Useful For Suggests clinical disorders or settings where the test may be helpful
Rapid detection of Mycobacterium tuberculosis complex DNA, preferred method
Detection of Mtuberculosis, when used in conjunction with mycobacterial culture
This test should not be used to determine bacteriologic cure or to monitor response to therapy.
This test is not intended for the detection of latent tuberculosis and must not be used as a substitute for tests intended for detection of latent tuberculosis such as the tuberculin skin test (TST/PPD) or an interferon gamma release assay (IGRA).
Testing Algorithm Delineates situations when tests are added to the initial order. This includes reflex and additional tests.
Clinical Information Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test
Each year, Mycobacterium tuberculosis accounts for approximately 1.4 million deaths and is responsible for 9 million newly diagnosed cases of tuberculosis worldwide. M tuberculosis is spread from person-to-person via respiratory transmission, and has the potential to become resistant to many or all of the antibiotics currently used if antimycobacterial treatment is not promptly initiated. Therefore, rapid and accurate detection of M tuberculosis in patient specimens is of clinical and public health importance.
Conventional culture methods can generally detect M tuberculosis in 2 to 3 weeks, although up to 8 weeks of incubation may be required in some instances. Developed at Mayo Clinic, this rapid PCR assay detects M tuberculosis complex DNA directly from respiratory specimens and other specimens without waiting for growth in culture and, therefore, the results are available the same day the specimen is received in the laboratory. A mycobacterial culture should always be performed in addition to the PCR assay. The PCR assay is rapid but the culture has increased sensitivity over the PCR assay. The PCR assay targets a unique sequence within the katG gene, which is present in members of the M tuberculosis complex. In addition, the assay can detect genotypic resistance to isoniazid mediated by mutations in the katG target, when present.
Reference Values Describes reference intervals and additional information for interpretation of test results. May include intervals based on age and sex when appropriate. Intervals are Mayo-derived, unless otherwise designated. If an interpretive report is provided, the reference value field will state this.
Interpretation Provides information to assist in interpretation of the test results
A positive result indicates the presence of Mycobacterium tuberculosis complex DNA. Members of the M tuberculosis complex detected by this assay include M tuberculosis, M bovis, M bovis bacillus Calmette-Guerin(BCG), M africanum, M canettii, and M microti. Other species within the M tuberculosis complex (eg, M caprae, M pinnipedii, and M mungi) should, in theory, be detected using the primer and probe sequences in this assay, but they have not been tested. This assay method does not distinguish between the species of the M tuberculosis complex.
A negative result indicates the absence of detectable M tuberculosis complex DNA.
Isoniazid (INH) resistance mediated through a katG variant will be reported when observed but lack of a katG variant does not imply that the isolate is susceptible to INH. There are other genetic loci in addition to katG that can contribute to resistance for this drug.
Cautions Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances
This rapid PCR assay detects Mycobacterium tuberculosis complex nucleic acid and, therefore, does not distinguish between viable, disease-related organisms and nucleic acid persisting from prior infection. Test results should be correlated with patient symptoms and clinical presentation before a definitive diagnosis is made.
A negative result does not rule out the presence of M tuberculosis complex or active disease because the organism may be present at levels below the limit of detection for this assay.
This test has not been studied for use with specimens from patients being treated with antituberculous agents and, therefore, should not be used to determine bacteriologic cure or to monitor response to therapy. It is not known how long the PCR assay can remain positive following treatment for M tuberculosis.
The sensitivity of this test with stool specimens is 80% and testing of additional stool specimens should be considered if the result from the first specimen is negative.
The analytical specificity of the assay was determined using Basic Local Alignment Search Tool (BLAST) analysis of the National Center for Biotechnology Information (NCBI) GenBank database and no sequences were detected that would interfere with the LightCycler PCR assay. Further, the assay was tested using a panel of 104 respiratory pathogens (bacteria and viruses) that were extracted and subjected to the LightCycler PCR assay. As predicted, only Mycobacterium tuberculosis complex was detected from this panel. In addition, nearly 100 species of nontuberculous mycobacteria were evaluated using this PCR assay and there was no cross-reactivity detected. The analytical sensitivity of the assay was determined to be 10 target copies/microliter using a dilution series of M tuberculosis spiked into respiratory specimens in triplicate.
The sensitivity and specificity of the assay for detection of M tuberculosis complex verses culture was found to be 100% and 100% respectively for 26 M tuberculosis-positive cultures and 266 M tuberculosis-negative cultures. The PCR inhibition rate of the assay was determined to be 0% by spiking 100 negative extracted respiratory specimens with M tuberculosis at 100 targets/microliter. The PCR assay was able to detect 100% of specimens spiked at the limit of detection for bronchoalveolar lavage (BAL) fluid, muscle/skin tissue, organ tissues, bone, cerebrospinal fluid (CSF), and urine. One of 30 (3%) of spiked respiratory tissues was inhibited and 3 of 30 (10%) of spiked sterile body fluids other than CSF were inhibited. Not surprising increased inhibition was seen in stool (24 of 30 spiked specimens were positive) and formalin-fixed, paraffin-embedded tissue (19 of 30 spiked specimens were positive).
Method comparison of the LightCycler PCR assay versus mycobacterial culture was done using 192 respiratory specimens. The results are shown in Table 1.
Table 1. Results for the LightCycler PCR assay versus mycobacterial culture for 192 respiratory specimens.
Culture positive for Mycobacterium tuberculosis complex
Table 2 provides a comparison of the LightCycler PCR assay versus the GEN-PROBE Mycobacterium tuberculosis Direct (MTD) assay, performed using 542 respiratory specimens (226 BAL fluids, bronchial washings and lung washings plus 316 sputa, induced sputa, and tracheal secretions). The kappa coefficient of 0.96 indicates excellent agreement between the 2 methods.
Table 2. Clinical sensitivity of the LightCycler PCR versus MTD for respiratory specimens.
Two melt peaks can be produced during this assay. A melt peak at a temperature of 64.0 degrees C + or - 2.5 degrees C can correspond to either isoniazid-susceptible or isonazid-resistant M tuberculosis and, therefore, no indication of isoniazid susceptibility is provided for these isolates. However, an isolate with melt peak occurring at a temperature of 58.0 degrees C + or - 2.5 degrees C correlated with isoniazid resistance determined using a broth reference method in 100% (26/26) of isolates tested. Isolates with a peak at a temperature of 58.0 degrees C + or - 2.5 degrees C are reported as "Positive, probable isoniazid resistance detected." The PCR result is available 7 to 14 days prior to the broth method and, therefore, may be helpful in selecting appropriate antibiotic therapy for these patients. Confirmation of isonaizid resistance must be done using a phenotypic method if the isolate grows in culture.
Clinical Reference Recommendations for in-depth reading of a clinical nature
1. Iseman MD: A clinicianâ€™s guide to tuberculosis. Philadelphia, PA. Lippincott Williams and Wilkins, 2000
2. Centers for Disease Control and Prevention: Treatment of Tuberculosis, American Thoracic Society, CDC, and Infectious Diseases Society of America. MMWR Morb Mortal Weekly Rep 2003;52(No. RR-11):1-88
Special Instructions Library of PDFs including pertinent information and forms related to the test
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