What is being tested

Acid-fast bacilli (AFB) are rod shaped bacteria. They get their name because they can be seen and counted under the microscope when smeared on a slide and treated with an "acid-fast" staining procedure that differs from the routine stain. The most common and medically important acid-fast bacilli are members of the genus Mycobacterium.

Mycobacterium tuberculosis is one of the most prevalent and infectious species of mycobacteria. Most samples that are submitted for AFB smears and cultures are collected because the doctor suspects that someone has a lung infection caused by M. tuberculosis (TB). Another group of bacteria referred to as non-tuberculous mycobacteria (NTM), can also cause infections. These organisms are common in the environment (including water and soil) however only a few of them cause infections in humans. They include:

• Mycobacteria avium-intracellulare complex (MAC) can cause a lung infection or a disseminated infection in immunosuppressed patients, such as the elderly and those with AIDS; this infection is not contagious, but it can be difficult to treat as it tends to be highly resistant to antibiotics.
• Cervical lymphadenitis can be seen in young children as a swollen lymph gland in the neck. It is most commonly caused by MAC, but also by M. tuberculosis or other NTM. Surgical excision is often required for treatment.
• Rapid growing mycobacterium (RGM) (M. abscessus, M. chelonae, M. fortuitum) may cause lung or non-pulmonary disease such as wound infection of prosthetic device infection.
• Mycobacterium ulcerans causes sporadic cases of non-healing ulcers (some names include Bairnsdale ulcer, Buruli ulcer, Daintree ulcer) that often require surgical treatment.
• Mycobacterium marinum grows in water, such as fish tanks, and can cause skin infections.
• Mycobacterium leprae causes leprosy.
• Some mycobacteria, such as Mycobacterium bovis, can sometimes be transferred from animal to human.

A definitive diagnosis requires the mycobacteria to be cultured. Mycobacteria grow more slowly than other types of bacteria so positive identification of the species that is/are present may take days to several weeks, while negative results (no mycobacterial growth) can take up to 6 to 8 weeks to confirm.

An AFB smear, which can provide presumptive results in a few hours, is a valuable tool in helping to make decisions about treatment while culture results are pending. Patient samples are processed for AFB cultures at the same time as the smears.

Typically, several AFB smears from different samples are screened for AFB since the number of bacilli may vary from sample to sample and day to day. If acid-fast bacilli are present on any of the smears, a mycobacterial infection is likely. A presumptive diagnosis of TB can be made if a patient has risk factors for disease, but other follow-up testing must be done to positively identify the acid-fast bacilli as either M. tuberculosis or another mycobacteria species.

Tests that may be done in addition to an AFB smear and culture include:

• Molecular tests for TB that detect the genetic components of mycobacteria have been developed to help decrease the amount of time necessary for a presumptive diagnose of tuberculosis. These include genetic probes and molecular TB testing. They amplify/replicate pieces of the microorganisms' genetic material to detect mycobacteria in body samples in less than 24 hours and can narrow the identification to a complex of mycobacteria (a combination, of which M. tuberculosis is the most common). They are fairly sensitive and specific when they are paired with positive AFB smears; when they are done on samples that are AFB negative by smear, they tend to be less accurate. These methods are approved for respiratory samples and must be confirmed with an AFB culture, but a positive result will be available more rapidly allowing the potentially infectious patient to be isolated to minimise the spread of the disease.
• Antibiotic susceptibility testing is complex and may take a long-time to return a result. It is generally performed on isolates of Mycobacterium tuberculosis. This testing is occasionally performed on other species of Mycobacteria, however there is no evidence that the results can predict the outcome of therapy with the drug tested, except in very specific cases.

Since TB is transmitted by airborne droplets from respiratory secretions it is a public health risk. It can spread in confined populations, such as correctional facilities, nursing homes, and schools. Those who are very young, elderly, or have diseases and conditions that compromise their immune systems tend to be especially vulnerable. AFB smears and cultures can help track and minimize the spread of TB in these populations and help determine the effectiveness of treatment.

How is it used?

An acid-fast stain (Ziehl-Neelsen or Kinyoun stain) is used on a sample from the site of suspected infection to look for acid fast bacilli (AFB). The sample is spread thinly onto a glass slide, treated with a special staining technique and examined under a microscope. This is a relatively quick way to determine if an infection may be due to one of the acid-fast bacilli, the most common of which is M. tuberculosis. Results of an AFB smear are typically available several hours to a day after a sample is collected, while an AFB culture typically takes several days to weeks.

A definitive diagnosis requires the mycobacteria to be cultured. Mycobacteria grow more slowly than other types of bacteria so positive identification of the species that is/are present may take days to several weeks, while negative results (no mycobacterial growth) can take up to 6 to 8 weeks to confirm.

AFB cultures are used to diagnose active M. tuberculosis infections. They are also used to diagnose infections due to another member of the Mycobacterium family and to determine whether TB-like symptoms are due to another cause. They are used to help determine whether the TB is confined to the lungs (pulmonary disease) or has spread to organs outside the lungs (extrapulmonary disease). AFB cultures can also be used to monitor the effectiveness of treatment and can help determine when a patient is no longer infectious.

Susceptibility testing may be ordered in conjunction with a culture to determine the most effective antibiotics to treat the infection. M. tuberculosis may be resistant to one or more drugs commonly used to treat TB. If the bacteria are resistant to isoniazid and rifampicin and perhaps others of the primary drugs used for therapy, the organisms are called multidrug-resistant TB (MDR-TB).  If the organisms are MDR-TB and also resistant to certain drugs that are second-line anti-TB treatment, they are called extensively drug-resistant tuberculosis (XDR-TB).

When is it requested?

AFB testing is requested when:

• Someone has symptoms that suggest pulmonary TB  or another mycobacterial lung infection
• Someone has symptoms associated with a TB or another 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 anaemia (mycobacteria in the bone marrow), altered mental state, headache, and coma (TB meningitis), joint pain or abdominal pain.
• A person likely to have been exposed to TB is at increased risk for active disease and/or has characteristic lung involvement as shown by X-ray.
• 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 that puts them at a much higher risk of contracting the disease such as HIV/AIDS. (Those with AIDS are more likely than other affected patients to have extrapulmonary TB with a few, vague symptoms.)
• Someone is being treated for TB. AFB smears and cultures are usually ordered at intervals, both when the doctor is evaluating the effectiveness of treatment and when he/she is attempting to determine whether or not a person is still infectious.
• An immunosuppressed person is systemically unwell and they are screened for unusual infections such as mycobacteria and fungi

What does the result mean?

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

Positive AFB smears indicate a probable mycobacterial infection. However, a culture must be performed to confirm a diagnosis.

AFB Culture
Positive AFB cultures identify the particular mycobacterium causing symptoms, and susceptibility testing on the identified organism gives the doctor 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. A positive culture 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 someone does not have an AFB infection or that mycobacteria were not present in that particular specimen (which is why multiple samples are often collected). Cultures are incubated for six to eight weeks before being reported as negative. If someone has TB, the infection may be in another part of the body and a different type of sample may need to be collected. 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.

Is there anything else I should know?

TB requires a lengthy course of multiple antibiotics to eradicate an active infection. Persons 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.

Common questions

• Can I be infected with TB and not be sick?

Yes. There are many people world wide, who have a latent form of TB infection. They have been exposed to the bacteria and their body's immune system has confined it to a localised 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. 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 is causing noticeable symptoms.

• What is the difference between MDR-TB and XDR-TB?

Both indicate strains of Mycobacteria tuberculosis that can be difficult to treat, but XDR-TB is resistant to more drug therapies. XDR-TB is currently defined by the US Centers for Disease Control and Prevention (CDC) and World Health Organization (WHO) as M. tuberculosis that is resistant to isoniazid and rifampicin 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 is being closely watched by the world medical community and measures are being taken in hopes of limiting its spread.

• Why is the doctor asking me to take my TB medication in the presence of a nurse?

The practice of taking TB medications in the presence of a health care provider is known as direct observed therapy (DOT). DOT ensures that patients 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 multiple drugs for several months. Patients 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 health care provider administer the medications weekly increases the likelihood that the entire regimen will be completed and decreases the likelihood that a patient will relapse with a more resistant strain of TB.

More information

RCPA Manual: Mycobacteria testing

Healthdirect Australia: Tuberculosis