Mycobacterium tuberculosis, the organism responsible for Tuberculosis (TB), a highly infectious bacterial disease, commonly seen as a respiratory illness, but is well established to disseminate into other disease progression. In recent years, TB has been identified in outbreaks related to healthcare related surgical interventions, further increasing its concern for public health intervention. TB was originally discovered in 1882 and remains a prevalent human pathogen today. CDC and public health collaborations work together towards the goal of eliminating M. tuberculosis infections. Although anyone can become infected with TB, individuals with comorbidities, such as Human Immunodeficiency Virus (HIV), along with individuals from geographical locations of underserved and/or overpopulated communities, are at higher risk than other of contracting TB. In 2021 88.1 percent of the TB cases in the United States were among minority groups. Of the national case total in 2021, 71.4 percent were non-U.S. born.

DE Public Health Lab serves the state of Delaware as a reference laboratory for identification and susceptibility testing of M. tuberculosis as well as identification testing of non-TB Mycobacteria species. DPHL continuously works closely with the CDC and our state clinics to help identify TB and work toward eliminating it.

TESTS OFFERED

• Smear for Acid Fast Bacteria (AFB)
• Culture for AFB
• Identification of AFB
• Antimicrobial Susceptibility Testing
• Special Requests and Reference Handling not limited to:
  • Molecular Detection of Drug Resistance (MDDR) testing
  • Pyrosequencing (POQ)
  • Mycobacterium bovis rule out.
  • Second-lone drug testing
• Interferon gamma release assay (IGRA)
• Whole Genome Sequencing - for mutation detection for predictive antimicrobial susceptibility or resistance

SAMPLE COLLECTION, HANDLING AND SHIPMENT

1. Obtain supplies for testing by contacting the laboratory, faxing orders or by emailing 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 pick up.
3. For collection of specimens, refer to directions listed below.
4. Label each container with a unique patient identifier, name, and collection date to match the requisition form. Make sure the cap is on the container tightly and each specimen is 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

Urine is not validated for culture in the MGIT 960 system. Because of this DPHL recommends sending these specimens to a commercial reference lab for testing for AFB. However, if a clinic sends a specimen to DPHL for emergency testing the results will be solid media based.

STERILE 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 appropriate mycobacteria enrichment media whenever possible. Liquid 7H9 proprietary broths are also acceptable (i.e., MGIT or BacT/Alert MP bottle). Include any preliminary identification on the test requisition form. Please contact DPHL if referral to CDC for specialized testing is requested.

BLOOD-IGRA Testing

DPHL performs IGRA testing using specific laboratory methods. Blood is to be collected in the collection kits supplied by the laboratory. Be sure to check the expiration dates prior to drawing blood. Within 16 hours of collection, tubes need to be stored in an incubator for 16-24 hours at 37 degrees C. Samples should be picked up by a courier the day after collection. Refer to DPHL for appropriate sample collection dates and times.

TESTS

DPHL utilizes an automated broth culture system for both detection of AFB in specimens, and susceptibility testing.

Identification from clinical specimens (i.e., pulmonary) as well as cultures (broth or grown isolates) is performed by laboratory designed test (LDT) using real-time polymerase chain reaction (qPCR). Non-M. tuberculosis complex (Mtbc) AFB identification is also performed on isolated colonies.

Conventional susceptibility testing is only performed on Mtbc. Primary drug susceptibility testing (DST) reports Rifampin, Isoniazid, Pyrazinamide, and Ethambutol (RIPE) Secondary drug testing is referred to the regional laboratory or Federal Laboratory (CDC) when warranted, or upon request. DPHL is expected to complete Whole Genome Sequencing validation in late 2024.

Further specialized testing, such as MDDR, second-line drug testing, pyrosequencing (PSQ), M. bovis rule out, can all be referred to both regional and/or national laboratory partners upon request.

RESULTS

In keeping with CDC and health People 2030 recommendations DPHL expects to report AFB results within the following timeframes:

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 positive specimens are expected to be reported to the submitter the same business day of receipt, if possible.

REJECTION

Samples will be rejected if they are:

• Unlabeled - All specimens MUST have a unique patient identifier.
• Date of collection not recorded on specimen container.
• Insufficient in quantity - No specimen received, no specimen in container, or insufficient specimen to perform testing.
• Blood- If collection tubes are expired or if blood is improperly collected and incubated prior to receipt.
• Sputums containing <2 mL of sample or appearing as saliva.
• Urine with <40 mL volume
• Damaged - Specimen leaked or broken in transit.
• Too old - Samples >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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This page was last updated 4/24

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