46 • CID 2009:49 (1 July) • Laraque et al.
M A J O R A R T I C L E
Performance of Nucleic Acid Amplification Tests
for Diagnosis of Tuberculosis in a Large Urban Setting
Fabienne Laraque,1,aAnne Griggs,2,aMeredith Slopen,1,aand Sonal S. Munsiff3,a
1Bureau of Tuberculosis Control, New York City Department of Health of Mental Hygiene, New York, New York; and
Service Program, Office of Workforce and Career Development, and
and Prevention, Atlanta, Georgia
2Public Health Prevention
3Division of Tuberculosis Elimination, Centers for Disease Control
(See the editorial commentary by Dorman on pages 55–7)
A diagnosis of tuberculosis (TB) relies on acid-fast bacilli (AFB) smear and culture results. Two
rapid tests that use nucleic acid amplification (NAA) have been approved by the US Food and Drug Administration
for the diagnosis of TB based on detection of Mycobacterium tuberculosis from specimens obtained from the
respiratory tract. We evaluated the performance of NAA testing under field conditions in a large urban setting
with moderate TB prevalence.
The medical records of patients with suspected TB during 2000–2004 were reviewed. Analysis was
restricted to the performance of NAA on specimens collected within 7 days after the initiation of treatment for
TB. The assay’s sensitivity, specificity, and positive and negative predictive values (PPV and NPV, respectively)
The proportion of patients with confirmed or suspected TB whose respiratory tract specimens were
tested by use of NAA increased from 429 (12.9%) of 3334 patients in 2000 to 527 (15.6%) of 3386 patients in
2004; NAA testing among patients whose respiratory tract specimens tested positive for AFB increased from 415
(43.6%) of 952 patients in 2000 to 487 (55.5%) of 877 patients in 2004 (
patients being evaluated for pulmonary TB, 4642 (28.1%) had specimens that tested positive for AFB on smear.
Of those 4642 patients, 2241 (48.3%) had NAA performed on their specimens. Of those 2241 patients, 1279
(57.1%) had positive test results. Of those 1279 patients, 1262 (98.7%) were confirmed to have TB. For 1861
(40.1%) of the 4642 patients whose specimens tested positive for AFB on smear, the NAA test had a sensitivity
of 96.0%, a specificity of 95.3%, a PPV of 98.0%, and an NPV of 90.9%. For 158 patients whose specimens tested
negative for AFB on smear, the NAA test had a sensitivity of 79.3%, a specificity of 80.3%, a PPV of 83.1%, and
an NPV of 76.0%, respectively. For the 215 specimens that tested positive for AFB by smear, we found a sensitivity,
specificity, PPV, and NPV of 97.5%, 93.6%, 95.1%, and 96.8%, respectively. A high-grade smear was associated
with a better test performance.
NAA testing was helpful for determining whether patients whose specimens tested positive for
AFB on smear had TB or not. This conclusion supports the use of this test for early diagnosis of pulmonary and
for both trends). Of the 16,511P ! .001
In the mid-1990s, the US Food and Drug Administra-
tion approved 2 rapid diagnostic tests—the Amplified
Received 26 November 2008; accepted 7 February 2009; electronically published
28 May 2009.
aPresent affiliation: Bureau of HIV Prevention and Control, New York City
Department of Health of Mental Hygiene, New York, NY (F.L., M.S., and S.S.M.);
and Malaria Consortium, Maputo, Mozambique (A.G.).
Reprints or correspondence: Dr. Fabienne Laraque, Office of Care, Treatment,
and Housing, Bureau of HIV Prevention and Control, New York City Dept. of Health
of Mental Hygiene, 40 Worth St., Rm. 1502, CN A-1, New York, NY 10013
Clinical Infectious Diseases2009;49:46–54
This article is in the public domain, and no copyright is claimed.
Mycobacterium tuberculosis Direct (MTD) test (Gen-
Probe) and the Amplicor M. tuberculosis test (Roche
Diagnostics)—that use nucleic acid amplification
(NAA) for the diagnosis of tuberculosis(TB)inpatients
whose sputum specimens tested positive for acid-fast
bacilli (AFB) on smear . In 1999, the US Food and
Drug Administration approved the MTD test for use
on specimens obtained from the respiratory tract that
tested negative for AFB on smear . The American
Thoracic Society deems that NAA tests are a significant
addition to the diagnostic tools available for the de-
tection of TB, particularly in patients with possible pul-
monary TB (i.e., patients whose sputum specimens
by guest on November 11, 2015
Performance of NAA Tests for Diagnosis of TB • CID 2009:49 (1 July) • 47
tested positive for AFB on smear) . The evaluation of rapid
diagnostic tests for TB is particularly critical in places like New
York City, because the city has a relatively high prevalence of
TB and is home to large immigrant populations, and as aresult,
decisions need to be made promptly with regard to the treat-
ment and/or respiratory isolation of patients with TB.
In small- to moderate-size studies [4–18], the use of NAA
testing for the rapid diagnosis of the M. tuberculosis complex
has been evaluated using specimens obtained from the respi-
ratory tract and from other body sites. For respiratory tract
specimens that tested positive for AFB on smear, some authors
have reported test sensitivities of 73%–100%, specificities of
96%–100%, positive predictive values (PPVs) of 86%–100%,
and negative predictive values (NPVs) of 199% [4–8, 10–13,
16, 17, 19, 20]. Although NAA testing had a lower sensitivity
for respiratory tract specimens that tested negative for AFB on
smear, compared with respiratory tract specimens that tested
positive for AFB on smear, the sensitivity was higher than that
reported using AFB microscopy and was close to that reported
using mycobacterial culture [4, 5, 10, 16, 19, 20].
In addition, several studies have shown the benefits of NAA
for testing specimens obtained from body sites other than the
respiratory tract. Evaluations of the NAA test for use on tissue
specimens (pleural, lung, lymph node, liver biopsies, and bone
marrow) and body fluids (central nervous system, pleural, as-
citic, synovial, pericardial, and peritoneal fluids) showed a sen-
sitivity of 64%–86%, a specificity of 97–100%, a PPV of 81.5%–
100%, and an NPV of 87.5–99.2% [5, 10–12, 14, 15, 18]. One
study found a lower sensitivity (52.6%) among pleural biopsy
The present study retrospectively evaluates the performance
of NAA tests among patients evaluated for TB in a large urban
setting by several mycobacteriology laboratories in a real-life
situation, using information entered in the New York City De-
partment of Health and Mental Hygiene TB registry. The ob-
jectives of our study were (1) to describe the characteristics of
patients whose specimens were tested by use of NAA and the
factors associated with the decision to perform NAA testing;
(2) to calculate the sensitivity, specificity, PPV, and NPV of
NAA tests; and (3) to examine the relationship between the
NAA test result, the AFB smear resultandgrade,andtheculture
result, per patient, for detection of the M. tuberculosis complex.
ated for TB and reported to the New York City Department
of Health and Mental Hygiene’sBureauofTuberculosisControl
during the period from 2000 through 2004. A diagnosis of TB
was confirmed using the Centers for Disease Control and Pre-
vention’s clinical or laboratory case definition .
Our analysis relied on routine surveillancedatafrom
The study population consisted of patients evalu-
the Bureau of Tuberculosis Control’s TB registry, which in-
cluded sociodemographic, clinical, and laboratory data. The
type of NAA test performed is not recorded in the TB registry.
Most NAA testing in New York City is performed with the
MTD test; the city and state public health laboratories use the
MTD test (37% and 21% of the time, respectively) and per-
formed the majority of the tests that occurred during the study
period; an additional 10% of the testing was done at several
private laboratories, all of which used the MTD test. The rest
of the testing was performed either in out-of-city laboratories
or in laboratories that were not recorded in the registry. All
New York State laboratories follow the New York State De-
partment of Health Mycobacteriology Standards for AFBsmear
and culture . NAA testing was assumed to have been per-
formed according to the manufacturer’s recommendation .
The analysis was restricted to NAA tests performed on speci-
mens collected within 7 days after the start of treatment for
TB. New York City laboratories are required to report all pos-
itive test results identifying AFB or the M. tuberculosis complex
and all negative test results associated with positive test results,
such as a negative NAA test result on a specimen that tested
positive for AFB on smear. In New York City, smears that are
interpreted as inconclusive are considered to be positive for
AFB, for the treatment of patients, and thus they were grouped
with smears interpreted as positive for AFB in our analysis of
Data analysis wasstratifiedonthebasis
of specimen source (i.e., sources from the respiratory tract vs.
sources from other body sites). The sources of specimens from
the respiratory tract included sputum, trachea, bronchus and/
or bronchioles, bronchial fluid, and lung tissue. The sources of
specimens from other body sites included cerebrospinal fluid,
lymphatic tissue, gastric aspirate, and pleural and peritoneal
fluid, of which at least 30 specimens were available for analysis
of test performance.
For cases of pulmonary TB, we analyzed the performance
of the NAA test by patient characteristics overall and then by
stratifying patients into smear-positive and smear-negative pa-
tients. Categorical data were compared by use of the x2test.
To determine which factors predicted the use of NAA testing
for patients with suspected pulmonary TB, multivariate logistic
regression models were constructed. The analysis was per-
formed by use of SAS, version 9 (SAS), and SPSS, version 14.0
The sensitivities, specificities, PPVs, and NPVs were calcu-
lated for the NAA tests performed for patients who had spec-
imens obtained from the respiratory tract, with the denomi-
nator being the number of patients, regardless of how many
specimens were obtained for testing. The performance of NAA
was evaluated by comparing NAA test results to 2 gold stan-
dards of TB diagnosis: (1) a culture positive for M. tuberculosis,
by guest on November 11, 2015
54 • CID 2009:49 (1 July) • Laraque et al.
congregate housing facility with a high prevalence of the human
immunodeficiency virus, caution should be exercised.
4. A negative NAA test result for a patient whose specimen
tested negative for AFB on smear and for whom the clinical
suspicion for TB is low indicates a low likelihood of active TB
and suggests that treatment for TB can be delayed.
5. A negative NAA test result for a patient whose sputum
specimen tested positive for AFB on smear can be used to
discontinue an investigation of exposed contacts for possible
TB transmission and to discontinue airborne isolation.
In summary, the findings from our study on the performance
of NAA tests for diagnosis of TB in this large urban setting
identify patients with pulmonary tuberculosis. The sensitivity,
specificity, and predictive values of the test in a real-life situation
were high for patients who had a specimen that tested positive
for AFB on smear, and they were at acceptable levels for patients
who had a specimen that tested negative for AFB on smear. To
improve the use of rapid testing for TB, in May 2006, the New
York State Department of Health updated its mycobacteriology
guidelines to mandate NAA testing on all specimensthatinitially
tested positive for AFB on smear . The Centers for Disease
Control and Prevention updated its guidelines to recommend
NAA testing on specimens obtained from the respiratory tract
of patients suspected of pulmonary TB .
We thank Ms. Shama Ahuja for reviewing the manuscript andMs.Yelena
Shuster for assistance in preparing the database.
Potential conflicts of interest.
All authors: no conflicts
1. Centers for Disease Control and Prevention (CDC). Nucleic acid am-
plification tests for tuberculosis. MMWR Morb Mortal Wkly Rep
2. Centers for Disease Control and Prevention (CDC). Update: nucleic
acid amplification tests for tuberculosis. MMWR Morb Mortal Wkly
3. American Thoracic Society. Rapid diagnostic tests for tuberculosis—
what is the appropriate use? Am J Respir Crit Care Med 1997;155:
4. Bradley SP, Reed SL, Catanzaro A. Clinical efficacy of the amplified
Mycobacterium tuberculosis direct test for the diagnosis of pulmonary
tuberculosis. Am J Respir Crit Care Med 1996;153:1606–10.
5. Coll P, Garrigo ´ M, Moreno C, Martı ´ N. Routine use of Gen-Probe
amplified Mycobacterium tuberculosis (MTD) direct test for detection
of Mycobacterium tuberculosis with smear-positive and smear-negative
specimens. Int J Tuberc Lung Dis 2003;7:886–91.
6. Conaty SJ, Claxton AP, Enoch DA, Hayward AC, LipmanMC,Gillespie
SH. The interpretation of nucleic acid amplification tests for tuber-
culosis: do rapid tests change treatment decisions? J Infect 2005;50:
7. Dalovisio JR, Montenegro-James S, Kemmerly SA, et al. Comparison
of the amplified Mycobacterium tuberculosis (MTD) direct test, Am-
plicor MTB PCR, and IS6110-PCR for detection of MTB in respiratory
specimens. Clin Infect Dis 1996;23:1099–106.
8. Gallina M, Troupioti P, Rocco G, Sensalari G, Libanori E. Predicting
culture results for Mycobacterium tuberculosis complex: amplified My-
cobacterium tuberculosis direct test and acid-fast bacilli microscopy.
9. Lim TK, Gough A, Chin N, Kumarasinghe G. Relation between esti-
mated pretest probability and accuracy of automated Mycobacterium
tuberculosis assay in smear-negative pulmonary tuberculosis. Chest
10. Maugein J, Fourche J, Vacher S, Grimond C, Bebear C. Evaluation of
the BDProbeTec ET DTB assay for direct detection of Mycobacterium
tuberculosis complex from clinical specimens. Diagn Microbiol Infect
11. Moore DF, Curry JI. Detection and identification of Mycobacterium
tuberculosis directly from sputum sediments by Amplicor PCR. J Clin
12. Pfyffer GE, Kissling, P, Jahn EM, Welscher HM, Salfinger M, Weber
R. Diagnostic performance of amplified Mycobacterium tuberculosis di-
rect test with cerebrospinal fluid,othernon-respiratory,andrespiratory
specimens. J Clin Microbiol 1996;34:834–41.
13. Pfyffer GE, Kissling P, Wirth R, Weber R. Direct detection of Myco-
bacterium tuberculosis complex in respiratory specimens by a target-
amplified test system. J Clin Microbiol 1994;32:918–23.
14. Ruiz-Manzano J, Manterola JM, Gamboa F, et al. Detection of My-
cobacterium tuberculosis in paraffin-embedded pleural biopsy speci-
mens by commercial ribosomal RNA and DNA amplification kits.
15. Shah S, Miller A, Mastellone A, et al. Rapid diagnosis of tuberculosis
in various biopsy and body fluid specimens by the Amplicor Myco-
bacterium tuberculosis polymerase chain reaction test. Chest 1998;113:
16. Smith MB, Bergmann JS, Harris SL, Woods, GL. Evaluation of the
Roche Amplicor MTB assay for the detection of Mycobacterium tu-
berculosis in sputum specimens from prison inmates. Diagn Microbiol
Infect Dis 1997;27:113–6.
17. Tan MF, Ng WC, Chan SH, Tan WC. Comparative usefulness of PCR
in the detection of Mycobacterium tuberculosis in different clinicalspec-
imens. J Med Microbiol 1997;46:164–9.
18. Wiener RS, Della-Latta P, Schluger NW. Effect of nucleic acid ampli-
fication for Mycobacterium tuberculosis on clinical decision making in
suspected extrapulmonary tuberculosis. Chest 2005;128:102–21.
19. Moore DF, Guzman JA, Mikhail LT. Reduction in turnaround time for
laboratory diagnosis of pulmonary tuberculosis by routine use of a
nucleic acid amplification test. Diagn Microbiol Infect Dis 2005;52:
20. Campos M, Quartin A, Mendes E, et al. Feasibility of shortening re-
spiratory isolation with a single sputum nucleic acid amplification test.
Am J Respir Crit Care Med 2008;178:300–5.
21. Centers for Disease Control and Prevention (CDC). Case definitions
for infectious conditions under public health surveillance. MMWR
Morb Mortal Wkly Rep 1997;46(RR10):1–55.
22. New York State Department of Health. Clinical laboratory standards
of practice. January 2008. Available at: http://www.wadsworth.org/
labcert/clep/files/Microbiology.pdf. Accessed 27 April 2009.
23. Amplied MTD Test (amplified Mycobacterium tuberculosis direct test
for in vitro diagnostic use) [package insert].SanDiego,CA:Gen-Probe,
24. Lim TK, Zhu D, Gough A, Lee KH, Kumarasinghe G. What is the
optimal approach for using a direct amplification test in the routine
diagnosis of pulmonary tuberculosis? A preliminary assessment. Res-
25. Centers for Disease ControlandPrevention(CDC).Updatedguidelines
for the use of nucleic acid amplification tests in the diagnosis of tu-
berculosis. MMWR Morb Mortal Wkly Rep 2009;58:7–10.
by guest on November 11, 2015