Pan-European study on culture-proven Legionnaires' disease: distribution of Legionella pneumophila serogroups and monoclonal subgroups.

This pan-European study included unrelated strains of Legionella pneumophila obtained from 1335 cases of Legionnaires' disease. The isolates were serotyped into the serogroups 1 to 15 by monoclonal antibodies (MAb) and/or rabbit antisera. Additionally, MAb subgrouping was undertaken for isolates belonging to serogroups 1, 4, and 5. Monoclonal types of serogroup 1 were subdivided as having, or not having, the virulence-associated epitope recognized by the MAb 3/1 (Dresden Panel). This epitope is not present on strains belonging to any other serogroups. Taking all Legionella incidents together, MAb 3/1-positive cases were most frequent (66.8%); 11.7% of the isolates belonged to MAb 3/1-negative serogroup 1 subgroups and 21.5% to other serogroups, with serogroups 3 and 6 predominating. Among all serotypes discriminated in this study, monoclonal subtype Philadelphia was the most frequent. If categories of infection were considered, the proportion of MAb 3/1-negative strains differed significantly ( P<0.0005) between community-acquired cases (139/510; 27.3%) and travel-associated (42/295; 14.2%) or hospital-acquired infections (176/329; 53.5%). Moreover, taking distribution in different European areas into account, the proportion of MAb 3/1-negative strains was significantly higher in the Scandinavian region than in the Mediterranean countries or the UK for both community-acquired (48.7% vs. 18.6% or 12.0%; P<0.0005) and nosocomial cases (87.7% vs. 32.6% or 52.6%; P

Leptospira strains kept at the National Centre for Leptospirosis in Rome, Italy.

Since the National Centre for Leptospirosis (Department of Bacteriology and Medical Mycology, Istituto Superiore di Sanità, Rome) was established in 1956 by B. Babudieri, efforts have been devoted to identifying new Leptospira isolates and maintaining a collection of strains that today comprises 670 strains, 550 of which have been totally or partially classified, and 120 are still under study. This collection includes 23 serogroups and 156 serovars of pathogenic leptospires, and 32 serogroups and 54 serovars of saprophytic leptospires. The conventional serogroup and serovar identification, mainly based on antigenic relatedness, is tedious and time-consuming, requiring the maintenance of a comprehensive collection of serovar reference strains and the preparation of the corresponding rabbit antisera. Although considerable difficulties are encountered in the classification of leptospires at the serogroup and serovar level, this classification system is essential to obtain information on the epidemiology of leptospirosis in the different geographical areas. Serovar identification has become faster with the introduction of pulsed-field gel electrophoresis (PFGE) of large DNA fragments obtained after digestion of leptospiral DNAs with rare-cutting restriction enzymes. This technique has been successfully utilized to discriminate between closely related serovars of the Leptospira interrogans complex. We have recently used PFGE to characterize several Italian leptospiral isolates, confirming that PFGE analysis combined with microscopic agglutination test (MAT) with monoclonal and polyclonal antibodies can be used as an accurate and reliable method to compare and classify leptospires.

Multiple types of Legionella pneumophila serogroup 6 in a hospital heated-water system associated with sporadic infections.

Five sporadic cases of nosocomial Legionnaires' disease were documented from 1989 to 1997 in a hospital in northern Italy. Two of them, which occurred in a 75-year-old man suffering from ischemic cardiopathy and in an 8-year-old girl suffering from acute leukemia, had fatal outcomes. Legionella pneumophila serogroup 6 was isolated from both patients and from hot-water samples taken at different sites in the hospital. These facts led us to consider the possibility that a single clone of L. pneumophila serogroup 6 had persisted in the hospital environment for 8 years and had caused sporadic infections. Comparison of clinical and environmental strains by monoclonal subtyping, macrorestriction analysis (MRA), and arbitrarily primed PCR (AP-PCR) showed that the strains were clustered into three different epidemiological types, of which only two types caused infection. An excellent correspondence between the MRA and AP-PCR results was observed, with both techniques having high discriminatory powers. However, it was not possible to differentiate the isolates by means of ribotyping and analysis of rrn operon polymorphism. Environmental strains that antigenically and chromosomally matched the infecting organism were present at the time of infection in hot-water samples taken from the ward where the patients had stayed. Interpretation of the temporal sequence of events on the basis of the typing results for clinical and environmental isolates enabled the identification of the ward where the patients became infected and the modes of transmission of Legionella infection. The long-term persistence in the hot-water system of different clones of L. pneumophila serogroup 6 indicates that repeated heat-based control measures were ineffective in eradicating the organism.

Legionnaires' disease on a cruise ship linked to the water supply system: clinical and public health implications.

The occurrence of legionnaires' disease has been described previously in passengers of cruise ships, but determination of the source has been rare. A 67-year-old, male cigarette smoker with heart disease contracted legionnaires' disease during a cruise in September 1995 and died 9 days after disembarking. Legionella pneumophila serogroup 1 was isolated from the patient's sputum and the ship's water supply. Samples from the air-conditioning system were negative. L. pneumophila serogroup 1 isolates from the water supply matched the patient's isolate, by both monoclonal antibody subtyping and genomic fingerprinting. None of 116 crew members had significant antibody titers to L. pneumophila serogroup 1. One clinically suspected case of legionnaires' disease and one confirmed case were subsequently diagnosed among passengers cruising on the same ship in November 1995 and October 1996, respectively. This is the first documented evidence of the involvement of a water supply system in the transmission of legionella infection on ships. These cases were identified because of the presence of a unique international system of surveillance and collaboration between public health authorities.

Molecular epidemiology of an outbreak of Legionnaires' disease associated with a cooling tower in Genova-Sestri Ponente, Italy.

Fatty acid profile analysis, monoclonal antibody (MAb) subtyping, pulsed-field gel electrophoresis (PFGE), arbitrarily primed polymerase chain reaction (AP-PCR), and ribotyping were used to compare clinical and environmental Legionella pneumophila serogroup 1 isolates from an outbreak of Legionnaires' disease presumptively associated with cooling towers. According to the Oxford subtyping scheme, the MAb subtype of patients' isolates and of two strains originating from a cooling tower was Pontiac, whereas the other isolates were subtype Olda. The strains showed no intrinsic strain-to-strain difference in fatty acid profiles, and ribotyping and length polymorphism of the 16S-23S rDNA intervening regions failed to reveal any differences between the isolates. Conversely, PFGE and AP-PCR appeared to be more discriminatory, as the same genomic profile was found for the clinical and some environmental strains. Meteorologic and epidemiological data and the results of molecular analysis of the Legionella pneumophila serogroup 1 isolates support the hypothesis that the infection was transmitted from one of the cooling towers to the indoor environment of the same building, to homes in proximity that had open windows, and to the streets. In fact, the outbreak diminished and later ended after a part in the tower was replaced. This investigation demonstrates the utility of combined molecular methods (i.e., phenotypic and genomic typing) in comparing epidemiologically linked clinical and environmental isolates. Finally, the outbreak confirms the risk of Legionnaires' disease posed by cooling towers, mainly when atmospheric thermal and humidity inversions occur. This finding emphasizes the need to determine whether the source of infection is in the living or working environment or somewhere else.

Effect of monensin on the invasiveness and multiplication of Legionella pneumophila.

The polyether antibiotic monensin exhibited bacteriostatic activity against a clinical isolate of Legionella pneumophila in vitro. Experiments designed to test the effect of the compound on the invasiveness and multiplication of L. pneumophila in HeLa cells showed that, in the presence of the antibiotic, legionellas that penetrated the cells did not multiply. However, monensin did not alter the characteristics of phagosomes that contained ingested legionellas. In the presence of monensin, infected cells exhibited extensive vacuolation and a noticeable reduction in the number of intracellular micro-organisms was evident a few hours after infection.

Genomic heterogenicity amongst phenotypically similar Legionella micdadei strains.

Nine unrelated Legionella micdadei strains isolated from clinical and environmental samples have been characterized biochemically, serologically using polyclonal and monoclonal antibodies and by macrorestriction analyses using pulsed-field gel electrophoresis. All strains were positive in the Bromocresol purple spot test and grew as blue colonies on dye-containing media. They were positive for catalase, weakly positive for oxidase, and negative for sodium-hippurate hydrolysis, beta-lactamase and gelatinase. None of the strains showed autofluorescence under long-wave ultraviolet light. A panel of six monoclonal antibodies raised against the ATCC strain TATLOCK revealed no significant differences in the surface antigen composition of the L. micdadei strains. None of these monoclonal antibodies reacted with L. maceachernii and L. longbeachae serogroup 2, the only species that cross-react with polyclonal antisera. Each of the nine L. micdadei strains showed individual restriction patterns of the genomic DNA when using both SfiI and NotI restriction enzymes in the pulsed-field gel electrophoresis. Macrorestriction analysis is a valuable tool for studies on the molecular epidemiology of L. micdadei.

Effect of sub-inhibitory concentrations of antibiotics on the hemolytic activity of Legionella.

The effect of sub-inhibitory concentrations of various antibiotics on the hemolytic activity of different strains of Legionella has been tested. By means of a gradient plate technique it was possible to demonstrate that in a limited range of sub-inhibitory concentrations, antibiotics did not affect bacterial growth but inhibited the hemolytic activity of the strains examined.

Six cases of travel-associated Legionnaires' disease in Ischia involving four countries.

The detection of travel-associated legionellosis can be extremely difficult; hence, an extensive case investigation is recommended in pneumonia-striken travellers and tourists, who are particularly at risk of acquiring the disease. On the Island of Ischia (Isola d'Ischia, Naples, Italy) a total of six cases of Legionnaires' disease occurred from 1986 to 1990. All patients (one man and two women from Germany, one Austrian woman, one Swiss man, and one Italian woman) had taken thermal baths and stayed in local hotels; they all experienced severe pneumonia, and three of them died. These cases were associated with hotels, and the hot-water supply was presumed to have transmitted the infection. Remedial procedures were applied to the hot-water plumbing of the hotels according to the WHO recommendations and were proved to be effective. The occurrences described in this paper stress the importance of rapid and accurate reporting of diagnosed cases to the country where the infection was probably acquired, in order to ensure early detection of endemic foci and emerging clusters of legionellosis.

[10 years of legionellosis in Italy (June 1980-June 1990)]. / Dieci anni di legionellosi in Italia (giugno 1980-giugno 1990).

Legionella infection was recognized as a cause of human disease in 1976, following the occurrence of an epidemic at Bellevue Stratford Hotel, Philadelphia, USA. In the subsequent years, various cases, both sporadic and epidemical, have been reported worldwide. In Italy, the first reports date from 1980. The compulsory notification of the disease was established in February 1983, and a few months later a National Surveillance Programme started operating at the Istituto Superiore di Sanità, Rome. Up to June 30, 1990, 542 confirmed cases of legionellosis were reported. For 249 of them, data of clinical and epidemiological significance are available. The incidence of the disease is higher in males (76% of total cases) and 51.5% of the patients are aged more than 50 years. During the clinical course, the occurrence of hepatic and/or renal dysfunction, cough, dyspnoea, diarrhoea and confusion was documented in a significant proportion of patients. The fatality rate was 12.4%. The disease occurred both sporadically and epidemically. Namely, clusters and outbreaks were detected all over the country, accounting for a total of 97 cases. Hospital patients should be regarded as an important category at risk for infection. The occurrence is also frequent among travellers and tourists, because of the stressing factors that may increase individual susceptibility, as well as the more frequent contact with infective sources. In Italy, the major risk of infection appears connected with water plumbing systems of hotels, residential houses and hospitals, where oxygen bubble-humidifiers have been found to represent an important source of infection. On the contrary, the association of human infection with air conditioning systems has not been evidenced up-to-now.

Legionella pneumophila serogroup 1 population in Italy by monoclonal subtyping.

The Oxford panel of monoclonal antibodies was used to subtype 83 strains of Legionella pneumophila serogroup 1 of human and environmental origin. The International panel was also used to subtype 50 of them. All the 18 patients' isolates were of the Pontiac subgroup, and 40/43 of the environmental strains of the Pontiac subgroup were associated with human infection. The remaining environmental strains were subgroups Olda (15 strains), Camperdown (5 strains), and Bellingham (2 strains). The Philadelphia subgroup was the commonest among the environmental strains tested with the international MABs panel. This study confirms previous findings that L. pneumophila serogroup 1 isolates with the Pontiac (Oxford panel) or MAB-2 (international panel) reacting antigen marker seem to be more virulent than the other subgroups.

Endemicity of Legionella pneumophila serogroup 3 in a hospital water supply.

A microbiological and epidemiological investigation at the Infectious Diseases Hospital in Turin, Italy, demonstrated Legionella pneumophila serogroup 3 at 10(2) to greater than 4 X 10(3) cfu l-1 from 24 of 32 hot water samples collected from hand-basins in six separate buildings. A sample taken from the public water supply, and a hot water sample (80 degrees C) collected from hot water tanks, did not yield legionellas. Legionella pneumophila serogroup 3 was found in samples taken at the first point of mixed hot and cold water (50 degrees C) at 3 X 10(2) cfu l-1. 12 of 26 samples from the shower-heads yielded 10(3) to 2.5 X 10(5) cfu l-1 and one of 12 water samples from oxygen bubble humidifiers tested yielded 1.6 X 10(4) cfu l-1. No other legionellas species or serogroups of Legionella pneumophila were isolated during the study. No cases of nosocomial pneumonia were detected among 3653 patients' records, nor was there serological evidence of Legionella infection in the 180 patients tested.

Legionella pneumophila in a hospital in Torino, Italy. A retrospective one-year study.

Legionella pneumophila serogroup 1 was isolated from post mortem specimens from 13 out of 58 patients with pneumonia diagnosed at autopsy. The results of a study undertaken in the hospital environment showed that the water plumbing system was colonized with L. pneumophila serogroup 1 which could also be isolated from respiratory devices filled with tap water. Control measures instituted are described.

A family cluster of Legionella pneumophila infections.

Three members of one single family of 4, the father, a son and a daughter, showed seroconversion against Legionella pneumophila serogroup 1 (Lp SG1). The son had a severe pneumonia, whereas the father and the daughter did not develop any other illness than mild and transient fever. A fourth member, the mother, remained seronegative. Lp SG1 was detected by a direct immunofluorescence test in water samples from the shower at home, in tap water in the family's butcher shop, and in condensation water from the ventilator of refrigerator cells in the shop. Two different sources of infection appear to have occurred: showering at home and an aerosol of contaminated condensation water. Reports of more than one case of legionella infection within a family seem to be extremely rare and have not been found in the literature.

Plasmid profiles of Legionella spp. isolates, Italy.

Plasmid analysis and restriction-endonuclease digestion were used to study 54 clinical and environmental Legionella strains. Plasmids with approximate molecular masses of 40, 50, 70, and 90 megadaltons (Mdal) have been isolated from L. pneumophila serogroup 1 strains. One L. jordanis strain contained two plasmids of 25 and 70 Mdal. Restriction analysis of clinical and related hospital-environmental isolates resulted in identical patterns. Geographic diversity is shown for strains of different origin.

Nosocomial legionellosis associated with use of oxygen bubble humidifiers and underwater chest drains.

In 1 year 12 of 48 patients who developed fatal pneumonia following admission with non-respiratory disorders to the Hospital Molinette, Torino, yielded Legionella pneumophila serogroup 1 from lung at autopsy. Patients were hospitalized on seven different wards for different conditions; only two of the wards had air conditioning but legionellas were not isolated from these. All patients were in poor health or immunocompromised. Some patients had inhaled humidified oxygen from piped supplies and three had undergone surgery. Legionella pneumophila serogroup 1 was detected in the water of oxygen bubble humidifiers and an underwater chest drain. The contaminated devices had been filled with tap or distilled water and the hospital water supply was found to be contaminated with L. pneumophila serogroup 1. Our findings suggest that filling bubble humidifiers or underwater chest drains with tap water is a potential hazard and should be avoided.

Comparative in vitro susceptibility studies of FCE 22250 and rifampicin on Legionella and Chlamydia trachomatis strains.

The in vitro activities of rifampicin and the new rifamycin FCE 22250 were evaluated against the intracellular pathogens Legionella and Chlamydia trachomatis. The data reported in this study give evidence that FCE 22250 shows excellent in vitro results, even better than those obtained with rifampicin, leading to the possibility of clinical applications of this new drug on Legionnaire's disease and chlamydial infections.

More experience on the microagglutination test in the diagnosis of Legionella pneumophila infection.

The sensitivity of the indirect immunofluorescence (IFA) test in Legionella pneumophila infection is said to be maximal when a plyimmunoglobulin conjugate is used. However commercially available non-class-specific fluorescent antisera are not always sensitive enough to detect IgM antibodies as class-specific conjugates do. IFA test's drawback is its inability to detect early stages of infection. We routinely performed the microagglutination (MA) test in order to check the reliability of this test alone in screening diagnostic work for L. pneumophila group 1 infections. The 252 sera tested were from suspected or confirmed legionellosis cases. Five-hundred and thirty sera from healthy-people, 49 sera from patients with serologically confirmed chlamydia, coxiella and mycoplasma pneumonia, and ten sera from patients with Pseudomonas aeruginosa infection were used as controls. There was a good agreement between IFA and MA tests, the MA proving almost as specific as, and in some cases more sensitive than the IFA test. This was particularly evident in early stages of infection. For these reasons, together with its low cost and the ease to perform, it appears that the MA test can be a useful screening test for presumptive cases of legionellosis even on a single serum specimen.