INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

The incidence of tuberculosis in the United States increased by 20% from 1985 to 1992 (8). Outbreaks of multidrug-resistant Mycobacterium tuberculosis raised the awareness of health care workers and the general public regarding the reemerging threat of tuberculosis (8, 10, 11). Both clinical and public health laboratories were called upon to provide faster means of definitive diagnosis of tuberculosis and to provide drug susceptibility results for all isolates (26, 27). The Centers for Disease Control and Prevention have recommended that clinical laboratories employ the most rapid methods available, including nucleic acid probes for identification of positive mycobacterial cultures, and that they consider even faster direct nucleic acid amplification techniques for direct detection of M. tuberculosis (9, 26). Two commercial methods for amplification of nucleic acids directly from acid-fast smear-positive respiratory specimens have been approved for use in the United States by the Food and Drug Administration (FDA). The first was the transcription-mediated amplification test (Amplified Mycobacterium Tuberculosis Direct [MTD] test) marketed by Gen-Probe (San Diego, Calif.), followed later by the Amplicor PCR assay (Roche Molecular Systems, Branchburg, N.J.) (9). The present communication describes our experience with several false-positive MTD test results due to M. kansasii and M. avium infections in patients with AIDS and in one human immunodeficiency virus (HIV)-negative patient with chronic lung disease due to M. avium.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Patients. All but one of the patients included in this study were in the advanced stage of AIDS and were suspected of having pulmonary tuberculosis. Salient features of each case are summarized as follows.

Mycobacterial culture procedures. Specimens were initially processed in one of the three laboratories by using the N-acetyl-L-cysteine NaOH procedure for digestion and decontamination (21). Fluorochrome smears were prepared from the sputum concentrates with the auramine-rhodamine stain (21). Concentrated sputum sediment was cultured with either a manual broth culture system, Septi-Chek or MGIT (Becton Dickinson Microbiology Systems), used according to the manufacturer's instructions, or an instrument broth culture system, the BACTEC radiometric or the MB BACT system (Organon Teknika, Durham, N.C.). The laboratories also inoculated either Middlebrook and Cohn 7H11 or Löwenstein-Jensen solid culture medium. Mycobacterial cultures were incubated for 4 to 6 weeks, depending on the individual laboratory's policy. Mycobacterial isolates were identified by use of the Accu-Probe (Gen-Probe) method according to the manufacturer's guidelines with probes for M. tuberculosis complex, M. avium complex, and M. kansasii. Broth cultures were probed for the presence of M. tuberculosis even if they were positive for M. kansasii or M. avium complex. In addition, solid culture media were incubated for an extended period and examined for the presence of multiple mycobacterial species.

Performance of the Gen-Probe MTD test. The direct Gen-Probe MTD test was performed in one of the laboratories (University Hospital) by following the manufacturer's procedures explicitly (13). All tests were performed by the same microbiologist, who had undergone training at the product manufacturer's site and was subsequently certified by Gen-Probe to perform the test on clinical samples. MTD tests were read in a Gen-Probe Leader 50 luminometer at the end of the probe selection step. Each MTD test run included positive and negative amplification controls and hybridization controls, and in addition, each patient's sputum sample was tested for potential MTD test inhibition by spiking a portion of the sample with the M. tuberculosis positive-control suspension. A positive MTD test result was defined by Gen-Probe as a luminescence reading of 30,000 RLU or greater. Cultures of several Mycobacterium species were tested by suspending the growth of patients' isolates or control strains in sterile 0.9% saline to the density of a McFarland opacity standard of 1. These suspensions were then used in place of sputum concentrate in the standard MTD test procedure and were tested at the standard 1:100 dilution specified in the package insert (called dilution 1). Colony counts of each of the mycobacterial culture suspensions tested in this manner were performed on 7H11 agar. Multiple MTD test kits with different lot numbers were used for the direct tests on patients' sputum samples and for the culture suspension tests described above.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

This report describes low-level-positive MTD test results from five patients' smear-positive sputum specimens that contained either M. kansasii or M. avium but were not found to contain M. tuberculosis. The MTD results of the patients' specimens ranged from 43,498 to 193,858 RLU (Table 1). Three of five specimens contained large numbers of acid-fast bacilli based upon the microscopic examination of fluorochrome-stained smears of concentrated sputum. However, two patients' specimens contained only 1 to 10 bacilli per high-power field. In one case an initial MTD test gave a negative result for a sputum specimen that contained only 1+ acid-fast bacilli based on examination of the fluorochrome smear, while a second specimen obtained 2 days later contained a slightly larger number of acid-fast bacilli (2+) and gave a low-level-positive MTD test result (Table 1). Patient 2 had a follow-up culture that was still positive for M. kansasii 10 months later, but the MTD test result for that specimen was negative.

The initial findings with the MTD test performed directly on sputum specimens from these patients led us to examine cultures of several mycobacterial species. When culture-grown suspensions of stock culture or of patient isolates of M. kansasii and M. simiae were tested at densities of 10⁵ CFU/ml, MTD test results of >100,000 RLU were obtained. However, culture suspensions of several other mycobacteria other than M. tuberculosis (MOTT) did not give rise to positive MTD test results, even at cell densities exceeding 10⁷ CFU/ml (Table 2).

Our laboratory (University Hospital) has performed MTD tests on smear-positive sputum specimens from a total of 82 separate patients since the test was incorporated into our standard procedure in 1996 (data not shown). Of the 48 patients whose specimens were culture-positive for M. tuberculosis, 43 have had MTD test results of >1,000,000 RLU (test sensitivity = 89.6%). Only two marginally smear-positive specimens that were culture-positive for M. tuberculosis yielded MTD test results lower than this number, specifically, 157,824 and 588,343 RLU. If these two specimens are included as positive, the overall sensitivity of the test in our series is 93.8%. There have been five false-positive results, those described herein, during this period, resulting in a test specificity of 85.3% in our experience. There have been 19 other patients with culture-documented MOTT infections whose MTD test results were lower than 30,000 RLU. These included 3 other patients infected with M. kansasii, 10 with M. avium, 4 with M. simiae, 1 with M. chelonae, and 1 with M. gordonae.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

The Centers for Disease Control and Prevention and many state, territorial, and local public health departments have focused considerable energy and resources on controlling the reemergence of tuberculosis that was noted to occur in the United States between the mid-1980s and 1992 (8, 10, 11, 27, 30). Their efforts have resulted in a 26% decrease in the number of cases reported between 1992 and 1997 (11). Contributing to the reversal of this trend have been measures such as improved case contact tracing, prompt initiation of appropriate antituberculosis therapy with four drugs, including directly observed therapy, and laboratory measures that allow for the prompt identification of persons with tuberculosis (10, 11). The resurgence of tuberculosis has provided a strong impetus to increase the efficiency and rapidity of laboratory methods for the detection of M. tuberculosis. The use of the fluorochrome stain, prompt performance of smears and prompt reporting of smear results, faster methods for the isolation of mycobacteria (i.e., use of broth culture media), rapid identification of isolates by nucleic acid probes, and faster drug susceptibility testing procedures (e.g., the radiometric method) have all contributed to an overall sharp reduction in the time required to document infection with M. tuberculosis (27, 30). However, nucleic amplification methods for direct testing of smear-positive sputum specimens, now available, represent the most rapid laboratory methods for the documentation of M. tuberculosis infection (9).

By use of the Gen-Probe MTD or Roche Amplicor amplification method, it is possible to determine within 8 h of specimen collection whether an acid-fast organism seen in a patient's sputum is M. tuberculosis (1, 3, 12, 16, 19, 23, 25). The MTD test was approved by the FDA and has been actively marketed in the United States starting in January 1996. The test amplifies 16S rRNA of Mycobacterium species by transcription-mediated amplification (2, 18). The resulting amplicons are then detected by a hybridization protection assay using a probe that purportedly is specific for M. tuberculosis (13, 18).

Shortly after initiating use of the MTD test in our setting, we began to experience the false-positive results described above. Four of the five patients with false-positive results had AIDS and were suspected of having pulmonary tuberculosis. Only patients 1 and 5 were known to have had previous infections with a MOTT, i.e., M. avium. None of the patients had a history of tuberculosis or were undergoing treatment for tuberculosis, which might lead to persistence of M. tuberculosis rRNA in sputum (20). Despite attempts to isolate M. tuberculosis from these patients' specimens and despite examination of their cultures by the Accu-Probe test, there was no evidence of concurrent infection with M. tuberculosis in addition to M. kansasii or M. avium. One patient's culture contained both M. kansasii and M. avium. However, subsequent cultures from three of the patients again revealed M. kansasii. During this period, a total of seven patients examined by us had acid-fast smear-positive sputum specimens due to M. kansasii (data not shown). Four of the seven patients had false-positive MTD test results, as described above. Furthermore, direct examination of the cultures of M. kansasii from our first three patients and of a type strain control culture provided evidence of false-positive MTD test readings due to the presence of that species and, in addition, of M. simiae.

Our findings now extend the list of Mycobacterium species that can potentially lead to false-positive MTD tests. M. celatum has been reported previously to represent a possible source of false-positive probe results (4) because strains of this species differ from M. tuberculosis by only 1 to 2 bp in the ~20-bp target sequence for the probe used by Gen-Probe for amplicon detection (5). Indeed, there have been several reports of infection with M. celatum in HIV-infected (3, 24), and immunocompetent patients (6). However, M. kansasii and M. simiae are more commonly encountered in specimens from immunocompromised patients (7, 22, 28, 29); they differ by only 4 bp from M. tuberculosis in the MTD probe region (2). In fact, M. gastri, M. scrofulaceum, M. kansasii, and M. simiae are all identical in the probe region of the 16S rRNA (2). However, false-positive probe results with M. avium complex isolates are unexpected because they have even fewer base pairs in common with M. tuberculosis in the probe region (2). Despite this difference, ours is the second report of false-positive MTD test results apparently due to infection with M. avium (17).

The findings described herein for the first two of our patients led to a revision of the MTD test package insert in August 1996. The possibility of false-positive results due to large numbers of M. kansasii organisms was included in the revised MTD test package insert (14). Gen-Probe then advised that readings of 30,000 to 500,000 RLU represented a low range of positivity that could be the result of large numbers of MOTT. It was recommended that test results in this range be viewed as "inconclusive" (14). However, our data do not entirely support the concept that only very high densities of MOTT can lead to low-level-positive results. In fact, our findings are in contrast with the statement in the package insert that the MTD test will not cross-react when fewer than 2 × 10⁶ to 4 × 10⁶ CFU per test are present (14). Moreover, two of our five patients had only 2+ acid-fast bacilli observed on their concentrated sputum fluorochrome smears. Only two specimens in our series that were culture-positive for M. tuberculosis yielded MTD readings lower than 1,000,000 RLU.

It is our belief that the Gen-Probe MTD test has substantial value in the early diagnosis of tuberculosis in patients with acid-fast smear-positive sputum specimens. However, we believe that MTD test results in the range of 30,000 to 500,000 RLU should be interpreted with caution but that readings higher than 500,000 RLU can be considered indicative of the presence of M. tuberculosis. This may be further refined by knowledge of the degree of smear positivity; i.e., low-level-positive MTD results from sputum specimens with 2+ to 4+ acid-fast bacilli probably represent false-positive results, whereas low-level-positive MTD results from sputa with scant numbers of bacilli may represent true-positive results for M. tuberculosis.

In our limited series, the sensitivity of the MTD test was 93.8% and the specificity was 85.3% when a positive test result was defined as 30,000 RLU. Our findings regarding the sensitivity of the MTD test are quite similar to those of previous investigators, but the specificity of the test observed by us is somewhat lower than that previously reported (1, 12, 16, 18, 19, 23, 24). The lower specificity observed by us may be a function of the number of pulmonary infections due to large numbers of MOTT in the sputa of our patients. Gen-Probe has recently released a second-generation MTD test (referred to as MTD 2) that incorporates several procedural changes, including a larger specimen volume, a shorter amplification phase, and a longer probe selection step (15). These changes may enhance the specificity of the test by limiting the number of amplicons produced and extending the probe selection phase. However, the package insert for the revised procedure still includes the possibility of results in an indeterminate range, which would require repeating the test (15). Thus, it remains to be determined if this "enhanced" MTD 2 test will make our recommendations for MTD test interpretation unnecessary.