MYCOBACTERIOLOGY INTRODUCTION The Central Tuberculosis Laboratory (CTBL) of the Department of Pathology provides laboratory diagnosis of tuberculosis (TB) and other mycobacterial diseases for all hospitals, clinics, laboratories and other healthcare facilities in Singapore. It has been the National Reference Laboratory for AFB* (Smear) Testing since 11 Feb 1998. In conjunction with the Licensing Unit of the Ministry of Health, Singapore, the Laboratory accreditates clinical laboratories performing AFB (acid-fast bacilli) smears in Singapore. RANGE OF LABORATORY SERVICES Services provided by the laboratory include: Acid-fast smear, Microscopic examination for acid-fast bacilli Culture for Mycobacterium species (AFB Culture) Direct detection for nucleic acid of M. tuberculosis complex * Identification of Mycobacterium tuberculosis complex Identification of M. bovis BCG Identification of non-tuberculous mycobacteria (NTM) Mycobacterial Viability Test Quantiferon Test Susceptibility testing for rapidly growing mycobacteria Susceptibility testing for M. avium complex (MAC) Susceptibility testing for M. kansasii Susceptibility testing for M. tuberculosis complex Susceptibility testing of M. tuberculosis complex for Pyrazinamide Strain Typing for TB * * This test is under validation at the time of publication. Contact laboratory (Tel: 6222 1391 or e-mail to sng.li.hwei@sgh.com.sg) for further details. Please refer to respective test for special specimen collection and delivery instructions. Clinical specimens for all other mycobacterial investigations should be sent to or collected by: Client Services Section, Department of Pathology, Singapore General Hospital (Tel: 63266113, fax: 6222 8924). 197 041-280_PathoH_SL.indd 197 4/3/08 1:03:06 PM
SECTION 4: SAMPLE COLLECTION & HANDLING SPECIAL INSTRUCTIONS & LAB TESTS SPECIAL INSTRUCTIONS ON SPECIMEN COLLECTION AND HANDLING Use the Request for Bacteriological Investigation form for SGH specimens and equivalent forms from other government, restructured and private hospitals, outpatient and private clinics and other organisations. Instructions for filling in the Request Form are given in the section Specimen Collection and Handling General Information. Indicate on the request form the type of the test to be performed. Please refer to the section Alphabetical Test Listing Mycobacteriology for the tests available. Verbal requests should be followed by laboratory request forms either faxed or sent to CTBL. For external clients, the forms should be sent through their respective laboratories to be referred to CTBL. Relevant clinical information including prior anti-mycobacterial treatment, previous smear, culture and susceptibility testing results should be included where possible. The attending doctor s name/mcr No. and contact number should be clearly indicated in the request form. Referring laboratories should include their 24-hour emergency number and contact person. COLLECTION AND TRANSPORTATION OF SPECIMENS 1. Collect specimen before starting patients on anti-mycobacterial drug therapy. 2. Collect specimens in sterile, screw-capped, leak-proof, disposable plastic containers with caps which should fit tightly and be of the type that cannot possibly become accidentally loose or cause leakage. Do not use waxed containers. 3. Label each container with the patient s name, NRIC number, nature of specimen, and date and time of collection. 4. Primary container/s for each specimen should be placed in a sealed biohazardlabelled plastic bag and accompanied by a request form with relevant patient and clinical data. Attach the request form to the outside of the plastic bag (Do not staple through the sealed portion of the bag). 5. Collect specimen aseptically, using standard precautions and minimising contamination with commensal organisms. These microbes will affect the smear and culture results. 6. Collect three first-morning sputum specimens for acid-fast smears and culture from patients with clinical and chest X-ray findings compatible with tuberculosis, on three separate days. 7. Avoid collecting specimens on swabs as these provide limited material for analysis. 8. Send specimens to the laboratory promptly, ideally within 30 minutes; at least within the day of collection. 9. Specimens should be refrigerated at 4 C within one hour of collection. Frozen specimens are unacceptable. 198 041-280_PathoH_SL.indd 198 4/3/08 1:03:07 PM
MYCOBACTERIOLOGY 10. Mycobacterial cultures or suspected mycobacterial isolates for identification and susceptibility must be submitted on solid media. Primary containers should be screw-capped tightly and sealed properly with tape or parafilm before placing them into secondary containers (see point 4). Known M. tuberculosis complex isolates must be packed and transported according to MOH-BATA requirements. 11. For regional laboratories, contact CTBL before sending the specimen. The packaging must conform to international standards (IATA). SPECIMEN REQUIREMENTS FOR MYCOBACTERIAL ISOLATION AND ACID- FAST STAIN Specimen type Specimen requirement Special instructions Abscess contents, aspirated fluid Blood Body fluids (pleural, pericardial, peritoneal, etc) Bone Bone marrow Bronchoalveolar lavage or bronchial washing/brushing CSF As much as possible in sterile disposable container. 5 ml of plain blood inoculated directly into Myco-F-Lytic vial.* As much as possible (minimum 10 15 ml) in sterile disposable container Bone in sterile disposable container with no fixative or preservative. Inoculate directly into Myco-F-Lytic vial* 5 ml in sterile disposable container 2 ml in sterile disposable container Disinfect skin with 70% alcohol before aspiration. Use swab to collect only if volume is insufficient for aspiration. Disinfect skin with 70% alcohol followed by iodine solution. Disinfect cap of Myco-F-Lytic vial with alcohol. Mix the contents immediately after collection. Mainly performed for immunocompromised patients, particularly those with AIDS. Please indicate such underlying condition on the form as prolonged culture incubation may be required. Disinfect skin with 70% alcohol if collecting by needle and syringe. Do not submit specimen in formalin. As for blood Avoid contaminating bronchoscope with tap water as free-living saprophytic mycobacteria may produce false-positive results. Disinfect skin before aspiration. 199 041-280_PathoH_SL.indd 199 4/3/08 1:03:07 PM
SECTION 4: SAMPLE COLLECTION & HANDLING SPECIAL INSTRUCTIONS & LAB TESTS Specimen type Specimen requirement Special instructions Gastric lavage 5 10 ml in sterile disposable container. Collect in the morning soon after patient awakens in order to obtain sputum Collect fasting early morning specimen on three consecutive days. Use sterile saline. Collect in container containing sodium carbonate. swallowed during sleep. Laryngeal swabs Not recommended for the following reasons: Difficulty in proper collection Limited amount of material collected on swabs Risk of sharps injury from broken glass containers High rejection rate from unlabelled specimens Lymph node Skin lesion material Node or portion of node in sterile disposable container with no fixative or preservative. Send biopsy specimen or aspirate in sterile disposable container with no fixative or preservative. Collect aseptically. Select caseous portion if available. Do not freeze, immerse in formalin or other preservatives or wrap in gauze. For cutaneous ulcers, collect biopsy sample from periphery of lesion or aspirate material from under margin of lesion. Swabs in transport medium (Amies or Stuarts) are acceptable only if biopsy sample or aspirate is not obtainable. If infection was acquired in Africa, Australia, Mexico, South America, Indonesia, New Guinea or Malaysia, indicate this on request form, because Mycobacterium ulcerans may require prolonged incubation for primary isolation. 200 041-280_PathoH_SL.indd 200 4/3/08 1:03:07 PM
MYCOBACTERIOLOGY Specimen type Specimen requirement Special instructions Sputum 5 10 ml in sterile, waxfree disposable container. Instruct patient to rinse mouth before sputum is collected. Collect an earlymorning specimen from deep productive cough on at least three consecutive days. Do not pool specimens (e.g. 24-hr pooled sputum); such samples and saliva are unacceptable. Induced sputum can be collected from patients who cannot or find it difficult to expectorate. For induced sputum, use sterile hypertonic saline. Avoid sputum contamination with nebuliser reservoir water. Indicate induced sputum on request form as these watery specimens resemble saliva. Stool Tissue biopsy sample Transtracheal aspirate 1 g in sterile disposable wax-free container 1 g of tissue, if possible, in sterile disposable container with no fixative or preservative As much as possible in a sterile disposable container Collect specimen directly into container or transfer from bedpan. Do not send frozen specimen. Recommended only for detection of Mycobacterium avium complex (MAC) involvement in the gastrointestinal tracts of patients with AIDS. Collect aseptically. Select caseous portion if available. Do not freeze, immerse in formalin or other preservatives or wrap in gauze. 201 041-280_PathoH_SL.indd 201 4/3/08 1:03:17 PM
SECTION 4: SAMPLE COLLECTION & HANDLING SPECIAL INSTRUCTIONS & LAB TESTS Specimen type Specimen requirement Special instructions Urine Collect at least 40 ml of mid-stream clean catch or catheterised urine in sterile disposable container. Collect as much as possible of suprapubic urine in a sterile disposable container. Collect first morning specimen on three consecutive days. 24-hour pooled specimens or urine from catheter bag are unacceptable. Specimens of < 40 ml are unacceptable unless a larger volume is not obtainable. The first morning void provides the best yield. Specimens collected at other times are diluted and not optimal. Wound material See biopsy or aspirate Due to limited material collected, swabs are acceptable only if biopsy or aspirate is not obtainable. If used, send the swab in transport medium (Amies or Stuarts). Available upon request from Client Services* 202 041-280_PathoH_SL.indd 202 4/3/08 1:03:18 PM
MYCOBACTERIOLOGY ALPHABETICAL TEST LISTING MYCOBACTERIOLOGY ACID-FAST SMEAR, MICROSCOPIC EXAMINATION OF AFB Type of test : Routine Sample required : Sputum, bronchoalveolar lavage, CSF, body fluid, tissue, pus etc : Smear prepared from digested, decontaminated and concentrated sample Screening: Auramine O stain Fluorescent microscopy Confirmatory: Ziehl Nielsen stain : Acid-fast bacilli seen (with quantitation) / No acid-fast bacilli seen Quantitation according to United States Centers for Disease Control and Prevention (CDC method): Number of AFB seen Report Fuchsin stain (1000x) Fluorochrome stain (450x) 0 0 No AFB seen 1-2/300F 1-2/70 F Doubtful* 1-9/100F 2-18/50F 1 + 1-9/10F 4-36/10F 2 + 1-9/F 4-36/F 3 + > 9/F > 36/F 4 + * Not considered positive. Smears are repeated from either the same or a second specimen. Turnaround time Day(s) test set up : 8 24 hours : Monday Saturday AFB CULTURE, MYCOBACTERIAL CULTURE (CLINICAL SPECIMENS) Test type : Routine Specimen required : Sputum, bronchoalveolar lavage, CSF, body fluids, gastric lavage, tissue, urine, pus, etc : Culture by MGIT non-radiometric method and LJ solid media. Alternate media include BACTEC 460 Radiometric or 7H10 solid media. Includes reflex drug testing : First-line susceptibility testing for M. tuberculosis (MGIT) for the first MTBC isolate cultured for new TB cases and upon follow-up at 6 8 weeks. Second-line panel automatically performed for isolates resistant to isonizid, rifampin or ethambutol. 203 041-280_PathoH_SL.indd 203 4/3/08 1:03:18 PM
SECTION 4: SAMPLE COLLECTION & HANDLING SPECIAL INSTRUCTIONS & LAB TESTS Special request and separate charges apply for the following: M. tuberculosis Isolates that do not meet the above criteria for reflex testing Pyrazinamide susceptibility testing of M. tuberculosis (MGIT method) Extended-line susceptibility testing for M. tuberculosis e.g. Capreomycin, clofazamine and PAS, (BACTEC 460 method) Identification of NTM isolated from sputa that do not meet ATS criteria for significance : (a) M. tuberculosis complex** isolated. Susceptibility test result pending / refer to previous cultures for susceptibility test results (b) Nontuberculous mycobacteria isolated (c) AFB culture overgrown with non-mycobacterial organism/s (d) No growth of AFB Turnaround time Day(s) test set up ** M. tuberculosis complex includes M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. canettii and M. microti. : (a) Receipt of specimen to detection of growth (average: 21 days). Affected by type of organism, specimen received and prior antibiotic treatment (b) Detection of growth to final identification (range of 2 days to 4 weeks, average 5 7 days for MTBC) Affected by slow growing mycobacteria, mixed cultures requiring additional processing and repeat cultures Negatives: 6 8 weeks : Monday Saturday DIRECT DETECTION FOR NUCLEIC ACID OF M. TUBERCULOSIS COMPLEX Type of test : Routine Specimen required : As for Mycobacterial Culture except that blood and bone marrow specimens should be collected in sterile screwcapped containers or plain Vacutainer tubes (NOT in Myco-F-Lytic vials) and delivered to the lab immediately. If delay is unavoidable, collect in EDTA or citrated tubes, though amplification results may be less consistent. Special instructions : Collect in a dedicated container. Do not order non- CTBL tests using the same request form and on the same specimen. Specimens should reach CTBL within 24 hours of collection. If transport delay is unavoidable, store at 4 C. Reflex testing : This test only provides preliminary information for empirical TB management. A reflex AFB smear and culture (both chargeable) is set up on each specimen. Written instruction from the doctor is necessary if these are not to be performed. 204 041-280_PathoH_SL.indd 204 4/3/08 1:03:18 PM
MYCOBACTERIOLOGY Turnaround time Day(s) test set up : ProbeTec Assay (BD ProbeTec ET system) using strand displacement amplification (SDA). The alternate method used is the Amplification Mycobacterium tuberculosis Direct test (AMTD) based on transcription-mediated amplification. : M. tuberculosis complex DNA detected / Not detected (BD ProbeTec) M. tuberculosis complex rrna detected / Not detected (AMTD) : 2 5 days : Once a week IDENTIFICATION OF MYCOBACTERIA Specimen required : Referred isolate from other laboratories. Please contact CTBL at telephone 6222 1391 before sending isolate. Fresh pure culture of isolate on slant in sealed, screwcapped plastic tube. Tube should be tightly closed, labelled with identifying lab number and placed in a biohazardlabelled ziplock bag. Pack and transport according to MOH-BATA requirements. Request form (stating name and address of referring laboratory, source of mycobacterial isolate, patient particulars with relevant clinical information, clinician in-charge and requester s contact number) is to be placed in separate sealed plastic bag outside the ziplock bag. The type of test should be specified as Mycobacterial Identification. For regional labs, packaging must conform to IATA standards. : AFB smear, culture, DNA probes, HPLC, sequencing and biochemical tests : Species identified reported Turnaround time : 4 weeks depending on the nature of the organism Day(s) test set up : Monday Saturday IDENTIFICATION OF M. BOVIS BCG AND NTM Specimen required : For mycobacteria isolated in CTBL or in other laboratories Referred M. tuberculosis complex isolates for identification of BCG from other laboratories please contact CTBL before sending isolate. Fresh pure culture isolate on slant in sealed screw-capped plastic tube. Tube should be tightly closed, labelled with identifying lab number and placed in a biohazard-labelled ziplock bag. Pack, transport and notify CTBL (in advance) and MOH of the transfer, according to MOH-BATA requirements for M. tuberculosis complex. Referred NTM for identification: Fresh pure culture isolate on slant in sealed screw-capped plastic tube. Tube should be tightly closed, labelled with identifying lab number and placed in a biohazard-labelled ziplock bag. 205 041-280_PathoH_SL.indd 205 4/3/08 1:03:18 PM
SECTION 4: SAMPLE COLLECTION & HANDLING SPECIAL INSTRUCTIONS & LAB TESTS Test report Turnaround time Day(s) test set up MYCOBACTERIAL VIABILITY Specimen required Turnaround time Day(s) test set up QUANTIFERON TEST Specimen required Special instructions Turnaround time Day(s) test set up Regional laboratories must make arrangements with the lab for the MOH-Import Permit before sending specimens and packaging must conform to international standards (IATA). The Request form should state the name and address of referring laboratory, organism identification, clinical source, patient particulars with relevant clinical information, clinician in-charge and requester s contact number. The type of test request should be specified as Identification of BCG or NTM. Forms are to be placed in a separate sealed plastic bag in the outer package. : HPLC, probes, molecular : Mycobacteria species identified : 1 week to 2 months, depending on species : Monday Friday : Post-inactivation samples : MGIT broth culture : Positive, Negative for AFB : 6 weeks for Negatives. 2 6 weeks for Positives : Upon request : Whole blood collected into Quantiferon Nil, TBAg and Mitogen tubes. 1 ml in each Nil (grey), TBAg (red) and mitogen (purple) Quantiferon tube. Acceptable range per tube = 0.8 1.2 ml : Quantiferon tubes are available from either SGH SOC Lab at Block 3 or CTBL and should be stored at 4 C till specimen collection. Do not collect on Saturdays and the eve of public holidays. Visually check that the blood level is approximately at the 1 ml marking on each tube before withdrawing the phlebotomy needle. Recollect in a fresh tube if there is over or underfilling. Shake the tubes 10 times after collection and transport upright at room temperature to SGH SOC Lab level 1 before 4 pm on the same day or hand-deliver directly to CTBL. : QuantiFERON TB-Gold In-tube. IFN-γ level detected by EIA : Positive, Negative, Indeterminate : Negatives: 2 5 days Positives and Indeterminates 4 7 days : 3 times a week 206 041-280_PathoH_SL.indd 206 4/3/08 1:03:19 PM
MYCOBACTERIOLOGY SUSCEPTIBILITY TESTING FOR MAC Specimen required : M. avium complex isolated and identified at CTBL. Isolates identified in referring labs must specify the organism identification and clinical source on the lab request form. For further instructions, refer under Mycobacterial Identification. Indication : Should be performed on isolates from blood or tissue (disseminated disease) and clinically significant respiratory specimens from patients who have failed prior macrolide therapy or prophylaxis Drug tested : Clarithromycin. For alternative drugs, please contact CTBL. : Radiometric. Minimum Inhibitory Concentration breakpoint testing : Susceptible, Resistant Turnaround time : 2 weeks Day(s) test set up : Monday Friday SUSCEPTIBILITY TESTING FOR M. KANSASII Specimen required : M. kansasii isolated and identified at CTBL. Isolates identified in referring labs must specify the organism identification and clinical source on the lab request form. For further instructions, refer under Mycobacterial Identification. Drug Tested : Rifampicin : Radiometric. Minimum Inhibitory Concentration breakpoint testing : Susceptible, Resistant Turnaround time : 2 weeks Day(s) test set up : Monday Friday SUSCEPTIBILITY TESTING FOR RAPIDLY GROWING MYCOBACTERIA Specimen required : Rapidly growing mycobacteria (RGM) isolated and identified at CTBL. Isolates identified in referring labs must specify the organism identification and clinical source on the lab request form. For further instructions, refer under Mycobacterial Identification. : Microbroth dilution method Drugs tested : Amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline, ertapenem, imipenem (not reported for M. abscessus chelonae complex), linezolid, levofloxacin, moxifloxacin, tigecycline, tobramycin (M. abscessus from eye cultures only) and sulfamethoxazole : Susceptible, Intermediate, Resistant according to NCCLS interpretive criteria Turnaround time : 1 2 weeks Days(s) Test set up : Once a week 207 041-280_PathoH_SL.indd 207 4/3/08 1:03:19 PM
SECTION 4: SAMPLE COLLECTION & HANDLING SPECIAL INSTRUCTIONS & LAB TESTS SUSCEPTIBILITY TESTING OF M. TUBERCULOSIS COMPLEX Specimen required : Referred M. tuberculosis complex isolates from other laboratories. Please contact CTBL before sending isolate. Fresh pure culture of M. tuberculosis isolate on slant in sealed screw-capped plastic tube. Tube should be tightly closed, labelled with identifying lab number and placed in a biohazard-labelled ziplock bag. Pack, transport and notify CTBL (in advance) and MOH of the transfer, according to MOH-BATA requirements. Regional laboratories must make arrangements with the lab for the MOH-Import Permit before sending specimens and packaging must conform to international standards (IATA). The request form should state the name and address of referring laboratory, organism identification, clinical source, patient particulars with relevant clinical information, clinician in-charge and requester s contact number. The type of test request should be specified as TB Culture for Susceptibility Testing. Forms are to be placed in a separate sealed plastic bag in the outer package. : MGIT non-radiometric for first line and BACTEC460 radiometric for second and third. Minimum Inhibitory Concentration breakpoint testing First line drugs: Streptomycin, Rifampicin, Isoniazid, Ethambutol Second line drugs: Ofloxacin, kanamycin, ethionamide Extended panel: Capreomycin, clofazamine and PAS : Susceptible, Resistant Turnaround time : 2 weeks. Affected by slow-growing isolates Day(s) test set up : Monday Saturday SUSCEPTIBILITY TESTING OF M. TUBERCULOSIS COMPLEX FOR PYRAZINAMIDE Specimen required : M. tuberculosis complex isolates from clinical specimens cultured at Central TB Laboratory. Test must be specified on lab request form. : MGIT. Minimum Inhibitory Concentration breakpoint testing : Susceptible, Borderline resistant, Resistant Turnaround time : 2 weeks Day(s) test set up : Monday Saturday 208 041-280_PathoH_SL.indd 208 4/3/08 1:03:19 PM