Mycobacteriology

Introduction

The Central Tuberculosis Laboratory (CTBL) of the Division 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 - BD MAX MDR-TB ASSAY
Direct detection for nucleic acid of M. tuberculosis complex - XPERT MTB/RIF ULTRA ASSAY*
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 for M. tuberculosis complex- Minimum inhibitory concentration (MIC)
Susceptibility testing for M. tuberculosis complex – Hain MTBDR for Rifampin and Isoniazid
Susceptibility testing for M. tuberculosis complex – Hain MTBDRsl for Fluoroquinolone and Aminoglycoside/Cyclic Peptide
Susceptibility testing of M. tuberculosis complex for Pyrazinamide
Identification and susceptibility testing for NTM – Hain NTM-DR for M. avium complex, M. abscessus-chelonae complex; Macrolide and Aminoglycoside resistance

*Please refer to respective test for special specimen collection and delivery instructions. Clinical specimens for all mycobacterial investigations should be sent to or collected by:Client & Specimen Management, Division of Pathology, Singapore General Hospital (Tel: 6321 4950, fax: 6326 5436).

Special Instructions on Specimen Collection and Handling

Use the CPOE on-line request or 'Request for Mycobacteriology 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 the section ALPHABETICAL TEST LISTING – MYCOBACTERIOLOGY for the tests available.

Verbal requests should be followed by laboratory request forms either faxed to 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 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

Collect specimen before starting patients on anti-mycobacterial drug therapy.
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.
Label each container with the patient's name, NRIC number, nature of specimen, and date and time of collection.
Primary container/s for each specimen should be placed in a sealed biohazard-labelled plastic bag and accompanied by a request form with relevant patient and clinical data. Staple the request form to the outside of the plastic bag (Do not staple through the sealed portion of the bag). Users are advised to avoid stapling forms to bags as these result in sharps injuries. Use pouched bags and place the laboratory request from in the exterior pouch of the bag.
Collect specimens aseptically, using standard precautions and minimising contamination with commensal organisms. These microbes will affect the smear and culture results.
Collect 2-3 consecutive sputum samples for acid-fast smears and culture in an 8 to 24 hour period; one of which should be a first morning specimen, for patients with clinical and chest x-ray findings compatible with tuberculosis.
The specimen should be collected in the morning before any oral intake.
The patient's mouth should be rinsed with clean water before starting to collect the specimen, in order to remove contaminating food particles and commensal bacteria.
Sputum specimens should be collected in a well-ventilated area and precautions should be taken to ensure that healthcare workers and others are not exposed to infectious aerosals and materials.
CTBL specimens should be collected in containers separate from non-CTBL specimens to ensure prompt delivery and lessen the likelihood of cross-contamination.
Avoid collecting specimens on swabs as these provide limited material for analysis.
Do not send the specimens in a syringe with needles as they pose a hazard to personnel. Dispense the specimen into a sterile, screw-capped container; minute amounts can be aspirated and flushed out into the containers with sterile saline. Otherwise, aspirate the specimen back into the syringe and replace the needle with a rubber stopper/cap. Discard the needle carefully using sharps precautions.
Specimens should be refrigerated at 4^°C within one hour of collection. Frozen specimens are unacceptable.
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. Myco-F-Lytic bottles that are culture-positive with unknown AFB should be submitted using triple packaging. For known M. tuberculosis complex isolates, CTBL and MOH must be notified in advance of the transfer before the isolates are packed and transported according to MOH-BATA requirements.
For regional laboratories, contact CTBL before sending the specimen. The packaging must conform to international standards (IATA) and the relevant MOH / AVA permits code.

Specimen Requirements

Specimen Type	Specimen Requirement	Special Instructions
Abscess contents, aspirated fluid	As much as possible in sterile disposable container.	Disinfect skin with 70% alcohol before aspiration. Use swab to collect only if the volume is insufficient for aspiration. Aspirate material from under the margin of the lesion/abscess. Laboratory may provide 7H9 broth for transport of small volumes of aspirates.
Blood	5ml of plain blood inoculated directly into Myco-F-Lytic vial.** On rare occasions, if blood culture vials are not available, blood and bone marrow may be submitted in Vacutainer tubes containing SPS, heparin or citrate. EDTA tubes are not acceptable	Disinfect skin with 70% alcohol. 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. Do not refrigerate or freeze blood specimens
Body fluids (pleural, pericardial, peritoneal, etc)	As much as possible (minimum 10 - 15ml) in a sterile leak-proof, screw-capped, single-use container.	Disinfect skin with 70% alcohol if collecting by needle and syringe.
Bone	Bone in sterile disposable container with no fixative or preservative.	Do not submit specimen in formalin.
Bone marrow	Inoculate directly into Myco-F-Lytic vial.** If direct molecular testing is required, then collect at least 3 ml in a sterile, screw-capped disposable container.	As for blood
Bronchoalveolar lavage or bronchial washing/brushing	≥ 5 ml in sterile disposable container	Avoid contaminating bronchoscope with tap water as free-living saprophytic mycobacteria may produce false-positive results.
CSF	≥2 ml in sterile disposable container	Disinfect skin before aspiration. Do not refrigerate or freeze.
Gastric lavage	≥5 - 10 ml in sterile disposable container. Collect in the morning soon after patient awakens in order to obtain sputum swallowed during sleep.	Collect a fasting early morning sample on 3 consecutive days. Unless contraindicated, use 25 to 50ml of chilled, sterile saline. Collect in container containing 100 mg of sodium carbonate per 50 ml of total fluid if delivery is expected to be delayed for > 4 hours.**
Laryngeal swabs	Unless the patient is suspected to have laryngeal TB, laryngeal swabs are not recommended for diagnosing pulmonary TB due to the following reasons: 1. Difficulty in proper collection. 2. The amount of material collected on the swab is limited. 3. Risk of sharps injury from broken glass containers.
Lymph node	Node or portion of node in sterile disposable container with no fixative or preservative. Minute amounts may be submitted in a 1-2 ml of sterile saline.	Collect aseptically. Select caseous portion if available. Do not freeze, immerse in formalin or other preservatives or wrap in gauze.
Skin lesion material	Send biopsy specimen or aspirate in sterile disposable container with no fixative or preservative.	For cutaneous ulcers, collect biopsy sample from periphery of lesion or aspirate material from under margin of lesion. Swabs in transport medium 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, state this on request form, because Mycobacterium ulcerans requires prolonged incubation for primary isolation.
Sputum	5 - 10ml in sterile, wax-free disposable specimen container.	Instruct patient to rinse mouth before sputum is collected. Collect 2-3 consecutive deep productive cough sputum samples in an 8 to 24 hour period; one of which should be a first morning specimen before any oral intake. 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	≥1g in sterile, disposable wax-free container	Collect the specimen directly into the container or transfer from the bedpan. Recommended only for the detection of Mycobacterium avium complex (MAC) involvement in the gastrointestinal tracts of patients with AIDS. Refrigerate if delay in transportation is expected, but do not freeze.
Tissue biopsy sample	1g of tissue, if possible, in sterile disposable container with no fixative or preservative. Minute amounts may be submitted in a 1-2 ml of sterile saline.	Collect aseptically. Select caseous portion if available. Do not freeze, immerse in formalin or other preservatives or wrap in gauze
Transtracheal aspirate	As much as possible in a sterile, leak-proof disposable container
Urine	Clean the urethral area with distilled or cooled boiled water (tap water may contain saprophytic bacteria). Mid-stream urine is not advisable. Collect at least 40ml in a sterile, leak-proof disposable container. For suprapubic urine, collect as much as possible in a sterile disposable container.	Collect first morning specimen on three consecutive days. The first morning void provides the best yield. Specimens collected at other times are diluted and not optimal. Specimens of < 40ml are unacceptable unless a larger volume is not obtainable. An early morning midstream urine is acceptable only if an early morning urine sample is not available. 24-hour pooled specimens or urine from a catheter bag are unacceptable. A small fraction of patients undergoing BCG immunotherapy for bladder cancer may have local or even disseminated disease from BCG. For 95% of such patients, serious side effects do not occur and BCG isolation from urine does not have clinical significance. Routine urine culture for mycobacteria in this setting is not recommended.
Wound material	See biopsy or aspirate	Due to limited material collected, swabs are acceptable only if biopsy or aspirate is not obtainable; negative results may not be reliable. If used, use a sterile swab moistened with sterile saline; do not use eSwab.
Specimens for nucleic acid amplification (BD Max MDR-TB or Xpert MTB/RIF Ultra Assay)	All specimens, as for culture. Specimens in transport swabs are not acceptable. Turbid and grossly bloody specimens may contain inhibitors or give rise to false-positives due to high background. If critical, blood/bone marrow specimens should be sent in plain tubes. Specimens sent in heparin-containing containers, Myco-F-Lytic or on slides are not suitable.	To avoid false positives from cross contamination, specimens should not be pre-processed in other labs. If delay in delivery is expected, store at 4ºC.
Blood for Quantiferon testing	The Quantiferon-Plus tubes may be obtained from Client & Specimen Management (CSM) as follows: Complete the CSM's "Media Request Form” (fill in under "other") and fax to CSM (62254965) a day before collecting the tubes. The fax should reach CSM on Mondays to Fridays, between 0800 to 1600 hrs. Collect the requested items from CSM, Academia Level 8, the following day between 0900 to 1200 hrs. Collect whole blood (1ml per tube, accepted range = 0.8-1.2 ml) in the Quantiferon-Plus tubes in correct order: Nil (grey cap with white ring), TB1 (green cap with white ring), TB2 (yellow cap with white ring) then Mitogen (Purple cap with a white ring). The blood volume in each tube should be within the black marking on the side. The black marking indicates the 0.8 ml to 1.2 ml fill volume, and the prevacuumed tubes have been validated by the manufacturer for volumes ranging from 0.8 to 1.2 ml.	Use a butterfly needle or vacutainer system - using hypodermic syringes increases the risk of sharps injury. Purge the contents of the butterfly tubing with a plain tube first. Labelling - 1. Label all 4 tubes with the patient’s particulars, taking care to avoid obscuring the area where the blood filling and the black volume marks can be observed. 2. Indicate on the tubes or request form the date and time of specimen collection. For CPOE orders, print the barcodes just prior to the collection. To avoid overfilling - 1. Before specimen collection, allow the tubes to equilibrate to room temperature (17-25ºC). 2. Do not choose a limb that has an intravenous drip attached or force blood into the tubes with a syringe. 3. Remove the tourniquet once blood enters the first tube. Note that the tubes draw blood relatively slowly; keep the tube on the needle for 2-3 seconds once the tube appears to have completed filling. 4. If the level of blood in any tube is not close to or exceeds the indicator line, another blood sample should be obtained. Mixing Post collection, mix the blood with the tube contents by shaking tubes 10 times, to ensure that the entire inner surface of the tubes are coated with blood. Foaming may be expected and this will not affect the result. The shaking should not be so hard as to disrupt the gel at the base of the tubes as this may cause aberrant results. Transportation Transport the tubes at room temperature (22 +/- 5ºC) to CSM before 1630 hrs on the same day of blood collection. It is not necessary for them to be transported upright. Timing Specimens must be received at CTBL within 16 hours of sample collection on Mon-Fri between 8.30 am - 5.00 pm. They will not be accepted on Saturday, Sunday, eve of public holidays and on public holidays.

Available upon request from CSM**

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