Although the virulences and host ranges differ among members of theafricanum, but further characterization resulted in profiles specific for all members. Although six RD regions were used in the analyses with the original 88 isolates, it was found that the use of RD 1, RD 9, and RD 10 was sufficient for initial screenings, followed by the use of RD 3, RD 5, and RD 11 if the results for any of the first three regions were negative. When 605 sequential clinical isolates were screened, 578 (96%) were identified as M. tuberculosis, 6 (1%) were identified as M. africanum, 8 (1%) were identified as M. bovis, and 13 (2%) were identified as M. bovis BCG. Since PCR-based assays can be implemented in most clinical mycobacteriology laboratories, this approach provides a rapid and simple means for the differentiation of members of TBC, especially M. bovis and M. tuberculosis, when it is important to distinguish between zoonotic sources (i.e., cattle and unpasteurized dairy products) and human sources of tuberculosis disease.The Mycobacterium tuberculosis complex (TBC) (4, 34) comprises the closely related organisms M. tuberculosis, M. africanum, M. bovis, the M. bovis BCG vaccine strain, and two rarely seen members, M. microti and M. canettii (35). Differentiation of the members of the TBC is necessary for the treatment of individual patients and for epidemiological purposes, especially in areas of the world where tuberculosis has reached epidemic proportions or wherever the transmission of M. bovis between animals or animal products and humans is a problem. In addition, it can be important to rapidly identify isolates of M. bovis BCG recovered from immunocompromised patients.Although no clear-cut means of differentiation of the members of the TBC was found in the past by using numerical classification (34), a few conventional methods have been useful. Those methods include assays for the ability to metabolize glycerol or pyruvate in Loewenstein-Jensen medium, oxygen preference (aerophilic versus microaerophilic), niacin accumulation, nitrate reductase activity, colony morphology, and resistance to two compounds, thiophen-2-carboxylic acid hydrazide (TCH) and pyrazinamide (PZA) (12,19,38). Partially due to the slow growth of the TBC, interpretation of the results of these assays can be highly subjective, especially interpretation of differences in colony morphology (19), which can be due to the loss of virulence or to mutations associated with drug resistance. An alternative approach is the use of high-performance liquid chromatography; however, only the profile for M. bovis BCG differs from those for the other members of the complex (10).Testing for resistance to TCH has been reported to be the only single test that assigned isolates to any specific member of the TBC; classical M. tuberculosis isolates are resistant to TCH, irrespective of their resistance to isoniazid (8). Alternatively, the Asian strain of M. tuberculosis and all other members of the TBC are TCH susceptible (39, 40). However, cross-resistance to TCH has ...

A PCR-restriction fragment length polymorphism (PCR-RFLP) procedure capable of rapidly identifying 28 species of clinically encountered mycobacteria was evaluated for use in the routine identification of acid-fast isolates growing in BACTEC 12B and 13A liquid media. PCR-RFLP identified 100 of 103 acid-fast isolates recovered from 610 patient specimens submitted for culture during the study. The three isolates unidentifiable by PCR-RFLP produced restriction patterns not included in the PCR-RFLP algorithm and could therefore not be assigned to a species. These isolates were characterized by their morphologic and biochemical characteristics. Two of the isolates were identified as M. terrae complex and M. gordonae. The third isolate could not be definitively identified and could only be characterized as a Mycobacterium sp. most closely resembling M. chelonae. PCR-RFLP identifications agreed with the conventional identifications for 96 of the 100 isolates identified by PCR-RFLP. Subsequent identification of the four discordant isolates by gas chromatography analysis supported the PCR-RFLP identification of each isolate. Amplification products were also obtained from isolates of Streptococcus albus and Rhodococcus equi recovered from patient specimens; however, the restriction patterns of these nonmycobacterial species did not resemble the patterns of any mycobacterial species included in the PCR-RFLP algorithm. PCR-RFLP seems to be a reliable procedure for the routine identification of mycobacteria and has the potential for providing identifications of mycobacterial isolates which are more accurate than conventional identification techniques based on morphologic and biochemical characteristics.

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Even in the 21st century, tuberculosis continues to be a problem. Although the number of cases continues gradually to decrease in the United States, cases get more difficult to treat, specifically those that are multiple-drug resistant. Infection of one-third of the world's population ensures that tuberculosis will not disappear in the near future. In light of this, it will be useful to know the goals for the health care system and how these goals may be accomplished. Laboratory testing in the mycobacteriology field is experiencing more changes today than ever before. Determining what assays will be most useful to the clinician is a challenge, and acceptance of the new technology by the medical community an even greater one. Clinicians must use the best available resources to determine the most appropriate care for their patients and work together with the laboratory to ensure that the communication channels are open. This review focuses on current state-of-the-art resources useful for accurate and rapid laboratory diagnosis of mycobacterial infections.

The inactivation of smears that contain Mycobacterium tuberculosis for microscopy before removal of the material from a biosafety cabinet is an important safety factor in preventing the potential transmission of tuberculosis to laboratory workers. The fixing and inactivating properties of heat flaming, 70% ethanol, and 1, 3, and 5% phenol in ethanol for smears containing M. tuberculosis were investigated. Heat flaming failed to inactivate the smear material, whereas 5% phenol in ethanol successfully fixed and inactivated all smears containing M. tuberculosis both from concentrated sputum samples and from culture material.The potential hazard of laboratory work with Mycobacterium tuberculosis is well recognized. There are numerous records of laboratory-acquired tuberculosis (TB) infections, with aerosols and skin punctures being the most common reported routes of transmission (10,13,15,18,19,24). The resurgence of TB in industrialized countries in the past decade has resulted in an increasing number of specimens and cultures being tested in laboratories, thereby enhancing the potential of accidental exposure for laboratory staff.Stringent safety precautions must be followed for laboratory work with M. tuberculosis (6,14). There remains one part of the routine laboratory process, however, where the hazard for potential exposure has not been addressed-unstained smears. Specimen and culture manipulations for mycobacteriological analyses, including the preparation of smears, are carried out within a biosafety cabinet (BSC). The smears are usually then heat fixed by passage of the slides through the flame of a gas burner or by placement on a hot plate. After the smears are fixed, they are removed from the BSC to a staining sink or they may be stored, transported to a reference laboratory, or used in proficiency testing panels.Several studies have shown that heat-fixed smear material, whether fixed by flaming or for 2 h at 65°C on a hot surface, still contains viable bacilli (1, 4; L. R. B. Giacomelli, S. R. Sespede, A. M. W. Barreto, and C. L. Cardoso, Abstr. Clin. Microbiol., abstr. C-129, p. 131, 1999). Our data from this study confirm that M. tuberculosis is still viable after flame fixing of smear material. Additional problems with heat fixing are that the use of a flame inside a BSC is not recommended and that 2 h of fixing on a hot plate is cumbersome and time-consuming when large numbers of slides must be fixed.When slides containing viable M. tuberculosis smear material are removed from a BSC, laboratory staff can be exposed to infective material if slides are broken, thereby possibly generating aerosols or skin penetration, or the smear material may flake off slides, thus potentially infecting the worker and contaminating the laboratory environment. Staining methods used for acid-fast bacilli (AFB), whether fluorochrome or carbol fuchsin based, are known to kill mycobacteria. However, staining is done over a sink outside the BSC, necessitating the prior removal of unstained smears from the cabinet.The purpose...