Diagnostics For Active Tuberculosis
Nucleic Acid Amplification (NAA) Tests
Nucleic Acid Amplification (NAA) tests amplify target nucleic acid regions that uniquely identify the M. tuberculosis complex. Because NAA tests can be directly used on clinical specimens (such as sputum), they are also called "direct amplification tests." NAA tests are categorized as commercial kits or in-house assays. Commercial kits include the Amplicor MTB tests (Roche Diagnostic Systems, Inc., NJ, USA), the Amplified M.
tuberculosis
Direct Test (MTD; Gen-Probe, Inc., San Diego, CA), and the BD ProbeTec ET assay
(Becton Dickinson Biosciences, Sparks, MD). The Amplicor and MTD tests are PDA
approved. In-house tests are laboratory-developed Polymerase Chain Reaction
(PCR) assays; they vary greatly in their design and laboratory methods.
In house NAA tests tend to be used predominantly in research settings and in developing countries where commercial NAA tests are expensive.4 At present NAA tests are most useful for the rapid confirmation of tuberculosis in persons with AFB-positive sputa. They may also have utility for the diagnosis of AFB-negative pulmonary and
extrapulmonary tuberculosis in selected patients. However, their applicability
is limited by low sensitivity and high cost.
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Amplicor MTB Tests & Amplified Mycoba-cterium Tuberculosis Direct Test (MTD)
The Amplicor is a DNA-based test that amplifies the 16S rRNA gene using genus-specific primers with detection in a colorimetric reaction. The MTD is based on amplification of 16S ribosomal transcripts, which are detected with a DNA probe. Both methods were PDA-approved for the direct detection of M. tuberculosis in smear-positive respiratory specimens. The enhanced MTD was recently
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approved also for smear-negative respiratory specimens of suspect patients.
Compared to culture and the clinical status, sensitivity and specificity for
smear-positive specimens have been higher than for smear-negative specimens,
precluding their use to rule out the disease.
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BD ProbeTec ET Assay
BD ProbeTec ET assay is a semi-automated system based on the Strand-Displacement Amplification (SDA) technique that uses the enzymatic replication of target sequences in IS61 10 and the 16S rRNA gene. The amplified products are detected with a luminometer. The major drawback of this method, however, have been the presence of false-positive results, which were reduced after the personnel gained experience with the technique, and the time required for sample preparation being at least 2 h.3
Real-time Polymerase Chain Reaction (PCR) Techniques
The real-time PCR technology is based on hybridization of amplified nucleic
acids with fluorescent-labeled probes spanning DNA regions of interest and
monitored inside thermal cyclers. The fluorescent signal increases in direct
proportion to the amount of amplified product in the reaction tube.
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Real-time PCR has been evaluated in several studies in culture material and more recently in clinical samples. The sensitivity in these studies has ranged from 71-98%, with specificity close to 100%. The main advantage of real-time PCR is its speed in giving results, 1.5-2 h after DNA extraction, and the decrease in the risk of contamination since both reaction and detection occurs in a single tube. However, due to the irregular distribution of bacilli in the clinical specimens, sensitivity of the test could be affected by the initial volume of DNA pres.
Loop-Mediated Isothermal.