Mycobacterial multiplexed molecular assay

Rapid detection of Mycobacterium tuberculosis complex and/or Mycobacteria spp. DNA.

(Supplemental assay to be used in conjunction with mycobacterial culture for enhanced diagnosis)

 

Method Name

Mycobacterial multiplexed molecular assay (Multiplexed Real-Time Polymerase Chain Reaction)

 

Aliases

Acid-Fast Bacilli (AFB)
AFB (Acid-Fast Bacilli)
MTB (Mycobacterium tuberculosis)

Mycobacteria species

Non-tuberculous mycobacteria
Mycobacterium tuberculosis (MTB)
TB (Tuberculosis)
Tuberculosis (TB)

Tuberculosis (TB) is among the top 10 causes of death worldwide and a leading killer of HIV-positive persons. Annually, Mycobacterium tuberculosis (MTB) causes approximately 1.5 million deaths and accounts for 10 million newly diagnosed cases globally. In 2017, 1,796 cases of active tuberculosis were reported in Canada (disproportionately higher rates in foreign born individuals and indigenous Canadians). MTB can be transmitted from person-to-person via aerosol, and has the potential to become resistant if proper antimycobacterial treatment is not administered. Rapid and accurate detection of M tuberculosis in patient specimens is a clinical and public health priority.

Nontuberculous mycobacteria (NTM) are responsible for the majority of mycobacterial infections in both immunocompromised and immunocompetent individuals in developed countries, where TB incidence is low. Rapid presumptive identification can facilitate focused empirical antibiotic therapy and aid in determining isolation measures.

Conventional culture methods typically detect MTB complex and slow growing NTM spp. in 2 to 3 weeks, although up to 8 weeks of incubation may be required. Developed at PHML, this multiplexed PCR assay detects MTB complex and/or Mycobacteria spp. DNA directly from non-fixed clinical specimens with results available within ~48 hours of specimen receipt (including specimens that are microscopically acid-fast smear staining negative). This method has a much higher sensitivity for MTB complex detection than prior method due to the use of a multi-copy target IS6110 gene, limit of detection ~5 CFU/ml. The target for pan detection of Mycobacteria spp. has a limit of detection ~ 100 CFU/ml.

 

Note: Mycobacterial culture (the more sensitive gold standard) should always be performed in addition to the PCR assay.

 

Interpretation

A positive MTBC DNA result indicates the presence of MTB complex DNA. This assay method does not distinguish between the species of the MTB complex.

 

A negative MTBC DNA (IS6110 gene not detected), but positive Mycobacteria spp. DNA result could represent a NTM infection. However, this result DOES NOT rule out MTBC. The results need to be interpreted with caution considering patients’ origin and travel history. 99% of global M. tuberculosis strains harbor the IS6110 gene, however rare isolates may not contain the target (subset of isolates from patients originating from South Asia and South East Asia).

 

A negative MTBC DNA and negative Mycobacteria spp. DNA result does not rule out MTBC or NTM infection. The organisms may be present at levels below the limit of detection for this assay. Culture remains the more sensitive and definitive gold standard for diagnosis of mycobacterial infection.

 

Caution

This rapid multiplexed assay detects MTB complex and/or Mycobacteria spp. 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.

 

Molecular detection should NOT be used on specimens collected from patients who have received antimicrobials for more than 7 days or have received such therapy in the last 12 months prior to collection. The use of this assay is NOT recommended for monitoring treatment responses.

 

*Non-respiratory specimens were underrepresented in the validation study of this PHML-developed test. Use results with caution.

Assay Offered

Mon-Sun

 

Assay Performance

26 confirmed M. tuberculosis positive, 15 NTM positive and 83 confirmed negative archived clinical specimens were included in the validation study. Diagnostic sensitivity for MTBC and/or Mycobacteria spp. detection was 89.7% (100% for MTBC, 69.2% NTM). Diagnostic specificity, positive predictive value, negative predictive value were 100%, 100% and 99.9% respectively.

 

MTBC limit of detection was calculated to be approximately 5 CFU/ml.

Pan Mycobacterium spp. limit of detection was 117 CFU/ml.

 

References

  1. Forbes et al. Practice Guidelines for Clinical Microbiology Laboratories: Mycobacteria, Clinical Microbiology Reviews, 2018.
  1. Canadian Tuberculosis Standards 7th Edition: 2014.
  1. Armand et al. Comparison of the Xpert MTB/RIF test with an IS6110-TaqMan real-time PCR assay for direct detection of Mycobacterium tuberculosis in respiratory and non-respiratory specimens, JCM 2011.
 

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