Acid-Fast Bacillus (AFB) Identification, Sequencing and Stain, Paraffin Block

Tuberculosis (TB) is caused by Mycobacterium tuberculosis, an acid-fast bacillus (AFB). TB is one of the most common diseases worldwide. This organism infects both compromised (eg, those with HIV infection) and noncompromised hosts. Rapid and accurate detection is important to prevent spread of the disease and for initiation of appropriate chemotherapy. Typically, TB infections produce a wide range of tissue changes including granulomatous inflammation. This inflammation may present as caseous necrosis, aggregates of epithelioid cells, and frequently multinucleated Langhans’ giant cells.

Normally, culture is essential for definitive diagnosis of AFB infections, including those caused by M tuberculosis, M avium complex, or M marinum. Sometimes, clinical lesions are biopsied and all the tissue is embedded in paraffin. When subsequent histological review reveals the presence of an AFB infection (AFB smear-positive tissue and/or presence of granulomatous changes), culture is no longer possible. But, identification of the AFB to the genus/species level can be performed directly from the fixed, paraffin-embedded tissue using pyrosequencing.

In contrast to traditional Sanger sequencing, pyrosequencing is a rapid method that is actually sequencing by synthesis. After amplification of specific 16S rRNA sequences using AFB PCR primers, short sequences of approximately 30-50 bases are identified and then compared to public or private databases to determine the AFB genus and species. Pyrosequencing is highly accurate for the identification of AFB.

In vitro studies indicate that the PCR-sequencing assay requires as little as 100 AFB to be present in order to produce a positive AFB sequencing result.

Normally, AFB culture can require up to 8 weeks for incubation. Results by PCR-sequencing may be available within the same week after submission of the paraffin block.

Currently, PCR-sequencing cannot assess antibiotic susceptibility. In most cases, antibiotic susceptibilities are performed on viable AFB recovered by culture.