Hospital washbasin water: risk of Legionella-contaminated aerosol inhalation.

The contamination of aerosols by washbasin water colonized by Legionella in a hospital was evaluated. Aerosol samples were collected by two impingement technologies. Legionella was never detected by culture in all the (aerosol) samples. However, 45% (18/40) of aerosol samples were positive for Legionella spp. by polymerase chain reaction, with measurable concentrations in 10% of samples (4/40). Moreover, immunoassay detected Legionella pneumophila serogroup 1 and L. anisa, and potentially viable bacteria were seen on viability testing. These data suggest that colonized hospital washbasins could represent risks of exposure to Legionella aerosol inhalation, especially by immunocompromised patients.

The characterization and certification of a quantitative reference material for Legionella detection and quantification by qPCR.

AIMS: The characterization and certification of a Legionella DNA quantitative reference material as a primary measurement standard for Legionella qPCR. METHODS AND RESULTS: Twelve laboratories participated in a collaborative certification campaign. A candidate reference DNA material was analysed through PCR-based limiting dilution assays (LDAs). The validated data were used to statistically assign both a reference value and an associated uncertainty to the reference material. CONCLUSIONS: This LDA method allowed for the direct quantification of the amount of Legionella DNA per tube in genomic units (GU) and the determination of the associated uncertainties. This method could be used for the certification of all types of microbiological standards for qPCR. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of this primary standard will improve the accuracy of Legionella qPCR measurements and the overall consistency of these measurements among different laboratories. The extensive use of this certified reference material (CRM) has been integrated in the French standard NF T90-471 (April 2010) and in the ISO Technical Specification 12 869 (Anon 2012 International Standardisation Organisation) for validating qPCR methods and ensuring the reliability of these methods.

[Molecular comparison of Legionella pneumophila serogroup 1 isolated in Tunisia]. / Comparaison moléculaire de Legionella pneumophila sérogroupe 1 isolées en Tunisie.

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.

Different growth rates in amoeba of genotypically related environmental and clinical Legionella pneumophila strains isolated from a thermal spa.

Two cases of legionellosis occurring 3 years apart were acquired in the same French thermal spa and were apparently due to the same strain of Legionella pneumophila serogroup 1, as shown by genomic macrorestriction analysis. Minor differences between the two isolates were found by random amplification PCR profiling which showed an additional band with one of the isolates. Analysis of 107 L. pneumophila strains isolated from the spa waters by genome macrorestriction failed to identify the infective strain, but a closely related L. pneumophila serogroup 3 strain differing from the clinical isolates by only one band was found. To determine if the clinical L. pneumophila serogroup 1 isolates was better adapted for intracellular multiplication than related serogroup 3 environmental isolates, the growth kinetics of six isolates were determined in co-culture with Acanthamoeba lenticulata. One clinical isolate failed to grow within amoeba, while the other clinical isolate yielded the highest increase in bacterial cell count per amoeba (1,200%) and the environmental isolates gave intermediate values. Genetic analysis of L. pneumophila isolates by DNA macrorestriction does not therefore appear to reflect their growth kinetics within amoeba, and is not sufficiently discriminatory to identify potentially virulent strains.

Legionella gresilensis sp. nov. and Legionella beliardensis sp. nov., isolated from water in France.

Novel Legionella-like isolates, strains Montbéliard A1T and Gréoux 11 D13T, isolated from two different French water sources, were studied taxonomically and phylogenetically. Morphological and biochemical characterization revealed that they were Gram-negative, aerobic, non-spore-forming bacilli with a cut-glass appearance that grew only on L-cysteine-supplemented buffered charcoal yeast extract agar. Phenotypic characterization using fatty acid and ubiquinone profiles and SDS-PAGE analysis confirmed that they were closely related, but distinct from, other species of the genus Legionella, since serotyping could not relate them to any existing serogroup. Genotypic profiles generated by randomly amplified polymorphic DNA and 16S-23S rDNA spacer region PCR analyses were unique for each of these isolates. DNA-DNA relatedness values of strains Montbéliard A1T and Gréoux 11 D13T to each other and to other Legionella type strains were less than 25%. Phylogenetic affiliation of these organisms obtained by 16S rDNA sequence comparisons confirmed that they were distinct from any other known Legionella species. All the above results confirm that these strains constitute two novel species for which the names Legionella gresilensis sp. nov. (type strain Gréoux 11 D13T = ATCC 700509T = CIP 106631T) and Legionella beliardensis sp. nov. (type strain Montbéliard A1T = ATCC 700512T = CIP 106632T) are proposed.

Single clonal origin of a high proportion of Legionella pneumophila serogroup 1 isolates from patients and the environment in the area of Paris, France, over a 10-year period.

Arbitrarily primed PCR with three primers and pulsed-field gel electrophoresis were used to characterize a set of 75 clinical Legionella pneumophila serogroup 1 isolates, with no apparent epidemiological link, obtained from 24 hospitals in Paris, France, from 1987 to 1997. Unexpectedly, 25 clinical isolates from 15 hospitals had an identical profile (termed type A) by both methods. The same profile was subsequently found in 16 of 64 randomly selected environmental L. pneumophila serogroup 1 isolates from 15 different sites in the Paris area. There was no evidence of geographic clustering or a peak incidence of type A isolation. Type A has not been found in France outside the Paris area, suggesting that a particular type of L. pneumophila serogroup 1 is specifically present in the Paris water distribution network.

Legionella taurinensis sp. nov., a new species antigenically similar to Legionella spiritensis.

A group of 42 Legionella-like organisms reacting specifically with Legionella spiritensis serogroup 1 antisera were collected throughout Europe by the Centre National de Référence (French National Reference Centre) for Legionella. This group of isolates differed somewhat from L. spiritensis in terms of biochemical reactions, ubiquinone content and protein profile. The latter two analyses revealed that one of these L. spiritensis-like isolates, Turin I no. 1T, was highly related, but not identical to any of the red autofluorescent species of Legionella. In fact, this strain was the first of these particular isolates recognized to emit a red autofluorescence when exposed to UV light. Profile analysis of randomly amplified polymorphic DNA established that the red autofluorescent L. spiritensis-like isolates constituted a homogeneous group distinct from Legionella rubrilucens and Legionella erythra. DNA-DNA hybridization studies involving the use of S1 nuclease confirmed that the indicated group of isolates are a new species of Legionella, for which the name Legionella taurinensis is proposed with strain Turin I no. 1T (deposited as ATCC 700508T) as the type strain.

Epidemic cluster of legionnaires disease in Paris, June 1998.

From 29 June to July 1998, four cases of legionnaires disease in British citizens were reported to the Reseau National de Sante Publique (RNSP) by the statutory notification system (declaration obligatoire (DO)) and by theEuropean Surveillance Scheme for

Species identification of Legionella via intergenic 16S-23S ribosomal spacer PCR analysis.

Species identification of Legionella in routine laboratory testing is hampered by the lack of highly discriminatory phenotypic tests. Amplification polymorphism of the intergenic 16S-23S spacer regions (ISR) has been previously developed for identification of species within the Legionellaceae [Hookey, J.V., Birtles, R.J. & Saunders, N.A. (1995). J Clin Microbiol 33, 2377-2381], but it did not provide enough resolution to distinguish all members of the bluish-white autofluorescent species and the red autofluorescent group of the Legionellaceae. By choosing new primers that target regions 4 (positions 1521-1541 of Escherichia coli 16S rRNA gene) and 6 (positions 114-132 of E.coli 23S rRNA gene) within the rDNA operon close to the 16S-23S intergenic spacer, 34 profiles were determined among the 79 type and reference strains representing 42 species that were tested. Analysis of the RFLP generated after Hinfl restriction digestion of the PCR products further improved the method, allowing complete discrimination among the species and subspecies of Legionella tested. Twenty-three well-identified strains from unrelated origins belonging to seven species gave amplification patterns identical to that of their type strain. The technique was also tested on 80 field isolates that could not be unequivocally assigned to groups by phenotypic methods. Seventy-two per cent (58/80) of these isolates had a profile identical to that of a type strain, while 27% (22/80) may correspond to new taxa since their ISR-PCR profiles did not match any of the known profiles.

Comparison of sample preparation methods for detection of Legionella pneumophila in culture-positive bronchoalveolar lavage fluids by PCR.

A prospective study was conducted on 25 Legionella pneumophila culture-positive and 98 culture-negative bronchoalveolar lavage fluid samples to compare two DNA preparation methods: a rapid modified Chelex-based protocol and a proteinase K method. PCR was found to be more sensitive with the Chelex-based method (P = 0.03). N difference was found concerning the inhibition rate.

Comparative analysis of infrequent-restriction-site PCR and pulsed-field gel electrophoresis for epidemiological typing of Legionella pneumophila serogroup 1 strains.

Two methods were compared for the analysis of 48 unrelated and epidemiologically related Legionella pneumophila serogroup 1 isolates. These are the infrequent-restriction-site PCR (IRS-PCR) assay with adapters designed for XbaI and PstI restriction sites and the pulsed-field gel electrophoresis (PFGE) analysis determined after DNA restriction with SfiI. Both methods demonstrated a high level of discrimination with a similar capacity for differentiating 23 of the 24 unrelated isolates. PFGE analysis and IRS-PCR assay were both able to identify epidemiologically related isolates of L. pneumophila from three outbreaks. Hence, IRS-PCR assay appears to be a reproducible (intergel reproducibility, 100%) and discriminative (discriminatory index, > or = 0.996) tool for typing of Legionella. Compared to PFGE, however, IRS-PCR presented an advantage through ease of performance and with attributes of rapidity and sensitivity of target DNA.

[Legionnaires' disease in travellers]. / Légionelloses du voyageur.

The outbreak of pneumonia involving delegates to the 1976 American Legion convention at a Philadelphia hotel was the first example of travel-associated legionnaires' disease. Travel is now well known as a common risk factor for legionnaires' disease. This travel-associated disease is a preoccupation among European countries because of morbidity among citizens of the European Union. The definition of the case of legionellosis is a patient who presents an acute lower respiratory tract infection with focal signs of pneumonia and/or radiological features, and microbiological evidence of Legionella infection. A case is considered to be travel associated if the patient has spent one or more nights away from home during the ten days before becoming ill. An European Surveillance Scheme for Travel-Associated Legionnaires' Disease was established in 1987 to identify clusters and outbreaks of cases of the disease. This group centralizes the case reports of twenty-nine collaborating centres in twenty-five countries. Outbreaks of legionnaires' disease were described in hotels, camps or cruise ships. In 1996, the number of travel-associated cases of legionnaires' disease represented 16% of the total number cases. The increase of the number of reported cases may reflect improved surveillance and increased ascertainment. In Europe in 1996, the diagnosis of legionellosis was confirmed by detection of Legionella pneumophila sero-group 1 antigen in urine (36%), seroconversion (fourfold rise in antibody titre, 33%) and culture of the organism (16%). Fifteen per cent of legionellosis was diagnosed by the identification of a single high antibody titre. In France a coordination between Public Health Institutions (Réseau National de Santé Public and DDASS), clinicians, laboratories and National Reference Center was established to improve prevention and control of legionnaires' disease outbreaks. Legislation obliges to report each case. When more two cases in the same area are notified an epidemiological investigation must be done. The knowing of the source of the contamination and its eradication allows to prevent new cases and outbreaks. Outbreaks of Legionnaires' disease are even now mediatic and this fact leads to maintain attention for the quality of diagnosis and epidemiology investigation due to touristic and economic consequences for the implicated countries.

The first clinical isolate of Legionella parisiensis, from a liver transplant patient with pneumonia.

A bluish white autofluorescent strain of Legionella was isolated from the tracheal aspirate of a female liver transplant patient who developed hospital-acquired pneumonia. This strain had biochemical characteristics compatible with those of L. cherrii, L. anisa, and L. parisiensis and could not be differentiated from L. bozemanii and L. parisiensis by the direct fluorescent-antibody assay. Phylogenetic analysis of partial 16S rRNA gene sequences of this strain (ATCC 700174) revealed the closest homology to the species L. parisiensis (99.5%). An L. parisiensis species-specific profile was also identified by a random amplified polymorphic DNA technique. This is the first report of L. parisiensis isolation from humans.

Outbreak of legionnaire s disease in two groups of tourists staying at camp sites in France.

On 11 June 1996, three suspected cases of legionnaires disease in a group of 42 Dutch tourists were reported to the local public health authority by Millau hospital in south west France. The group (group 1) had been touring with caravans and staying at d

Legionella jordanis pneumonia unresponsive to fluoroquinolones in a non-immunocompromised host.

Legionella jordanis has seldom been reported as a cause of infection in humans. We describe a case of pneumonia due to L. jordanis that occurred in a non-immunocompromised 74-year-old patient and failed to respond to a combination of ceftriaxone and ofloxacin. Cure was achieved only after an erythromycin-rifampin combination was started.

Distribution of mip-related sequences in 39 species (48 serogroups) of Legionellaceae.

The macrophage infectivity potentiator gene (mip) from Legionella pneumophila is a major virulence factor of the species. Thus, mip-detection by amplification has been proposed to assess the presence of L. pneumophila in clinical and environmental samples. The distribution of mip-related sequences within the Legionellaceae was studied by DNA amplification using mip-specific primers followed by Southern blot hybridization with an internal probe. Thirty-nine species (48 serogroups) of Legionellaceae were screened in this attempt. Using this approach, sequences related to mip were observed in 89% of the tested species including the most recently described L. fairfieldensis, L. lansingensis and L. shakespearei. In several cases, cloning and sequencing of the amplified products confirmed the high levels of similarity between the sequence found in non-pneumophila species with that of the L. pneumophila mip gene. This confirms previous reports that mip related genes are widespread among Legionellaceae and therefore specific detection of the species L. pneumophila cannot be based on mip-targeted amplification.

Molecular typing of nosocomial strains of Legionella pneumophila by arbitrarily primed PCR.

Arbitrarily primed PCR with two different primers was compared with ribotyping and monoclonal antibody analysis for typing Legionella strains. Applied to 11 epidemiologically unrelated strains, arbitrarily primed PCR resulted in an index of discrimination of 100% with both primers. It was found able to identify an epidemic clone of Legionella pneumophila serogroup 1 that was isolated from both patients and a hot water circuit of the same hospital.