Detection and identification of mycobacteria in fixed stained smears and formalin-fixed paraffin-embedded tissues using PCR.

OBJECTIVE

To determine the feasibility of using polymerase chain reaction to amplify DNA from methanol-fixed, Romanowsky-stained and Ziehl-Neelsen-stained smears to confirm the presence of mycobacteria.

MATERIALS AND METHODS

Tissue was obtained from 10 archival slides and 27 slides from a prospective series of consecutive cases. Phosphate buffered saline (500 μL) was pipetted onto a stained smear (on a glass slide) using a disposable filtered pipette tip. The material adherent to the slide was scraped from its surface and drawn up into the saline. Routine DNA extraction and purification was carried out before nested polymerase chain reaction testing targeting the 16S-23S internal transcribed spacer region or a TaqMan real-time polymerase chain reaction. The real-time polymerase chain reaction was also used on thick sections cut from formalin-fixed paraffin-embedded tissue blocks from 24 canine leproid granulomas.

RESULTS

Mycobacterial DNA was detected in 34 of 37 slides. Polymerase chain reaction products could not be amplified from three archived smears stained using the Ziehl-Neelsen acid-fast method, probably because its harsher fixation damaged the DNA. With the nested polymerase chain reaction, species identification using internal transcribed spacer sequence analysis was achieved in all instances, diagnosing a wide range of mycobacteria. The real-time polymerase chain reaction detected Mycobacterium sp. CLG DNA within all 24 formalin-fixed paraffin-embedded specimens tested.

CLINICAL SIGNIFICANCE

This technique should provide a non-invasive and cost-effective means of diagnosing mycobacterial infections.