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Education Materials
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Curriculum > Chapter 5
Core Curriculum on Tuberculosis, 2000
Chapter 5
Diagnosis of TB
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.
2, 3
Once the mycobacteria have been grown in culture, nucleic acid
probes can identify the species in 2 to 4 hours. 4
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. 5
A test for inhibition by r-nitro-a-acetylamino-b-hydroxypropiophenone
(NAP or NAP test) can identify M. tuberculosis in 3 to 4
days. 4
If a solid medium and conventional biochemical tests are used, the
isolation and identification of the organism can take 6 to 12 weeks.
3
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 (see Community
TB Control).
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.
2, 3
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.
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