Acid-Fast Culture

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Acid-Fast Culture

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 and other immunosuppressed 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, 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, amongst new immigrants, in prison inmates, and in persons infected with the human immunodeficiency 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 10-100 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 non-tuberculous 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 multi-drug-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 airborn 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 immunocompromised patients, such as patients infected with HIV and patients with preexisting 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, alcohol abusers with pre-existing 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 immunocompromised 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 immunosuppressed patients with AIDS, Hodgkins's disease, 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 slow and rapid growing, and only rarely implicated as a cause of human infection.

Specimen collection

Specimens to be processed for the recovery of mycobacteria are obtained and handled using specific guidelines to ensure successful growth, isolation and identification of the causative organism. Containers must be sterile, leak-proof and labeled properly. After collection, if the specimen cannot be processed within one hour, refrigeration is required but no longer than overnight. However, blood samples must be placed in the proper media and incubated immediately at 95-98.6 °F (35-37 °C).

The most often requested specimens are pulmonary specimens (secretions) which must be obtained before any treatment (antibiotic therapy) is given. Pulmonary specimens may be obtained in several ways: spontaneously produced (expectorated) sputum; aerosol-induced sputum; bronchioscopic aspirations, washings and brushings; gastric aspirates, and lavages (washings) from patients who have swallowed sputum through the night. Saliva is not acceptable as a specimen for the recovery of mycobacteria and is usually rejected as a contaminated specimen. A series of early morning sputum specimens are recommended over a three-day period. The ideal amount of sputum specimen for processing and recovery of mycobacteria is 5-10 mL of sputum. Upon rising in the morning, the patient is instructed to cough deeply to produce sputum (expectorated sputum). A patient who is unable to bring up any sputum is given an aerosol treatment (aerosol-induced sputum) by a respiratory therapist in order to recover a sufficient amount of sputum for culture.

Other specimens requested for culture and recovery of mycobacteria are early morning, voided urine specimens; fecal specimens; tissue and body fluids (pleural, pericardial and peritoneal fluids), cerebrospinal fluid (CSF), bone marrow aspirates, and blood. Blood and stool specimens are usually cultured from AIDS patients. These specimens reveal numerous mycobacteria, when infection is present in these patients. Wound or skin lesions (abscesses) require a technique using aspiration of the specimen into a syringe rather than the use of a swab to obtain the specimen.

Specimens not suitable for culture and usually rejected are 24-hour urine specimens, pooled sputum, saliva, and swabs containing pulmonary secretions. The high rate of contamination as well as the reduced rate of mycobacteria recovery in these specimens renders them unsuitable.

Specimen processing

Decontamination and digestion of sputum specimens is necessary to recover mycobacteria for culture and identification. The process of decontamination (removing unwanted bacteria) and digestion (breaking down mucous and protein) of sputum specimens is necessary to release the mycobacteria that may be present but are trapped in the mucous, and also to kill the unwanted bacteria (normal flora). Specimens from sterile body sites (blood, tissue and body fluids, etc.) do not need the process of decontamination and digestion as do sputum samples. If the process of decontamination and digestion is not done or done improperly, recovery of mycobacteria from sputum samples is inhibited causing a false-negative report. Mucous, cells, and normal bacterial flora (from the oral cavity) entrap and enmesh the mycobacteria in sputum. A common decontaminant is sodium hydroxide (4%) which is also used as a mucolytic agent (for liquifaction or digestion of mucous). A combination is often used which consists of N-acetyl-L-cysteine (NALC) and a lower concentration (2%) of sodium hydroxide. This combination gives a better recovery rate when used together as a mucolytic-decontaminant. Liquifaction of the thick mucous in sputum is necessary to free the mycobateria trapped in it without harming the mycobacteria and decontamination kills the normal flora (bacteria from the mouth, throat and oral cavity) which interfere with the recovery of mycobacteria. The final product is reduced (concentrated) from the original 5-10 mL volume and a portion of the resulting specimen is transferred by sterile technique to either sterile solid, tube or plate media, and liquid media while another portion is used to make several smears on glass slides for staining.

Acid fast and fluorescent staining

The smears made after the process of decontamination and digestion of sputum are stained using either an acid-fast staining procedure or a fluorochrome stain. Mycobacteria do not stain well with the Gram staining procedure used routinely in the microbiology laboratory. Specimens obtained from sterile sites (bone marrow, tissue, etc.) do not need processing and smears are made directly from the specimen onto glass microscope slides. Mycobacteria are slightly curved or straight bacilli, about 0.2 to 0.6 by 1.0 to 10 micrometers in size. The cell wall of mycobacteria contains a high lipid content, and is made up of long-chain, multiply cross-linked fatty acids (mycolic acids). In the acid-fast staining procedure, a basic dye, carbolfuchsin stain is used to stain the cell wall. The long-chain mycolic acids and waxes in the mycbacteria cell wall serve to complex the carbolfuchsin. The Ziehl-Neelsen acid fast stain for mycobacteria, uses heat to fix the dye in the cell wall, while the Kinyoun staining method uses an increased concentration of basic fuchsin and phenol eliminating the heat requirement. In the Ziehl-Neelsen procedure, the carbolfuchsin stain is left on the smear for five minutes while heat is applied under the slide by a bunsen burner or a hot plate. The carbolfuchsin dye penetrates the cell wall and the excess stain is washed off with a 3% acid-alcohol mixture (95% ethanol and 3% hydrochloric acid). The mycobacteria cell wall retains the dye (a red-purple color) and will not be decolorized (washed out) by the acid-alcohol, thus the term acid-fast. A second dye, methylene blue, is used to stain any background material including any other bacteria that may be present. This dye results in a light background providing good contrast to the redpurple stain of the carbolfuchsin dye, thus aiding in the detection of acid-fast bacilli. If mycobacteria are present in the smear, the appearance of red-purple short or long bacilli are observed at 1000 X magnification. Some species of mycobacteria appear "beaded" while others may appear pleomorphic (a mixture of coccoid and rod shapes), or filamentous (branching of the bacillus).

Another staining method used for the detection of mycobaceria is the auramine-rhodamine fluorochrome stain. This method requires a fluorescent microscope. Smears are scanned at a lower magnification (250 X to 400 X). The fluorochrome dyes used in this procedure complex to the mycolic acids in acidfast cell walls. The fluorescing mycobacteria are seen as bright yellow-orange bacilli against a dark background. Fluorescent stained smears can be read more rapidly than acid-fast stains, but there are drawbacks. (Mycobacteria spp.) that are rapid-growers may not appear fluorescent with these stains; artifacts may fluoresce; material on the oil objective may have floated off a previous positive smear causing a falsepositive reading for the next smear examined. All positive smears from the auramine-rhodamine fluorochrome method should be confirmed using the Ziehl-Neelsen method for acid-fast bacilli.

Acid-fast bacillus (AFB) smear report

Laboratories performing staining procedures and reporting smear results must adhere to guidelines from the U.S. Department of Health and Human Services (Public Health Service, Centers for Disease Control, Atlanta). The rule for reporting acid-fast smears for mycobacteria requires scanning the smear for a minimum of 15 minutes (at least 300 oil immersion fields) before calling the slide negative for acid-fast bacilli or "No AFB seen." The following are recommended interpretations and ways to report smear results:

A request for another specimen or a doubtful report is the result of seeing AFB of 1-2/300 fields for the Ziehl-Neelsen (Z-N) stain and AFB of 1-2/70 fields for the auramine-rhodamine (fluorochrome) stain.
A "1+" report for AFB seen = 1-9/100 fields for the Z-N method and 2-18/50 fields for the fluorochrome stain.
A "2+" report for AFB seen = 1-9/10 fields for the Z-N method and 4-36/10 fields for the fluorochrome stain.
A "3+" report for AFB seen = 1-9/field for the Z-N method and 4-36/field for the fluorochrome stain.
A "4+" report for AFB seen = less than 9/field for the Z-N method and less than 36/field for the fluorochrome stain.

Culture media and isolation methods

Several types of media are used for the cultivation of mycobacteria and each facility determines which ones are most appropriate for them. A combination of culture media is often used to optimize recovery of mycobacteria as well as inhibit the growth of contaminants. Mycobacteria require a pH of 6.5-6.8 for growth and grow best at higher humidity. Commercially prepared solid culture media (in tubes with screw-top caps) consist of bovine serum albumin agar-based media (Middlebrook 7H10 and 7H11) and egg-based media (Lowenstein-Jensen). Liquid media (Middlebrook 7H9) is used to subculture stock strains or as part of a system (e.g., BACTEC 12B medium, Septi-Chek AFB) to cultivate and detect growth of acid-fast bacilli. Mycobacterium spp. grow more rapidly in liquid media; solid media takes approximately 17 days for the isolation of acid-fast bacilli whereas liquid media takes only about 10 days. The following are descriptions of three general types of media that are most often used.

Lowenstein-Jensen media (L-J) is an egg-potato base solid media containing malachite green (an inhibitory agent). The use of L-J media is excellent for the recovery of M. tuberculosis from sterile-site specimens as well as decontaminated-digested sputum specimens.
Petragnani media is an egg-milk-potato solid medium also containing malachite green. It is primarily used for specimens from highly contaminated areas (e.g., fecal material).
Middlebrook 7H10 media is a liquid based media containing salts, vitamins, cofactors, oleic acid, albumin, catalase, glycerol, and glucose. This media enhances the recovery of MAC organisms (Mycobacteria avium complex).

Each culture medium described above represents a nonselective formulation, but selective formulations are also used which contain antibiotics to enhance the growth of mycobacteria and suppress the growth of contaminating bacteria. The enhanced formulas are used for specimens that are highly contaminated.

All culture tubes are incubated in an atmosphere of 5-10% CO₂ (for growth enhancement) even though mycobacteria are strict aerobes. The tubed media are kept in a high humidity incubator at 95 °F (35 °C) in the dark in a slanted position with the caps loosened (in order for CO₂ to enter the tubes and excess fluid to evaporate). For specimens obtained from skin or superficial lesions, a lower temperature 77-86 °F (25-30 °C) is required for the recovery of M. marinum and M. ulcerans. A nutritional requirement of hemin and a temperature of 30 °C are needed for the recovery of M. haemophilum (cultured from skin nodule specimens). If M. xenopi is suspected, a temperature of 107.6-113 °F (42-45 °C) is required (cultured from hospital hot water tanks).

AFB cultures are held for six to eight weeks before reporting "No growth of AFB." Cultures are observed daily for the first two weeks, checking for any growth or colony formation. Rapid-growing mycobacteria usually appear on non-selective media in two to three days at temperatures between 68-104 °F (20-40 °C). The slow-growing mycobacteria associated with disease require four to six weeks of incubation on selective media. Since the use of liquid media allows mycobacteria to grow more rapidly and is considered the most sensitive primary isolation media, the Becton Dickinson Diagnostic Instrument Systems developed the BACTEC System. The BACTEC System utilizes Liquid Middlebrook 7H12 and 7H13 in an automated radiometric culture system. The broth is placed in commercially prepared vials containing a 14C-labeled substrate (palmitic acid) used by mycobacteria, liberating radioactive carbon dioxide (¹⁴C0₂) into the upper part of the vial. The ¹⁴C0₂ liberated is detected by the BACTEC 460 (instrument) and is recorded as a "growth index" denoting growth of mycobacteria in the vial of broth. This method of growth significantly improves the isolation rate of mycobacteria compared with conventional isolation using solid tubed media. The BACTEC vials must be checked within four days of inoculation. This method detects Mycobacteria spp. growth in clinical specimens in less than two weeks compared to four to six weeks for conventional methods.

Non-radiometric automated systems are also available for the detection of growth and recovery of mycobacteria from clinical specimens. An example is the Septi-Chek AFB system (BBL-Becton Dickinson Microbiology Systems) that detects, isolates, rapidly identifies, and performs antibiotic susceptibility testing. This is a biphasic media system (a bottle containing liquid media and solid media) that uses growth enhancing factors and antimicrobial agents in the liquid and three different solid media on a paddle inserted in the top of the vial. This system rapidly grows, isolates and presumptively identifies M. tuberculosis (i.e., differentiates it from other mycobacteria).

Identification

Based on the volume of specimens submitted, the ability of performance, and the expertise of the clinical laboratory personnel, the American Thoracic Society (ATS) and the College of American Pathologists (CAP) have recommended levels of service for clinical laboratories testing of mycobacteria. The ATS recommends four levels of testing while the CAP lists three levels. The three levels of service recommended by CAP are:

Level I. Specimen collection only; no identification procedures performed with all specimens sent to other qualified laboratories.
Level II. Perform microscopy; isolate and identify and sometimes perform susceptibility tests for M. tuberculosis.
Level III. Perform microscopy; isolate, identify and perform susceptibility testing for all species of Mycobacterium.

Identification of Mycobacteria spp. by qualified clinical laboratories entails several of the following:

Confirmation that the isolate recovered in broth or on solid media is an acid-fast organism.
Categorize (presumptively) the isolate by phenotypic characteristics, such as colony morphology, photoreactivity, growth rate, and optimum growth temperature.
Identification through tests based on enzyme systems of the organism, metabolic by-products, and inhibition of growth by exposure to selected biochemicals.
Chromatographic detection of mycolic acid.
Identification by DNA hybridization (e.g., Gen-Probe-San Diego, California).
Identification by PCR (polymerse chain reaction) tests.

The biochemical tests most often utilized are niacin accumulation, nitrate reduction, TCH (inhibition of growth when exposed to thiophene-2-carboxylic acid hydrazide); growth in 5% NaCl, tellurite reduction,; growth on MacConkey agar, catalase, hydrolysis of Tween 80, iron uptake, and tests for the enzymes arylsulfatase; urease; and pyrazinamidase. Biochemical testing is time consuming and may take several weeks to obtain results. Molecular methods (DNA and PCR) are becoming increasingly available commercially and allow for identification and detection of mycobacteria faster, with less cost and more specificity.

Antibiotic susceptibility testing for tuberculosis

The susceptibility testing for Mycobacteria tuberculosis is done on a pure culture which may take two to three weeks to prepare after the initial culture has grown. Thus, a total of five to seven weeks is not uncommon before the physician finally receives an antibiotic susceptibility report for a patient with a positive MTB culture. However, rapid testing systems mentioned previously may be used for susceptibility testing which reduces the time considerably.

Once the physician receives the initial smear report (i.e., positive AFB on smear) and the initial culture report (presumptive M. tuberculosis isolated), the patient is given two or more primary drugs (first-line drugs) to initiate treatment that may require six to nine months of drug therapy. The first line (primary drugs) drugs tested in vitro include isoniazid (INH), rifampin, pyrazinamide, ethambutol, and streptomycin. After three months of therapy, patients are again cultured. If the cultures are still positive, retesting of different or secondary drugs is done. The second-line drugs include ethionamide, capreomycin, cycloserine, kanamycin, pyrazinamide, amikacin, ciprofloxacin, ofloxacin, rifabutin, and para-aminosalicylic acid.

The methods used for susceptibility testing are: radiometric (BACTEC System); proportional; resistance ratio (agar dilution and disk elution); and absolute concentration methods. It is important to isolate and determine the susceptibility pattern for M. tuberculosis because of the increase in multidrug-resistant cases in the United States.

Preparation

Prior to breakfast, the patient will be asked to provide a 5-10 mL specimen of sputum delivered into a sterile cup with a screw top lid. Obtaining an appropriate sample will require that the patient cough deeply several times to bring up the sputum. Failure to do so will result in a specimen containing saliva or post-nasal drip, which are both considered sample contaminants.

Aftercare

There are not specific requirements for care after obtaining the specimen.

Complications

There are no complications associated with this test.

KEY TERMS

Bronchiectasis— The formation of dilated, enlarged bronchi that results from lower respiratory tract infection.

Granuloma— Encapsulation of infected tissue caused by phagocytic cells that surround the foci of infection.

Nosocomial— An infection acquired in a hospital setting.

Results

The acid-fast smear report will indicate "no AFB seen" if results are negative. If positive, the report should be documented as described above. For cultures, "no growth of AFB" on any medium after eight weeks is considered a negative test. Growth on any medium is tested for acid-fastness and if positive, a preliminary report of a positive culture for Mycobacterium spp. is submitted. A final report of the mycobacterium species identified and antibiotic susceptibility is submitted as soon as results are available. The antibiotic susceptibility report indicates one of three conditions for each drug: sensitive, equivocal, or resistant.

Health care team roles

A physician orders and interprets the report for an acid-fast culture. A nurse, physician assistant, or respiratory therapist assists in sputum or sample collection. A clinical laboratory scientist/medical technologist who is specially trained in mycobacteriology performs the microbiological testing.

Resources

BOOKS

Chernecky, Cynthia C, and Berger, Barbara J. Laboratory Tests and Diagnostic Procedures. 3rd ed. Philadelphia, PA: W. B. Saunders Company, 2001.

Fischback, Francis. A Manual of Laboratory and Diagnostic Tests, 5th Edition. Philadelphia: J. B. Lippincott Company, 1996, p.327 335.

Forbes, BA, Sahm, DF, and Weissfeld, AS. Baily and Scott's Diagnostic Microbiology. 10th Edition. Mosby, St. Louis, 1998.

Kee, Joyce LeFever. Handbook of Laboratory and Diagnostic Tests. 4th ed. Upper Saddle River, NJ: Prentice Hall, 2001.

Metchock, BG, Nolte, FS, and Wallace RJ. "Mycobacterium." In Manual of Clinical Microbiology. 7th ed. Murray, P, Baron EJ, Pfaller, MA, et. al. Editors. Washington, DC: American Society for Microbiology, 1999.

Vossler JL. "Mycobacterium tuberculosis and other nontuberculous mycobacteria." In Textbook of Diagnostic Microbiology. 2nd ed. Mahon, CR, Manuselis, G, Editors. Philadelphia: Saunders, 2000.