MycobacteriologyRoutine use of the gen-probe MTD2 amplification test for detection of Mycobacterium tuberculosis in clinical specimens in a large public health mycobacteriology laboratory1
Introduction
The rapid and accurate detection of Mycobacterium tuberculosis (MTB) in clinical specimens plays an essential role in the control and prevention of the transmission of tuberculosis (TB) Doern 1996, Tenover et al 1993. The initiation of appropriate chemotherapy and contact tracing is dependant on the timely identification of infected individuals.
Conventional laboratory testing for the detection of MTB is dependant on the acid-fast stain, which is rapid but is neither sensitive nor specific, and on culture and identification tests that are sensitive and specific, but can require 2 to 8 weeks to complete. The introduction of nucleic acid amplification technology to the mycobacteriology laboratory allows the sensitive and specific detection of MTB directly from clinical specimens in a few hours. A number of different amplification techniques have been incorporated into commercially produced test systems such as transcription-mediated amplification, MTD test (GenProbe Inc., San Diego, CA), polymerase chain reaction (PCR), Amplicor M. tuberculosis (Roche Diagnostic Systems, Somerville, NJ) and the ligase chain reaction, LCx MTB (Abbott Diagnostics Division, Abbott Park, IL).
The MTD test by Gen-Probe has been in use in our laboratory since May 1996. Several published studies Bradley et al 1996, Pfyffer et al 1994, Piersimoni et al 1997 have evaluated the performance of the original MTD test, with the sensitivity and specificity compared with culture reported in the range of 93.6 to 95.9% and 97.8 to 98.9% respectively, when the test is used on acid-fast, smear-positive respiratory specimens from untreated patients.
The MTD test is an isothermal transcription-mediated amplification system based on specific mycobacterial rRNA targets using DNA intermediates. The RNA amplicons produced are identified by a hybridization protection assay using an acridinium ester-labeled MTB complex-specific DNA probe. The original MTD test was FDA approved in 1995. Gen-Probe subsequently modified the test, making it faster and simpler to perform, and using a larger sample volume to increase sensitivity (Bodmer et al. 1996). The new enhanced MTD test, the MTD2, (FDA approved, May 1998), was used during the 1-year period of this study from May 1, 1997 to April 30, 1998.
On average, 25,000 clinical specimens are processed annually in this laboratory for isolation of mycobacteria. Specific specimens from our laboratory and from regional laboratories throughout the province of Ontario are selected for MTD2 testing. An efficient specimen selection protocol is necessary to make the test both cost and labor effective. In this report, we review the selective application of the MTD2 amplification test to the regular testing algorithm for the detection of MTB in a large, public health mycobacteriology laboratory.
Section snippets
Specimen selection
Clinical specimens with the following specifications were selected for testing by MTD2:
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All acid-fast, smear-positive specimens from new patients from both respiratory and nonrespiratory sources.
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One acid-fast, smear-positive specimen from any patient with a previous history of infection with a nontuberculosis mycobacteria (NTM)
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Any specimen (or concentrate from a specimen) from another laboratory that met the above criteria. Laboratories were asked to submit both specimen and concentrate for
Results
A total of 823 clinical specimens, from unique patients, which included 616 respiratory and 207 nonrespiratory specimens, were tested by MTD2 during the 1-year study. The types of specimens with the corresponding AFB smear, culture, and MTD2 results are shown in TABLE 1, TABLE 2 . Table 3 provides details of 15 specimens where results of culture and MTD2 were discrepant. Thirteen of these results were resolved retrospectively on investigation of clinical findings. The two remaining discrepant
Discussion
The regular use of the MTD2 amplification test for the direct detection of MTB in clinical specimens has become an important component of our mycobacteriology laboratory service. During the 1-year review period, 202 cases of respiratory TB and 56 cases of extrapulmonary TB, including 4 cases of tuberculous meningitis, were detected by the MTD2 test within 0 to 4 days of specimen arrival in the laboratory.
The diagnostic utility of a direct amplification test for MTB is greater in areas with a
Acknowledgements
We thank the staff of the Mycobacteriology Laboratory of the Central Public Health Laboratory, Etobicoke for their expert technical assistance and Ms. Suzanne Lombardi for excellent secretarial help.
References (26)
Rapid diagnostic tests for tuberculosis. Progress but no gold standard
Am J Respir Crit Care Med
(1997)- et al.
Enhanced amplified mycobacterium tuberculosis direct test for detection of Mycobacterium tuberculosis complex in positive BACTEC 12B broth cultures of respiratory specimens
J Clin Microbiol
(1999) - et al.
Improved performance of Gen-Probe amplified Mycobacterium tuberculosis direct test when 500 instead of 50 microliters of decontaminated sediment is used
J Clin Microbiol
(1996) - et al.
Clinical efficacy of the amplified Mycobacterium tuberculosis direct test for the diagnosis of pulmonary tuberculosis
Am J Respir Crit Care Med
(1996) - et al.
The application of molecular techniques to the diagnosis and epidemiology of mycobacterial diseases
J Appl Bacteriol
(1996) - et al.
Identification of mycobacteria by HPLC
J Clin Microbiol
(1991) - et al.
Rapid diagnostic tests for tuberculosis. What is the appropriate use?
Am J Respir Crit Care Med
(1997) - et al.
Changing patterns of mycobacterial disease at a teaching community hospital
Infection Control and Hospital Epidemiology
(1994) Diagnostic MycobacteriologyWhere are we today?
J Clin Microbiol
(1996)- et al.
Diagnosis of extrapulmonary tuberculosis by Gen-Probe amplified Mycobacterium tuberculosis direct test
J Clin Microbiol
(1996)
Current and future applications for mycobacterial amplification assays
Clin Microbiol Newsletter
Comparative evaluation of initial and new versions of the Gen-Probe amplified mycobacterium tuberculosis direct test for direct detection of Mycobacterium tuberculosis in respiratory and nonrespiratory specimens
J Clin Microbiol
Evaluation of acridinium-ester-labeled DNA probes for identification of Mycobacterium tuberculosis and Mycobacterium avium-Mycobacterium intracellulare complex in culture
J Clin Microbiol
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The MTD2 kit “for export,” as used in Canada and this study does not have an indeterminate cut-off value. The recently FDA approved (1998) MTD2 kit for use in the United States, recommends an indeterminate cut-off level of 500,000 RLUs. Our experience with the first MTD1 kit was that some results gave a low amplification of 30,000 to 300,000 RLUs, which were subsequently negative on repeat testing. On discussion wth Gen-Probe Inc., San Diego, in 1997, it was agreed that we would interpret results in the range of 30,000 to 300,000 RLUs as indeterminate.