Detection of Legionella bacteria in clinical material
such as tissue or body fluids by immunofluorescence
microscopy (e.g. direct fluorescent antibody (DFA)
microscopy or indirect fluorescent antibody test (IFAT))
Detection of Legionella bacteria DNA using qualitative
polymerase chain reaction (PCR) or quantitative real-time
PCR methods.
In recent years the application of diagnostic tests
for legionellosis has changed significantly. Urinary
antigen detection has now largely replaced serology
as the primary diagnostic method (see executive summary
of laboratory survey Appendix B) but serology remains
an important tool for case finding during outbreak
investigations and for late or retrospective diagnosis.
Culture continues to play an important role (see below)
and while PCR is not yet available for routine use,
it is likely that genus-specific assays based on this
technology will be available in the near future.
Since many laboratories now rely almost exclusively
on urinary antigen testing, the detection of L. pneumophila
serogroup 1 is increasing and all other serogroups
are probably underdiagnosed. The antigen detection
test is substantially more sensitive for community-acquired
and travel-associated Legionnaires disease than for
nosocomial infection because the tests are more sensitive
for Pontiac L. pneumophila serogroup 1 than for non-Pontiac
strains of Legionella. Pontiac strains cause the majority
of community-acquired and travel-associated Legionnaires
disease cases but are significantly less common in
nosocomial cases. For this reason culture of sputum
(or other respiratory specimens such as bronchial
washings, when available) is recommended whenever
possible.
It is important that healthcare facilities have policies
in place to test for Legionnaires disease in patients
with nosocomial pneumonia. Effective diagnosis and
evaluation of results are crucial for the adequate
and prompt management of incidents and outbreaks,
for the control of clusters of infection and for the
protection of other patients.
The UK Health Protection Agency guidance note 'Laboratory
Diagnosis of Legionella Infections in the HPA' gives
advice on the selection and usefulness of tests on
clinical specimens. It also provides a testing algorithm
for the diagnosis of legionnaires disease.
Methods used for clinical specimens should be based
on recognised reference procedures. In Ireland, the
most commonly used reference methods are based on
National Standard Methods issued by the UK HPA. The
HPA National Standard Method for Legionella species
is BSO 47.25
All laboratories performing this testing should be
accredited for the methods used and participate in
an appropriate external proficiency scheme. The subcommittee
recommends that an external proficiency scheme is
developed for Ireland.
2.3 Water and environmental samples
2.3.1 Introduction
The usefulness and quality of results on water and
environmental samples is dependent on the appropriateness
and quality of the sampling procedures and plans.
It is therefore important that before any samples
are taken for either surveying or the investigation
of an outbreak the staff involved are appropriately
trained and have a thorough knowledge of the guidelines
to be followed. The UK Environment Agency booklet
'The determination of Legionella in waters and other
environmental samples (2005) Part 1 - rationale of
surveying and sampling' gives guidance on the factors
to be considered before samples are taken.
Considerable laboratory work and resources are required
for the laboratory testing of environmental samples
so it is important that only appropriate samples are
taken and that sampling is carried out in accordance
with the above guidelines. The subcommittee recommends
that laboratory facilities for environmental testing
are available in each Health Service Executive (HSE)
area.
2.3.2 Environmental testing laboratory methods
Methods used for testing environmental samples should
be based on the International Organization for Standardization
(ISO) standard reference methods. ISO 11731:1998 is
the appropriate method.This method has been divided
into 2 parts: the latest part, Part 2 (ISO 11731 -
2:2004)28 and the original ISO 11731:1998 which is
currently under revision and when revision is complete
will subsequently be called ISO 11731 Part 1. All
laboratories performing this testing should also be
accredited for these methods and participate in an
appropriate external proficiency scheme.
2.4 Application of PCR for the detection and enumeration
of Legionella species
Culture on solid agar media in the laboratory is
considered the 'gold standard' for the detection and
enumeration of viable legionellae. However, this approach
is time-consuming because of the slow growth rates
of these organisms and can take up to ten days. Furthermore,
standard culture techniques will not detect viable
non-culturable legionellae in a somnicell state. This
is further complicated by difficulties in isolating
legionellae in samples containing high background
levels of other microorganisms (some of which can
inhibit Legionella growth) or in situations where
legionellae are protected within amoebae or protozoa.
Additionally, some non-L. pneumophilia species grow
poorly on conventional solid media used for the routine
isolation of legionellae.
A brief overview of the application and potential
advantages of PCR technology to the detection and
enumeration of legionellae is provided below, together
with selected references. The reference list is not
intended to be exhaustive but provides a good introduction
to the subject field and relevant literature.
Over the last two decades, the application of PCR
technology has revolutionised the diagnosis of infections
caused by a wide variety of microorganisms, especially
organisms that are slow growing or difficult to grow
in the laboratory. Indeed, PCR represents one of the
few diagnostic tests with the potential to detect
the presence of all known microorganisms, including
Legionella. PCR involves the highly specific amplification
of particular target DNA sequences from the microorganism
under investigation. The target sequences are usually
species-specific. Thermostable enzymes (e.g. Taq polymerase)
that can copy DNA sequences are used to generate millions
of copies of the target sequence in a matter of a
few hours. The highly amplified target sequences can
then be visualised in agarose gels or can be detected
by a variety of other means. Determining the nucleotide
sequence of the amplified target DNA can be used to
validate the specificity of amplification. In this
way, PCR assays can be developed and validated for
the rapid detection of any target DNA sequence and
thus any microorganism.
Over the last ten years a wide variety of PCR tests
have been developed to detect legionellae in environmental
samples (e.g. water samples or samples from cooling
towers),29-34 and from clinical specimens (e.g. broncho-alveolar
lavage, respiratory secretions, lung biopsy samples,
pharyngeal swabs, nasopharyngeal swabs, peripheral
blood mononuclear cells, urine and serum).29;35-44
Several PCR tests have been developed to detect all
Legionella species or specifically just L. pneumophila,
that target DNA sequences from the 16S rRNA gene,45
the 5S rRNA gene,46;47 the 23S-5S spacer region,40
the macrophage infectivity potentiator gene mip30;47-49
and the dotA gene.32 Many PCR tests were found to
be highly sensitive and highly specific, but by their
very nature qualitative, and provided little information
on the relative risk of legionellosis in the case
of environmental samples (i.e. the tests indicated
the presence or absence of Legionella DNA only, with
no information as to the presence of whole cells or
whether they were alive or dead). Furthermore, environmental
and clinical samples may contain PCR inhibitors that
prevent amplification of target sequences and can
result in false-negative results. The problem of PCR
inhibition, particularly that caused by iron compounds
(e.g. rust), fulvic acid (a natural acidic organic
polymer found in soil, sediment, or aquatic environments)
humic acids (major constituents of soil organic matter
that can be found in streams) frequently present in
environmental water samples, as well as other inhibitors,
can limit the usefulness of PCR-based tests unless
effective DNA purification methods are employed and
PCR inhibition controls are routinely included (i.e.
positive amplification controls) in the PCR tests.32;34
Qualitative PCR with high sensitivity and high specificity
has been used to successfully detect legionellae in
environmental and respiratory samples in a matter
of hours and has proven to be a valuable adjunct to
culture, serology and urinary antigen detection. However,
its usefulness with environmental samples is limited
by its failure to distinguish between live, viable
non-culturable or dead Legionella cells.
Several research groups have described the development
of quantitative real-time PCR assays for detecting
legionellae in environmental and clinical samples.29;30;32;33;40;41;45
This approach provides information on the number of
Legionella genome units in the samples tested, but
equivalence with the number of colony forming units
(CFU) has not yet been established robustly. Usually,
the number of genome units is higher than the number
of CFU, probably due to the presence of viable non-culturable
and dead Legionella cells in the samples tested. Nonetheless,
recently developed quantitative real-time PCR assays
have shown immense potential for the detection and
enumeration of Legionella in both clinical and water
samples, with many benefits including speed (results
within a few hours), high-specificity, high-sensitivity,
stability and cost-effectiveness. Multiplex real-time
PCR assays capable of the simultaneous detection of
multiple Legionella species have also been described.50
These assays are ideally suited to routine surveillance
of water samples and for clinical specimens. However,
it is important to emphasise that these assays have
to be rigorously validated and controlled to obtain
meaningful and informative results. Some researchers
have combined real-time PCR Legionella detection with
immunogenetic separation of L. pneumophila from water
samples. Immunogenetic separation involves the interaction
of Legionella-specific antibodies attached to paramagnetic
beads and Legionella surface antigens, permitting
separation of Legionella cells from water samples
by placing a bead-water suspension in a strong magnetic
field.32 This helps to specifically enrich Legionella
recovery from water samples contaminated with different
bacterial species. The DNA from Legionella recovered
by immunogenetic separation can then be used as a
template for quantitative real-time PCR detection.
The development of standardised real-time PCR protocols
and reagents for detecting Legionella will go a long
way to making this technology more accessible and
applicable in the clinical laboratory and for the
routine surveillance of water supplies and water distribution
networks in buildings. One potential approach to standardisation
is the use of commercial kits for the identification
and enumeration of Legionella and for DNA purification
from samples. Several such kits are currently available
(e.g. AquaScreen, Minerve Biolabs, Germany; iO-Check
legionella, BioRad, USA) but large-scale comprehensive
independent comparative studies on their sensitivity,
specificity and accuracy have yet to be undertaken.
Qualitative PCR and real-time PCR have become important
investigative tools in many clinical and environmental
microbiology laboratories for a wide variety of applications.
The equipment required is expensive to purchase and
maintain and requires considerable technical expertise.
However, use of PCR technology is cost-effective when
applied to microorganisms that are slow growing or
difficult to grow in the laboratory, such as Legionella,
and is ideal when accurate and rapid detection is
required. PCR will often detect the presence of a
microorganism in a sample when culture results are
negative, which may occur in a patient being treated
with antibiotics.
Finally, DNA microarrays for detecting Legionella
in water and clinical samples are very likely to be
developed as alternative molecular tools for Legionella
detection. This technology involves immobilising species-specific
oligonucleotides on to the surface of microarrays
that hybridise with target DNA in test samples permitting
fluorescent signal detection.51 This technology has
the potential for the simultaneous detection of multiple
microbial species and is ideally suited to pathogen
and opportunistic pathogen detection.
In conclusion, PCR assays (especially real-time PCR)
have immense potential for the accurate, rapid and
cost-effective detection and enumeration of legionellae
in environmental samples. PCR assays also have immense
potential to enhance our ability to rapidly and accurately
diagnose Legionella infections. The development of
standardised and validated PCR protocols and procedures
involving the integration of efficient and rapid sample
preparation techniques with rapid PCR technologies
in coming years should significantly improve the detection,
prevention and management of Legionella infection.
This approach should also be invaluable for evaluating
the effectiveness of water treatment regimes. Currently,
culture on solid media remains the 'gold standard'
for Legionella detection and enumeration.
