A real-time PCR for specific detection of the Legionella pneumophila serogroup 1 ST1 complex.

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.

Genomic sequence investigation Streptococcus pyogenes clusters in England (2010-2015).

OBJECTIVES: To analyse genomic sequence data of referred Streptococcus pyogenes isolates and those pertaining to selected elderly/nursing care or maternity clusters from 2010 to 2015 to ascertain genomic differences between epidemiologically related isolates and unrelated isolates from outbreaks of disease. METHODS: The genomic sequences of 134 S. pyogenes isolates from 21 clusters of infection in elderly care or maternity settings from 2010 to 2015 were analysed using bioinformatics to ascertain genomic phylogeny, single nucleotide polymorphism (SNP) differences and statistical outliers from epidemiologically defined outbreaks. Analysis was undertaken within clusters and compared with sporadic isolates from geographically distinct outbreaks of S. pyogenes infection. RESULTS: Genomic sequence analysis of 21 outbreaks of S. pyogenes infection ranged in size from a single patient (with colonized healthcare worker link) to 18 patient cases of group A streptococcus (GAS) infection in a single setting. Seventeen healthcare workers were identified in 8 of 21 outbreaks with the associated outbreak strain, with multiple staff in 2 of 21 outbreaks. Genomic sequences from epidemiologically linked isolates from patients, staff and healthcare environmental settings were highly conserved, differing by 0-1 SNP in some cases and mirrored geographical data. Four of 21 outbreaks had environmental contamination with the outbreak strain, indistinguishable or of limited SNP difference to the patient isolates. Genomic SNP analysis enabled exclusion of ten isolates from epidemiological outbreaks. CONCLUSIONS: Genomic discrimination can be applied to assist outbreak investigation. It enabled confirmation or exclusion of GAS cases from epidemiologically defined outbreaks. Colonization of healthcare workers and environmental contamination with the outbreak strain was demonstrated for several outbreaks.

Antibiotic susceptibility of Legionella pneumophila strains isolated in England and Wales 2007-17.

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.

Low genomic diversity of Legionella pneumophila within clinical specimens.

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.

Heated birthing pools as a source of Legionnaires' disease.

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.

International Mycoplasma pneumoniae typing study: interpretation of M. pneumoniae multilocus variable-number tandem-repeat analysis.

Typing of Mycoplasma pneumoniae by multiple-locus variable-number tandem repeat analysis (MLVA) is increasingly in use. However, no specific internationally agreed guidance is available. Thirty M. pneumoniae DNA samples including serial dilutions of a type strain were sent to six international laboratories to perform MLVA and results were compared. Good correlation was observed, indicating that this methodology can be robustly performed in multiple sites. However, differences due to interpretation of fragment size, repeat sequence identification and repeat numbering led to inconsistency in the final profiles assigned by laboratories. We propose guidelines for interpreting M. pneumoniae MLVA typing and assigning the number of repeats.

Design and validation of a qPCR assay for accurate detection and initial serogrouping of Legionella pneumophila in clinical specimens by the ESCMID Study Group for Legionella Infections (ESGLI).

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.

Late-onset prosthetic valve endocarditis caused by Mycoplasma hominis, diagnosed using broad-range bacterial PCR.

We report what is believed to be the first case of late-onset prosthetic valve endocarditis caused by Mycoplasma hominis in a case of blood culture-negative endocarditis. The objective of this report is to emphasize the use of a broad-range PCR technique for bacterial 16S rRNA genes in identifying the causative pathogen, thus enabling targeted antimicrobial treatment.

Mycoplasma pneumoniae infection in primary care investigated by real-time PCR in England and Wales.

Real-time PCR was employed to detect a conserved region of the P1 cytadhesin gene of Mycoplasma pneumoniae in combined nose and throat swabs collected from patients attending GP surgeries during 2005-2009 with symptoms of respiratory tract infection (RTI). Samples were collected as part of an annual winter epidemiological and virological linked study in England and Wales. A total of 3,987 samples were tested, 65 (1.7%, 95%CI 1.3-2.1) had detectable M. pneumoniae DNA. Positive patients were detected of both gender, aged from 9 months to 78 years, who had clinical signs of upper RTI, fever and/or myalgia, an influenza-like illness to lower RTI. Mixed infections were identified in four cases, two with influenza A H1, one with H3 and one with influenza B. Children aged 5-14 years were more likely to have detectable M. pneumoniae in samples than all other age groups (Fishers p = 0.03), attributed to the 2005-2006 season in which 6.0% (12/200, 95%CI 3.4-10.3) of 5-14 year olds had detectable M. pneumoniae in comparison to 2.2% in 2006-2007 (3/141 95%CI 0.5-6.4), 2.2% in 2007-2008 (2/89 95%CI 0.1-8.3) and 0% in 2008-2009 (0/151 95%CI 0-2.9).

External quality assessment for the molecular detection of Hepatitis C virus.

UNLABELLED: BACKGROUND, OBJECTIVES AND STUDY DESIGN: External quality assessment (EQA) panels were distributed internationally by UK NEQAS for Microbiology to 159 participants for the detection, quantification and genotyping of Hepatitis C virus (HCV) in freeze-dried plasma from 2000 to 2004. The results were analysed to determine the level of standardisation of qualitative detection, quantitative detection and genotyping. RESULTS: The accurate detection of HCV in the panels varied from 86.9% to 100%. Four genotypes were distributed with the panels and there was no significant difference in the detection of different genotypes of HCV by participants. Further analysis indicated most variation occurred in quantification of HCV at lower concentrations and from 0% to 14.8% reported quantitative values outside 0.5 log(10) of the median value. In addition, three negative specimens were distributed and false positives were found to be rare (0.9-2.2%) with all methods included in the study. CONCLUSION: The laboratory detection of HCV in plasma EQA specimens was varied, with decreasing parity of quantification at lower concentrations of HCV. False positives and negatives were rare, irrespective of the genotype under test.

Canine mycoplasmas.

This review aims to summarise our current understanding of the role of mycoplasmas in domestic dogs. Canine mycoplasmology is a small field, with less than 50 publications in the past 40 years. In this time we have gained knowledge about the number of species and have made associations with infections in dogs. However much evidence is still lacking. The importance of all canine mycoplasmas remains unknown, yet certain species are associated with canine anaemia (Mycoplasma haemocanis), respiratory disease (Mycoplasma cynos) and urogenital tract infections (Mycoplasma canis). Mycoplasmas can be isolated in pure culture from canine clinical specimens and it is hoped that this review will stimulate veterinarians to consider mycoplasmas as a potential cause of disease in dogs, especially when antibiotic therapy is failing.

External quality assessment for detection of Chlamydia trachomatis.

The use of molecular methods for detection of Chlamydia trachomatis is increasing in clinical laboratories. External quality assessment enables unbiased monitoring of the performance of laboratories in the detection of specific pathogens. This study details the results of molecular and enzyme immunosorbent assay (EIA) testing for C. trachomatis detection in simulated endocervical swab specimens recently distributed internationally by United Kingdom National External Quality Assessment Scheme for Microbiology (UK NEQAS for Microbiology) external quality assessment panels. The frequency of accurate detection of C. trachomatis in the panels ranged from 32 to 100%. Participants using molecular methods were significantly more likely to detect C. trachomatis in specimens than those using an EIA. Two strains were distributed with the panels: an L2 laboratory-adapted strain and an uncharacterized primary isolate. Further analysis indicated a difference in detection of C. trachomatis between specific methods only with the L2 strain at lower concentrations. In addition, eight negative specimens were distributed, and false positives were found to be rare by all methods included in the study.

The LuxM homologue VanM from Vibrio anguillarum directs the synthesis of N-(3-hydroxyhexanoyl)homoserine lactone and N-hexanoylhomoserine lactone.

Vibrio anguillarum, which causes terminal hemorrhagic septicemia in fish, was previously shown to possess a LuxRI-type quorum-sensing system (vanRI) and to produce N-(3-oxodecanoyl)homoserine lactone (3-oxo-C10-HSL). However, a vanI null mutant still activated N-acylhomoserine lactone (AHL) biosensors, indicating the presence of an additional quorum-sensing circuit in V. anguillarum. In this study, we have characterized this second system. Using high-pressure liquid chromatography in conjunction with mass spectrometry and chemical analysis, we identified two additional AHLs as N-hexanoylhomoserine lactone (C6-HSL) and N-(3-hydroxyhexanoyl)homoserine lactone (3-hydroxy-C6-HSL). Quantification of each AHL present in stationary-phase V. anguillarum spent culture supernatants indicated that 3-oxo-C10-HSL, 3-hydroxy-C6-HSL, and C6-HSL are present at approximately 8.5, 9.5, and 0.3 nM, respectively. Furthermore, vanM, the gene responsible for the synthesis of these AHLs, was characterized and shown to be homologous to the luxL and luxM genes, which are required for the production of N-(3-hydroxybutanoyl)homoserine lactone in Vibrio harveyi. However, resequencing of the V. harveyi luxL/luxM junction revealed a sequencing error present in the published sequence, which when corrected resulted in a single open reading frame (termed luxM). Downstream of vanM, we identified a homologue of luxN (vanN) that encodes a hybrid sensor kinase which forms part of a phosphorelay cascade involved in the regulation of bioluminescence in V. harveyi. A mutation in vanM abolished the production of C6-HSL and 3-hydroxy-C6-HSL. In addition, production of 3-oxo-C10-HSL was abolished in the vanM mutant, suggesting that 3-hydroxy-C6-HSL and C6-HSL regulate the production of 3-oxo-C10-HSL via vanRI. However, a vanN mutant displayed a wild-type AHL profile. Neither mutation affected either the production of proteases or virulence in a fish infection model. These data indicate that V. anguillarum possesses a hierarchical quorum sensing system consisting of regulatory elements homologous to those found in both V. fischeri (the LuxRI homologues VanRI) and V. harveyi (the LuxMN homologues, VanMN).