Acid-Fast Culture Health Article

Definition

The term acid-fast refers to a type of organism not readily decolorized by acid after staining. An acid-fast culture is the microbiological analysis of such an organism. An acid-fast culture refers to the process of detection, growth, isolation, identification, and antibiotic susceptibility testing of mycobacteria that cause pulmonary tuberculosis and other infections such as skin, abdominal, and disseminated (widely spread throughout many organs).

Purpose

The acid-fast culture is used to isolate Mycobacterium tuberculosis when tuberculosis (TB) is suspected. More recently the test has become important for the identification of other acid-fast organisms including Mycobacterium avium complex (MAC), Mycobacterium bovis, and Mycobacterium africanum responsible for causing tuberculosis in AIDS patients and other immuno-suppressed persons. Antibiotic sensitivity testing performed when cultures are positive or when patients are known to have tuberculosis determines the appropriate drugs for treatment. This is essential because of the emergence of tuberculosis strains that are resistant to many of the antibiotics that were once effective in treating this disease. The test is also used to differentiate tuberculosis from carcinoma and bronchiectasis that may appear similar on x ray.

Precautions

Antibiotics and some sulfonamides may interfere with test results, causing the results to be falsely negative. Sufficient organisms may not be recovered to diagnose infection when a single culture sample is collected. Therefore, sputum cultures should be collected on three consecutive mornings.

Special safety precautions

Health care workers involved with collection and handling of specimens from patients suspected of having tuberculosis or other mycobacterial infections should observe universal precautions for the prevention of transmission of bloodborne pathogens. In addition, health care personnel working with patients and handling specimens from patients suspected of having tuberculosis must be given a skin test (e.g. Mantoux or PPD test) on a regular basis. Precautions must be followed closely when handling mycobacterial specimens. The laboratory personnel who process and handle the infectious material from the patient are at greatest risk (about three times higher than other laboratory personnel) for tuberculosis infection or skin test positivity. The hazard of working in a laboratory that handles mycobacterial specimens is greatly reduced if the personnel follow proper procedures when handling and processing the specimens. All processing should take place in a biologic safety cabinet (BSC). The biologic safety cabinets used in the clinical mycobacterial laboratory are of two types: Class I, or negative-pressure cabinets, and Class II, or vertical-laminar-flow cabinets. Correct operation of these safety devices along with proper maintenance and testing of the air flow are essential to their performance. Yearly inspection of the cabinets by trained individuals is required.

Processing specimens, testing organisms, and transferring viable cultures must be carried out within the BSC. After processing specimens or working under the BSC, the area inside the cabinet is disinfected and a UV (ultraviolet) light located within the cabinet is turned on to kill any organisms on the surface of the work area as well as any airborne bacteria. After performing a procedure, the work area must be decontaminated with a disinfectant solution (e.g., the use of a phenol-soap mixture containing orthophenol or phenolic derivitives with an effective contact time of 10-30 minutes).

Protective clothing including gloves, fluid-proof gowns, goggles, and face mask or respirator is recommended for laboratory personnel working in the mycobacterial laboratory. Incinerators (no bunsen burners) are used within the BSC to reduce aerosoling of bacteria from infectious material while processing and culturing.

Description

Tuberculosis is an infection caused by Mycobacterium tuberculosis, a disease which is a major health problem worldwide. Mycobacterium tuberculosis is a rod-shaped bacterium characterized by acid-fastness. It is commonly transmitted via the air to the lungs, where it thrives, causing fever, cough, and hemoptysis (coughing up blood-tainted secretions). Tuberculosis is highly contagious.

Disease is spread when persons cough, releasing an aerosol of organisms that are easily inhaled by others. Although deaths from tuberculosis in the United States had declined since the 1950s, recently there has been a resurgence of the disease, with the higher incidence of infection seen in certain races, in poor socioeconomic conditions, among new immigrants, in prison inmates, and in persons infected with the human immuno-deficiency virus.

Because it takes several weeks for most Mycobacteria to grow in a culture, the laboratory performs an acid-fast smear first to aid in early diagnosis; however, the acid-fast smear should not be used in place of culture, as a culture is far more sensitive. An acid-fast culture can detect as few as 10 to 100 CFU/mL of sputum. The smear can provide a presumptive diagnosis of mycobacterial disease; confirm that cultures growing on media are acid-fast; and demonstrate that antibiotic treatment is effective pending follow-up culture results.

The genus Mycobacterium includes organisms that are obligate parasites, saprophytes (i.e., organisms that live off dead tissue), and opportunistic pathogens. Mycobacteria cause tuberculosis as well as non-tuberculous clinical conditions; therefore, mycobacteria are divided into two major groups based upon whether they cause tuberculosis (M. tuberculosis complex) or nontuberculous infections (NTM). The principle pathogen causing tuberculosis in humans is Mycobacterium tuberculosis. It is estimated that about one third of the world's population is infected with M. tuberculosis. The World Health Organization reports an estimated eight million new cases and three million deaths attributable to tuberculosis each year. Tuberculosis is a leading cause of death in developing countries.

Other organisms causing human tuberculosis that are included in the M. tuberculosis complex are: M. bovis (the cause of tuberculosis in cattle and humans, as well as other carnivores); M. bovis BCG (a strain used as a vaccine against tuberculosis in many parts of the world); and M. africanum (the cause of human tuberculosis in tropical Africa). Mycobacterium tuberculosis causes an infection that may mimic other diseases such as pneumonia, neoplasm, or fungal infections. Patients may be symptomatic or asymptomatic with signs of pulmonary and other organ involvement. Symptoms include night sweats, low-grade fever, anorexia, fatigue, weight loss, and a productive cough or coughing of blood in pulmonary tuberculosis infections. Patients with HIV are more likely to develop active tuberculosis.

It is necessary to identify the tuberculosis-causing mycobacteria by species and determine the antibiotic sensitivity or resistance-pattern for epidemiologic and public health information as well as for the effective treatment of infected persons. As stated earlier, about one-third of the world's population (1.7 billion persons) are infected with M. tuberculosis. Therefore, it is of great concern that the emergence of epidemic multidrug-resistant strains of M. tuberculosis has increased at the same time as the increase in HIV infections in the United States.

The primary routes of transmission for the M. tuberculosis complex are via inhalation of airborne droplets from an infected person; through infectious aerosols produced when processing clinical specimens for the recovery of Mycobacteria spp.; and by ingestion of contaminated milk from cows (or goats) infected with M. bovis. M. africanum is also transmitted by the inhalation of droplets containing infecting organisms. In all cases, close contact with infected individuals leads to the acquisition of tuberculosis infection.

The nontuberculous mycobacteria (NTM) group, which are not transmitted by person to person contact as is the M. tuberculosis complex, are differentiated by rate of growth (slow-growing or rapid-growing) as well as color pigmentation (the ability or inability of the colonies to change color when exposed to light). Growth patterns are divided into two main groups: slow-growers and rapid growers. Slow growers take more than seven days to grow and form colonies on solid media; rapid-growers produce colonies on solid media within three to five days. This method of classification for the NTM, by growth patterns and exposure to light, is referred to as the Runyon Classification. Some organisms in this group are considered pathogenic, and others are potentially pathogenic or non-pathogenic.

One of the most often recovered mycobacterium species in the United States belongs to the NTM group and is referred to as the Mycobacterium avium complex (MAC). The MAC group consists of two main species, M. avium and M. intracellulare. These two mycobacteria are very similar and are differentiated by DNA tests. The MAC organisms are frequently isolated from immuno-compromised patients, such as patients infected with HIV and patients with pre-existing pulmonary disease. MAC infections have been found to be the most common cause of NTM (nontuberculous mycobacteria) infections in humans. The NTM organisms are found in the environment (frequently recovered from water, soil, house dust, and plants) and are sometimes found colonized in the respiratory or gastrointestinal tract of healthy individuals. In AIDS patients, MAC infections may be focal or disseminated. It is theorized that the MAC organisms, acquired from the environment, colonize the respiratory tract or gastrointestinal tract before disseminating in an HIV-positive patient. Sputum and stool samples from HIV infected patients often contain MAC organisms.

Pulmonary disease in AIDS patients due to MAC cannot be distinguished clinically or by x ray from those caused by M. tuberculosis. Infections caused by disseminated MAC organisms in AIDS patients usually occur about one year after the diagnosis of AIDS. Also, non-AIDS patients who are white males, 45-60 years of age, typically heavy smokers, or alcohol abusers with preexisting lung disease are good candidates for a tuberculosis-like disease also caused by MAC organisms.

An NTM, which will not grow in vitro (non-cultivatable), is M. leprae. Mycobacterium leprae is the cause of leprosy, or Hansen's disease. This organism causes a chronic, debilitating, and disfiguring disease involving the skin, mucous membranes, and nerve tissue. There is often extensive damage to the skin (lesions) and nerves. Infectivity is low and transmission can occur from person to person through contact with infected skin; however, inhalation of nasal secretions from the infected person (close contact) appears to be the predominant mode of transmission. Leprosy in North America is rare, and most of the cases are acquired from exposure to the organism while in a tropical country. Mycobacterium leprae cannot be cultured on solid or liquid media in vitro; therefore, it is diagnosed by DNA amplification tests such as the polymerase chain reaction (PCR) using infected tissue, or mucous membrane secretions, and by observing acid-fast bacilli (using acid-fast staining procedures) in the tissue preps or skin biopsies of infected patients.

Several other NTM (non-tuberculous mycobacteria) organisms are considered potential pathogens for humans while others are rarely implicated in disease. The following NTM are considered potential pathogens and should be identified especially if recovered from immuno-compromised patients:

• Mycobacterium kansasii: A slow grower, causing a chronic pulmonary disease resembling classic tuberculosis as well as cervical lymphadenitis and cutaneous diseases; tap water is the main reservoir for humans.
• Mycobacterium haemophilum: A slow grower, causing skin nodules and disseminated disease in immuno-suppressed patients with AIDS, Hodgkins's disease, and kidney and bone marrow transplants, as well as cervical lymphadenitis in children.
• Mycobacterium marinum: A slow grower, causing cutaneous infections such as "swimming pool granuloma" and "fish tank granuloma" with its natural reservoir being fresh and salt water from infected fish and other marine life.
• Mycobacterium ulcerans: A slow grower, infecting the skin (usually after some trauma) causing nodules and ulcers to form; infection occurs mainly in tropical and temperate climates (Africa and Australia) and is rare in the United States.
• Mycobacterium xenopi: A slow grower, causing pulmonary infections in adults (resembling MTB complex and MAC complex). The infection is considered nosocomial, since it is recovered from hospital water storage systems and hot and cold taps quite often.
• Mycobacterium scrofulaceum: A slow grower responsible for cervical adenitis in children, recovered from raw milk, soil, water, and dairy products.
• Mycobacterium szulgai: A slow grower causing pulmonary disease similar to M. tuberculosis.
• Mycobacterium fortuitum complex: Rapid growing microorganisms which include M. fortuitum, M. abscessus, and M. chelonae causing infections involving surgical wounds, post-traumatic wound infection, otitis media, and chronic pulmonary disease.

Mycobacterium gordonae is the non-pathogenic mycobacterium most commonly recovered from patient specimens. It is found in the environment and is called the "tap water bacillus." It is only rarely implicated as a cause of human infection.