Rapid detection and differentiation of mobile colistin resistance (mcr-1 to mcr-10) genes by real-time PCR and melt-curve analysis.

BACKGROUND: The emergence of multi-drug-resistant (MDR) micro-organisms prompted new interest in older antibiotics, such as colistin, that had been abandoned previously due to limited efficacy or high toxicity. Over the years, several chromosomal-encoded colistin resistance mechanisms have been described; more recently, 10 plasmid-mediated mobile colistin resistance (mcr) genes have been identified. Spread of these genes among MDR Gram-negative bacteria is a matter of serious concern; therefore, reliable and timely mcr detection is paramount. AIM: To design and validate a multiplex real-time polymerase chain reaction (PCR) assay for detection and differentiation of mcr genes. METHODS: All available mcr alleles were downloaded from the National Center for Biotechnology Information Reference Gene Catalogue, aligned with Clustal Omega and primers designed using Primer-BLAST. Real-time PCR monoplexes were optimized and validated using a panel of 120 characterized Gram-negative strains carrying a wide range of resistance genes, often in combination. Melt-curve analysis was used to confirm positive results. FINDINGS: In-silico analysis enabled the design of a 'screening' assay for detection of mcr-1/2/6, mcr-3, mcr-4, mcr-5, mcr-7, mcr-8 and mcr-9/10, paired with an internal control assay to discount inhibition. A 'supplementary' assay was subsequently designed to differentiate mcr-1, mcr-2, mcr-6, mcr-9 and mcr-10. Expected results were obtained for all strains (100% sensitivity and specificity). Melt-curve analysis showed consistent melting temperature results. Inhibition was not observed. CONCLUSIONS: The assay is rapid and easy to perform, enabling unequivocal mcr detection and differentiation even when more than one variant is present. Adoption by clinical and veterinary microbiology laboratories would aid the surveillance of mcr genes amongst Gram-negative bacteria.

Rapid detection of OXA-23-like, OXA-24-like, and OXA-58-like carbapenemases from Acinetobacter species by real-time PCR.

BACKGROUND: Carbapenemase-producing Acinetobacter species, especially A. baumannii, are frequently associated with treatment failures and hospital outbreaks; thus, rapid and reliable detection of specific resistance markers is paramount. The most common carbapenemases found in A. baumannii, namely OXA-23-like, OXA-24-like, and OXA-58-like, belong to the oxacillinase group (class D ß-lactamases) which is notoriously difficult to identify phenotypically due to the lack of specific inhibitors. AIM: To design and validate a multiplex real-time polymerase chain reaction (PCR) assay to detect and differentiate the above three oxacillinases. METHODS: All available variants of the above three oxacillinase subfamilies were downloaded (as of November 2019) from the Beta-Lactamase DataBase (http://bldb.eu/) aligned with Clustal Omega and oligonucleotides designed using Primer-BLAST. A multiplex real-time PCR assay that included an internal control to discount inhibition was optimized on the Rotor-Gene Q (Qiagen) using the Rotor-Gene Multiplex PCR Kit (Qiagen) and validated using a panel of 122 previously characterized strains carrying a wide range of ß-lactamases, often in combination. FINDINGS: The in-silico approach enabled the design of oligonucleotides in conserved regions of the OXA-24-like and OXA-58-like alignments. Among the 42 described OXA-23-like variants, a single nucleotide polymorphism (SNP) was present in one of the oligonucleotide binding sites of OXA-27, OXA-166, OXA-811, OXA-812, and OXA-816. The assay was 100% sensitive and highly specific. Inhibition was not observed. CONCLUSION: The assay is easy to perform with results available in about 70 min. It enables unequivocal detection and differentiation of OXA-23-like, OXA-24-like, and OXA-58-like carbapenemases even when more than one is simultaneously present.

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.

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.

Rapid detection and evolutionary analysis of Legionella pneumophila serogroup 1 sequence type 47.

OBJECTIVES: Legionella pneumophila serogroup 1 (Lp1) sequence type 47 is the leading cause of legionellosis in north-western Europe, but, surprisingly, it is rarely isolated from environmental samples. Comparative genomics was applied to develop a PCR assay and to better understand the evolution of this strain. METHODS: Comparative analysis of 36 genomes representative of the Lp species was used to identify specific PCR targets, which were then evaluated in silico on 545 sequenced genomes and in vitro on 436 Legionella strains, 106 respiratory samples, and three environmental samples from proven ST47 sources. Phylogenetic analyses were performed to understand the evolution of ST47. RESULTS: The gene LPO_1073 was characterized as being 100% conserved in all 129 ST47 genomes analysed. A real-time PCR designed to detect LPO_1073 was positive for all 110 ST47 strains tested and agreed with culture and typing results previously obtained for 106 respiratory samples. The three environmental samples were also positive. Surprisingly, 26 of the 44 ST109 strains tested among 342 non-ST47 strains scored positive for LPO_1073. SNP-based phylogenetic analysis was undertaken to understand this result: the PCR-positive ST109 genomes were almost identical to ST47 genomes, with the exception of a recombined region probably acquired by ST47 from a ST62(-like) strain. CONCLUSION: The genomic analysis allowed the design of a highly specific PCR assay for rapid detection of ST47 strains. Furthermore, it allowed us to uncover the evolution of ST47 strains from ST109 by homologous recombination with ST62. We hypothesize that this recombination generated the leading cause of legionellosis in north-western Europe.

High prevalence of Legionella in non-passenger merchant vessels.

There is a paucity of information on the risk from potable water in non-passenger merchant vessels (NPMVs) particularly with regard to Legionella and other bacteria. This retrospective study examined water samples from 550 NPMVs docked in eight UK ports. A total of 1027 samples from 412 NPMVs were examined for total aerobic colony counts (ACC), coliforms, Escherichia coli and enterococci; 41% of samples yielded ACC above the action level (>1 × 103 c.f.u./ml) and 4·5% contained actionable levels (>1 c.f.u./100 ml) of faecal indicator bacteria. Eight hundred and three samples from 360 NPMVs were cultured specifically for Legionella and 58% of vessels proved positive for these organisms with 27% of samples showing levels greater than the UK upper action limit of 1 × 103 c.f.u./l. Cabin showers (49%) and hospital shower (45%) were frequently positive. A subset of 106 samples was analysed by quantitative polymerase chain reaction for Legionella and identified a further 11 Legionella-positive NPMVs, returning a negative predictive value of 100%. There was no correlation between NPMV age or size and any microbial parameters (P > 0·05). Legionella pneumophila serogroup 1 was isolated from 46% of NPMVs and sequence-based typing of 17 isolates revealed four sequence types (STs) previously associated with human disease. These data raise significant concerns regarding the management of microbial and Legionella risks on board NPMVs and suggest that better guidance and compliance are required to improve control.

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.

Molecular epidemiology of Legionnaires' disease in Israel.

National surveillance of Legionnaires' disease (LD) is important to inform control measures and facilitate international networking for timely reporting. This study is the first to describe the molecular epidemiology of LD in Israel. Case notifications for 2006-2011, collated through mandatory reporting, were identified and demographic, clinical and laboratory data were extracted. Unrelated clinical and environmental Legionella pneumophila strains were characterized using standard procedures, Dresden panel of monoclonal antibodies and the ESCMID Study Group for Legionella Infections (ESGLI) Sequence-Based Typing scheme. In all, 294 cases were reported (crude incidence 0.67 cases/100 000; age-standardized incidence 1/100 000). LD epidemiological trends and features largely resembled those of the EU, except for a larger proportion of nosocomial cases. Of 28 clinical and 23 environmental strains analysed, 71.4% and 21.7% were serogroup (sg) 1 and the most common immunological subgroup was OLDA/Oxford (64%). Of the clinical strains, OLDA/Oxford, ST1 was the most common (43%) followed by Allentown/France, ST40 (14%). The unusual sg 3 ST338 was found in 17.4% of environmental strains. Novel STs were detected amongst 23.5% of strains. These findings warrant further molecular investigation. Molecular epidemiology data generated from neighbouring countries newly adopting the ESGLI typing scheme for L. pneumophila contribute to understanding of regional strain diversity.

Extension of the Legionella pneumophila sequence-based typing scheme to include strains carrying a variant of the N-acylneuraminate cytidylyltransferase gene.

Sequence-based typing (SBT) combined with monoclonal antibody subgrouping of Legionella pneumophila isolates is at present considered to be the reference standard during epidemiological investigation of Legionnaires' disease outbreaks. In some isolates of L. pneumophila, the seventh allele of the standard SBT scheme, neuA, is not amplified, because a homologue that is refractory to amplification with the standard neuA primers is present. Consequently, a complete seven-allele profile, and hence a sequence type, cannot be obtained. Subsequently, primers were designed to amplify both neuA and the homologue, but these yielded suboptimal sequencing results. In this study, novel primers specific for the neuA homologue were designed and internationally validated by members of the ESCMID Study Group for Legionella Infections at national and regional Legionella reference laboratories with a modified version of the online L. pneumophila sequence quality tool. To date, the addition of the neuAh target to the SBT protocol has allowed full typing data to be obtained for 108 isolates of 11 different serogroups, namely 1, 2, 3, 4, 5, 6, 7, 8, 10, 13, and 14, which could not previously be typed with the standard SBT neuA primers. Further studies are necessary to determine why it is still not possible to obtain either a neuA or a neuAh allele from three serogroup 11 isolates.

Humidifier-associated paediatric Legionnaires' disease, Israel, February 2012.

We report a fatal case of community-acquired Legionnaires' disease in an infant aged under six months. Epidemiological and microbiological investigations suggested that a free-standing cold water humidifier using domestic tap water contaminated with Legionella pneumophila serogroup 1 served as a vehicle for infection. These findings were corroborated by sequence-based typing (SBT). Humidifier-associated Legionnaires' disease can be prevented by appropriate control measures. This case also illustrates the emerging role of SBT in the investigation of legionellosis.

Travel-associated Legionnaires disease in residents from England and Wales travelling to Corfu, Greece, August to October 2011.

Fourteen cases of Legionnaires' disease were confirmed in residents from England and Wales with history of travel to Corfu, Greece, in the 14 days before symptom onset. These cases were reported to the Health Protection Agency national surveillance scheme for Legionnaires' disease in residents of England and Wales between August and October 2011. In addition, one case in a Greek national and a case of non-pneumonic legionellosis in a resident from Scotland were also reported. Few cases shared the same accommodation site in Corfu during their incubation period. Epidemiological investigations and microbiological analysis of clinical and environmental samples excluded a single source but rather implicated several accommodation sites as sources of sporadic infection. Control measures have since been implemented at these accommodation sites and no further cases have been reported. This incident highlights the value of epidemiological typing and the importance of effective international response to control and prevent legionella infection.

Application of Legionella pneumophila-specific quantitative real-time PCR combined with direct amplification and sequence-based typing in the diagnosis and epidemiological investigation of Legionnaires' disease.

The detection of Legionella pneumophila DNA in clinical specimens using quantitative real-time polymerase chain reaction (qPCR) combined with direct sequence-based typing (SBT) offers rapid confirmation and timely intervention in the investigation of cases of Legionnaires' disease (LD). We assessed the utility of a specific L. pneumophila qPCR assay targeting the macrophage infectivity potentiator (mip) gene and internal process control with three clinical specimen types from confirmed LD cases. The assay was completely specific for L. pneumophila, as demonstrated by positive results for 39/39 strains from all subspecies and 16 serogroups. No cross-reaction was observed with any of the 54 Legionella non-pneumophila (0/69 strains) or 21 non-Legionella (0/58 strains). All L. pneumophila culture-positive respiratory samples (81/81) were qPCR-positive. Of 80 culture-negative samples tested, 47 (58.8%) were qPCR-positive and none were inhibitory. PCR was significantly more sensitive than culture for samples taken ≤ 2 days of hospitalisation (94.7% vs. 79.6%), with the difference being even more marked for samples taken between 3 and 14 days (79.3% vs. 47.8%). Overall, the sensitivity of the qPCR was ∼30% greater than that of culture and direct typing on culture-negative PCR-positive samples resulted in full 7-allele profiles from 23/46, 5 to 6 alleles from 8/46 and ≥ 1 allele from 43/46 strains.

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).

Application of multiplex single nucleotide primer extension (mSNuPE) to the identification of bacteria: the Burkholderia cepacia complex case.

Burkholderia cepacia complex (BCC) is characterized by a complex taxonomy constituted by seventeen closely related species of both biotechnological and clinical importance. Several molecular methods have been developed to accurately identify BCC species but simpler and effective strategies for BCC classification are still needed. A single nucleotide primer extension (SNuPE) assay using gyrB as a target gene was developed to identify bacteria belonging to the B. cepacia (BCC) complex. This technique allows the successful detection and distinction of single nucleotide polymorphisms (SNPs) and is effectively applied in routine medical diagnosis since it permits to analyze routinely many samples in a few times. Seven SNuPE primers were designed analyzing the conserved regions of the BCC gyrB sequences currently available in databases. The specificity of the assay was evaluated using reference strains of some BCC species. Data obtained enabled to discriminate bacteria belonging to the species B. multivorans, B. cenocepacia (including bacteria belonging to recA lineages III-A, III-C, and III-D), B. vietnamiensis, B. dolosa, B. ambifaria, B. anthina and B. pyrrocinia. Conversely, identification failed for B. cepacia, B. cenocepacia III-B and B. stabilis. This study demonstrates the efficacy of SNuPE technique for the identification of bacteria characterized by a complex taxonomical organization as BCC bacteria.

Transcatheter embolization of hypogastric arteries as an aid or alternative to surgery in the management of life-threatening pelvic haemorrhage.

Transcatheter embolization for uncontrollable haemorrhage from pelvic organs was performed in 27 patients (12 women, 15 men) as an aid or alternative to surgery. The advantages and peculiarities of different embolic materials employed, and methods for managing intractable haemorrhage are discussed.

[The combined treatment of Parkinson's disease with L-dopa plus decarboxylase inhibitors (carbidopa, benserazide) (author's transl)]. / Zur Behandlung des Parkinson-Syndroms mit Kombinationspräparaten von L-Dopa mit Dekarboxylasehemmern (Carbidopa, Benserazid).

An open cross-over study of 20 patients with Parkinson's disease performed with two drugs containing L-dopa and a peripheral aromatic amino acid decarboxylase inhibitor (benserazide, carbidopa) confirmed the conclusions reached in other clinical trials that this combined treatment of Parkinson's disease is the most effective form of drug therapy available at present. With both drugs, Madopar or Sinemet, an optimum therapeutic result was obtained with relatively small doses of L-dopa (the reduction in L-dopa dosage amounting to about 80%). A loss of efficacy with both drugs, which has observed during long-term treatment of patients with Parkinson's disease, could be avoided by switching the patients from Sinemet to Madopar and vice versa. Determination of L-dopa in the plasma demonstrated that with either drug similar plasma levels of L-dopa were achieved during clinically effective treatment.