Acid-Fast Bacillus (AFB) Testing

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.

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
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.

Interpretation of smear and NAAT results are summarized in the following table. Again, all results must be confirmed by results from culture.

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).

TB requires a lengthy course of multiple antibiotics to cure an active infection. People with inactive (latent) infections, although asymptomatic, may be treated with a single drug to reduce the risk of having an active infection in the future.

A faster lab method to culture Mycobacterium tuberculosis has been developed. Culturing the sample in a liquid broth-based medium allows the organisms to be detected sooner. Some of the broth cultures require an automated instrument to detect the presence of the mycobacteria, while other methods can be read manually. A liquid culture method, called Microscopic-Observation Drug-Susceptibility (MODS) assay, takes only about 7 days to diagnose TB and detects bacterial resistance to antibiotics at the same time. Since this method can recognize the presence of multidrug-resistant TB (MDR-TB) much more quickly than conventional culture, it can help health practitioners diagnose and treat the disease at an earlier stage and has the potential to help control the spread of infectious TB. The benefits and limitations of this non-automated test are still being evaluated in resource-limited countries with high prevalence of TB.

There are other new methods being used in some laboratories that can identify M. tuberculosis very rapidly and accurately once the mycobacteria is growing in culture.

Yes. There are many people in the United States and worldwide who have a latent form of TB infection. They have been exposed to the bacteria, but their body’s immune system has confined it to a localized area in their lungs, in an inactive form. People with latent TB infections are not sick and they are not infectious, but the bacteria are still there and still alive. If those with latent infections are tested, most would have a positive TB skin test or IGRA test. The majority of people with latent TB infection, about 90%, will never progress to active tuberculosis disease.

Those who do have active TB may not feel ill at first. Early symptoms may be subtle and, if the TB is extrapulmonary (outside of the lungs in organs such as the kidney and bone), the tuberculosis may be fairly advanced by the time it causes noticeable symptoms.

Both indicate strains of Mycobacteria tuberculosis that can be difficult to treat, but XDR-TB is resistant to more drug therapies. MDR-TB is resistant to the two most powerful drugs, isoniazid and rifampin. XDR-TB is currently defined by the Centers for Disease Control and Prevention and the World Health Organization as M. tuberculosis that is resistant to isoniazid and rifampin plus resistant to any fluoroquinolone and to at least one of three injectable “second-line” drugs (amikacin, kanamycin, or capreomycin). The emergence of XDR-TB represents a public health risk and is being closely watched by the world medical community and measures are being taken in hopes of limiting its spread.

The practice of taking TB medications in the presence of a health care practitioner is known as direct observed therapy (DOT). DOT ensures that people are taking their medications and continuing their therapy for the required length of time. Unlike other bacterial infections that can be cured in 7-10 days, TB must be treated with two or more drugs for several months. People tend to forget to take their medication when they are feeling better. Since TB medications must be taken for many months, the risk of non-compliance is high. Having a healthcare practitioner administer the medications weekly increases the likelihood that the entire regimen will be completed and decreases the likelihood that someone will relapse with a more resistant strain of TB.

Examples of other mycobacteria that can cause infections and are detected using AFB tests include:

• Mycobacterium avium-intracellulare complex (MAC)—can cause a lung infection in people with weakened immune systems, such as those with AIDS; this infection is not contagious but it can be difficult to treat as it tends to be highly resistant to antibiotics.
• Mycobacterial species, such as Mycobacterium marinum, grow in water, such as fish tanks, and can cause skin infections.
• Mycobacterium fortuitum, Mycobacterium abscessus and Mycobacterium chelonae, and other rapidly growing mycobacteria, cause skin and wound infections following cosmetic surgery, prosthetic device implantation, and visits to nail salons.
• A few mycobacteria, such as Mycobacterium bovis, can sometimes be transferred from animals to humans.

Nocardia species are not a type of mycobacteria but can be detected using some AFB laboratory tests. Nocardia can cause infections of the lungs, brain, or skin.