Travel-associated Legionnaires' disease: would changing cluster definition lead to the prevention of a larger number of cases?

According to European Guidelines for Legionnaires' Disease prevention and control, travel-associated Legionnaires' disease (TALD) cases are managed differently if classified as sporadic or as part of a cluster and more stringent control measures are deployed after clusters are identified. In this study, we propose to modify the current cluster definition: 'two or more cases of Legionnaires' disease (LD) who stayed at, or visited, the same commercial accommodation site 2-10 days before onset of illness and whose onset is within the same 2-year period' with a new cluster definition, i.e. accommodation sites associated with multiple cases regardless of the time elapsed between them. TALD cases occurred in Italy and in the Balearic Islands between 2005 and 2015 were analysed applying the current European Legionnaires' Disease Surveillance Network (ELDSNet) cluster definition. In a sample of selected accommodation sites with multiple cases, a microbiological study was also conducted. Using the new definition, 63 additional sites (16.4% increase) and 225 additional linked cases (19.5% increase) were identified. Legionella pneumophila sg1 was isolated from 90.7% of the selected accommodation sites. The use of the here proposed TALD cluster definition would warrant a full investigation for each new identified case. This approach should therefore increase the number of sites that will require a risk assessment and, in the presence of an increased risk, the adoption of LD control measures to hopefully prevent additional cases.

Water safety in healthcare facilities. The Vieste Charter.

The Study Group on Hospital Hygiene of the Italian Society of Hygiene, Preventive Medicine and Public Health (GISIO-SItI) and the Local Health Authority of Foggia, Apulia, Italy, after the National Convention "Safe water in healthcare facilities" held in Vieste-Pugnochiuso on 27-28 May 2016, present the "Vieste Charter", drawn up in collaboration with experts from the National Institute of Health and the Ministry of Health. This paper considers the risk factors that may affect the water safety in healthcare facilities and reports the current regulatory frameworks governing the management of installations and the quality of the water. The Authors promote a careful analysis of the risks that characterize the health facilities, for the control of which specific actions are recommended in various areas, including water safety plans; approval of treatments; healthcare facilities responsibility, installation and maintenance of facilities; multidisciplinary approach; education and research; regional and national coordination; communication.

An unusually long-lasting outbreak of community-acquired Legionnaires' disease, 2005-2008, Italy.

An unusually long-lasting community-acquired outbreak of Legionnaires' disease (LD) occurred in the inhabitants of a town in northern Italy from 2005 to 2008. Overall, 43 cases were diagnosed including five deaths. Hundreds of water samples were collected for Legionella isolation but only two clinical samples were obtained. Clinical strains were ST23 as were environmental isolates detected in most Legionella-positive patients' homes and those from a public fountain. Although no Legionella was found in the municipal water mains, a continuous chlorination was applied in 2008. This action resulted in a halving of cases, although incidence remained tenfold higher than the Italian average incidence until the end of 2013, when it dropped to the expected rate. Retrospective analyses of prevalent wind direction suggested that a hidden cooling tower could have been the main cause of this uncommon outbreak, highlighting the importance of implementation of cooling tower registers in supporting LD investigations.

Legionnaires' disease in Italy: results of the epidemiological surveillance from 2000 to 2011.

According to the Italian Surveillance System for Legionnaires' disease (LD), physicians must fill in a form for every case and send it through the Local Health Units to the National Institute of Health (Istituto Superiore di Sanità, ISS). Forms reported in the period from 2000 to 2011 were analysed and discussed. A total of 9,803 cases of LD were reported to ISS during the study period. The median age of cases was 63 years, with a ratio male/female of 2.6 and a case fatality rate of 11.8%. The number of cases has been steadily increasing from 192 cases in 2000 to 1,235 in 2010 and 1,008 cases in 2011. The reported cases showed a geographical gradient, with the highest number notified in the north and the lowest in the south. The majority of cases (73.0%) were community-acquired, followed by travel-associated (13.5%) and healthcare-associated cases (9.3%), cases acquired in long-term care facilities (2.1%), and other types of exposure (2.1%). Even though the increasing trend of LD in Italy indicates an improvement in the ability to detect and report cases, the geographical gradient highlights the existence of low reporting areas where the epidemiological surveillance of LD should be further strengthened.

An international trial of quantitative PCR for monitoring Legionella in artificial water systems.

AIMS: To perform an international trial to derive alert and action levels for the use of quantitative PCR (qPCR) in the monitoring of Legionella to determine the effectiveness of control measures against legionellae. METHODS AND RESULTS: Laboratories (7) participated from six countries. Legionellae were determined by culture and qPCR methods with comparable detection limits. Systems were monitored over ≥10 weeks. For cooling towers (232 samples), there was a significant difference between the log mean difference between qPCR (GU l(-1) ) and culture (CFU l(-1) ) for Legionella pneumophila (0·71) and for Legionella spp. (2·03). In hot and cold water (506 samples), the differences were less, 0·62 for Leg. pneumophila and 1·05 for Legionella spp. Results for individual systems depended on the nature of the system and its treatment. In cooling towers, Legionella spp. GU l(-1) always exceeded CFU l(-1) , and usually Legionella spp. were detected by qPCR when absent by culture. The pattern of results by qPCR for Leg. pneumophila followed the culture trend. In hot and cold water, culture and qPCR gave similar results, particularly for Leg. pneumophila. There were some marked exceptions with temperatures ≥50°C, or in the presence of supplementary biocides. Action and alert levels for qPCR were derived that gave results comparable to the application of the European Guidelines based on culture. Algorithms are proposed for the use of qPCR for routine monitoring. CONCLUSIONS: Action and alert levels for qPCR can be adjusted to ensure public health is protected with the benefit that remedial actions can be validated earlier with only a small increase in the frequency of action being required. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms it is possible to derive guidelines on the use of qPCR for monitoring the control of legionellae with consequent improvement to response and public health protection.

m-bonacci metamaterial multilayers: location of the zero-average index bandgap edges.

We examine quasiperiodic multilayers arranged in m-bonacci sequences, which combine ordinary positive-index materials and dispersive metamaterials with negative index in a certain frequency range. When the volume-averaged refractive index of the nonperiodic multilayer equals zero, the structure does not propagate light radiation and exhibits a forbidden band. We identify some analytical expressions to determine the upper and lower limits of the above zero-average refractive-index bandgap. We recognize that these limits are not explicitly dependent on the geometrical parameters of the stack of layers.

Persistence of the same strain of Legionella pneumophila in the water system of an Italian hospital for 15 years.

In 2004, an outbreak of legionnaires disease occurred in a hospital in northern Italy with a water system that had been disinfected multiple times since 1990 and equipped with a continuous disinfecting system. Molecular typing linked the outbreak to contamination of the hospital water system and demonstrated the persistence of a predominant strain of Legionella pneumophila for 15 years.

Comparison of three molecular methods used for subtyping of Legionella pneumophila strains isolated during an epidemic of Legionellosis in Rome.

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.

Legionnaires' disease outbreak in Rome, Italy.

Between August and October 2003, 15 cases of Legionnaires' disease were detected in the 9th district of Rome. To identify possible sources of Legionella exposure, a matched case-control study was conducted and environmental samples were collected. Hospital discharge records were also retrospectively analysed for the period July-November 2003, and results were compared with the same period during the previous 3 years. The case-control study revealed a significantly increased risk of disease among those frequenting a specific department store in the district (OR 9.8, 95% CI 2.1-46.0), and Legionella pneumophila was isolated from the store's cooling tower. Genotypic and phenotypic analysis of human and environmental isolates demonstrated that the cluster was caused by a single strain of L. pneumophila serogroup 1, and that the cooling tower of the store was the source of infection. The increased number of hospital admissions for microbiologically undiagnosed pneumonia during the study period may indicate that some legionellosis cases were not identified.

Diphtheria antibody levels in the Italian Population.

Immunity to diphtheria was assessed in serum samples obtained from 3111 healthy Italian males and females aged 0-84 years. Diphtheria antitoxin was tested using a double-antigen, time-resolved fluorescence immunoassay (DA-DELFIA). According to internationally accepted criteria, antitoxin concentrations < 0.01 IU/ml indicate susceptibility to diphtheria, those > or = 0.01-0.09 IU/ml provide basic or inadequate protection, and concentrations > or =0.1 IU/ml are protective. By these criteria, 9.9% (95% CI 8.9 to 11.18) of the participants were susceptible to diphtheria, 30.2% (95% CI, 28.6 to 31.9) had basic protection, and 59.9% (95% CI, 58.1 to 61.6) were protected. The prevalence of unprotected individuals showed an age-related increase, up to the 45-49-year-old age group for females and the 50-54-year-old age group for males (34.9% and 31.3% of individuals, respectively). The prevalence of immunity did not significantly differ in relation to sex in any of the age groups. These results indicate that booster shots should be routinely provided to the adult population in order to maintain a protective level of diphtheria antibodies.

Upregulation of p75 tumor necrosis factor alpha receptor in Mycobacterium avium-infected mice: evidence for a functional role.

The bacterial growth and the production of tumor necrosis factor alpha (TNF-alpha) and TNF receptors (TNF-Rs) in the spleen and blood of BALB/c mice challenged with Mycobacterium avium complex (MAC) were monitored. Infection developed in two phases: the first, up to day 21, was associated with rapid MAC multiplication in the spleen and a drop in the mycobacteremia, and the second was associated with control of the infection in both compartments. In the spleen, TNF-alpha and TNF-RII mRNA levels peaked on day 21 and then slowly decreased; however, no increase in the level of TNF-RI mRNA was observed throughout these experiments. The level of circulating soluble TNF-RII (sTNF-RII) was transiently increased after day 21. In a model in which overproduction of bioactive TNF-alpha was triggered in response to a second infection with MAC, an increased production of sTNF-RII by cultured splenocytes was also observed. Administration of an antagonist anti-TNF-RII monoclonal antibody (MAb 6G1) to infected mice inhibited the bacterial growth in the spleen, suggesting that the TNF-RII and/or sTNF-RII was functionally involved in the mechanisms that control the infection. Overall, these observations suggest that upregulation of TNF-RII or sTNF-RII contributes to modulation of the TNF-alpha antibacterial activity in MAC infections.

rpoB mutations in multidrug-resistant strains of Mycobacterium tuberculosis isolated in Italy.

Mutations of rpoB associated with rifampin resistance were studied in 37 multidrug-resistant (MDR) clinical strains of Mycobacterium tuberculosis isolated in Italy. At least one mutated codon was found in each MDR strain. It was always a single-base substitution leading to an amino acid change. Nine different rpoB alleles, three of which had not been reported before, were found. The relative frequencies of specific mutations in this sample were different from those previously reported from different geographical areas, since 22 strains (59.5%) carried the mutated codon TTG in position 531 (Ser-->Leu) and 11 (29.7%) had GAC in position 526 (His-->Asp).

Activity of 16 antimicrobial agents against drug-resistant strains of Mycobacterium tuberculosis.

The in vitro activity of 16 antimicrobial agents against 46 drug-resistant strains of Mycobacterium tuberculosis recently isolated from Italian patients was determined. As for first-line antituberculosis drugs, while isoniazid was ineffective against all the strains tested, resistance to streptomycin, rifampicin, pyrazinamide, and ethambutol was 80.4%, 71.7%, 39.1%, and 8.7%, respectively. Among second-line antituberculous drugs, resistance to ciprofloxacin, ofloxacin, and sparfloxacin and to amikacin and kanamycin was around 20%. About 10% of the strains were resistant to capreomycin and cycloserine and 4.3% were resistant to ethionamide; no strain was found to be resistant to thiacetazone, para-aminosalicylic acid, and viomycin. Although all strains displayed a rather continuous distribution of minimal inhibitory concentrations (MICs), a bimodal distribution was observed for rifampicin, amikacin, and kanamicin, with very high MIC values for resistant strains; relatively low MICs were found for fluoroquinolone-resistant strains. Among the small number of strains resistant to second-line agents, low resistant levels were observed. Restriction fragment length polymorphism analysis showed few strain clusters with resistance to first-line antituberculous drugs and aminoglycosides, fluoroquinolones, or both. Altogether, these results showed that second-line agents were still active against the isoniazid-resistant and multiply first-line resistant strains tested, with none or low resistance levels; these observations can be of importance for the treatment of multidrug-resistant tuberculosis in Italy.

Activities of isoniazid alone and in combination with other drugs against Mycobacterium avium infection in beige mice.

Monotherapy with isoniazid or amikacin or clarithromycin or combinations of two of these drugs showed nil to modest therapeutic activity in beige mice infected with Mycobacterium avium. However, the combination of all three, isoniazid-amikacin-clarithromycin, markedly reduced CFUs in both spleens and lungs after 91 days of infection.

Protective activity of a murine monoclonal antibody against acute and chronic experimental infection with type IV group B streptococcus.

A murine IgM monoclonal antibody (MAb H11) was developed against the type polysaccharide capsular antigen of group B streptococcus (GBS), serotype IV, after intraperitoneal immunisation of BALB/c mice with heat-killed bacteria. MAb H11 reacted in immunodiffusion with the purified polysaccharide in both its sialylated and desialylated form, giving a line of identity, and opsonised type IV GBS strains in an in vitro assay. When administered at the time of intraperitoneal lethal challenge with homologous GBS, or 4 h earlier, MAb H11 protected 90% of the mice. Protection was still observed when MAb H11 was given 4 h after the challenge. This MAb was strongly effective in preventing septic arthritis induced by type IV GBS.

In vitro and ex vivo activities of antimicrobial agents used in combination with clarithromycin, with or without amikacin, against Mycobacterium avium.

MICs of clarithromycin, amikacin, isoniazid, rifabutin, ciprofloxacin, sparfloxacin, ethambutol, and clofazimine were determined for six isolates of Mycobacterium avium complex (MAC) from AIDS patients both by the radiometric method and by an ex vivo model of infection in human macrophages. The median MICs in macrophages were similar or slightly lower than values found in broth, except for amikacin, which had slightly higher MICs inside the cells. Combinations of clarithromycin with other antimicrobial agents showed that clarithromycin-clofazimine and clarithromycin-rifabutin were synergistic on five of six strains while clarithromycin-amikacin and clarithromycin-isoniazid were antagonistic on one and two strains, respectively. The addition of amikacin made the combinations of clarithromycin-clofazimine and clarithromycin-ethambutol synergistic against all the MAC strains. In the macrophage model, the combination of clarithromycin-clofazimine (mean survival, 21%) and clarithromycin-rifabutin (mean survival, 29%) showed a strong reduction in viable counts compared with single drugs, while clarithromycin-amikacin was less active than single drugs alone. In general, the addition of amikacin did not improve the activity of the combinations, except for clarithromycin-isoniazid-amikacin (mean survival, 19%), which was significantly more active than either clarithromycin-isoniazid or clarithromycin-amikacin. The use of the macrophage model can suggest new combinations of antimicrobial agents with anti-MAC activity which, on the basis of their in vitro effectiveness, would probably be disregarded for assay in animal models.

Electrotransformation of Streptococcus agalactiae with plasmid DNA.

A protocol for efficient electrotransformation of Streptococcus agalactiae (group B streptococcus) Lancefield's strain O90R (NTCT 9993) (an unencapsulated derivative of type Ia strain O90) was developed. The Escherichia coli-Streptococcus shuttle vector pDP28 (7.8 kb) carrying the ermB gene for resistance to erythromycin was used as donor DNA. Frozen 'electrocompetent' cells were prepared by repeated washes in 10% glycerol. A 50-microliters aliquot containing about 5 x 10(9) colony forming units of bacteria was subjected to the electric pulse. Optimal conditions for electrotransformation were determined using different media, harvesting cells at different points of the growth curve, and using different field strengths. The dose-response curve for transformation of S. agalactiae with pDP28 showed one-hit kinetics as donor DNA varied between 0.01 and 3 micrograms. The efficiency of electrotransformation for this range of amounts of donor DNA was 1.2 x 10(4) cfu micrograms-1. The transformation frequencies obtained with this electroporation protocol are high enough to allow both subcloning and shotgun cloning of streptococcal DNA in S. agalactiae.

Adherence of glucan-positive and glucan-negative strains of Streptococcus bovis to human epithelial cells.

Adherence to buccal epithelial cells (BEC) and the role played in the binding by lipoteichoic acid (LTA) and other superficial components have been studied in reference and clinical strains of Streptococcus bovis either glucan-positive biotype I or glucan-negative biotype II. To avoid the synthesis of glucan by biotype I strains, adherence was studied in bacteria grown in Todd-Hewitt broth, a sucrose deficient medium. Both biotypes were shown to bind to BEC and clinical isolates, irrespective of biotype attached to the same degree but in greater numbers than reference strains. Inhibition studies suggest that at least two mechanisms,--LTA and protein-mediated--are responsible for the adherence of both glucan-positive and negative strains of S. bovis. Moreover, in glucan-positive strains capsular polysaccharides may be also involved.

Lack of activity of transferrins towards Streptococcus spp.

Clinically relevant Streptococcus spp. were tested for their susceptibility towards human serum transferrin (TR) and lactoferrin (LF). Neither clinical isolates or type strains were inhibited by transferrins (5 mg/ml). All species tested were shown to be able to grow under iron-limiting conditions (< 0.1 microM) and this might account for the lack of TR or LF activity towards streptococci. Even if not sensitive to LF and TR, some species were shown to bind LF in the apo-form.

Adherence of S. bovis to adult buccal epithelial cells.

The ability of S. bovis 83/5364, glucan-positive (biotype I) and S. bovis R 81/536 glucan-negative (biotype II) to adhere to buccal epithelial cells (BEC) by lipoteichoic acid (LTA) was examined. LTA from both biotypes was prepared by cold phenol extraction from supernatants of penicillin supplemented cultures and partially purified by Sepharose CL-6B chromatography. Both glucan-positive and glucan-negative S. bovis strains adhered to BEC, but biotype I seemed to be more adhesive. For both biotypes the adhesion was not significantly inhibited by treatment of the bacteria with anti-LTA serum, while the preincubation of BEC with LTA, extracted from S. agalactiae, or cardiolipin strongly decreased the S. bovis binding.