Self-Study Modules on Tuberculosis
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3: Diagnosis of Tuberculosis Infection and Disease
The Bacteriologic Examination
The next step in diagnosing TB disease is the bacteriologic
examination. This is done in a laboratory that specifically
deals with M. tuberculosis and other mycobacteria
(a mycobacteriology laboratory). There are four
parts to a bacteriologic examination:
- Obtaining a specimen
- Examining the specimen under a
- Culturing the specimen
- Doing drug susceptibility testing
- Obtaining a specimen.
Specimens that will be sent to the laboratory can be obtained
in several ways. Usually, patients who are suspected of having
pulmonary TB disease simply cough up sputum (phlegm
from deep in the lungs) into a sterile container for processing
and examination (Figure 3.7). This is the cheapest and easiest
Figure 3.7 A TB patient has coughed up sputum and is spitting
it into a sterile container. (The patient is sitting in a special
sputum collection booth that prevents the spread of tubercle
bacilli.) This is a picture of a TB patient sitting in a special
sputum collection booth that prevents the spread of tubercle
bacilli. The patient is coughing up sputum and is spitting it
into a sterile container.
If a patient cannot cough up sputum on his or her own, other
techniques can be used to obtain a specimen. An induced
sputum sample can be obtained by having the patient
inhale a saline (salt water) mist, which causes the patient
to cough deeply. This procedure is easily done, and it should
be used to help patients cough up sputum if they cannot do so
on their own. Induced specimens are often clear and watery,
so they should be labeled "induced specimen" so that
they will not be confused with saliva. (Laboratories will not
accept saliva as a specimen.)
Another procedure, bronchoscopy, can be used
to obtain pulmonary secretions or lung tissue. In this procedure,
an instrument called the bronchoscope is passed through the
mouth directly into the diseased portion of the lung, and some
sputum or lung tissue is removed. Bronchoscopy should be used
only when patients cannot cough up sputum on their own and an
induced specimen cannot be obtained.
A fourth procedure, gastric washing, involves
inserting a tube through the patient's nose and passing it into
the stomach. The idea is to get a sample of sputum that has
been coughed into the throat and then swallowed. Gastric washings
are done in the morning because patients usually swallow sputum
during the night. This procedure is usually used only when patients
cannot cough up sputum on their own, an induced specimen cannot
be obtained, and bronchoscopy cannot be done. However, gastric
washings are often used for obtaining sputum from children.
Most children produce little or no sputum when they cough.
It is very important for health care workers to use precautions
to control the spread of tubercle bacilli during these procedures
and any other procedures that may cause persons who have pulmonary
TB disease to cough. This is discussed further in
Module 5, Infectiousness and Infection Control.
In patients who have extrapulmonary TB disease,
specimens other than sputum are obtained. The
specimen obtained from these patients depends on the part of
the body that is affected. For example, urine samples are obtained
from patients suspected of having TB disease of the kidney,
and fluid samples are obtained from the area around the spine
in patients suspected of having TB meningitis (TB disease in
the membranes surrounding the brain).
The methods of obtaining a sputum specimen are summarized in
|Case Study 3.5
Mr. Lee has a cough and
other symptoms of TB disease, and he is evaluated with a
chest x-ray. However, he is unable to cough up any sputum
on his own for the bacteriologic examination.
- + under a microscope, it is smeared onto a glass slide and stained
with a dye. This is called a smear. Then laboratory
personnel use the microscope to look for acid-fast bacilli
(AFB) on the smear (Figure 3.8). AFB are mycobacteria that stay
stained even after they have been washed in an acid solution.
Tubercle bacilli are one kind of AFB.
Figure 3.8 AFB smear. In this photograph, the AFB (shown
in red) are tubercle bacilli. This is a photograph of an AFB
When AFB are seen in a smear, they are counted. There is a
system for reporting the number of AFB that are seen at a certain
magnification. According to the number of AFB seen, the smears
are classified as 4+, 3+, 2+, or 1+. In smears classified at
4+, 10 times as many AFB were seen as in smears classified as
3+; in 3+ smears, 10 times as many as in 2+ smears; and in 2+
smears, 10 times as many as in 1+ smears.
Smears that are classified as 4+ and 3+ are considered strongly
positive; 2+ and 1+ smears are considered moderately positive.
If very few AFB are seen, the smear is classified by the actual
number of AFB seen (no plus sign). For example, if only 4 AFB
were seen in the entire smear, the smear is classified as "4
AFB seen." Smears classified in this way are considered
weakly positive. Finally, if no AFB are seen, the smear is called
negative. But a negative smear does not rule out
the possibility of TB because there can be AFB in the smear
that were not seen.
It takes only a few hours to prepare and examine a smear. Therefore,
the results of the smear examination should be available to
the clinician within 1 day.
The results of the smear examination can be used to help determine
the infectiousness (contagiousness) of the patient. Patients
who have many tubercle bacilli in their sputum have a positive
smear. Patients who have positive smears are considered infectious
because they can cough many tubercle bacilli into the air. (This
is discussed in more detail in
Module 5, Infectiousness and Infection Control.) However,
because AFB are not always tubercle bacilli, patients who have
positive smears do not necessarily have TB. Furthermore, as
mentioned previously, patients who have negative smears may
The classification of smears is summarized in Table 3.4.
* The criteria for determining whether a patient may be considered
noninfectious are discussed in
Module 5, Infectiousness and Infection Control.
|Study Questions 3.27-3.29
are the four ways to collect sputum specimens? Indicate
which procedure is the cheapest and easiest to perform.
3.28. What do laboratory personnel look for in a smear?
3.29. What does a positive smear indicate about a patient's
|Case Study 3.6
Ms. Thompson gave three
sputum specimens, which were sent to the laboratory for
smear examination and culture. The smear results were reported
as 4+, 3+, and 4+.
- What do these results tell you about Ms. Thompson's
diagnosis and her infectiousness?
|Case Study 3.7
Mr. Sagoo has symptoms
of TB disease and a cavity on his chest x-ray, but all of
his sputum smears are negative for acid-fast bacilli.
- Does this rule out the diagnosis of pulmonary TB disease?
- Why or why not?
- Culturing the specimen.
Culturing the specimen means growing the mycobacteria on media,
substances that contain nutrients, in the laboratory (Figure 3.9).
When the mycobacteria have formed colonies (groups),
they can be identified. All specimens should be cultured, regardless
of whether the smear is positive or negative.
Culturing the specimen is necessary to determine whether
the specimen contains M. tuberculosis and to confirm
a diagnosis of TB disease. (However, in some cases,
patients are diagnosed with TB disease on the basis of their
signs and symptoms, even if their specimen does not contain
Figure 3.9 Colonies of M. tuberculosis growing on media.
This is a photograph of colonies of M. tuberculosis growing
The first procedure in culturing the specimen is to detect
the growth of the mycobacteria. Mycobacteria grow very slowly.
When solid media are used to culture the specimen, it can take
as long as 2 to 8 weeks for the growth of the mycobacteria to
be detected. However, rapid culturing methods that involve liquid
media can decrease this time to 4 to 8 days.
The second procedure is to identify the organism that has grown.
All types of mycobacteria will grow in solid or liquid media.
For this reason, laboratory tests must be done to determine
whether the organism is M. tuberculosis or one of the
nontuberculous mycobacteria. Traditional tests require an additional
3 to 6 weeks from the time the cultures have grown. However,
other tests have been developed to shorten the time it takes
to identify the type of mycobacteria present in clinical specimens.
When M. tuberculosis is identified in a patient's
culture, the patient is said to have a positive culture
for M. tuberculosis. A positive culture for M.
tuberculosis, also called an M. tuberculosis isolate,
confirms the diagnosis of TB disease.
When M. tuberculosis is NOT identified in a patient's
culture, the patient is said to have a negative culture
for M. tuberculosis. A negative culture does not necessarily
rule out the diagnosis of TB disease; as mentioned earlier,
some patients with negative cultures are diagnosed with TB disease
on the basis of their signs and symptoms.
The differences between sputum smears and cultures are summarized
in Table 3.5.
Criteria for Reporting TB Cases
All 50 states, the District of Columbia, New York City, U.S.
dependencies and possessions, and independent nations in free
association with the U.S.* report TB cases to the federal
Centers for Disease Control and Prevention (CDC) based on
certain criteria. Each reported TB case is checked to make
sure that it meets the criteria. All cases that meet the criteria,
called verified TB cases, are counted each
Cases that meet one of these three sets
of criteria are counted as verified TB cases:
- The patient has a positive culture for M. tuberculosis
- The patient has a positive smear for AFB, but a culture
has not been done or cannot be done
- The patient has a positive tuberculin skin test reaction,
has other signs and symptoms of TB disease, is being treated
with two or more TB drugs, and has been given a complete
In addition, cases that do not meet any of these sets of
criteria (for example, a patient who is anergic and has
a negative culture for M. tuberculosis but who
has signs and symptoms of TB disease) may be counted as
a verified TB case if a health care provider has reported
the case and decided to treat the patient for TB disease.
| *The dependencies, possessions, and independent
nations include Puerto Rico, the U.S. Virgin Islands, Guam,
American Samoa, the Republic of the Marshall Islands, the
Commonwealth of the Northern Mariana Islands, and the Federated
States of Micronesia.
- Doing drug susceptibility testing.
Drug susceptibility tests, the final part of the bacteriologic
examination, are done to determine which drugs will kill the tubercle
bacilli that are causing disease in a particular patient. Tubercle
bacilli that are killed by a particular drug are said to be susceptible
to that drug, whereas those that can grow even in the presence
of a particular drug are said to be resistant
to that drug. The drug susceptibility pattern
of a strain of tubercle bacilli is the list of drugs to which
the strain is susceptible and to which it is resistant.
The results of drug susceptibility tests can help clinicians
choose the appropriate drugs for each patient. This is very
important. Patients with TB disease who are treated with drugs
to which their strain of TB is resistant may not be cured. In
fact, their strain of TB may become resistant to additional
Drug susceptibility tests should be done when a patient is
first found to have a positive culture for M. tuberculosis
(that is, the first isolate of M. tuberculosis). In
addition, drug susceptibility tests should be repeated if a
patient has a positive culture for M. tuberculosis
after 2 months of treatment or if a patient does not seem to
be getting better. That way, the clinician can find out whether
the patient's strain of TB has become resistant to certain drugs;
if necessary, the clinician may change the drugs used for treating
In the laboratory, drug susceptibility testing can be done
using solid media. Organisms that grow in media containing a
specific drug are considered resistant to that drug (Figure
3.10). This technique is slow, taking as long as 8 to 12 weeks.
Rapid methods for drug susceptibility testing can shorten this
time to 3 weeks.
Figure 3.10 Drug susceptibility testing on solid media. Organisms
are resistant to the drug in the upper right compartment and
susceptible to the drugs in the lower compartments. Upper left
contains no drugs. This is a photograph of drug susceptibility
testing on solid media.
|Study Questions 3.30-3.33
is it necessary to culture a specimen?
3.31. What does a positive culture for M. tuberculosis
mean? How is this important for the TB diagnosis?
3.32. Why are drug susceptibility tests done?
3.33. How often should drug susceptibility tests be done?
|Case Study 3.8
In the public health
clinic, you see a patient, Ms. Sanchez, who complains of
weight loss, fever, and a cough of 4 weeks' duration. When
questioned, she reports that she has been treated for TB
disease in the past and that she occasionally injects heroin.
- What parts of Ms. Sanchez's medical historylead you
to suspect TB disease?
- What diagnostic tests should be done?
Released October 2008
Centers for Disease Control and Prevention
National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention
Division of Tuberculosis Elimination - http://www.cdc.gov/tb
Please send comments/suggestions/requests
CDC/Division of Tuberculosis Elimination
Communications, Education, and Behavioral Studies Branch
1600 Clifton Rd., NE - Mailstop E-10, Atlanta, GA 30333