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

Abstract

<jats:sec><jats:title>Aims</jats:title><jats:p>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.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>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.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>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 <jats:italic>Mycobacterium tuberculosis</jats:italic> complex (14 cases), <jats:italic>Mycobacterium leprae</jats:italic> (3), <jats:italic>Mycobacterium genavense</jats:italic> (2), <jats:italic>Mycobacterium marinum-ulcerans</jats:italic> 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.</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>The PCR-Seq method provided specific identification of various AFB species or complexes from FFPE tissues.</jats:p></jats:sec>

Upper Gastrointestinal Tract Involvement in Inflammatory Bowel Diseases: Histologic Clues and Pitfalls

“… 67 , 68 Given its superior sensitivity, if available, polymerase chain reaction analysis should be preferred over the labor-intensive but frequently unrewarding examination of acid-fast stains. 69 …”

“…Unfortunately, the epithelioid granulomata do not always harbor the distinguishing histologic feature of caseous necrosis 67,68. Given its superior sensitivity, if available, polymerase chain reaction analysis should be preferred over the labor-intensive but frequently unrewarding examination of acid-fast stains 69…”

Comparison of One-Tube Nested-PCR and PCR-Reverse Blot Hybridization Assays for Discrimination of Mycobacterium tuberculosis and Nontuberculous Mycobacterial Infection in FFPE tissues

“…Globally, the most widely used conventional method for detecting MTB in formalin-fixed paraffin-embedded (FFPE) tissue is an acid-fast bacilli (AFB) stain. This is a rapid and simple method; however, FFPE specimens can contain low bacterial load, and the granuloma formation and necrosis caused by mycobacteria can be also found in other bacterial and fungal infections (Bao et al, 2018). Additionally, AFB staining displays low sensitivity in the detection of MTB and the method cannot differentiate MTB from nontuberculous mycobacteria (NTM) species (Lee et al, 2011).…”