Legionella Laboratory Case Definition (LCD)

The Public Health Laboratory Network have developed a standard case definition for the diagnosis of diseases which are notifiable in Australia. This page contains the laboratory case definition for Legionella.

Page last updated: 16 August 2019

Authorisation: PHLN

Consensus Date: 15 November 2018


1 PHLN Summary Laboratory Definition

1.1 Condition:

Legionella infections (Legionnaires’ Disease)

1.1.1 Definitive Criteria

  1. Isolation by culture of Legionella species from any clinical specimen; or
  2. Detection of Legionella species-specific target by Polymerase Chain Reaction (PCR); or
  3. Detection of Legionella species-specific antigen in urine; or
  4. Seroconversion or significant increase in serum Legionella antibody level.

1.1.2 Suggestive Criteria

  1. Single high antibody level to L. pneumophila (Lp), L. longbeachae or other member of Genus Legionella.

2 Introduction

Legionnaires’ Disease is an acute infection caused by bacteria belonging to the genus Legionella that usually occurs in adults and is often associated with underlying disease. Legionnaires’ disease usually presents as pneumonia. Systemic features are common and may predominate, and Legionella wound infection occasionally occurs. Legionella pneumophila serogroup 1 (Lp1) is the most common cause of Legionnaires’ Disease. However, in Australia up to half of all cases are due to L. longbeachae serogroup 1, which is much commoner in some Australian jurisdictions than others7. The source of Legionnaires’ Disease is environmental, although an environmental source is rarely identified with confidence in sporadic cases. Outbreaks or case clusters of Legionnaires’ Disease (usually due to Lp serogroup 1 (Lp1)) occur sporadically and may be traced to a common source, such as an air-conditioner cooling tower, showers or other potable water outlets18. The use of biocides may prevent culture of Legionella species from environmental samples collected in these case clusters, resulting in greater reliance on nucleic acid amplification tests8. Cases of infection with L. longbeachae serogroup 1 (Ll1) are usually associated with contaminated commercial potting soils or similar garden products17. Infections due to other serogroups and species are less common, usually sporadic and more likely to occur in individuals with underlying disease associated with immune deficiency or respiratory pathology. The diagnosis of Legionnaires’ Disease is made by

  1. isolation of bacteria belonging to the genus Legionella from a clinical specimen; usually respiratory, but occasionally blood or wound swab;
  2. a significant change in the level of serum antibody (or seroconversion) against a Legionella species, using a suitably validated test;
  3. detection of specific urinary antigen (Lp1 only) or
  4. specific Legionella nucleic acid amplification test in an appropriate specimen.

3 Tests

3.1 Culture1,14

Legionellae are fastidious bacteria requiring essential growth promoting factors including cysteine for their successful isolation from clinical material. Legionella grows aerobically and requires a high level of humidity.

3.1.1 Suitable Specimens

Bronchial washings and broncho-alveolar lavage specimens are the best choice and, if possible, should be collected before commencing antibiotic therapy. Pleural aspirates, lung or other relevant tissue specimens are suitable if available. Avoid using saline during collection of specimens since this inhibits the growth of Legionella species. Where this is unavoidable, such as with bronchoscopy specimens, the specimen should be cultured promptly. If delays setting up cultures are expected the specimen can be centrifuged and resuspended in a growth medium such as trypticase soy broth (TSB).

Expectorated sputum and tracheal aspirates are less satisfactory due to heavy contamination with oral flora and a relatively low Legionella content. These specimens should be cultured to both non- selective and selective media. Additional agar media can be inoculated after acid or heat treatment to reduce the growth of contaminants.

3.1.2 Media

Most laboratories use buffered charcoal yeast extract agar (BCYE) with added ferric pyrophosphate, L -cysteine and alpha-ketoglutarate. More selective agar media in common used are BCYE with added vancomycin, polymyxin B and pimafucin (BCYE VPP), and BCYE with cefamandole, polymyxin B and anisomycin (BMPA). Another alternative Legionella selective agar is modified Wadowsky and Yee medium (MWY) which contains anisomycin, polymyxin B and vancomycin.

Legionella species usually require 48 hours’ incubation before growth is visible. Colonies may not be clearly visible for up to five days or more. Culture plates should be incubated and examined daily for typical colonies for up to 10 days’ incubation using a dissecting microscope with side lighting. This technique highlights the iridescence of early colony growth, and reveals the ground glass appearance of mature colonies.

3.1.3 Suitable sensitivity

The quality of the specimen determines the sensitivity of culture methods. Patients with milder disease may have a lower bacterial load in their lower respiratory tract. Delayed inoculation will reduce the efficiency of culture. The presence of contaminating commensal bacteria may inhibit the growth of Legionella, particularly when the specimen contains low numbers. Antibiotic treatment may also inhibit growth.

3.1.4 Test specificity

The isolation of any Legionella species from a clinical specimen is considered to be clinically significant.

3.1.5 Predictive values

A negative culture does not exclude the diagnosis of Legionnaires’ Disease.

3.1.6 Suitable test acceptance criteria

Legionella species is a pale staining gram negative rod, often pleomorphic, which grows only on BCYE and not on Blood Agar or BCYE without cysteine.

3.1.7 Suitable internal controls

It is necessary to check that each batch of media used for isolation of Legionella species supports their growth under suitable incubation conditions by inoculating samples of each media batch with L. pneumophila and L. longbeachae.

3.1.8 Suitable test validation criteria

Isolation of Legionella species is the reference standard.

3.1.9 Suitable external QC program

None in Australia.

3.1.10 Special consideration

L. oakridgensis requires cysteine only for primary isolation. On subculture it will grow without cysteine. Francisella tularensis has similar growth characteristics, but is an unusual isolate in Australia.

3.2 Identification of Legionella species

In routine laboratory practice serotyping is used to identify presumptive L. pneumophila and L. longbeachae but is unreliable for other species owing to the high degree of cross-reactivity among them. For these species, rapid Legionella species identification from culture can be performed by reacting whole bacterial cells with antibody raised in rabbits to known Legionella species. The reaction can be performed on a slide using sera bound to latex particles to demonstrate agglutination, or by UV microscopy with fluorescent labelled antisera (DFA). While serological identification by latex agglutination is acceptable for most sporadic clinical isolates of L. pneumophila and L. longbeachae, all isolates should be kept and/or sent to a reference laboratory and fully identified if

  1. there is a suspected outbreak or
  2. the isolate is unusual or
  3. the diagnosis of Legionnaires’ Disease is uncertain but clinically relevant, or
  4. there is a possible medicolegal issue not covered in (a) – (c).

Legionella species identification can be completed quickly from suspected isolates on selective media by MALDI-TOF procedures4,5, which can be performed in larger clinical laboratories with MALDI-TOF mass spectrometer bacterial identification capability.

The following Legionella species can be differentiated by MALDI-TOF: L. anisa, L. birminghamensis, L. bozemanae, L. cincinnatiensis, L. erythra, L. feeleii, L. jamestowniensis, L. jordanis, L. londiniensis, L. longbeachae, L. oakridgiensis, L. parisiensis, L. pneumophila, L. rubrilucens, L. sainthelensi, and L. tourinensis.

Species level identification using mip gene sequencing can rapidly confirm the identification of commoner species although the full description of unusual species still requires an extended range of definitive tests.15 A database for mip sequences may be accessed at Legionnaires' disease: guidance, data and analysis (www.hpa.org.uk/Topics/InfectiousDiseases/InfectionsAZ/LegionnairesDisease/).

Definitive identification of Legionella species is classically based on DNA/DNA hybridisation, gas chromatography (GC) for long chain fatty acids and high performance liquid chromatography (HPLC) for ubiquinones.

3.3 Antigen Detection Tests

3.3.1 Direct fluorescent antigen (DFA)

DFA may be used directly on specimens including paraffin sections after dewaxing. However it is rarely used except during outbreak investigations or when unfixed tissue is not available because of the laborious nature of the test, its lack of sensitivity and the low rate of positives seen routinely. As DFA has given way to more rapid, specific and consistent test methods in major Australian laboratories, it will not be considered further.

3.3.2 Urinary antigen test

Antigen to L. pneumophila serogroup 1 in urine may be detected by immunoassay and rapid format immunochromatographic kits6,10,11. In Australia the most commonly used test is a qualitative immunochromatographic test for detection of L. pneumophila serogroup 1 antigen in urine (Binax NOW ICT). Reference laboratories may also perform a Legionella Urinary Antigen Enzyme Immunoassay (Binax), which uses microtitre trays coated with polyclonal rabbit antibody specific for a heat stable lipopolysaccharide antigen. For this test, urine specimens can be concentrated by ultrafiltration to enhance low or borderline results. The ICT is faster than the EIA format (15 vs 90 minutes) and more suitable for use with smaller numbers of specimens.

3.3.2.1 Suitable specimen

Urine.

3.3.2.2 Test sensitivity

High (85% to 94%)10 in community acquired Legionnaires’ Disease, and 50% in nosocomial infections depending on time of collection relative to onset of disease (refer below). The Binax EIA is slightly more sensitive than the Binax NOW ICT test. Whilst the Binax NOW ICT has good specificity and sensitivity for Legionella infection, occasional borderline or false negative results occur. If any ICT result is in doubt, laboratories can refer the urine specimen to another laboratory which also performs the Binax EIA. As antigen excretion can be intermittent and may persist for months a repeat urine specimen can be requested, following the infection.

3.3.2.3 Test specificity

Close to 100% to Legionella spp.

3.3.2.4 Predictive values

Positive: ~ 100% for Legionella spp.

Negative: 85% for community-acquired L. pneumophila serogroup 1 infections.

3.3.2.5 Suitable test acceptance criteria

Refer to the manufacturer’s instructions.

3.3.2.6 Suitable internal controls

Positive and negative controls are supplied in the test kit

Or positive control: human urine containing Lp1antigen

Negative control: normal human urine

3.3.2.7 Suitable validation criteria

As described by the test kit manufacturer.

3.3.2.8 Suitable external QC program

An EQA programme is available in Australia through the RCPAMicroQA Programme. Additional QA is available from Europe through the European Legionnaires' Disease Surveillance Network (ELDSNet) - formerly European Working Group on Legionella infections (EWGLI): (ecdc.europa.eu/en/about-us/partnerships-and-networks/disease-and-laboratory-networks/eldsnet).

3.3.2.9 Special considerations

An antigen peak is found at 5–10 days after onset of symptoms and usually diminishes rapidly to barely detectable 21 days after onset of symptoms. In rare cases antigen may continue to be excreted for months after infection has resolved.11 These kits are very sensitive for detecting most subtypes isolated from community-acquired Lp1 infections, which are usually caused by Pontiac strains. However, they are less sensitive when infections are caused by non-Pontiac strains, particularly the Bellingham subtype. This may have implications for their use in hospital settings.11 These tests cannot detect other serogroups of L. pneumophila and other Legionella spp. Therefore positive urinary antigen results should always be verified by culture or PCR if possible. In addition, if Legionnaires’ Disease is strongly suspected on clinical grounds, additional tests such as culture or PCR should be performed because a negative urinary antigen test does not exclude infection due to other Legionella species.

3.4 Nucleic Acid (NA) Based Tests

Highly sensitive PCR assays are capable of detecting Legionella in serum and urine.2,11,13 PCR has the advantage over urinary antigen assays of detecting Legionella other than L. pneumophila serogroup 1. Most in-house assays described in the literature target the genes for rRNA or mip (macrophage infectivity potentiator). Becton Dickenson Biosciences’ BD ProbeTec ET L. pneumophila (LP) Amplified DNA Assay has been approved by the US Food and Drug Administration for sputum specimens but is not marketed in Australia. Multiple products for Legionella nucleic acid-based tests are listed on the Australian Register of Therapeutic Goods (ARTG). Specific details can be obtained from vendors of ARTG-listed tests.

3.4.1 Suitable Specimens

Respiratory tract specimens, urine, and serum.

3.4.2 Test sensitivity

This is measured in patients who fit the laboratory case definition (culture positive or urinary antigen positive or seroconversion or significant increase in antibody level).

All targets1

Urine 49.7% (95% CI 26.5–73.0)

Blood 48.9% (95% CI 38.4–59.5)

Respiratory samples 97.4% (95% CI 91.1–99.2)

S rRNA: 54.4%–80% in serum, 80% in respiratory specimens, 46%–86% in urine specimens,13

16S rRNA: 30.9% in serum, 72 to 95% in respiratory specimens

mip: 52.9% in serum, 78 to 98% in respiratory specimens

Sensitivity is highest early in disease and assay may be positive up to one month post disease onset. PCR in serum may be positive for longer than the urinary antigen test.

3.4.3 Test specificity

Specificity varies with primer and probe design and approaches 100% in some studies. There is marginal cross-reactivity of some Lp PCR assays with L.fairfieldensis and L worsleiensis. A positive Legionella PCR assay, irrespective of intended target gene sequence, cannot distinguish live from dead Legionella but false positive reactions due to laboratory sources of contamination can be excluded by internal quality control. In the presence of clinical features of LD, a positive Legionella PCR result on an extract from a clinical specimen would be adequate evidence of a need for specific treatment.

3.4.4 Predictive values

Positive predictive value depends on primer and assay design and can be high. Negative predictive value depends on the site of specimen collection and time after onset of disease.

3.4.5 Suitable test acceptance criteria

Results for control samples obtained as expected.

3.4.6 Suitable internal controls

Controls should be designed to detect sample inhibitory activity and external contamination of reagents or clinical samples by environmental Legionella, as recommended in NPAAC guidelines: Laboratory Accreditation Standards and Guidelines for Nucleic Acid Detection and Analysis.

3.4.7 Suitable validation criteria

Consistent with NPAAC Guidelines:

Requirements for the Development and Use of In-House In Vitro Diagnostic Medical Devices (IVDs).

For commercial IVDs the assay should be listed with the Australian Register of Therapeutic Goods and the certificate and validation data should be available on request from the IVD sponsor.

3.4.8 Suitable external QC program

Quality Control for Molecular Diagnostics (QCMD)  offer an external quality assessment and proficiency testing program for L. pneumophila that is available to Australian participants.

3.4.9 Special considerations

PCR can be used to detect Legionella in tissue in paraffin blocks after dewaxing and may be useful for diagnosis when unfixed specimens are not available. Since Legionella bacteria are ubiquitous in the environment caution should be exercised in using these tests to confirm cases of Legionnaires’ Disease if there is any likelihood of environmental contamination of the specimen. Some commercial DNA (spin column) extraction kits have been contaminated with Legionella DNA, producing false positives when used for DNA extraction from specimens.13

3.5 Serological Tests14,16

Different public health and hospital laboratories use different test methods, with a correspondingly different range of Legionnaires’ Disease and interpretive cut off points. These reflect genuine and substantial differences in the epidemiology of Legionnaires’ Disease across Australia.

Serum antibodies produced in response to Legionella infection can be measured by a variety of methods including the indirect immunofluorescent assay (IFA) and enzyme immunoassay (EIA). In Australia the majority of laboratories use either in-house or commercial IFA assays. A commercial, automated CFT for L. pneumophila serogroups 1-6 is also available in Australia.

IFA

In the IFA assay, dilutions of patient sera are added to killed Legionella cells fixed to microscope slides. Antihuman immunoglobulin conjugated with fluorescein isothiocyanate is then added. If Legionella antibody is present in the serum sample, the Legionella cells fluoresce when the slides are viewed with a UV microscope.3

EIA

There are several commercial Legionella EIA assays overseas although none is presently in use in Australia. Published results9 and local surveys show that commercial assays are generally inferior to in house EIA assays. EIA assays are designed to provide a sensitive screen for Legionnaires’ Disease and detect IgM using sonicated whole Lp1 or Ll1 cells as their source of antigen. A single positive IgM assay should be interpreted with caution in sporadic cases because IgM to Legionella species can remain elevated for years. Numerous cross-reactions occur among the non-pneumophila legionellae and other organisms.

3.5.1 Suitable specimens

A minimum of paired serum samples should be collected as soon as possible after the onset of illness and then 3-6 weeks later. There may be up to nine weeks’ delay before seroconversion can be detected.

3.5.2 Test sensitivity

IFA

Seroconversion is defined as a four-fold increase in titre of immunofluorescent antibody against heat killed Lp1, to ≥ 1:128. Sensitivity is 70–80% overall, and up to 90% if collection of convalescent serum is delayed to six weeks after onset of symptoms.

Similar sensitivity has been found for antibody seroconversion to the heat killed Ll1 antigen. Four fold rises to ≥ 1:512 were detected in 11/12 patients with culture-confirmed L. longbeachae infection.11

A single high titre of 1:512 or higher in either Ll or Lp is a sensitive indicator of infection with Legionella but may represent past infection or more rarely, infection with another species (refer below).

EIA

In published reports commercial EIA sensitivities vary widely from less than 20% when compared with IFA in randomly selected sera12 to ~70% when tested in patients from a single outbreak.16 EIA assays developed in house using high quality antigen preparations have higher sensitivities (82%10) than commercial test kits. IgM measured by EIA can become positive earlier in the course of illness than IFA.

3.5.3 Test specificity

IFA

Specificity of IFA tests, using Lp1 antigens, is reported from 95 to 99.9%.11

Specificity of IFA for other Legionella species is variable and results should be interpreted with caution because of the numerous cross reactions which occur among Legionella and other bacteria. For example, cross reactions in IFA tests using non-Lp1 antigens have been reported in patients with pneumonia or bacteraemia caused by Pseudomonas, Haemophilus, Mycobacteria, Bordetella, Chlamydia, Rickettsia, Campylobacter and Bacteroides species, Enterobacteriaceae and Coxiella burneti.3

EIA

IgM EIA based tests have been reported to be slightly less specific (97%) than IFA when acute and convalescent sera are tested for Lp1.16 There are no published data for specificity of IgM EIA for Ll, but, as with IFA, cross-reactions among Legionellas and other organisms are highly likely to affect specificity. Therefore, single positive results should be interpreted with caution.

3.5.4 Predictive values

IFA:

Positive tests: four-fold rise in antibody titre is highly predictive of Legionnaires’ Disease (L. pneumophila to 1:128 or higher and L. longbeachae to 1:256 or higher). Serological tests for Legionella species other than Lp1 are predictive of Legionnaires’ Disease caused by the Legionella species antigen used or a related Legionella species.

Negative tests: In one study approximately 10% of patients with culture proven L. longbeachae failed to seroconvert after 3 months16 and up to 20% may not seroconvert to L. pneumophila 1 antigen in culture proven cases.19

A single high antibody titre of ~512 or greater against L. pneumophila is rarely seen in control subjects and, if detected in a patient with suspected Legionnaires’ Disease, may be significant and should be reported.

Similarly, single high antibody titres against L. longbeachae of ~512 or greater are rare (1–2 %) in healthy controls and may be significant in a patient with compatible illness. Actual cut-off titres vary in different laboratories, based on local test evaluation using representative samples of control sera.

EIA:

Positive tests: Single high levels to Lp 1 are highly predictive of disease but may represent past infection.

Single high levels to L. longbeachae may represent past infection or cross-reaction. The significance of positive EIA results should be determined using another serological test or test method such as urinary antigen assays.

Negative tests: predictive values of commercial kits may be low, but much higher in well designed and validated in- house assays.

3.5.5 Suitable test acceptance/validation criteria

Consistent with NPAAC Guidelines:

Requirements for the Development and Use of In-House In Vitro Diagnostic Medical Devices (IVDs).

Commercial kits: according to manufacturer’s instructions.

IFA: Positive control/s should be within one doubling dilution of the established endpoint.

3.5.6 Suitable internal controls

IFA: a positive control for each antigen tested should be titrated to at least two dilutions past the established endpoint.

3.5.7 Suitable external QC program

RCPA QAP P/L Serology and Microbiology QAPs

3.5.8 Special considerations

The endpoint titre for IFA is the reciprocal of the highest serum dilution giving 1+ fluorescence in at least 80% Legionella cells. A suitable means of determining the percentage of fluorescing cells is to use a fluorescent microscope equipped with a dark field condenser. The practice of running the IFA method using pooled antigens, containing multiple serogroups of L. pneumophila may give misleading results owing to the variability of fluorescent staining often seen with this species. The use of a fluorescence-labelled conjugate that detects all classes of antibody is recommended for optimum sensitivity. IgM antibody can persist for a long time and is not reliable for distinguishing new from old disease.

All in-house assays should be validated with an adequate number of culture-confirmed cases. Laboratories should establish L. pneumophila and L. longbeachae reactivity with sera from healthy controls in their region and if possible from patients with bacteriologically confirmed pneumonia not caused by Legionella species. These studies may identify patients whose antibody levels suggest infection and require further action, including notification of the local Public Health Unit.

To avoid unnecessary testing and potentially false negative results, acute serum specimens (i.e. collected within 2 weeks of onset of illness) should be stored until convalescent sera are received for testing in parallel. An exception can be made for cases in which the first serum specimen is collected relatively late in the illness.

4 Subtyping

4.1 Methods

Various genotyping methods are in use for subtyping Legionella species including pulsed field gel electrophoresis (PFGE), restriction fragment length polymorphisms (RFLP) using Southern hybridisation, random amplified polymorphic DNA (RAPD), AFLP, MLST and consensus sequence based typing (SBT). The choice of method depends on the preference of the laboratory performing the test. The UK public health agency (Public Health England) hosts a Legionella website (www.hpa-bioinformatics.org.uk/legionella/legionella_sbt/php/sbt_homepage.php) containing links to a database for consensus sequence based typing (SBT) profiles to provide standardised subtyping schemes throughout the EU.

Subtyping is most helpful in demonstrating differences between isolates. A common genotypic identity among isolates does not prove that they have originated from the same source but may provide helpful molecular epidemiological support for epidemiological and clinical surveillance data.

A panel of seven monoclonal antibodies for subtyping L. pneumophila 1 was developed through collaboration between British, US, Canadian and French laboratories in 1987 and is used extensively overseas.9 The full panel distinguished ten main subtypes of L. pneumophila 1: the Pontiac subtypes, Philadelphia, Knoxville, Benidorm, Allentown, France, and the non- Pontiac subtypes, Olda, Heysham, Camperdown, Oxford and Bellingham.

These place names were allocated according to where each subtype was first isolated. Only three monoclonal antibodies from this panel have been used in Australia, and were developed at CDC by McKinney. These are available from SA Pathology on request. Monoclonal antibody 2 is particularly useful for identifying Pontiac strains, which have caused all known community outbreaks of Lp1 and most sporadic cases in Australia. Non-Pontiac strains do not react with Mab 2 but may cause nosocomial infection. Mab 2 can be used to identify Pontiac strains and therefore provide evidence for pathogenic subtypes in environmental samples from sources implicated in community outbreaks where there is not a human isolate to compare.

4.2 Special considerations

Subtyping to identify a possible environmental source of Legionnaires’ Disease caused by any Legionella species is only informative when supported by unambiguous epidemiological evidence to implicate that source. Most jurisdictions now have more than a decade’s worth of Lp1 profiles. Comparison of these profiles demonstrates that each jurisdiction has its own distinct pattern of strain/s that causes the majority of sporadic and outbreak-associated infections.

There is evidence that commercial potting soils are a source of Ll1 infection. Potting soils from the homes of Legionnaires’ disease cases have been shown to contain genotypically similar strains to isolates from the patients.17

5 Laboratory nomenclature for national data dictionary

SNOMED CT concept Code
Legionella infection (disorder) 26726000
Legionella genus (organism) 7527002
Legionella pneumophila (organism) 80897008
Legionella pneumophila serogroup 1 (organism) 103463005
Legionella longbeachae (organism) 89605004
Legionella species culture (procedure) 122216005
Polymerase chain reaction analysis (procedure) 9718006
Legionella antigen (substance) 709241004
Legionella species DNA (substance) 121186005
Legionella species antibody (procedure) 120743008
Legionella IgM (substance) 720071002
Legionella IgG (substance) 710472002
Polymerase chain reaction analysis for genomic fingerprinting (procedure) 252370006
6 References
  1. Avni T et al. (2016) Diagnostic Accuracy of PCR Alone and Compared to Urinary Antigen Testing for Detection of Legionella spp.: a Systematic Review. J Clin Microbiol. 54: 401-411.
  2. Diederen BMW et al. (2007). Evaluation of real-time PCR for the early detection of Legionella pneumophila DNA in serum samples. J Med Microbial 56: 94-101.
  3. Edelstein, PH, (1997) Manual of Clinical Laboratory Immunology. 5th ed, Rose NR et al., (Ed). American Society for Microbiology, Washington, DC.
  4. Gaia V, Casati S, Tonolla M. (2011) Rapid identification of Legionella spp. by MALDI-TOF MS based protein mass fingerprinting. Syst Appl Microbiol. 34:40-44.
  5. He Y, Chang TC, Li H, Shi G, Tang YW. (2011) Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and database for identification of Legionella species. Can J Microbiol. 57:533-538.
  6. Helbig JH et al. (2001) Detection of Legionella pneumophila antigen in urine samples by the Binax NOW immunochromatographic assay and comparison with both Binax Legionella Urinary Enzyme Immunoassay (EIA) and Biotest Legionella URIN Antigen EIA. J. Med. Microbiol. 50:509-516.
  7. Inglis TJJ et al. (2002). The epidemiology of Legionella infection in Western Australia. Ch 72. In Marre R et al. Legionella ASM Press, Washington, DC. 353-355.
  8. Inglis TJJ et al. (2018) Legionnaires’ disease outbreak on a non-passenger merchant vessel. Emerg Infect Dis 24:1345-1348.
  9. Joly JR et al. (1986). Development of a standardized subgrouping scheme for Legionella pneumophila serogroup 1 using monoclonal antibodies. J Clin Microbiol 23:768-771.
  10. Kazandjian et al. (1997) Rapid diagnosis of Legionella pneumophila serogroup 1 infection using enzyme immunoassay for detection of urinary antigen. J Clin Microbiol; 35: 954-956.
  11. Lindsay DSJ et al. (2004) Laboratory diagnosis of legionnaires’ disease due to Legionella pneumophila serogroup 1: comparison of phenotypic and genotypic methods. J. Med. Microbiol. 53:183-187.
  12. Malan AK et al. (2003) Comparison of two commercial enzyme-linked immunosorbent assays with an immunofluorescence assay for detection of Legionella pneumophila types 1 to 6. J Clin Microbiol. 41:3060-3063.
  13. Diederen BMW, Kluytmans JAJW, Vandenbroucke-Grauls CM, Peeters MF. (2008) Utility of Real-Time PCR for Diagnosis of Legionnaires’ Disease in Routine Clinical Practice J Clin Microbiol. 46(2): 671–677.
  14. Pierre DM et al. (2017) Diagnostic testing for Legionnaires' disease. Ann Clin Microbiol Antimicrob. 16: 59.Ratcliff et al (1998). Sequence-based classification scheme for the Genus Legionella targeting the mip gene. J Clin Microbiol. 36:1560-1567.
  15. Rojas A et al. (2005). Value of serological testing for diagnosis of Legionnaires’ Disease in outbreak patients. J Clin Microbiol 43:4022-4025.
  16. Steele, TW. (1997) Control of Legionella spp particularly Legionella longbeachae in potting mixes. Horticultural Research & Development Corporation. Report no. RDC NT234.
  17. van Heijnsbergen E et al. (2015) Confirmed and Potential Sources of Legionella Reviewed. Environ Sci Technol. 49: 4797-815.
  18. Winslow, WE, Steele, TW. (1993). Indirect immunofluorescent antibody tests with Legionella longbeachae serogroup 1 antigen in confirmed infections. In Legionella: Current Status and Emerging Perspectives. Barbaree et al Ed. American Society for Microbiology, Washington, DC.

Further references concerning assay characteristics for commercial kits can be found in the package inserts.