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Previous Article | Next Article 
Journal of Clinical Microbiology, February 2001, p. 747-749, Vol. 39, No. 2
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.2.747-749.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Clinical Evaluation of the Gen-Probe Amplified
Mycobacterium Tuberculosis Direct Test for Rapid Detection of
Mycobacterium tuberculosis in Select Nonrespiratory
Specimens
Gail L.
Woods,*
John
S.
Bergmann, and
Natalie
Williams-Bouyer
Department of Pathology, University of Texas
Medical Branch, Galveston, Texas
Received 22 September 2000/Returned for modification 5 November
2000/Accepted 1 December 2000
 |
ABSTRACT |
The performance of the Amplified Mycobacterium Tuberculosis Direct
Test (MTD; Gen-Probe, Inc., San Diego, Calif.) for rapid diagnosis of
extrapulmonary tuberculosis was evaluated by testing 178 nonrespiratory
specimens from 158 patients. Criteria for specimen inclusion were (i) a
positive smear for acid-fast bacilli (n = 54) and (ii)
the source if the smear was negative (tissue biopsies and aspirates and
abscess material were tested; n = 124). Results were
compared to those of mycobacterial culture; clinical history was
reviewed when MTD and culture results disagreed. Forty-eight specimens
(27.0%) were positive for mycobacteria, including 23 Mycobacterium tuberculosis complex specimens; of which 21 were smear positive. Twenty-five specimens were MTD positive; 20 of these grew M. tuberculosis complex. All of the five
MTD-positive, M. tuberculosis complex culture-negative
specimens were considered truly positive, based on review of the
medical record. Of the three MTD-negative, M. tuberculosis
complex culture-positive specimens, two contained inhibitory
substances; one of the two was smear positive. Excluding the latter
specimen from analysis, after chart review, the sensitivity,
specificity, and positive and negative predictive values of the MTD
were 92.6, 100, 100, and 98.7%, respectively, by specimen and 89.5, 100, 100, and 98.6% by patient. Given the few smear-negative samples
from patients with extrapulmonary tuberculosis in our study, additional
similar studies that include more smear-negative, M. tuberculosis complex culture-positive specimens to confirm our
data are desirable.
| |
TEXT |
Tuberculosis remains a public health
problem in the United States, despite a continual decline in incidence
in each of the past several years. A key aspect of tuberculosis control
is rapid diagnosis, which for many years has been based on the staining of smears for acid-fast bacilli (AFB) and culturing for mycobacteria. AFB smear results should be available within 24 h (7), but the test lacks sensitivity and is not specific for tuberculosis (8). Mycobacterial culture and identification results,
which provide a specific diagnosis, often are not available for 2 to 3 weeks or longer. In response to the need for a more rapid diagnostic test, nucleic acid amplification tests specific for Myocbacterium tuberculosis complex have been developed. Currently in the United States, two such tests are commercially available for testing of
respiratory specimens: the Amplified Mycobacterium Tuberculosis Direct
Test (MTD; Gen-Probe, Inc., San Diego, Calif.), which is approved for
use regardless of the AFB smear result, and the AMPLICOR Mycobacterium tuberculosis Test (Roche Molecular Systems,
Branchburg, N.J.), which is approved for smear-positive specimens only.
Despite the fact that the nucleic acid amplification tests are approved
only for respiratory specimens, many investigators have used them to
test specimens from other sites (2, 3, 5, 6). Most such
studies, however, have included a mixture of specimen types, and in
many cases the number of any one type of specimen is insufficient for
test validation. In our institution, the rate of recovery of M. tuberculosis complex from nonrespiratory specimens is low.
Therefore, testing of all such specimens by nucleic acid amplification
would be cost prohibitive and would contribute little to patient
management. The purpose of our study, therefore, was to evaluate the
performance of the MTD assay with those nonrespiratory specimens that
in our institution are most likely to be culture positive for M. tuberculosis complex
i.e., those that are AFB smear positive and
tissue biopsies, fine-needle aspirates of tissues (predominantly lymph
nodes), and abscesses or wounds that are smear negative.
Clinical specimens.
One hundred seventy-eight nonrespiratory
specimens (from 158 patients) received in the laboratory for detection
of mycobacteria between November 1998 and May 2000 were selected for
evaluation based on two criteria: the smear result and, for AFB
smear-negative specimens, the source. All smear-positive nonrespiratory
specimens were tested; this included feces (n = 20);
tissue biopsies (n = 17) and aspirates (n = 8); wound, drainage, or abscess material (n = 8);
and pleural fluid (n = 1). Of the smear-negative
specimens, only tissue biopsies, fine-needle aspirates of tissues
(predominantly lymph nodes), and those labeled abscess, drainage, or
wound were tested (n = 124). All specimens were
processed within 24 h of receipt in the laboratory.
Specimen processing and culture.
Specimens that were
considered contaminated (i.e., feces and specimens labeled wound,
drainage, or abscess) were decontaminated with 1% (final
concentration) sodium hydroxide-N-acetylcysteine and concentrated by
centrifugation at 3,000 × g for 20 min in accordance
with standard procedures (4). Protocols to limit the
potential for cross-contamination during processing included the use of
single-use containers for phosphate buffer and digestant, removal and
replacement of the cap of each specimen tube sequentially during the
addition of specimens and reagents, and allowing tubes to stand for a
few minutes after agitation on a vortex mixer (9). Tissues
were ground with a disposable sterile tissue grinder, and liquid
specimens (i.e., pleural fluid and some aspirates) were concentrated
prior to smear preparation and culture. Smears were stained with
auramine O. For culture, 0.5 ml of the decontaminated or fresh specimen
was inoculated into an ESP II bottle (Trek Diagnostics, Westlake, Ohio)
or MGIT tube (BD Biosciences, Sparks, Md.) and 0.2 ml was inoculated
onto each side of a Middlebrook 7H11/7H11 selective biplate (BD
Biosciences). The remainder of the specimen was reserved for MTD
testing: one 500-µl aliquot was stored at 2 to 8°C and tested
within 72 h of processing; if the volume was sufficient, a second
500-µl aliquot was stored frozen at 
70°C for future analysis, if necessary.
ESP II bottles and MGIT tubes were incubated in the ESP II and BACTEC
960 instruments, respectively, where they were continuously monitored
for growth for 6 weeks. Plates were incubated at 37°C in 8%
CO2 and examined weekly for growth for 6 weeks: Isolates of
mycobacteria were identified by using DNA probes (AccuProbe [Gen-Probe, Inc.] for M. tuberculosis complex, M. avium complex; and M. kansasii) or conventional
biochemical tests (for rapidly growing mycobacteria) in accordance with
the standard protocol (4). Isolates not identified by
these procedures were referred to the Texas Department of Health for
identification by high-performance liquid chromatography and/or
conventional biochemical tests.
MTD.
MTD was performed in accordance with the manufacturer's
protocol for respiratory specimens as described in detail elsewhere (1). Interpretation of results was as follows: <30,000
relative light units (RLU), negative; 
500,000 RLU, positive; 30,000 to 499,999 RLU, equivocal. For samples yielding an equivocal result, a
second aliquot of a decontaminated or fresh specimen was tested; if the
second result was 30,000 RLU, the sample was considered positive,
whereas if it was <30,000 RLU, it was negative. MTD results were
acceptable when the negative control was <20,000 RLU and the positive
control was 500,000 RLU (>1,000,000 after September 1999, at which
time the manufacturer's directions changed). MTD results were not
reported because the test was not approved by the Food and Drug
Administration for this purpose.
Protocol for detection of inhibitory substances.
Specimens
that grew M. tuberculosis complex but were MTD negative were
analyzed for the presence of inhibitors or interfering substances as
follows. The specimen lysate was thawed, and 25 µl was added to an
amplification tube containing 50 µl of amplification reagent and 200 µl of oil. The tube was then seeded with 5 µl of the positive cell
control lysate, placed in a 95°C heat block for 15 min, and tested in
accordance with the manufacturer's protocol. Positive and negative
controls were processed and tested. Additionally, 5 µl of the
positive cell control lysate was added to a second, negative control
tube to serve as a positive control for the seeded lysates.
Interpretation of the results was as follows: 30,000 RLU, negative
for inhibitory substances that prevent amplification; <30,000 RLU,
substances that inhibit amplification present.
Resolution of discrepancies.
For specimens with discordant
M. tuberculosis complex culture and MTD results, the medical
record (including initial symptoms, Mantoux test result [if the test
was performed and the result was recorded], chest radiograph findings,
response to therapy, and admission and discharge diagnoses) was
reviewed, and laboratory records were examined to investigate the
possibility of cross-contamination. For MTD-negative specimens that
grew M. tuberculosis complex, the lysate was tested for
inhibitory substances.
Evaluation of the MTD.
Forty-eight (27.0%) of the 178 specimens were positive for mycobacteria, including 23 that were
M. tuberculosis complex positive. Twenty-one M. tuberculosis complex and all 25 nontuberculous mycobacterial strains (18 M. avium complex, 3 M. fortuitum-chelonae complex, 2 M. kansasii, and 2 M. simiae) were recovered from smear-positive specimens.
M. tuberculosis complex culture and MTD results are summarized in Table 1. On initial
testing, 24 specimens were MTD positive, 1 (a pleural fluid specimen)
was equivocal but yielded a positive result when retested, and the
remainder were negative. Twenty of the 25 MTD-positive specimens were
M. tuberculosis complex culture positive. Of the five
specimens that were MTD positive and M. tuberculosis complex
culture negative, four (two smear positive and two smear negative) were
from patients who had been previously diagnosed with tuberculosis and
were receiving appropriate therapy. The fifth specimen (smear-positive
feces) grew M. avium complex; however, 42 days after that
specimen had been collected, a sputum specimen from the patient grew
M. tuberculosis complex. Therefore, the MTD result for each
of these five specimens was considered truly positive. Two (one smear
positive and one smear negative) of the three specimens that were
M. tuberculosis complex culture positive and MTD negative
contained inhibitory substances.
View this table:
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|
TABLE 1.
Summary of M. tuberculosis complex culture and
MTD results for smear-positive and smear-negative specimens
|
|
After chart review, the sensitivity, specificity, and positive and
negative predictive values of the MTD were 89.3, 100, 100, and 98.0%,
respectively, by specimen and 85, 100, 100, and 97.9% by patient.
Based on our usual laboratory practice for respiratory specimens of
testing all smear-positive, MTD-negative specimens for inhibitors and
reporting MTD results for those with a positive inhibitory assay as
uninterpretable, deleting the smear-positive specimen with inhibitors
from analysis is reasonable. Excluding that specimen, the sensitivity,
specificity, and positive and negative predictive values of the MTD
were 92.6, 100, 100, and 98.7%, respectively, by specimen and 89.5, 100, 100, and 98.6% by patient.
Rapid diagnosis is critical to the control of tuberculosis, especially
pulmonary disease. Rapid diagnosis of extrapulmonary tuberculosis has a
greater impact on patient management than on limiting spread of the
disease. It allows prompt initiation of appropriate therapy (and
discontinuation of potentially toxic, unnecessary drugs) and may
obviate the need for additional invasive diagnostic tests and decrease
hospital stays. For many years the AFB smear was the only rapid
diagnostic test available. Because the smear lacks sensitivity and
specificity, a laboratory test with superior performance
characteristics but a similar turnaround time is needed. Theoretically,
nucleic acid amplification tests can fulfill these requirements;
however, those currently available are approved for respiratory
specimens only.
Studies evaluating the performance of the current version of the MTD
with nonrespiratory specimens have included a mixture of specimen
types, in various proportions. Sensitivity has ranged from 79 to 100%,
and specificity has ranged from 99 to 100% (2, 3, 5, 6).
However, because the performance of the MTD may not be the same for all
types of specimens, comparison of the results of studies in which the
proportion of different specimens varies considerably may not be
appropriate. Our study design and results are most similar to those of
Chedore and Jamieson, who reported a sensitivity and specificity of
100% for smear-positive specimens and 100% and 98.9% for
smear-negative specimens (2). They included all AFB
smear-positive specimens, as did we, and any smear-negative specimen
when the physician specifically requested the test based on a high
clinical suspicion of tuberculosis. Our goals were to optimize patient
management and achieve cost effectiveness; therefore, we tested those
nonrespiratory specimens that in our experience were most likely to
yield M. tuberculosis complex. Additionally, testing of a
reasonable number of specimens with similar characteristics allows test
validation. To our knowledge, a universally accepted number deemed
appropriate for validation does not exist. However, we believe that our
study included an adequate number of tissue biopsies, fine-needle
aspirates, and smear-positive specimens (assuming that it is reasonable
to consider smear result a specimen characteristic).
Three specimens in our study had false-negative MTD results. Two of
these contained inhibitors, which explains the negative results. The
third specimen was included in a run (performed prior to September
1999) in which the result of the positive control was 690,400 RLU. This
was in the acceptable range but was much lower than the typical
positive control results in our laboratory, i.e., 3,000,000 RLU. The
kit reagents used on the day when the low acceptable positive control
value was obtained were on day 30 of their 30-day expiration period.
Therefore, fresh reagents from a new kit were prepared and the specimen
with the false-negative result was retested and tested for inhibitors.
The repeat result was positive, and no inhibitors were detected; the
positive control for the run was >3,000,000 RLU. Based on this
observation, we modified our procedure for respiratory specimens such
that if the positive control result were considerably less than the
usual value, the patient results would not be reported and the run
would be repeated. Then, in September 1999, when the MTD was approved for testing regardless of the AFB smear result, the manufacturer changed the cutoff for the positive control to >1,000,000. This modification likely will decrease the possibility of false-negative patient results.
In conclusion, the performance of the MTD with nonrespiratory
smear-positive specimens and smear-negative tissue biopsies, fine-needle aspirates, and abscess material was very good. Based on our
results, we currently perform the MTD on any smear-positive nonrespiratory specimen if it is specifically requested by the physician and we test those with negative MTD results for inhibitors. We also test smear-negative tissue biopsies and aspirates with the
approval of an infectious disease or pulmonary specialist. Testing of
all smear-negative, MTD-negative specimens for inhibitors is cost
prohibitive; therefore, in our institution it requires a specific
request by an infectious disease or pulmonary specialist and approval
of the microbiology laboratory director. Although performance of the
MTD was excellent with our smear-negative tissue biopsies and aspirates
and abscess material, only 4 (3.2%) of the 124 smear-negative
specimens tested were from patients with tuberculosis. Therefore,
additional, similar studies that include more smear-negative, M. tuberculosis complex culture-positive specimens should be
conducted to confirm our findings.
| |
ACKNOWLEDGMENTS |
MTD kits were kindly provided by Gen-Probe, Inc. We acknowledge the
technical expertise of Lisa Hinson, Geoff Fish, Alice Gatson, and Cindy Tholcken.
G.L.W. is supported in part by a Tuberculosis Academic Award from the
National Heart, Lung, and Blood Institute (K07 HL03335).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Pathology, University of Texas Medical Branch, Galveston, TX
77555-0740. Phone: (409) 772-4851. Fax: (409) 772-5683. E-mail:
gwoods{at}utmb.edu.
| |
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Journal of Clinical Microbiology, February 2001, p. 747-749, Vol. 39, No. 2
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.2.747-749.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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