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Objective: Legionella pneumophila serogroup 1 (Lp1) sequence type (ST) 1 is globally widespread in the environment and accounts for a significant proportion of Legionella infections, including nosocomial Legionnaires' disease (LD). This study aimed to design a sensitive and specific detection method for Lp ST1 that will underpin epidemiological investigations and risk assessment., Methods: A total of 628 Lp genomes (126 ST1s) were analyzed by comparative genomics. Interrogation of more than 900 accessory genes revealed seven candidate targets for specific ST1 detection and specific primers and hydrolysis probes were designed and evaluated. The analytical sensitivity and specificity of the seven primer and probe sets were evaluated on serially diluted DNA extracted from the reference strain CIP107629 and via qPCR applied on 200 characterized isolates. The diagnostic performance of the assay was evaluated on 142 culture-proven clinical samples from LD cases and a real-life investigation of a case cluster., Results: Of seven qPCR assays that underwent analytical validation, one PCR target (lpp1868) showed higher sensitivity and specificity for ST1 and ST1-like strains. The diagnostic performance of the assay using respiratory samples corresponded to a sensitivity of 95% (19/20) (95% CI (75.1-99.9)) and specificity of 100% (122/122) (95% CI (97-100)). The ST1 PCR assay could link two out of three culture-negative hospitalized LD cases to ST1 during a real-time investigation., Conclusion: Using whole genome sequencing (WGS) data, we developed and validated a sensitive and specific qPCR assay for the detection of Lp1 belonging to the ST1 clonal complex by amplification of the lpp1868 gene. The ST1 qPCR is expected to deliver an added value for Lp control and prevention, in conjunction with other recently developed molecular assays., (Copyright © 2019 European Society of Clinical Microbiology and Infectious Diseases. All rights reserved.)

Objectives: Antibiotic susceptibility of Legionella pneumophila is poorly understood, with treatment of Legionnaires' disease often based on empirical choice. The aim of this study was to determine the antibiotic susceptibility of L. pneumophila strains., Methods: Antibiotic susceptibility of 92 L. pneumophila strains isolated in England and Wales between 2007 and 2017 was determined using a microbroth dilution methodology for each agent tested. MICs and MBCs were determined and compared with published intracellular concentrations of each agent tested., Results: The MIC range of erythromycin was 0.06-1 mg/L, the MIC range of rifampicin was 0.0001 mg/L, the MIC range of ciprofloxacin was 0.004-0.25 mg/L and the MIC range of levofloxacin and moxifloxacin was 0.03-0.25 mg/L. The MBC range of erythromycin was 1-32 mg/L, but the MBC range of ciprofloxacin was the same as the MIC range. For levofloxacin and moxifloxacin the MBC range was elevated by one dilution and two dilutions, respectively. Typically, intracellular bronchial secretion concentrations of erythromycin might be expected to reach a suitable level to exceed the MIC range; however, 91 of 92 (98.9%) isolates had an MBC below the expected intracellular concentrations, which indicated erythromycin may have variable efficacy. MIC and MBC values of ciprofloxacin, levofloxacin and moxifloxacin were below achievable intracellular levels within bronchial secretions. Comparison of the MIC/MBC correlation showed very little clustering for erythromycin, but strong clustering for levofloxacin and to a lesser extent ciprofloxacin., Conclusions: Use of the MIC/MBC linkage analysis seems an appropriate way forward for antimicrobial susceptibility testing and supports current guidance recommending levofloxacin for the treatment of Legionnaires' disease.

Objectives: Legionella pneumophila is the leading cause of Legionnaires' disease, a severe form of pneumonia acquired from environmental sources. Investigations of both sporadic cases and outbreaks rely mostly on analysis of a single to a few colony pick(s) isolated from each patient. However, because of the lack of data describing diversity within single patients, the optimal number of picks is unknown. Here, we investigated diversity within individual patients using sequence-based typing (SBT) and whole-genome sequencing (WGS)., Methods: Ten isolates of L. pneumophila were obtained from each of ten epidemiologically unrelated patients. SBT and WGS were undertaken, and single-nucleotide polymorphisms (SNPs) were identified between isolates from the same patient., Results: The same sequence type (ST) was obtained for each set of ten isolates. Using genomic analysis, zero SNPs were identified between isolates from seven patients, a maximum of one SNP was found between isolates from two patients, and a maximum of two SNPs was found amongst isolates from one patient. Assuming that the full within-host diversity has been captured with ten isolates, statistical analyses showed that, on average, analysis of one isolate would yield a 70% chance of capturing all observed genotypes, and seven isolates would yield a 90% chance., Conclusions: SBT and WGS analyses of multiple colony picks obtained from ten patients showed no, or very low, within-host genomic diversity in L. pneumophila, suggesting that analysis of one colony pick per patient will often be sufficient to obtain reliable typing data to aid investigation of cases of Legionnaires' disease., (Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Legionella pneumophila is the leading cause of Legionnaires' disease, a severe pneumonia that can occur as sporadic cases or point-source outbreaks affecting multiple patients. The infection is acquired by inhalation of aerosols from contaminated water systems. In order to identify the probable source and prevent further cases, clinical and environmental isolates are compared using phenotypic and genotypic methods. Typically up to 10 days are required to isolate L. pneumophila prior to the application of standard typing protocols. A rapid protocol using a real-time PCR specific for L. pneumophila and serogroup 1, combined with nested direct molecular typing, was adopted by Public Health England in 2012 to reduce reporting time for preliminary typing results. This rapid protocol was first used to investigate an outbreak that occurred in July/August 2012 and due to the positive feedback from that investigation, it was subsequently applied to other incidents in England and Wales where faster typing results would have aided incident investigation. We present here results from seven incidents that occurred between July 2012 and June 2015 where the use of this rapid approach provided preliminary characterization of the infecting strain in an average 1.58 days (SD 1.01) after sample receipt in contrast to 9.53 days (SD 3.73) when standard protocols were applied.

In June 2014 Public Health England confirmed a case of Legionnaires' disease (LD) in a neonate following birth at home in a hired birthing pool incorporating a heater and a recirculation pump which had been filled in advance of labour. The case triggered a public health investigation and a microbiological survey of an additional ten heated birthing pools hired or recently hired to the general public across England. The birthing pool used by the parent of the confirmed case was identified as the source of the neonate's infection following detection of Legionella pneumophila ST48 in both patient and environmental samples. Legionella species were detected by quantitative polymerase chain reaction but not culture in a further three pools together with other opportunistic pathogens identified by culture and matrix-assisted laser desorption ionization-time of flight (MALDI-ToF) mass spectrometry. A Patient Safety Alert from NHS England and Public Health England was issued stating that heated birthing pools filled in advance of labour should not be used for home births. This recommendation remains in place. This investigation in conjunction with other recent reports has highlighted a lack of awareness regarding the microbiological safety of heated birthing pools and their potential to be a source of LD and other opportunistic infections. Furthermore, the investigation raised important considerations with regards to microbiological sampling and testing in such incidents. Public health authorities and clinicians should consider LD in the differential diagnosis of severe respiratory infection in neonates within 14 days of a water birth.

Prompt detection of Legionella pneumophila is essential for rapid investigation of legionellosis. Furthermore, as the majority of L. pneumophila infections are caused by serogroup 1 (sg1) strains, rapid identification of such strains can be critical in both routine and outbreak scenarios. The ESCMID Study Group for Legionella Infections (ESGLI) was established in 2012 and immediately identified as a priority the validation of a reliable, easy to perform and interpret, cost-effective qPCR assay to standardise the detection of L. pneumophila DNA amongst members. A novel L. pneumophila assay targeting the mip gene was designed and combined with previously published methodologies amplifying the sg1 marker (wzm) and the green fluorescent protein gene (gfp) internal process control. The resulting triplex assay was validated internationally on the three qPCR platforms used by the majority of European Legionella reference laboratories: ABI 7500 (Life Technologies), LightCycler 480 Instrument II (Roche) and Rotor-Gene Q (Qiagen). Clinical and EQA specimens were tested together with a large panel of strains (251 in total) to validate the assay. The assay proved to be 100% specific for L. pneumophila and sg1 DNA both in silico and in vitro. Efficiency values for mip and wzm assays ranged between 91.97 and 97.69%. Limit of detection values estimated with 95% confidence were adopted for mip and wzm assays on all three qPCR platforms. Inhibition was not observed. This study describes a robust assay that could be widely implemented to standardise the molecular detection of L. pneumophila among ESGLI laboratories and beyond.

Prompt detection of Legionella pneumophila is essential for rapid investigation of legionellosis. Furthermore, as the majority of L. pneumophila infections are caused by serogroup 1 (sg1) strains, rapid identification of such strains can be critical in both routine and outbreak scenarios. The ESCMID Study Group for Legionella Infections (ESGLI) was established in 2012 and immediately identified as a priority the validation of a reliable, easy to perform and interpret, cost-effective qPCR assay to standardise the detection of L. pneumophila DNA amongst members. A novel L. pneumophila assay targeting the mip gene was designed and combined with previously published methodologies amplifying the sg1 marker ( wzm) and the green fluorescent protein gene ( gfp) internal process control. The resulting triplex assay was validated internationally on the three qPCR platforms used by the majority of European Legionella reference laboratories: ABI 7500 (Life Technologies), LightCycler 480 Instrument II (Roche) and Rotor-Gene Q (Qiagen). Clinical and EQA specimens were tested together with a large panel of strains (251 in total) to validate the assay. The assay proved to be 100 % specific for L. pneumophila and sg1 DNA both in silico and in vitro. Efficiency values for mip and wzm assays ranged between 91.97 and 97.69 %. Limit of detection values estimated with 95 % confidence were adopted for mip and wzm assays on all three qPCR platforms. Inhibition was not observed. This study describes a robust assay that could be widely implemented to standardise the molecular detection of L. pneumophila among ESGLI laboratories and beyond. [ABSTRACT FROM AUTHOR]