Updated Guidelines for the Use of Nucleic Acid Amplification Tests in
the Diagnosis of Tuberculosis
Guidelines for the use of nucleic acid amplification (NAA) tests for the diagnosis of tuberculosis (TB) were published
in 1996 (1) and updated in 2000 (2). Since then, NAA testing has become a routine procedure in many settings because
NAA tests can reliably detect Mycobacterium
tuberculosis bacteria in specimens 1 or more weeks earlier than culture
(3). Earlier laboratory confirmation of TB can lead to earlier treatment initiation, improved patient outcomes, increased opportunities
to
interrupt transmission, and more effective public health interventions
(4,5). Because of the increasing use of NAA tests
and the potential impact on patient care and public health, in June 2008, CDC and the Association of Public Health
Laboratories (APHL) convened a panel of clinicians, laboratorians, and TB control officials to assess existing guidelines
(1,2) and make recommendations for using NAA tests for laboratory confirmation of TB. On the basis of the panel's report and
consultations with the Advisory Council for the Elimination of TB (ACET),* CDC recommends that NAA testing be performed on at
least one respiratory specimen from each patient with signs and symptoms of pulmonary TB for whom a diagnosis of TB is
being considered but has not yet been established, and for whom the test result would alter case management or TB
control activities, such as contact investigations. These guidelines update the previously published guidelines
(1,2).
Background
Conventional tests for laboratory confirmation of TB include acid-fast bacilli (AFB) smear microscopy, which can
produce results in 24 hours, and culture, which requires 2--6 weeks to produce results
(5,6). Although rapid and inexpensive, AFB smear microscopy is limited by its poor sensitivity (45%--80% with culture-confirmed pulmonary TB cases) and its
poor positive predictive value (50%--80%) for TB in settings in which nontuberculous mycobacteria are commonly
isolated (3,6,7).
NAA tests can provide results within 24--48 hours. The Amplified
Mycobacterium tuberculosis Direct Test (MTD,
Gen-Probe, San Diego, California) was approved by the Food and Drug Administration (FDA) in 1995 for use with AFB
smear-positive respiratory specimens, and in a supplement application, an enhanced MTD test was approved in 1999 for use
with AFB smear-negative respiratory specimens from patients suspected to have TB. In addition, the Amplicor
Mycobacterium tuberculosis Test (Amplicor, Roche Diagnostics, Basel, Switzerland) was approved by FDA in 1996 for use with AFB
smear-positive respiratory specimens from patients suspected to have TB. NAA tests for TB that have not been FDA-approved
also have been used clinically (e.g., NAA tests based on analyte specific reagents, often called
"home-brew" or "in-house"
tests) (8,9).
Compared with AFB smear microscopy, the added value of NAA testing lies in its 1) greater positive predictive
value (>95%) with AFB smear-positive specimens in settings in which nontuberculous mycobacteria are common and 2) ability
to confirm rapidly the presence of M.
tuberculosis in 50%--80% of AFB smear-negative, culture-positive specimens
(3,7--9). Compared with culture, NAA tests can detect the presence of
M. tuberculosis bacteria in a specimen weeks earlier than
culture for 80%--90% of patients suspected to have pulmonary TB whose TB is ultimately confirmed by culture
(3,8,9). These advantages can impact patient care and TB control efforts, such as by avoiding unnecessary contact investigations
or respiratory isolation for patients whose AFB smear-positive specimens do not contain
M. tuberculosis.
Despite being commercially available for more than a decade
(1), NAA tests for TB have not been widely used in the
United States largely because of 1) an uncertainty as to whether NAA test results influence case-management decisions or TB
control activities; 2) a lack of information on the overall cost-effectiveness of NAA testing for TB; and 3) a lack of demand
from clinicians and public health authorities. However, recent studies showed that 1) clinicians already rely on the NAA test
result as the deciding factor for the initiation of therapy for 20%--50% of TB cases in settings where NAA testing is a
routine practice (4,7) and 2) overall cost savings can be achieved by using NAA test results for prioritizing contact
investigations, making decisions regarding respiratory isolation, or reducing nonindicated TB treatment
(4,7).
In response to the increasing demand for NAA testing for TB and recognition of the importance of prompt
laboratory results in TB diagnosis and control, ACET requested that APHL and CDC convene a panel to evaluate the
available information (e.g., current practices, existing guidelines, and publications) and to propose new guidelines for the use of
NAA tests for TB diagnosis. The panel met in June 2008 and included TB clinicians; TB control officials; laboratory directors
or supervisors from small, medium, and large public health laboratories, hospital laboratories, and commercial laboratories;
and representatives from the TB Regional Training and Medical Consultation Centers, ACET, APHL, and CDC. In brief,
the panel recommended† that NAA testing become a standard practice in the United States to aid in the initial diagnosis
of patients suspected to have TB, rather than just being a reasonable approach, as suggested in previously published
guidelines (1,2). On the basis of the panel's report and consultations with ACET, CDC developed revised guidelines.
Updated Recommendation
NAA testing should be performed on at least one respiratory specimen from each patient with signs and symptoms
of pulmonary TB for whom a diagnosis of TB is being considered but has not yet been established, and for whom the test
result would alter case management or TB control activities. The following testing and interpretation algorithm is proposed.
Revised Testing and Interpretation Algorithm
Routinely collect respiratory specimens (e.g., sputum), process (liquefy, decontaminate, and concentrate), and test by
AFB smear microscopy and culture as previously recommended
(6). Specimen collection and microbiologic testing should
not be delayed to await NAA test results.
At least one specimen, preferably the first diagnostic specimen, from each patient to be tested by NAA should
be processed, suspended in a sufficient volume of buffer to ensure adequate sample volume for all planned tests
(e.g., microscopy, culture, and NAA), and tested using an NAA test for TB. NAA testing should be performed in
accordance with the manufacturer's instructions or a validated standard operating procedure.
Interpret NAA test results in correlation with the AFB smear results.
If the NAA result is positive and the AFB smear result is
positive, presume the patient has TB and begin anti-TB treatment
while awaiting culture results. The positive predictive value of FDA-approved NAA tests for TB is >95%
in AFB smear-positive cases (8).
If the NAA result is positive and the AFB smear result is negative, use clinical judgment whether to begin
anti-TB treatment while awaiting culture results and determine if additional diagnostic testing is needed. Consider testing
an additional specimen using NAA to confirm the NAA result. A patient can be presumed to have TB, pending
culture results, if two or more specimens are NAA positive.
If the NAA result is negative and the AFB smear result is positive, a test for inhibitors should be performed and
an additional specimen should be tested with NAA. Sputum specimens (3%--7%) might contain inhibitors that prevent
or reduce amplification and cause false-negative NAA results
(8,9).
i. If inhibitors are detected, the NAA test is of no diagnostic help for this specimen. Use clinical judgment to
determine whether to begin anti-TB treatment while awaiting results of culture and additional diagnostic testing.
ii. If inhibitors are not detected, use clinical judgment to determine whether to begin anti-TB treatment while
awaiting culture results and determine if additional diagnostic testing is needed. A patient can be presumed to have an
infection with nontuberculous mycobacteria if a second specimen is smear positive and NAA negative and has no
inhibitors detected.
If the NAA result is negative and the AFB smear result is negative, use clinical judgment to determine whether to
begin anti-TB treatment while awaiting results of culture and additional diagnostic tests. Currently available NAA tests are
not sufficiently sensitive (detecting 50%--80% of AFB smear-negative, culture-positive pulmonary TB cases) to exclude
the diagnosis of TB in AFB smear-negative patients suspected to have TB
(8,9).
Cautions
Culture remains the gold standard for laboratory confirmation of TB and is required for isolating bacteria for
drug-susceptibility testing and genotyping. In accordance with current recommendations
(6), sufficient numbers and portions of specimens should always be reserved for culturing. Nonetheless, NAA testing should become standard practice for
patients suspected to have TB, and all clinicians and public health TB programs should have access to NAA testing for TB to
shorten the time needed to diagnose TB from 1--2 weeks to 1--2 days
(3). More rapid laboratory results should lead to
earlier treatment initiation, improved patient outcomes, and increased opportunities to interrupt transmission
(4,5). Rapid laboratory confirmation of TB also can help reduce inappropriate use of fluoroquinolones as empiric monotherapy
of pneumonias, a practice which is suspected to lead to development of fluoroquinolone-resistant
M. tuberculosis and delays in initiating appropriate anti-TB therapy
(10).
To maximize benefits of NAA testing, the interval from specimen collection to communication of the laboratory report
to the treating clinician should be as brief as possible. NAA test results should be available within 48 hours of
specimen collection. Laboratorians should treat an initial positive NAA test result as a critical test value, immediately report the result
to
the clinician and public health authorities, and be available for consultation regarding test interpretation and the possible
need for additional testing.
Although NAA testing is recommended to aid in the initial diagnosis of persons suspected to have TB, the
currently available NAA tests should not be ordered routinely when the clinical suspicion of TB is low, because the positive
predictive value of the NAA test is <50% for such cases
(8). Clinicians, laboratorians, and TB control officials should be aware of
the appropriate uses of NAA tests.
Clinicians should interpret all laboratory results on the basis of the clinical situation. A single negative NAA test
result should not be used as a definitive result to exclude TB, especially when the clinical suspicion of TB is moderate to
high. Rather, the negative NAA test result should be used as additional information in making clinical decisions, to expedite
testing for an alternative diagnosis, or to prevent unnecessary TB treatment. Consultation with a TB expert should be considered
if the clinician is not experienced in the interpretation of NAA tests or the diagnosis and treatment of TB.
Although FDA-approved NAA tests for TB are eligible for Medicare or Medicaid reimbursement, the costs of adding
NAA testing to the routine testing of respiratory specimens from patients suspected to have TB might be considerable
(e.g., operating costs exceed $100 per MTD test)
(8). However, NAA testing has the potential to provide overall cost savings to
the treatment center and TB control program through reduced costs for isolation, reduced costs of contact investigations
of persons who do not have TB, and increased opportunities to prevent transmission. Within the parameters of these
guidelines, each TB control or treatment program should evaluate the overall costs and benefits of NAA testing in deciding the value
and optimal use of the test in their setting.
Because the testing algorithm includes NAA testing of AFB smear-negative specimens, laboratories must use an
FDA-approved test for such specimens or a test produced and validated in accordance with applicable FDA and Clinical
Laboratory Improvement Amendments (CLIA)
regulations.§ However, the performance of in-house tests or FDA-approved tests used
for nonapproved indications (off-label use) is variable
(8,9), and insufficient information is available to provide
recommendations on the use of such tests for the diagnosis of TB. Their use should be guided by the clinical context, and the results of such
tests should be interpreted on the basis of performance in the local laboratory and in validation studies.
For procedural and economic reasons, NAA testing might be impractical in laboratories with a small volume of
testing. Referral of samples for NAA testing to high-volume laboratories might be preferable to improve cost-efficiency,
proficiency, and turnaround times. The New York and Florida Fast Track Programs are successful NAA testing services that could serve
as models for a regional service (5).
Information is limited regarding NAA test performance for nonrespiratory specimens or specimens from patients
under treatment (8). NAA results often remain positive after culture results become negative during therapy. Further research
is needed before specific recommendations can be made on the use of NAA testing in the diagnosis of extrapulmonary TB
and TB in children who cannot produce sputum; however, evidence exists for the utility of such testing in individual cases
(8).
These guidelines do not address the use of molecular tests for detecting drug resistance, which is an urgent public health
and diagnostic need. No molecular drug-susceptibility tests (DSTs) have been approved by FDA for use in the United
States, although well-characterized molecular DSTs are commercially available in Europe and
elsewhere.¶ Nonetheless, a
proposed revision of the Diagnostic Standards and Classification of Tuberculosis in Adults and Children
(6) is likely to support the use of molecular DSTs for AFB smear-positive sputum sediments from TB patients who are suspected to have
drug-resistant disease or who are from a region or population with a high prevalence of drug resistance.
Reported by: Div of Tuberculosis Elimination, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, CDC.
Acknowledgments
This report is based on contributions of an ad hoc panel of external consultants convened by CDC and the Assoc of Public
Health Laboratories (D Alland, MD, New Jersey Medical School; J Bernardo, MD, Boston Univ School of Medicine, Massachusetts; B
Hanna, PhD, New York Univ School of Medicine; RL Kaplan, PhD, Quest Diagnostics, Madison, New Jersey; M Kawamura, MD, TB
Control Section, San Francisco Dept of Public Health; S Liska, DrPH, San Francisco Public Health Laboratory; C Nivens, Missouri
State Tuberculosis Laboratory; M Salfinger, MD, Bur of Laboratories, Florida Dept of Health; B Seaworth, MD, Heartland National
TB Center, San Antonio, Texas; D Warshauer, PhD, Wisconsin State Laboratory of Hygiene; and KE Wroblewski, MPH, Assoc of
Public Health Laboratories) and K Castro, MD, L Diem, J Jereb, MD, P Lobue, MD, S Marks, MPH, J Mazurek, MD, B Metchock,
DrPH, T Shinnick, PhD, and A Vernon, MD, Div of Tuberculosis Elimination, National Center for HIV/AIDS, Viral Hepatitis, STD, and
TB Prevention, CDC.
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