1. Water systems
44
Health Estate Journal
August 2011
A
round 300-400 cases of
Legionnaires’ disease are reported
every year in England and Wales,
and, while the incidence is low in
hospitals, those affected will be the most
susceptible – the immuno-compromised,
ICU patients, transplant and oncology
patients, diabetics, smokers, and
alcoholics – and the most likely to die.
While the mortality rate for the general
population is around 13%, the nosocomial
rate has reached 32%.
Legionnaires’ disease is the severest
form of infection caused by Legionella
bacteria, opportunistic waterborne
pathogens which occur naturally in the
environment. Of the 50-plus species, only
20 appear to be associated with disease
in humans, with Legionella pneumophila
by far and away the most significant.
Victims are infected by inhaling organisms
suspended in air from an aerosol, or
sometimes by aspiration, particularly in
the case of hospital patients. Although
less than 5% of exposed individuals will
develop Legionnaires’ disease, up to
95% may contract a milder form of
legionellosis known as Pontiac Fever, a
short, influenza-like illness that does
not require treatment. However, some
exposed individuals will remain completely
symptomless.
Dormant at low temperatures
Dormant at low temperatures, Legionella
multiplies to large numbers in water
between temperatures of 20˚C and 45˚C,
and cannot survive at temperatures above
50˚C. Other risk factors for outbreaks are
water stagnation, for example in pipework
“dead legs”, leading to build-up of biofilm
which harbours pathogenic bacteria, and
lack of appropriate biocide concentrations.
Although 27% of Legionnaires’ disease
outbreaks are associated with cooling
towers, hot and cold water systems are
also major culprits, with spa pools the
third most significant source.
In new buildings, warmer weather and
energy conservation requirements are
also making cold water systems more
vulnerable to microbial contamination.
Heat is now better retained in buildings,
and is transferred to the cooler parts of
the building – normally the cold water
system. Even well-insulated pipes may be
inappropriately “warmed” by hot pipes
running alongside cold pipes in service
ducts, or above ceiling-mounted radiant
heat panels.
In the UK, there is a legal requirement
to follow the “L8” guidelines1
to prevent
Legionnaires’ disease, which includes
sampling for Legionella species to monitor
the effectiveness of control measures
against the organism.
Faster Legionella
testing on horizon
While the “traditional” way to measure Legionella quantitatively in water is based on a complex culture method
where results can take up to 14 days, the last few years have seen the availability of very rapid real-time
monitoring of the bacterium in water systems, with the development of quantitative polymerase chain reaction
(qPCR), a process which gives results “within hours”. To date, however, a lack of consensus on how to
interpret such results in relation to those from culture has been a stumbling block, although, as Susan
Pearson, a freelance journalist and public relations consultant specialising in medicine and the environment,
reports, the positive results of a recent multi-centre European study mean this could soon all change.
In the UK, there is a legal requirement to follow the
‘L8’ guidelines1
to prevent Legionnaires’ disease,
which includes sampling for Legionella species
Target DNA strand taken from microorganism.

2. Water systems
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Health Estate Journal
August 2011
A ‘complex
culture method’
The traditional way of measuring
Legionella quantitatively in
water is based on a complex
culture method, which involves
concentrating bacteria from
water by filtration and/or
centrifugation, followed by
heat and acid treatments, and
culture on selective media,
including nutrients and
antibiotics such as Vancomycin
and Cycloheximide. Although
currently the “gold standard”,
the culture method can take
up to 14 days to produce
results. It also has an
inconsistent recovery rate for
bacterial cells, from between
20% to 70%, in turn giving
variable results.
However, a much faster solution has
now come into sight. In the last few
years, very rapid real-time monitoring of
Legionella in water systems has become
available with the development of
quantitative polymerase chain reaction
(qPCR), which can produce results within
a matter of hours. This has proved a
specific and sensitive method, yet so far
could not be adopted alongside culture
as a method for compliance testing for
Legionella counts because the action
levels detailed in legislation and guidance
such as L8 have been based solely on
the standard culture method.
Until now there has existed no
consensus on how qPCR results might
be interpreted in relation to results from
culture. However, this could be about to
change.
Positive results from a multi-centre
European study to define alert and action
guidelines for the use of a qPCR protocol
for monitoring the control of legionellas,
and an evaluation trial in the UK at the
Brighton and Sussex University Hospitals
NHS Trust’s Environmental Microbiology
Unit, have recently made major steps
forward in resolving this problem.
An international effort
Led by Dr John Lee, an international
Legionella expert based in the UK,
the European study involved seven
laboratories from six countries (France,
Germany, Italy, Spain, Switzerland,
and the UK); it came to the very positive
conclusion that “it is possible to derive
guidelines on the use of qPCR for
monitoring the control of legionellas
with consequent improvement to
response and public health protection”.
“Action and alert levels can be adjusted,”
the researchers said, and “remedial
actions can be validated earlier, with
only a small increase in the frequency
of action being required.”2
Each of the participating laboratories
sampled at least six systems weekly for
a minimum of six weeks, including both
cooling towers and hot and cold water
systems. The systems selected were
expected to produce some positive
results. Each sample was divided into
two equal parts, one of which was tested
using qPCR for Legionella species and
L. pneumophila, while the other was
tested for Legionella species using the
culture method.
The qPCR system used, from Pall
GeneDisc Technologies (Bruz, France),
includes separate tests for L. pneumophila
and Legionella species. This system
simplifies PCR and minimises
contamination issues by using a closed
disk incorporating reagents for the
analysis of five DNA extracts from
water samples and one negative control,
arranged in six analytical sectors.
The GeneDisc plates were analysed
using Pall’s GeneDisc Cycler. To make
sure that all the participating laboratories
were able to use the qPCR method
reliably, a ring trial performed at the
beginning of the study established that
all the laboratories performed well, with a
deviation of only <0.2 between samples.
The practical upshot of this trial was
that the researchers were able to propose
algorithms for use with cooling tower
samples, with another set for healthcare
settings and outbreak investigations.
Adoption in future
In the meantime, following its own
validation trial, the Brighton and
Sussex University Hospitals NHS Trust’s
laboratory is planning to adopt the
GeneDisc qPCR test to supplement, or
even replace, culture as
the first test they offer for
Legionella monitoring once
accreditation has been
approved by UKAS (United
Kingdom Accreditation
Service) for this service.
Based at the Princess
Royal Hospital in Haywards
Heath, West Sussex, the food,
water, and environmental
microbiology laboratory, once
an HPA unit, is now managed
as a commercial laboratory
by the NHS Trust. Although
it supports the Trust’s
microbiology diagnostics
services, the bulk of its work
is analysis of food, water,
and environmental samples
to provide compliance
monitoring for food, leisure,
and other industries in the East and
West Sussex, West Kent, and South
Surrey areas.
The laboratory’s manager, Clare
Reynolds, explains how they first became
aware of the GeneDisc test 18 months
ago: “There were already PCR tests
around for Legionella in water, but these
could only indicate the presence or
absence of the organism – what is
significant about this test is that it is
able to quantify the amount of Legionella
DNA present. This is essential to allow
the guidelines on whether action needs
to be taken to be followed. For example,
in a sample from a cooling tower,
100 Legionella per litre is considered
‘under control’, while 100-1,000 organisms
per litre equates to an ‘amber alert’
requiring a risk assessment, and
over 1,000 Legionella per litre would
necessitate immediate corrective action.
“This is what caught our eye, along with
the rapid timescale that could allow a
sample to be taken in the morning, and
a result to be available by the afternoon.
We believe this could be a huge
advantage to our clients. The lengthy
culture process can mean economic
disaster if a facility has to be shut down
while waiting for results.”
The straightforwardness of the test
was also a bonus, Clare Reynolds added,
although she also emphasised that
“expertise in carrying out PCR protocols
(which we have) is essential”.
The laboratory ran the test alongside
its current culture method to check for
correlation. Staff checked sterile water to
calibrate for a negative result, and tested
around 1,000 samples where it was
Researchers were able to propose algorithms for
use with cooling tower samples, with another set
for healthcare settings and outbreak investigations
The GeneDisc Cycler.

3. Water systems
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Health Estate Journal
August 2011
unknown whether they were positive
for Legionella, and further “spiked”
samples containing known amounts.
The evaluation was based on types of
water samples that the laboratory would
be most likely to be presented with
for testing, such as from local mains,
hospital waters, and spa pools. Samples
from the areas the facility covers are all
hard water.
“Our biggest problem,” Clare Reynolds
said, “was that there were no published
algorithms on how to interpret results,
and the levels which require action.
This work is ongoing. In the meantime
the publication of the European study is
hugely significant for us – this is what
we have been waiting for. We now have
algorithms we can compare our work to,”
she says, “and, significantly, our findings
show very similar results.”
Both trials show how it is possible to
quantify L. pneumophila, but the results
for the test for Legionella species are
less clear. While qPCR gives a high
reading when Legionella species are
present in high numbers, indicating clear
“failures”, the significance of low level
results is less clear, and would need
confirmation by culture.
Clare Reynolds is, however, very positive
about the test: “We are very pleased to
be ‘piloting’ this test in the South East,
and plan to offer it as an initial screen
once we have completed our UKAS
accreditation, which is imminent.
We anticipate that the speed of this test
means we will be able to more than
double our throughput of samples, as
I think probably the vast majority will be
able to be reported on the PCR results
alone, with very few needing culture to
help interpret the results.”
References
1 Health and Safety Executive (HSE):
Legionnaires’ disease: the control
of Legionella bacteria in water
systems. Approved Code of Practice
Guidance L8. ISBN 0 7176.
2 Lee J V, Lai S, Exner M, Lenz J, Gaia V,
Casati S, Hartemann P, Lück C, Pangon
B, Ricci ML, Scaturro M, Fontana S,
Sabria M, Sánchez I, Assaf S,
Surman-Lee S. An international trial
of quantitative PCR for monitoring for
Legionella in artificial water systems.
J Appl Microbiol 2011; 110: 1032-44.
Susan Pearson
Susan Pearson (susan@wordways.co.uk)
is a freelance writer, editor, and PR
consultant, specialising in medical and
environmental issues. A biology graduate
with a degree from Bristol University,
she worked in medical research in the
Department of Immunology at St Mary’s
Hospital Medical School in London before
moving into publishing – as the editor
of the clinical laboratory industry’s
Medical Laboratory World.
Since then, wide-ranging roles have
included project editor for a life sciences
encyclopaedia for schoolchildren, while
environmental clients have included
Bristol Friends of the Earth, Campaign
to Protect Rural England, and the
Soil Association. She is a frequent
contributor to the Institute of Biomedical
Science’s journal, The Biomedical
Scientist, and has also written for
the Clinical Services Journal.
We anticipate that the
speed of this test means
we will be able to
more than double our
throughput of samples