Polymerase chain reaction detection of Mycobacterium tuberculosis in formalin-fixed tissue

Abstract

Use of the polymerase chain reaction (PCR) to detect Mycobacterium tuberculosis in formalin-fixed, paraffin-embedded tissue would be of great diagnostic value. However, formaldehyde has been reported to decrease the efficiency of amplification by structurally altering the polynucleotide chain. We sought to determine the ability of the PCR assay to detect M. tuberculosis in formalin-fixed, paraffin-embedded tissue in a mouse experimentally infected with the H37Rv strain of M. tuberculosis. Lung tissue from the infected mouse was cultured to determine the number of organisms per gram of tissue. The remaining lung tissue was divided into eight portions, seven of which were fixed in 10% neutral buffered formalin for 24-h intervals over periods lasting from 1 to 7 d, and a control portion was placed in isotonic saline. The tissue samples were then paraffin-embedded, and sections were obtained from each tissue block for PCR analysis. We show that the PCR assay can detect as few as nine organisms in a 5-micron section of tissue, and that up to 7 d of fixation in 10% neutral buffered formalin has a negligible effect on the assay. The PCR assay can detect low numbers of M. tuberculosis organisms in formalin-fixed, paraffin-embedded tissue.