany. mho@gbf.de

Appl Environ Microbiol. 2010 Sep;76(18):6186-95.

ABSTRACT: Central to the understanding of infections by the waterborne pathogen Legionella pneumophila is its detection at the clonal level. Currently, multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) of L. pneumophila isolates can be used as a tool for high-resolution genotyping. Since L. pneumophila is difficult to isolate, the isolation of outbreak strains often fails due to a viable but nonculturable (VBNC) state of the respective environmental population. Therefore, we developed a cultivation-independent approach to detect single clones in drinking water. This approach is based on the extraction of DNA from drinking water followed by PCR using a set of eight VNTR primer pairs necessary for MLVA genotyping of L. pneumophila. The PCR amplicons were analyzed by single-strand conformation polymorphism (SSCP) and capillary electrophoresis to obtain the respective MLVA profiles. Parallel to the high-resolution analysis, we used the same environmental DNA to quantify the number of L. pneumophila cells in drinking water using real-time PCR with 16S rRNA gene-targeted primers. We used a set of drinking water samples from a small-scale drinking water network to test our approach. With these samples we demonstrated that the developed approach was directly applicable to DNA obtained from drinking water. We were able to detect more L. pneumophila MLVA genotypes in drinking water than we could detect by isolation. Our approach could be a valuable tool to identify outbreak strains even after the outbreak has occurred and has the potential to be applied directly to clinical material.

 

Repetitive element-polymerase chain reaction for genotyping of clinical and environmental isolates of Legionella spp

Haroon A, Koide M, Higa F, Hibiya K, Tateyama M, Fujita J.

Department of Infectious, Respiratory, and Digestive Medicine, Control and Prevention of Infectious Diseases, Faculty of Medicine, University of the Ryukyus, Okinawa, Japan.koide-mi@med.u-ryukyu.ac.jp

Diagn Microbiol Infect Dis. 2010 Sep;68(1):7-12.

ABSTRACT: Genotyping of Legionella strains is important for the epidemiologic survey of Legionnaires' disease infections. In this study, we investigated the potential of repetitive element-polymerase chain reaction (rep-PCR) for differentiating various isolates of Legionella spp. We used 38 Legionella pneumophila isolates (collected in clinics all over Japan between 1980 and 2007), 19 environmental Legionella anisa isolates (collected in Okinawa, Nara, Osaka, and Hyogo prefecture between 1987 and 2007), and 2 Legionella-type strains. We extracted bacterial genomic DNA and applied it to rep-PCR. PCR products were then converted into bands by agarose gel electrophoresis. The L. pneumophila serogroup (SG) 1 displayed very diverse patterns. Different bands were produced for each species of Legionella, and each species was clearly distinct. Phylogenetic analysis displayed 1 cluster of L. anisa isolates, while other Legionella spp. were present at discrete levels. Our findings show that rep-PCR is an effective, rapid, and simple technique for differentiation of L. pneumophila strains as well as Legionella spp.

 

Investigation of plasma-functionalized multiwalled carbon nanotube film and its application of DNA sensor for Legionella pneumophila detection

Park EJ, Lee JY, Kim JH, Lee CJ, Kim HS, Min NK.

Department of Biomicrosystem Technology, Korea University, Seoul 136-701, Republic of Korea. parkeunjin@korea.ac.kr

Talanta. 2010 Aug 15;82(3):904-11.

ABSTRACT: A novel multiwall carbon nanotube (MWCNT) electrode functionalized with oxygen plasma treatment was prepared and characterized, and its DNA sensing ability for Legionella pneumophila (L. pneumophila) detection was examined using electrochemical measurement. A well-patterned MWCNT working electrode (WE) on a Pt track was fabricated using photolithography, transfer methods and an etching technique. The MWCNT WE was functionalized by oxygen plasma treatment prior to applying for DNA sensor. The surface morphology of the plasma-functionalized MWCNT (pf-MWCNT) WEs were observed by scanning electron microscope (SEM) and the change of chemical composition was characterized by X-ray photoelectron spectroscopy (XPS), and electrochemical measurements were performed using CV with ferricyanide/ferrocyanide redox couple. Effective areas of working electrodes were calculated to be 0.00453 cm(2) for pristine MWCNT electrode and 0.00747-0.00874 cm(2) for pf-MWCNT electrodes with different plasma treatment times. Differential pulse voltammetry (DPV) was carried out in methylene blue solution for DNA sensing. The pf-MWCNT based DNA sensor was successfully operated in a target concentration range of 10 pM to 100 nM and had a lower detection limit than a pristine MWCNT based DNA sensor.

 

Rapid quantification of viable legionellae in water and biofilm using ethidium monoazide coupled with real-time quantitative PCR

Chen NT, Chang CW.

Institute of Environmental Health, College of Public Health, National Taiwan University, Taipei, Taiwan. chingwenchang@ntu.edu.tw

J Appl Microbiol. 2010 Aug;109(2):623-34.

ABSTRACT: AIMS: To optimize ethidium monoazide (EMA) coupled with real-time quantitative PCR (qPCR) and to evaluate its environmental applicability on quantifying viable legionellae in water and biofilm of cooling towers and hot water systems.

METHODS AND RESULTS: EMA (0.9-45.5 microg ml(-1)) and propidium monoazide (PMA, 0.9 and 2.3 microg ml(-1)) combined with qPCR (i.e. EMA-qPCR and PMA-qPCR, respectively) were applied to unheated and heated (70 degrees C for 30 min) Legionella pneumophila to quantify viable cells, which was also simultaneously determined by BacLight Bacterial Viability kit with epifluorogenic microscopic enumeration (BacLight-EM). The effects of nontarget microflora and sample matrix on the performance of EMA-qPCR were also evaluated. In comparison with BacLight-EM results, qPCR with EMA at 2.3 microg ml(-1) was determined as the optimal EMA-qPCR assay, which performed equally well as PMA-qPCR for unheated Leg. pneumophila but better than PMA-qPCR for heated Leg. pneumophila (P < 0.05). Moreover, qPCR with EMA at 2.3 microg ml(-1) accurately quantified viable Leg. pneumophila, Legionella anisa and Legionella-like amoebal pathogens 6 (LLAP 6) without interferences by heated legionellae, unheated nonlegionellae cells and cooling tower water matrix (P > 0.05). As for water and biofilm samples collected from cooling towers and hot water systems, the viable legionellae counts determined by EMA-qPCR were mostly greater than the culturable counts by culture assay but consistently lower than the total cell counts quantified by qPCR.

CONCLUSIONS: The qPCR with EMA at 2.3 microg ml(-1) may accurately quantify viable legionellae (including fastidious LLAP 6) and Leg. pneumophila pretreated with superheating and is applicable for water and biofilm samples obtained from cooling towers and hot water systems.

SIGNIFICANCE AND IMPACT OF THE STUDY: The EMA-qPCR assay may be useful in environmental surveillance for viable legionellae and in evaluation of superheating efficacy against legionellae.

 

Isolation and identification of Acanthamoeba from Taiwan spring recreation areas using culture enrichment combined with PCR

Huang SW, Hsu BM.

Department of Earth and Environmental Sciences, National Chung Cheng University, Chiayi, Taiwan, ROC. bmhsu@ccu.edu.tw

Acta Trop. 2010 Sep;115(3):282-7.

ABSTRACT: In the study, 52 spring water samples were collected from three hot spring recreation areas in northern Taiwan and Acanthamoebae were isolated from 11 samples (21.2%) on two hot spring recreation areas and mainly present in the hot spring water, hot tubs and wastewater. The most frequently identified Acanthamoeba genotype was T15, followed by T6, and then T5. Genotype T1, T2, T3 and T4 were detected once, respectively. The presence or absence of Acanthamoeba within the spring water samples showed significant difference with the levels of heterotrophic plate counts (HPC). Genotype T2-T6 and genotype T15, the organism responsible for Acanthamoeba keratitis, and the Acanthamoeba species organism, retained pathogenic Legionella, and should be considered a potential health threat associated with human activities in spring recreation areas.

 

Evaluation of Legionella V-TesT for the detection of Legionella pneumophila antigen in urine samples

Bruin JP, Peeters MF, Ijzerman EP, Diederen BM.

Regional Laboratory of Public Health, Haarlem, The Netherlands. bramdiederen@gmail.com

Eur J Clin Microbiol Infect Dis. 2010 Jul;29(7):899-900.

ABSTRACT: We evaluated a new immunochromatographic assay (Legionella V-TesT, Coris BioConcept, Gembloux, Belgium) for its ability to detect Legionella pneumophila serogroup 1 antigen in urine. Test devices were read at various time points to determine the optimum incubation time regarding performance. The results were compared with those obtained with the BinaxNOW urinary antigen test. The sensitivity and specificity were 82.2% and 98.6%, respectively, for the Legionella V-TesT and 83.9% and 100%, respectively, for the BinaxNOW urinary antigen test after 15 min of incubation. When tests were examined after 60 min, the sensitivity for both tests increased to 91.5%.

 

Legionella feeleii serotype 2 pneumonia in a man with chronic lymphocytic leukemia: a challenging diagnosis

Siegel MO, Fedorko DP, Drake SK, Calhoun LB, Holland SM.

George Washington Medical Center, Division of Infectious Disease, 2150 Pennsylvania Ave. NW, Washington, DC 20037, USA. msiegel@mfa.gwu.edu

J Clin Microbiol. 2010 Jun;48(6):2294-7.

ABSTRACT: Legionella feeleii has rarely been reported as causing pneumonia in patients with hematologic malignancies. We present a case of Legionella feeleii serotype 2 pneumonia with empyema in a man with chronic lymphocytic leukemia and describe the methods of identifying this organism using both standard methods and newer diagnostic techniques.

 

Characterization of Legionella pneumophila isolates from patients in Japan according to serogroups, monoclonal antibody subgroups and sequence types

Amemura-Maekawa J, Kura F, Helbig JH, Chang B, Kaneko A, Watanabe Y, Isobe J, Nukina M, Nakajima H, Kawano K, Tada Y, Watanabe H; Working Group for Legionella in Japan.

Department of Bacteriology I, National Institute of Infectious Diseases, Tokyo, Japan. jmaekawa@nih.go.jp

J Med Microbiol. 2010 Jun;59(Pt 6):653-9.

ABSTRACT: We collected 86 unrelated clinical Legionella pneumophila strains that were isolated in Japan during the period 1980-2008. Most (80.2%) belonged to serogroup 1, followed by serogroups 5, 3 and 2. Interestingly, the patients with L. pneumophila serogroup 1 had a significantly higher male-to-female ratio (12.4) than the patients with other L. pneumophila serogroups (2.0) (OR, 10.5; 95% CI, 2.5-44.5). When the serogroup 1 strains were analysed by monoclonal antibody (mAb) typing, the most prevalent subgroup was Benidorm (34.9% of all isolates). Moreover, 79.7% of the serogroup 1 isolates were bound by mAb 3/1, which recognizes the virulence-associated epitope. When all 86 isolates were subjected to sequence-based typing (SBT) using seven loci, they could be divided into 53 sequence types (STs). The ST with the most isolates (seven) was ST1, to which most isolates from patients and environments around the world belong. However, six of the seven ST1 isolates were isolated before 1994. Other major STs were ST306 (n=6), ST120 (n=5) and ST138 (n=5). All ST306 and ST138 isolates, except for one isolate (ST306), were suspected or confirmed to be derived from bath water, which suggests that these strains prefer bath habitats. The sources of all ST1 and ST120 isolates remain unclear. By combining the SBT and mAb data, the 86 isolates could be divided into 59 types (discrimination index, 0.984). This confirms the usefulness of this combination in epidemiological studies.

 

Comparison of the plating efficiencies and shelf lives of three different commercial buffered charcoal yeast extract media supplemented with alpha-ketoglutaric acid

Edelstein PH, Edelstein MA.

University of Pennsylvania School of Medicine and Department of Pathology and Laboratory Medicine, and Clinical Microbiology Laboratory, 4 Gates, Hospital of the University of Pennsylvania, 3400 Spruce St., Philadelphia, PA 19104-4283, USA. phe@mail.med.upenn.edu

J Clin Microbiol. 2010 May;48(5):1882-3.

ABSTRACT: The plating efficiencies and shelf lives of locally made buffered charcoal yeast extract medium supplemented with alpha-ketoglutaric acid (BCYEalpha) were compared to those of media made by BD, Hardy, and Remel. Lung homogenates from guinea pigs infected with Legionella pneumophila were plated monthly onto different medium lots. All media performed equally well and had shelf lives of at least 12 months.

 

Development of triplex SYBR green real-time PCR for detecting Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. without extraction of DNA

Kerdsin A, Uchida R, Verathamjamrus C, Puangpatra P, Kawakami K, Puntanakul P, Lochindarat S, Bunnag T, Sawanpanyalert P, Dejsirilert S, Oishi K.

National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Nonthaburi, Thailand. ryuryu1_u@yahoo.co.jp

Jpn J Infect Dis. 2010 May;63(3):173-80.

ABSTRACT: Although Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. are prevalent causes of community-acquired pneumonia, rapid and sensitive diagnosis is difficult. Real-time PCR provides rapid and sensitive diagnosis, however, DNA extraction is still required, which is time-consuming, costly and includes a risk of contamination. Therefore, we aimed to develop triplex real-time PCR without DNA extraction. AmpDirect(R) Plus which inhibits PCR inhibitors was used as the PCR buffer. Melting temperatures of the PCR products for the three bacteria were analyzed by SYBR green triplex real-time PCR and were found to be significantly different. Detection limits of bacteria cells diluted in nasopharyngeal aspirates (NPAs) were comparable with the detection limits of previously reported real-time PCR. Our PCR without DNA extraction and probe real-time PCR with DNA extraction showed identical results for the detection of the three bacteria from 38 respiratory specimens (sputum, endotracheal aspirates, and NPAs) collected from patients with pneumonia. No cross-reaction with other bacteria was observed. Our triplex real-time PCR successfully detected and differentiated the three bacteria. Although further field tests are required, our assay is a promising method for the rapid and cost-effective detection of the three bacteria.

 

Survey of Naegleria and its resisting bacteria-Legionella in hot spring water of Taiwan using molecular method

Huang SW, Hsu BM.

Department of Earth and Environmental Sciences, National Chung Cheng University, 168, University Rd, Min-Hsiung, Chiayi, Taiwan, Republic of China. bmhsu@ccu.edu.tw

Parasitol Res. 2010 May;106(6):1395-402.

ABSTRACT: Naegleria is a free-living amoebae existing in soil and aquatic environments. Within the genus Naegleria, N. fowleri is most recognized as potential human pathogen causing primary amoebic meningoencephalitis (PAM). Furthermore, the Naegleria spp. can serve as vehicles for facultative pathogens, such as Legionella. In this study, we identified Naegleria and Legionella based on the PCR amplification with a genus-specific primer pair and investigated the distribution of Naegleria and Legionella at five spring recreation areas in Taiwan. In this study of hot spring and other water sources in Taiwan, five Naegleria spp. were detected in 15 (14.2%) of the water samples. The most frequently detected was N. lovaniensis (n = 6), followed by N. australiensis (n = 5), and then N. clarki (n = 2). N. americana and N. pagei were detected once, respectively. The pathogenic species N. fowleri was not detected; however, N. australiensis considered to be a potential pathogen species in humans was found. Legionella spp., an endosymbiont of Naegleria, was detected in 19 (17.9%) of the water samples in this study. Overall, 5.7% of the water samples contained both Naegleria and Legionella. The Legionella spp. identified were L. pneumophila and L. erythra. Results of this survey confirm the existence of Naegleria and Legionella in Taiwan spring recreation areas. It should be considered a potential threat for health associated with human activities in spring recreation areas of Taiwan.

 

Quantitative real-time PCR tests for diagnostic and prognostic purposes in cases of legionellosis

Maurin M, Hammer L, Gestin B, Timsit JF, Rogeaux O, Delavena F, Tous J, Epaulard O, Brion JP, Croizé J.

Laboratoire de Bactériologie, Centre Hospitalier Universitaire de Grenoble, Université Joseph Fourier, Grenoble. mmaurin@chu-grenoble.fr

Clin Microbiol Infect. 2010 Apr;16(4):379-84.

ABSTRACT: The usefulness of two quantitative real-time PCR assays (qrt-PCRmip targeting Legionella pneumophila, and qrt-PCR16S targeting all Legionella species) performed on lower respiratory tract (LRT) samples for diagnostic and prognostic purposes in 311 patients hospitalized for community-acquired pneumonia (CAP) in Rhône-Alpes (France) was evaluated. The Now Legionella urinary antigen test (UAT) from Binax (Portland, ME, USA) was used as a reference test. Samples were divided into two groups. Group A included 255 CAP patients admitted to Chambery hospital in 2005 and 2006. The Now Legionella UAT was positive in 14 patients. Sensitivities, specificities, positive predictive and negative predictive values for both qrt-PCR tests were 63.6, 98.7, 77.7 and 97.4%, respectively. Group B included 56 consecutive legionellosis patients diagnosed during a 4-year period (2003-2006) at the Grenoble University Hospital. The qrt-PCR16S and qrt-PCRmip displayed a sensitivity of 82.14 and 80.4%, respectively. Among the 70 legionellosis cases, L. pneumophila serogroup 1 was isolated in 15; qrt-PCRmip was positive in another 36, suggesting L. pneumophila infection, whereas the Legionella species involved could not be determined in the remaining 19 cases. The Legionella burden in LRT samples at the time of admission was determined in 46 patients using qrt-PCR16S tests, 44 for qrt-PCR mip groups A and B patients. It varied from 1.9 to 8.35 log(10) DNA copies/mL of LRT sample for qrt-PCR16S and from 1.9 to 8.11 log(10) DNA copies/mL of sample for qrt-PCRmip. High bacterial loads in LRT samples at hospital admission were significantly associated with higher Fine classes, the need for hospitalization in an intensive care unit and for prolonged hospitalization.

 

Variable genetic element typing: a quick method for epidemiological subtyping of Legionella pneumophila

Pannier K, Heuner K, Lück C.

Institute of Medical Microbiology and Hygiene, TU Dresden, Dresden, Germany. Christian.Lueck@tu-dresden.de

Eur J Clin Microbiol Infect Dis. 2010 Apr;29(4):481-7.

ABSTRACT: A total of 57 isolates of Legionella pneumophila were randomly selected from the German National Legionella strain collection and typed by monoclonal antibody subgrouping, seven-gene locus sequence-based typing (SBT) scheme and a newly developed variable element typing (VET) system based on the presence or absence of ten variable genetic elements. These elements were detected while screening a genomic library of strain Corby, as well as being taken from published data for PAI-1 (pathogenicity island) from strain Philadelphia. Specific primers were designed and used in gel-based polymerase chain reaction (PCR) assays. PCR amplification of the mip gene served as a control. The end-point was the presence/absence of a PCR product on an ethidium bromide-strained gel. In the present study, the index of discrimination was somewhat lower than that of the SBT (0.87 versus 0.97). Nevertheless, the results obtained showed as a 'proof of principle' that this simple and quick typing assay might be useful for the epidemiological characterisation of L. pneumophila strains.

 

Evaluation of the usefulness of a new direct immunofluorescence assay (ScanVIT-Legionella) for monitoring hospital water systems contaminated with Legionella spp

Ditommaso S, Giacomuzzi M, Gentile M, Zotti CM.

Dipartimento di Sanità Pubblica e di Microbiologia, Università degli Studi di Torino, Via Santena, Torino, Italy. savina.ditommaso@unito.it

Lett Appl Microbiol. 2010 Apr;50(4):341-6.

ABSTRACT: AIMS: To compare the efficiency of the ScanVIT-Legionella test (Vermicon, Munich, Germany) vs a conventional culture method for the quantification of Legionella spp. in hospital water samples in daily hospital practice. METHODS AND RESULTS: The detection of Legionella spp. takes place on a cultivated filter brought into contact with dye-marked gene probes. The results are analysed under fluorescence microscopy. Bacteria that light up green belong to the genus Legionella; those that light up both green and red belong to the species Legionella pneumophila. Our results showed that the ScanVIT test has a sensitivity of 90%; agreement between the two methods was 82%. In the 48 samples that tested positive with both methods, the Legionella concentration detected by the culture method was consistently higher. A statistically significant difference between the results obtained with the two test methods emerged at the Wilcoxon test (P < 0.001). CONCLUSION: The ScanVIT test may be recommended for investigating the presence of Legionella by qualitative testing. SIGNIFICANCE AND IMPACT OF THE STUDY: Given the simplicity of colony identification by fluorescence, the ScanVIT test can be used in laboratories where staffs are not experienced in identifying typical colonies of Legionella.

 

Legionella species and serogroups in Malaysian water cooling towers: identification by latex agglutination and PCR-DNA sequencing of isolates

Yong SF, Goh FN, Ngeow YF.

School of Biomedical Science, Taylor's University College, No. 1, Jalan SS15/8, 47500 Sabang Jaya, Selangor, Malaysia. stacey.yong@taylors.edu.my

J Water Health. 2010 Mar;8(1):92-100.

ABSTRACT: In this study, we investigated the distribution of Legionella species in water cooling towers located in different parts of Malaysia to obtain information that may inform public health policies for the prevention of legionellosis. A total of 20 water samples were collected from 11 cooling towers located in three different states in east, west and south Malaysia. The samples were concentrated by filtration and treated with an acid buffer before plating on to BCYE agar. Legionella viable counts in these samples ranged from 100 to 2,000 CFU ml(-1); 28 isolates from the 24 samples were examined by latex agglutination as well as 16S rRNA and rpoB PCR-DNA sequencing. These isolates were identified as Legionella pneumophila serogroup 1 (35.7%), L. pneumophila serogroup 2-14 (39%), L. pneumophila non-groupable (10.7%), L. busanensis, L. gormanii, L. anisa and L. gresilensis. L. pneumophila was clearly the predominant species at all sampling sites. Repeat sampling from the same cooling tower and testing different colonies from the same water sample showed concurrent colonization by different serogroups and different species of Legionella in some of the cooling towers.

 

A quick and easy method to identify bacteria by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry

Pennanec X, Dufour A, Haras D, Réhel K.

IPL Bretagne, 4 rue de Galilée, 56270 Ploemeur, France. xaviera.pennanec@univ-ubs.fr

Rapid Commun Mass Spectrom. 2010 Feb;24(3):384-92.

ABSTRACT: Concerns with water quality have increased in recent years, in part due to the more frequent contamination of water by pathogens like E. coli and L. pneumophila. Current methods for the typing of bacteria in water samples are based on culture of samples on specific media. These techniques are time-consuming, subject to the impact of interferents and do not totally meet all the requirements of prevention. There is a need for accurate and rapid identification of these microorganisms. This report deals with the detection of bacteria, more precisely of Legionella spp., and the development of an analytical strategy for a rapid and unambiguous identification of these pathogens in water from diverse origins. Therefore, a protein mass mapping using matrix-assisted laser desorption/ionisation mass spectrometry (MALDI MS) of whole bacteria combined with a home-made database of bacteria spectra is applied. A large variety of different bacteria and microorganisms is used to approach the actual composition of samples with numerous interferents. The objective is to propose a universal method for sampling preparation before MALDI MS analysis and optimised spectrometric conditions for reproducible intense peaks. Several experimental factors known to influence signal quality such as time and media of culture have been studied. The proposed method gives promising results for a sure differentiation of Legionella species and subspecies and a rapid identification of bacteria which are the most dangerous or difficult to eradicate. This method is easy to perform with an excellent reproducibility. The analytical protocol and the corresponding database were validated on samples from different origins (cooling tower, plumbing hot water).

 

Evaluation of three Immunochromatographic Assays for Detection of Legionella pneumophila serogroup 1 Antigen in Urine Samples

Muñoz MJ, Martínez MC, Yagüe G, Segovia M.

Hospital Virgen de la Arrixaca, 30120 El Palmar, Murcia, Spain. mariajose.munoz5@alu.um.es

Rev Esp Quimioter. 2009 Dec;22(4):207-209.

ABSTRACT: The Uni-Gold, the SAS and the Binax NOW immunochromatographic test (ICT) urinary antigen assays for the qualitative detection of Legionella pneumophila serogroup 1 were compared using 39 unfrozen and nonconcentrated urine samples from patients with Legionnaires disease (LD). The Uni-Gold antigen test detected the urinary antigen in 41% (16/39), the SAS antigen test in 61.5% (24/39), and the Binax NOW antigen test in 74.3% (29/39). The Binax NOW ICT assay showed the best results when detecting L. pneumophila urinary antigen.

 

Rapid diagnostic testing for community-acquired pneumonia: can innovative technology for clinical microbiology be exploited?

Yu VL, Stout JE.

University of Pittsburgh and Special Pathogens Laboratory, Pittsburgh, PA 15219, USA. vly@pitt.edu

Chest. 2009 Dec;136(6):1618-21.

ABSTRACT: Two nonsynchronous events have affected the management of community-acquired pneumonia (CAP): spiraling empiricism for CAP and the "golden era" of clinical microbiology. The development of broad-spectrum antibiotics has led to widespread empiric use without ascertaining the etiology of the infecting microbe. Unfortunately, this approach clashes with the second event, which is the advent of molecular-based microbiology that can identify the causative pathogen rapidly at the point of care. The urinary antigen is a most effective rapid test that has allowed targeted therapy for Legionnaire disease at the point of care. The high specificity (> 90%) allows the clinician to administer appropriate anti-Legionella therapy based on a single rapid test; however, its low sensitivity (76%) means that a notable number of cases of Legionnaire disease will go undiagnosed if other tests, especially culture, are not performed. Further, culture for Legionella is not readily available. If a culture is not performed, epidemiologic identification of the source of the bacterium cannot be ascertained by molecular fingerprinting of the patient and the putative source strain. We recommend resurrection of the basic principles of infectious disease, which are to identify the microbial etiology of the infection and to use narrow, targeted antimicrobial therapy. To reduce antimicrobial overuse with subsequent antimicrobial resistance, these basic principles must be applied in concert with traditional and newer tests in the clinical microbiology laboratory.

 

Investigation on strains of Legionella pneumophila, isolated from a hospital of Milano, with three genotyping methods

Bianchi A, Tesauro M, Consonni M, Galli MG.

Dipartimento di Sanità Pubblica, Microbiologia e Virologia, Università degli Studi di Milano, Milano. annalisa.bianchi@unimi.it

Ann Ig. 2009 Sep-Oct;21(5):517-22.

ABSTRACT: Various techniques have been developed in recent years for the molecular typing of microorganisms. Remains particularly difficult to isolate clinical strains for the low availability of cases and even more problematic matching clinical / environmental strains. We investigated 13 strains of Legionella pneumophila of clinical and environmental origin, isolated in 3 Health Facilities in Milan (2003-2006), using three molecular typing methods: Pulse-Field Gel Electrophoresis, Amplified Fragment Length Polymorphism and Sequence-Based Typing. PFGE and AFLP showed the correlation between a clinical case with only one of the environmental isolates taken from the places frequented by the patient, demonstrating with certainty the nosocomial origin of the case and identifying the source of infection in the shower water (Clin. 1 and Env. 1N). Two clinical samples from patients admitted to different wards presented an identical profile, which suggests that the nosocomial origin assumed an epidemic form, even without having isolated the environmental strain due to the absence of samples drawn during the period under consideration (Clin. 2 and 3). Finally, the comparison between the isolated environmental strains demonstrated a heterogeneous presence of strains, not correlated to each other although they belong to the same serum-group, having profiles that are clearly different regarding number and position of bands (Env. 2 and 4). The profile 2,10,18,10,1,1 had never been isolated and typed previously in Europe. The SBT has proved a better technique for reproducibility and interpretation of results than PFGE and AFLP To complete studies on SBT method, now considered gold standard, is currently being the EWGLI 5th Proficiency Panel, in which we are actively involved with the genotyping of five strains according to the latest version of the protocol (4.1).

  

Diagnostic and typing methods for investigating Legionella infection

Blyth CC, Adams DN, Chen SC.

Centre for Infectious Diseases and Microbiology Laboratory Services, Sydney West Area Health Service. Sharon.chen@swahs.health.nsw.gov.au

N S W Public Health Bull. 2009 Sep-Oct;20(9-10):157-61.

ABSTRACT: Legionella infection is an important cause of community-acquired pneumonia in Australia. Morbidity and mortality is significant. Diagnosis remains a challenge with infection often unrecognised, particularly early in the course of illness. An understanding of available diagnostic methods and their limitations is important to public health practitioners and clinicians alike.

 

Inhibition of Legionella pneumophila PCR in respiratory samples: a quantitative approach

Kern M, Böhm S, Deml L, Wolf H, Reischl U, Niller HH.

Institute for Medical Microbiology and Hygiene, University of Regensburg, Franz-Josef-Strauss-Allee 11, Regensburg, Germany. monika.kern@klinikuni-regensburg.de

J Microbiol Methods. 2009 Nov;79(2):189-93.

ABSTRACT: Impurities in complex biological samples that persist through nucleic acid preparation can inhibit PCR or reduce the sensitivity and efficiency of PCR amplification and thus affect reliable results in PCR diagnostics. To obtain information on the incidence of inhibition events and on the magnitude of the loss of sensitivity, respectively, we devised a relative inhibition assay and examined 100 samples from patients with respiratory tract infections. As a reference, samples were spiked with Legionella (L.) pneumophila. Detection was by standard nucleic acid purification and subsequent real-time PCR. By comparing the crossing points of the fluorescent curves to those of an L. pneumophila standard dilution series, we were able to quantify the respective degrees of inhibition into several categories. We found complete inhibition in 2% of the samples. 12% were not reliably detected. 65% of the tested samples showed moderate to strong inhibition, but were still reliably detected, whereas in 21% of the samples no inhibition was observed. Except for a significantly higher inhibition in tracheal aspirates than in BAL samples, the degree of inhibition did not correlate with the physical properties of the respective sample. The relative inhibition assay established an unexpectedly broad distribution of the inhibition-degrees in inflammatory respiratory materials.

 

Detection of IgM antibodies against Legionella pneumophila serogroup 1 in Malaysian blood donors and patients with respiratory illnesses: evaluation of enzyme-linked immunosorbent assay and indirect immunofluorescence assay

Tay ST, Lakhbeer Singh HK, Ramasame SD, Vadivelu J.

Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia. tayst@um.edu.my

Jpn J Infect Dis. 2009 Sep;62(5):409-10.

NO ABSTRACT

 

Direct sequencing of Legionella pneumophila from respiratory samples for sequence-based typing analysis

Coscollá M, González-Candelas F.

Institut Cavanilles de Biodiversitat i Biologia Evolutiva, University of Valencia, Apartado Oficial 22085, Valencia 46071, Spain. mireia.coscolla@uv.es

J Clin Microbiol 2009 Sep;47(9):2901-5.

ABSTRACT: We have developed a procedure to test the efficiency and reliability of sequencing of Legionella pneumophila genes directly from respiratory samples and have compared the results with those derived from cultured isolates. We tried to obtain the nucleotide sequences of six protein-coding loci included in the sequence-based typing scheme for Legionella pneumophila and three intergenic regions from 132 samples corresponding to 106 patients positive for urine antigen. A seminested PCR approach was used to amplify and sequence these nine loci directly from respiratory samples. Nucleotide sequences were directly obtained for 23 Legionella isolates and also for 66 respiratory secretions from a total of 69 patients. The efficiency of sequencing from respiratory secretions was higher than that of sequencing after the isolation of the Legionella isolates. Moreover, the perfect match between the sequences obtained by both approaches when respiratory samples and cultured isolates from the same patient were available corroborates the suitability of the direct sequencing approach for the identification of Legionella species and molecular epidemiology studies with Legionella species.

 

Comparison of the sensitivity of the Legionella urinary antigen EIA kits from Binax and Biotest with urine from patients with infections caused by less common serogroups and subgroups of Legionella

Olsen CW, Elverdal P, Jørgensen CS, Uldum SA.

Unit of Atypical Pneumonia, Department of Bacteriology, Mycology and Parasitology (ABMP), Statens Serum Institut, Artillerivej 5, 2300, Copenhagen S, Denmark. cwo@ssi.dk

Eur J Clin Microbiol Infect Dis. 2009 Jul;28(7):817-20.

ABSTRACT: The detection of urinary antigen is the most widely used method to diagnose Legionnaires' disease (LD), so it is important that these assays have a high sensitivity for the disease. In this study, we compare two kits for their ability to detect urinary antigen in urine samples from patients infected with Legionella species and L. pneumophila sero- and subgroups not considered as the most common causes of LD. Urine samples (n = 33) from 30 culture-proven cases of L. pneumophila serogroup (sg) 1, subgroup non-Pontiac infection, and urine samples (n = 35) from 32 cases of non-L. pneumophila species or non-sg 1 infection were examined using the Binax EIA and Biotest EIA kits. For both groups, the overall diagnostic sensitivity of the Binax kit was significantly better than the sensitivity of the Biotest kits (P < 0.0001). For the non-Pontiac group, the sensitivity was 81.8 and 42.4%, respectively, and for the non-sg1 group, it was 51.4 and 28.6%, respectively. It was concluded that the Binax kit was more suitable for the general diagnosis of LD than the Biotest kit, but we still need urinary antigen detection methods with higher sensitivity for non-sg1 LD.

 

Evaluation of the Oxoid Xpect Legionella Test Kit for Detection of Legionella pneumophila Serogroup 1 Antigen in Urine

Diederen BM, Bruin JP, Scopes E, Peeters MF, Ijzerman EP.

The Regional Laboratory of Public Health, Boerhaavelaan 26, Haarlem 2035 RC, The Netherlands. bramdiederen@gmail.com.

J Clin Microbiol. 2009 Jul;47(7):2272-4.

ABSTRACT: We evaluated a new immunochromatographic assay (Oxoid Xpect Legionella test kit) for the ability to detect Legionella pneumophila serogroup 1 antigen in urine. The results were compared with those obtained with the Binax NOW urinary antigen test by following the manufacturers' instructions. The sensitivities and specificities were estimated to be 89 and 100%, respectively, for the Oxoid Xpect Legionella test kit and 86 and 100%, respectively, for the Binax NOW test.

  

The rapid and specific real-time detection of Legionella pneumophila in water samples using Optical Waveguide Lightmode Spectroscopy

Cooper IR, Meikle ST, Standen G, Hanlon GW, Santin M.

School of Pharmacy and Biomolecular Sciences, University of Brighton, Lewes Road, Brighton, BN2 4GJ, United Kingdom. m.santin@bton.ac.uk

J Microbiol Methods. 2009 Jul;78(1):40-4.

ABSTRACT: The detection of Legionella pneumophila in water samples using standard microbiological culture techniques is both prolonged and problematic. The bacterium is slow-growing and nutritionally fastidious, such that other indigenous species can out-compete the Legionella even when using antibiotic supplemented media. Optical Waveguide Lightmode Spectroscopy (OWLS) is a real-time analytical system whereby a change to a higher coupling angle where the refractive index of a bacterial cell is higher than that of the covering medium. In this study an aqueous suspension of L. pneumophila was passed across the surface of waveguides functionalised with a specific anti-Legionella antibody. The binding between the bacterial cells and the antibody specific for that cell resulted in an increase in the refraction indices of the transverse electric and transverse magnetic photoelectric currents. We report the optimisation of a rapid and sensitive (1.3 x 10(4) CFU mL-1) detection method for L. pneumophila contamination in a water sample in less than 25 min. This is a significant reduction in the time taken to determine the presence of the bacterium which with conventional techniques normally takes up to fourteen days. In addition, the specificity of the technique to L. pneumophila was demonstrated. The OWLS results were validated by conventional microbiology screening and atomic force microscopy of the surface of the waveguide, showing its species specificity and potential applications in environmental and clinical analysis.

 

Validation of SYTO 9/propidium iodide uptake for rapid detection of viable but noncultivable Legionella pneumophila

Gião MS, Wilks SA, Azevedo NF, Vieira MJ, Keevil CW.

Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal. salome.giao@deb.uminho.pt

Microb Ecol. 2009 Jul;58(1):56-62.

ABSTRACT: Legionella pneumophila is an ubiquitous environmental microorganism that can cause Legionnaires' disease or Pontiac fever. As a waterborne pathogen, it has been found to be resistant to chlorine disinfection and survive in drinking water systems, leading to potential outbreaks of waterborne disease. In this work, the effect of different concentrations of free chlorine was studied (0.2, 0.7, and 1.2 mg l(-1)), the cultivability of cells assessed by standard culture techniques (buffered charcoal yeast extract agar plates) and viability using the SYTO 9/propidium iodide fluorochrome uptake assay (LIVE/DEAD BacLight). Results demonstrate that L. pneumophila loses cultivability after exposure for 30 min to 0.7 mg l(-1) of free chlorine and in 10 min when the concentration is increased to 1.2 mg l(-1). However, the viability of the cells was only slightly affected even after 30 min exposure to the highest concentration of chlorine; good correlation was obtained between the rapid SYTO 9/propidium iodide fluorochrome uptake assay and a longer cocultivation with Acanthamoeba polyphaga assay, confirming that these cells could still recover their cultivability. These results raise new concerns about the assessment of drinking water disinfection efficiency and indicate the necessity of further developing new validated rapid methods, such as the SYTO 9/propidium iodide uptake assay, to assess viable but noncultivable L. pneumophila cells in the environment.

 

Viability PCR, a culture-independent method for rapid and selective quantification of viable Legionella pneumophila cells in environmental water samples

Delgado-Viscogliosi P, Solignac L, Delattre JM.

Département Eaux et Environnement, Institut Pasteur de Lille, France. pilar.viscogliosi@pasteur-lille.fr

Appl Environ Microbiol. 2009 Jun;75(11):3502-12.

ABSTRACT: PCR-based methods have been developed to rapidly screen for Legionella pneumophila in water as an alternative to time-consuming culture techniques. However, these methods fail to discriminate between live and dead bacteria. Here, we report a viability assay (viability PCR [v-PCR]) for L. pneumophila that combines ethidium monoazide bromide with quantitative real-time PCR (qPCR). The ability of v-PCR to differentiate viable from nonviable L. pneumophila cells was confirmed with permeabilizing agents, toluene, or isopropanol. v-PCR suppressed more than 99.9% of the L. pneumophila PCR signal in nonviable cultures and was able to discriminate viable cells in mixed samples. A wide range of physiological states, from culturable to dead cells, was observed with 64 domestic hot-water samples after simultaneous quantification of L. pneumophila cells by v-PCR, conventional qPCR, and culture methods. v-PCR counts were equal to or higher than those obtained by culture and lower than or equal to conventional qPCR counts. v-PCR was used to successfully monitor in vitro the disinfection efficacy of heating to 70 degrees C and glutaraldehyde and chlorine curative treatments. The v-PCR method appears to be a promising and rapid technique for enumerating L. pneumophila bacteria in water and, in comparison with conventional qPCR techniques used to monitor Legionella, has the advantage of selectively amplifying only viable cells.

 

Identification of legionella species by use of an oligonucleotide array

Su HP, Tung SK, Tseng LR, Tsai WC, Chung TC, Chang TC.

Department of Health, Centers for Disease Control, Taipei, Taiwan. tsungcha@mail.ncku.edu.tw

J Clin Microbiol. 2009 May;47(5):1386-92.

ABSTRACT: The genus Legionella contains a diverse group of motile, asaccharolytic, nutritionally fastidious gram-negative rods. Legionella pneumophila is the most important human pathogen, followed by L. micdadei, L. longbeachae, L. dumoffii, and other rare species. Accurate identification of Legionella spp. other than L. pneumophila is difficult because of biochemical inertness and phenotypic identity of different species. The feasibility of using an oligonucleotide array for identification of 18 species of Legionella was evaluated in this study. The method consisted of PCR amplification of the macrophage infectivity potentiator mip gene, followed by hybridization of the digoxigenin-labeled PCR products to a panel of 30 oligonucleotide probes (16- to 24-mers) immobilized on a nylon membrane. A collection of 144 target strains (strains we aimed to identify) and 50 nontarget strains (44 species) were analyzed by the array. Both test sensitivity (144/144 strains) and specificity (50/50 strains) of the array were 100%. The whole procedure for identification of Legionella species by the array can be finished within a working day, starting from isolated colonies. It was concluded that species identification of clinically relevant Legionella spp. by the array method is very reliable and can be used as an accurate alternative to conventional or other molecular methods for identification of Legionella spp.

 

PCR-based 'serotyping' of Legionella pneumophila

Thürmer A, Helbig JH, Jacobs E, Lück PC.

Institute of Medical Microbiology and Hygiene, Technische Universität Dresden, Fiedlerstrasse 42, D-01307 Dresden, Germany. christian.lueck@tu-dresden.de

J Med Microbiol. 2009 May;58(Pt 5):588-95.

ABSTRACT: Currently, several PCR assays based on 16S rRNA and virulence-associated genes are available for detection of Legionella pneumophila. So far, no genotyping method has been published that can discriminate between serogroups and monoclonal subgroups of the most common L. pneumophila serogroup 1. Our first approach was to analyse LPS-associated genes of seven L. pneumophila serogroup 1 strains, and we developed two PCR-based methods specific for serogroup 1. Specific DNA fragments could be amplified from all the serogroup 1 strains (n=43) including the strains from the American Type Culture Collection. In contrast, none of the strains from serogroups 2-15 (n=41) contained these specific gene regions. In a second approach, primers specific for the lag-1 gene, encoding an O-acetyltransferase, which is responsible for the presence of the LPS epitope recognized by mAb 3/1, were designed and tested for their ability to differentiate between mAb 3/1-positive and -negative strains. All mAb 3/1-positive strains (n=30) contained the lag-1 gene, but in turn 4 of 13 tested mAb 3/1-negative strains were also positive in the PCR. Thus, the discrimination between mAb 3/1-positive and mAb 3/1-negative subgroups could not be achieved for all strains. In a third approach, two intergenic regions expected to be specific for monoclonal subgroup Knoxville and closely related subgroups Benidorm/Bellingham were identified and used for selective genotyping. These intergenic regions could not only be amplified in every tested strain belonging to the subgroups Knoxville, Benidorm and Bellingham, but also in some strains of other unrelated subgroups. The two PCR approaches with primers specific for serogroup 1 genes definitely represent a valuable tool in outbreak investigations and for risk assessment. They also might be used for culture-independent diagnosis of legionellosis caused by L. pneumophila serogroup 1.

 

PCR-coupled electrochemical sensing of Legionella pneumophila

Miranda-Castro R, de-Los-Santos-Alvarez N, Lobo-Castañón MJ, Miranda-Ordieres AJ, Tuñón-Blanco P.

Departamento de Química Física y Analítica, Universidad de Oviedo, Julián Clavería, 8, 33006, Oviedo, Principado de Asturias, Spain. ptb@uniovi.es

Biosens Bioelectron. 2009 Apr 15;24(8):2390-6.

ABSTRACT: Human infections with Legionella pneumophila represent a public health problem. Current culture assays for surveillance and control of L. pneumophila in water are time-consuming and limited by the sensitivity, especially when samples also contain microorganisms that inhibit Legionella growth. In this work, an electrochemical method, different from real-time polymerase chain reaction (PCR) approaches, for semiquantitative evaluation of L. pneumophila is presented. A PCR assay targeting the 16S-rRNA gene of L. pneumophila giving rise to a 95-mer amplicon was established. Amplicons were hybridized to a biotin-labeled reporter sequence and then to a thiolated stem-loop structure immobilized onto gold electrodes as a reporter molecule with 1-naphthyl phosphate as a substrate. 1-Naphthol enzymatically generated was determined by differential pulse voltammetry (DPV). For a constant number of amplification cycles, results show that the voltammetric signal is related to the number of copies in the sample thus achieving a useful semiquantitative estimation of L. pneumophila. After 40 cycles of PCR amplification this methodology has a limit of detection of 10 genomes, allowing the reliable detection of 10(2) genomes of L. pneumophila as well as distinguishing 10(3) and 10(4) genomes of the pathogen, values related to corrective actions in water systems in buildings, in accordance with the legislation currently in force.

 

Legionella pneumophila DNA in serum samples during Legionnaires' disease in relation to C-reactive protein levels

van de Veerdonk FL, de Jager CP, Schellekens JJ, Huijsmans CJ, Beaumont F, Hermans MH, Wever PC.

Department of Internal Medicine, Jeroen Bosch Hospital, 's-Hertogenbosch, The Netherlands. f.veerdonk@aig.umcn.nl

Eur J Clin Microbiol Infect Dis. 2009 Apr;28(4):371-6.

ABSTRACT: Legionella pneumophila DNA can be detected in serum from patients with Legionnaires' disease (LD). We explored this observation studying the kinetics of L. pneumophila DNA in serum samples in relation to C-reactive protein (CRP). Eleven hospitalized patients with LD were studied. Diagnosis was made by Legionella urinary antigen test in 8 patients and seroconversion in 3 patients. A macrophage infectivity potentiator (MIP) real-time PCR was performed on 31 serum samples, including 20 follow-up serum samples. Serum samples obtained on the day of admission were MIP PCR-positive in 7 (64%) and MIP PCR-negative in 4 (36%) patients. Three (75%) of the 4 patients with a MIP PCR-negative serum sample on the day of admission became positive during follow-up. Overall, L. pneumophila DNA was detected in serum samples from 10 of the 11 patients (91%). CRP levels in the 7 patients with a positive MIP PCR serum sample on day of admission (499 +/- 144 mg/l; median +/- SD) were significantly higher than those in the 4 patients with a negative MIP PCR serum sample on the day of admission (244 +/- 97 mg/l). No difference in the severity of the disease on the day of admission was found between these patients. The presence of L. pneumophila DNA in serum is a common phenomenon in hospitalized patients with LD, although in some cases it is not yet present on the day of admission. L. pneumophila DNA in serum on the day of admission correlates with high CRP levels, but not with the severity of the disease.

 

Evaluation of a nested-PCR-derived sequence-based typing method applied directly to respiratory samples from patients with Legionnaires' disease

Ginevra C, Lopez M, Forey F, Reyrolle M, Meugnier H, Vandenesch F, Etienne J, Jarraud S, Molmeret M.

Université de Lyon, Lyon, France. maelle.molmeret@univ-lyon1.fr

J Clin Microbiol. 2009 Apr;47(4):981-7.

ABSTRACT: Sequence-based typing (SBT) is a powerful method based on the sequencing of seven genes of Legionella pneumophila isolates. SBT performed directly on clinical samples has been used only in a limited number of cases. In our study, its efficiency was tested with 63 legionellosis respiratory samples. Sixty-three clinical samples, which included 23 samples from sporadic cases and 40 collected during four French outbreaks, confirmed by culture or urinary antigen testing and all positive by L. pneumophila quantitative PCR were subtyped by SBT according to the European Working Group for Legionella Infections standard scheme. Only 28.6% of the samples provided nucleotide sequences by SBT. Nested-PCR-based SBT (NPSBT) applied to the same respiratory samples was thus evaluated with new PCR primers surrounding the first set of primers used for the SBT. Sequencing results were obtained with 90.5% of the samples. Complete allelic profiles (seven genes sequenced) were obtained for 3.2% versus 53.9% of the samples by SBT and NPSBT, respectively. More importantly, of the 28 culture-negative samples, only 4 did not give any sequencing results. Taken together, NPSBT applied directly to clinical specimens significantly improved epidemiological typing compared to the initial SBT, in particular when no isolates are available.

 

 

Specific detection of viable Legionella cells by combined use of photoactivated ethidium monoazide and PCR/real-time PCR

Chang B, Sugiyama K, Taguri T, Amemura-Maekawa J, Kura F, Watanabe H.

Department of Bacteriology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan. haruwata@nih.go.jp

Appl Environ Microbiol. 2009 Jan;75(1):147-53.

ABSTRACT: Legionella organisms are prevalent in manmade water systems and cause legionellosis in humans. A rapid detection method for viable Legionella cells combining ethidium monoazide (EMA) and PCR/real-time PCR was assessed. EMA could specifically intercalate and cleave the genomic DNA of heat- and chlorine-treated dead Legionella cells. The EMA-PCR assay clearly showed an amplified fragment specific for Legionella DNA from viable cells, but it could not do so for DNA from dead cells. The number of EMA-treated dead Legionella cells estimated by real-time PCR exhibited a 10(4)- to 10(5)-fold decrease compared to the number of dead Legionella cells without EMA treatment. Conversely, no significant difference in the numbers of EMA-treated and untreated viable Legionella cells was detected by the real-time PCR assay. The combined assay was also confirmed to be useful for specific detection of culturable Legionella cells from water samples obtained from spas. Therefore, the combined use of EMA and PCR/real-time PCR detects viable Legionella cells rapidly and specifically and may be useful in environmental surveillance for Legionella.

 

Use of flow cytometry to monitor Legionella viability

Allegra S, Berger F, Berthelot P, Grattard F, Pozzetto B, Riffard S.

Groupe Immunité des Muqueuses et Agents Pathogènes, EA3064, Faculté de Médecine J. Lisfranc, Université Jean Monnet, Saint-Etienne, France. serge.riffard@univ-st-etienne.fr

Appl Environ Microbiol. 2008 Dec;74(24):7813-6.

ABSTRACT: Legionella viability was monitored during heat shock treatment at 70 degrees C by a flow cytometric assay (FCA). After 30 min of treatment, for 6 of the 12 strains tested, the FCA still detected 10 to 25% of cells that were viable but nonculturable (VBNC). These VBNC cells were able to produce ATP and to be resuscitated after culture on amoebae.

 

Comparison of the new InoDiag automated fluorescence multiplexed antigen microarray to the reference technique in the serodiagnosis of atypical bacterial pneumonia

Gouriet F, Levy PY, Samson L, Drancourt M, Raoult D.

Unité des Rickettsies, Faculté de Médecine, Université de la Méditerranée, Marseille, France. didier.raoult@medecine.univ-mrs.fr

Clin Microbiol Infect. 2008 Dec;14(12):1119-27.

ABSTRACT: The aetiological diagnosis of pneumonia depends largely on culture-, antigen- or PCR-based tests. Atypical agents of pneumonia include Coxiella burnetii, Chlamydophila pneumoniae, Chlamydia psittaci, Legionella pneumophila, Francisella tularensis and Mycoplasma pneumoniae. In these cases, serological tests are commonly used for diagnosis. All of the above species were comparatively screened for by using the InoDiag multiplexed automatic immunofluorescence assay and established reference techniques. The InoDiag assay required 5 microL of serum, took 76 min per serum sample, and required an incubator, a fluorescence reader and interpretation software. In total, 248 single sera from patients were tested, for the diagnosis of pneumonia, and the results obtained with selected serum samples were compared with results obtained with the reference method. It was shown that, for the detection of Coxiella burnetii IgM, the automated assay had a sensitivity and specificity of 100%. For the detection of M. pneumoniae IgM, sensitivity was 100% and specificity was 98%. For the detection of Chlamydophila pneumoniae and Chlamydia psittaci IgG, sensitivity was 81% and specificity was 94%. For the detection of L. pneumoniae IgG, sensitivity was 63% and specificity was 98%. For the detection of F. tularensis IgG and IgM, sensitivity was 100% for both, and specificity was 95% and 100%, respectively. The performance of this serological assay was comparable to that of other assays reported in the literature. This preliminary study shows that the automatic InoDiag assay opens the way to immunofluorescence assay standardization.

 

Multiplexed whole bacterial antigen microarray, a new format for the automation of serodiagnosis: the culture-negative endocarditis paradigm

Gouriet F, Samson L, Delaage M, Mainardi JL, Meconi S, Drancourt M, Raoult D.

Unité des Rickettsies, CNRS UMR, Faculté de Médecine, Université de la Méditerranée, Marseille, France. didier.raoult@medecine.univ-mrs.fr

Clin Microbiol Infect. 2008 Dec;14(12):1112-8.

ABSTRACT: The serological diagnosis of blood culture-negative endocarditis due to Coxiella burnetii, Bartonella spp., Brucella melitensis and Legionella pneumophila is based on a manual immunofluorescence assay (IFA), which is taken to be the reference method. The automated IFA InoDiag multiplexed antigenic microarray, which includes a slide with all the above bacteria and four internal controls, an incubator, a fluorescent reader and software with an algorithm of interpretation for infectious endocarditis (IE) was evaluated. A single serum dilution at 1/128 was used. Eleven patients with Bartonella spp. IE and ten with C. burnetii IE, diagnosed using the modified Duke criteria, as well as one patient with B. melitensis infection and three patients with L. pneumophila IE were tested. In total, 236 sera were used as negative controls, with the reference method. The results of IgG detection were: C. burnetii phase I, 'sensitivity (Se) = 88% and specificity (Sp) = 99%', and C. burnetii phase II, Se = 88% and Sp = 99%; for Bartonella henselae, Se = 100% and Sp = 100%; for Bartonella quintana, Se = 78% and Sp = 96%; for B. melitensis, Se = 100% and Sp = 99%; and for L. pneumophila, Se = 100% and Sp = 99%. With the algorithm interpretation, the negative and positive predictive values of the test 'were 100% for the diagnosis of IE caused by the four bacteria tested. These results were confirmed by two other assays, one using triplicate testing and one blind testing performed by another centre. This multiplexed test is therefore a valuable tool for the rapid diagnosis of blood-culture negative IE.

 

Detection of culturable and nonculturable Legionella species from hot water systems of public buildings in Japan

Edagawa A, Kimura A, Doi H, Tanaka H, Tomioka K, Sakabe K, Nakajima C, Suzuki Y.

Department of Environment and Water, Osaka Prefectural Institute of Public Health, Higashinari-ku, Osaka, Japan. akkimura@iph.pref.osaka.jp

J Appl Microbiol. 2008 Dec;105(6):2104-14.

ABSTRACT: AIMS: To investigate the prevalence of culturable and nonculturable Legionella species in hot water systems of public buildings in Japan and assess the risk factors associated with Legionella contamination in hot water systems. METHODS AND RESULTS: Legionella species were detected by conventional culture and molecular methods in 130 water samples collected from 40 buildings. A total of 26 (20.0%) water samples from 17 (42.5%) buildings were positive by culture, qualitative PCR or both methods: Legionella pneumophila and Leg. anisa were detected in four samples by a culture method, whereas 23 samples were positive by qualitative PCR, with the presence of various Legionella species confirmed by sequencing. Of these 23 samples, bacterial counts were quantifiable in 21 by real-time PCR (from 1.7 x 10(5) to 2.6 x 10(11) cells per litre). Phylogenetic analysis of amplified partial 16S rRNA gene showed close relations to various species of Legionella, including Leg. anisa and Leg. micdadei, all of which have been associated with respiratory diseases or increased antibody titres in human sera. Assessment of risk factors showed that turbidity, free chlorine concentration, iron concentration and heterotrophic plate count (HPC) were significantly associated with Legionella contamination (P < 0.05). CONCLUSIONS: Contamination of hot water systems of public buildings with culturable and nonculturable Legionella species may be a potential risk factor for Legionella infection in Japan. Adequate levels of chlorine, low levels of iron and HPC are important maintenance measures in the reduction of Legionella contamination in hot water systems. SIGNIFICANCE AND IMPACT OF THE STUDY: More than 40% of hot water systems in the Japanese public buildings examined were contaminated by not only culturable Leg. pneumophila and Leg. anisa but also by nonculturable pathogenic species. To our knowledge, this is the first report of both culturable and nonculturable Legionella contamination in hot water systems of public buildings in Japan.

 

Characteristics of Legionella pneumophila serogroup 2 strains by colony morphology

Koide M, Furugen M, Haranaga S, Higa F, Tateyama M, Yamane N, Fujita J.

Department of Medicine and Therapeutics, Control and Prevention of Infectious Diseases (First Department of Internal Medicine), University of the Ryukyus, Okinawa, Japan. koide-mi@med.u-ryukyu.ac.jp

Jpn J Infect Dis. 2008 Nov;61(6):487-9.

ABSTRACT: The isolation rate of Legionella pneumophila serogroup 2 from clinical samples is low. In 2007, we encountered the second case known to occur in Japan. As the L. pneumophila serogroup 2 type strain, the Kobe strain isolated in 1988, and the Okinawa strain isolated from the present patient could not be differentiated using the usual biochemical and serological tests, we tried to achieve differentiation by observing colony morphology. In Oxoid BCYEalpha medium, colonies of the Kobe strain developed multiple protuberances on the surface, but these did not develop on the other two strains. In Becton-Dickinson BCYEalpha medium, colonies of the Okinawa strain had several outgrowths from the margin, but the type strain and the Kobe strain did not have any outgrowths. The Okinawa strain isolated from the present case showed intermediate characteristics between the type strain and the Kobe strain in the appearance of colony morphology. It may be useful to conduct an investigation of this rare serogroup.

 

Design and implementation of a protocol for the detection of Legionella in clinical and environmental samples

Nazarian EJ, Bopp DJ, Saylors A, Limberger RJ, Musser KA.

Wadsworth Center, New York State Department of Health, Albany, NY 12208, USA. musser@wadsworth.org

Diagn Microbiol Infect Dis. 2008 Oct;62(2):125-32.

ABSTRACT: Our laboratory has developed a novel real-time polymerase chain reaction (PCR) assay for the detection of Legionella pneumophila and differentiation from other Legionella spp. in clinical and environmental samples. The 23S rRNA gene was used as a target to detect all Legionella spp., and the mip gene was targeted for the specific detection of L. pneumophila in this multiplex Taqman(R) real-time PCR assay. The 23S rRNA gene is a novel target for Legionella testing; it detects all species and serogroups of Legionella without the contamination issues that accompany the use of the 16S rRNA gene as a target. This assay provides an analytical sensitivity of <1 colony-forming unit and a specificity of 100%. Because culture is important and provides a means for molecular typing via pulsed-field gel electrophoresis (PFGE), we developed a testing algorithm that includes both the new real-time PCR assay and culture for clinical and environmental samples and applied this algorithm during a period of 3 years. Of the 64 clinical samples received by our laboratory for Legionella testing during this period, PCR was found to be an essential diagnostic tool because only 13.3 % (2/15) clinical samples that were determined to be L. pneumophila were detected by culture during this period. Of the 276 environmental samples received for Legionella testing during this period, 140 were found to be positive for L. pneumophila. Of these 140 samples, 69.3% were detected by both PCR and culture methods, 29.3% were positive by PCR alone, and 1.4% were positive by culture methods alone. We feel these results indicate that our algorithm, including both PCR and culture, should be used for environmental samples. Among both the clinical and environmental Legionella samples identified by PCR, a subset was not suitable for culture because of issues of lengthy transport, antimicrobial treatment, or bacterial overgrowth. Samples like these are commonly submitted to our laboratory, so the use of our testing algorithm combining these methods is critical. We conclude that molecular and culture methods must be used in combination to provide the best and most comprehensive approach to laboratory detection and investigation of legionellosis.

 

Antibody detection and cross-reactivity among species and serogroups of Legionella by indirect immunofluorescence test

Ditommaso S, Giacomuzzi M, Gentile M, Zotti CM.

Dipartimento di Sanità Pubblica e di Microbiologia, Università degli Studi di Torino, Via Santena 5 bis 10126 Torino, Italy. savina.ditommaso@unito.it

J Microbiol Methods. 2008 Oct;75(2):350-3.

ABSTRACT: The purpose of the present study was to determine possible cross-reactivity between different serogroups or between different species by means of bacterial smears of individual L. pneumophila serogroups and several non-pneumophila Legionella spp. Using BIOCHIP slide we found a considerable frequency of cross-reactions with L. pneumophila serogroup 12 (88.4%) therefore BIOCHIP slide proved as useful method for identification of cross-reactivity between members of the Legionnellaceae.

 

Detection of Escherichia coli O157:H7, Salmonella typhimurium, and Legionella pneumophila in water using a flow-through chemiluminescence microarray readout system

Wolter A, Niessner R, Seidel M.

Institute of Hydrochemistry, Technische Universität München, Marchioninistrasse 17, D-81377 München, Germany. seidel@ch.tum.de

Anal Chem. 2008 Aug 1;80(15):5854-63.

ABSTRACT: Fast, sensitive, and especially, multianalyte test systems are currently of high interest for the monitoring and quality control of drinking water, since traditional microbiological methods are labor intensive and can take days until a response is achieved. In this study, the first flow-through chemiluminescence microarray was developed and characterized for the rapid and simultaneous detection of Escherichia coli O157:H7, Salmonella typhimurium, and Legionella pneumophila in water samples using a semiautomated readout system. Therefore, antibody microarrays were produced on poly(ethylene glycol)-modified glass substrates by means of a contact arrayer. For capturing bacteria, species-specific polyclonal antibodies were used. Cell recognition was carried out by binding of species-specific biotinylated antibodies. Chemiluminescence detection was accomplished by a streptavidin-horseradish peroxidase (HRP) catalyzed reaction of luminol and hydrogen peroxide. The chemiluminescence reaction that occurred was recorded by a sensitive charge-coupled device (CCD) camera. The overall assay time was 13 min, enabling a fast sample analysis. In multianalyte experiments, the detection limits were 3 x 10(6), 1 x 10(5), and 3 x 10(3) cells/mL for S. typhimurium, L. pneumophila, and E. coli O157:H7, respectively. Quantification of samples was possible in a wide concentration range with good recoveries. The presented system is well suited for quick and automatic water analysis.

  

A PCR-based method for monitoring Legionella pneumophila in water samples detects viable but noncultivable legionellae that can recover their cultivability

Dusserre E, Ginevra C, Hallier-Soulier S, Vandenesch F, Festoc G, Etienne J, Jarraud S, Molmeret M.

Université de Lyon, Lyon, France. maelle.molmeret@univ-lyon1.fr

Appl Environ Microbiol. 2008 Aug;74(15):4817-24.

ABSTRACT: Legionella pneumophila is the causative agent of Legionnaires' disease. This bacterium is ubiquitous in aqueous environments and uses amoebae as an intracellular replicative niche. Real-time PCR has been developed for rapid detection of Legionella DNA in water samples. In addition to culturable bacteria, this method may also detect dead and viable but noncultivable (VBNC) legionellae. In order to understand the significance of positive PCR results in this setting, we prepared water samples containing known concentrations of L. pneumophila and analyzed them comparatively by means of conventional culture, real-time PCR, viability labeling, and immunodetection (solid-phase cytometry). We also examined the influence of chlorination on the results of the four methods. The different techniques yielded similar results for nonchlorinated water samples but not for chlorinated samples. After treatment for 24 h with 0.5 and 1 ppm chlorine, all cultures were negative, PCR and immunodetection showed about 10(6) genome units and bacteria/ml, and total-viable-count (TVC) labeling detected 10(5) and 10(2) metabolically active bacteria/ml, respectively. Thus, PCR also detected bacteria that were VBNC. The recoverability of VBNC forms was confirmed by 5 days of coculture with Acanthamoeba polyphaga. Therefore, some TVC-positive bacteria were potentially infective. These data show that L. pneumophila PCR detects not only culturable bacteria but also VBNC forms and dead bacterial DNA at low chlorine concentrations.

 

Detection of Legionella pneumophila serogroup 1 antigen in respiratory samples using an immunochromatographic membrane test

Higa F, Koide M, Furugen M, Akamine M, Hibiya K, Haranaga S, Yara S, Tateyama M, Yamane N, Fujita J.

Department of Medicine and Therapeutics, Control and Prevention of Infectious Diseases, University of the Ryukyus, Okinawa 903-0215, Japan. fhiga@med.u-ryukyu.ac.jp

J Microbiol Methods. 2008 Aug;74(2-3):121-2.

ABSTRACT: The immunochromatographic membrane test (ICT) efficacy of Legionella antigen detection (Binax Now Legionella) was evaluated using respiratory samples, including bronchial washings (44 cases) and sputum (128 cases), from suspected Legionella pneumonia patients. The ICT results using respiratory samples agreed well with isolation of L. pneumophila SG1 and ICT using urines.

 

Real-time PCR assay for the detection and quantification of Legionella pneumophila in environmental water samples: utility for daily practice

Morio F, Corvec S, Caroff N, Le Gallou F, Drugeon H, Reynaud A.

Laboratoire de Bacteriologie-Virologie-Hygiene Hospitaliere, Institut de Biologie des Hopitaux de Nantes, 9 quai Moncousu, 44093 Nantes Cedex 01, France. stephane.corvec@chu-nantes.fr

Int J Hyg Environ Health. 2008 Jul;211(3-4):403-11.

ABSTRACT: We developed a quantitative real-time PCR assay targeting the mip gene of Legionella pneumophila for a prospective study from September 2004 to April 2005. It was compared with a standard culture method (French guideline AFNOR T90-431), analysing 120 water samples collected to monitor the risk related to Legionellae at Nantes hospital and to investigate a case of legionellosis acquired from hospital environment. Samples from six distinct water distribution systems were analysed by DNA extraction, amplification and detection with specific primers and FRET probes. The detection limit was 100 genomic units of L. pneumophila per liter (GU/l), the positivity threshold about 600 GU/l and the quantification limit 800 GU/l. PCR results were divided into three groups: negative (n=63), positive but non-quantifiable (n=22) or positive (n=35). PCR showed higher sensitivity than culture, whereas four culture-positive samples appeared negative by PCR (PCR inhibitor detected for two of them). Although no correlation was observed between both methods and real-time PCR cannot substitute for the reference method, it represents an interesting complement. Its sensitivity, reproducibility and rapidity appear particularly interesting in epidemic contexts in order to identify the source of contamination or to evaluate critical points of contamination in water distribution systems.

 

Molecular comparison of Legionella pneumophila serogroup 1 isolated in Tunisia

Mehiri-Zghal E, Essalah L, Ghariani A, Mahjoubi W, Reyrolle M, Meugnier H, Forey F, Jarraud S, Freney J, Etienne J, Slim-Saidi L.

Laboratoire de microbiologie, hôpital Abderahman-Mami de pneumologie, Ariana, Tunis 2080, Tunisie. emna.mehiri@tunet.tn

Pathol Biol (Paris). 2008 Jul;56(5):279-82.

ABSTRACT: Legionella pneumophila is a common cause of hospital and community-acquired pneumonia, being transmitted by inhalation of aqueous aerosols. Most outbreaks are linked to contaminated hot water systems and cooling towers. Our study was about the molecular typing of 35 strains of L. pneumophila including four clinical isolates and 31 environmental strains isolated from the distribution systems of 14 hotels. Among the clinical strains, two have the same pattern, however, all were different from the studied environmental strains. For the 31 environmental strains, ten patterns were obtained. Among which, a same pulsotype was found for four strains isolated from four different establishments. In addition, two different pulsotypes were found for strains isolated from the same establishment. The pulsed-field gel electrophoresis showed the existence of various patterns. Although cases of legionellosis were declared in these hotels, there are no epidemiological links between the clinical and environmental strains.

 

Severe Legionella pneumonia: rapid presumptive clinical diagnosis with Winthrop-University Hospital's weighted point score system (modified)

Cunha BA.

Infectious Disease Division, Winthrop-University Hospital, Mineola, NY 11501, USA.

Heart Lung. 2008 Jul-Aug;37(4):311-20.

ABSTRACT: Legionnaires' disease is a systemic infection involving the lungs and accompanied by a characteristic pattern of extrapulmonary organ involvement. Legionnaires' disease is one of the non-zoonotic causes of atypical community-acquired pneumonia (CAP). Legionnaires' disease commonly presents as severe CAP requiring hospitalization and intensive care. Each atypical CAP has its own characteristic pattern of extrapulmonary laboratory clinical findings and abnormalities that are the basis of clinical syndromic diagnosis. Studies have been unsuccessful in identifying individual clinical and laboratory parameters that are specific for Legionella. Individually, clinical and laboratory abnormalities lack diagnostic specificity. The diagnostic specificity of clinical and laboratory findings is increased when combined and are the basis of a clinical syndromic diagnosis. The importance of serial nonspecific laboratory abnormalities with Legionnaires' disease is emphasized. The sensitivity and specificity of a clinical syndromic diagnosis are enhanced if they are based on a weighted point score system. A diagnostic weighted point score system is based on the varying diagnostic importance of clinical and laboratory diagnostic findings. The Winthrop-University Hospital's Infectious Disease Division's rapid clinical diagnostic weighted point system is based on a weighted point score of clinical and laboratory findings. The case presented is that of a 55-year-old man with severe CAP who required hospitalization and intensive care admission. The presumptive clinical diagnosis of Legionella CAP was based on the Winthrop-University Hospital Infectious Disease Division's weighted point score system, which permitted early empiric anti-Legionella antimicrobial therapy and prompted specific Legionella testing. Legionnaires' disease is definitively diagnosed by serology or a urinary Legionella antigen test. This case of severe Legionnaires' CAP was confirmed by urinary antigen test reported on hospital day 6. The Winthrop-University Hospital is weighted point score system (modified) permits a rapid clinical presumptive diagnosis of Legionnaires' disease and is an accurate predictor of Legionella CAP.

 

Monitoring Legionella species in hospital water systems. Link with disease and evaluation of different detection methods

Koziol-Montewka M, Magrys A, Stojek N, Palusinska-Szysz M, Danielak M, Wojtowicz M, Niewiedziol J, Koncewicz R, Niedzwiadek J, Paluch-Oles J, Trzeciak H, Drozanski W, Dutkiewicz J.

Department of Medical Microbiology, Medical University of Lublin, Chodzki 1, 20-093 Lublin, Poland. magrysa@yahoo.pl

Ann Agric Environ Med. 2008 Jun;15(1):143-7.

ABSTRACT: The aim of this work was to evaluate three currently available isolation methods for Legionella using water samples and swabs of a single pediatric hospital water system. Additionally, high risk patients were screened for the presence of Legionella pneumophila antigen in urine. Fifteen water samples and 11 swab samples were collected from distal sites at 18 sampling locations. The International Standard Method (PN-ISO11731-2) based on membrane filtration and direct culture of bacteria on selective media were compared with amoebic co-culture. The numbers of legionellae detected exceeded 10(2) cfu/100 ml in 50% of the samples. All the positive samples contained L. pneumophila SGs 2-14. Urine samples were obtained from 57 immunosuppressed children and screened for the presence of L. pneumophila serogroup (SG) 1 antigen by Legionella urinary antigen EIA. Of the 57 urine samples tested for the presence of Legionella pneumophila SG 1 antigen, none were positive. Our results highlight the value of combined membrane filtration and amoebic co-culture methods in detecting viable L. pneumophila strains. Direct plating of 0.2 ml water is a useful screening method for samples containing large bacterial amount.

 

Evaluation of different nucleic acid amplification techniques for the detection of M. pneumoniae, C. pneumoniae and Legionella spp. in respiratory specimens from patients with community-acquired pneumonia

Loens K, Beck T, Ursi D, Overdijk M, Sillekens P, Goossens H, Ieven M.

Department of Microbiology, Vaccine and Infectious Disease Institute, University of Antwerp UIA, Antwerp, Belgium. Katherine.loens@ua.ac.be

J Microbiol Methods. 2008 Jun;73(3):257-62.

ABSTRACT: The number of pathogens involved in community-acquired pneumonia, with varying susceptibilities to antimicrobials, is numerous constituting an enormous challenge for diagnostic microbiology. Differentiation of infections due to Streptococcus pneumoniae and those due to Mycoplasma pneumoniae, Chlamydophila pneumoniae, or L. pneumophila as well as those due to viruses is essential to allow correct decisions concerning the antibiotics to be administered. The sensitivity and specificity of real-time simplex and multiplex nucleic acid sequence-based amplification (NASBA), and simplex PCR were compared for the detection of M. pneumoniae, C. pneumoniae and Legionella spp. in respiratory specimens from hospitalized and outpatients with community-acquired pneumonia (CAP). Two hundred fifty one respiratory specimens were collected from 147 patients with CAP. NASBA was done using the NucliSens Basic Kit (bioMérieux). PCR for M. pneumoniae and C. pneumoniae was done as described earlier [Ieven, M., Ursi, D., Van Bever, H., Quint, W., Niesters, H. G. M., and Goossens, H. 1996. Detection of Mycoplasma pneumoniae by two polymerase chain reactions and role of M. pneumoniae in acute respiratory tract infections in pediatric patients. J. Infect. Dis. 173, 1445-14452.; Ursi, D., Ieven, M., Van Bever, H. P., and Goossens, H. 1998. Construction of an internal control for the detection of Chlamydia pneumoniae by PCR. Mol. Cellul. Probes. 12, 235-238.]. A real-time PCR was developed to detect L. pneumophila whereas a real-time NASBA was designed to detect Legionella spp. All samples with discordant results were re-analysed. Compared to an expanded gold standard the sensitivities of the different techniques, were 77.8%, 100%, and 100% for detection of M. pneumoniae; and 50%, 100%, and 50% for detection of L. pneumophila by PCR, real-time simplex NASBA, and real-time multiplex NASBA, respectively. C. pneumoniae was detected in two samples only. Simplex real-time NASBA proved to be more sensitive than simplex PCR and was also more sensitive than real-time multiplex NASBA, as previously found with spiked clinical specimens. It's practical attractiveness pleads for further optimalisation of the multiplex approach.

 

The simple and rapid detection of specific PCR products from bacterial genomes using Zn finger proteins

Osawa Y, Ikebukuro K, Motoki H, Matsuo T, Horiuchi M, Sode K.

Department of Biotechnology and Life Science, Tokyo University of Agriculture & Technology, 2-24-16 Naka-cho, Koganei, 184-8588 Tokyo, Japan. ikebu@cc.tuat.ac.jp

Nucleic Acids Res. 2008 Jun;36(11):e68.

ABSTRACT: A novel method of rapid and specific detection of polymerase chain reaction (PCR) products from bacterial genomes using Zn finger proteins was developed. Zn finger proteins are DNA-binding proteins that can sequence specifically recognize PCR products. Since Zn finger proteins can directly detect PCR products without undergoing dehybridization, unlike probe DNA, and can double check the specific PCR amplification and sequence specificity of the PCR products, this novel method would be quick and highly accurate. In this study, we tried to detect Legionella pneumophila using Sp1. It was found that a 49 bp L. pneumophila-specific region containing the Sp1 recognition site is located on the flhA gene of the L. pneumophila genome. We succeeded in specifically detecting PCR products amplified from L. pneumophila in the presence of other bacterial genomes by ELISA, and demonstrated that Sp1 enables the discrimination of L. pneumophila-specific PCR products from others. By fluorescence depolarization measurement, these specific PCR products could be detected within 1 min. These results indicate that the rapid and simple detection of PCR products specific to L. pneumophila using a Zn finger protein was achieved. This methodology can be applied to the detection of other bacteria using various Zn finger proteins that have already been reported.

 

Evaluation of different nucleic acid amplification techniques for the detection of M. pneumoniae, C. pneumoniae and Legionella spp. in respiratory specimens from patients with community-acquired pneumonia

Loens K, Beck T, Ursi D, Overdijk M, Sillekens P, Goossens H, Ieven M.

Department of Microbiology, Vaccine and Infectious Disease Institute (VIDI), University of Antwerp UIA, Antwerp, Belgium. Katherine.loens@ua.ac.be

J Microbiol Methods. 2008 Jun;73(3):257-62.

ABSTRACT: The number of pathogens involved in community-acquired pneumonia, with varying susceptibilities to antimicrobials, is numerous constituting an enormous challenge for diagnostic microbiology. Differentiation of infections due to Streptococcus pneumoniae and those due to Mycoplasma pneumoniae, Chlamydophila pneumoniae, or L. pneumophila as well as those due to viruses is essential to allow correct decisions concerning the antibiotics to be administered. The sensitivity and specificity of real-time simplex and multiplex nucleic acid sequence-based amplification (NASBA), and simplex PCR were compared for the detection of M. pneumoniae, C. pneumoniae and Legionella spp. in respiratory specimens from hospitalized and outpatients with community-acquired pneumonia (CAP). Two hundred fifty one respiratory specimens were collected from 147 patients with CAP. NASBA was done using the NucliSens Basic Kit (bioMérieux). PCR for M. pneumoniae and C. pneumoniae was done as described earlier [Ieven, M., Ursi, D., Van Bever, H., Quint, W., Niesters, H. G. M., and Goossens, H. 1996. Detection of Mycoplasma pneumoniae by two polymerase chain reactions and role of M. pneumoniae in acute respiratory tract infections in pediatric patients. J. Infect. Dis. 173, 1445-14452.; Ursi, D., Ieven, M., Van Bever, H. P., and Goossens, H. 1998. Construction of an internal control for the detection of Chlamydia pneumoniae by PCR. Mol. Cellul. Probes. 12, 235-238.]. A real-time PCR was developed to detect L. pneumophila whereas a real-time NASBA was designed to detect Legionella spp. All samples with discordant results were re-analysed. Compared to an expanded gold standard the sensitivities of the different techniques, were 77.8%, 100%, and 100% for detection of M. pneumoniae; and 50%, 100%, and 50% for detection of L. pneumophila by PCR, real-time simplex NASBA, and real-time multiplex NASBA, respectively. C. pneumoniae was detected in two samples only. Simplex real-time NASBA proved to be more sensitive than simplex PCR and was also more sensitive than real-time multiplex NASBA, as previously found with spiked clinical specimens. It's practical attractiveness pleads for further optimalisation of the multiplex approach.

 

Multigenome analysis identifies a worldwide distributed epidemic Legionella pneumophila clone that emerged within a highly diverse species

Cazalet C, Jarraud S, Ghavi-Helm Y, Kunst F, Glaser P, Etienne J, Buchrieser C.

Unité de Génomique des Microorganismes Pathogènes, Institut Pasteur, 75724 Paris Cedex 15, France. cbuch@pasteur.fr

Genome Res. 2008 Mar;18(3):431-41.

ABSTRACT: Genomics can provide the basis for understanding the evolution of emerging, lethal human pathogens such as Legionella pneumophila, the causative agent of Legionnaires' disease. This bacterium replicates within amoebae and persists in the environment as a free-living microbe. Among the many Legionella species described, L. pneumophila is associated with 90% of human disease and within the 15 serogroups (Sg), L. pneumophila Sg1 causes over 84% of Legionnaires' disease worldwide. Why L. pneumophila Sg1 is so predominant is unknown. Here, we report the first comprehensive screen of the gene content of 217 L. pneumophila and 32 non-L. pneumophila strains isolated from humans and the environment using a Legionella DNA-array. Strikingly, we uncovered a high conservation of virulence- and eukaryotic-like genes, indicating strong environmental selection pressures for their preservation. No specific hybridization profile differentiated clinical and environmental strains or strains of different serogroups. Surprisingly, the gene cluster coding the determinants of the core and the O side-chain synthesis of the lipopolysaccaride (LPS cluster) determining Sg1 was present in diverse genomic backgrounds, strongly implicating the LPS of Sg1 itself as a principal cause of the high prevalence of Sg1 strains in human disease and suggesting that the LPS cluster can be transferred horizontally. Genomic analysis also revealed that L. pneumophila is a genetically diverse species, in part due to horizontal gene transfer of mobile genetic elements among L. pneumophila strains, but also between different Legionella species. However, the genomic background also plays a role in disease causation as demonstrated by the identification of a globally distributed epidemic strain exhibiting the genotype of the sequenced L. pneumophila strain Paris.

 

Legionnaires' disease: evaluation of a quantitative microbial risk assessment model

Armstrong TW, Haas CN.

ExxonMobil Biomedical Sciences, Inc., 1545Rt. 22 East, Rm LG 340, Annandale, NJ 08801 - 0971, USA. thomas.w.armstrong@ExxonMobil.com

J Water Health. 2008 Jun;6(2):149-66.

ABSTRACT: Background: The quantities of Legionella vary considerably from natural waters to water in contaminated domestic hot water supplies, whirlpool spas and cooling towers, with the risk for LD rising as the Legionella counts grow. We currently report the results from our Quantitative Microbial Risk Assessment (QMRA) model evaluation. We developed the LD QMRA model to better understand Legionella exposure risks. Methods: Using an animal data derived model for LD, we calculated risks from estimated exposures for a whirlpool spa outbreak, two hot spring spa outbreaks and compared the results to the reported LD risks. Results: The QMRA model shows agreement (generally less than an order of magnitude discrepancy) with the reported Legionnaires' disease sub-clinical severity infection, clinical severity infection, and mortality risks. Conclusions: The LD QMRA model may lead to risk based limits to supplement the current guidance on Legionella control in cooling towers, whirlpool spas and other potential exposure sources. The verification of QMRA for LD also suggests the techniques, given suitable animal model data, may be useful in quantifying human response to other airborne pathogens.

 

Evaluation of a Legionella urinary antigen enzyme immunoassay for rapid detection of Legionella pneumophila in water samples

Blanco S, Prat C, Sánchez MD, Ferrer D, Pellicer T, Haba L, Latorre I, Vilaplana C, Ausina V, Domínguez J.

Servei de Microbiologia, Hospital Universitari Germans Trias i Pujol, Carretera del Canyet s/n, 08916 Badalona, Departament de Genètica i Microbiologia, Universitat Autònoma de Barcelona, Barcelona, Spain. jadominguez.igtp.germanstrias@gencat.net

Int J Hyg Environ Health. 2008 Mar;211(1-2):168-71.

ABSTRACT: Despite advances in medium formulations and pretreatment techniques, recovery of Legionella from water samples can still be quite low, difficult and time consuming. The aim of this study was to evaluate the utility of a Legionella urinary antigen enzyme immunoassay (Bartels ELISA, Trinity Biotech, Ireland) for the detection of Legionella in water samples. Reference ATCC Legionella strains were used to spike water samples to a final concentration of 10(4)-10(5)cfu/ml. The lower detection limit of the test for all Legionella pneumophila serogroups was assessed by serial dilutions of spiked water samples. Legionella antigen was detected in all filtered samples except for those spiked with L. bozemanii and L. longbeachae. The lower detection limit for soluble L. pneumophila serogroup 1 antigen was 780cfu/ml. Bartels ELISA could be a useful method for antigen detection in water samples when a high recovery of L. pneumophila is suspected. The test could be used as a rapid screening method for the detection of Legionella in a large number of samples. However, the low sensitivity of the test requires to keep on performing conventional culture for isolation and for further studies on isolated bacteria.

 

Development of Real-Time Multiplex Nucleic Acid Sequence-Based Amplification for Detection of Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. in Respiratory Specimens

Loens K, Beck T, Ursi D, Overdijk M, Sillekens P, Goossens H, Ieven M.

Department of Medical Microbiology, University of Antwerp, Universiteitsplein 1 S009a, B-2610 Wilrijk, Belgium. Katherine.loens@ua.ac.be

J Clin Microbiol. 2008 Jan;46(1):185-91.

ABSTRACT: Real-time multiplex isothermal nucleic acid sequence-based amplification (NASBA) was developed to detect Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. in respiratory specimens using the NucliSens Basic Kit (bioMérieux, Boxtel, The Netherlands). Oligonucleotide primers were derived from the M. pneumoniae, C. pneumoniae, and Legionella pneumophila 16S rRNA. For real-time detection, molecular beacons were used. Specificity was established on a panel of bacterial strains. The analytical sensitivity of the assay was determined by testing dilutions of wild-type in vitro-generated RNA in water and dilutions of reference strains in lysis buffer or added to pools of respiratory specimens. Subsequently, a limited number of M. pneumoniae-, C. pneumoniae-, and L. pneumophila-positive and -negative clinical specimens were analyzed. Specific detection of the 16S rRNA of the three organisms was achieved. The analytical sensitivity of the multiplex NASBA on spiked respiratory specimens was slightly diminished compared to the results obtained with the single-target (mono) real-time assays. We conclude that the proposed real-time multiplex NASBA assay, although less sensitive than the real-time mono NASBA assay, is a promising tool for the detection of M. pneumoniae, C. pneumoniae, and Legionella spp. in respiratory specimens, regarding handling, speed, and number of samples that can be analyzed in a single run.

 

Legionella in clinical specimens and hospital water supply facilities: molecular detection and genotyping of the isolates

Qasem JA, Mustafa AS, Khan ZU.

Department of Applied Medical Sciences, College of Health Sciences, The Public Authority for Applied Education and Training, Kuwait University, Kuwait. qasemj@paaet.edu.kw

Med Princ Pract. 2008;17(1):49-55.

ABSTRACT: To evaluate genus- and species-specific polymerase chain reactions (PCRs) for the detection of the genus Legionella and the species Legionella pneumophila in clinical specimens and hospital water supplies, and to establish a simple and reproducible random amplification of polymorphic DNA (RAPD)-PCR technique for genotyping of Legionella. MATERIALS AND METHODS: A total of 70 respiratory tract specimens(bronchoalveolar lavage: n = 46; endotracheal secretions: n = 9; sputum: n = 15) from patients with atypical pneumonia, and 283 environmental samples (water: 20; swabs: 263) collected from water storage and supply facilities of the Mubarak Al-Kabeer Hospital, Kuwait, were tested by culture and genus-specific PCR for the detection of Legionella. The L. pneumophila isolates were subsequently typed by serology and RAPD-PCR using serotype-specific sera and arbitrary primers, respectively. RESULTS: Of the 70 clinical samples, culture yielded 2 (2.9%) whereas genus-specific PCR detected Legionella in 20 (28.6%) samples. The 2 culture-positive specimens were also positive for L.-pneumophila-specific PCR. Testing of swab and water samples by culture and genus-specific PCR yielded 61 (21.6%) and 67 (23.7%) positive samples, respectively. All of the 61 culture-positive samples were also positive by genus-specific PCR and 45 of them were positive for L.-pneumophila-specific PCR. Serological typing of 43 L. pneumophila isolates showed that 8 of these belonged to serotype 1 and 35 to serotype 3; however, RAPD-PCR analyses demonstrated polymorphisms among the isolates of both serotypes. CONCLUSION: A higher association between PCR and culture was observed for the environmental samples than for the clinical samples. The application of genus- and species-specific PCRs and RAPD is useful in the detection and typing of Legionella in clinical and environmental samples.

 

External quality assessment of a DNA sequence-based scheme for epidemiological typing of Legionella pneumophila by an international network of laboratories

Afshar B, Fry NK, Bellamy W, Underwood AP, Harrison TG; Members of the European Working Group for Legionella Infections.

Respiratory and Systemic Infection Laboratory, Health Protection Agency, Centre for Infections, 61 Colindale Avenue, London NW9 5EQ, United Kingdom. Baharak.Afshar@HPA.org.uk

J Clin Microbiol. 2007 Oct;45(10):3251-6.

ABSTRACT: We report the results of an international external quality assessment (EQA) program to assess the performance of laboratories in genotyping Legionella pneumophila isolates using the standard European Working Group for Legionella Infections sequence-based typing protocol. Three coded distributions of L. pneumophila isolates were sent to laboratories in 12, 14, and 20 countries, respectively. The data were returned by 11 of 16, 18 of 19, and 27 of 29 centers, respectively. Incomplete submission of data resulted in exclusion from certain aspects of the analyses. The number of centers achieving 100% score, for all loci tested, rose successively from 50% (5 of 10) for the first EQA distribution, to 56% (9 of 16) for the second EQA distribution, to 76% (19 of 25) for the third EQA distribution. A number of additional centers made only a few errors (one to three) in each distribution. Sequence data from the first two distributions were collected in flat text file format and using specially developed software, the sequence quality tool (SQT), in the third distribution. The SQT allows users to upload trace files in standard file formats, automates basecalling using phred and phrap software, contig assembly, trimming, and matching against a reference library. The program described here allow users an independent measure of sequence quality, and such schemes are vital in order to identify strengths and weakness in centers responsible for the generation of genotyping data in legionella outbreak investigation. The present study demonstrates that DNA sequence data can be highly reproducible but, when independently assessed, in practice frequently falls short of this goal. However, experience and training in the methodology results in increased performance.

 

Multicenter comparison of molecular methods for detection of Legionella spp. in sputum samples

Bencini MA, van den Brule AJ, Claas EC, Hermans MH, Melchers WJ, Noordhoek GT, Salimans MM, Schirm J, Vink C, van der Zee A, Jansen R.

Regional Public Health Laboratory Kennemerland, Boerhaavelaan 26, 2035 RC Haarlem, The Netherlands. rolf.jansen@helmholtz-hzi.de

J Clin Microbiol. 2007 Oct;45(10):3390-2

ABSTRACT: Legionellosis can be diagnosed by PCR using sputum samples. In this report, the methods of nine laboratories for 12 sputum samples with Legionella pneumophila and Legionella longbeachae are compared. We conclude that (i) liquefaction prevents PCR inhibition, (ii) the employed mip gene PCRs detected L. pneumophila only, and (iii) the 16S rRNA gene PCR detected both Legionella species and is preferred for the diagnosis of legionellosis.

 

Culture-independent identification of the source of an infection by direct amplification and sequencing of Legionella pneumophila DNA from a clinical specimen

Lück PC, Ecker C, Reischl U, Linde HJ, Stempka R.

Institute of Medical Microbiology and Hygiene, TU Dresden, Fiedlerstrasse 42, Dresden, 01307, Germany.  Christian.Lueck@tu-dresden.de

J Clin Microbiol. 2007 Sep;45(9):3143-4.

NO ABSTRACT

 

Sensitivity of Legionella pneumophila DNA detection in serum samples in relation to disease severity

Diederen BM, Bruin JP, den Boer JW, Peeters MF, Yzerman EP.

Regional Laboratory of Public Health Haarlem, Boerhaavelaan 26, 2035 RC Haarlem, The Netherlands. bramdiederen@gmail.com

J Med Microbiol. 2007 Sep;56(Pt 9):1255.

NO ABSTRACT

 

Increased sensitivity of a direct fluorescent antibody test for Legionella pneumophila in bronchoalveolar lavage samples by immunomagnetic separation based on BioMags

Sethi S, Gore MT, Sethi KK.

Institute of Medical Microbiology and Hygiene, University of Saarland Hospital, Kirrberger Strasse, Building No.43, 66421 Homburg/Saar, Germany. shneh.sethi@uniklinikum-saarland.de

J Microbiol Methods. 2007 Aug;70(2):328-35.

ABSTRACT: In the present study, immunomagnetic separation of Legionella pneumophila from mock bronchoalveolar lavage (BAL) fluid samples, which were artificially spiked with L. pneumophila, and culture positive patient BAL fluid samples, was achieved using BioMags (superparamagnetic particles) loaded with purified rabbit immunoglobulin G specific for L. pneumophila. Bacteria binding onto BioMag-immunomatrix were directly stained with a L. pneumophila species-specific DFA reagent and examined under a fluorescence microscope. BioMag-based immunomagnetic separation (BIMS) followed by DFA staining (BIMS-DFA) could correctly identify all the 20 (100%) BAL samples which were spiked with low numbers (2x10(2) CFU) of L. pneumophila. Cultures could be recovered from 15 (75%) of these 20 spiked BAL samples, 5 (25%) of the samples failed to yield positive cultures. Both culture and BIMS-DFA methods showed 100% positive results when spiked BAL samples containing high bacterial load (10(4) CFU) were tested. The findings with true patient culture positive BAL specimens which were examined retrospectively indicated that BIMS-DFA is significantly more sensitive for detecting L. pneumophila than conventional cytospin method of DFA staining (cytospin-DFA). Out of the 25 culture positive BAL specimens tested, 7 (28%) proved negative by cytospin-DFA whereas BIMS-DFA correctly detected all the 25 (100%) specimens. It is suggested that the BIMS-DFA procedure increases the sensitivity of DFA testing for L. pneumophila in large volume samples such as BAL fluids.

 

Quantitative microbial risk assessment model for Legionnaires' disease: assessment of human exposures for selected spa outbreaks

Armstrong TW, Haas CN.

Occupational and Public Health Division, Exxon-Mobil Biomedical Sciences Inc, Annandale, NJ 08801-0971, USA.  thomas.w.armstrong@ExxonMobil.com

J Occup Environ Hyg. 2007 Aug;4(8):634-46.

ABSTRACT: Evaluation of a quantitative microbial risk assessment (QMRA) model for Legionnaires' disease (LD) required Legionella exposure estimates for several well-documented LD outbreaks. Reports for a whirlpool spa and two natural spring spa outbreaks provided data for the exposure assessment, as well as rates of infection and mortality. Exposure estimates for the whirlpool spa outbreak employed aerosol generation, water composition, exposure duration data, and building ventilation parameters with a two-zone model. Estimates for the natural hot springs outbreaks used bacterial water to air partitioning coefficients and exposure duration information. The air concentration and dose calculations used input parameter distributions with Monte Carlo simulations to estimate exposures as probability distributions. The assessment considered two sets of assumptions about the transfer of Legionella from the water phase to the aerosol emitted from the whirlpool spa. The estimated air concentration near the whirlpool spa was 5 to 18 colony forming units per cubic meter (CFU/m(3)) and 50 to 180 CFU/m(3) for each of the alternate assumptions. The estimated 95th percentile ranges of Legionella dose for workers within 15 m of the whirlpool spa were 0.13-3.4 CFU and 1.3-34.5 CFU, respectively. The modeling for hot springs Spas 1 and 2 resulted in estimated arithmetic mean air concentrations of 360 and 17 CFU/m(3), respectively, and 95 percentile ranges for Legionella dose of 28 to 67 CFU and 1.1 to 3.7 CFU, respectively. The Legionella air concentration estimates fall in the range of limited reports on air concentrations of Legionella (0.33 to 190 CFU/m(3)) near showers, aerated faucets, and baths during filling with Legionella-contaminated water. These measurements may provide some indication that the estimates are of a reasonable magnitude, but they do not clarify the exposure estimates accuracy, since they were not obtained during LD outbreaks. Further research to improve the data used for the Legionella exposure assessment would strengthen the results. Several of the primary additional data needs include improved data for bacterial water to air partitioning coefficients, better accounting of time-activity-distance patterns and exposure potential in outbreak reports, and data for Legionella-containing aerosol viability decay instead of loss of capability for growth in culture.

 

Limited applicability of direct fluorescent-antibody testing for Bordetella sp. and Legionella sp. specimens for the clinical microbiology laboratory

She RC, Billetdeaux E, Phansalkar AR, Petti CA.

Department of Pathology, University of Utah, 30 North 1900 East, Salt Lake City, UT 84132, USA. rosemary.she-bender@hsc.utah.edu

J Clin Microbiol. 2007 Jul;45(7):2212-4.

ABSTRACT: The rapid diagnosis of infections with Bordetella and Legionella species is important for patient management. With observed increases in direct fluorescent-antibody (DFA) testing volumes, we retrospectively compared the performance characteristics of DFA testing to those of culture and PCR. For Bordetella sp., samples were classified as positive by DFA testing (184 [3%] of 6,195 samples) and culture (150 [2%] of 6,251 samples) significantly less often than by PCR (2,557 [10%] of 26,929 samples). Of 360 samples tested by both DFA and PCR methods, 81 (16 by DFA testing and 79 by PCR) were determined to be positive for Bordetella, with a sensitivity and specificity of DFA testing of 18% and 99%, respectively. Of 1,426 samples tested by both DFA and culture methods, 48 (44 by DFA testing and 15 by culture) were determined to be positive for Bordetella, with a sensitivity and specificity of DFA testing of 73% and 98%, respectively. For Legionella sp., samples were identified as positive by DFA testing (31 [0.25%] of 12,597 samples) and culture (85 [0.6%] of 13,572 samples) significantly less often than by PCR (27 [4%] of 716 samples). Of 62 samples tested by both DFA and PCR methods, none were positive for Legionella sp. by DFA testing and 3 were positive by PCR. Of 3,923 samples tested by both DFA and culture methods, 22 (3 by DFA testing and 21 by culture) were positive for Legionella sp., with a sensitivity and specificity of DFA testing of 9.5% and 100%. Overall, DFA testing for Bordetella sp. and Legionella sp. is an insensitive method, and despite its continued popularity, clinical microbiology laboratories should not offer it when more sensitive tests like PCR are available.

 

Validation of a new seminested PCR-based detection method for Legionella pneumophila

Yáñez MA, Barberá VM, Catalán V.

Applus+ LABAQUA, Pol Ind. Atalayas, C) Del Dracma 16-18, 03114 Alicante, Spain. vicente.catalan@labaqua.com

J Microbiol Methods. 2007 Jul;70(1):214-7.

ABSTRACT: We report a new seminested PCR method for detection of Legionella pneumophila based on the simultaneous amplification of a 387 bp fragment of the dotA gene and a 827 bp recombinant fragment that serves as an internal positive control. This new detection system was validated and its specificity and detection limit were determined. Parallel analysis of 90 environmental samples to compare this method, a real-time PCR method and the standard culture isolation method, demonstrated that this seminested method is useful for the study of L. pneumophila.

 

Comparative evaluation of Duopath Legionella lateral flow assay against the conventional culture method using Legionella pneumophila and Legionella anisa strains

Koide M, Haranaga S, Higa F, Tateyama M, Yamane N, Fujita J.

Department of Medicine and Therapeutics, Faculty of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan. koide-mi@med.u-ryukyu.ac.jp

Jpn J Infect Dis. 2007 Jul;60(4):214-6. 

ABSTRACT: Duopath Legionella is a new immunochromatographic assay for the identification of Legionella pneumophila and Legionella spp. As excellent specificity has been previously reported for this kit, we attempted an evaluation of its sensitivity using L. pneumophila serogroup 1 and Legionella anisa strains. Bacterial suspensions of L. pneumophila at concentrations of 1.2 x 10(8) cfu/ml and 1.2 x 10(7) cfu/ml were detected, but those below 1.2 x 10(6) cfu/ml were not recognized by the Duopath kit. After centrifugation and the sediment resuspension, a 2.8 x 10(7) cfu/ml concentration of bacterial suspension showed a positive result, but negative results were obtained below 2.8 x 10(6) cfu/ml. Also, bacterial suspension with concentrations of 3.2 x 10(9) cfu/ml and 1.4 x 10(9) cfu/ml after centrifugation of L.anisa were detected, but those below 3.2 x 10(8) cfu/ml and 1.4 x 10(8) cfu/ml after centrifugation were not recognized by the Duopath kit. Meanwhile, this kit was less sensitive to the L. pneumophila serogroup 1 suspension, and was more sensitive to the L. pneumophila serogroup 2 and L.anisa than the Binax NOW immunochromatographic kit. It was realized that the sensitivity of this kit is too low for determining the presence of Legionella in water samples. Although this kit may have excellent specificity, it has low sensitivity.

 

Hairpin-DNA probe for enzyme-amplified electrochemical detection of Legionella pneumophila

Miranda-Castro R, de-Los-Santos-Alvarez P, Lobo-Castañón MJ, Miranda-Ordieres AJ, Tuñón-Blanco P.

Departamento de Química Física y Analítica, Universidad de Oviedo, C/JuliAn Clavería, 8. 33006. Oviedo, Principado de Asturias, Spain. ptb@uniovi.es

Anal Chem. 2007 Jun 1;79(11):4050-5.

ABSTRACT: An electrochemical genosensor for the detection of nucleic acid sequences specific of Legionella pneumophila is reported. An immobilized thiolated hairpin probe is combined with a sandwich-type hybridization assay, using biotin as a tracer in the signaling probe, and streptavidin-alkaline phosphatase as reporter molecule. The activity of the immobilized enzyme was voltammetrically determined by measuring the amount of 1-naphthol generated after 2 min of enzymatic dephosphorylation of 1-naphthyl phosphate. The sensor allows discrimination between L. pneumophila and L. longbeachae with high sensitivity under identical assay conditions (no changes in stringency). A limit of detection of 340 pM L. pneumophila DNA, and a linear relationship between the analytical signal and the logarithm of the target concentration to 2 muM were obtained. Experimental results show the superior sensitivity and selectivity of the hairpin-based assay when compared with analogous sandwich-type assays using linear capture probes.

  

Integrated real-time PCR for detection and monitoring of Legionella pneumophila in water systems

Yaradou DF, Hallier-Soulier S, Moreau S, Poty F, Hillion Y, Reyrolle M, André J, Festoc G, Delabre K, Vandenesch F, Etienne J, Jarraud S.

Centre National de Référence des Légionelles, INSERM E-0230, Faculté de Médecine Laennec, Laboratoire de Bactériologie, 7 rue Guillaume-Paradin, 69372 Lyon cedex 08, France. sophie.jarraud@univ-lyon1.fr

Appl Environ Microbiol. 2007 Mar;73(5):1452-6.

ABSTRACT: We evaluated a ready-to-use real-time quantitative Legionella pneumophila PCR assay system by testing 136 hot-water-system samples collected from 55 sites as well as 49 cooling tower samples collected from 20 different sites, in parallel with the standard culture method. The PCR assay was reproducible and suitable for routine quantification of L. pneumophila. An acceptable correlation between PCR and culture results was obtained for sanitary hot-water samples but not for cooling tower samples. We also monitored the same L. pneumophila-contaminated cooling tower for 13 months by analyzing 104 serial samples. The culture and PCR results were extremely variable over time, but the curves were similar. The differences between the PCR and culture results did not change over time and were not affected by regular biocide treatment. This ready-to-use PCR assay for L. pneumophila quantification could permit more timely disinfection of cooling towers.

 

Molecular typing of a Legionella pneumophila outbreak in Ontario, Canada

Gilmour MW, Bernard K, Tracz DM, Olson AB, Corbett CR, Burdz T, Ng B, Wiebe D, Broukhanski G, Boleszczuk P, Tang P, Jamieson F, Van Domselaar G, Plummer FA, Berry JD.

National Microbiology Laboratory, Public Health Agency of Canada, 1015 Arlington Street, Winnipeg, Manitoba R3E 3R2, Canada.

Matthew_Gilmour@phac-aspc.gc.ca

J Med Microbiol. 2007 Mar;56(Pt 3):336-41.

ABSTRACT: An outbreak of Legionnaires' disease at a long-term care facility in Ontario, Canada from September to October 2005 resulted in the death of 23 residents and the illness of 112 other people. In response, molecular methods were developed to detect Legionella pneumophila in clinical lung samples and to subtype isolates from clinical and environmental samples. The targeted genetic loci included Legionella-specific virulence determinants (mip, icmO, sidA and lidA) and core bacterial determinants (ftsZ, trpS and dnaX). An established amplified fragment length polymorphism typing method provided the first indication of genetic relatedness between strains recovered from clinical samples and strains cultured from environmental samples taken from the outbreak site. These associations were verified using the European Working Group for Legionella Infections sequence-based typing protocol targeting the flaA, pilE, asd, mip, mompS and proA loci. These molecular typing methods confirmed the outbreak source as a contaminated air conditioning cooling tower.

 

Vircell assays for detection of antibodies against Legionella pneumophila

Rojas A, Rojas J, Mendoza J.

Vircell S.L., Pza. Domínguez Ortiz 1, 18320 Santa Fé, Granada, Spain. immunology@vircell.com

Clin Vaccine Immunol. 2007 Feb;14(2):208-9.

Letter.

 

A large, travel-associated outbreak of legionellosis among hotel guests: utility of the urine antigen assay in confirming Pontiac fever

Burnsed LJ, Hicks LA, Smithee LM, Fields BS, Bradley KK, Pascoe N, Richards SM, Mallonee S, Littrell L, Benson RF, Moore MR; Legionellosis Outbreak Investigation Team.

Communicable Disease Div., Oklahoma State Department of Health, Oklahoma City, OK 73117-1299, USA. laurence@health.ok.gov

Clin Infect Dis. 2007 Jan 15;44(2):222-8.

BACKGROUND: During March 2004, a large outbreak of legionnaires disease and Pontiac fever occurred among hotel guests in Oklahoma. An investigation was conducted to identify the source and evaluate the utility of the Legionella urine antigen assay and serologic testing for the identification of Pontiac fever. METHODS: A retrospective cohort investigation of hotel guests and employees and an environmental evaluation were performed. Participants were interviewed, and clinical specimens were collected from consenting individuals. RESULTS: Six cases of legionnaires disease and 101 cases of Pontiac fever were identified. Exposure to the indoor pool and hot tub area was associated with legionellosis (relative risk, 4.4; 95% confidence interval, 2.8-6.9). Specimens from the pool and hot tub tested positive for Legionella pneumophila serogroup 1 by polymerase chain reaction. For Pontiac fever, the sensitivity and positive predictive value were 35.7% and 100%, respectively, for the urine antigen assay, and 46.4% and 90%, respectively, for serologic testing. The specificity and negative predictive value were 100% and 47.8%, respectively, for the urine antigen assay, and 89.3% and 45.5%, respectively, for serologic testing. CONCLUSIONS: Urine antigen testing, with or without serologic testing, can be used to confirm outbreak-associated cases of Pontiac fever caused by L. pneumophila serogroup 1.

 

Evaluation of the SAS Legionella Test, a new immunochromatographic assay for the detection of Legionella pneumophila serogroup 1 antigen in urine

Diederen BM, Peeters MF.

Laboratory for Medical Microbiology and Immunology, St Elisabeth Hospital, Tilburg, The Netherlands. bramdiederen@gmail.com

Clin Microbiol Infect. 2007 Jan;13(1):86-8. 

ABSTRACT: This study evaluated a new immunochromatographic assay (SAS Legionella Test) for its ability to detect Legionella pneumophila serogroup 1 antigen in urine. Results were compared with those obtained using the Binax Now urinary antigen test. Sensitivity and specificity were estimated as 82.9% and 99.0%, respectively, for the SAS Legionella Test, and 91.4% and 100%, respectively, for the Binax Now urinary antigen test. The sensitivity of both tests increased to 97.1% (p 0.009) and 94.2% (p 0.7), respectively, if the tests were examined after 1 h.

 

Identification and serotyping of atypical Legionella pneumophila strains isolated from human and environmental sources

Helbig JH, Benson RF, Pelaz C, Jacobs E, Lück PC.

Medizinische Fakultät TU Dresden, Institut Medizinische Mikrobiologie und Hygiene, Dresden, Germany. j.helbig@mailbox.tu-dresden.de

J Appl Microbiol. 2007 Jan;102(1):100-5.

ABSTRACT: AIMS: To validate identification methods for Legionella pneumophila strains that cannot be serotyped into the known serogroups and to characterize their antigenic diversity. METHODS AND RESULTS: Fifty L. pneumophila strains that could not be serogrouped, but which had been confirmed as L. pneumophila by mip gene sequencing, were further identified phenotypically. We used (i) MONOFLUO anti-Legionella Staining Reagent (Bio-Rad) (50/50), (ii) an in-house prepared immunoblot assay for the detection of L. pneumophila- specific Mip protein epitope (50/50), (iii) fatty acid analysis using the Microbial Identifications System (MIDI) (47/50) and (iv) Oxoid agglutination tests (44/50). The serological diversity was further characterized by testing with five serogroup-cross-reactive monoclonal antibodies, resulting in nine phenons. CONCLUSIONS: The division of L. pneumophila into 15 serogroups does not reflect the serogroup heterogeneity. Results of these tests indicate that there are more serogroups. SIGNIFICANCE AND IMPACT OF THE STUDY: MONOFLUO anti-Legionella Staining Reagent is the only commercially available tool for identifying atypical strains of L. pneumophila. If necessary for epidemiological purposes, the antigenic heterogeneity of these strains can be analysed by monoclonal antibodies.

 

Use of partial 16S rRNA gene sequencing for identification of Legionella pneumophila and non-pneumophila Legionella spp

Wilson DA, Reischl U, Hall GS, Procop GW.

The Cleveland Clinic, Ohio, USA. gprocop@med.miami.edu

J Clin Microbiol. 2007 Jan;45(1):257-8.

ABSTRACT: We examined 49 Legionella species, 26 L. pneumophila and 23 non-pneumophila Legionella spp., using partial 16S rRNA gene sequencing. This approach accurately identified all the L. pneumophila isolates, characterized all non-pneumophila Legionella isolates as such within this genus, and classified most (20/23; 87%) of the non-pneumophila Legionella isolates to the species level.

 

Computed tomographic features of Legionella pneumophila pneumonia in 38 cases

Sakai F, Tokuda H, Goto H, Tateda K, Johkoh T, Nakamura H, Matsuoka T, Fujita A, Nakamori Y, Aoki S, Ohdama S.

Department of Diagnostic Radiology, Tokyo Metropolitan Komagome Hospital, Tokyo, Japan. fmksakai@yahoo.co.jp

J Comput Assist Tomogr. 2007 Jan-Feb;31(1):125-31.

ABSTRACT: PURPOSE: To characterize the imaging features of Legionella pneumophila pneumonia (LPP). SUBJECTS AND METHODS: Imaging findings of computed tomography (CT) in 38 cases of microbiologically or serologically determined LPP were analyzed and compared with those of 35 cases of Streptococcus pneumoniae pneumonia. RESULTS: In cases with LPP, abnormal opacities were distributed in a single lobe in 5 cases, in multiple lobes unilaterally in 10 cases, and multifocally and bilaterally in 23 cases. All cases showed consolidation and/or ground glass opacity in lung fields. Sharply demarcated peribronchovascular foci of consolidation intermingled with ground glass opacity were noted in 24 cases (24 of 38, 63%), whereas imaging features were seen in only 3 cases (3 of 35, 9%) of Streptococcus pneumoniae pneumonia. These CT patterns have nothing to do with clinical features such as age, sex, severity of disease, and time between onset of disease and CT examination. CONCLUSIONS: Imaging features of LPP on CT include bilateral and unilateral single and multifocal consolidation and ground opacity. Sharply demarcated peribronchovascular foci of consolidation intermingled with ground glass opacity seem to be one of the most frequent CT appearances of LPP.

 

Detection of airborne Legionella while showering using liquid impingement and fluorescent in situ hybridization (FISH)

Deloge-Abarkan M, Ha TL, Robine E, Zmirou-Navier D, Mathieu L.

Département Environnement et Santé Publique, INSERM ERI no 11, Faculté de Médecine, 9 avenue de la Forêt de Haye, BP 184, F-54 505, Vandoeuvre-lès-Nancy, France. laurence.mathieu@medecine.uhp-nancy.fr

J Environ Monit. 2007 Jan;9(1):91-7.

ABSTRACT: Aerosols of water contaminated with Legionella bacteria constitute the only mode of exposure for humans. However, the prevention strategy against this pathogenic bacteria risk is managed through the survey of water contamination. No relationship linked the Legionella bacteria water concentration and their airborne abundance. Therefore, new approaches in the field of the metrological aspects of Legionella bioaerosols are required. This study was aimed at testing the main principles for bioaerosol collection (solid impaction, liquid impingement and filtration) and the in situ hybridization (FISH) method, both in laboratory and field assays, with the intention of applying such methodologies for airborne Legionella bacteria detection while showering. An aerosolization chamber was developed to generate controlled and reproducible L. pneumophila aerosols. This tool allowed the identification of the liquid impingement method as the most appropriate one for collecting airborne Legionella bacteria. The culturable fraction of airborne L. pneumophila recovered with the liquid impingement principle was 4 and 700 times higher compared to the impaction and filtration techniques, respectively. Moreover, the concentrations of airborne L. pneumophila in the impinger fluid were on average 7.0 x 10(5) FISH-cells m(-3) air with the fluorescent in situ hybridization (FISH) method versus 9.0 x 10(4) CFU m(-3) air with the culture method. These results, recorded under well-controlled conditions, were confirmed during the field experiments performed on aerosols generated by hot water showers in health institutions. This new approach may provide a more accurate characterization of aerobiocontamination by Legionella bacteria.

 

Evaluation of real-time PCR for the early detection of Legionella pneumophila DNA in serum samples

Diederen BM, de Jong CM, Marmouk F, Kluytmans JA, Peeters MF, Van der Zee A.

Laboratory of Medical Microbiology and Immunology, St Elisabeth Hospital, PO Box 747, 5000 AS Tilburg, The Netherlands. bramdiederen@gmail.com

J Med Microbiol. 2007 Jan;56(Pt 1):94-101. 

ABSTRACT: Legionella pneumonia can be difficult to diagnose. Existing laboratory tests all have shortcomings, especially in the ability to diagnose Legionnaires' disease (LD) at an early stage of the disease in a specimen that is readily obtainable. The aim of this study was to assess the performance of PCR as a rapid diagnostic method and to compare the results of different PCR assays of serum samples from patients with LD. Samples included 151 serum samples from 68 patients with proven LD and 60 serum samples from 36 patients with respiratory tract infections other than Legionella. PCR assays were based on the 5S rRNA gene, 16S rRNA gene and the mip gene. The samples from patients with infections caused by pathogens other than Legionella all tested negative in PCR. Among the patients with proven LD 54.4 % (37/68) tested positive in 5S rRNA PCR, 52.9 % (36/68) in mip gene PCR and 30.9 % (21/68) in 16S rRNA PCR in the first available serum sample. The association between threshold cycle value in 5S PCR positive serum samples (n=49) and C-reactive protein value was determined, and showed a strong negative correlation (Pearson correlation coefficient r=-0.63, P<0.0001). In addition to existing tests for the diagnosis of LD, detection of Legionella DNA in serum could be a useful tool for early diagnosis of LD caused by any Legionella species and serogroup, and has the potential to provide a diagnosis in a time frame that could affect initial infection management.

 

Development and evaluation of a Taqman duplex real-time PCR quantification method for reliable enumeration of Legionella pneumophila in water samples

Behets J, Declerck P, Delaedt Y, Creemers B, Ollevier F.

Laboratory of Aquatic Ecology, Zoological Institute, Katholieke Universiteit Leuven,Charles Deberiotstraat 32, 3000 Leuven, Belgium. Priscilla.declerck@bio.kuleuven.be

J Microbiol Methods. 2007 Jan;68(1):137-44.

ABSTRACT: This study describes the development and evaluation of a specific Legionella pneumophila Taqman duplex real-time PCR (qPCR) for fast and reliable quantification of this human pathogen in suspected man-made water systems. The qPCR assay was 100% specific for all L. pneumophila serogroups 1-15 with a sensitivity of 60 genome units/l and an amplification efficiency of 98%. Amplification inhibitors were detected via an exogenous internal positive control, which was amplified simultaneously with L. pneumophila DNA using its own primer and probe set. Mean recovery rates of the qPCR assay for tap water and cooling circuit water, spiked with a known number L. pneumophila bacteria, were 93.0% and 56.3%, respectively. Additionally, by using the Ultraclean Soil DNA isolation kit, we were able to remove amplification inhibitors ubiquitously present in cooling water. The practical value of our qPCR assay was evaluated through analysis of 30 water samples from showers, taps, eyewash stations, fire sprinklers and recirculation loops with qPCR and traditional culture. In conclusion, the described L. pneumophila Taqman duplex real-time assay proved to be specific, sensitive and reproducible. This makes it a promising method complementing the current time-consuming culture standard method.

 

Nosocomial Legionnaires' disease caused by Legionella pneumophila serogroup 6: implication of the sequence-based typing method (SBT)

Fendukly F, Bernander S, Hanson HS.

Department of Clinical Microbiology, Karolinska University Hospital. Uppsala, Stockholm, Sweden. faiz.fendukly@capio.se

Scand J Infect Dis. 2007;39(3):213-6.

ABSTRACT: Sequence-based typing (SBT) was used to determine the allelic profiles of 3 sporadic clinical isolates as well as 7 environmental isolates of Legionella pneumophila serogroup 6, isolated at the Karolinska Hospital during 2004. The clinical isolates were cultured from patients with nosocomial Legionnaires' disease (LD), while the environmental isolates were cultured from potable water sources of the hospital wards in the close vicinity of the 3 patients being investigated. The genes sequenced for the construction of the SBT profile included flaA, pilE, asd, mip, mompS and proA, in this pre-determined order and the allelic profile of the 10 isolates was identical (3, 13, 1, 28, 14, 9). Furthermore, 2 of the isolates, 1 clinical and 1 environmental, were analysed using the amplified fragment length polymorphism analysis (AFLP). The AFLP genotype of both isolates was congruent. Eight of 9 control L. pneumophila serogroup 6 isolates had the same SBT profile as the study isolates. We conclude that the environmental strain isolated from our hospital's drinking water is indistinguishable genotypically from the 3 clinical isolates of Legionella. However, this genotype of L. pneumophila is geographically widespread. Thus, results of genotyping must be evaluated in conjunction with the clinical and epidemiological data.

 

Use of sequence-based typing for investigation of a case of nosocomial legionellosis

Wong S, Pabbaraju K, Burk VF, Broukhanski GC, Fox J, Louie T, Mah MW, Bernard K, Tilley PA.

Provincial Laboratory for Public Health (Microbiology), Calgary site, Calgary, Alberta, Canada. p.tilley@provlab.ab.ca

J Med Microbiol. 2006 Dec;55(Pt 12):1707-10.

ABSTRACT: A fatal case of nosocomial legionellosis in a low prevalence region (Calgary, Alberta, Canada) prompted investigation into the source of infection. Hospital water systems contaminated with Legionella pneumophila have been shown to pose a risk to compromised patients. Typing of an L. pneumophila serogroup 1 strain isolated from the patient using sequence-based typing (SBT) and amplified fragment length polymorphism (AFLP) analysis linked it to a persistent and widespread strain isolated from the hospital water system establishing a nosocomial mode of acquisition. Different SBT and AFLP patterns were determined for non-epidemiologically linked cases and isolates from different hospitals.

 

Development of conventional and real-time NASBA for the detection of Legionella species in respiratory specimens

Loens K, Beck T, Goossens H, Ursi D, Overdijk M, Sillekens P, Ieven M.

Department of Microbiology, University of Antwerp UA, Antwerp, Belgium. katherine.loens@ua.ac.be

J Microbiol Methods. 2006 Dec;67(3):408-15.

ABSTRACT: Isothermal nucleic acid sequence-based amplification (NASBA) was applied to detect Legionella 16S rRNA. The assay was originally developed as a Legionella pneumophila conventional NASBA assay with electrochemiluminescence (ECL) detection and was subsequently adapted to a L. pneumophila real-time NASBA format and a Legionella spp. real-time NASBA using molecular beacons. L. pneumophila RNA prepared from a plasmid construct was used to assess the analytical sensitivity of the assay. The sensitivity of the NASBA assay was 10 molecules of in vitro wild type L. pneumophila RNA and 0.1-1 colony-forming units (CFU) of L. pneumophila. In spiked respiratory specimens, the sensitivity of the NASBA assays was 1-10000 CFU of L. pneumophila serotype 1 depending on the background. After dilution of the nucleic acid extract prior to amplification, 1-10 CFU of L. pneumophila serotype 1 could be detected with both detection methods. Finally, 27 respiratory specimens, well characterized by culture and PCR, collected during a L. pneumophila outbreak, were tested by conventional and real-time NASBAs. All 11 PCR positive samples were positive by conventional NASBA, 9/11 and 10/11 were positive by L. pneumophila real-time NASBA and Legionella spp. real-time NASBA, respectively.

 

Detection of legionella species in clinical samples: Comparison of polymerase chain reaction and urinary antigen detection kits

Koide M, Higa F, Tateyama M, Nakasone I, Yamane N, Fujita J.

Dept. of Medicine and Therapeutics, Control and Prevention of Infectious Diseases (First Department of Internal Medicine), Faculty of Medicine, University of the Ryukyus, 207 Uehara, Okinawa 903-0215, Japan. koide-mi@med.u-ryukyu.ac.jp

Infection. 2006 Oct;34(5):264-8.

BACKGROUND: Recently, two excellent methods have been used for the diagnosis of Legionnaires' disease: urinary antigen detection and PCR. The purpose of the present study is to analyze and evaluate the sensitivity and specificity of three different urinary antigen detection kits as well as PCR. MATERIALS AND METHODS: A total of 148 samples were collected from 33 patients between 1993 and 2004. These consisted of 73 urine samples obtained from 33 patients, 57 serum samples provided by 29 patients, and 18 respiratory tract specimens from 13 patients. Three commercially available kits were used to detect urinary antigen. For the 5S PCR reaction, primers L5SL2 and L5SR84 were used. RESULTS: Positive results were shown in all patients' urine (representing 79.5% of total samples) using the Binax EIA kit, in 93.9% patients (representing 75.3% samples) using the Binax NOW immunochromatographic kit, and in 90.9% (representing 72.6% samples) using the Biotest EIA kit. Urine samples from 12.1% patients (representing 6.8% of total samples), serum samples from 41.4% patients (representing 35.1% of total samples), and respiratory samples from 84.6% patients (representing 88.9% of total samples) showed positive results with PCR. CONCLUSION: In testing urine of legionellosis patients, it was suggested that three kits were all valuable tools for diagnosis of legionellosis. Since over one-third of patients' serum samples and most respiratory specimens showed positive results with PCR, the addition of PCR for testing of these samples might be useful, particularly in cases of culture negative and serum antibody negative patients.

 

Identification and differentiation of Legionella pneumophila and Legionella spp. with real-time PCR targeting the 16S rRNA gene and species identification by mip sequencing

Stølhaug A, Bergh K.

Department of Medical Microbiology, Laboratory Center, St. Olavs Hospital, N-7006 Trondheim, Norway.kare.bergh@ntnu.no

Appl Environ Microbiol. 2006 Sep;72(9):6394-8.

ABSTRACT: Fluorescent resonance energy transfer probes targeting the 16S rRNA gene were constructed for a sensitive and specific real-time PCR for identification and differentiation of Legionella pneumophila from other Legionella spp. For identification of non-L. pneumophila spp. by direct amplicon sequencing, two conventional PCR assays targeting the mip gene were established.

 

Evaluation of two new immunochromatographic assays (Rapid U Legionella antigen test and SD Bioline Legionella antigen test) for detection of Legionella pneumophila serogroup 1 antigen in urine

Diederen BM, Peeters MF.

Laboratory of Medical Microbiology and Immunology, St Elisabeth Hospital, P.O. Box 747, 5000 AS Tilburg, The Netherlands. b.diederen@elisabeth.nl

J Clin Microbiol. 2006 Aug;44(8):2991-3.

ABSTRACT: We evaluated two new immunochromatographic assays for their abilities to detect Legionella pneumophila serogroup 1 antigen in urine. The results were compared with those obtained by the Binax NOW urinary antigen test. The sensitivities and specificities were estimated to be 71.2% and 96.6%, respectively, for the Rapid U test; 31.5% and 98.9%, respectively, for the SD Bioline test; and 91.8% and 100%, respectively, for the Binax NOW test.

Evaluation of the Duopath Legionella Lateral Flow Assay for Identification of Legionella pneumophila and Legionella Species Culture Isolates

Helbig JH, Luck PC, Kunz B, Bubert A.

Institute of Medical Microbiology and Hygiene, Medical Faculty of the Technical University Dresden, Fetscherstr. 74, D-01307 Dresden, Germany.

Appl Environ Microbiol. 2006 Jun;72(6):4489-91.

ABSTRACT: Duopath Legionella (Merck KGaA, Darmstadt, Germany) is a new immunochromatographic assay for the simultaneous identification of cultured L. pneumophila and Legionella species other than L. pneumophila. In tests of 89 L. pneumophila strains and 87 Legionella strains other than L. pneumophila representing 41 different species, Duopath and a widely used latex agglutination assay detected L. pneumophila with 100% and 98% accuracy, respectively, whereas the percentages differed significantly for other Legionella spp. (93% versus 37% [P < 0.001]). Since many countries' regulations require the identification of Legionella spp. in water and environmental samples, the use of Duopath Legionella to comply with those regulations could contribute to significantly fewer false-negative results.

 

Development of conventional and real-time NASBA(R) for the detection of Legionella species in respiratory specimens

Loens K, Beck T, Goossens H, Ursi D, Overdijk M, Sillekens P, Ieven M.

Department of Microbiology, University of Antwerp UA, Antwerp, Belgium.

J Microbiol Methods. 2006 Dec;67(3):408-15.

ABSTRACT: Isothermal nucleic acid sequence-based amplification (NASBA) was applied to detect Legionella 16S rRNA. The assay was originally developed as a Legionella pneumophila conventional NASBA assay with electrochemiluminescence (ECL) detection and was subsequently adapted to a L. pneumophila real-time NASBA format and a Legionella spp. real-time NASBA using molecular beacons. L. pneumophila RNA prepared from a plasmid construct was used to assess the analytical sensitivity of the assay. The sensitivity of the NASBA assay was 10 molecules of in vitro wild type L. pneumophila RNA and 0.1-1 colony-forming units (CFU) of L. pneumophila. In spiked respiratory specimens, the sensitivity of the NASBA assays was 1-10000 CFU of L. pneumophila serotype 1 depending on the background. After dilution of the nucleic acid extract prior to amplification, 1-10 CFU of L. pneumophila serotype 1 could be detected with both detection methods. Finally, 27 respiratory specimens, well characterized by culture and PCR, collected during a L. pneumophila outbreak, were tested by conventional and real-time NASBAs. All 11 PCR positive samples were positive by conventional NASBA, 9/11 and 10/11 were positive by L. pneumophila real-time NASBA and Legionella spp. real-time NASBA, respectively.

 

Sensitive, real-time PCR detects low-levels of contamination by Legionella pneumophila in commercial reagents.
Shen H,
Rogelj S, Kieft TL.
Department of Biology, New Mexico Tech., 801 Leroy Place, Socorro, 87801, USA.

Mol Cell Probes. 2006 Jun-Aug;20(3-4):147-53.

In a real-time PCR assay of Legionella pneumophila (targeting the L. pneumophila-specific mip gene and using SYBR Green dye for DNA detection in conjunction with the iCycler system) we detected as few as 1.3 copies of a mip gene in a 50-microl reaction from serially diluted L. pneumophila genomic DNA. However, cycle threshold (C(T)) were yielded and DNA product detected in our no-template negative controls and the phenomenon persisted when two separate batches of PCR reagents and water from two different biochemical companies were tested. Since L. pneumophila can be widespread in municipal water supplies, the commercial reagents, especially the reagent water (80% of the reaction volume), could be the source of contamination. To test this hypothesis, we treated Millipore Milli-Q water by filtering through a 0.2 microm-pore-size polycarbonate filter to remove bacteria prior to autoclaving. Real-time PCR using this water had no contamination. Our finding is indirect evidence that commercially available purified water can harbor low level contamination by L. pneumophila DNA that has escaped purification processes. This presents a challenge when developing a sensitive DNA-based bacterial detection method if the target organism or its DNA is a common contaminant of necessary reagents.

 

Development of an improved PCR-ICT hybrid assay for direct detection of Legionellae and Legionella pneumophila from cooling tower water specimens.
Horng YT, Soo PC, Shen BJ, Hung YL, Lo KY, Su HP, Wei JR, Hsieh SC, Hsueh PR, Lai HC.
Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University College of Medicine, No. 1. Chan-Dar Street 100, Taipei, Taiwan, ROC; Tyson Bioresearch Inc., Taiwan, ROC.

Water Res. 2006 Jun;40(11):2221-9.

A novelly improved polymerase chian reaction and immunochromatography test (PCR-ICT) hybrid assay comprising traditional multiplex-nested PCR and ICT, (a lateral-flow device) was developed for direct detection of Legionella bacteria from environmental cooling tower samples. The partial 16S rDNA (specific for Legionella spp.) and dnaJ (specific for Legionella pneumophila) genes from Legionella chromosome were first specifically amplified by multiplex-nested PCR, respectively, followed by detection using ICT strip. Reading of results was based on presence or absence of the two test lines on the strips. Presence of test line 1 indicated existence of Legionella spp. specific 16S rDNA and identified Legionella spp. Presence of test line 2 further indicated existence of dnaJ and thus specifically identified L. pneumophila. In contrast, for non-Legionellae bacteria no test line formation was observed. Results of direct detection of Legionella bacteria and L. pneumophila from water tower specimens by this assay showed 100% sensitivity, and 96.6% and 100% specificity, respectively compared with traditional culture, biochemical and serological identification methods. The PCR-ICT hybrid assay does not require sophisticated equipment and was proved to be practically useful in rapid and direct Legionellae detection from environmental water samples.

 

Characterization of the Legionella anisa population structure by pulsed-field gel electrophoresis.
Akermi M, Doleans A, Forey F, Reyrolle M, Meugnier H, Freney J, Vandenesch F, Etienne J, Jarraud S.
Centre National de reference des Legionella, Laboratoire de Bacteriologie INSERM E-0230, Faculte de Medecine, Lyon, France.

FEMS Microbiol Lett. 2006 May;258(2):204-7.

We analysed 38 French isolates of Legionella anisa by means of pulsed-field gel electrophoresis (PFGE) with single or double digestion. Double digestion was more discriminatory than single digestion, and can thus be useful for epidemiological studies of L. anisa. Several isolates from different parts of France clustered together on the basis of their PFGE patterns (similarity cutoff of 80%), suggesting that the L. anisa population structure is homogenous or that a few clones of L. anisa strains have spread widely in France.

 

Detection of Legionella spp. by fluorescent in situ hybridization in dental unit waterlines.
Dutil S, Tessier S, Veillette M, Laflamme C, Meriaux A, Leduc A, Barbeau J, Duchaine C.
Centre de recherche, Hopital Laval, Institut universitaire de cardiologie et de pneumologie de l'Universite Laval, Ste-Foy, Quebec, Canada.

J Appl Microbiol. 2006 May;100(5):955-63.

AIMS: To confirm the presence of viable Legionella spp. in dental unit waterlines (DUWL) using fluorescent in situ hybridization (FISH) and compare this method with culture approach and also to validate the utility of an enrichment to increase FISH sensitivity. METHODS AND RESULTS: Water samples from 40 dental units were analysed. Three different techniques for detecting Legionella spp. were compared: (i) culture approach, (ii) direct FISH and (iii) FISH with a previous R2A medium enrichment (R2A/FISH). The FISH detection was confirmed by PCR. The use of the direct FISH does not improve significantly the detection of legionellae when compared with the culture. On the contrary, when R2A/FISH was performed, sensitivity was, respectively, two- and threefold higher than that with the direct FISH and culture approach. Using R2A/FISH, 63% of water samples analysed showed a contamination by legionellae. CONCLUSIONS: Legionellae detection by direct FISH and R2A/FISH in dental unit water is possible but is more rapid and more sensitive (R2A/FISH) than the culture approach. SIGNIFICANCE AND IMPACT OF THE STUDY: R2A/FISH showed that several pathogens present in DUWL are viable but may not be culturable. Unlike PCR, R2A/FISH is designed to detect only metabolically active cells and therefore provides more pertinent information on infectious risk.

 

Detection and quantification of Legionella pneumophila DNA in serum: case reports and review of the literature.
Diederen BM, de Jong CM, Kluytmans JA, van der Zee A, Peeters MF.
Laboratory of Medical Microbiology and Immunology, St Elisabeth Hospital, PO Box 747, 5000 AS Tilburg, The Netherlands.

J Med Microbiol. 2006 May;55(Pt 5):639-42

Legionella pneumonia can be difficult to diagnose. Existing laboratory tests all have shortcomings, especially the ability to diagnose all Legionella spp. at an early stage. Detection of Legionella DNA in serum can be a valuable tool for the diagnosis of Legionnaires' disease (LD). This report describes two patients with LD diagnosed by PCR using serum samples. In addition, quantification of L. pneumophila DNA using real-time PCR during the course of illness was carried out. The results obtained mirrored both the clinical condition and C-reactive protein values during the course of the illness. Quantification of Legionella DNA in serum using real-time PCR could be a valuable tool to monitor the effects of antimicrobial therapy in patients with LD.

 

Sensitivity of three serum antibody tests in a large outbreak of Legionnaires' disease in the Netherlands.
Yzerman EP, den Boer JW, Lettinga KD, Schel AJ, Schellekens J, Peeters M.
Regional Laboratory of Public Health Haarlem, Boerhaavelaan 26, 2035 RC Haarlem, The Netherlands.

J Med Microbiol. 2006 May;55(Pt 5):561-6.

In 1999, an outbreak involving 188 patients with Legionnaires' disease (LD) occurred at a flower show in the Netherlands. This large outbreak provided the opportunity to evaluate serum antibody tests to assay anti-Legionella pneumophila, since limited data are available on the sensitivity of these tests. The sensitivities of an indirect serotype 1-6 immunofluorescence antibody test (IFAT), a rapid micro-agglutination test (RMAT) IgM serotype 1 antibody assay, and an ELISA to detect IgM and IgG serotype 1-7 antibodies, were evaluated using serum samples from LD patients related to the 1999 outbreak. Sensitivity was calculated using positive culture and/or a positive urinary antigen test as the gold standard in outbreak-related patients with radiographically confirmed pneumonia who fulfilled the epidemiological criteria. The IFAT, RMAT and ELISA showed sensitivities of 61, 44 and 64%, respectively. The sensitivity of the three tests combined was 67%. In epidemic situations, however, high standing titres may be included in the laboratory evidence of LD cases. In the study population, high standing titres were found in 16% of cases. If the presence of high standing antibody titres was added to the criteria of a positive test, the sensitivities of IFAT, RMAT and ELISA were 86, 48 and 75%, respectively. The sensitivity was 91% for all tests combined. The higher sensitivity for the combined use of tests is offset by a reduction in specificity to 97.6%. The results of this study indicate that using a combination of serologic tests in pneumonia patients suspected to have LD does not substantially improve sensitivity. The results suggest that in the microbiological diagnosis of LD, both IFAT and ELISA are reasonably sensitive assays. In an epidemic situation, both tests are highly sensitive, the IFAT more so than the ELISA.

 

Quantitative real-time Legionella PCR for environmental water samples: data interpretation.
Joly P, Falconnet PA, Andre J, Weill N, Reyrolle M, Vandenesch F, Maurin M, Etienne J, Jarraud S.
Centre National de Reference des Legionella, INSERM E-0230, Faculte de Medecine, IFR 62, 7 rue Guillaume-Paradin, 69372 Lyon Cedex 08, France.

Appl Environ Microbiol. 2006 Apr;72(4):2801-8.

Quantitative Legionella PCRs targeting the 16S rRNA gene (specific for the genus Legionella) and the mip gene (specific for the species Legionella pneumophila) were applied to a total of 223 hot water system samples (131 in one laboratory and 92 in another laboratory) and 37 cooling tower samples (all in the same laboratory). The PCR results were compared with those of conventional culture. 16S rRNA gene PCR results were nonquantifiable for 2.8% of cooling tower samples and up to 39.1% of hot water system samples, and this was highly predictive of Legionella CFU counts below 250/liter. PCR cutoff values for identifying hot water system samples containing >10(3) CFU/liter legionellae were determined separately in each laboratory. The cutoffs differed widely between the laboratories and had sensitivities from 87.7 to 92.9% and specificities from 77.3 to 96.5%. The best specificity was obtained with mip PCR. PCR cutoffs could not be determined for cooling tower samples, as the results were highly variable and often high for culture-negative samples. Thus, quantitative Legionella PCR appears to be applicable to samples from hot water systems, but the positivity cutoff has to be determined in each laboratory.

 

Evaluation of Vircell Enzyme-Linked Immunosorbent Assay and Indirect Immunofluorescence Assay for Detection of Antibodies against Legionella pneumophila

Diederen BM, Kluytmans JA, Peeters MF.

Laboratory for Medical Microbiology and Immunology, St. Elisabeth Hospital, P.O. Box 747, 5000 AS Tilburg, The Netherlands.

Clin Vaccine Immunol. 2006 Mar;13(3):361-4.

ABSTRACT: We evaluated the abilities of the Vircell immunoglobulin G (IgG) and IgM indirect immunofluorescence assay (IFA) for Legionella pneumophila serogroup 1, the IgM and IgG enzyme-linked immunosorbent assay (ELISA) for Legionella pneumophila serogroup 1, and the IgM-plus-IgG ELISA for Legionella pneumophila serogroups 1 to 6 to diagnose Legionnaires' disease (LD) in a well-described sample of patients with and without LD. Also, we determined the agreements, sensitivities, and specificities of the different Vircell assays in comparison to a validated ELISA (Serion classic ELISA). Clinical sensitivity and specificity were 74.6% and 96.6%, respectively, for the IgM IFA, 65.1% and 88.0% for the IgG IFA, 92.3% and 100% for the IgM ELISA, 43.3% and 96.6% for the IgG ELISA, and 90.8% and 100% for the IgM-plus-IgG ELISA. Compared to Serion classic ELISA, agreement, sensitivity, and specificity were 80.0%, 83.1%, and 78.4%, respectively, for the IgM IFA, 75.2%, 66.0%, and 79.5% for the IgG IFA, 89.5%, 82.0%, and 97.6% for the IgM ELISA, 81.9%, 88.9%, and 78.0% for the IgG ELISA, and 93.5%, 90.0%, and 96.6% for the IgM-plus-IgG ELISA. The value of a positive diagnostic result obtained by the Vircell IgM IFA, the Vircell IgG IFA, and the Vircell IgG ELISA might not be acceptable for a diagnostic assay. Both the high specificities and sensitivities of the Vircell IgM ELISA and the IgM-plus-IgG ELISA and the high correlation with the Serion classic ELISA indicate that they are useful in the diagnosis of LD.

 

Deja Vu All Over Again: Rapid Enumeration of Legionella pneumophila in Water

Edelstein PH.

Appl Environ Microbiol. 2006 Jan;72(1):980.

NO ABSTRACT

 

Legionella confirmation using real-time PCR and SYTO9 is an alternative to current methodology

Giglio S, Monis PT, Saint CP.

Australian Water Quality Centre, PMB 3, Salisbury, South Australia 5108, Australia.

Appl Environ Microbiol. 2005 Dec;71(12):8944-8.

ABSTRACT: The currently accepted culture techniques for the detection of Legionella spp. in water samples (AS/NZS 3896:1998 and ISO 11731 standard methods) are slow and laborious, requiring from 7 to 14 days for a result. We describe a fully validated rapid confirmation technique that uses real-time PCR incorporating the intercalating dye SYTO9 for the direct identification of primary cultures, significantly decreasing turnaround time and allowing faster remedial action to be taken by the industry

 

Detection of Legionella pneumophila in water samples by species-specific real-time and nested PCR assays

Fiume L, Bucca Sabattini MA, Poda G.

ARPA, Regional Agency for Environmental Protection and Health Prevention, Emilia Romagna Region, Bologna, Italy.

Lett Appl Microbiol. 2005;41(6):470-5.

ABSTRACT: Legionella pneumophila is a contaminant of man-made water systems, including potable water, cooling towers, water systems of large buildings, etc. It is the most common causative agent of legionellosis, a respiratory infection, which may give rise to restricted outbreaks. To survey environmental water samples from hospitals and private habitations in Bologna, we developed a species-specific nested and a TaqMan real-time PCR for the detection of L. pneumophila. We compared the two assays and both to cultural isolation. Methods and Results: The targeted gene was macrophage infectivity potentiator (mip), conserved in L. pneumophila, and divergent in other legionellae. One assay was based on a nested PCR and the other on a TaqMan real-time PCR protocol. Their sensitivities were 14 % or 5% higher than that of cultural isolation respectively. The detection limits were 1-2 genome equivalents per 50-mul reaction. Specificity was assessed using DNA from nine target and 20 nontarget organisms. Conclusions: When applied to water samples, both assays detected L. pneumophila at 80% or higher frequency. Significance and Impact of the Study: The species-specific molecular diagnosis of L. pneumophila by means of nested PCR does not require a specific instrumentation, exhibits a high sensitivity, and is advantageous over the cultural isolation and real-time PCR detection. It allows to quickly monitor water samples for the risk assessment of environmental contaminations.

 

Comparison of three Legionella urinary antigen assays during an outbreak of legionellosis in Belgium

Dirven K, Ieven M, Peeters MF, van der Zee A, De Schrijver K, Goossens H.

Laboratory of Medical Microbiology, University of Antwerp UA, Wilrijk, Belgium.

J Med Microbiol. 2005 Dec;54(Pt 12):1213-6.

ABSTRACT: During an outbreak of legionellosis in Belgium, urine samples of 32 legionellosis patients were tested with three Legionella urinary antigen assays: the Biotest enzyme immunoassay (EIA) kit, the Binax EIA kit and the Binax NOW Immunochromatographic Test kit. The three tests were concomitantly compared. The test sensitivities on the first urine samples were 65.6 % for the Biotest EIA, 50.0 % for the Binax EIA and 56.3 % for the Binax NOW. Testing of a second urine sample increased the sensitivities to 71.9 %, 59.4 % and 65.6 %, respectively. The differences were not statistically significant. In outbreak settings, testing second samples from patients presenting with symptoms but initially testing negative and/or concentrating urine samples for testing might be valuable additions to the urinary antigen test to increase the sensitivities of the tests.

 

 

Micro- and macromethod assays for the ecological study of Legionella pneumophila

Guerrieri E, Bondi M, Ciancio C, Borella P, Messi P.

Department of Biomedical Science, University of Modena and Reggio Emilia, Modena, Italy.

FEMS Microbiol Lett. 2005 Nov 1;252(1):113-9

ABSTRACT: The survival of a strain of Legionella pneumophila (Lp-1) inoculated in artificial water microcosms was investigated with and without an amoebal host and varying environmental conditions, such as biofilm formation, amount of nutrients and incubation temperature. The results obtained using short (micromethod) and long (macromethod) term methods showed that L. pneumophila Lp-1 dies rapidly at 4 degrees C in the "macromethod" assay. When the same temperature (4 degrees C) was applied to the "micromethod" assay, L. pneumophila Lp-1 survived for three weeks, although it progressively decreased. At an incubation temperature of 30 degrees C, the aquatic environment was more favourable and better survival emerged in the "macromethod"; in contrast, this favourable temperature condition did not improve the survival of L. pneumophila Lp-1 cultured with the "micromethod". The role of the protozoa Acanthamoeba polyphaga proved to be indispensable for legionella survival only when environmental conditions become unfavourable.

 

A multiplex PCR for detection of Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, and Bordetella pertussis in clinical specimens

McDonough EA, Barrozo CP, Russell KL, Metzgar D.

Department of Defense Center for Deployment Health Research, Naval Health Research Center, P.O. Box 85122, San Diego, CA 92186-5122, USA.

Mol Cell Probes. 2005 Oct;19(5):314-22.

ABSTRACT: A multiplex PCR was developed that is capable of detecting four of the most important bacterial agents of atypical pneumonia, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, and Bordetella pertussis in uncultured patient specimens. These organisms cause similar symptomologies and are often not diagnosed because they are difficult to identify with classical methods such as culture and serology. Given this, the overall impact of these pathogens on public health may be grossly underestimated. The molecular test presented here provides a simple method for identification of four common, yet diagnostically challenging, pathogens.

 

Impact of rapid urine antigen tests to determine the etiology of community-acquired pneumonia in adults

Andreo F, Dominguez J, Ruiz J, Blanco S, Arellano E, Prat C, Morera J, Ausina V.

Department of Pneumology, Hospital Germans Trias i Pujol, Badalona, Barcelona, Spain.

Respir Med. 2005 Oct 11; [Epub ahead of print]

ABSTRACT: STUDY OBJECTIVES: To evaluate the rapid urine antigen tests, including a new rapid immunochromatographic test (ICT) for the detection of the Streptococcus pneumoniae antigen and an enzyme immunoassay (EIA) for the detection of the Legionella antigen, in order to improve the diagnosis of community-acquired pneumonia (CAP) in adults. DESIGN: Prospective study. SETTING: A tertiary hospital in Spain. PATIENTS: We consecutively recruited 107 adults with CAP evaluated at our hospital. INTERVENTIONS: The analyses included blood and sputum cultures, pleural fluid culture (if present) and serologic studies. The detection of the Legionella pneumophila urinary antigen was performed by EIA, and the detection of S. pneumoniae antigen in urine samples was performed by counterimmunoelectrophoresis (CIE) and a rapid ICT. RESULTS: Using conventional microbiologic tests we succeeded in performing the etiologic diagnosis of 39 out of the 107 cases (36.4%). The inclusion of rapid antigen detection techniques increased the percentage of diagnosis to 54.2%, which represents a total increase of 17.8% (P=0.034). CONCLUSIONS: The data obtained in this study indicate that rapid urine antigen tests are very useful to determine CAP etiology in adults and, consequently, to quickly identify a group of patients in whom narrow spectrum antibiotics may be used.

 

Comparison of Three Molecular Methods Used for Subtyping of Legionella pneumophila Strains Isolated during an Epidemic of Legionellosis in Rome

Scaturro M, Losardo M, De Ponte G, Ricci ML.

Department of Infectious Parasitic and Immunomediate Diseases, Istituto Superiore di Sanita, Viale Regina Elena, 299-00161 Rome, Italy.

J Clin Microbiol. 2005 Oct;43(10):5348-50.

ABSTRACT: In the summer of 2003 a community-acquired outbreak of Legionella pneumophila occurred in Rome, Italy. Three molecular typing methods, pulse-field gel electrophoresis, amplified fragment length polymorphism analysis, and sequence-based typing (SBT), were used to establish the clonal correlation among the isolates of the epidemic cluster. By comparison of the methods, SBT was the most rapid and the easiest to perform and provided unambiguous results.

 

Contribution of systematic serological testing in diagnosis of infective endocarditis

Raoult D, Casalta JP, Richet H, Khan M, Bernit E, Rovery C, Branger S, Gouriet F, Imbert G, Bothello E, Collart F, Habib G.

CNRS UMR 6020, IFR 48, Universite de la Mediterranee, 27 Boulevard Jean Moulin, 13385 Marseille cedex 05, France.

J Clin Microbiol. 2005 Oct;43(10):5238-42.

ABSTRACT: Despite progress with diagnostic criteria, the type and timing of laboratory tests used to diagnose infective endocarditis (IE) have not been standardized. This is especially true with serological testing. Patients with suspected IE were evaluated by a standard diagnostic protocol. This protocol mandated an evaluation of the patients according to the modified Duke criteria and used a battery of laboratory investigations, including three sets of blood cultures and systematic serological testing for Coxiella burnetii, Bartonella spp., Aspergillus spp., Legionella pneumophila, and rheumatoid factor. In addition, cardiac valvular materials obtained at surgery were subjected to a comprehensive diagnostic evaluation, including PCR aimed at documenting the presence of fastidious organisms. The study included 1,998 suspected cases of IE seen over a 9-year period from April 1994 to December 2004 in Marseilles, France. They were evaluated prospectively. A total of 427 (21.4%) patients were diagnosed as having definite endocarditis. Possible endocarditis was diagnosed in 261 (13%) cases. The etiologic diagnosis was established in 397 (93%) cases by blood cultures, serological tests, and examination of the materials obtained from cardiac valves, respectively, in 348 (81.5%), 34 (8%), and 15 (3.5%) definite cases of IE. Concomitant infection with streptococci and C. burnetii was seen in two cases. The results of serological and rheumatoid factor evaluation reclassified 38 (8.9%) possible cases of IE as definite cases. Systematic serological testing improved the performance of the modified Duke criteria and was instrumental in establishing the etiologic diagnosis in 8% (34/427) cases of IE.

 

Analysis of the genetic diversity of Legionella by sequencing the 23S-5S ribosomal intergenic spacer region: from phylogeny to direct identification of isolates at the species level from clinical specimens

Grattard F, Ginevra C, Riffard S, Ros A, Jarraud S, Etienne J, Pozzetto B.

Laboratoire de Bacteriologie-Virologie, GIMAP, Faculte de Medecine Jacques Lisfranc, 42023 Saint-Etienne, France.

Microbes Infect. 2005 Sep 26; [Epub ahead of print]

ABSTRACT: This study focuses on the interest of the hypervariable 23S-5S ribosomal intergenic spacer region (ISR) of the genus Legionella to analyze the phylogenic diversity of Legionella at the species and subspecies levels and to identify isolates directly from clinical specimens. The method, using a real-time PCR assay with a single primer pair followed by sequencing, was able to identify correctly 49 reference strains of Legionella belonging to 37 different species, including those implicated in human infections, and to clearly differentiate the three subspecies of L. pneumophila. Based on sequence similarities, the 23S-5S ISR sequences were much more variable than the rpoB and mip sequences (P<0.0001 by the Wilcoxon signed rank test). The 23S-5S ISR method was able to cluster Legionella species in accordance with phenotypic traits, such as autofluorescence or fatty acid membrane composition. Using maximum parsimony methods, the rpoB and 23S-5S ISR data sets were shown to be incongruent (P<0.001). In contrast, the 23S-5S ISR and the mip data sets were found to be congruent (P=0.313), suggesting the interest of combining these two regions to demonstrate phylogenetic links between Legionella species. This molecular assay was shown able to both detect Legionella DNA directly in respiratory specimens from patients exhibiting a Legionella infection and provide accurate identification of the bacterium at the species level in the tested specimens. These properties open a wide range of applications to the 23S-5S ISR sequencing method, from taxonomic analyses to clinical and epidemiological investigations.

 

Persistent culture-positive Legionella infection in an immunocompetent adult

Glaser S, Weitzel T, Schiller R, Suttorp N, Luck PC.

Clin Infect Dis. 2005 Sep 1;41(5):765-6.

Letter

 

Components of the Legionella pneumophila flagellar regulon contribute to multiple virulence traits, including lysosome avoidance and macrophage death

Molofsky AB, Shetron-Rama LM, Swanson MS.

Department of Microbiology and Immunology, University of Michigan Medical School, 6734 Medical Sciences Building II, Ann Arbor, MI 48109-0620, USA.

Infect Immun. 2005 Sep;73(9):5720-34.

ABSTRACT: Legionella pneumophila is a motile intracellular pathogen of macrophages and amoebae. When nutrients become scarce, the bacterium induces expression of transmission traits, some of which are dependent on the flagellar sigma factor FliA (sigma(28)). To test how particular components of the L. pneumophila flagellar regulon contribute to virulence, we compared a fliA mutant with strains whose flagellar construction is disrupted at various stages. We find that L. pneumophila requires FliA to avoid lysosomal degradation in murine bone marrow-derived macrophages (BMM), to regulate production of a melanin-like pigment, and to regulate binding to the dye crystal violet, whereas motility, flagellar secretion, and external flagella or flagellin are dispensable for these activities. Thus, in addition to flagellar genes, the FliA sigma factor regulates an effector(s) or regulator(s) that contributes to other transmissive traits, notably inhibition of phagosome maturation. Whether or not the microbes produced flagellin, all nonmotile L. pneumophila mutants bound BMM less efficiently than the wild type, resulting in poor infectivity and a loss of contact-dependent death of BMM. Therefore, bacterial motility increases contact with host cells during infection, but flagellin is not an adhesin. When BMM contact by each nonmotile strain was promoted by centrifugation, all the mutants bound BMM similarly, but only those microbes that synthesized flagellin induced BMM death. Thus, the flagellar regulon equips the aquatic pathogen L. pneumophila to coordinate motility with multiple traits vital to virulence.

 

Involvement of fractalkine/CX3CL1 expression by dendritic cells in the enhancement of host immunity against Legionella pneumophila

Kikuchi T, Andarini S, Xin H, Gomi K, Tokue Y, Saijo Y, Honjo T, Watanabe A, Nukiwa T.

Department of Respiratory Oncology and Molecular Medicine, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryomachi, Aobaku, Sendai 980-8575, Japan.

Infect Immun. 2005 Sep;73(9):5350-7.

ABSTRACT: Legionnaires' disease is clinically manifested as severe pneumonia caused by Legionella pneumophila. However, the dendritic cell (DC)-centered immunological framework of the host defense against L. pneumophila has not been fully delineated. For this study, we focused on a potent chemoattractant for lymphocytes, fractalkine/CX3CL1, and observed that the fractalkine expression of DCs was somewhat up-regulated when they encountered L. pneumophila. We therefore hypothesized that fractalkine expressed by Legionella-capturing DCs is involved in the induction of T-cell-mediated immune responses against Legionella, which would be enhanced by a genetic modulation of DCs to overexpress fractalkine. In vivo immunization-challenge experiments demonstrated that DCs modified with a recombinant adenovirus vector to overexpress fractalkine (AdFKN) and pulsed with heat-killed Legionella protected immunized mice from a lethal Legionella infection and that the generation of in vivo protective immunity depended on the host lymphocyte subsets, including CD4(+) T cells, CD8(+) T cells, and B cells. Consistent with this, immunization with AdFKN/Legionella/DC induced significantly higher levels of serum anti-Legionella antibodies of several isotypes than those induced by control immunizations. Further analysis of spleen cells from the immunized mice indicated that the AdFKN/Legionella/DC immunization elicited Th1-dominated immune responses to L. pneumophila. These observations suggest that fractalkine may play an important role in the DC-mediated host defense against intracellular pathogens such as L. pneumophila.

 

Incomplete activation of macrophage apoptosis during intracellular replication of Legionella pneumophila

Abu-Zant A, Santic M, Molmeret M, Jones S, Helbig J, Abu Kwaik Y.

Department of Microbiology, University of Louisville College of Medicine, Louisville, KY 40292, USA.

Infect Immun. 2005 Sep;73(9):5339-49.

ABSTRACT: The ability of the intracellular bacterium Legionella pneumophila to cause disease is totally dependent on its ability to modulate the biogenesis of its phagosome and to replicate within alveolar cells. Upon invasion, L. pneumophila activates caspase-3 in macrophages, monocytes, and alveolar epithelial cells in a Dot/Icm-dependent manner that is independent of the extrinsic or intrinsic pathway of apoptosis, suggesting a novel mechanism of caspase-3 activation by this intracellular pathogen. We have shown that the inhibition of caspase-3 prior to infection results in altered biogenesis of the L. pneumophila-containing phagosome and in an inhibition of intracellular replication. In this report, we show that the preactivation of caspase-3 prior to infection does not rescue the intracellular replication of L. pneumophila icmS, icmR, and icmQ mutant strains. Interestingly, preactivation of caspase-3 through the intrinsic and extrinsic pathways of apoptosis in both human and mouse macrophages inhibits intracellular replication of the parental stain of L. pneumophila. Using single-cell analysis, we show that intracellular L. pneumophila induces a robust activation of caspase-3 during exponential replication. Surprisingly, despite this robust activation of caspase-3 in the infected cell, the host cell does not undergo apoptosis until late stages of infection. In sharp contrast, the activation of caspase-3 by apoptosis-inducing agents occurs concomitantly with the apoptotic death of all cells that exhibit caspase-3 activation. It is only at a later stage of infection, and concomitant with the termination of intracellular replication, that the L. pneumophila-infected cells undergo apoptotic death. We conclude that although a robust activation of caspase-3 is exhibited throughout the exponential intracellular replication of L. pneumophila, apoptotic cell death is not executed until late stages of the infection, concomitant with the termination of intracellular replication.

 

Assessment of fluorescent amplified fragment length polymorphism analysis for epidemiological genotyping of Legionella pneumophila serogroup 1

Fry NK, Afshar B, Visca P, Jonas D, Duncan J, Nebuloso E, Underwood A, Harrison TG.

Health Protection Agency, Respiratory & Systemic Infection Laboratory, Centre for Infections, London, UK.

Clin Microbiol Infect. 2005 Sep;11(9):704-12.ABSTRACT: This study assessed the reproducibility and epidemiological concordance of double-enzyme fluorescent amplified fragment length polymorphism (fAFLP) analysis for genotyping of Legionella pneumophila serogroup (sg) 1. fAFLP fragment analysis was performed on three different sequencing platforms (one gel- and two capillary-based) in different laboratories with a well-characterised set of 50 strains of L. pneumophila sg 1. fAFLP data were analysed with the Pearson correlation similarity coefficient, using a range of parameters, and dendrogram outputs were converted to arbitrary types after selection of a specified percentage similarity threshold. The results obtained were compared with those obtained by the standard non-fluorescent AFLP method and were found to be broadly concordant. Using optimised settings for each fAFLP method to analyse the panel of 50 strains, epidemiological concordance (E) and reproducibility (R) values of 1.00 were obtained, and the number of types ranged from nine to 15, compared with E=1.00 and R=1.00, with 16 types, for the non-fluorescent AFLP protocol. The study demonstrated the potential of fAFLP for typing strains of L. pneumophila sg 1 on all three platforms; however, inter-platform comparison of fAFLP data was not achieved. fAFLP analysis may have a role in the fingerprinting of multiple isolates during Legionella outbreak investigations, but further work is required before type designations and identification libraries can be developed.

 

Sensitive genus-specific detection of Legionella by a 16S rRNA based sandwich hybridization assay

Leskela T, Tilsala-Timisjarvi A, Kusnetsov J, Neubauer P, Breitenstein A.

Bioprocess Engineering Laboratory, Department of Process and Environmental Engineering and Biocenter Oulu, University of Oulu, PO Box 4300, FI-90014 University of Oulu, Finland. peter.neubauer@oulu.fi

J Microbiol Methods. 2005 Aug;62(2):167-79.

ABSTRACT: The aim of this study was to develop a sensitive, cultivation-independent analytical method for Legionella in man-made water systems which can be performed within one day in crude sample extracts. The new assay for the genus Legionella is a paramagnetic bead based fluorescence sandwich hybridization assay (SHA) for the 16S rRNA based on two oligonucleotide probes which makes the method highly specific. An advantage over RT-PCR is the exclusive detection of viable cells and, due to the high number of 16S RNA molecules, the possibility to apply the method directly in crude cell extracts without prior purification of the nucleic acids. A high sensitivity was obtained by modifying the probe chemistry and hybridization conditions. The most sensitive assay uses a 3'-end biotin-labelled capture probe and a 3'-end DIG tailed detection probe and has a detection limit of 20 amol target molecules corresponding to 1.2x10(7) molecules of 16S rRNA and approximately 1800 Legionella cells. Using this assay type the number of Legionella cells was determined in Legionella contaminated water samples. The results show that the developed SHA can be applied for estimation of the approximate number of Legionella cells based on the number of 16S rRNA molecules in a water sample.

 

Value of serological testing for diagnosis of legionellosis in outbreak patients

Rojas A, Navarro MD, Fornes FE, Serra E, Simarro E, Rojas J, Ruiz J.

Vircell S. L., Plaza Dominguez Ortiz 1, 18320 Santa Fe, Granada, Spain. immunology@vircell.com

J Clin Microbiol. 2005 Aug;43(8):4022-5.

ABSTRACT: Serum antibody detection tests and a urine antigen detection technique were compared in samples from 116 patients epidemiologically characterized as belonging to a legionellosis outbreak. Sera were tested by enzyme-linked immunosorbent assays (ELISAs) for immunoglobulin M (IgM) and IgG plus IgM and by immunofluorescent assays (IFAs) for IgG, IgM, IgA, and polyimmunoglobulin using commercial kits (Vircell); concentrated urines were tested with the Binax NOW Legionella test. ELISA for IgM, ELISA for IgG plus IgM, antigenuria detection, and IFA for IgM were able to diagnose 72.3%, 60.5%, 53.3%, and 51.4%, respectively, of patients. Antigenuria was present in 53.8% of first samples, ELISA detected IgM in 29.7%, ELISA detected IgG plus IgM in 7.9%, and IFA detected IgM in 3.9%. Ten antigenuria-negative first samples tested serologically positive, 9 of them to IgM by ELISA. Despite the single source of the samples included in the study, detection of IgM using a sensitive technique such as ELISA seems to be a suitable complement to antigenuria detection for the diagnosis of legionellosis.

 

Evaluation of nested PCR assays for the detection of Legionella pneumophila in a wide range of aquatic samples

Devos L, Clymans K, Boon N, Verstraete W.

Laboratory of Microbial Ecology and Technology (LabMET), Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium. willy.verstraete@ugent.be

J Appl Microbiol. 2005;99(4):916-25.

ABSTRACT: AIMS: To compare the sensitivities of two nested PCR assays for the detection of Legionella pneumophila to each other and to the plate counting technique (ISO 11731) in a wide range of aquatic samples. METHODS AND RESULTS: The nested PCR assay with the primer set LEG 225-LEG 858 revealed 56% of the 46 analysed aquatic samples as being positive for Legionella spp., while the primer set JFP-JRP yielded 98% positive samples. The detection was confirmed by sequencing the PCR products. These results are considerably higher than the result obtained with the plate counting technique (41%), indicating the higher sensitivity of PCR-based diagnostic methods. As the PCR assay with the LEG 225-LEG 858 primer set resulted in a lower number of positive samples, it is considered not sensitive enough for aquatic samples. Similar results for the respective primer sets were obtained for the detection of the species L. pneumophila, responsible for 90% of all human Legionella infections, in the aquatic samples analysed. Both microbial community analysis by PCR-denaturing gradient gel electrophoresis and the analysis of biotic and abiotic water quality parameters revealed no relation between L. pneumophila-positive and -negative samples and the physico-chemical and bacteriological characteristics of the aquatic samples. CONCLUSIONS: The results show the additional value of the PCR assay with the JFP-JRP primer set compared with the plate counting technique, as well as its applicability in a wide range of aquatic samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the importance of comparing different primer sets for nested PCR assays for the detection of L. pneumophila in aquatic samples, as well as the lower sensitivity of the widely accepted plate counting technique (ISO 11731).

 

Community-acquired bacteria frequently detected by means of quantitative polymerase chain reaction in nosocomial early-onset ventilator-associated pneumonia

Apfalter P, Stoiser B, Barousch W, Nehr M, Kramer L, Burgmann H.

Department of Clinical Microbiology, Institute of Hygiene and Medical Microbiology, Vienna General Hospital, Medical University of Vienna, Austria.

Crit Care Med. 2005 Jul;33(7):1492-8.

ABSTRACT: OBJECTIVE: To test whether real-time polymerase chain reaction allows for rapid quantitative detection of Streptococcus pneumoniae, Chlamydia pneumoniae, Mycoplasma pneumoniae, and Legionella pneumophila in bronchoalveolar lavage fluids and to determine the prevalence of these pathogens in nosocomial ventilator-associated pneumonia. DESIGN: Prospective epidemiologic study applying a new molecular biology-based diagnostic tool during a 27-month period. SETTING: Three medical intensive care units of a tertiary care university hospital. PATIENTS: One hundred patients suffering from nosocomial ventilator-associated pneumonia, hospitalized for > or =14 days, intubated for reasons other than pneumonia, and mechanically ventilated for >48 hrs. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: S. pneumoniae, M. pneumoniae, and C. pneumoniae were detected in bronchoalveolar lavage fluids of 100 patients in 20 (20%), three (3%), and two (2%) cases, respectively. There of 17 (71%) revealed no growth or no significant growth by conventional culture. In one patient, S. pneumoniae and M. pneumoniae were detected simultaneously. Corresponding colony-forming units/mL were partly up to 10 CFU/mL with Gram stainings showing signs of acute inflammation in 80%. A significant temporary correlation between the number of days on ventilator, development of nosocomial pneumonia, and the frequency of detection of these pathogens was found for day 4. CONCLUSIONS: S. pneumoniae, M. pneumoniae, and C. pneumoniae should be considered as causative agents in critically ill patients who develop early-onset nosocomial ventilator-associated pneumonia. Thus, empirical antimicrobial regimens should cover S. pneumoniae, Chlamydia, and Mycoplasma alike. Quantitative polymerase chain reaction is a fast diagnostic tool allowing for detection of these bacteria within 3 hrs in pretreated patients.

 

Rapid method for enumeration of viable Legionella pneumophila and other Legionella spp. in water

Delgado-Viscogliosi P, Simonart T, Parent V, Marchand G, Dobbelaere M, Pierlot E, Pierzo V, Menard-Szczebara F, Gaudard-Ferveur E, Delabre K, Delattre JM.

Departement Eaux et Environnement, Institut Pasteur de Lille, F-59019 Lille Cedex, France. pilar.viscogliosi@pasteur-lille.fr

Appl Environ Microbiol. 2005 Jul;71(7):4086-96.

ABSTRACT: A sensitive and specific method has been developed to enumerate viable L. pneumophila and other Legionella spp. in water by epifluorescence microscopy in a short period of time (a few hours). This method allows the quantification of L. pneumophila or other Legionella spp. as well as the discrimination between viable and nonviable Legionella. It simultaneously combines the specific detection of Legionella cells using antibodies and a bacterial viability marker (ChemChrome V6), the enumeration being achieved by epifluorescence microscopy. The performance of this immunological double-staining (IDS) method was investigated in 38 natural filterable water samples from different aquatic sources, and the viable Legionella counts were compared with those obtained by the standard culture method. The recovery rate of the IDS method is similar to, or higher than, that of the conventional culture method. Under our experimental conditions, the limit of detection of the IDS method was <176 Legionella cells per liter. The examination of several samples in duplicates for the presence of L. pneumophila and other Legionella spp. indicated that the IDS method exhibits an excellent intralaboratory reproducibility, better than that of the standard culture method. This immunological approach allows rapid measurements in emergency situations, such as monitoring the efficacy of disinfection shock treatments. Although its field of application is as yet limited to filterable waters, the double-staining method may be an interesting alternative (not equivalent) to the conventional standard culture methods for enumerating viable Legionella when rapid detection is required.

 

Quantitative detection of Legionella pneumophila in water samples by immunomagnetic purification and real-time PCR amplification of the dotA gene

Yanez MA, Carrasco-Serrano C, Barbera VM, Catalan V.

Labaqua, Pol. Ind. Las Atalayas, c/Del Dracma, 16-18, 03114 Alicante, Spain. vicente.catalan@labaqua.es

Appl Environ Microbiol. 2005 Jul;71(7):3433-41.

ABSTRACT: A new real-time PCR assay was developed and validated in combination with an immunomagnetic separation system for the quantitative determination of Legionella pneumophila in water samples. Primers that amplify simultaneously an 80-bp fragment of the dotA gene from L. pneumophila and a recombinant fragment including a specific sequence of the gyrB gene from Aeromonas hydrophila, added as an internal positive control, were used. The specificity, limit of detection, limit of quantification, repetitivity, reproducibility, and accuracy of the method were calculated, and the values obtained confirmed the applicability of the method for the quantitative detection of L. pneumophila. Moreover, the efficiency of immunomagnetic separation in the recovery of L. pneumophila from different kinds of water was evaluated. The recovery rates decreased as the water contamination increased (ranging from 59.9% for distilled water to 36% for cooling tower water), and the reproducibility also decreased in parallel to water complexity. The feasibility of the method was evaluated by cell culture and real-time PCR analysis of 60 samples in parallel. All the samples found to be positive by cell culture were also positive by real-time PCR, while only eight samples were found to be positive only by PCR. Finally, the correlation of both methods showed that the number of cells calculated by PCR was 20-fold higher than the culture values. In conclusion, the real-time PCR method combined with immunomagnetic separation provides a sensitive, specific, and accurate method for the rapid quantification of L. pneumophila in water samples. However, the recovery efficiency of immunomagnetic separation should be considered in complex samples.

 

Development and evaluation of Chlamylege, a new commercial test allowing simultaneous detection and identification of Legionella, Chlamydophila pneumoniae, and Mycoplasma pneumoniae in clinical respiratory specimens by multiplex PCR

Ginevra C, Barranger C, Ros A, Mory O, Stephan JL, Freymuth F, Joannes M, Pozzetto B, Grattard F.

Laboratoire de Bacteriologie-Virologie, GIMAP, Faculte de Medecine Jacques Lisfranc, Saint-Etienne, France. florence.grattard@univ-st-etienne.fr

J Clin Microbiol. 2005 Jul;43(7):3247-54.

ABSTRACT: This study describes the development and evaluation of a new commercial test, Chlamylege (Argene Inc.), which allows the simultaneous detection in respiratory samples of Chlamydophila pneumoniae, Mycoplasma pneumoniae, and most Legionella species, as well as PCR inhibitors, by using a multiplex PCR and microplate hybridization. The sensitivities of Chlamylege were 1 x 10(-3) IFU, 5 x 10(-2) color-changing units, and 1 CFU per reaction tube for C. pneumoniae, M. pneumoniae, and Legionella pneumophila, respectively. A cohort of 154 clinical samples from patients with documented respiratory infections was analyzed by the kit, including 2 samples from patients with C. pneumoniae infection, 9 samples from patients with M. pneumoniae infection, 19 samples from patients with Legionella species infection, and 114 samples that tested negative for the three pathogens. All the positive specimens were correctly detected and identified by the Chlamylege kit, and no false-positive result was observed with the negative samples. The kit was then evaluated in a pediatric prospective study that included 220 endotracheal aspirates, and the results were compared with those obtained by three single in-house PCR assays. Four specimens were found to be positive for C. pneumoniae and six were found to be positive for M. pneumoniae by using both strategies. The Chlamylege kit detected two additional samples positive for M. pneumoniae and one additional sample positive for a Legionella species other than L. pneumophila; these three samples were shown to be true positive by other techniques. These overall results demonstrate that the Chlamylege assay is sensitive, specific, and convenient for the rapid detection and identification of atypical pathogens in clinical samples from patients with respiratory infections.

 

Sensitivity of urinary antigen test in relation to clinical severity in a large outbreak of Legionella pneumonia in Spain

Blazquez RM, Espinosa FJ, Martinez-Toldos CM, Alemany L, Garcia-Orenes MC, Segovia M.

Section of Infectious Diseases, Hospital J.M. Morales Meseguer, Avenida Marques de los Velez s/n., Murcia, 30007, Spain. rblazquezg@yahoo.es

Eur J Clin Microbiol Infect Dis. 2005 Jul;24(7):488-91.

ABSTRACT: Presented here are the results of Legionella urinary antigen testing correlated with patient characteristics and severity of pneumonia (Fine score) in 295 patients diagnosed with Legionella pneumonia in connection with a large outbreak in Murcia, Spain. Overall, the sensitivity of the urinary antigen test was 47.7% (141/295). A statistically significant association was found between the clinical severity of pneumonia and test sensitivity; 85.7% for patients with severe pneumonia versus 37.9% for patients with mild pneumonia (risk ratio, 2.3). Variables significantly associated with test positivity in multivariate analysis were as follows: pre-existing pulmonary disease, body temperature >40 degrees C, leukocytosis and multilobar infiltrates. Patients with mild pneumonia may go undiagnosed if the urinary antigen test is used alone.

 

Comparison of three kinds of commercially available kits for detecting antigen for Legionella in the urine from patients with legionellosis

Koide M, Shinzato T, Higa F, Tateyama M, Sakugawa H, Saito A.

Department of Internal Medicine, Division of Infectious Diseases, Graduate School and Faculty of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215.

Intern Med. 2005 Jun;44(6):673-4.

NO ABSTRACT

 

 

The pneumoplex assays, a multiplex PCR-enzyme hybridization assay that allows simultaneous detection of five organisms, Mycoplasma pneumoniae, Chlamydia (Chlamydophila) pneumoniae, Legionella pneumophila, Legionella micdadei, and Bordetella pertussis, and its real-time counterpart

Khanna M, Fan J, Pehler-Harrington K, Waters C, Douglass P, Stallock J, Kehl S, Henrickson KJ.

Prodess Inc., Waukesha, Wisconsin, Medical College of Wisconsin, Milwaukee, Wisconsin, USA. kellyj@mcw.edu

J Clin Microbiol. 2005 Feb;43(2):565-71.

ABSTRACT: Respiratory disease caused by atypical bacteria remains an important cause of morbidity and mortality for adults and children, despite the widespread use of effective antimicrobials agents. Culture remains the "gold standard" for the detection of these agents. However, culture is labor-intensive, takes several days to weeks for growth, and can be very insensitive for the detection of some of these organisms. Newer singleplex PCR diagnostic tests are sensitive and specific, but multiple assays would be needed to detect all of the common pathogens. Therefore, we developed the Pneumoplex assays, a multiplex PCR-enzyme hybridization assay (the standard assay) and a multiplex real-time assay to detect the most common atypical pathogens in a single test. Primer and probe sequences were designed from conserved regions of specific genes for each of these organisms. The limits of detection were as follows: for Bordetella pertussis, 2 CFU/ml; for Legionella pneumophila (serotypes 1 to 15) and Legionella micdadei, 9 and 80 CFU/ml, respectively; for Mycoplasma pneumoniae, 5 CFU/ml; and for Chlamydia (Chlamydophila) pneumoniae, 0.01 50% tissue culture infective doses. Recombinant DNA controls for each of these organisms were constructed, and the number of copies for each DNA control was calculated. The Pneumoplex could detect each DNA control down to 10 copies/ml. The analytical specificity demonstrated no cross-reactivity between 23 common respiratory pathogens. One hundred twenty-five clinical bronchoalveolar lavage fluid samples tested by the standard assay demonstrated that the Pneumoplex yielded a sensitivity and a specificity of 100 and 98.5%, respectively. This test has the potential to assist clinicians in establishing a specific etiologic diagnosis before initiating therapy, to decrease hospital costs, and to prevent inappropriate antimicrobial therapy.

 

Single-run, parallel detection of DNA from three pneumonia-producing bacteria by real-time polymerase chain reaction

Raggam RB, Leitner E, Berg J, Muhlbauer G, Marth E, Kessler HH.

Molecular Diagnostics Laboratory, Institute of Hygiene, Medical University Graz, Universitaetsplatz 4, A-8010 Graz, Austria. harald.kessler@meduni-graz.at.

J Mol Diagn. 2005 Feb;7(1):133-8.

ABSTRACT: A molecular assay for parallel detection of three bacteria, Chlamydia (C.) pneumoniae, Legionella (L.) spp., and Mycoplasma (M.) pneumoniae, in clinical specimens by a set of real-time polymerase chain reactions (PCRs) in a single run was evaluated. Bacterial DNAs were extracted by an automated DNA extraction protocol on the MagNA Pure LC System. Amplification and detection were done by real-time PCR on the LightCycler (LC) instrument. For amplification, specific oligonucleotides derived from the 16s rRNA genes of C. pneumoniae, L. spp., and M. pneumoniae were used. The three assays were complemented with an internal control (IC), a specially designed DNA fragment which contains the specific primer binding sites for the three PCRs. The IC was added to the samples, co-extracted, and co-amplified. Primers and hybridization probes were designed to suit one LC PCR program. LC PCRs were established, detection limits were determined, and clinical samples were tested. The detection limits were found between 5.0 and 0.5 IFU/CFU per PCR reaction for each of the bacteria. A total number of 100 clinical specimens were tested for validation of the molecular assay. Tested samples included 63 bronchoalveolar lavages (BALs) and 37 induced sputa specimens. The internal control was detected in all negative and low-positive samples; no inhibition was found throughout the whole study. Additionally, samples underwent testing by culture for L. spp., and M. pneumoniae; for C. pneumoniae, the serological microimmunofluorescence (MIF) test was used. In conclusion, the developed set of LC PCR assays permits parallel detection of C. pneumoniae, L. spp., and M. pneumoniae in a single LC run. This molecular assay may lead to accurate and early diagnosis of pneumonia produced by these three types of bacteria. The assay proved to be suitable for the high-throughput routine diagnostic laboratory.

 

 

Diagnostic test for etiologic agents of community-acquired pneumonia

Bartlett JG.

Division of Infectious Diseases, Department of Medicine, Johns Hopkins University School of Medicine, Johns Hopkins Hospital , 600 North Wolfe Street, Baltimore, MD 21205-2191, USA. jb@jhmi.edu

Infect Dis Clin North Am. 2004 Dec;18(4):809-27.

ABSTRACT: Diagnostic tests for the detection of the etiologic agent of pneumonia are neither recommended nor done for most outpatients with CAP (Table 4).Most of these patients have no clear diagnosis but seem to do well with empiric antibiotic treatment, which often costs less than the diagnostic tests. For hospitalized patients, a pre-treatment blood culture and an expectorated sputum gram stain and culture should be done. Testing for Legionella spp is appropriate in hospitalized patients, especially those who are seriously ill.New tests that merit use in selected patients are the urinary antigen assay for S pneumoniae and the PCR test for L pneumophila. Anticipated developments in the near future are PCR tests for detection of C pneumoniae and M pneumoniae.

 

Comparison of five methods for extraction of Legionella pneumophila from respiratory specimens

Wilson D, Yen-Lieberman B, Reischl U, Warshawsky I, Procop GW.

Section of Clinical Microbiology, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195, USA. procopg@ccf.org

J Clin Microbiol. 2004 Dec;42(12):5913-6.

ABSTRACT: The efficiencies of five commercially available nucleic acid extraction methods were evaluated for the recovery of a standardized inoculum of Legionella pneumophila in respiratory specimens (sputum and bronchoalveolar lavage [BAL] specimens). The concentrations of Legionella DNA recovered from sputa with the automated MagNA Pure (526,200 CFU/ml) and NucliSens (171,800 CFU/ml) extractors were greater than those recovered with the manual methods (i.e., Roche High Pure kit [133,900 CFU/ml], QIAamp DNA Mini kit [46,380 CFU/ml], and ViralXpress kit [13,635 CFU/ml]). The rank order was the same for extracts from BAL specimens, except that for this specimen type the QIAamp DNA Mini kit recovered more than the Roche High Pure kit.

 

Molecular genetic methods in the diagnosis of lower respiratory tract infections

Murdoch DR .

Department of Pathology, Christchurch School of Medicine and Health Sciences, and Microbiology Unit, Canterbury Health Laboratories, Christchurch , New Zealand . david.murdoch@cdhb.govt.nz

APMIS. 2004 Dec;112(11-12):713-27.

ABSTRACT: Molecular diagnostic techniques, such as PCR, have become useful tools for the rapid etiological diagnosis of lower respiratory tract infections. Nucleic acid amplification tests (NAATs) have been evaluated for detecting most respiratory pathogens, and commercial assays are available for some pathogens. However, standardized protocols are needed before these assays are introduced into routine diagnostic use. For pneumonia, NAATs offer advantages over conventional tests for the detection of Mycoplasma pneumoniae, Legionella spp. and Chlamydia pneumoniae. For pneumococcal pneumonia in adults, PCR adds little to existing diagnostic tests, and is unable to distinguish pneumococcal colonization from infection when testing respiratory samples. Although less sensitive than culture-based methods, several commercial molecular diagnostic assays have been developed for tuberculosis and are useful rapid tests for selected patients. PCR can now be considered the rapid diagnostic test of choice for pertussis and some respiratory virus infections. Further work is required to better characterize the role of molecular diagnostic tests for diagnosing lower respiratory tract infections, and to develop standard assays that can be readily adopted by routine diagnostic laboratories.

 

Diagnosis of Legionella infection in Legionnaires' disease

Den Boer JW, Yzerman EP.

Municipal Health Service Kennemerland, Haarlem , The Netherlands . e.yzerman@streeklabhaarlem.nl

Eur J Clin Microbiol Infect Dis. 2004 Dec;23(12):871-8.

ABSTRACT: Since 1977, the diagnostic tools for Legionnaires' disease have been culture and serological investigation. Both methods require considerable time to produce results and have low to reasonable sensitivity. Since the introduction of urinary antigen tests in the mid 1990s, underdiagnosis has diminished and mortality has declined, thanks to early diagnosis. To obtain the most accurate diagnosis, culture, serological investigation, and urinary antigen testing should all be performed. In the last decade, much effort has been directed toward the development of assays detecting Legionella nucleic acid. Thus far, only widely varying results with small patient series have been reported. Furthermore, these assays are labour intensive and complicated. As a result, these assays are not yet suitable for the average medical microbiological laboratory.

 

Evaluation of different primers for DNA fingerprinting of Legionella pneumophila serogroup 1 strains by polymerase chain reaction

Wiese J, Helbig JH, Luck C, Meyer HG, Jansen B, Dunkelberg H.

Medical Institute of General Hygiene and Environmental Health, Gottingen , Germany . jwiese@ifm-geomar.de

Int J Med Microbiol. 2004 Oct;294(6):401-6.

ABSTRACT: A DNA fingerprinting method for the characterization of Legionella pneumophila serogroup 1 strains was established. This method was based on the DNA extraction using Chelex 100 and subsequent PCR analysis using primers under conditions of low stringency. Sixteen single primers were tested for the typing of the 10 epidemiologically unrelated reference strains of L. pneumophila serogroup 1 as well as patient isolates and environmental strains isolated from the water system of a hospital where patients with legionellosis were treated. In addition, a combination of two primers (Lpm-1 and Lpm-2) originally established for the specific detection of Legionella strains was tested. The PCR results were compared with two further subtyping methods, i.e. monoclonal antibody analysis and pulsed-field gel electrophoresis. The type strains Philadelphia 1, Knoxville 1, Allentown 1, Benidorm 0303E, Bellingham 1, and France 5811 could be distinguished clearly in experiments using all of the primers. Depending on the primer used, Heysham 1 and Oxford 4032E showed different DNA profiles. The strains Olda and Camperdown 1 were nearly indistinguishable. In contrast, the analysis by PFGE and MAb subtyping revealed distinct types for all 10 reference strains. The discrimination of the patient isolates from two suspected cases of nosocomial legionellosis and environmental isolates was not possible with the 16 single primers used in the study. However, the PCR assay with the combination of Lpm-1 and Lpm-2 as well as the PFGE and MAb analysis were able to differentiate distinct types. The use of the sequence-specific primers under low-stringency annealing conditions allowed both simultaneous gene detection as well as epidemiological typing of Legionella strains.

 

 

Rapid identification of Legionella pneumophila serogroups by latex agglutination

Reyrolle M, Ratat C, Leportier M, Jarraud S, Freney J, Etienne J.

Laboratoire de Bacteriologie, Faculte de Medecine Laennec IFR 62, Centre National de Reference des Legionella (CNRL) INSERM E-230, 7 rue Guillaume Paradin, 69372, Lyon Cedex 08, France. monique.reyrolle@chu-lyon.fr

Eur J Clin Microbiol Infect Dis. 2004 Nov;23(11):864-6.

NO ABSTRACT

 

Contribution of urinary pneumococcal antigen detection combined with the research of legionella antigen for diagnosis of pneumonia in hospitalized patients [Article in French]

Honore S, Trillard M, Ould-Hocine Z, Lesprit P, Deforges L, Legrand P.

Laboratoire de microbiologie-virologie-hygiene, hopital Henri-Mondor, 51, avenue du Marechal-de-Lattre-de-Tassigny, 94010 Creteil cedex, France. patrick.legrand@hmn.ap-hop-paris.fr

Pathol Biol (Paris). 2004 Oct;52(8):429-433.

ABSTRACT: Aim of the study. - Bacteriological confirmation of pneumonia (PNM) in hospitalized patients is often erratic or belated. Because of importance of prognosis, early adaptation of treatment requires an empirical antimicrobial therapy (generally aminopenicillin and macrolide combination). The starting therapeutic strategy should profit by a fast and reliable test asserting a pneumococcal etiology. The Binax Now S. pneumoniae((R)) (BNP) test allows an urinary pneumococcal antigen (UPA) detection using an immunochromatographic membrane assay within 15 minutes. Materials and methods. - We first evaluated the BNP test for 28 patients with pneumococcal PNM proved by culture, and 118 negative control patients without PNM. The BNP test was then evaluated by testing urine from 158 hospitalized patients with a clinical picture of PNM (community-acquired: 90, nosocomial: 68) for whom a research of urinary Legionella antigen (Binax Now) was prescribed and was positive for only two cases. 57 patients (36.1%) were hospitalized in ICU. Results. - The sensitivity was 71.4% (85.7% for the 21 bacteriemic PNM), and the specificity was 98.3%; that is consistent with previous published data. Among the 158 patients with PNM, UPA was detected in 17 cases (10.8%): 15 within the community-acquired PNM (16.7%) and 2 (2.9%) within the nosocomial cases. The pneumococcal etiology was confirmed by bacteriological samples in 7/17 patients (6 by blood cultures). The 10 others showed clinical and radiological features in agreement with a pneumococcal PNM. Among the 141 patients with negative AUP, S. pneumoniae was isolated from 6 of them (2 in blood cultures). Conclusion. - The Binax Now S. pneumoniae((R)) test allowed a fast and reliable etiological diagnosis in 10.8% of hospitalized PNM (16.7% of the community-acquired cases) having a research of urinary Legionella antigen (conceiving with severity factors). So it could conduce to an improved adjustment of the starting antimicrobial therapy of hospitalized adult patients with PNM.

 

Centrifugal ultrafiltration method for rapid concentration of Legionella pneumophila urinary antigen

Blanco S, Prat C, Pallares MA, Matas L, Dominguez J.

crisprat@ns.hugtip.scs.es

J Clin Microbiol. 2004 Sep;42(9):4410.

NO ABSTRACT

 

A predominant and virulent Legionella pneumophila serogroup 1 strain detected in isolates from patients and water in Queensland, Australia, by an amplified fragment length polymorphism protocol and virulence gene-based PCR assays

Huang B, Heron BA, Gray BR, Eglezos S, Bates JR, Savill J.

Molecular Microbiology R&D Unit, Public Health Microbiology Laboratory, Queensland Health Scientific Services, 39 Kessels Rd., Coopers Plains, QLD 4108, Australia. Ben_Huang@health.qld.gov.au

J Clin Microbiol. 2004 Sep;42(9):4164-8.

ABSTRACT: In epidemiological investigations of community legionellosis outbreaks, knowledge of the prevalence, distribution, and clinical significance (virulence) of environmental Legionella isolates is crucial for interpretation of the molecular subtyping results. To obtain such information for Legionella pneumophila serogroup 1 isolates, we used the standardized amplified fragment length polymorphism (AFLP) protocol of the European Working Group on Legionella Infection to subtype L. pneumophila SG1 isolates obtained from patients and water sources in Queensland, Australia. An AFLP genotype, termed AF1, was predominant in isolates from both patients (40.5%) and water (49.0%). The second most common AFLP genotype found in water isolates was AF16 (36.5%), but this genotype was not identified in the patient isolates. When virulence gene-based PCR assays for lvh and rtxA genes were applied to the isolates from patients and water, nearly all (65 of 66) AF1 strains had both virulence genes, lvh and rtxA. In contrast, neither the lvh nor the rtxA gene was found in the AF16 strains, except for one isolate with the rtxA gene. It appears that this may explain the failure to find this genotype in the isolates from patients even though it may be common in the environment. In view of the evidence that the AF1 genotype is the most common genotype among strains found in patients and water sources in this region, any suggested epidemiological link derived from comparing the AF1 genotype from patient isolates with the AF1 genotype from environmental isolates must be interpreted and acted on with caution. The use of virulence gene-based PCR assays applied to environmental samples may be helpful in determining the infection potential of the isolates involved.

 

Mycoplasma pneumoniae and Legionella pneumophila in Community-acquired Lower Respiratory Tract Infections among Hospitalized Children: Diagnosis by Real Time PCR

Maltezou HC, La-Scola B, Astra H, Constantopoulou I, Vlahou V, Kafetzis DA, Constantopoulos AG, Raoult D.

Unite des Rickettsies, Faculte de Medecine Universite de la Mediterranee CNRS UMR 6020, IFR 48, 27 Boulevard Jean Moulin, 13385 Cedex 05 Marseille. didier.raoult@medecine.univ-mrs.fr

Scand J Infect Dis. 2004;36(9):639-42.

ABSTRACT: Mycoplasma pneumoniae and Legionella pneumophila are increasingly recognized as important agents of community-acquired lower respiratory tract infections (LRTI). Mycoplasma pneumoniae has been also recognized as a cause of nosocomial infections. The aim of this study was to investigate the role of real time polymerase chain reaction (PCR) for the rapid diagnosis of these infections among hospitalized children with community-acquired LRTI. During 2001, 65 children were prospectively studied. Microbiological investigation consisted of capillary PCR with a LightCycler for M. pneumoniae and L. pneumophila in induced sputum or throat swab specimens, IgM enzyme immunoassay for M. pneumoniae and immunofluorescence for L. pneumophila in paired sera. Serology testing showed acute M. pneumoniae infection in 18 (27.5%) patients and L. pneumophila in 1 (1.5%). M. pneumoniae was also detected in sputum specimen by capillary PCR in 9 (50%) serologically diagnosed cases, including 4 (22%) with non-diagnostic IgM levels in the acute phase. Capillary PCR and IgM enzyme immunoassay diagnosed together 15 (83%) M. pneumoniae cases in the acute phase. It is concluded that M. pneumoniae is an important cause of LRTI necessitating hospitalization among children in Greece. Capillary PCR in sputum may diagnose M. pneumoniae LRTI in the acute setting and direct therapy and isolation of patients.

 

Mixed lung infection by Legionella pneumophila and Legionella gormanii detected by fluorescent in situ hybridization

Buchbinder S, Leitritz L, Trebesius K, Banas B, Heesemann J.

Max von Pettenkofer Institute for Hygiene and Medical Microbiology, Munich, Germany. buchbins@medizin.uni-leipzig.de

Infection. 2004 Aug;32(4):242-5.

ABSTRACT: A mixed infection by Legionella pneumophila and a nonpneumophila Legionella species was detected in a lung biopsy specimen obtained from a patient with atypical pneumonia by fluorescent in situ hybridization (FISH). This result was confirmed by polymerase chain reaction (PCR). Sequencing of PCR products confirmed mixed infection by L. pneumophila and L. gormanii. Culture for Legionella spp. was negative and serology showed a rise only in IgG anti- Legionella pneumophila titer. To our knowledge, this is the first report of a mixed infection by L. pneumophila and a non-pneumophila Legionella species detected by FISH. Because FISH is a rapid and culture independent method that detects specific microorganisms in biopsy specimens it is recommended, in particular, for the detection of fastidious bacteria.

 

Legionella pneumophila serogroup 1 antigen can be detected in sputum samples by an immunochromatographic assay

Stralin K, Alriksson I, Tornqvist E.

Department of Infectious Diseases, Örebro University Hospital, SE-70185 Örebro, Sweden.

kristoffer.stralin@orebroll.se

J Clin Microbiol. 2004 Jul;42(7):3377.

NO ABSTRACT

 

Legionella micdadei pneumonia diagnosed by culture isolation and DNA-dNA hybridization from bronchial lavage fluid

Kitahara Y, Nishino R, Kohara T, Daga H, Taooka Y, Ohashi N, Arita K.

Department of Respiratory Disease, Hiroshima Red Cross Hospital and Atomic Bomb Survivors Hospital, Hiroshima.

Intern Med. 2004 Jun;43(6):503-7.

ABSTRACT: An 80-year-old man was admitted because of dyspnea on effort. We suspected an acute exacerbation of chronic heart failure and idiopathic interstitial pneumonia caused by right-sided pneumonia. A nodular shadow in right upper lobe spread and consolidated into the airspace, and it failed to improve despite administration of meropenem trihydrate, vancomycin hydrochloride and clindamycin. A definitive diagnosis of Legionella micdadei pneumonia was made on the basis of this organism being isolated in culture from bronchial lavage fluid and subsequent identification of Legionella micdadei using DNA-DNA hybridization. The airspace consolidation gradually improved following treatment with intravenous erythromycin and minocycline hydrochloride.

 

 

 

Multiplex PCR for the simultaneous detection of Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella pneumophila in community-acquired pneumonia

Miyashita N, Saito A, Kohno S, Yamaguchi K, Watanabe A, Oda H, Kazuyama Y, Matsushima T; CAP Study Group.

Division of Respiratory Diseases, Department of Internal Medicine, Kawasaki Medical School , 577 Matushima, Kurashiki City , Okayama 701-0192, Japan . nao@med.kawasaki-m.ac.jp

Respir Med. 2004 Jun;98(6):542-50.

ABSTRACT: A multiplex polymerase chain reaction (PCR) was developed for the simultaneous detection of Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella pneumophila. Oligonucleotide primers for the amplification of the DNA of these three organisms were optimized for use in combination in the same reaction. PCR products were detected by the Micro-Chip Electrophoresis Analysis System. Clinical samples were obtained from 208 community-acquired pneumonia (CAP) patients who were participants in a multicenter CAP surveillance study performed at seven medical schools and their affiliate hospitals in Japan . No significant differences in the sensitivity of each primer set were observed when tested in both the multiplex and monoplex PCR assays. Our multiplex PCR was able to reliably detect 10 copies/100 microl of each of the three pathogen DNAs. Of the panel of 208 samples, 14 of 15 C. pneumoniae, 10 of 10 M. pneumoniae, eight of eight L. pneumophila and 165 of 176 negative samples were correctly identified. Eleven cases who were the multiplex PCR positive and conventional method negative were observed. The PCR findings were of possible significance in at least four of these patients. Our multiplex PCR assay could potentially be used as a diagnostic and epidemiological tool. Further prospective studies are needed to establish its clinical usefulness.

 

 

 

Investigation of atypical bacteria and virus antigens in respiratory tract infections by use of an immunofluorescence method

Kaygusuz S, Koksal I, Aydin K, Caylan R.

Department of Infectious Disease and Clinical Microbiology, Kirikkale University Medical Faculty, 71200 Kirikkale, Turkey. skaygysuz@yahoo.com

Jpn J Infect Dis. 2004 Apr;57(2):33-6.

ABSTRACT: In this study an immunofluorescence (IF) method was used to investigate the antigens of viruses and atypical bacteria in respiratory tract infections (RTI) in pediatric and adult age groups. In this prospective study of 2 years (1998-2000), IF was used to investigate the antigens of 7 viral and 3 atypical bacteria to be used for the etiological diagnosis of RTI. Sputum (33.6%) and nasopharyngeal aspirate specimens were obtained from pediatric patients (Group I, 76 cases) and adults (Group II, 135 cases) with RTI symptoms. Antigen detection rates were found to be 44.7% in Group I and 67.4% in Group II (P < 0.05). The following rates for specific antigens in Groups I and II, respectively, were as follows: Chlamydia pneumoniae, 17.1 and 13.3% (P > 0.05); Mycoplasma pneumoniae, 0 and 9.6% (P < 0.05); influenza A virus, 3.9 and 16.3% (P < 0.05); adenovirus, 3.9 and 14.8% (P < 0.05); parainfluenza virus type 1, 5.3 and 7.4% (P > 0.05); respiratory syncytial virus, 9.2 and 1.5% (P < 0.05); parainfluenza virus type 2, 3.9 and 3%(P > 0.05); and influenza B virus, 1.3 and 1.5% (P > 0.05). Mixed agents were found at a rate of 2.6 and 3.7% (P > 0.05) in Groups I and II, respectively. Parainfluenza virus type 3 and Legionella pneumophila antigens were not found. Since detecting etiological agents provides an important guide for determining the most appropriate antibiotic therapy, this IF method could be applied in clinical practice for arriving at a correct diagnosis and administration of effective treatment.

 

 

 

Rapid detection of bacterial atypical pneumonia agents by multiplex PCR

Pinar A, Bozdemir N, Kocagoz T, Alacam R.

Hacettepe University, Faculty of Medicine, Department of Microbiology and Clinical Microbiology, Sihhiye, Ankara, Turkey. pinar-a@tr.net

Cent Eur J Public Health. 2004 Mar;12(1):3-5.

ABSTRACT: Approximately one third of community acquired pneumonia cases are caused by atypical pneumonia agents, Mycoplasma pneumoniae, Legionella pneumophila, and Chlamydophila pneumoniae (formerly Chlamydia pneumoniae). The laboratory diagnosis of these organisms is difficult and time-consuming by conventional microbiological techniques. Polymerase chain reaction (PCR) is one of the important tools which can circumvent this problem. A multiplex PCR assay was developed to achieve the diagnosis of these three organisms in a single tube. Primers used in PCR were selected in a way that they amplified different length DNA fragments from different agents but they all worked at the same amplification conditions. Therefore the organisms could be diagnosed according to the length of amplified products by agarose gel electrophoresis without using any hybridization probes. After development of the multiplex PCR method, totally 309 clinical samples which were sent to our laboratory for single-agent PCR, were also evaluated by this technique. The results showed that the multiplex PCR assay is a sensitive, useful, cheap, and rapid diagnostic tool for the management of pneumonia patients.

 

 

 

Laboratory diagnosis of legionnaires' disease due to Legionella pneumophila serogroup 1: comparison of phenotypic and genotypic methods

Lindsay DS, Abraham WH, Findlay W, Christie P, Johnston F, Edwards GF.

Scottish Legionella Reference Laboratory, Department of Microbiology, Stobhill Hospital , Balornock Road, Glasgow G21 3UW , Scotland , UK . diane.lindsay@northglasgow.scot.nhs.uk

J Med Microbiol. 2004 Mar;53(Pt 3):183-7.

ABSTRACT: Laboratory results of 67 cases of legionnaires' disease caused by Legionella pneumophila serogroup (Sg) 1 spanning a 6-year period were analysed by both phenotypic and genotypic methods. The methods compared were urinary antigen enzyme immunoassay (EIA), an immunofluorescent antibody (IFA) test, direct fluorescent antibody (DFA), culture and a 5S rRNA PCR with Southern blotting confirmation. Urine was available in 53 cases, of which 35 (66%) were positive, with an antigen peak observed at 5-10 days after onset of disease symptoms. The IFA test was positive in 62 (92.5%) cases, with 56 (90.3%) cases producing a greater than fourfold rise in titre and 6 (9.7%) giving presumptive high titres of > or =1:128. There were two antibody peaks, one at 10-15 days and another at >25 days after onset. In 23 cases where samples were available, DFA and culture were respectively positive in 5 (22%) and 10 (48%) cases. There was a peak in culture-positives 5-10 days after onset of disease. A Legionella-specific 5S rRNA PCR on patient serum was positive in 54 (80.5%) cases, with a peak in PCR positivity at 6-10 days after disease onset. In 22 of the 67 cases, the full panel of diagnostic methods was available for comparison. The relative sensitivity and specificity of the urinary antigen EIA and the serum PCR was 100%. The IFA gave relative sensitivity and specificity values of 93.8 and 95%. DFA and culture, although 100% specific, produced only low sensitivities, of 19 and 42.8%, respectively. This study has shown that urinary antigen and serum PCR are valuable tests in the acute phase of disease, with excellent sensitivity and specificity values. At present, the Legionella species causing infection requires to be verified by IFA serology and/or culture, but this could become unnecessary as new antigen and L. pneumophila Sg 1-specific PCR tests become available.

 

 

Rapid detection and enumeration of Legionella pneumophila in hot water systems by solid-phase cytometry

Aurell H, Catala P, Farge P, Wallet F, Le Brun M, Helbig JH, Jarraud S, Lebaron P.

Centre National de Reference des Legionella, INSERM E-0230, Laboratoire de Bacteriologie, Faculte de Medecine Laennec IFR 62, 69372 Lyon , France. lebaron@obs-banyuls.fr

Appl Environ Microbiol. 2004 Mar;70(3):1651-7.

ABSTRACT: A new method for the rapid and sensitive detection of Legionella pneumophila in hot water systems has been developed. The method is based on an IF assay combined with detection by solid-phase cytometry. This method allowed the enumeration of L. pneumophila serogroup 1 and L. pneumophila serogroups 2 to 6, 8 to 10, and 12 to 15 in tap water samples within 3 to 4 h. The sensitivity of the method was between 10 and 100 bacteria per liter and was principally limited by the filtration capacity of membranes. The specificity of the antibody was evaluated against 15 non-Legionella strains, and no cross-reactivity was observed. When the method was applied to natural waters, direct counts of L. pneumophila were compared with the number of CFU obtained by the standard culture method. Direct counts were always higher than culturable counts, and the ratio between the two methods ranged from 1.4 to 325. Solid-phase cytometry offers a fast and sensitive alternative to the culture method for L. pneumophila screening in hot water systems.

 

 

 

Evaluation of an automated complement-fixation test (Seramat) for diagnosis of acute respiratory infections caused by viruses and atypical bacteria

de Ory F, Guisasola ME, Coccola F, Tellez A, Echevarria JM.

Servicio de Microbiologia Diagnostica, Centro Nacional de Microbiologia, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain. fory@iscii.es

Clin Microbiol Infect. 2004 Mar;10(3):220-3.

ABSTRACT: The complement-fixation test (CFT) permits low-cost screening of serum samples for different agents within a single assay, and is a useful tool for the serological diagnosis of acute respiratory infections. This study evaluated the automated Seramat CFT system with 160 paired serum samples taken from 80 patients with acute respiratory infection in comparison with in-house CFTs against a panel of agents, including influenza A and B, adenovirus, respiratory syncitial virus, cytomegalovirus, Mycoplasma pneumoniae, Coxiella burnetti and Chlamydia spp., and in comparison with indirect immunofluorescence (IIF) against Legionella pneumophila. Overall, the Seramat system identified 75 (88.2%) of the 85 seroconversions recognised by in-house CFTs or IIF. In comparison to the in-house CFTs, the correlation was 89.2% (66/74). For L. pneumophila, the Seramat system detected nine (81.8%) of the 11 cases diagnosed by IIF. The Seramat system also identified eight additional seroconversions that were not detected by the in-house assays; none of these seroconversions was detected by the in-house assay on retesting. The Seramat system represents a significant technical improvement that may enable many clinical laboratories to use the CFT as a routine diagnostic tool.

 

 

 

False-positive result with BinaxNOW Legionella Antigen immunochromatographic (ICT) assay: response to Helbig et al. (2001)

Bailleul E, Magerman K, Mewis A, Peeters V, Rummens JL, Cartuyvels R.

Department of Clinical Laboratory, Virga Jesseziekenhuis, Stadsomvaart 11, B-3500 Hasselt, Belgium koen.magerman@virgajesse.be

J Med Microbiol. 2004 Feb;53(Pt 2):173.

NO ABSTRACT

 

 

 

Comparison of diagnostic sensitivities of three assays (Bartels enzyme immunoassay [EIA], Biotest EIA, and Binax NOW immunochromatographic test) for detection of Legionella pneumophila serogroup 1 antigen in urine
Guerrero C, Toldos CM, Yague G, Ramirez C, Rodriguez T, Segovia M.
Servicio de Microbiologia, Hospital "J M Morales Meseguer," 30008 Murcia, Spain. micromorales@hotmail.com.

J Clin Microbiol. 2004 Jan;42(1):467-8.

ABSTRACT: The Bartels enzyme immunoassay (EIA), Biotest EIA, and Binax NOW immunochromatographic test (ICT) urinary antigen kits for the detection of Legionella pneumophila serogroup 1 were compared using 178 frozen urine samples. When nonconcentrated urine samples were used, the sensitivity levels of both enzyme EIAs were significantly higher than the sensitivity level of the ICT (Bartels EIA, 71.3%; Biotest EIA, 65.1%; Binax NOW ICT, 37% [P < 0.001]). After concentration of the urine samples, no significant differences in sensitivity were found among the three tests.

 

 

 

Development of a membrane strip immunosensor utilizing ruthenium as an electro-chemiluminescent signal generator
Yoon CH, Cho JH, Oh HI, Kim MJ, Lee CW, Choi JW, Paek SH.
Graduate School of Biotechnology, Korea University, 302 Biotechnology Building, 1, 5-ka, Anam-dong, Sungbuk-ku, Seoul 136-701, South Korea. shpaek@korea.ac.kr

Biosens Bioelectron. 2003 Dec 15;19(4):289-96.

ABSTRACT: A photometric immunosensor that can be used for on-site diagnosis has been constructed. The sensor system was assembled by partially superimposing a nitrocellulose membrane strip (the lower) containing an immobilized antigen on the surface with a glass fiber membrane strip (the upper) including two electrodes on the opposite surfaces. To amplify the signal, we introduced a liposome, containing ruthenium molecules trapped in the core, chemically coupled to an antibody specific to the analyte (e.g. Legionella antigen). In the presence of the analyte, immune complexes were formed by antigen-antibody reactions upon addition of the immuno-liposome into a sample. This mixture was then absorbed by the capillary action from the bottom of the membrane strip. The liposome particles in the complexes were carried by a medium through the antigen pad without interaction, while free immuno-liposome was trapped by immune reactions on the pad surfaces. The aqueous medium influx into the glass pad dissolved a detergent pre-located within the compartment and the liposome rupture thereby released ruthenium molecules into the solution. The molecules were oxidized on the electrode surfaces and produced an electro-chemiluminescence (ECL) in proportion to the analyte concentration. The signal generation based on ECL resulted in an exponential dose-response pattern and the analyte detection limit of 2 ng/ml was approximately 10-fold more sensitive than that obtained from a conventional system.

 

 

Reference Values for the SERION classic ELISA for Detecting Legionella pneumophila Antibodies
Boshuizen HC, Den Boer JW, De Melker H, Schellekens JF, Peeters MF, Van Vliet JA, Conyn-Van Spaendonck MA.
IMA, National Institute for Public Health and the Environment, P.O. Box 1, 3720 BA, Bilthoven, The Netherlands. Hendriek.Boshuizen@rivm.nl

Eur J Clin Microbiol Infect Dis. 2003 Nov;22(11):706-8.

NO ABSTRACT

 

 

 

Characterization of a lipoprotein common to Legionella species as a urinary broad-spectrum antigen for diagnosis of Legionnaires' disease

Kim MJ, Sohn JW, Park DW, Park SC, Chun BC.

Division of Infectious Diseases, Department of Internal Medicine, College of Medicine, Korea University, Seoul 136-705, Republic of Korea. macropha@chollian.net

J Clin Microbiol. 2003 Jul;41(7):2974-9.

ABSTRACT: We have previously identified the Legionella 19-kDa peptidoglycan-associated lipoprotein (PAL) as a species-common immunodominant antigen. We describe here for the first time the excretion and detection of the PAL antigen in infected urine specimens, which is useful for the diagnosis of Legionnaires' disease. Rabbit anti-PAL immunoglobulin G (IgG) antibody was produced by immunization with the purified, recombinant PAL of Legionella pneumophila serogroup 1 and used in the PAL antigen capture enzyme-linked immunosorbent assay (ELISA) to detect urinary PAL antigen. A soluble-antigen capture ELISA using rabbit IgG antibodies against Legionella soluble antigens was prepared independently and used as a broad-spectrum standard test to detect soluble antigens of several Legionella species. Urine samples were obtained from guinea pigs experimentally infected with each of L. pneumophila serogroups 1, 3, and 6, and other Legionella species. The absorbance values of the PAL antigen ELISA highly correlated with those of the soluble-antigen ELISA in infected urine samples, with a correlation coefficient of 0.84 (P < 0.01). When applied to 17 infected urine samples and 67 negative controls from guinea pigs, the sensitivity and specificity of the PAL antigen capture ELISA were 88.2 and 95.5%, respectively. Compared to the commercial Biotest enzyme immunoassay, the PAL antigen ELISA was more efficient for detecting pneumophila non-serogroup 1 and nonpneumophila species. None of the 161 control human urine specimens obtained from healthy adults and patients with either non-Legionella pneumonia or urinary tract infections tested positive in the PAL antigen ELISA. The present study shows that the Legionella PAL is a very useful broad-spectrum antigen for urinary diagnostic testing. Moreover, since recombinant PAL antigen can be produced more efficiently than the soluble antigens, the development of a broad-spectrum diagnostic immunoassay based on the detection of the PAL antigen appears to be warranted.

 

 

Comparison of two commercial enzyme-linked immunosorbent assays with an immunofluorescence assay for detection of Legionella pneumophila types 1 to 6

Malan AK, Martins TB, Jaskowski TD, Hill HR, Litwin CM.
J Clin Microbiol. 2003 Jul;41(7):3060-3.

Associated Regional and University Pathologists Institute for Clinical and Experimental Pathology, Salt Lake City , Utah 84108 , USA . malanak@aruplab.com

ABSTRACT: Members of the genus Legionella are characterized as gram-negative, motile, freshwater-dwelling bacteria that were responsible for a pneumonia outbreak among American Legion members in 1976. Because clinicians routinely order serologic testing for Legionella pneumophila serogroups 1 to 6 as a screen for possible L. pneumophila infections, we evaluated the Wampole Laboratories L. pneumophila type 1 to 6 immunoglobulin G (IgG) and IgM combined enzyme-linked immunosorbent assay (ELISA) and the Zeus Scientific L. pneumophila type 1 to 6 IgG-IgM-IgA multispecific combined ELISA systems and compared them to an IgG-specific immunofluorescence assay (IFA) for L. pneumophila serogroups 1 to 6. The Centers for Disease Control and Prevention recommends that the positive titer cutoff for an IFA be 1:256. Regardless of where the positive IFA cutoff titer is placed, however, the sensitivity of both commercial assays was below what would be acceptable for a screening assay. With a 1:256 IFA titer as the positive cutoff, the agreement, sensitivity, and specificity of the Wampole ELISA were 74.6, 21.4, and 98.4%, respectively. The agreement, sensitivity, and specificity of the Zeus ELISA were 72.6, 10.5, and 100.0%, respectively. We recommend that any laboratories attempting to replace an IFA type 1 to 6 screen with an alternative ELISA carefully investigate the sensitivity of the replacement assay.

 

 

 

Detection of Legionella pneumophila in water samples by quantitative culture and an antigen detection assay

Luck PC, Liebscher B.

Institut fur Medizinische Mikrobiologie und Hygiene, TU Dresden, Fiedlerstr. 42, D-01307 Dresden, Germany.  Christian.Lueck@mailbox.tu-dresden.de

Int J Hyg Environ Health. 2003 Jun;206(3):201-4.

ABSTRACT: We evaluated the new Legionella pneumophila antigen detection assay Binax Equate for quantitative determination of legionellae in potable water samples. Seventy-seven water samples from different sources were investigated by Binax Equate and quantitative culture. Our culture assay is able to detect 20 to 40 cfu per 100 ml water. The rates of detection of legionellae were 1% (1 of 77) for the antigen detection assay and 25% (19 of 77) by culture. We were able to detect antigen in one water sample with 28 cfu per ml L. pneumophila serogroup 1. In in-vitro experiments the antigen assay had a sensitivity of about 333 cfu per ml when the bacteria were added directly to the test tubes and about 1000 cfu per ml when a simulated water sample was investigated. None of the water samples positive for L. pneumophila serogroup 2 to 14 was positive in the Binax Equate. The new antigen assay proved to be a valuable tool for investigating heavy L. pneumophila Serogroup 1 contamination in potable water systems but lacks sufficient sensitivity to be used in the surveillance of water supplies.

 

 

 

New biochip technology for label-free detection of pathogens and their toxins

Grow AE, Wood LL, Claycomb JL, Thompson PA.

Biopraxis, Inc., P.O. Box 910078, San Diego, CA 92191-0078, USA. agrow@biopraxis.com

J Microbiol Methods. 2003 May; 53(2): 221-33.

ABSTRACT: microSERS is a new biochip technology that uses surface-enhanced Raman scattering (SERS) microscopy for label-free transduction. The biochip itself comprises pixels of capture biomolecules immobilized on a SERS-active metal surface. Once the biochip has been exposed to the sample and the capture biomolecules have selectively bound their ligands, a Raman microscope is used to collect SERS fingerprints from the pixels on the chip. SERS, like other whole-organism fingerprinting techniques, is very specific. Our initial studies have shown that the Gram-positive Listeria and Gram-negative Legionella bacteria, Bacillus spores and Cryptosporidium oocysts can often be identified at the subspecies/strain level on the basis of SERS fingerprints collected from single organisms. Therefore, pathogens can be individually identified by microSERS, even when organisms that cross-react with the capture biomolecules are present in a sample. Moreover, the SERS fingerprint reflects the physiological state of a bacterial cell, e.g., when pathogenic Listeria and Legionella were cultured under conditions known to affect virulence, their SERS fingerprints changed significantly. Similarly, nonviable (e.g., heat- or UV-killed) microorganisms could be differentiated from their viable counterparts by SERS fingerprinting. Finally, microSERS is also capable of the sensitive and highly specific detection of toxins. Toxins that comprised as little as 0.02% by weight of the biomolecule-toxin complex produced strong, unique fingerprints when spectra collected from the complexes were subtracted from the spectra of the uncomplexed biomolecules. For example, aflatoxins B(1) and G(1) could be detected and individually identified when biochips bearing pixels of antibody or enzyme capture biomolecules were incubated in samples containing one or both aflatoxins, and the spectra were then collected for 20 s from an area of the biomolecule pixel approximately 1 microm in diameter. In the future, we plan to investigate the use of hyperspectral imaging Raman microscopy for collecting fingerprints from all the pixels on the biochip, individually yet simultaneously, to enable the rapid detection of diverse pathogens and their toxins in a sample, using a single biochip.

 

 

 

Immunosensor for detection of Legionella pneumophila using surface plasmon resonance

Oh BK, Kim YK, Lee W, Bae YM, Lee WH, Choi JW.

Department of Chemical Engineering, Sogang University, C.P.O. Box 1142, 100-611, Seoul, South Korea.

Biosens Bioelectron. 2003 May; 18(5-6): 605-11.

jwchoi@ccs.sogang.ac.kr

ABSTRACT: Immunosensor using surface plasmon resonance (SPR) onto self-assembled protein G layer was developed for the detection of Legionella pneumophila. A self-assembled protein G layer on gold (Au) surface was fabricated by adsorbing a mixture of 11-mercaptoundecanoic acid (MUA) and hexanethiol (molar ratio of 1:2) and the activation process for chemical binding between free amine (-NH(2)) of protein G and 11-(MUA) using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) in series. The formation of self-assembled protein G layer on Au substrate and the binding of antibody and antigen in series were confirmed by SPR spectroscopy. The surface morphology analyses of self-assembled protein G layer on Au substrate and monoclonal antibody against L. pneumophila immobilized on protein G were performed by atomic force microscope (AFM). The immunosensor for detection of L. pneumophila using SPR was developed and its detection limit could find up to 10(5) cells/ml.

 

 

 

Nucleic acid amplification tests for the diagnosis of pneumonia

Murdoch DR.

Microbiology Unit, Canterbury Health Laboratories, and Department of Pathology, Christchurch School of Medicine and Health Sciences, Christchurch, New Zealand.

david.murdoch@cdhb.govt.nz

Clin Infect Dis. 2003 May; 36(9): 1162-70.

ABSTRACT: Molecular diagnostic techniques, such as polymerase chain reaction (PCR), are promising tools for the rapid etiological diagnosis of pneumonia. PCR offers potential advantages over conventional tests for the detection of Mycoplasma pneumoniae, Legionella species, and Chlamydia pneumoniae. For pneumococcal pneumonia in adults, PCR adds little to existing diagnostic tests and is unable to distinguish pneumococcal colonization from infection when testing respiratory samples. Although PCR is probably more sensitive than are conventional microscopy-based methods for diagnosing Pneumocystis carinii pneumonia, the specificity is uncertain, because P. carinii can occasionally be detected in the absence of clinical symptoms. PCR is useful for the diagnosis of viral pneumonia in immunocompromised patients. Further work is required to better characterize the role of PCR versus the role of other tests for diagnosing pneumonia and to develop standard PCR assays that can be readily adopted by routine diagnostic laboratories.

 

 

Detection of Legionella pneumophila serogroup 1 antigen in bronchoalveolar lavage fluid by an immunochromatografic assay

Wever PC, Notermans DW, Tulevski II, Eeftinck-Schattenkerk JK, Jong MD.

Departments of Medical Microbiology and Internal Medicine, Academic Medical Center , University of Amsterdam , Amsterdam , The Netherlands .

J Clin Microbiol. 2003 May; 41(5): 2265.  

p.c.wever@amc.uva.nl

NO ABSTRACT

 

 

A detection method for Legionella spp in (cooling) water: fluorescent in situ hybridisation (FISH) on whole bacteria
Declerck P, Verelst L, Duvivier L, Van-Damme A, Ollevier F.
Laboratory of Aquatic Ecology, Zoological Institute, Catholic University Leuven, Charles De Beriotstraat 32, B-3000 Leuven, Belgium. aquabio@bio.kuleuven.ac.be

Water Sci Technol. 2003; 47(3): 143-6.

ABSTRACT: Although traditional culture methods are appropriate for detection of Legionella species, such culture takes several days. Rapid detection (< 24 h) of individual Legionella is possible using fluorescent in situ hybridisation (FISH) on whole bacteria. Water samples were filtered and the concentrated bacteria were immediately detected (without culture) with a fluorescence microscope following appropriate labelling. The detection level was very high and quantification was possible. For the detection of all Legionella spp. the probe LEG705 was used, complementary to a 16S rRNA sequence conserved in all Legionella spp. For specific detection of L. pneumophila the probe LEGPNE1 was used. This probe is designed against a variable domain of the 16S rRNA sequence from L. pneumophila. CY3 and FLUOS labels were tested and CY3 showed clearly detectable bacteria with minimum background staining. This FISH technique is very sensitive, fast, reliable and individual bacteria are easily detected.

 

 

 

PCR as a test for the presence or absence of Legionella in (cooling) water
Declerck P, Verelst L, Duvivier L, Van-Damme A, Ollevier F.
Laboratory of Aquatic Ecology, Zoological Institute, Catholic University Leuven, Charles De Beriotstraat 32, B-3000 Leuven, Belgium. aquabio@bio.kuleuven.ac.be

Water Sci Technol. 2003; 47(3): 103-7.
ABSTRACT: Although traditional culture methods are appropriate for detection of Legionella species, such culture takes several days. Rapid detection (< 24 h) of individual Legionella is possible using fluorescent in situ hybridisation (FISH) on whole bacteria. Water samples were filtered and the concentrated bacteria were immediately detected (without culture) with a fluorescence microscope following appropriate labelling. The detection level was very high and quantification was possible. For the detection of all Legionella spp. the probe LEG705 was used, complementary to a 16S rRNA sequence conserved in all Legionella spp. For specific detection of L. pneumophila the probe LEGPNE1 was used. This probe is designed against a variable domain of the 16S rRNA sequence from L. pneumophila. CY3 and FLUOS labels were tested and CY3 showed clearly detectable bacteria with minimum background staining. This FISH technique is very sensitive, fast, reliable and individual bacteria are easily detected.

Legionella pneumophila serogroup 1 antibody kinetics in patients with Legionnaires' disease: implications for serological diagnosis
Darelid J, Lofgren S, Malmvall BE, Olinder-Nielsen MA, Briheim G, Hallander H.
Department of Infectious Diseases, Ryhov Hospital, Jonkoping, Sweden.
Scand J Infect Dis. 2003; 35(1): 15-20.
ABSTRACT: To evaluate current serological criteria for Legionella pneumophila serogroup 1 (Lp1), the antibody response was prospectively studied in all patients hospitalized for Legionnaires' disease in a Swedish county during 11 y (n = 62). A 4-fold or greater rise in antibody titre to > or = 128 (the minimum convalescent antibody level for diagnosis, as recommended by the Centers for Disease Control and Prevention), using the indirect immunofluorescence antibody test, was found in 21/52 (40%) of tested patients. By referring to the titre levels in healthy residents from the local population (World Health Organization criteria), 45/52 (87%) cases were confirmed serologically. In 21 patients followed longitudinally for 10 y, the median antibody titre fell from 256 (range 32-1024) to 16 (range 2-128) in 3 y. No booster reactions were observed in any patient. After 10 y, the geometric mean titre of this clinical cohort had reached the same level as observed in the background population 5 y earlier. Titre levels in subjects exposed to Legionella from a municipal water system indicate that only 1 out of 10 of all infections are identified clinically. Indirect immunofluorescent antibody testing with local reference sera is a sensitive method for laboratory confirmation of Lp1 in an unselected pneumonia population.

 

Rapid detection of biofilms and adherent pathogens using scanning confocal laser microscopy and episcopic differential interference contrast microscopy
Keevil CW.
Environmental Healthcare Unit, School of Biological Sciences, University of Southampton, Southampton, UK.
Water Sci Technol. 2003; 47(5): 105-16.
ABSTRACT: Knowledge of biofilm structure and function has changed significantly in the last few years due to advances in light microscopy. One pertinent example is the use of scanning confocal laser microscopy (SCLM) to visualise corrosion pits caused by the biofilm mosaic footprint on corroding metal surfaces. Nevertheless, SCLM has some limitations as to its widespread use, including cost, inability to observe motile bacteria and eukaryotic grazers within biofilms, and difficulty to scan a curved surface. By contrast, episcopic differential interference contrast (EDIC) microscopy has provided a rapid, real time analysis of biofilms on opaque, curved, natural or man-made surfaces without the need for cover slips and oil. EDIC, coupled with epi-fluorescence (EDIC/EF), microscopy has been used successfully to visualise the 3-D biofilm structure, physiological niches, protozoal grazing and iron biomineralization, and the location of specific pathogens such as Legionella pneumophila, Campylobacter jejuni and Cryptosporidium parvum. These species were identified using gold nanoparticles or fluorophores coupled to monoclonal antibodies or 16S rRNA probes, respectively. Among its many potential uses, the EDIC technique will provide a rapid procedure to facilitate the calibration of the modern generation of biofilm-sensing electrodes.