Testing Available/Specimen Requirements

Tuberculosis

Acid-fast smears:
Routine smears are performed by a fluorochrome method and read on fluorescent microscopes. Smear reports are completed within 24 hours of processing the specimen and are faxed and mailed to health care providers. Positive smear results are telephoned on the same day they are tested to a responsible party at the submitting facility and to the respective local public health agency.

Nucleic acid amplification (NAA) – direct specimen testing:
The MSTBL offers nucleic acid amplification testing (Gen-Probe Amplified Mycobacterium Tuberculosis Direct) of clinical specimens for the detection of M. tuberculosis complex rRNA. The specimens submitted for testing may be processed sediments or unprocessed (raw) samples. The raw or processed samples should be forwarded to MSTBL as soon as possible and must be received within 5 days of collection. All supplies necessary to expedite the shipping process will be provided: submission forms, IATA approved diagnostic specimen kits and Fed-Ex clinical specimen envelops with billable stamps. There is no charge to the submitter for this test, as funding is provided through CDC’s TB Elimination Cooperative Grant and through a Memorandum of Understanding between the MSTBL and The Florida Department of Health (FDOH), Bureau of Laboratories, where testing is performed. Turn-around time for nucleic acid amplification (TB) test results is approximately 48 hours from receipt of specimen at MSTBL until test result is faxed to the submitter.

The nucleic acid amplification testing is targeted at specimens from patients showing signs and symptoms consistent with active pulmonary tuberculosis and who have received no antituberculous therapy, less than 7 days of such therapy or have not received such therapy in the last 12 months. The clinical specimens tested are limited to acid-fast bacilli (AFB) smear positive respiratory specimens. This test is to be used as an adjunctive test – it does NOT replace the need for AFB smear and culture.

Culture:
Processed material for culture for mycobacteria are inoculated to a series of media formulated to enhance rapid recovery of organisms. Both solid and liquid media are employed for mycobacterial recovery. Mycobacterial isolates are identified to species level, primarily by high performance liquid chromatography (HPLC) which yields overnight identification results. When necessary, genetic probes and conventional biochemical methods may be utilized to further characterize the organism. A sample of each positive M. tuberculosis isolate is frozen at -70°C as a permanent archive for epidemiological purposes. Mycobacteria other than tuberculosis (MOTT) isolates are also frozen at -70°C and maintained for at least two years.

Genotyping:
Missouri participates in CDC’s Genotyping Program. One isolate from every patient with a culture-positive specimen is forwarded to CDC for genotyping. Two PCR genotyping methods (spoligotyping and MIRU analysis) are performed on all submitted isolates. Selected isolates will also be analyzed with RFLP analysis. The genotyping fingerprint of an isolate is essential for epidemiological research of TB cases.

Susceptibilities:
Susceptibilities to the five primary drugs (streptomycin, isoniazid, rifampin, ethambutol, and pyrazinamide) are performed on M. tuberculosis isolates from every patient. These tests are done by the rapid BACTEC methodology and results are usually available in five to eight days (from the time the procedure is started). Secondary drug susceptibilities are automatically performed on multi-drug resistant isolates of M. tuberculosis. Physicians may also call and discuss specific patient needs for secondary drugs. The MSTBL currently has the capability to test for levofloxacin, amikacin, tehionamide, rifabutin, capreomycin, and clofazimine sensitivity/resistance. Drug testing on mycobacteria other than tuberculosis (MOTT) is available upon request. Guidelines published by the American Thoracic Society and by NCCLS (Diagnosis and Treatment of Disease Caused by Nontuberculous Mycobacteria, Respir & Crit Care Med. Supplement August 1997, Vol. 156) are followed.

For rapidly growing mycobacteria, The MSTBL offer sensitivities performed by the E-test method; results are available in two to six days. Currently isolates are tested for clarithromycin, amikacin, levofloxacin, doxycycline, imipenem, and cefoxitin sensitivity/resistance. The MSTBL follows scientific and clinical recommendations in the selection of antimicrobials for susceptibility testing.

Specimen Requirements

Sputum: Instruct the patient in proper oral hygiene and collect 2 – 10 ml of first morning sputum produced by deep cough. No saliva or nasal secretions please! Place the specimen in the sterile centrifuge provided and place inside the metal container. Complete the requisition form and wrap around the outside of the metal container and place in the outside cardboard mailer. We recommend collecting specimens on at least three consecutive days. We do not accept 24-hour specimens. Deliver by FedEx promptly; if delays are unavoidable, refrigerate until shipped.

Bronchial lavage: Aseptically place 2 – 10 ml of specimen in the sterile centrifuge tube provided and place inside the metal container. Handle as per sputum instructions above.

Body fluids (Pleural fluid, Knee fluid, CSF, Bone marrow, etc.): Aseptically place 2 – 10 ml of specimen in the sterile centrifuge tube provided and place inside the metal container. Wrap the completed requisition form around the metal can and place in the outside mailer. Alternately, the specimen can also be placed in a sterile tube of your choosing and placed in the centrifuge provided along with adequate packaging to prevent breakage, and then placed in the mailing container. Deliver by FedEx promptly; if delays are unavoidable, refrigerate until shipped.

Wounds: Wounds will normally be collected on swabs. Aseptically place the swab along with 2 ml of sterile saline in the sterile centrifuge tube provided and place in the metal container. Wrap the completed requisition form around the metal can, place in the outside mailer, and ship according to above instructions.

Tissue: Aseptically place small pieces of tissue in the sterile centrifuge tube provided along with 2 ml of sterile saline and place in the metal can. Ship according to above instructions.

Urine: Place approximately 40 ml of urine in the sterile centrifuge tube provided and place in the metal can. Ship according to above instructions. Early morning, mid stream urine is preferred. We do not accept 24-hour urine collections.

Blood: Blood should be aseptically drawn into anticoagulant, preferably a liquid anticoagulant such as heparin. 10 ml is an adequate sample. Place the tube of blood, along with adequate packing to prevent breakage, in the sterile centrifuge tube provided, ship according to above directions.

Stool: We do not accept raw stool specimens for culturing or smears. Stool specimens must be pretreated before being submitted to us.

Pretreatment procedure:

To one volume of stool, add 2 volumes of sterile distilled water. Stir thoroughly and filter through gauze to remove coarse particles. Add enough NaCl crystals to make a saturated solution. Mix thoroughly, and allow to stand for several hours. Mycobacteria will float to the surface and may be skimmed off. Place the skimmed stool material in the sterile centrifuge tube provided and place in the metal can. Ship according to above instructions.

Gastric lavage: Specimens from gastric lavage are frequently compromised for many reasons. We feel every effort should be made to replace gastric specimens with those obtained by postural drainage, induced sputum, or bronchoscopy. If gastric fluid must be submitted, it is your responsibility to stabilize the pH at the time the specimen is collected. Untreated specimens will have to be rejected. Ship according to above directions.