Peripheral blood based C-PCR assay for diagnosing extra-pulmonary tuberculosis

Area of Science:

• Medical Microbiology
• Infectious Diseases
• Molecular Diagnostics

Background:

• Extra pulmonary tuberculosis (EPTB) accounts for 20% of tuberculosis cases in India.
• Conventional diagnostic methods for EPTB have limitations in sensitivity and specificity.
• Polymerase chain reaction (PCR) offers improved detection but lacks quantitative capabilities.

Purpose of the Study:

• To evaluate peripheral blood as a diagnostic specimen for EPTB.
• To assess the utility of a novel Competitive PCR (C-PCR) assay for quantifying Mycobacterium tuberculosis in blood.
• To compare PCR-based methods with conventional mycobacterial culture for EPTB diagnosis.

Main Methods:

• Analysis of peripheral blood samples from 38 EPTB patients and 89 non-tuberculous controls using MPB 64 gene-based PCR.
• Evaluation of an in-house Competitive PCR (C-PCR) assay to determine mycobacterial load (genome equivalents/mL).
• Comparison of diagnostic performance metrics (sensitivity, negative predictive value) against mycobacterial culture.

Main Results:

• The MPB 64 gene-based PCR showed a sensitivity of 60.53% and NPV of 76.92%, outperforming mycobacterial culture (10.53% sensitivity, 72.36% NPV).
• A significant proportion (43.82%) of non-tuberculous subjects tested positive with the standard PCR, limiting its clinical utility.
• C-PCR successfully quantified mycobacterial load in peripheral blood, with distinct ranges observed for culture-positive EPTB, culture-negative EPTB, and controls.

Conclusions:

• Peripheral blood is a viable specimen for EPTB diagnosis.
• Competitive PCR (C-PCR) offers a quantitative advantage for diagnosing EPTB.
• C-PCR provides valuable insights into mycobacterial load, aiding in EPTB diagnosis.