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NATIONAL STANDARD METHOD

INVESTIGATION OF SPECIMENS FOR LEGIONELLA SPECIES

BSOP 47 Issued by Standards Unit, Evaluations and Standards Laboratory Specialist and Reference Microbiology Division

INVESTIGATION OF SPECIMENS FOR LEGIONELLA SPECIES Issue no: 4.1 Issue date 03.05.05 Issued by: Standards Unit, Evaluations and Standards Laboratory Page 1 of 16 Reference no: BSOP 47i4.1 This SOP should be used in conjunction with the series of other SOPs from the Health Protection Agency www.evaluations-standards.org.uk Email: standards@hpa.org.uk

Evaluations and Standards Laboratory Page 2 of 16 Reference no: BSOP 47i4. The representatives participate in the development of the National Standard Methods but their views are not necessarily those of the entire organisation of which they are a member. These procedures are intended solely as a general resource for practising professionals in the field. Investigation of specimens for Legionella species. The methods also provide a reference point for method development.05. Please note the references are now formatted using Reference Manager software.org. The Health Protection Agency (HPA) should at all times be acknowledged. If you alter or delete text without Reference Manager installed on your computer. It seeks to take every possible precaution to prevent unauthorised disclosure of patient details and to ensure that patient-related records are kept under secure conditions1. The HPA aims to be a fully Caldicott compliant organisation. The current list of participating organisations can be obtained by emailing standards@hpa. development and audit and promotes public health and patient confidence in their healthcare services.uk Email: standards@hpa. the references will not be updated automatically.org. it must be made clear where changes have been made to the original document. This in turn facilitates standardisation of surveillance underpinned by research. Inclusion of an organisation’s logo on the front cover implies support for the objectives and process of preparing standard methods. http://www. More details can be found on the website at www. are members of the working groups which develop National Standard Methods.org.1 This SOP should be used in conjunction with the series of other SOPs from the Health Protection Agency www. INVESTIGATION OF SPECIMENS FOR LEGIONELLA SPECIES Issue no: 4.uk.uk/pdf_bacteriology. Contributions to the development of the documents can be made by contacting standards@hpa.hpa-standardmethods.hpa. Internal and external quality assurance procedures should also be in place. and specialist advice should be obtained where necessary. in using National Standard Methods. The performance of standard methods depends on the quality of reagents.STATUS OF NATIONAL STANDARD METHODS National Standard Methods.org.asp.uk. Whereas every care has been taken in the preparation of this publication. promote high quality practices and help to assure the comparability of diagnostic information obtained in different laboratories. National Standard Method BSOP 47 Issue 4. the Health Protection Agency or any supporting organisation cannot be responsible for the accuracy of any statement or representation made or the consequences arising from the use of or alteration to any information contained in it. Representatives of several professional organisations. The HPA is an independent organisation dedicated to protecting people’s health. Laboratories should ensure that these have been validated and shown to be fit for purpose.uk. reviewed and updated through an open and wide consultation process where the views of all participants are considered and the resulting documents reflect the majority agreement of contributors. laboratories should take account of local requirements and may need to undertake additional investigations.org. National Standard Methods are developed. equipment.1 Issue date 03.evaluations-standards.org. operating in the UK. However.org. More information about the HPA can be found at www.uk. If you make any changes to this publication.05 Issued by: Standards Unit. algorithms and guidance notes.uk . Suggested citation for this document: Health Protection Agency (2004). It brings together the expertise formerly in a number of official organisations.evaluations-standards. commercial and in-house test procedures. The methods are well referenced and represent a good minimum standard for clinical and public health microbiology. including those whose logos appear on the front cover. which include standard operating procedures (SOPs).

.............................................................................................................................................. 7 LIMITATIONS..................................................................................................................................................................................................................6 5..............................................................2 1..............1 2........................................................................................................................5 4...................2 5....................................................................................................................... 7 1................ 9 MICROSCOPY .................................... 5 RISK FACTORS PREDISPOSING TO LEGIONELLOSIS....................................0 5..........org............................................................4 6..........................................................................................................................................................................................................................0 4.............0 3........................................... 14 INVESTIGATION OF SPECIMENS FOR LEGIONELLA SPECIES Issue no: 4........................1 5....1 1...................... 5 ISOLATION OF LEGIONELLA SPECIES ......................... 13 REFERENCES ....... 12 REPORTING PROCEDURE .............................................org................................................................. 11 ANTIBIOTIC SUSCEPTIBILITY TESTING ....................................................................... 10 IDENTIFICATION ................. 12 ANTIBIOTIC SUSCEPTIBILITY TESTING ......................................................................uk ............... 9 TIME BETWEEN SPECIMEN COLLECTION AND PROCESSING ......................................................................................... 8 SPECIMEN COLLECTION .................................... 3 AMENDMENT PROCEDURE ............................................................4 4................................. 9 TEST SELECTION .....................0 1..................................................... 12 URINE FOR ANTIGEN DETECTION ....................3 5............. 8 CORRECT SPECIMEN TYPE AND METHOD OF COLLECTION .........1 This SOP should be used in conjunction with the series of other SOPs from the Health Protection Agency www............................................................ 9 SPECIMEN PROCESSING.....................................................................uk Email: standards@hpa.............................................................................................................................................................1 Issue date 03.................................................................................................................................................... Evaluations and Standards Laboratory Page 3 of 16 Reference no: BSOP 47i4.... 8 SPECIMEN COLLECTION ..... 8 SPECIMEN TRANSPORT AND STORAGE ....................... 8 OPTIMAL TIME OF SPECIMEN COLLECTION ...................... 12 REPORTING TO THE HPA (LOCAL AND REGIONAL SERVICES AND CDSC CENTRE FOR INFECTIONS).....................................................................................................3 2............................................................................ 5 CLINICAL CHARACTERISTICS OF LEGIONELLA INFECTIONS.....................................05............ 4 SCOPE OF DOCUMENT .................................................................................................. 12 CULTURE ................................................................................................................................................................................ 8 SPECIMEN PROCESSING.....................................................................................................05 Issued by: Standards Unit..............0 SAFETY CONSIDERATIONS..1 3...................................................... 9 CULTURE AND INVESTIGATION ......................2 4................1 4............................ 9 APPEARANCE ....................... 12 MICROSCOPY ........................................................2 4................... 8 SPECIMEN TRANSPORT AND STORAGE............................................................................................................................................................... 6 TECHNICAL INFORMATION ............ 5 INTRODUCTION .... 2 INDEX.3 4....................2 2................................................ 9 SPECIAL CONSIDERATIONS TO MINIMISE DETERIORATION ................................................0 2.........................................INDEX STATUS OF NATIONAL STANDARD METHODS ..............................................................evaluations-standards................................................ 8 ADEQUATE QUANTITY AND APPROPRIATE NUMBER OF SPECIMENS .3 3......................................

org.org. Discarded 4 Insert Issue no.1 Page Section(s) involved Amendment 1 2 4 Front page Status of document Amendment page Redesigned Reworded Redesigned INVESTIGATION OF SPECIMENS FOR LEGIONELLA SPECIES Issue no: 4.05 Issue no.uk Email: standards@hpa.org.05. Evaluations and Standards Laboratory Page 4 of 16 Reference no: BSOP 47i4. Amendment Number/ Date 5/ 03.1 This SOP should be used in conjunction with the series of other SOPs from the Health Protection Agency www. The amendment history is available from standards@hpa.05 Issued by: Standards Unit.AMENDMENT PROCEDURE Controlled document reference Controlled document title BSOP 47 Standard Operating Procedure for the investigation of specimens for Legionella species Each National Standard Method has an individual record of amendments. 4.uk.05.uk .evaluations-standards.1 Issue date 03. The current amendments are listed on this page. On issue of revised or new pages each controlled document should be updated by the copyholder in the laboratory.

pneumophila8. In the UK most (95%) Legionella infections are caused by Legionella pneumophila2 principally serogroup 13.uk Email: standards@hpa.14. increasing age. Evaluations and Standards Laboratory Page 5 of 16 Reference no: BSOP 47i4. Legionnaires’ disease. Legionella species have not been isolated by culture and the disease is diagnosed serologically or by urinary antigen detection. The causative organism. requiring L-cysteine and iron for growth. immunosuppression and surgery13. Clusters of cases may occur and all suspected cases should be INVESTIGATION OF SPECIMENS FOR LEGIONELLA SPECIES Issue no: 4. Legionella species are nutritionally fastidious.05 Issued by: Standards Unit. bronchoalveolar lavage. the most serious manifestation of Legionella infection.uk .org. Transplant patients are at particularly high risk15. which support their growth.org. These embrace Legionnaires’ disease. lung biopsy/tissue. Pontiac fever (a self limiting flu-like illness) and other less common clinical manifestations. either from an environmental source or occasionally iatrogenically following a respiratory tract manipulation such as humidification or nebulisation of infected material. and are commonly found in the presence of other micro-organisms includingamoebae. without pneumonic involvement12. Non-pneumonic disease or Pontiac fever is an acute febrile illness occurring 24 . CLINICAL CHARACTERISTICS OF LEGIONELLA INFECTIONS Transmission is by inhalation of an aerosol of the organism.1 This SOP should be used in conjunction with the series of other SOPs from the Health Protection Agency www. Onset is usually abrupt with pyrexia.48 hours after exposure to any species.1 Issue date 03. was first recognised in 19764 following investigation of a large outbreak of pneumonia (221 cases with 34 fatalities) amongst delegates attending an American Legion Convention at a hotel in Philadelphia. transtracheal aspirate SCOPE OF DOCUMENT This SOP describes the culture procedures for the isolation of Legionella species from clinical material INTRODUCTION Currently. Watery diarrhoea may be present6 and neurological symptoms ranging from mild headache to encephalopathy may also occur. Legionella micdadei10 and Legionella anisa11. Superficially. Chest X-rays show pulmonary infiltrates progressing to consolidation often with pleural effusion7.made aquatic reservoirs such as wet cooling towers and water distribution systems. myalgia. the disease resembles influenza and is usually self-limiting. lakes) and man. Legionella feeleii9. pleural fluid. particularly to Mediterranean holiday resorts17. Legionella species are widely distributed in nature and are found in aquatic environments. chronic lung disease. the Legionellaceae family comprises 48 species and in excess of 60 serogroups. sputum. Most cases in the UK are associated with recent travel abroad.STANDARD OPERATING PROCEDURE FOR THE INVESTIGATION OF SPECIMENS FOR LEGIONELLA SPECIES Types of specimens: Bronchial/tracheal aspirate.16.threatening disease. headache and nonproductive cough following a 2-10 day incubation period5. but particularly to L.05. Pneumonia is the most common manifestation of Legionella infections. USA. was isolated from autopsy lung specimens taken from affected individuals.evaluations-standards. both natural (eg rivers. subsequently named Legionella pneumophila. Severity varies from mild to severe life. Legionellosis refers to the clinical syndromes produced by infection with organisms of the genus Legionella. RISK FACTORS PREDISPOSING TO LEGIONELLOSIS Factors that have been shown to predispose to Legionella infection include cigarette smoking.

the antibody binding to any L. Plate cultures should be examined using a low power binocular microscope and oblique incident lighting to demonstrate the typical ground glass. specificities have been reported to be as high as 95%28. its sensitivity being similar to that of culture (80-85%)24. Equivocal EIA results should be examined by a second person and repeated26.25. the immunofluorescent techniques are relatively insensitive methods (2570%)28. Heat treatment and specimen dilution are the simplest to perform and the recommended methods for clinical specimens20. pneumophila antibody (preferably a monoclonal antibody reactive against all serogroups) labelled with fluorescent dye is applied to the smear. Culture is reported as being slightly more sensitive than immunofluorescent techniques and is highly specific (>99%)19. heating to 50°C is simpler to control. As the volume of the specimen and the container dimensions affect the time required to heat to 60°C. then applying a fluoresceinisothiocyanate-conjugated (FITC) immunoglobulin.uk . Sputum with squamous cell contamination may be positive for L. There are currently four techniques routinely used for the diagnosis of legionellosis18. Two temperature/time combinations may be used for decontamination. Direct fluorescent antibody (DFA) microscopy is a rapid method for the detection of Legionella.1 This SOP should be used in conjunction with the series of other SOPs from the Health Protection Agency www. Culture should be performed whenever possible because of the importance of epidemiological investigations in establishing the source in an outbreak2. Anti-L.05 Issued by: Standards Unit.05. Antigen becomes detectable soon after onset of symptoms and the test may remain positive for several weeks.org. The indirect fluorescent antibody test (IFAT) is a two-stage test. Direct or indirect Immunofluorescence microscopy Immunofluorescence microscopy may be used for the demonstration of organisms in clinical material.29. pneumophila present. pneumophila so specimens should be processed regardless of purulence22. This SOP therefore recommends heat decontamination is performed at 50°C for 30 minutes. These tagged organisms may then be visualised by fluorescence microscopy. iridescent colonial appearance. ISOLATION OF LEGIONELLA SPECIES Isolation of the organism is the definitive method of diagnosis of Legionnaires’ disease18. Evaluations and Standards Laboratory Page 6 of 16 Reference no: BSOP 47i4. first incubating the smear with a hyperimmune antiserum and washing. Smears of almost any type of lower respiratory tract specimen are appropriate for this type of test. provided an experienced microbiologist performs the test meticulously. INVESTIGATION OF SPECIMENS FOR LEGIONELLA SPECIES Issue no: 4. However. even after other tests have become negative23.1 Issue date 03. Detection of urinary antigen Urinary antigen detection is a very convenient method of diagnosing Legionnaires’ disease.29.org.evaluations-standards. They are 50oC for 30 minutes or 60°C for 1-3 minutes.uk Email: standards@hpa. Antigen detection is a highly specific method (>99%) of diagnosing legionellosis. The fastidious nature of Legionella species makes special media and selective techniques essential for optimum isolation. Other methods of decontamination include acid treatment21 (which is mainly used for environmental samples).reported to the CCDC for further investigation. Factors to consider with immunofluorescent techniques are: • sensitivity can be affected by the area of the well used for the smear • appropriate controls are essential • a negative result may not exclude infection • detection of a minimum of five fluorescing rods are required to report a positive result27 • cross reactions with other bacteria have been reported Compared with culture. Heavily contaminated specimens may be processed to remove contaminants by a variety of methods.

ferric pyrophosphate. Various agents (listed) have been added to produce a range of selective media. LIMITATIONS This SOP describes the laboratory diagnostic procedure of culture only for the demonstration of Legionella species in clinical material.05 Issued by: Standards Unit. Primary isolation plates are incubated at 35-37°C for up to 10 days in a moist atmosphere.uk . Buffered cefamandole. α-ketoglutarate medium (BMPA α)35 Cefamandole Polymyxin B Anisomycin Buffered charcoal yeast extract. an IFA test and enzyme immunoassays (EIA)31-33.org. Enrichment and antibiotic supplements are available commercially.(2-acetamido)-2aminoethanesulfonic acid (ACES) buffer. Pseudomonas species and other bacteria can occur15-17. Cross-reactions with antibodies to Campylobacter species. has now largely been displaced by urinary antigen testing. polymyxin.05. traditionally the most widely used technique for diagnosis30.uk Email: standards@hpa. anisomycin agar (BCYEA)2 Polymyxin B Cefamandole Anisomycin Vancomycin BMPAα is recommended for clinical specimens although there have been reports of cefamandole35 being inhibitory to some Legionella species. A single high titre in combination with a suggestive clinical history affords a presumptive diagnosis of Legionnaires’ disease29. Evaluations and Standards Laboratory Page 7 of 16 Reference no: BSOP 47i4.evaluations-standards. INVESTIGATION OF SPECIMENS FOR LEGIONELLA SPECIES Issue no: 4. or the detection of L. Techniques include a latex agglutination screening test. TECHNICAL INFORMATION Besides supporting the growth of Legionella species. L-cysteine hydrochloride and αketoglutarate (BCYE).org. Vancomycin sensitive strains have also been detected. However it is still a useful test in outbreak investigations or to establish a disgnosis retrospectively. pneumophila antigen in urine are diagnostic of Legionnaires’ disease. the primary isolation media should also be suitable for use with contaminated specimens and be stable during storage. The basic medium employed is charcoal yeast extract (CYE) supplemented with 1% N.1 Issue date 03. anisomycin.34. For the detection of antigen and antibody in urine and in serum refer to the manufacturers instructions of the commercially available kits.1 This SOP should be used in conjunction with the series of other SOPs from the Health Protection Agency www. Paired sera showing a four-fold rise in titre.Antibody detection Antibody.

05.2 SPECIMEN TRANSPORT AND STORAGE Sterile leakproof container in a sealed plastic bag 1.uk .1 OPTIMAL TIME OF SPECIMEN COLLECTION Before antimicrobial therapy when possible Culture for Legionella species may still be successful after antimicrobial therapy has started 2. at least 1mL Tissue and biopsies: specimens should ideally be large enough to carry out all microscopic preparations and cultures INVESTIGATION OF SPECIMENS FOR LEGIONELLA SPECIES Issue no: 4.3 ADEQUATE QUANTITY AND APPROPRIATE NUMBER OF SPECIMENS Bronchoalveolar lavage (BAL): as large a volume as possible Pleural fluid: preferably.evaluations-standards.1 SPECIMEN COLLECTION N/A 1. Evaluations and Standards Laboratory Page 8 of 16 Reference no: BSOP 47i4. Then place in a rack or other suitable holder NOTE 1: Heat-fixing may not kill all Mycobacterium species42 Centrifugation must be carried out in sealed buckets which are subsequently opened in a microbiological safety cabinet Specimen containers must be placed in a suitable holder Tissues and biopsies Homogenisation of all specimens must be undertaken in a microbiological safety cabinet Wherever possible.0 SPECIMEN COLLECTION 2.uk Email: standards@hpa.0 SAFETY CONSIDERATIONS36-41 1.2 CORRECT SPECIMEN TYPE AND METHOD OF COLLECTION Specialist collection according to local protocols NOTE 2: Sputum specimens should be processed regardless of purulence 2. fix smeared material by placing the slide on an electric hotplate (65 to 75°C) under the hood.05 Issued by: Standards Unit. until dry.3 SPECIMEN PROCESSING All respiratory specimens for culture must be processed in a microbiological safety cabinet in a Containment Level 3 room Prior to staining.1 This SOP should be used in conjunction with the series of other SOPs from the Health Protection Agency www.1.org.1 Issue date 03. at least 1mL Sputum: preferably.org. the use of sterile scissors is recommended in preference to a scalpel blade The above guidance should be supplemented with local COSHH and risk assessments 2.

as it is known to be inhibitory to Legionella species2 It is recommended that all specimens of tissue and biopsy from suspected cases of legionellosis are stored at -20°C.uk . until the final report is issued.4.evaluations-standards. Evaluations and Standards Laboratory Page 9 of 16 Reference no: BSOP 47i4.05.uk Email: standards@hpa. cut the tissue to give a clean fresh surface Scrape the blade across the surface to create a slurry of cellular tissue Prepare a thin smear of this material on a microscope slide for fluorescent staining INVESTIGATION OF SPECIMENS FOR LEGIONELLA SPECIES Issue no: 4.1 3. Delays of over 48h are undesirable Tissue and biopsies If specimen is small place it in sterile water to prevent desiccation43.org. as overgrowth with nonlegionella bacteria may necessitate retesting of the original specimen 4.Consideration should be given to use of chain of evidence forms in view of the potential for legal action in the event of acquisition of Legionella species 3.2 Supplementary Fluorescent staining technique Homogenised specimens Using a sterile pipette place one drop of homogenised specimen (see Section 4.2 APPEARANCE N/A 4.org.1) on to a clean PTFE microscope slide Spread the drop with a sterile loop to make a thin smear for fluorescent staining Tissues and biopsies Using sterile scissors or a scalpel blade.1 MICROSCOPY Standard N/A 4. refrigeration for up to 48h is preferable to storage at ambient temperature.3.2 TIME BETWEEN SPECIMEN COLLECTION AND PROCESSING Specimens should be transported and processed as soon as possible43 SPECIAL CONSIDERATIONS TO MINIMISE DETERIORATION If processing is delayed.3.1 Issue date 03. NOTE 3: This would not be appropriate for specimens undergoing processing for diagnosis by molecular methods NOTE 4: Avoid the use of saline.0 SPECIMEN PROCESSING 4.1 TEST SELECTION N/A 4.05 Issued by: Standards Unit.0 SPECIMEN TRANSPORT AND STORAGE 3.3 4.1 This SOP should be used in conjunction with the series of other SOPs from the Health Protection Agency www.

If fluorescence is satisfactory examine the test specimens 4.1% in phosphate buffer) Agitate gently for approximately 10 secs Incubation at 35-37°C for approximately 15mins followed by gentle agitation for approximately 15 secs will assist homogenisation. pseudomonads and Proteus species and then recultured INVESTIGATION OF SPECIMENS FOR LEGIONELLA SPECIES Issue no: 4.4.org.4.05 Issued by: Standards Unit.05.1mL of digested sputum Bronchoalveolar lavages Centrifuge at a minimum of 2000 x g for 15 mins.1 This SOP should be used in conjunction with the series of other SOPs from the Health Protection Agency www. conical-bottomed container at a minimum of 2000 x g for 15 mins Using a sterile pipette transfer all but 0.resulting in false positive reactions Follow kit manufacturers’ instructions Examine under a high power objective (X40) Examine the control slide first.1) on to a clean microscope slide Spread the drop with a sterile loop to make a thin smear for fluorescent staining Direct and indirect immunofluorescence NOTE 5: Include a positive and a negative control.4 CULTURE AND INVESTIGATION 4.1. capped.org. Evaluations and Standards Laboratory Page 10 of 16 Reference no: BSOP 47i4.Fluids and BAL Using a sterile pipette place one drop of centrifuged deposit (see Section 4.evaluations-standards.uk Email: standards@hpa.5mL of the supernatant to another sterile container for additional testing if required (eg virology) Resuspend the deposit in the remaining fluid Tissue/biopsy Tissues may be homogenised in sterile water using a sterile tissue grinder (Griffiths tube or preferably an unbreakable alternative) Supplementary Heavily contaminated specimens should be heat-treated and diluted to decrease the numbers of yeasts.uk . Inoculate plates directly with 0. and a known positive specimen (if possible) in every batch NOTE 6: Care must be taken to prevent the transfer of bacilli from the positive control to the test slides .1 Issue date 03. if present. Use the deposit as the inoculum For other respiratory tract specimens select any milky or bloodstained portion. Pre-treatment Standard Sputum Add an equal volume of dithiothreitol (0. for use as the inoculum Fluids Centrifuge in a sterile.

1 IDENTIFICATION Minimum level in the laboratory Legionella to genus level 4.2 Referral to Reference Laboratory Legionella species obtained from clinical material must be referred for identification and serotgrouping All specimens testing positive for urinary antigen should be sent to the reference laboratory for confirmation Respiratory and Systemic Infection Laboratory Atypical Pneumonia Reference Unit SRMD 61.5.org. spread inoculum with a sterile loop Whenever possible include a positive control plate 4.1 Issue date 03. Specimen processing Inoculate each agar plate with specimen (see BSOP 54) For the isolation of individual colonies.uk .evaluations-standards.1 This SOP should be used in conjunction with the series of other SOPs from the Health Protection Agency www. conditions and organisms For all specimens: Clinical details/ conditions Standard media Temp °C When requested ?pneumonia Legionella selective agar* 35-37 Incubation Atmos air (moist) Time 10 days at 3.4. A sample should be retained at -20°C in the event that re-testing may be required regarding legal action 4. Colindale Avenue London NW9 5HT Telephone: 020 8200 4400 INVESTIGATION OF SPECIMENS FOR LEGIONELLA SPECIES Issue no: 4. Evaluations and Standards Laboratory Page 11 of 16 Reference no: BSOP 47i4.05. 7 and 10 days Legionella species Cultures read Target organism(s) *Refer to MSOP 29 Legionella selective agar 4.org.3 Culture media.4.05 Issued by: Standards Unit.2.Heat treatment Place the specimens in a water bath at 50°C for 30 mins (or 60°C for 1-3 mins) Culture both the heated and unheated specimens Dilution Dilute the original specimen 1:100 in distilled water and reculture Urine for antigen detection Refer to kit manufacturers’ instructions Positive urine samples should be forwarded to the Reference laboratory for confirmatory testing.uk Email: standards@hpa.5.5 4. 5.

4.2 CULTURE Positives Legionella species isolated Negatives Legionella species NOT isolated Culture reporting time Urgent culture results to be telephoned or sent electronically Written report.org. 16 .1 MICROSCOPY Immunofluorescence Legionella pneumophila detected by immunofluorescence or Legionella pneumophila NOT detected by immunofluorescence Microscopy reporting time Urgent microscopy results to be telephoned or sent electronically Written report.1 This SOP should be used in conjunction with the series of other SOPs from the Health Protection Agency www.0 REPORTING PROCEDURE 5.05.uk Email: standards@hpa.05 Issued by: Standards Unit.72h 5.evaluations-standards.3 URINE FOR ANTIGEN DETECTION Positives Legionella antigen detected Negatives Legionella antigen NOT detected 5.4 ANTIBIOTIC SUSCEPTIBILITY TESTING N/A INVESTIGATION OF SPECIMENS FOR LEGIONELLA SPECIES Issue no: 4. that a further report will be issued 5. 3 . Evaluations and Standards Laboratory Page 12 of 16 Reference no: BSOP 47i4. if appropriate.uk .org.1 Issue date 03.6 ANTIBIOTIC SUSCEPTIBILITY TESTING N/A 5.10 days stating.

org. Evaluations and Standards Laboratory Page 13 of 16 Reference no: BSOP 47i4.1 Issue date 03.uk .org.evaluations-standards.6.0 REPORTING TO THE HPA44 (LOCAL AND REGIONAL SERVICES AND CDSC CENTRE FOR INFECTIONS) Individual SOPs on organism identification Health Protection Agency publications: "Reporting to the CDR: A guide for laboratories" "Hospital infection control: Guidance on the control of infection in hospitals" Refer to current guidelines on CDSC and COSURV reporting Local guidelines Report all isolates of Legionella species INVESTIGATION OF SPECIMENS FOR LEGIONELLA SPECIES Issue no: 4.uk Email: standards@hpa.05.05 Issued by: Standards Unit.1 This SOP should be used in conjunction with the series of other SOPs from the Health Protection Agency www.

December 1997. Emslie JA. Arisumi D. Jr. Toma S. In: Harrison TG. Carratala J. N Engl J Med 1977.05 Issued by: Standards Unit. Kapoor W.uk Email: standards@hpa. A prospective multicenter study of 359 cases. Calpe JL. Gillespie SH. 1988. 14. 6. Dolin R. Yu VL. Am J Respir Crit Care Med 1994. Fine M. Bruun BG. Legionella feeleii species nova. 12.26:191-202. A Laboratory Manual for Legionella. Wewalka G.21:712-3. 9. Martinez C. Orenstein W. Medicine (Baltimore) 1990. Clin Infect Dis 1995. Hum Pathol 1981. Isolation of legionellae from clinical specimens. N Engl J Med 1997.114:337-47. Jr. 11. Orloff J. Hawkey PM. et al. Legionella. Mandell. Am J Epidemiol 1978. In: Mandell GL.337:682-7. Gorman GW.REFERENCES 1. Pallares R.69:307-16. 7. editors. Uldum S. p. Jensen JS. Tsai TR. Wrench JG. Clinical and epidemiologic aspects. 1995. Yu VL.1:316-8. Dournon E. INVESTIGATION OF SPECIMENS FOR LEGIONELLA SPECIES Issue no: 4. Ann Intern Med 1984.14:99-104.. Hightower AW. Ariza J.evaluations-standards. and Bennett's Principles and Practice of Infectious Diseases. Fraser DW. Masia MM. 3. Harrison TG.7:377-92. Douglas. Chiner E.149:625-9. Prodinger WM. The pathology of the Legionella pneumonias. 17. 15. Mallison G. Ortiz dlT. Fallon RJ.297:1189-97. Allerberger F. Fang GD. Sharrar RG. Goldberg DJ. Aichberger C.org. Parkin WE. Glick TH. Legionella and the clinical microbiologist. et al. Eur J Clin Microbiol Infect Dis 1995. Kaufmann AF. Winn WC. London. Edinburgh: Churchill Livingstone. Legionella pneumophila (Legionnaires' disease).05. 4.1 Issue date 03. 349-66. Legionnaires' disease in patients infected with human immunodeficiency virus. Stout JE. An epidemic of unknown etiology in a health department: I. 13. Feeley JC. Winn WC. Chichester: John Wiley & Sons. causes Pontiac fever in an automobile plant.1 This SOP should be used in conjunction with the series of other SOPs from the Health Protection Agency www. Risk factors for nosocomial Legionella pneumophila pneumonia. Herwaldt LA.107:149-60. Bangsborg JM. Report on the review of patientidentifiable information. Verdaguer R. Legionellosis. Jr. 2087-97. J Hosp Infect 1994. Pontiac fever. Kassanoff I. A new Legionella species. Nosocomial legionellosis in three heart-lung transplant patients: case reports and environmental observations. p. Legionella pneumonia in transplant recipients: a cluster of cases of eight years' duration. New and emerging etiologies for community-acquired pneumonia with implications for therapy. Lancet 1989. In: Emmerson AM. 2. Department of Health NHS Executive: The Caldicott Committee. Berman B. editors. et al. Skaliy P. McGrath T. p. Collier PW.12:401-22. editors. V.100:333-8. 8. Evaluations and Standards Laboratory Page 14 of 16 Reference no: BSOP 47i4. et al. Myerowitz RL. Dorca J.org. Patton CM. Taylor AG. A review of 74 cases and the literature. Brake B. Principles and Practice of Clinical Bacteriology. Gudiol F. Gregg MB. Pontiac fever: isolation of the etiologic agent (Legionella pneumophilia) and demonstration of its mode of transmission. et al. Beecham HJ. Bennett JV. Legionnaires' disease: description of an epidemic of pneumonia.. et al. Am J Epidemiol 1981. Lochgoilhead fever: outbreak of non-pneumonic legionellosis due to Legionella micdadei. Chichester: John Wiley. 10. 16. Gutierrez RF. Bonatti H. 1997. Bennett JE. Forbes GI.uk . McDade JE. 13-30. Infect Dis Clin North Am 1993. 5. Yu VL. et al. Rhodes WW. 4th ed. Harrison TG.

The Diagnosis of Legionnaires' Disease by Estimation of Antibody Levels. J Clin Pathol 1990. Edelstein PH. Legionella: current status and emerging perspectives. J Microbiol Methods 1998. Wreghitt TG.C. Evaluations and Standards Laboratory Page 15 of 16 Reference no: BSOP 47i4. Benson RF. 32. Edelstein PH. Harrison TG. J Clin Microbiol 1997. Ingram JG. Laboratory methods for the Diagnosis of Legionella infections.34:1579-80. Plouffe JF. Kudesia G. Tenover FC. Washington D. An ELISA test for the detection of antibodies to Legionella pneumophila. 27. Isolation of Legionella spp. 21. 9. 34.50:509-16. Chichester: John Wiley & Sons Ltd. Plouffe JF. In: Murray PR. editors. 20. 22.33:59-79. Bopp CA.: American Society for Microbiology. Breiman RF. 26. 29. J Med Microbiol 2001.1 This SOP should be used in conjunction with the series of other SOPs from the Health Protection Agency www. Chiew R. 19. False positive legionella serology in campylobacter infection: campylobacter serotypes. J Clin Microbiol 1996. Harrison TG. p. Helbig JH. 28. Laboratory Diagnosis of Legionnaires Disease: an Update from 1984. Marshall LE. Edelstein PH. 30. 533-44.35:9546. Yolken RH. Comparison of Binax Legionella Urinary Antigen EIA kit with Binax RIA Urinary Antigen kit for detection of Legionella pneumophila serogroup 1 antigen. p.evaluations-standards. 1995. Gray J.13:714-9. Legionella. Evaluation of sensitivity of two serological tests for diagnosing pneumonia caused by Legionella pneumophila serogroup 1. Washington D. A Laboratory Manual for Legionella. Clin Infect Dis 1993.6:4-10. Maiwald M.18. Dufour AP.43:685-90.org. In: Isenberg HD. editor.8. Wells JG. Sumner JW. 25. Fields BS. 31.43:860-2. Boswell TC. from environmental water samples by low-pH treatment and use of a selective medium. J Clin Microbiol 1981. Legionnaires' disease. duration of antibody response and elimination of cross-reactions in the indirect fluorescent antibody test. Gilbert GL. p. INVESTIGATION OF SPECIMENS FOR LEGIONELLA SPECIES Issue no: 4. Dournon E. J Clin Pathol 1982. Taylor AG.: American Society for Microbiology. Winn WC J.05. 1993. Use of latex agglutination technique for detecting Legionella pneumophila (serogroup 1) antibodies. Taylor AG. Evaluation of urinary antigen ELISA for diagnosing Legionella pneumophila serogroup 1 infection. Danger of sputum purulence screens in culture of Legionella species.112:347-57. 1992. editors. Laboratory diagnosis of infections caused by legionellae. 6th ed. Rapid diagnosis of Legionella pneumophila serogroup 1 infection with the Binax enzyme immunoassay urinary antigen test. Benson RF. Uldum SA. Detection of Legionella pneumophila antigen in urine samples by the BinaxNOW immunochromatographic assay and comparison with both Binax Legionella Urinary Enzyme Immunoassay (EIA) and Biotest Legionella Urin Antigen EIA.Vol 2. editors. 24. Holliday MG. Eur J Clin Microbiol 1987. Clinical Microbiology Procedures Handbook. Kazandjian D.32:209-10. 1988. Manual of Clinical Microbiology.05 Issued by: Standards Unit. Pfaller MA. Samuel D.1 Issue date 03. Washington D. Hackman BA. 7-11. Breiman RF. Harrison TG. Luck PC. J Clin Pathol 1987. Morris GK. Nagington J. Ward KW. 33. Direct immunofluorescent antibody examination for Legionella species.uk .C: American Society for Microbiology. Birtles RJ. Baron EJ. J Clin Pathol 1990. p.5. J Clin Microbiol 1994.uk Email: standards@hpa. Harrison TG.35:657-60.16:741-7. Helbig JH. Taylor AG. 23. Epidemiol Infect 1994. In: Harrison TG.org. 113-32.40:7782.C. Taylor AG. Luck PC. In: Babaree J.

Hausler WJ J. Health and Safety Executive. 4th ed. In: Balows A.hse. May. Evaluations and Standards Laboratory Page 16 of 16 Reference no: BSOP 47i4. 36. Doern GV. 1991.14:298-303. Health Services Advisory Committee. Safe working and the prevention of infection in clinical laboratories and similar facilities. 42. Washington D. J Clin Microbiol 1981.uk . PHLS. 2002. L5. Improved semiselective medium for isolation of Legionella pneumophila from contaminated clinical and environmental specimens. Washington JA II. Public Health Laboratory Service Standing Advisory Committee on Laboratory Safety.05. 38. 40. Health and Safety Executive.34:719-22. 2001. INVESTIGATION OF SPECIMENS FOR LEGIONELLA SPECIES Issue no: 4. http://www. Suffolk: HSE Books. 5 steps to risk assessment: a step by step guide to a safer and healthier workplace. Amsterdam D. London: Public Health Laboratory Service (PHLS). Allen BW.uk Email: standards@hpa. Suffolk: HSE Books. Herrmann KL.05 Issued by: Standards Unit. IND (G) 218 (L). Survival of tubercle bacilli in heat-fixed sputum smears. 2002.evaluations-standards. 2003. General COSHH Approved Code of Practice and Guidance. 1-17 37. 1993.gov. Isenberg HD.: American Society for Microbiology.uk/pubns/misc208.35. 5th ed.C. A guide to risk assessment requirements: common provisions in health and safety law. editors. 2002. Advisory Committee on Dangerous Pathogens 2004 Approved List of Biological Agents. Safety Precautions: Notes for Guidance. CDSC.1 Issue date 03.org. 2nd ed. Suffolk: HSE Books. Control of Substances Hazardous to Health Regulations 2002.1 This SOP should be used in conjunction with the series of other SOPs from the Health Protection Agency www. Shadomy HJ. 41. 39. IND (G) 163 (REVL). p.pdf. 15-28. J Clin Pathol 1981. Specimen collection and handling. Edelstein PH. Isenberg HD.org. Manual of Clinical Microbiology. Safety in Health Service laboratories. p. Reporting to the PHLS Communicable Disease Surveillance Centre: a reference for laboratories. Suffolk: HSE Books. 44. 43.