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Education Materials > Publications > Self-Study Modules on TB > Module 3 > Diagnosis of TB > Bacteriologic Examination

Self-Study Modules on Tuberculosis

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Module 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:

  1. Obtaining a specimen
  2. Examining the specimen under a microscope
  3. Culturing the specimen
  4. Doing drug susceptibility testing

  1. 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 procedure.

    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 Table 3.3.

Table 3.3
Methods of Obtaining a Sputum Specimen
Method Description Advantage Disadvantage
Coughing up sputum Patient coughs up sputum Inexpensive

Easy to do

Patient may not be able to cough up sputum on his or her own, or may spit up saliva instead of sputum
Inducing sputum Patient inhales a saline mist, causing him or her to cough deeply Easy to do Specimens may be watery and may be confused with saliva (should be labeled "induced specimen")

Requires special equipment

Bronchoscopy Bronchoscope is passed through the mouth directly into the diseased portion of the lung, and some sputum or lung tissue is removed Useful for obtaining sputum when coughing or inducing sputum does not work More expensive

Requires special equipment

Must be done by a specialized physician in a hospital or clinic

Gastric washing Tube is inserted through the patient's nose and passed into the stomach to get a sample of sputum that has been coughed into the throat and then swallowed Useful for obtaining sputum in children, who usually produce little or no sputum when they cough Must be done as soon as patient wakes up in the morning; patient may be required to stay in hospital

 
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.

  • What should be done?

Answer

  1. + 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 smear.

    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 have TB.

    The classification of smears is summarized in Table 3.4.

Table 3.4
Smear Classifications and Results
Classification of Smear Smear Result Infectiousness of Patient
4+ Strongly positive Probably very infectious
3+ Strongly positive Probably very infectious
2+ Moderately positive Probably infectious
1+ Moderately positive Probably infectious
Actual number of AFB seen (no plus sign) Weakly positive Probably infectious
No AFB seen Negative May not be infectious*

* 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

3.27. What 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 infectiousness?

Answers


 
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?

Answer


 
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?

Answers

  1. 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 M. tuberculosis.)

    Figure 3.9 Colonies of M. tuberculosis growing on media. This is a photograph of colonies of M. tuberculosis growing on media.

    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.

Table 3.5
Differences Between Sputum Smears and Cultures
Feature Smears
(see Figure 3.8)
Cultures
(see Figure 3.9)
Equipment needed Microscope, glass slides, special dyes Incubators, safety cabinet, culture plates or tubes, culture media, biochemicals for tests
Time needed to make report 1 day 2 to 8 weeks
Basis of procedure Looking for AFB on slide under microscope Growth of tubercle bacilli or other mycobacteria on culture media in incubator
Significance of a negative report Patient is less likely to be infectious

Does not rule out TB disease (culture may be positive)

No live tubercle bacilli found in the specimen

Does not rule out TB disease (live tubercle bacilli may be in other specimens and/or in the patient)

Significance of a positive report Patient is more likely to be infectious (if AFB are tubercle bacilli)

AFB could be nontuberculous mycobacteria

Confirms diagnosis of TB disease

 

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 year.

Cases that meet one of these three sets of criteria are counted as verified TB cases:

  1. The patient has a positive culture for M. tuberculosis

    or

  2. The patient has a positive smear for AFB, but a culture has not been done or cannot be done

    or

  3. 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 diagnostic evaluation

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.
  1. 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 drugs.

    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 the patient.

    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

3.30. Why 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?

Answers


 
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?

Answers

 


Released October 2008
Centers for Disease Control and Prevention
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