Test ID: MTBRP
Mycobacterium tuberculosis Complex, Molecular Detection, PCR
Secondary ID 
A test code used for billing and in test definitions created prior to November 2011
NY State Approved Indicates the status of NY State approval and if the test is orderable for NY State clients.
Useful For Suggests clinical disorders or settings where the test may be helpful
Rapid detection of Mycobacterium tuberculosis complex, preferred method
Diagnosis of tuberculosis, when used in conjunction with mycobacterial culture
Method Name A short description of the method used to perform the test
Real-Time Polymerase Chain Reaction (PCR)
(PCR is utilized pursuant to a license agreement with Roche Molecular Systems, Inc.)
Reporting Name A shorter/abbreviated version of the Published Name for a test; an abbreviated test name
Aliases Lists additional common names for a test, as an aid in searching
AFB (Acid-Fast Bacilli)
Bacillus, Acid-Fast
MTB (Mycobacterium tuberculosis)
Mycobacterium tuberculosis (MTB)
TB (Tuberculosis)
Tubercle Bacilli: Mycobacterium tuberculosis
Tuberculosis (TB)
Specimen Type Describes the specimen type needed for testing
Specimen Required Defines the optimal specimen. This field describes the type of specimen required to perform the test and the preferred volume to complete testing. The volume allows automated processing, fastest throughput and, when indicated, repeat or reflex testing.
The high sensitivity of amplification by PCR requires the specimen to be processed in an environment in which contamination of the specimen by Mycobacterium tuberculosis DNA is not likely.
Forms: If not ordering electronically, submit a Microbiology Request Form (Supply T244) with the specimen.
Preferred Specimens: Body fluid, cerebrospinal fluid, respiratory (eg, bronchoalveolar lavage fluid, bronchial washing, sputum), stool, tissue (fresh or paraffin) or bone, urine
Acceptable Specimens: If no fresh specimen is available, some digested respiratory specimens treated with N-acetyl-L-cysteine (NALC)/NaOH are acceptable (eg, bronchoalveolar lavage fluid, bronchial washing, gastric washing, respiratory fluid, sputum, or tracheal secretion)
Unacceptable Specimens: Blood, bone marrow, specimen in anaerobe vial or viral transport medium (including but not limited to M4, M5, BD viral transport media, thioglycolate broth), swab, tissue in formalin fluid, specimen >7 days old (with the exception of paraffin tissue)
Specimen must arrive within 7 days of collection; specimen >7 days will be rejected.
Specimen source is required.
Submit only 1 of the following specimens:
Preferred:
Specimen Type: Body fluid
Sources: Body fluid or cerebrospinal fluid
Container/Tube: Sterile container
Specimen Volume: 1 mL
Specimen Stability Information: Refrigerated (preferred) 7 days/Ambient 7 days/Frozen 7 days
Additional Information: Only fresh, non-NALC/NaOH-digested body fluid is acceptable.
Specimen Type: Gastric Washing
Container/Tube: Sterile container
Specimen Volume: 10 mL
Collection Instructions: Neutralize specimen within 4 hours of collection with 100 mg of sodium carbonate per 5-10 mL of gastric washing.
Specimen Stability Information: Refrigerated (preferred) 7 days/Ambient 7 days/Frozen 7 days
Specimen Type: Respiratory
Sources: Bronchoalveolar lavage fluid, bronchial washing, or sputum
Container/Tube: Sterile container
Specimen Volume: 3 mL
Specimen Stability Information: Refrigerated (preferred) 7 days/Ambient 7 days/Frozen 7 days
Specimen Type: Stool
Container/Tube: Sterile container
Specimen Volume: 5-10 g
Specimen Stability Information: Refrigerated (preferred) 7 days/Ambient 7 days/Frozen 7 days
Additional Information: Only fresh, non-NALC/NaOH-digested stool is acceptable.
Specimen Type: Tissue
Sources: Fresh, paraffin, or bone
Container/Tube:
Preferred: Sterile container
Acceptable: Biopsy specimen of tissue fixed with formalin in a paraffin block
Specimen Volume: 5-10 mm
Collection Instructions: Block must be sent for sectioning.
Specimen Stability Information:
Fresh tissue: Refrigerated (preferred) 7 days/Ambient 7 days/Frozen 7 days
Fixed tissue (paraffin): Ambient
Additional Information: Only fresh, non-NALC/NaOH-digested tissue is acceptable.
Specimen Type: Urine
Container/Tube: Sterile container
Specimen Volume: 2 mL
Collection Instructions: Collect a random urine specimen.
Specimen Stability Information: Refrigerated (preferred) 7 days/Ambient 7 days/Frozen 7 days
Additional Information: Only fresh, non-NALC/NaOH-digested urine is acceptable.
Acceptable:
NALC/NaOH-digested specimen must arrive within 7 days of digestion.
Specimen Type: NALC/NaOH-digested respiratory specimens
Sources: Lavage fluid, bronchial washing, gastric washing, respiratory fluid, sputum, or tracheal secretion
Container/Tube: Sterile container
Specimen Volume: 2 mL
Collection Instructions:
1. Submit digested specimen treated with NALC/NaOH.
2. Clearly indicate on container and order form that specimen is a digested specimen.
Specimen Stability Information: Refrigerated (preferred) 7 days/Ambient 7 days/Frozen 7 days
Additional Information: If a single specimen is being shared between mycobacteria culture, acid-fast smear, and/or Mycobacterium tuberculosis PCR, a minimum volume of 1.5 mL for body fluid, 3 mL for respiratory specimen, or a pea-sized piece of tissue should be obtained. Specimen volumes less than indicated may decrease sensitivity of testing. If insufficient volume is submitted, test or tests will be canceled.
Specimen Minimum Volume Defines the amount of specimen required to perform an assay once, including instrument and container dead space. Submitting the minimum specimen volume makes it impossible to repeat the test or perform confirmatory or perform reflex testing. In some situations, a minimum specimen volume may result in a QNS (quantity not sufficient) result, requiring a second specimen to be collected.
Reject Due To Identifies specimen types and conditions that may cause the specimen to be rejected
| Hemolysis | NA |
| Lipemia | NA |
| Icterus | NA |
| Other | Blood, bone marrow, specimen in anaerobe vial or viral transport medium (including but not limited to M4, M5, BD viral transport media, thioglycolate broth), swab, tissue in formalin fluid, specimen >7 days old (with the exception of paraffin tissue) |
Specimen Stability Information Provides a description of the temperatures required to transport a specimen to the laboratory. Alternate acceptable temperature(s) are also included.
| Specimen Type | Temperature | Time |
|---|---|---|
| Varies | Varies | |
Clinical Information Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test
Each year, Mycobacterium tuberculosis accounts for nearly 2 million deaths and is responsible for 9 million newly diagnosed cases of tuberculosis worldwide. Mycobacterium 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 Mycobacterium tuberculosis in patient specimens is of clinical and public health importance.
Conventional culture methods can generally detect Mycobacterium 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 Mycobacterium 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. The assay targets a unique sequence within the katG gene, which is present in members of the Mycobacterium 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.
Not applicable
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 Mycobacterium tuberculosis complex detected by this assay include Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis Bacillus Calmette-Guerin, Mycobacterium africanum, Mycobacterium canetti, and Mycobacterium microti. The other species within the Mycobacterium tuberculosis complex (Mycobacterium bovis subspecies caprae and Mycobacterium pinnepedi) should, in theory, be detected using the primer and probe sequences in this assay, but they have not been tested at this time. This assay method does not distinguish between the species of the Mycobacterium tuberculosis complex.
A negative result indicates the absence of detectable Mycobacterium tuberculosis complex DNA.
Isoniazid (INH) resistance mediated through a katG mutation will be reported when observed but lack of a katG mutation 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 test should always be performed in conjunction with mycobacterial culture.
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 Mycobacterium 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 Mycobacterium tuberculosis.
The sensitivity of this test from stool is 80% and from formalin-fixed, paraffin-embedded tissue is 63%, therefore testing of a second specimen from these sources should be considered to increase sensitivity if the result from the first specimen is negative.
Supportive Data
The analytical specificity of the assay was determined using 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/L using a dilution series of Mycobacterium tuberculosis spiked into respiratory specimens in triplicate.
The sensitivity and specificity of the assay for detection of Mycobacterium tuberculosis complex from culture was found to be 100% and 100% respectively using 26 positive and 266 negative cultures for Mycobacterium tuberculosis that were originally identified using the GEN-PROBE (San Diego, CA) AccuProbe nucleic acid hybridization probe for Mycobacterium tuberculosis complex. The PCR inhibition rate of the assay was determined to be 0% by spiking 100 negative extracted respiratory specimens with Mycobacterium tuberculosis at 100 targets/L. The PCR assay was able to detect 100% of specimens spiked at the limit of detection for BAL fluid, muscle/skin tissue, organ tissues, bone, cerebrospinal fluid (CSF), and urine. One of 30 (3%) of spiked respiratory tissues were inhibited and 3 of30 (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 culture for 192 respiratory specimens.
|
| Culture positive for M. tuberculosis complex |
| |||
| + | - | Sensitivity | Specificity | ||
| LightCycler PCR | + | 33 | 1 | 87% | 99% |
| - | 5 | 153 | |||
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 vs MTD for respiratory specimens.
|
Assay
| MTD |
| ||||
| + | - | Agreement (%) | kappa coefficient | |||
| LightCycler PCR | + | 49 | 1 | 538/542 (99.3%) | 0.96 | |
| - | 3 | 489 | ||||
Two melt peaks can be produced during this assay. A melt peak at a Tm 64.5 degrees C +/- 2.5 degrees C can correspond to either isoniazid-susceptible or isonazid-resistant Mycobacterium tuberculosis and therefore no indication of isoniazid susceptibility is provided for these isolates. However, an isolate with melt peak occurring at a Tm of 58.0 degrees C +/- 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 Tm of 58.0 degrees C +/- 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 ordered if the isolate grows in culture.
The intra-day precision for wild-type Mycobacterium tuberculosis with a Tm at 64.5 degrees +/- 2.5 degrees C was evaluated using positive clinical specimens with high, intermediate and low-levels of Mycobacterium tuberculosis assayed in triplicate. The intra-day precision demonstrated a standard deviation of < or =0.06 degrees C and a coefficient of variation of < or =0.09%. For the isoniazid-resistant peak with a Tm of 58.0 degrees +/- 2.5 degrees C, the standard deviation was < or =0.10 degrees C with a coefficient of variation of < or =0.17%.
The inter-day precision for wild-type Mycobacterium tuberculosis with a Tm at 64.5 degrees +/- 2.5 degrees C was evaluated over 20 days using 2 LightCycler instruments and 7 technologists to perform the assay. The inter-day precision studies demonstrated a standard deviation of < or =0.23 degrees C and a coefficient of variation of < or =0.4%. For the isoniazid-resistant peak with a Tm of 58.0 degrees +/- 2.5 degrees C, the standard deviation was < or =0.28 degrees C with a coefficient of variation of < or =0.5%.
Clinical Reference Provides recommendations for further in-depth reading of a clinical nature
1. Iseman MD: A clinician’s guide to tuberculosis. Philadelphia, PA. Lippincott Williams & 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
Method Description Describes how the test is performed and provides a method-specific reference
Following specimen digestion and decontamination using N-acetyl cysteine and sodium hydroxide, genomic DNA is extracted using the MagNA Pure Compact (Roche Applied Sciences) extraction platform. The purified genomic DNA is placed on the LightCycler instrument, which amplifies and monitors, by fluorescence, the development of target nucleotide sequences after each PCR cycle. A specific target sequence from a portion of the katG gene from Mycobacterium tuberculosis complex is amplified and the resulting segment is detected by melt-curve analysis using sequence-specific fluorescence resonance energy transfer hybridization probes. The LightCycler PCR assay is a closed PCR system that greatly reduces the potential for false-positive results due to specimen cross-contamination as compared with traditional open-system PCR or other amplification methods like transcription-mediated amplification. (Unpublished Mayo method)
Day(s) and Time(s) Test Performed Outlines the days and times the test is performed. This field reflects the day and time the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time required before the test is performed. Some tests are listed as continuously performed, which means assays are performed several times during the day.
Monday through Friday
Analytic Time Defines the amount of time it takes the laboratory to setup and perform the test. This is defined in number of days. The shortest interval of time expressed is "same day/1 day," which means the results may be available the same day that the sample is received in the testing laboratory. One day means results are available 1 day after the sample is received in the laboratory.
Maximum Laboratory Time Defines the maximum time from specimen receipt at Mayo Medical Laboratories until the release of the test result
Specimen Retention Time Outlines the length of time after testing that a specimen is kept in the laboratory before it is discarded
Performing Laboratory Location The location of the laboratory that performs the test
Test Classification Provides information regarding the medical device classification for laboratory test kits and reagents. Tests may be classified as cleared or approved by the US Food and Drug Administration (FDA) and used per manufacturer's instructions, or as products that do not undergo full FDA review and approval, and are then labeled as an Analyte Specific Reagent (ASR), Investigation Use Only (IUO) product, or a Research Use Only (RUO) product.
CPT Code Information Provides guidance in determining the appropriate Current Procedural Terminology (CPT) code(s) information for each test or profile. The listed CPT codes reflect Mayo Medical Laboratories interpretation of CPT coding requirements. It is the responsibility of each laboratory to determine correct CPT codes to use for billing.
87556-Mycobacterium tuberculosis, complex, molecular detection, PCR
87015-Mycobacteria culture, concentration (if appropriate)
LOINC® Code Information Provides guidance in determining the Logical Observation Identifiers Names and Codes (LOINC) values for the result codes returned for this test or profile.
| Result ID | Reporting Name | LOINC Code |
|---|---|---|
| SRC62 | Specimen Source | 31208-2 |
| 56044 | Result | 48174-7 |
| 56045 | Special Information | 48767-8 |
| 56046 | Report Status | N/A |
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