Diagnostic multiplex PCR assay for the identification of the Liverpool Midlands 1 and Manchester CF epidemic strains of

Division of Medical Microbiology, University of Liverpool, Daulby Street, Liverpool L69 3GA, United Kingdom.
Journal of Cystic Fibrosis (Impact Factor: 3.48). 06/2008; 7(3):258-61. DOI: 10.1016/j.jcf.2007.09.002
Source: PubMed


Individual PCR amplification tests have been developed for three UK CF epidemic strains, the Liverpool epidemic strain (LES), Midlands 1 and the Manchester epidemic strain (MES). We report a simple diagnostic multiplex PCR test that can be used to screen for all three of these strains. To evaluate the test, we screened collections of LES, MES and Midlands 1 isolates, along with various CF and non-CF non-epidemic Pseudomonas aeruginosa strains. The test was 100% sensitive and 100% specific in the identification of these UK CF epidemic strains.

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    • "The presence of OXA-type carbapenemases in A. baumannii was determined using the assay described by Woodford et al. [11] with the addition of bla OXA-143 specific primers [12], whilst other ␤-lactamase genes were sought using the series of multiplex PCR assays described by Dallenne et al. [13]. Clinical isolates of E. coli and P. aeruginosa were assigned to epidemic lineages using the PCR-based assays described by Clermont et al. [14] and Fothergill et al. [15] "
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    • "Accessory genome profiling. PS21 and LESF9 (both markers for the Liverpool strain) were sought using previously published primers (Fothergill et al., 2008). Some targets used in the ArrayTube (AT) microarray (Wiehlmann et al., 2007) (PA0636, PA0722, PA0728, PA2185, PA2221, PA3835 and orfl) were sought using primers described inTable 1. "
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    ABSTRACT: Variable Number Tandem Repeat (VNTR) analysis at nine loci of isolates of Pseudomonas aeruginosa submitted to the national reference laboratory from UK hospitals and from over 2,000 patients between June 2010 and June 2012 revealed four widely found types that collectively were received from approximately a fifth of patients, including from those with cystic fibrosis. These types were also prevalent among related submissions from the clinical environment and were received from up to 54 (out of 143) hospitals. MLST and blaOXA-50-like sequencing confirmed the clonal relationship within each cluster, and representatives from multiple centres clustered within about 70% by pulsed-field gel electrophoresis. Illumina sequencing of 12 isolates of 'cluster A' of VNTR profile 8, 3, 4, 5, 2, 3, 5, 2, x' (where the repeat number at the last, most discriminatory locus is variable) revealed a large number of variably present targets in the accessory genome and seven of these were sought by PCR among a larger set of isolates. Representatives from patients within a single centre mostly had distinct accessory gene profiles, suggesting that these patients acquired the strain independently, while those with clear epidemiological links shared the same profile. Profiles also varied between representatives from different centres. Epidemiological investigations of widely found types such as these require the use of finer-typing methods, which increasingly will be informed by next generation sequencing.
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    • "2008–2009: In March 2008, by combining the specific primers and modifying the product mix we identified LES, Manchester and Midlands1 strains from one test (combined multiplex PCR).26 Primer F9 added to the PCR mix increased the specificity of the test to identify LES. "
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    ABSTRACT: To assess if cohort segregation policies are effective in preventing cross-infection in cystic fibrosis (CF) clinics. A prospective cohort study. A large adult CF centre in Northwest England. All CF patients cared for at the Liverpool adult CF centre 2003-2009. Regular sputum sampling with genotyping of pseudomonas aeruginosa (Psa) isolates led to a policy of inpatient and outpatient segregation by microbiological group. Prevalence and cross-infection/super-infection rates of a transmissible Psa strain, i.e. the Liverpool epidemic strain (LES) in adult CF patients at the Liverpool adult CF centre from 2003 to 2009. There was a decline in the proportion of patients with LES (71-53%) and an increase in those with unique strains (23-31%) and without Psa infection (6-17%) from 2003 to 2009. There were two cases of LES super-infection and one case of new chronic Psa infection (with a unique strain). There were no cases of transmissible strain infection in patients previously uninfected by Psa. Our segregation policy has halted the spread of the commonest highly transmissible strain in the UK (LES) in our clinic, without endangering patients who were not previously infected with Psa. It confirms that if genotypic surveillance is used, it is unnecessary to segregate patients infected with unique strains from those without Psa infection.
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