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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. [ABSTRACT FROM AUTHOR]

Botulinum toxin is effective to treat post-stroke lower limb''s spasticity. However, the impact of BTX therapy on the quality of life (QoL) of patients is not known. We aimed to evaluate the effect of Botulinum toxin type A (Dysport®) on QoL in post-stroke calf spasticity and to correlate the functional outcome measures with eventual changes in QoL. We included 24 patients (16M, 8F) who had suffered a stroke at least 3 months earlier. All the patients presented equinovarus deformity for spasticity, were able to walk for at least 10m with orthesis but showed a speed of walking <90% of normal over a distance of 10m. QoL was measured by SF-36. Outcome measures of effectiveness were: 2-min walking test; FAC, RMA, active and passive ROM of the affected ankle; Modified Ashworth Scale (MAS) for assessment of spasticity severity; pain. After infiltration with BTX-A, all outcome measures of effectiveness improved significantly (ANOVA repeated measures). After treatment, spasticity decreased (reduction MAS p<0.001), FAC and RMA improved (p<0.001), 2-min walking distance increased (p<0.006) and pain''s score decreased (p<0.001). Participants in the study compared to the general population had a lower score for SF-36 and had a worse perception of their health. After administration of BTX-A there was no significant amelioration for the physical and mental health scores of SF-36. [Copyright &y& Elsevier]

Legionnaires' disease (LD) is a serious type of pneumonia, typically contracted by susceptible people through the inhalation of aerosols contaminated with Legionella pneumophila (Lp) . In this report, the first case of coinfection with Lp - Bordetella bronchiseptica (Bb) is described. A possible source of the Lp infection may be the hotel in Paris (France) where the patient had stayed before developing the symptoms. The Bb infection may have been transmitted by the dog with which he had constant contact, although this has not been proven., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 La Sorda, Palucci, Natalini, Fillo, Giordani, Paglione, Monte, Lista, Mancini, Girolamo, Rota, Caporali, Ricci, Ginevra, Jarraud, Sanguinetti, Scaturro and Ricci.)

The pandemic marked the beginning of an era of dynamic and rapid changes in the diagnosis of respiratory infections. Herein we describe Legionnaires' disease trend in the years 2016-2023 in a large Italian hospital showing how improvements in diagnostic algorithms impact on its detection., (© 2024. The Author(s).)

Background and Aim: Legionnaires' disease is a severe form of pneumonia caused by the inhalation or aspiration of water droplets contaminated with Legionella pneumophila and other Legionella species. These bacteria are commonly found in natural habitats and man-made water systems. Legionnaires' disease is a significant public health problem, especially in healthcare settings where patients may be exposed to contaminated environmental sources. Nosocomial outbreaks have been reported worldwide, leading to high morbidity and mortality rates, and increased healthcare costs. This study aimed to compare, the clonal relationship of clinical L. pneumophila strains from two different hospitals with L. pneumophila strains isolated from the water supply., Methods: In the period from 2019 to 2021, clinical and environmental strains involved in three cases of legionellosis were compared by means of pulsed field gel electrophoresis and sequence based typing techniques., Results: Our findings highlight the persistence of clonally distinct strains within each hospital examined. Furthermore, the L. pneumophila strains detected from hospital environmental sources were related to the clinical strains isolated, demonstrating the nosocomial origin of these cases., Conclusions: Therefore, it is important to implement more accurate surveillance systems both for epidemiological studies and to check the effectiveness of remediation procedures. (www.actabiomedica.it).

Introduction: Collaborative computing has attracted great interest in the possibility of joining the efforts of researchers worldwide. Its relevance has further increased during the pandemic crisis since it allows for the strengthening of scientific collaborations while avoiding physical interactions. Thus, the E4C consortium presents the MEDIATE initiative which invited researchers to contribute via their virtual screening simulations that will be combined with AI-based consensus approaches to provide robust and method-independent predictions. The best compounds will be tested, and the biological results will be shared with the scientific community., Areas Covered: In this paper, the MEDIATE initiative is described. This shares compounds' libraries and protein structures prepared to perform standardized virtual screenings. Preliminary analyses are also reported which provide encouraging results emphasizing the MEDIATE initiative's capacity to identify active compounds., Expert Opinion: Structure-based virtual screening is well-suited for collaborative projects provided that the participating researchers work on the same input file. Until now, such a strategy was rarely pursued and most initiatives in the field were organized as challenges. The MEDIATE platform is focused on SARS-CoV-2 targets but can be seen as a prototype which can be utilized to perform collaborative virtual screening campaigns in any therapeutic field by sharing the appropriate input files.

Legionella pneumophila ( Lp ), responsible for a severe pneumonia called Legionnaires' disease, represents an important health burden in Europe. Prevention and control of Lp contamination in warm water systems is still a great challenge often due to the failure in disinfection procedures. The aim of this study was to evaluate the in vitro activity of Terpinen-4-ol (T-4-ol) as potential agent for Lp control, in comparison with the essential oil of Melaleuca alternifolia (tea tree) (TTO. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of T-4-ol were determined by broth micro-dilution and a micro-atmosphere diffusion method to investigate the anti- Lp effects of T-4-ol and TTO vapors. Scanning Electron Microscopy (SEM) was adopted to highlight the morphological changes and Lp damage following T-4-ol and TTO treatments. The greatest antimicrobial activity against Lp was shown by T-4-ol with a MIC range of 0.06-0.125% v / v and MBC range of 0.25-0.5% v / v . The TTO and T-4-ol MIC and MBC decreased with increasing temperature (36 °C to 45 ± 1 °C), and temperature also significantly influenced the efficacy of TTO and T-4-ol vapors. The time-killing assay showed an exponential trend of T-4-ol bactericidal activity at 0.5% v / v against Lp . SEM observations revealed a concentration- and temperature- dependent effect of T-4-ol and TTO on cell surface morphology with alterations. These findings suggest that T-4-ol is active against Lp and further studies may address the potential effectiveness of T-4-ol for control of water systems.

Legionella spp. are Gram-negative bacteria that inhabit freshwater environments representing a serious risk for human health. Legionella pneumophila ( Lp ) is the species most frequently responsible for a severe pneumonia known as Legionnaires' disease. Lp consists of 15 serogroups (Sgs), usually identified by monoclonal or polyclonal antibodies. With regard to Lp serogrouping, it is well known that phenotyping methods do not have a sufficiently high discriminating power, while genotypic methods although very effective, are expensive and laborious. Recently, mass spectrometry and infrared spectroscopy have proved to be rapid and successful approaches for the microbial identification and typing. Different biomolecules (e.g., lipopolysaccharides) adsorb infrared radiation originating from a specific microbial fingerprint. The development of a classification system based on the intra-species identification features allows a rapid and reliable typing of strains for diagnostic and epidemiological purposes. The aim of the study was the evaluation of Fourier Transform Infrared Spectroscopy using the IR Biotyper® system (Bruker Daltonik, Germany) for the identification of Lp at the serogroup (Sg) level for diagnostic purposes as well as in outbreak events. A large dataset of Lp isolates ( n = 133) and ATCC reference strains representing the 15 Lp serogroups were included. The discriminatory power of the instrument's classifier, was tested by Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA). All isolates were classified as follows: 12/133 (9.0%) as Lp Sg1 and 115/133 (86.5%) as Lp Sg 2-15 (including both ATCC and environmental Lp serogroup). Moreover, a mis-classification for 2/133 (1.5%) isolates of Lp Sg 2-15 that returned as Lp Sg1 was observed, and 4/133 (3.0%) isolates were not classified. An accuracy of 95.49% and an error rate of 4.51% were calculated. IR Biotyper® is able provide a quick and cost-effective reliable Lp classification with advantages compared with agglutination tests that show ambiguous and unspecific results. Further studies including a larger number of isolates could be useful to implement the classifier obtaining a robust and reliable tool for the routine Lp serogrouping. IR Biotyper® could be a powerful and easy-to-use tool to identify Lp Sgs, especially during cluster/outbreak investigations, to trace the source of the infection and promptly adopt preventive and control strategies., Competing Interests: MC was employed by Bruker Daltonic GmbH (the manufacturer of IR-Biotyper®). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pascale, Bisognin, Mazzotta, Girolamini, Marino, Dal Monte, Cordovana, Scaturro, Ricci and Cristino.)

Legionella pneumophila serogroup 1 (Lp1) sequence type (ST) 23 is one of the most commonly detected STs in Italy where it currently causes all investigated outbreaks. ST23 has caused both epidemic and sporadic cases between 1995 and 2018 and was analysed at genomic level and compared with ST23 isolated in other countries to determine possible similarities and differences. A core genome multi-locus sequence typing (cgMLST), based on a previously described set of 1,521 core genes, and single-nucleotide polymorphisms (SNPs) approaches were applied to an ST23 collection including genomes from Italy, France, Denmark and Scotland. DNAs were automatically extracted, libraries prepared using NextEra library kit and MiSeq sequencing performed. Overall, 63 among clinical and environmental Italian Lp1 isolates and a further seven and 11 ST23 from Denmark and Scotland, respectively, were sequenced, and pangenome analysed. Both cgMLST and SNPs analyses showed very few loci and SNP variations in ST23 genomes. All the ST23 causing outbreaks and sporadic cases in Italy and elsewhere, were phylogenetically related independent of year, town or country of isolation. Distances among the ST23s were further shortened when SNPs due to horizontal gene transfers were removed. The Lp1 ST23 isolated in Italy have kept their monophyletic origin, but they are phylogenetically close also to ST23 from other countries. The ST23 are quite widespread in Italy, and a thorough epidemiological investigation is compelled to determine sources of infection when this ST is identified in both LD sporadic cases and outbreaks., (© 2022 Ricci et al.)

Legionella pneumophila is ubiquitous in aquatic environments and responsible for severe pneumonia in humans through inhalation of aerosol containing Legionella spp. Macrolides and fluoroquinolones are frequently used antimicrobials, but treatment failures are increasingly being reported. As susceptibility testing is not routinely performed, this study aimed to determine the minimum inhibitory concentrations (MICs) on 58 environmental Legionella pneumophila strains (24 of serogroup 1 and 34 of non-serogroup 1) isolated in Northern Italy. MICs of azithromycin, erythromycin, ciprofloxacin, levofloxacin, and rifampicin were determined by the microdilution method using buffered yeast extract broth supplemented with α-ketoglutarate (BYEα). Seventy-five percent of Legionella pneumophila isolates showed MIC values below the tentative highest MICs indicated by the European Committee on Antimicrobial Susceptibility Testing (EUCAST); rifampicin was the most active agent with MIC 90 values below 0.008 mg/L. Interestingly, one isolate was tested and found to be PCR-positive for the azithromycin LpeAB active efflux system, further confirmed by the reserpine/resazurin microtiter assay. In conclusion, this study has provided additional susceptibility data for environmental Legionella pneumophila isolates from Northern Italy demonstrating, in general, low MICs values for the tested antimicrobials, although one strain tested was shown to possess the LpeAB resistance determinant, indicating that future surveillance studies are warranted.

The collection and storage of water-related matrices such as biofilm from collection to processing are critical for the detection of Legionella pneumophila by cultural and molecular tests. SRK™ is a liquid medium that acts both as an antimicrobial neutralizing agent and a transport medium for bacterial culture enumeration and is useful to maintain the stability of the sample from collection to analysis. The aims of this study were to evaluate Legionella pneumophila viability and bacterial nucleic acids' stability in SRK™ medium over time at different storage conditions. Artificial bacterial inoculates with an approximate concentration of 10 4 , 10 3 and 10 2 CFU/mL were made using Legionella pneumophila certified reference material suspended in SRK™ medium. Bacteria recovery was analyzed by cultural and molecular methods at time 0, 24 and 48 h at room temperature and at 0, 24, 48 and 72 h at 2-8 °C, respectively. SRK™ medium supported Legionella pneumophila culture viability with CFU counts within the expected range. The recovery after 72 h at 2-8 °C was 83-100% and 75-95% after 48 h at room temperature. Real-time PCR appropriately detected Legionella pneumophila DNA at each temperature condition, dilution and time point. Results demonstrated a good performance of SRK™ medium for the reliable recovery of environmental Legionella .

Legionnaires' disease (LD) is a severe pneumonia caused by bacteria belonging to the genus Legionella . This is a major public health concern and infections are steadily increasing worldwide. Several sources of infection have been identified, but they have not always been linked to human isolates by molecular match. The well-known Legionella contamination of private homes has rarely been associated with the acquisition of the disease, although some patients never left their homes during the incubation period. This study demonstrated by genomic matching between clinical and environmental Legionella isolates that the source of an LD cluster was a private building. Monoclonal antibodies and sequence-based typing were used to type the isolates, and the results clearly demonstrated the molecular relationship between the strains highlighting the risk of contracting LD at home. To contain this risk, the new European directive on the quality of water intended for human consumption has introduced for the first time Legionella as a microbiological parameter to be investigated in domestic water systems. This should lead to a greater attention to prevention and control measures for domestic Legionella contamination and, consequently, to a possible reduction in community acquired LD cases.

In September 2018 in Brescia province, northern Italy, an outbreak of Legionnaires' disease (LD) caused by Legionella pneumophila serogroup 2 ( Lp2 ) occurred. The 33 cases (two fatal) resided in seven municipalities along the Chiese river. All cases were negative by urinary antigen test (UAT) and most were diagnosed by real-time PCR and serology. In only three cases, respiratory sample cultures were positive, and Lp2 was identified and typed as sequence type (ST)1455. In another three cases, nested sequence-based typing was directly applied to respiratory samples, which provided allelic profiles highly similar to ST1455. An environmental investigation was undertaken immediately and water samples were collected from private homes, municipal water systems, cooling towers and the river. Overall, 533 environmental water samples were analysed and 34 were positive for Lp . Of these, only three samples, all collected from the Chiese river, were Lp2 ST1455. If and how the river water could have been aerosolised causing the LD cases remains unexplained. This outbreak, the first to our knowledge caused by Lp2 , highlights the limits of UAT for LD diagnosis, underlining the importance of adopting multiple tests to ensure that serogroups other than serogroup 1, as well as other Legionella species, are identified.

Background: Pandemic coronavirus disease 2019 (COVID-19) disease represents a challenge for healthcare structures. The molecular confirmation of samples from infected individuals is crucial and therefore guides public health decision making. Clusters and possibly increased diffuse transmission could occur in the context of the next influenza season. For this reason, a diagnostic test able to discriminate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from influenza viruses is urgently needed., Methods: A multiplex real-time reverse-transcription polymerase chain reaction (PCR) assay was assessed using 1 laboratory protocol with different real-time PCR instruments. Overall, 1000 clinical samples (600 from samples SARS-CoV-2-infected patients, 200 samples from influenza-infected patients, and 200 negative samples) were analyzed., Results: The assay developed was able to detect and discriminate each virus target and to intercept coinfections. The limit of quantification of each assay ranged between 5 and 10 genomic copy numbers, with a cutoff value of 37.7 and 37.8 for influenza and SARS-CoV-2 viruses, respectively. Only 2 influenza coinfections were detected in COVID-19 samples., Conclusions: This study suggests that multiplex assay is a rapid, valid, and accurate method for the detection of SARS-CoV-2 and influenza viruses in clinical samples. The test may be an important diagnostic tool for both diagnostic and surveillance purposes during the seasonal influenza activity period., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America.)

The good installation, as well as commissioning plan, of a water network is a crucial step in reducing the risk of waterborne diseases. The aim of this study was to monitor the microbiological quality of water from a newly built pavilion before it commenced operation. Overall, 91 water samples were tested for coliforms, Escherichia coli , enterococci, Pseudomonas aeruginosa and Legionella at three different times: T0 (without any water treatment), T1 (after treatment with hydrogen peroxide and silver ions at initial concentration of 20 mg/L and after flushing of water for 20 min/day for seven successive days) and T2 (15 days later). Coliforms were detected in 47.3% of samples at T0, 36.3% at T1 and 4.4% at T2. E. coli was isolated in 4.4% of the samples only at T1, while enterococci appeared in 12.1% of the samples at T1 and in 2.2% at T2. P. aeruginosa was isolated in 50.5% of the samples at T0, 29.7% at T1 and 1.1% at T2. Legionella pneumophila serogroup 8 was isolated in 80.2% of the samples at T0, 36.3% at T1 and 2.2% at T2. Our results confirmed the need for a water safety plan in new hospital pavilions to prevent the risk of waterborne diseases.

The COVID-19 pandemic started in China in early December 2019, and quickly spread around the world. The epidemic gradually started in Italy at the end of February 2020, and by May 31, 2020, 232,664 cases and 33,340 deaths were confirmed. As a result of this pandemic, the Italian Ministerial Decree issued on March 11, 2020, enforced lockdown; therefore, many social, recreational, and cultural centers remained closed for months. In Apulia (southern Italy), all non-urgent hospital activities were suspended, and some wards were closed, with a consequent reduction in the use of the water network and the formation of stagnant water. This situation could enhance the risk of exposure of people to waterborne diseases, including legionellosis. The purpose of this study was to monitor the microbiological quality of the water network (coliforms, E. coli, Enterococci, P. aeruginosa, and Legionella) in three wards (A, B and C) of a large COVID-19 regional hospital, closed for three months due to the COVID-19 emergency. Our study revealed that all three wards' water network showed higher contamination by Legionella pneumophila sg 1 and sg 6 at T1 (after lockdown) compared to the period before the lockdown (T0). In particular, ward A at T1 showed a median value = 5600 CFU/L (range 0-91,000 CFU/L) vs T0, median value = 75 CFU/L (range 0-5000 CFU/L) (p-value = 0.014); ward B at T1 showed a median value = 200 CFU/L (range 0-4200 CFU/L) vs T0, median value = 0 CFU/L (range 0-300 CFU/L) (p-value = 0.016) and ward C at T1 showed a median value = 175 CFU/L (range 0-22,000 CFU/L) vs T0, median value = 0 CFU/L (range 0-340 CFU/L) (p-value < 0.001). In addition, a statistically significant difference was detected in ward B between the number of positive water samples at T0 vs T1 for L. pneumophila sg 1 and sg 6 (24% vs 80% p-value < 0.001) and for coliforms (0% vs 64% p-value < 0.001). Moreover, a median value of coliform load resulted 3 CFU/100 ml (range 0-14 CFU/100 ml) at T1, showing a statistically significant increase versus T0 (0 CFU/100 ml) (p-value < 0.001). Our results highlight the need to implement a water safety plan that includes staff training and a more rigorous environmental microbiological surveillance in all hospitals before occupying a closed ward for a longer than one week, according to national and international guidelines., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Legionella spp are the causative agents of Legionnaires' diseases, which is a pneumonia of important public health concern. Ubiquitous freshwater and soil inhabitants can reach man-made water systems and cause illness. Legionella enumeration and quantification in water systems is crucial for risk assessment and culture examination is the gold standard method. In this study, Legionella recovery from potable water samples, at presumably a low concentration of interfering microorganisms, was compared by plating on buffered charcoal yeast extract (BCYE) and glycine, vancomycin, polymyxin B, cycloheximide (GVPC) Legionella agar media, according to the International Standard Organization (ISO) 11731: 2017. Overall, 556 potable water samples were analyzed and 151 (27.1%) were positive for Legionella . Legionella grew on both BCYE and GVPC agar plates in 85/151 (56.3%) water samples, in 65/151 (43%) on only GVPC agar plates, and in 1/151 (0.7%) on only BCYE agar plates. In addition, GVPC medium identified Legionella species other than pneumophila in six more samples as compared with the culture on BCYE. Although the medians of colony forming units per liter (CFU/L) detected on the BCYE and GVPC agar plates were 2500 and 1350, respectively ( p -value < 0.0001), the difference did not exceed one logarithm, and therefore is not relevant for Legionella risk assessment. These results make questionable the need to utilize BCYE agar plates to analyze potable water samples.

Detection and enumeration of Legionella in water samples is of great importance for risk assessment analysis. The plate culture method is the gold standard, but has received several well-known criticisms, which have induced researchers to develop alternative methods. The purpose of this study was to compare Legionella counts obtained by the analysis of potable water samples through the plate culture method and through the IDEXX liquid culture Legiolert method. Legionella plate culture, according to ISO 11731:1998, was performed using 1 L of water. Legiolert was performed using both the 10 mL and 100 mL Legiolert protocols. Overall, 123 potable water samples were analyzed. Thirty-seven (30%) of them, positive for L. pneumophila , serogroups 1 or 2-14 by plate culture, were used for comparison with the Legiolert results. The Legiolert 10 mL test detected 34 positive samples (27.6%) and the Legiolert 100 mL test detected 37 positive samples, 27.6% and 30% respectively, out of the total samples analyzed. No significant difference was found between either the Legiolert 10 mL and Legiolert 100 mL vs. the plate culture ( p = 0.9 and p = 0.3, respectively) or between the Legiolert 10 mL and Legiolert 100 mL tests ( p = 0.83). This study confirms the reliability of the IDEXX Legiolert test for Legionella pneumophila detection and enumeration, as already shown in similar studies. Like the plate culture method, the Legiolert assay is also suitable for obtaining isolates for typing purposes, relevant for epidemiological investigations.