Diagnostic Tests for Cystic Fibrosis Epidemic Strains
Submitting InstitutionsUniversity of Liverpool,
Liverpool School of Tropical Medicine
Unit of AssessmentClinical Medicine
Summary Impact TypeHealth
Research Subject Area(s)
Medical and Health Sciences: Cardiorespiratory Medicine and Haematology, Medical Microbiology, Oncology and Carcinogenesis
Summary of the impact
Chronic lung infections due to Pseudomonas aeruginosa are the major cause of morbidity and
mortality associated with cystic fibrosis. Some strains of P. aeruginosa transmit between patients
(epidemic strains). The Winstanley group, at the University of Liverpool (UoL) since 1999, in
collaboration with clinicians in Liverpool, has developed diagnostic PCR assays for identification of
the most widely reported UK epidemic strains of P. aeruginosa. The NHS clinicians use these
tests to make informed decisions about patient segregation leading to markedly reduced incidence
of LES infections. Researchers internationally have adopted the UoL research results and strategy
to tackle other transmissible strains and modify clinical procedures.
Cystic Fibrosis (CF) affects over 9000 people in the UK alone, and CF patients require lifelong
healthcare. An estimated £110m pa is spent on treating UK CF patients. Chronic lung infections
due to Pseudomonas aeruginosa are the major cause of morbidity and mortality associated with
CF. It is very important that CF patients are kept free from infection with P. aeruginosa for as long
as possible because (i) once a chronic infection is established, it is never eradicated and (ii) the
earlier the infection, the worse the patient prognosis. The UoL has played the main role in
recognising the important contribution in CF of a limited number of epidemic (transmissible) strains
of P. aeruginosa, in particular the Liverpool Epidemic Strain (LES), which is the most prevalent in
the UK, and has also been reported in North America. Patients infected with the LES suffer greater
morbidity than patients infected with other strains.
Research at the UoL (2002-2013) carried out by the Winstanley group (specialising in microbiology
and infection) has included the design, development and publication of PCR assays to identify the
LES and Midlands1 epidemic strains, and a multiplex PCR assay for the identification of the three
most widely reported UK epidemic strains of P. aeruginosa (LES, Midlands1 and Manchester
strain). The work involved collaboration with clinical colleagues, particularly Dr Martin Walshaw
(Head of the Adult CF Unit, Liverpool and Honorary status in UoL). The clinical collaboration
ensured the supply of relevant clinical samples and strains to enable testing of the assays, and
facilitated the introduction into diagnostic laboratories, initially in Liverpool. The Winstanley group
published a PCR assay for the LES in 2002 and further improvements to the assay in 2006, along
with a PCR assay for another prominent epidemic strain (Midlands1). A multiplex PCR assays for
three epidemic strains was published in 2008. Other publications by the Winstanley group were
used to demonstrate the utility of the assays and generate the basic science used to design and
improve the assays.
The assays were designed by using various genetic / genomic techniques to identify regions of the
genomes of P. aeruginosa epidemic strains that could be targeted for the design of specific PCR
assays (ie. regions present only in these strains).
Since being introduced into the routine analysis of strains from CF patients in Liverpool, these tests
have been used to guide clinicians with respect to decisions regarding segregation of patients.
Craig Winstanley is Professor of Bacteriology in the UoL and has worked at the University
continuously since 1999. There were various PhD students and postdoctoral researchers (under
the supervision of Winstanley) involved in the work, as well as clinical collaborators (particularly Dr
Martin Walshaw Head of the Adult CF Unit in Liverpool and honorary UoL academic), who were
responsible for providing samples and clinical input. Dr Jo Fothergill (Research Fellow) also
played a prominent role in the work (since 2005). Along with Winstanley, she was responsible for
developing the multiplex PCR assay.
References to the research
The impacts stem from a whole series of research papers published in peer-reviewed journals
identifying, characterising and developing / refining diagnostic tests for epidemic strains, especially
the LES. Some relevant publications are listed below.
1. Mowat E, Paterson S, Fothergill JL, Wright EA, Ledson MJ, Walshaw MJ, Brockhurst
MA and Winstanley C (2011). Pseudomonas aeruginosa population diversity and turnover
in cystic fibrosis chronic infections. American Journal of Respiratory and Critical Care
Medicine 183:1674-1679. Citations: 39 Impact Factor: 11.041
2. Winstanley C, Langille MGI, Fothergill JL, Kukavica-Ibrulj I, Paradis-Bleau C,
Sanschagrin F, Thompson NR, Winsor GL, Quail MA, Lennard N, Bignell A, Clarke L,
Seeger K, Saunders D, Harris D, Parkhill J, Hancock REW, Brinkman FSL and Levesque
RC (2009). Newly introduced genomic prophage islands are critical determinants of in vivo
competitiveness in the Liverpool Epidemic Strain of Pseudomonas aeruginosa. Genome
Research 19:12-23. Citations: 85 Impact Factor: 14.397
3. Fothergill JL, Upton A, Pitt TL, Hart CA and Winstanley C (2008). Diagnostic multiplex
PCR assay for the identification of the Liverpool, Midlands 1 and Manchester epidemic
strains of Pseudomonas aeruginosa. Journal of Cystic Fibrosis 7:258-261. Citations: 16
Impact Factor: 2.873
4. Salunkhe P, Smart CH, Morgan JAW, Panagea S, Walshaw MJ, Hart CA, Geffers R,
Tümmler B and Winstanley C (2005). A cystic fibrosis epidemic strain of Pseudomonas
aeruginosa displays enhanced virulence and antimicrobial resistance. Journal of
Bacteriology 187:4908-4920. Citations: 94 Impact Factor: 3.177
5. Al-Aloul M, Crawley J, Winstanley C, Hart CA, Ledson MJ and Walshaw MJ (2004).
Increased morbidity associated with colonization by an epidemic Pseudomonas aeruginosa
strain in cystic fibrosis patients. Thorax 59:334-336. Citations: 80 Impact Factor: 8.376
6. Parsons YN, Panagea S, Smart CHM, Walshaw MJ, Hart CA and Winstanley C (2002)
Use of subtractive hybridization to identify a diagnostic probe for a cystic fibrosis epidemic
strain of Pseudomonas aeruginosa. Journal of Clinical Microbiology 40: 4607-4611.
Citations: 34 Impact Factor: 4.068
7. Fothergill JL, White J, Foweraker JE, Walshaw MJ, Ledson MJ, Mahenthiralingam, E. and
Winstanley C (2010). Impact of Pseudomonas aeruginosa genomic instability on the
application of typing methods in chronic cystic fibrosis infections. Journal of Clinical
Microbiology 48:2053-2059. Citations: 13 Impact Factor: 4.068
Grant awards relating to this work
2013-2016. Cystic Fibrosis Trust. Clinical Exploitation of Genomics Data Produced by the
Pseudomonas International Consortium, £99,743, PI C Winstanley
2013-2016. Cystic Fibrosis Canada. Clinical Exploitation of Genomics Data Produced by
the Pseudomonas International Consortium, C$316,000 (£204,000) CoI C Winstanley
2011-2014. Wellcome Trust. Genetic diversification and within-host evolution of chronic
Pseudomonas aeruginosa infections in cystic fibrosis patients, £249,795, S Paterson, C
Winstanley and M Brockhurst
2009-2012. Wellcome Trust. Understanding the role of temperate bacteriophages in the
ecology and evolution of Pseudomonas aeruginosa in the cystic fibrosis lung environment,
£223,208 C Winstanley and M Brockhurst
2008-2011. Dr. Hadwen Trust. Response of populations of P. aeruginosa to antimicrobial
challenge during chronic infections in CF, £134,954, C Winstanley
2008-2012. NIHR. Virulent epidemic strains of Pseudomonas aeruginosa in cystic fibrosis,
NIHR, £153,762 (approx.) C Winstanley (A component of the Biomedical Research Centre
in Microbial Diseases c.£20 million total grant)
2005-2008. CF Trust/Big Lottery Fund. Genetic factors contributing to the success of CF
"superbugs" in the UK, £93,867, C Winstanley and CA Hart
2002-2005. Cystic Fibrosis Trust. Use of subtractive hybridisation to identify novel
genomic regions of an epidemic strain of Pseudomonas aeruginosa, £62,705, C
Winstanley and CA Hart
Details of the impact
Building on the work of the Winstanley group prior to this impact period, when the research led to
wider recognition that transmissible strains of P. aeruginosa, and the LES in particular, are
important in the context of CF, the group has achieved the following impacts:
- The multiplex PCR test for transmissible strains (combining Winstanley group designed PCR
tests for the LES and Midlands1 strains with a published PCR assay for the Manchester
strain) has been used routinely in the NHS diagnostic laboratories serving both Adult
(Liverpool Heart & Chest Hospital) and Children's (Alder Hey Hospital) CF Units from 2008.
This therefore represents a new clinical product.
- The clinical uptake of the multiplex PCR test has extended from local NHS to national
level, as HPA Colindale used it as its main assay to test P.aeruginosa isolates from CF
Units across the UK for the majority of the period Jan 2008 to July 2013. The PCR tests
developed in Liverpool have helped in the recognition of the presence of the LES in other
CF centres. HPA Colindale receives thousands of P. aeruginosa isolates each year from CF
Units across the UK for strain typing. Although they have more recently switched to wider
use of an alternative typing method (VNTR), the PCR assays are still used occasionally by
Public Health England, Colindale . The LES remains the most common clone of P.
aeruginosa associated with CF infections in the UK (Martin et al. 2013 J Med Microbiol on-
line ahead of print).
- The use of the multiplex PCR test has allowed clinicians to make informed decisions about
patients with transmissible strains of P. aeruginosa with respect to segregation of patients,
because patients are cohorted for treatment depending on their microbiological status (ie.
LES-positive or LES-negative). This has had an impact on the health of CF patients
because the use of this test has contributed to the fact that new cases of LES infections are
extremely rare in Liverpool. The main beneficiaries are CF patients who are not yet infected
with P. aeruginosa. Prior to the use of PCR tests to identify patients infected with
transmissible strains, there was no patient segregation. The consequence of this can be
seen from the fact that prior to its discovery and the development of a simple test, the LES
was already present in 80% of P. aeruginosa-infected patients in the Liverpool paediatric
unit (Cheng et al 1996 Lancet 348:639-642, Panagea et al. 2004 Molecular Diagnosis
7:195-200). Most of these patients have now moved on to the adult unit. Because of
effective segregation measures, there are now only small numbers of LES-infected patients
at the Liverpool paediatric unit, and there have been no new cases for several years. Cases
in the paediatric CF Unit in Liverpool have decreased from 47 LES-positive patients in 2003
to 8 LES-positive patients in 2009. In the adult unit there was a reduction in the proportion
of patients with LES (2003 to 2009) from 71% to 53% [12,13]. Since 2009, there have been
no new cases of LES-infected patients in the Adult CF Unit other than patients new to the
unit (ie. transferred from the paediatric centre or new to the region). There is a clear clinical
benefit to restricting the spread of the LES, which is associated with increased patient
morbidity (Al-Aloul et al. 2004 Thorax 59:334-336). This increased morbidity has been
shown to be true also for patients in North America infected with the LES (Aaron et al. 2010
JAMA 304:2145-2153). It is important to note that the impact of segregation based on
genotyping is ongoing. Patients, including new patients and first-time P. aeruginosa infected
patients, are regularly monitored using the PCR tests, enabling LES-positive patients to be
treated apart from non-LES, and minimising the threat of further spread of the strain. It is
likely that there has also been impact at other CF centres. Public Health England has
confirmed that "Professor Winstanley's research has played an important role in the
significant extensions of lifespan and quality of life improvements experienced by CF
patients in England in the last few years" .
- In addition, the research highlights the existence of transmissible strains of P. aeruginosa
(thought unlikely before Liverpool's initial LES report in 1996) has impacted on
researchers worldwide and contributed to the discovery of other transmissible strains
(Fothergill, Walshaw & Winstanley 2012 Eur Resp J 40:227-238), leading to changes in
clinical procedures aimed at control of cross infection.
- The use of our PCR assay is recommended in guidelines published by the UK Cystic
Fibrosis Trust Microbiology Laboratory Standards Group designed to underpin the
development of National Standard Operating procedures for the processing of respiratory
samples submitted from people with Cystic Fibrosis . This was circulated to all diagnostic
laboratories in the UK serving CF units. Hence, the assay has impacted on policy change.
Sources to corroborate the impact
Each source listed below provides evidence for the corresponding numbered claim made in section
4 (details of the impact).
- "Laboratory Standards for Processing Microbiological Samples from People with Cystic
4.3.1 gives details of a multiplex-PCR test developed in Liverpool for three epidemic
strains (LES, Midlands1 and Manchester), quoting two UoL studies [3,7] (Fothergill et al.
2008; Fothergill et al. 2010).
- Contact: Alder Hey Children's Hospital, CF paediatric unit, can confirm that the PCR tests
have been used routinely to screen CF patients in Liverpool and corroborate the clinical
impact in terms of reduced incidence of LES infection.
- Letter: Public Health England, Colindale.
- Contact: Papworth, can confirm that the PCR targets devised by us for the LES (PS21 and
LES-F9) are used in PCR assays.
- The publication Ashish et al. 2013 Journal of the Royal Society of Medicine 4:1 includes
data supporting the clinical impact in terms of reduced incidence of LES infection in
- Morris D, Fallon L, Heaf L, Burrows EF, Wallace H, McNamara PS, Winstanley C, Southern
KW. (2009) A reducing prevalence of the Liverpool Epidemic Strain of Pseudomonas
aeruginosa in children attending the index paediatric clinic. Ped Pulmonol vol 32 pp 325