Acid-fast (Mycobacteria) Smear and Culture With Reflex to Identification

First morning sputum (not saliva) (three separate specimens with each one collected and submitted on three separate days is recommended), fasting gastric aspirate, induced sputum, whole blood, tissue, biopsy, bronchial aspirate, urine, skin, cerebrospinal fluid (CSF), bone marrow, body fluid, stool. Swabs of exudate from skin sources are acceptable, otherwise swab specimens should not be submitted. Collect aspirate using sterile, nonbacteriostatic saline or other noninhibitory medium. Do not send syringe; it will be rejected.

5-10 mL sputum, bronchoalveolar lavage (BAL) or other respiratory specimen; 10 mL whole blood; 2 cm³ tissue; 5 mL CSF; 5-10 mL bone marrow (or as much as possible); 5-10 mL body fluid (pleural, pericardial, chronic peritoneal dialysate); 5-10 mL gastric aspirate/lavage (neutralized to pH 7 with sodium carbonate within 4 hours of collection); 20-40 mL urine (first morning specimen); 5 mL or 1 g stool; biopsy of skin

2 mL (respiratory specimen, body fluid, CSF, gastric aspirate/lavage); 20 mL urine

Sterile container with tight screw-cap seal or green-top (sodium heparin) tube or yellow-top (SPS) tube or Para-Pak® White

This test is used to isolate and identify mycobacteria.

Biopsy or body fluid: Transbronchial biopsy cultures may be of assistance in diagnosing tuberculosis in sputum smear-negative cases; however, sputum and bronchial washing cultures have a higher yield.^1,2 In one study, only 2 out of 12 (16%) transbronchial biopsies were positive, and in those cases the biopsy was not the only source of culture-positive material.¹

Mycobacterium marinum may cause a localized cutaneous lesion that may be nodular, verrucous, ulcerative or sporotrichoid and that may rarely involve deeper structures. If it is suspected, the laboratory must be notified so that the culture may be incubated at an appropriate temperature (30°C).³ Mycobacterium marinum infection occurs in patients who have been exposed to the organism following cutaneous abrasion or penetrating injury while cleaning aquariums, clearing barnacles and with other aquatic exposures.

Likewise, there are two additional mycobacterial pathogens requiring special conditions for laboratory culture. Mycobacterium haemophilum, which causes a cutaneous, joint or pulmonary infection in immunocompromised patients and lymphadenitis in children, requires special growth media and incubation temperature. Mycobacterium genavense has been recovered from disseminated infections in AIDS patients. It requires special acidulated media and extended incubation conditions. If either of these mycobacteria is suspected, the laboratory must be notified so that appropriate cultivation conditions can be initiated.

Sputum: Bronchial washings are frequently diluted with topical anesthetics and irrigating fluids, but bronchoscopy still provides a high yield of positive specimens. Postbronchoscopy expectorated specimens may provide a better yield of organisms than those obtained during the procedure. The yield of prebronchoscopy sputum was 75%, bronchial washings 66% and postbronchoscopy sputum 58%. Bronchoscopy can, however, be an important adjunct to serial sputum collection in the definitive diagnosis of pulmonary infection due to mycobacteria.^4,5 Gastric aspirates yield organisms in <50% of cases of M. tuberculosis infection in children. Acid-fast stain of gastric aspirate has a sensitivity of 30% and provides a useful clinical diagnosis if positive.⁶

Urine: Positive acid-fast stained smears with low numbers of organisms are not diagnostic, because of the presence of Mycobacterium smegmatis in genital secretions of normal patients.

Stool: M. avium complex is commonly isolated from the stool of patients with AIDS and may contribute to diarrheal disease, but other agents must also be ruled out. Stool is rarely the specimen of choice for the primary diagnosis of mycobacterial infection.

Concentrated smears are stained with auramine/rhodamine and read by fluorescence microscopy, broth-based and/or agar-based culture. Culture is held for six weeks before negative is reported. Organisms from culture are identified by use of real-time polymerase chain reaction (PCR) and/or MALDI-TOF and/or nucleic acid sequencing.

This test is set up in the Labcorp computer as a sequential series of tests to comply with customer needs and bill for testing that is performed. Smear, when appropriate, is a separate test along with appropriate specimen processing needed to perform the smear and culture. The culture portion of the test is also separate, and, once growth of acid-fast organisms (or other aerobic actinomycetales) is detected, the culture portion is reported and identification testing begins with PCR-based identification. The results of the PCR identification will be reported whether positive or negative. If negative, identification will proceed by MALDI-TOF and/or DNA sequencing.

Biopsy or body fluid: Occult infections with nontuberculous mycobacteria, particularly Mycobacterium avium and Mycobacterium intracellulare, occur in patients with acquired immune deficiency syndrome (AIDS).⁷ In some institutions, the incidence of isolation of non-Mycobacterium tuberculosis species, specifically M. avium complex, may exceed the rate of isolation of M. tuberculosis. Mycobacteria have been recovered from culture of Kaposi sarcoma and bone marrow specimens in which the characteristic granulomatous reaction has been absent.^8,9 Optimal isolation of mycobacteria from tissue is accomplished by processing as much tissue as possible for culture. Swabs should not be submitted.

Tuberculous spondylitis represents 50% to 60% of all cases of skeletal tuberculosis. It is seen in children in developing countries and adults older than 50 years of age in the United States and Europe. Frequently, several vertebrae are involved and adjacent psoas muscle abscesses or paravertebral abscesses are not uncommon ("cold abscesses"). Colony counts obtained from bone biopsies are low; however, >90% are culture positive. The diagnosis of vertebral tuberculosis should be considered in all cases of unexplained spondylitis.

Cases of sternal wound infection, early prosthetic valve endocarditis, infections complicating mammary augmentation surgery and other cutaneous/subcutaneous infections have been attributed to rapidly growing mycobacteria.^10,11 M. fortuitum is the most commonly implicated Mycobacterium in these infections, which are thought to be caused by local environmental strains rather than contaminated commercial surgical materials or devices. Rapidly growing mycobacteria often grow on routine bacterial culture media within the time allotted to incubating routine bacterial cultures. Such organisms may be misidentified as "diphtheroids" and disregarded as contaminants.

Pleural effusions frequently yield positive cultures in cases of pulmonary tuberculosis. The diagnosis of peritoneal tuberculosis is difficult and is usually made at laparotomy or after a considerable delay. Tuberculosis should be considered in any patient with ascitic fluid and chronic abdominal pain.¹² Peritoneal tuberculosis accounted for 11% of a series of cases of extrapulmonary tuberculosis reported by Alvarez and McCabe.¹³

Pericardial tuberculosis accounts for <5% of extrapulmonary tuberculosis and frequently requires biopsy for diagnosis. See table.

Sputum: Tuberculosis decreased in incidence in the United States in the 1970s and 1980s, but the incidence of tuberculosis in the United States increased from 1986-1994. High-incidence populations exist in depressed inner city areas, some rural areas, amongst new immigrants, in prison inmates, and in HIV-positive patients. The emergence of M. tuberculosis and M. avium complex infections complicating the acquired immunodeficiency syndrome was striking. When tuberculosis occurs as a first or case-defining opportunistic infection, 75% to 100% of patients of HIV-positive patients have pulmonary disease. After the diagnosis of AIDS has been made, 25% to 70% of HIV-associated tuberculosis patients have an extrapulmonary site of infection.¹⁴

In an ambulatory inner city population, two specimens processed for acid-fast stain and culture identified all cases of active tuberculosis within the time required for culture. The most infectious cases were identified immediately by the acid-fast stain. Tuberculin tests and chest x-rays were also performed but did not significantly increase the number of cases identified in this population.¹⁵

M. kansasii is uncommon as an environmental contaminant. Implication of M. avium complex as a pathogen usually requires at least one of the following criteria:

• Clinical evidence of a disease process that can be explained by nontuberculous mycobacterial infection

• Repeated isolation of the same mycobacterial species from sputum over a period of weeks to months

• Exclusion of other possible etiologies

• Biopsy demonstrating acid-fast bacilli or diagnostic histopathologic changes¹⁶

Endobronchial tuberculosis has been increasingly recognized because of its incidence in association with the acquired immunodeficiency syndrome and because it may mimic carcinoma.^17,18

Nosocomial transmission of multidrug-resistant Mycobacterium tuberculosis has been noted to occur from patient to patient and from patient to health care worker. Acid-fast bacilli isolation precautions and adherence to appropriate infection control procedures are recommended until at least three smears from specimens collected on different days are negative.^19,20

While M. tuberculosis is contagious and is usually transmitted from person to person, most of the other disease-causing mycobacteria are not characterized by person-to-person spread, are found in the environment and are considered opportunistic pathogens. They may be called potentially pathogenic environmental (PPE) mycobacteria. They include M. avium, M. intracellulare, M. asiaticum, M. flavescens M. fortuitum complex, M. haemophilum, M. kansasii, M. malmoense, M. marinum, M. scrofulaceum, M. simiae, M. genavense and M. xenopi. They are correlated with HIV.^16,21

Urine: Although it has been thought that tuberculosis of the urinary tract should be suspected when hematuria and pyuria (sterile pyuria) occur without recovery by routine culture of usual urinary tract pathogens, concomitant infections with ordinary pathogens are not rare. Mycobacteria cultures of the urine are approximately 90% sensitive. The kidney is the most frequent site of infection; prostate, salpinx, and endometrial involvement also occurs. Continuing tuberculous bacilluria may cause cystitis with frequency. Genitourinary infections with PPE mycobacteria, particularly M. kansasii and M. avium complex, occur.¹⁶ Recovery of M. bovis BCG from the urine of patients undergoing BCG treatment for bladder cancer can be expected.

Stool: The increasing recognition of mycobacterial infections in patients with the acquired immunodeficiency syndrome (AIDS) has resulted in increased awareness of the potential to recover clinically significant mycobacteria from stool. Seven of 132 AIDS patients studied for intestinal infection were found to harbor M. avium complex.²² Isolation of mycobacteria from stool indicates disseminated disease, and cultures from blood, bone marrow and lymph nodes are usually also positive for the same mycobacterial isolate.²³