A novel diagnostic method of Pneumocystis carinii. In situ hybridization of ribosomal ribonucleic acid with biotinylated oligonucleotide probes.

The reactivity and specificity of the in situ hybridization of ribosomal RNA in the diagnosis of Pneumocystis carinii were investigated. Three complementary oligonucleotide probes, 22 and 25 nucleotides long, corresponding to the species specific regions of 5S and 18S ribosomal RNA of Pneumocystis carinii were synthesized and labeled with biotinylated dUPT at the 3' termini. In situ hybridization was performed on formalin-fixed paraffin-embedded human lung tissues using the mixture of these probes and detected with the avidin-biotin peroxidase complex method. The reactions were positive in all 12 cases of Pneumocystis carinii pneumonia, but in none of the infections with other pathogenic agents, including virus (6 cases), mycobacteria (4 cases), protozoa (4 cases) and fungi (8 cases). The reactivity and specificity of this method was comparable with that of immunohistochemistry using a monoclonal anti-human Pneumocystis carinii antibody. Because the specificity of in situ hybridization is based on nucleotide sequences of ribosomal RNAs, that are constant among species, contrary to morphology of protista or expression of antigens, it should complement conventional staining and immunohistochemical methods, and provide a useful tool for the diagnosis of Pneumocystis carinii.