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Acid-fast bacterium detection and identification from paraffin-embedded tissues using a PCR-pyrosequencing method
  1. Jian R Bao,
  2. Richard B Clark,
  3. Ronald N Master,
  4. Kileen L Shier,
  5. Lynn L Eklund
  1. Department of Microbiology, Quest Diagnostics Nichols Institute Chantilly, Chantilly, Virginia, USA
  1. Correspondence to Dr Richard B Clark, Department of Microbiology, Quest Diagnostics Nichols Institute Chantilly, Chantilly, Virginia 20151, USA; richard.b.clark{at}


Aims Acid-fast bacterium (AFB) identification from formalin-fixed paraffin-embedded (FFPE) tissues is challenging and may not be readily available to the clinical laboratory. A method to detect and identify AFB from FFPE tissues using PCR and pyrosequencing (PCR-Seq) was developed and evaluated.

Methods The method was validated using spiked cell-clotted paraffin blocks before use with patients’ specimens. DNA was extracted from tissue sections, and a 16S rRNA gene fragment was amplified and a signature sequence was produced on a PyroMark ID system. Sequences were aligned to established databases for AFB identification. Additional tissue sections were stained and examined for AFB.

Results Both sensitivity and specificity were 100% on spiked cell-clotted blocks without cross-reactivity with non-AFB. Of 302 FFPE tissues from patients, 116 (38%) were AFB-stain positive; 83 (72%) of these had AFB identified. The 21 AFB identified included Mycobacterium tuberculosis complex (14 cases), Mycobacterium leprae (3), Mycobacterium genavense (2), Mycobacterium marinum-ulcerans group (3) and 17 other AFB (61). Thirteen cases were AFB-stain indeterminate and 4 were positive by the PCR-Seq method. Of the AFB stain-negative cases, 167 were negative and 6 were positive by PCR-Seq.

Conclusions The PCR-Seq method provided specific identification of various AFB species or complexes from FFPE tissues.

  • paraffin-embedded tissue
  • acid-fast bacterial identification
  • PCR-sequencing

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  • Handling editor Cheok Soon Lee.

  • Contributors JRB, RBC, RNM and KLS designed the study. JRB conducted the assays and wrote the manuscript. RBC, RNM and KLS provided technical assistance and reviewed the manuscript. LLE read most AFB staining results and provided technical assistance.

  • Competing interests None declared.

  • Provenance and peer review Not commissioned; externally peer reviewed.