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AFB Specimen Collection and Transport

General Considerations: AFB Specimens

 

Proper collection and handling of specimens is imperative for accurate isolation and identification of Mycobacteria.
  1. General Considerations
    1. Use a sterile, leak-proof, disposable plastic container. Do not use waxed containers.
    2. Label the container with the patient's name, MRN, specimen type, and date and time of collection.
    3. Collect initial specimens before antimicrobial therapy is started.
    4. Swabs are not recommended for isolation of mycobacteria.
    5. Collect sufficient material for the tests requested (See Specific Specimen Requirements).
    6. Do not use any fixatives or preservatives.
  2. Transportation of specimens
    1. Transport specimens to the laboratory in as short a time as practical to avoid overgrowth by contaminating indigenous microbiota.
    2. Refrigerate specimens whenever possible if transport to the laboratory or specimen processing will be delayed more than 1 hour. 
    3. If blood is collected in an Isolator tube, hold at room temperature, Do Not Refrigerate
  3. Frequency with which specimens should be submitted:
    1. On smear-negative patients, up to six sputum’s and three urines from different days may be submitted.
    2. As necessary per providers request.
    3. For known smear-positive patients, after initial set of three specimens from the positive source, no more than one specimen per week from the positive source should be submitted during the first three months of therapy. After three months of therapy, up to three specimens per month from the positive source can be submitted for smear and culture as a test of the efficacy of the drug treatment. Specimens from negative sources on the same patient will be accepted as for smear-negative patients.

 

Limitations of AFB Testing Procedures

  1. Limitations of procedure:

    1. Specimens containing cetylpyridinium chloride or Zephiran must be neutralized by being washed with buffer if inoculation to an agar-based medium is desired. This neutralization is unnecessary if only an egg-based medium is used.

    2. Collect sufficient material for the tests requested. Use of an insufficient quantity of specimen will result in false-negative results.

    3. Avoid contamination with normal microbiota and with tap water or other fluids that may contain either viable or nonviable environmental mycobacteria. Saprophytic mycobacteria may produce false-positive culture or smear results.

    4. Saprophytic mycobacteria may be recovered from gastric lavage fluids.

    5. Specimens obtained for initial diagnosis after the initiation of antimicrobial therapy may produce false-negative results. Even a few days of antimicrobial therapy may kill or inhibit sufficient numbers of mycobacteria to leave confirmation of disease in doubt.

Specific Specimen Requirements: AFB Testing

 

Specimen Type

Specimen Requirements

Special Instructions

Unacceptable Specimens

Abcess contents, Aspirated Fluid

As much as possible in syringe with Luer tip cap or sterile container. “No Needle”

Cleanse Skin with alcohol before aspirating samples

Collect Specimen on swab and place in transport medium only if volume is insufficient for aspiration by needle and syringe

Dry Swab

Blood

Preferred Dupont 10ml Isolator Tube.

Acceptable: 10mL green top tube (heparinized)

Disinfect Site as For Routine Blood Culture.
Mix tube contents immediately after collection.

Blood Collected in EDTA, which greatly inhibits mycobacterial growth even in trace amounts.

Coagulated Blood, immediately after collection

Short Draw Specimen

Body Fluids: Pleural, Pericardial, Peritoneal, etc

As much as possible (10-15mL minimum) in sterile container or syringe with Luer tip cap (No Needle) or sterile container.

Collect grossly bloody specimens into 10mL Isolator tube

Disinfect site with alcohol if collecting by needle and syringe

Since many of these fluids may contain fibrinogen it may be necessary to add anticoagulant (SPS or Heparin) to collection containers.

  

Bone

Bone in sterile container without fixative or preservative

  

Specimen submitted in formalin

Bone Marrow

Green top tube

Collect aseptically. Mix tube contents immediately following collection.

  

Broncoalveolar lavage or bronchial washings

>5mL in sterile container

Avoid contaminating bronchoscope with tap water. Saprophytic mycobacteria may produce false positive culture or smear results.

  

Bronchial Brushings

Submit in sterile container

    

Spinal Fluid (CSF)

>2mL in sterile container

Use maximum volume attainable

  

Gastric Lavage fluid

>5mL in sterile container

Collect in the morning soon after patient awakens, in order to obtain sputum swallowed during sleep.

Collect fasting early-morning specimen on 3 consecutive days. Use sterile saline.

Adjust to neutral pH with 100mg of sodium carbonate immediately following collection.

Specimen that has not been neutralized

Lymph Node

Node or portion in sterile container without fixative or preservative

Collect aseptically, and avoid indigenous microbiota

Select caseous portion if available

Do not immerse in saline or other fluid or wrap in gauze.

Freezing decreases yield.

Specimen submitted in formalin

Skin Lesion Material

Submit biopsy specimen in sterile container without fixative or preservative

Submit aspirate in syringe with Luer tip cap (No Needle), or sterile container.  

Swabs in transport medium (Amies or Stuarts) are acceptable only if biopsy sample or aspirate is not obtainable.

For cutaneous ulcer, collect biopsy sample from periphery of lesion or aspirate material from under margin of lesion.

If infection was acquired in Africa, Australia, Mexico, South America, Indonesia, New Guinea, or Malaysia not on request because Mycobacterium ulcerans may require prolonged incubation for primary isolation.

Dry Swab

Smear on Slides

Smear specimen over 1.5x1.5cm area of clear slide

Heat fix smears. Transport in slide container taped closed and labeled BIOHAZARD

Sixty Percent of Acid-Fast Bacilli Present On Smear Will Survive Heating For 2 Hours At 65C

  

Sputum

>3 ml in sterile, wax-free, disposable container

Collect an early-morning specimen from deep, productive cough on at least 3 consecutive days.

For follow-up of patients on therapy, collect at weekly intervals after initiation of therapy.

For expectorated sputum: Instruct patient on how to produce sputum specimens, as distinct from saliva or nasopharyngeal discharge. Have patient rinse mouth with water before collecting sputum, to minimize contaminating specimen with food particles, mouth wash, or oral drugs, which may inhibit the growth of mycobacteria.

For induce sputum: Use sterile hypertonic saline. Avoid sputum contamination with nebulizer reservoir water. Saprophytic mycobacteria in tap water may produce false-positive culture or smear results. Indicate on request if specimen is induced sputum.

24-hour pooled specimens; saliva, less than 3mL of sputum

Stool

>1 gram in sterile wax-free, disposable container

Collect specimen directly into container, or transfer from bedpan or plastic wrap stretched over toilet bowl (Wax from container may produce false positive smear).

Frozen Specimen

Utility of culturing stool for acid-fast bacilli remains controversial. Discuss with Microbiology before collection.

Tissue Biopsy sample

1 gram of tissue, if possible, in sterile container without fixative or preservative

Collect aseptically, and avoid indigenous microbiota

Select caseous portion if available

Do not immerse in saline or other fluid or wrap in gauze. Freezing decreases yield

Specimen submitted in formalin

Transtracheal aspirate

As much as possible in syringe with Luer tip cap ( No Needle)   or other sterile container

    

Urine

As much as possible (Minimum 40ml) of first morning specimen obtained by catheterization or of midstream clean catch in sterile container

For suprapubic tap, as much as possible in syringe with Luer tip cap (No Needle)  or other sterile container.

Collect first morning specimen on 3 consecutive days. Organisms accumulate in bladder over night, so first morning void provides best yield. Specimens collected at other times are dilute and not optimal

Accept only on specimen/day

24 hour pooled specimens

Urine from catheter bag

specimens of <40ml (unless larger volume is not obtainable)

Wound Material

See Biopsy or Aspirate

Swabs are acceptable only if biopsy or aspirate is not obtainable. If used, they must be placed in transport medium (Amies or Stuarts). Negative results are not reliable.

Dry Swab

 

Unacceptable Specimens

  1. Unacceptable Specimens: (See also Specific Specimen Requirements for individual specimen type rejection criteria.)

    1. Swabs are not recommended for the isolation of mycobacteria, since they provide limited material. They are acceptable only if a specimen cannot be collected by other means. State in report that negative results obtained from specimens submitted on swabs are not reliable.

    2. Waxed containers may produce false-positive smear results.

    3. Do not use 24-hour collections. They are likely to be dilute and contaminated.

    4. Freezing of specimens may decrease the yield.

    5. QNS Samples