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.

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.

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.

Cluster of travel-associated Legionnaires disease in Lazise, Italy, July to August 2011.

Since 18 August 2011, 17 cases of travel-associated Legionnaires' disease have been reported. They were tourists from five European countries who had stayed in five accommodation sites in Lazise, Italy. The dates of symptom onset ranged from 18 July to 25 August 2011. Control measures were implemented and no further cases associated with stays at the sites have been reported after disinfection. Timely notification of any further cases potentially associated with stay in Lazise is recommended.

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.

H1(0) and H3.3B mRNA levels in developing rat brain.

Two overlapping rat cDNAs, covering a continuous region of 1107 base pairs, have been isolated and sequenced. The clones contain identical open reading frames, encoding a 136 amino acid long polypeptide which exhibits 100% identity to other mammalian H3.3 histone variants. We show that the inserts derive, in particular, from the H3.3B gene. We used these inserts and an insert from an H1(0) encoding clone, previously described (6), as probes to study the accumulation of mRNAs encoding the corresponding histone replacement variants (namely, H1(0) and H3.3) during rat brain development. We found that the concentration of both H1(0) and H3.3B mRNAs decreases from the embryonal day 18 (E18) to the postnatal day 10 (P10), with inverse correlation to protein accumulation.

Posttranscriptional regulation of H1 zero and H3.3B histone genes in differentiating rat cortical neurons.

Accumulation of mRNAs encoding H1 zero and H3.3, two histone replacement variants, was studied in differentiating cortical neurons, cultured in a serum-free medium, with or without triiodothyronine (T3) supplementation. We found that the levels of both H1 (zero) and H3.3B mRNAs decrease in isolated neurons between the 2nd and 5th day of culture to the same extent as in vivo. At the same time, an active synthesis of the corresponding proteins was evidenced. The effects of transcription inhibition by actinomycin D and the results of nuclear run-on experiments suggest that H1 zero and H3.3 expression is regulated mainly at the posttranscriptional level. Concerning T3, only marginal effects were noticed, apart from up-regulation of both histone mRNAs at 2 days in culture. We propose one model for posttranscriptional regulation of the analyzed genes and discuss potential relationships to remodelling of chromatin.

PIPPin, a putative RNA-binding protein specifically expressed in the rat brain.

In looking for genes encoding RNA-binding factors, we prepared an expression library in lambda gt11, by cloning cDNAs corresponding to the polyadenylated fraction of RNA from rat brain at the embryonal day 18. The library was then screened by binding to a radioactive RNA, transcribed in vitro from a cDNA encoding the rat histone variant H3.3. Here we report some findings concerning a cDNA for a protein which contains two putative double stranded RNA-binding domains (dsBD). The corresponding message is specifically expressed in the brain. Moreover, Southern blot analysis showed that the gene is highly conserved from Drosophila melanogaster to man.

H1(0) RNA-binding proteins specifically expressed in the rat brain.

During brain maturation, histone H1(0) accumulates in both nerve and glial cells. The expression of this "linker" histone, the role of which still remains unclear, is a complex process, having both transcriptional and post-transcriptional regulatory components. In particular, the expression of H1(0) in rat cortical neurons is regulated mainly at the post-transcriptional level, and unknown cellular proteins are likely to affect H1(0) mRNA stability and/or translation. In looking for such factors, we tested the ability of rat brain extracts to protect H1(0) RNA probe from degradation by T1 RNase. The results reported here demonstrate that rat brain contains at least one major (p40) and two minor (p110 and p70) binding factors, specific for H1(0) RNA, all of which are much more or exclusively expressed in adult rat brain, when compared with other tissues. The binding of the factors is confined to a portion of the 3'-untranslated region (3'-UTR), which is highly conserved among murine and human H1(0) mRNAs. These findings suggest that the proteins identified play a critical role in regulating the expression of H1(0) histone in the brain of mammals.

PIPPin is a brain-specific protein that contains a cold-shock domain and binds specifically to H1 degrees and H3.3 mRNAs.

During maturation of mammalian brain, variants of both linker (i.e. H1 degrees) and core (i.e. H3.3) histone proteins accumulate in nerve cells. As the concentration of the corresponding transcripts decreases, in postmitotic cells, even if the genes are actively transcribed, it is likely that regulation of variant histone expression has relevant post-transcriptional components and that cellular factors affect histone mRNA stability and/or translation. Here we report that PIPPin, a protein that is highly enriched in the rat brain and contains a cold-shock domain, binds with high specificity to the transcripts that encode H1 degrees and H3.3 histone variants. Both mRNAs are bound through the very end of their 3'-untranslated region that encompasses the polyadenylation signal. Although PIPPin is present both in the cytoplasm and the nucleus of nerve cells, PIPPin-RNA complexes can be obtained only from nuclear extracts. The results of two-dimensional electrophoretic analysis suggest that a relevant proportion of nuclear PIPPin is more acidic than expected, thus suggesting that its RNA binding activity might be modulated by post-translational modifications, such as phosphorylation.

Expression of synapsin I gene in primary cultures of differentiating rat cortical neurons.

Synapsin I is a neuron-specific protein which is present in two isoforms, Ia and Ib. In the last few years this protein has been demonstrated to play a central role in the regulation of neurotransmitter release and synaptic plasticity. In this paper the developmental expression of this protein has been investigated in primary neuronal cultures from fetal rat brain cortices. The presence of thyroid hormone in the culture medium stimulates an early expression of the protein without exerting any effect at the level of mRNA transcription and accumulation. These observations implicate a T3-dependent regulation of this neuron-specific gene at the level of mRNA translation.

Selection of functional variants of the NS3-NS4A protease of hepatitis C virus by using chimeric sindbis viruses.

The NS3-NS4A serine protease of hepatitis C virus (HCV) mediates four specific cleavages of the viral polyprotein and its activity is considered essential for the biogenesis of the HCV replication machinery. Despite extensive biochemical and structural characterization, the analysis of natural variants of this enzyme has been limited by the lack of an efficient replication system for HCV in cultured cells. We have recently described the generation of chimeric HCV-Sindbis viruses whose propagation depends on the NS3-NS4A catalytic activity. NS3-NS4A gene sequences were fused to the gene coding for the Sindbis virus structural polyprotein in such a way that processing of the chimeric polyprotein, nucleocapsid assembly, and production of infectious viruses required NS3-NS4A-mediated proteolysis (G. Filocamo, L. Pacini, and G. Migliaccio, J. Virol. 71:1417-1427, 1997). Here we report the use of these chimeric viruses to select and characterize active variants of the NS3-NS4A protease. Our original chimeric viruses displayed a temperature-sensitive phenotype and formed lysis plaques much smaller than those formed by wild-type (wt) Sindbis virus. By serially passaging these chimeric viruses on BHK cells, we have selected virus variants which formed lysis plaques larger than those produced by their progenitors and produced NS3-NS4A proteins different in size and/or sequence from those of the original viruses. Characterization of the selected protease variants revealed that all of the mutated proteases still efficiently processed the chimeric polyprotein in infected cells and also cleaved an HCV substrate in vitro. One of the selected proteases was expressed in a bacterial system and showed a catalytic efficiency comparable to that of the wt recombinant protease.