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Diagnostic Microbiology

Author(s): Centers for Disease Control

CDC's Core Curriculum on Tuberculosis

Excerpts from "Guidelines from the Centers for Disease Control Core Curriculum on Tuberculosis," 4th ed. 2000.

Diagnostic Microbiology

 


Diagnostic Microbiology

Specimen Collection
Persons suspected of having pulmonary or laryngeal TB should have at least three sputum specimens examined by smear and culture. It is best to obtain a series of early-morning specimens collected on 3 consecutive days. Specimens should be obtained in an isolated, well-ventilated area or a sputum collection booth.

A health care worker should coach and directly supervise the person at least the first time sputum is collected. Persons should be properly instructed in how to produce a good specimen. Patients should be informed that sputum is the material brought up from the lungs and that mucus from the nose or throat and saliva are not good specimens. Coaching patients individually on how to expectorate can facilitate sputum collection. Unsupervised patients are seldom successful in providing an adequate specimen, especially the first time. The amount of coaching required on later visits will depend on individual patient needs.

For patients unable to cough up sputum, deep coughing may be induced by inhalation of an aerosol of warm, hypertonic (5%-15%) saline. Patients should be given time — 15 minutes is usually sufficient — to produce sputum, which is usually brought up by a deep cough. Because induced sputum is very watery and resembles saliva, it should be labeled "induced" to ensure that the laboratory staff do not discard it.

Bronchoscopy can be done if there is suspicion of TB and the patient cannot cough up sputum. Adequate infection control precautions should be taken when performing a bronchoscopy for the purpose of diagnosing TB disease (see Infection Control in Health Care Facilities). Bronchial washings, brushings, and biopsy specimens may be obtained, depending on the diagnostic possibilities and findings. Sputum collected after bronchoscopy may also be useful for a diagnosis.

Gastric aspiration can also be used to obtain specimens of swallowed sputum. Although it is uncomfortable, it is more cost effective and less invasive then bronchoscopy. It is the best way to obtain specimens from infants and some young children who cannot produce sputum even with aerosol inhalation. When using gastric aspiration to obtain specimens from children, it should be done in the morning before the patient gets out of bed or eats.

During specimen collection, patients produce an aerosol that may be hazardous to health care workers or other patients in close proximity. For this reason, precautionary measures for infection control must be followed during sputum induction, bronchoscopy, and other common diagnostic procedures (see Infection Control in Health Care Facilities).

Because TB can occur in almost any anatomical site, a variety of clinical specimens other than sputum (e.g., urine, cerebrospinal fluid, pleural fluid, pus, or biopsy specimens) may be submitted for examination when extrapulmonary TB disease is suspected. Tissue specimens for the culture of M. tuberculosis should be placed in a transport medium (e.g., Dubos) or a normal saline solution. Formalin or other preservatives should not be used because these solutions kill or inhibit the growth of M. tuberculosis. Tissue specimens should be delivered to the laboratory promptly.

Laboratory Examination

Detection of acid-fast bacilli (AFB) in stained smears examined microscopically may provide the first bacteriologic clue of TB. Fluorochrome staining with auramine-rhodamine is the preferred staining method because it is faster than the traditional methods in which Ziehl-Neelsen or Kinyoun (basic fuchsin dye) stains are used. Smear examination is an easy and quick procedure; results should be available within 24 hours of specimen collection. However, smear examination permits only the presumptive diagnosis of TB because the AFB in a smear may be mycobacteria other than M. tuberculosis. Furthermore, many TB patients have negative AFB smears.

Positive cultures for M. tuberculosis confirm the diagnosis of TB; however, TB may also be diagnosed on the basis of clinical signs and symptoms in the absence of a positive culture. Culture examinations should be done on all specimens, regardless of AFB smear results. The BACTEC Radiometric System or other recently developed liquid medium systems allow detection of mycobacterial growth in 4 to 14 days.

Once the mycobacteria have been grown in culture, nucleic acid probes can identify the species in 2 to 4 hours. Nucleic acid probes specific for the M. tuberculosis complex, for M. avium, and for M. intracellulare provide a rapid method of species identification. High-performance liquid chromatography (HPLC), which detects differences in the spectrum of mycolic acids in the cell wall, is equally rapid and can identify most pathogenic mycobacterial species. A test for inhibition by r-nitro-a-acetylamino-b-hydroxypropiophenone (NAP or NAP test) can identify M. tuberculosis in 3 to 4 days. If a solid medium and conventional biochemical tests are used, the isolation and identification of the organism can take 6 to 12 weeks.

Nucleic acid amplification (NAA) tests, such as polymerase chain reaction (PCR) and other methods for amplifying DNA and RNA, may facilitate rapid detection of microorganisms. Commercial NAA kits for the identification of M. tuberculosis complex have been approved by the Food and Drug Administration (FDA) for use on processed clinical specimens. These tests perform worst where needed most. Specificity is inadequate when applied to smear-negative specimens and sensitivity is inadequate when applied to smear-positive specimens. The test is approved for use in conjunction with culture for respiratory specimens that are positive for AFB on microscopy and were obtained from untreated patients. When used as approved, a positive NAA test result indicates a high likelihood of TB, but a negative result does not exclude TB. However, a reformulated AMPLIFIED Mycobacterium Tuberculosis Direct (MTD) Test for the detection of M. tuberculosis in both smear-positive and smear-negative clinical specimens has recently been approved. This is the first NAA test approved for this indication. Decisions about when and how to use NAA tests for TB diagnosis should be individualized. NAA tests cannot replace clinical judgment or be relied on as the only guide for therapy or isolation practices. The tests may enhance diagnostic certainty, but should be interpreted in a clinical context and on the basis of local laboratory performance.

Follow-up bacteriologic examinations are important for assessing the patient’s infectiousness and response to therapy. At a minimum, specimens should be obtained at monthly intervals until the culture results convert to negative. Culture conversion is the most important objective measure of response to treatment. Conversion is documented by the first negative culture in a series of cultures (i.e., all subsequent culture results must remain negative).

Laboratories should report positive smears and positive cultures within 24 hours by telephone or fax to the primary health care provider. Out-of-state laboratories must contact the health care provider in the patient’s state of origin. Follow-up results may be reported by mail. It is the responsibility of the primary health care provider to promptly report all suspected or confirmed cases of TB to the health department so that a contact investigation can be initiated as quickly as possible.

For all patients, the initial M. tuberculosis isolate should be tested for drug resistance. It is crucial to identify drug resistance as early as possible in order to ensure appropriate treatment. Susceptibility results from laboratories should be promptly forwarded to the health department. Drug susceptibility patterns should be repeated for patients who do not respond adequately or who have positive culture results despite 2 months of therapy.

The BACTEC radiometric method, which uses a liquid medium, is faster than conventional methods for determining susceptibility to first-line TB medications. Usually, susceptibility results can be obtained within 7-14 days of BACTEC inoculation; conventional methods, which use solid media for growth, can take as long as 21 days after inoculation.

Groups at an increased risk for drug resistance include

  • Persons who have a history of treatment with TB drugs;
  • Contacts of persons known to have drug-resistant TB;
  • Foreign-born persons from areas where the prevalence of drug-resistant TB is high;
  • Persons whose smears or cultures remain positive despite 2 months of therapy with TB drugs;
  • Persons receiving inadequate treatment regimens for > 2 weeks.

Restriction fragment length polymorphism (RFLP), a method of DNA fingerprinting, can be used to identify specific strains of M. tuberculosis and thus track TB transmission during outbreaks. The restriction enzymes used in this technique cut DNA at certain sites to produce fragments of various lengths. These fragments are separated by size to produce a pattern, or "fingerprint," that is specific for each strain. Related isolates show the same pattern. In addition, DNA fingerprinting can be used to detect lab contamination by determining if the isolates from the contaminating source culture and the suspect culture are related. If the isolates have differing DNA fingerprint patterns, cross-contamination is very unlikely to have taken place.