Mycobacterium tuberculosis, the organism responsible for tuberculosis (TB) is the primary public health concern in the United States. However, with the increase in the homeless, injectable drug use, and the appearance of HIV-infected patients, a significant increase in both TB and non-tuberculosis mycobacterial (NTM) disease has occurred in many areas of the country.

DE Public Health Lab serves the state of Delaware as a referral center for identification and/or susceptibility testing of M. tuberculosis and other mycobacterial isolates. The rapid techniques recommended by CDC for improving turnaround time in diagnoses of TB, as well as the specialized identification techniques required for the non-tuberculous Mycobacterium species, may not be feasible in local laboratories.

TESTS OFFERED

• Smear for Acid Fast Bacteria (AFB)
• Culture for AFB
• Identification of AFB
• Antimicrobial Susceptibility Testing
• Special Requests and Reference Handling

SAMPLE COLLECTION, HANDLING AND SHIPMENT

1. Obtain supplies by requesting individual components from the warehouse of this laboratory. Supplies can be obtained by contacting the laboratory  or faxing orders or email Labsupplies@delaware.gov.
2. Complete the Test Requisition Form including patient information or order request on-line through our Laboratory information management system (LIMS). Specimen and forms should be placed in a designated location at each site for the lab courier to pickup.
3. Obtain specimen, referring to directions listed below.
4. Label each container with a unique patient identifier, name, and collection date to match the requisition form. Make sure cap is on container tightly and bagged separately.
5. Place the specimen and requisition form if needed in the biohazard bag to transport to the laboratory. Be sure specimen is sealed inside double bag, and form is inserted into outer pocket, separated from the specimen itself. Transport to lab using courier system. DO NOT mail.

SPUTUM (EXPECTORATED)

Three early morning specimens obtained on different days should be submitted. A volume of 5 to 10 mL is adequate and there is no advantage in collecting a larger volume. The sample should contain recently discharged material from the bronchial tree with minimal saliva content.

SPUTUM (INDUCED)

If the patient has difficulty producing a sputum specimen, then induction should be considered. Sputum production may be induced by the inhalation of a warm aerosol of sterile 5-10% sodium chloride in water produced by a nebulizer.

The specimen should be clearly marked "INDUCED" on the request slip since nebulized sputa is watery in consistency and could be mistaken for saliva.

GASTRIC LAVAGE

This procedure can be employed where sputum production is unsuccessful. This technique requires professional attention and should only be attempted in the hospital. Gastric lavage is performed early in the morning before eating and at least 8 hours after the patient has eaten or taken oral drugs. A specimen volume of 5-10mL is required and must be neutralized with 100 mg of sodium carbonate.

URINE

An early morning midstream specimen should be collected. Send entire specimen. Multiple specimens over several days may be required to obtain a positive specimen. Due to contamination and deterioration, 24 hour urine specimens are NOT acceptable. Keep specimen refrigerated until transport.

BLOOD

Specimens should be collected in Isolator tubes available from the kit room of this laboratory on request. Check expiration date prior to drawing blood. Store tubes at room temperature. Transport the same day of collection.

FLUIDS

Body fluids (spinal, pleural, pericardial, synovial, ascitic, blood, pus, and bone marrow) must be aseptically collected and submitted in sterile containers. Keep refrigerated until transport.

TISSUE

Any tissue to be cultured must be collected aseptically into sterile a container without fixatives or preservatives. If the specimen may dry, add sterile saline to keep moist. Do not place tissue specimen for culture into formalin. Keep refrigerated until transport.

ISOLATES OR CULTURES FOR IDENTIFICATION AND/OR SUSCEPTIBILITY

Isolates should be submitted as pure, fresh subcultures on Middlebrook 7H10 or Lowenstein-Jensen media whenever possible, but liquid 7H9 proprietary broths are acceptable (i.e. MGIT or BacT/Alert MP bottle). Include preliminary identification on the test requisition form. If referral to CDC is likely or specifically requested, a completed CDC dash form must accompany the specimen.

TESTS

This laboratory utilizes the Bactec MGIT 960 TB System for detection of AFB in specimens, and performance of susceptibility testing.

Direct identification from direct specimens (i.e. pulmonary) as well as grown cultures (broth or grown isolates) is performed by laboratory designed test (LDT) using real-time polymerase chain reaction (qPCR).

Conventional susceptibilities only apply to M. tuberculosis complex (Mtbc).

Primary drug susceptibility testing (DST): Isoniazid, Rifampin, Ethambutol, Pyrazinamide is performed at DPHL (when warranted). Secondary drug panels or rapid primary DST is referred to CA Microbial Disease Laboratory (MDL) or CDC when warranted or requested.

Tests requiring further specialized techniques can be referred to CDC or CA MDL

RESULTS

In keeping with CDC recommendations to decrease the spread of M. tuberculosis, this laboratory provides AFB results as follows:

1. Fluorescent acid fast stain on original specimen: 24-72 hours from receipt in laboratory.
2. Identification of M. tuberculosis from culture: As soon as possible, but within 14-21 days from specimen receipt.
3. Susceptibility results for M. tuberculosis: As soon as possible, but within 15-30 days of specimen receipt.

NOTE: All positives will be called the same day of detection.

REJECTION

Samples will be rejected if they are:

• Unlabeled - All specimens MUST have a unique patient identifier.
• Have no collection date indicated
• Insufficient in Quantity - No specimen received, no specimen in container, or insufficient specimen to perform testing.
  • Sputums containing <2 mL of sample or appearing as saliva
  • Urines with <40 mL volume
• Damaged - Specimen leaked or broken in transit.
• Too Old - Samples greater than 3 days old are unreliable specimens for testing.
• Contaminated - Isolates or subcultures will be rejected if unable to isolate test organism.

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