AFB Stain Susmita Chakrabarty

Why Get Tested?

To help diagnose tuberculosis (TB) and infections caused by other Mycobacterium species, which are known as acid-fast bacilli (AFB), in people at risk of developing mycobacterial infections; to monitor the effectiveness of treatment

When To Get Tested?

When you have signs and symptoms of a lung infection, such as a chronic cough, weight loss, fever, chills, and weakness, that may be due to TB or a nontuberculous mycobacterial (NTM) infection; when you have a positive IGRA blood test or Tuberculin skin test (TST) and you are in a high-risk group for progressing to active TB; when you have a skin or other body site infection that may be due to mycobacteria; when you are undergoing treatment for TB

Sample Required?

For suspected cases of tuberculosis lung infections, usually three sputum samples are collected early in the morning on different days. If you are unable to produce sputum, a bronchoscope may be used to collect fluid during a procedure called a bronchoscopy. In children, gastric washings/aspirates may be collected. Depending on symptoms, urine, an aspirate from the site of suspected infection, cerebrospinal fluid (CSF), other body fluids, or biopsied tissue samples may be collected for AFB testing.

Test Preparation Needed?


What is being tested?

Most samples that are submitted for acid-fast bacilli (AFB) testing are collected because the healthcare practitioner suspects that a person has tuberculosis (TB), a lung infection caused by Mycobacterium tuberculosis. Mycobacteria are called acid-fast bacilli because they are a group of rod-shaped bacteria (bacilli) that can be seen under the microscope following a staining procedure where the bacteria retain the color of the stain after an acid wash (acid-fast). AFB laboratory tests detect the bacteria in a person's sample and help identify an infection caused by AFB.

There are several types of AFB that may be detected with this testing; however, the most common and medically important ones are members of the genus Mycobacterium. Mycobacterium tuberculosis is one of the most prevalent and infectious species of mycobacteria.

Since TB is transmitted through the air when an infected person sneezes, coughs, speaks, or sings, it is a public health risk. It can spread in confined populations, such as in the home and schools, correctional facilities, and nursing homes. Those who are very young, elderly, or have preexisting diseases and conditions, such as AIDS, that compromise their immune systems tend to be especially vulnerable. AFB testing can help diagnose, track, and minimize the spread of TB in these populations and help determine the effectiveness of treatment.

Another group of mycobacteria referred to as nontuberculous mycobacteria (NTM) can also cause infections. However, only a few of the more than 60 species of mycobacteria that have been identified cause infections in humans. Some examples include Mycobacterium avium-intracellulare complex (MAC), which can cause lung infection and disseminated disease in people with weakened immune systems. (See the article on Nontuberculous Mycobacteria for more details on different types). In addition to TB, AFB testing can help identify infections caused by these nontuberculous mycobacteria.

How is the sample collected for testing?

Sputum is the most commonly tested sample. Sputum is phlegm, thick mucus that is coughed up from the lungs. Preferably, three early morning samples obtained by deep cough are collected on consecutive days in individual sterile cups to increase the likelihood of detecting the bacteria.

Sterile Cups / Sputum Container

If a person is unable to produce sputum, a healthcare practitioner may collect respiratory samples using a procedure called a bronchoscopy. Bronchoscopy allows the healthcare practitioner to look at and collect samples from the bronchi and bronchioles. Once a local anesthetic has been sprayed onto the patient's upper airway, the practitioner can insert a tube into the bronchi and smaller bronchioles and aspirate fluid samples for testing. Sometimes, the healthcare practitioner will introduce a small amount of saline through the tubing and into the bronchi and then aspirate it to collect a bronchial washing.

Since young children cannot produce a sputum sample, gastric washings/aspirates may be collected. This involves introducing saline into the stomach through a tube, followed by fluid aspiration.

If the healthcare practitioners suspect TB is present outside of the lungs (extrapulmonary), they may test the body fluids and tissues most likely affected. For instance, one or more urine samples may be collected if the practitioner suspects TB has infected the kidneys. A needle may be used to collect fluid from joints or from other body cavities, such as the pericardium or abdomen. Occasionally, the practitioner may collect a sample of cerebrospinal fluid (CSF) or perform a minor surgical procedure to obtain a tissue biopsy.

Is any test preparation needed to ensure the quality of the sample?

No test preparation is needed, except to rinse the mouth with water before collecting the sputum sample.

How is it used?

AFB testing may be used to detect several different types of acid-fast bacilli, but it is most commonly used to identify an active tuberculosis (TB) infection.

Mycobacteria are called acid-fast bacilli because they are rod-shaped bacteria (bacilli) that can be seen under the microscope following a staining procedure in which the bacteria retain the color of the stain after an acid wash (acid-fast).

A few different tests may be used to help identify AFB as the cause of an infection:

  • AFB smear—a microscopic examination of a person's sputum or other specimen that is stained to detect acid-fast bacteria. It is a rapid test used to provide presumptive results within one to two days. It is valuable in helping to make decisions about treatment while waiting for culture results. However, AFB smears must be confirmed with AFB cultures.
  • Molecular tests for TB (nucleic acid amplification test or NAAT) detect the genetic material of mycobacteria. These tests are often used when the AFB smear is positive or TB is highly suspected. Like AFB smears, they can provide a presumptive diagnosis, which can aid in the decision of whether to begin treatment and isolate potentially infectious people before culture results are available. Results of NAAT are typically available in one to three days after a sample is collected. Molecular methods are approved for use with respiratory samples but must be confirmed with an AFB culture. Guidelines from the Centers for Disease Control and Prevention recommend that people with signs and symptoms of TB have at least one sample tested using nucleic acid amplification with AFB smear and culture. The NAAT test currently available is done directly on sputum samples and can simultaneously detect TB and its resistance to rifampicin in less than two hours. Rifampicin is a common treatment for TB.
  • AFB cultures are used to diagnose active M. tuberculosis infections as well as infections due to nontuberculous mycobacteria. AFB cultures can also be used to monitor the effectiveness of treatment and can help determine when a person is no longer infectious. Though culture is more sensitive than an AFB smear, positive results may take days to several weeks, while negative culture results (no mycobacterial growth) can take up to 6 to 8 weeks to confirm.
  • Susceptibility testing is usually ordered in conjunction with an AFB culture to determine the most effective antibiotic to treat the mycobacterial infection. M. tuberculosis may be resistant to one or more drugs commonly used to treat TB.

When is it ordered?

AFB testing is ordered when:

  • Someone has signs and symptoms that suggest an active TB lung infection (pulmonary) or other mycobacterial lung infection, such as:
  • Lingering, chronic cough that produces phlegm or sputum, sometimes with bloody streaks
  • Fever, chills
  • Night sweats
  • Loss of appetite
  • Unexplained weight loss
  • Weakness, fatigue
  • Chest pain
  • A person has symptoms associated with a TB or other mycobacterial infection located outside of the lungs (extrapulmonary); the symptoms vary depending on the area of the body that is affected. Some examples include back pain and paralysis (spinal TB), weakness due to anemia (TB in the bone marrow), altered mental state, headache, and coma (TB meningitis), joint pain or abdominal pain.
  • A TB screening test is positive and the person is at increased risk for active disease and/or characteristic signs are seen in an X-ray of the lung; there are two types of tests that are used to determine if a person has had contact or is infected with M. tuberculosis: the tuberculin test (TST) and the blood test (IGRA).
  • Someone has been in close contact with a person who has been diagnosed with TB and the exposed person either has symptoms or has a condition or disease, such as HIV, that puts the person at a much higher risk of contracting active TB.
  • An individual is being treated for TB; AFB testing is usually ordered at intervals, both for evaluating the effectiveness of treatment and for determining whether or not a person is still infectious.
  • An individual has a chronic skin infection that does not respond to the usual antibiotics given for a bacterial infection; NTM may be the cause of the infection since they do not respond to the same antibiotics used to treat a staphylococcal or streptococcal infection.

AFB Smear and NAAT

AFB Smear

A negative AFB smear may mean that no infection is present, that symptoms are caused by something other than mycobacteria, or that the mycobacteria were not present in sufficient numbers to be seen under the microscope. Usually three samples are collected to increase the probability that the organisms will be detected.

Nevertheless, if AFB smears are negative and there is still a strong suspicion of a mycobacterial infection, then additional samples may be collected and tested on different days. A smear negative sample may still grow mycobacteria since the culture media allows low numbers of bacteria that cannot be seen in a microscopic examination to multiply and be detected.

Positive AFB smears indicate a probable mycobacterial infection. However, a culture must be performed to confirm a diagnosis and identify the species of mycobacteria present.

For people with signs and symptoms of an active TB infection, AFB smear results are considered together with results from NAAT for TB, as recommended by the Centers for Disease Control and Prevention. Though definitive diagnosis requires results from a culture, results from the smear and NAAT may be helpful in deciding what to do. For example, if there is a presumptive diagnosis of TB based on rapid test results, most health practitioners would treat.

AFB Culture

Positive AFB cultures identify the particular mycobacterium causing symptoms, and susceptibility testing on the identified organism gives the healthcare practitioner information about how resistant it may be to treatment.

A positive AFB smear or culture several weeks after drug treatment has started may mean that the treatment regimen is not effective and needs to be changed. It also means that the person is still likely to be infectious and can pass the mycobacteria to others through coughing or sneezing.

A negative culture means that the person tested does not have an active AFB infection or that mycobacteria were not present in that particular sample (which is why multiple samples are often collected) or were present in numbers too low to be detected. Cultures are held for six to eight weeks before being reported as negative. The person tested may have a latent infection that caused a TB screening test to be positive but does not have active TB.

If it is suspected that someone has a TB infection that has spread to another part of the body, a sample from the site of suspected infection may need to be collected and tested to identify the infection.

A negative culture several weeks after treatment indicates that the TB infection is responding to drug treatment and that the person is no longer infectious.

Susceptibility Testing

Susceptibility testing results will list the antibiotics that will likely be most effective in treating the infection. Isoniazid and rifampin are two drugs commonly used to treat TB. If the bacteria are resistant to more than one or the primary drugs used for therapy, the organisms are called multidrug-resistant TB (MDR-TB), and if the organisms are resistant to multiple drugs approved for first and second lines of therapy, they are called extensively drug-resistant tuberculosis (XDR-TB).