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

Test code(s) 90870

Question 1. Why perform AFB PCR-pyrosequencing on paraffin-embedded tissue samples?

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.

Question 2. What is 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.

Question 3. How sensitive is the PCR-sequencing technique for detecting 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.

Question 4. How rapid is the PCR-sequencing technique for detection AFB?

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.

Question 5. Will antibiotic susceptibilities be available when PCR-sequencing detects an AFB on the paraffin block?

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

References

  1. Bao JR, Master RN, Schwab DA, et al. Identification of acid-fast bacilli using pyrosequencing analysis. Diagn Microbiol Infect Dis. 2010;67:234-238.
  2. Luo RF, Seahill MD, Banaei N. Comparison of single and multicopy real-time PCR for detection of Mycobacterium tuberculosis in paraffin-embedded tissue.J Clin Microbiol. 2010;48:2569-2570.
  3. Mahaisavariya P, Chaiprasert A, Manonukul J, et al. Detection and identification of Mycobacterium species by polymerase chain reaction (PCR) from paraffin-embedded tissue compared to AFB staining in pathological sections. J Med Assoc Thai. 2005;88:108-113.
  4. Marchetti G, Gori A, Catozzi L, et al. Evaluation of PCR in detection of Mycobacterium tuberculosis from formalin-fixed, paraffin-embedded tissues: comparison of four amplification assays. J Clin Microbiol. 1998;36:1512-1517.
  5. Srinivasulu R, Brown T, Drobniewski F. Detection of Mycobacterium tuberculosis from paraffin-embedded tissues by INNO-LIPA Rif.TB assay: retrospective analyses of Health Protection Agency National Mycobacterium Reference Laboratory data. J Med Microbiol. 2010;59:563-566.
This FAQ is provided for informational purposes only and is not intended as medical advice. A physician’s test selection and interpretation, diagnosis, and patient management decisions should be based on his/her education, clinical expertise, and assessment of the patient.
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