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Diagnostic mycobacteriology: where are we today?

Tuberculosis has reemerged as a significant public health problem in certain parts of the United States. In addition, strains of Mycobacterium tuberculosis with various patterns of resistance to antituberculosis chemotherapeutic agents have been recovered with increasing frequency (17, 18). The continuation of the human immunodeficiency virus (HIV) epidemic has contributed to both of these problems. In an attempt to address the concurrent problems of the increasing prevalence of tuberculosis and more frequent recognition of multidrug-resistant strains of M. tuberculosis, the Centers for Disease Control and Prevention (CDC) in Atlanta, Ga., has recently developed and now promulgates recommendations for controlling the transmission of tuberculosis (2, 3). Obviously, an essential component of any tuberculosis control program is rapid and accurate identification of infected individuals, especially those most likely to transmit viable organisms. In this regard, the CDC has also developed recommendations for standards of laboratory practice regarding detection, identification, and susceptibility testing of M. tuberculosis in clinical specimens (17). The three most fundamental aspects of these recommendations are provision of acid-fast bacillus smear results within 24 h of specimen collection, isolation and identification ofM. tuberculosis within 10 to 14 days, and provision of susceptibility test results within a total of 15 to 30 days. All three of these objectives significantly impact on the function of mycobacteriology sections of clinical microbiology laboratories. Hours of operation, staffing patterns, technology decisions, and the cost of providing service are all profoundly influenced by these proposed standards of laboratory practice. In particular, the second recommendation of the CDC, isolation and identification ofM. tuberculosis within 10 to 14 days, would have profound ramifications on the function of clinical microbiology laboratories. A question arises: can this standard be reasonably achieved using current “state-of-the-art” diagnostic methods? An important corollary to this question pertains to the recent licensure of a commercial molecular diagnostic test that will make it possible for even the smallest laboratory to detect and identify M. tuberculosis directly in clinical specimens in a matter of hours. What will be the role of direct molecular diagnostic tests in mycobacteriology? What are the performance characteristics of molecular detection assays? Use of such technologies would clearly permit laboratories to meet the standard of 10 to 14 days for the isolation and definitive species identification of M. tuberculosis. Will molecular-probe based methods become broadly applied in clinical microbiology laboratories? Should they be used routinely or selectively? What are the fiscal implications of direct molecular detection and identification? These issues serve as the basis for this guest commentary. Question: Are current CDC recommendations defining 10to 14-day limits on the recovery and definitive species identification of M. tuberculosis achievable by methods other than direct nucleic acid amplification methods? Answer: Probably not, at least in most laboratories. Currently, the fastest and most reliable approach to detecting and identifying M. tuberculosis in clinical specimens other than use of a direct molecular detection test is use of a brothbased radiometric or continuous monitoring detection system with identification of clinical isolates recovered in culture using a nucleic acid probe-based culture confirmation identification system (8). Seen in Table 1 are the lengths of times to recovery and identification of M. tuberculosis obtained in 10 U.S. laboratories during 1993. All 10 laboratories employed a radiometric detection system and nucleic acid probes for identification of isolates. Only 2 of these 10 laboratories, laboratories E and G, met the CDC standard of 10 to 14 days for isolation and identification of M. tuberculosis. The wide ranges observed for mean lengths of time to detection and species identification among the 10 laboratories described in Table 1 were explained by differing staffing patterns, variable scheduling of mycobacteria specimen processing and workup of positive cultures, and whether probe-based identification was applied directly to growth in primary cultures or used only to characterize organisms following at least one subculture. Both of the laboratories that met the CDC’s recommended standard processed mycobacteriology specimens 7 days per week and aggressively attempted probe-based identification whenever possible directly on growth from primary cultures. In addition, laboratory E worked up positive cultures with probe identification tests 7 days per week. Question: Would application of nucleic acid amplification assays for detection and identification of M. tuberculosis directly in clinical specimens permit laboratories to meet the CDC’s recommended standard? Answer: Possibly, at least for certain specimens. One nucleic acid amplification assay for the detection and identification ofM. tuberculosis directly in clinical material, the MTD test (Gen-Probe, Inc., San Diego, Calif.), has recently received Food and Drug Administration (FDA) approval and is now available commercially. The MTD assay is a 4-h procedure based on transcription-mediated amplification of a 16S rRNA target sequence. Results are read with a luminometer. A second amplification method, the Amplicor MTB procedure (Roche Diagnostics Systems, Somerville, N.J.), is currently awaiting FDA approval. The Amplicor MTB assay is predicted on PCR amplification of a 584-bp segment of 16S rRNA; it utilizes a thermocycler and requires 6 h to complete. In addition to the MTD test and the Amplicor MTB assay, three other direct nucleic acid amplification methods for detecting M. tuberculosis are in various stages of development. * Mailing address: University of Massachusetts Medical Center, Clinical Microbiology Laboratories, Worcester, MA 01655-0219. Phone: (508) 856-6417. Fax: (508) 856-1537.