Acid-fast bacterium detection and identification from paraffin-embedded tissues using a PCR-pyrosequencing method.

Area of Science:

• Microbiology
• Molecular Biology
• Pathology

Background:

• Acid-fast bacterium (AFB) identification in formalin-fixed paraffin-embedded (FFPE) tissues presents diagnostic challenges for clinical laboratories.
• Current methods may lack the sensitivity or specificity required for accurate AFB detection in FFPE samples.

Purpose of the Study:

• To develop and evaluate a novel Polymerase Chain Reaction and pyrosequencing (PCR-Seq) method for detecting and identifying AFB directly from FFPE tissues.
• To assess the performance of the PCR-Seq method compared to traditional AFB staining techniques.

Main Methods:

• DNA extraction from FFPE tissue sections.
• Amplification of a 16S rRNA gene fragment specific to AFB.
• Sequencing and identification of amplified DNA using the PyroMark ID system and alignment to established databases.
• Validation using spiked paraffin blocks and comparison with AFB staining on patient FFPE tissues.

Main Results:

• The PCR-Seq method demonstrated 100% sensitivity and specificity on spiked samples without cross-reactivity.
• In patient FFPE tissues, 38% were AFB-stain positive, with PCR-Seq identifying AFB in 72% of these.
• The method successfully identified various AFB, including Mycobacterium tuberculosis complex, Mycobacterium leprae, and others, with high accuracy even in AFB-stain indeterminate or negative cases.

Conclusions:

• The developed PCR-Seq method provides specific and reliable identification of diverse AFB species and complexes from FFPE tissues.
• This molecular approach enhances the diagnostic capabilities for AFB detection in challenging FFPE specimens, offering a valuable tool for clinical laboratories.