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Detection of Pneumocystis carinii by in situ hybridization in the lungs of immunosuppressed rats
J Kim,*1J R Yu,2S T Hong,3 and C S Park1
1Department of Pathology, Chonnam University Medical School, Kwangju 501-190, Korea.
Received July 31, 1996; Accepted August 26, 1996.
Abstract
In situ hybridization was performed to detect rat Pneumocystis carinii in the lung sections. Rats were immunosuppressed by weekly subcutaneous injection of 10 mg/kg methylprednisolone. On the 6th, 8th and 9th week of immunosuppression, the lungs were removed and fixed in 10% neutral formalin. A 22 base oligonucleotide probe complementary to P. carinii 5S ribosomal RNA was commercially synthesized and its 3' terminal was labeled with biotin. In situ hybridization was performed utilizing manual capillary action technology on the Microprobe system. P. carinii were detected along the luminal surface of alveolar pneumocytes, in exudate of alveolar cavities, and also in secretory material of bronchioles. In the 6th week group, positive reaction was observed focally in the peripheral region of the lung sections, but the reaction was observed diffusely in the 8th or 9th week groups. In comparison with Grocott's methenamine silver stain, In situ hybridization technique can detect the organism rapidly, and can detect trophic forms very well. Furthermore, no nonspecific reaction with other pathogenic fungi and protozoa was recognized. Therefore, In situ hybridization can be a good technique to detect P. carinii in the lungs of infected rats.
Figures
Figs. 1-5 Fig. 1.In situ hybridization using red chromogen. a mixture of naphthol AS-MX phosphate and fast red TR salt (NASFRTR). A. Focal positive reaction, 6 week group (× 40); B. Diffuse positive reaction, 9 week group (× 20). Fig. 2. Hematoxylin and eosin stain. Typical microscopic appearance of P. carinii pneumonia showing alveolar interstitial thickening and frothy exudate in the alveolar lumens, 8 week group (× 200). Fig. 3. Grocott's methenamine silver stain. Numerous cysts are seen apparently, but trophic forms are not stained, 8 week group (× 200). Fig. 4.In situ hybridization. Granular positive reaction within alveolar exudate, 8 week group. A. Red chromogen, NASFRTR; B. Brown chromogen, diaminobenzidine (× 200). Fig. 5.In situ hybridization. Linear positive reaction along the luminal surface of alveoli, 8 seek group. A. Red chromogen, NASFRTR; B. Brown chromogen, diaminobenzidine (× 200).
Fig. 6-10 Fig. 6.In situ hybridization. Positive signals gradually increased within the secretory material of bronchiole and within alveolar exudate according to the lapse of immunosuppression time. A. Control group; B. 6 week group; C. 8 week group; D. 9 week gorup (× 100). Fig. 7. Aspergillosis in human lung. A. PAS stain; B.In situ hybridization with P. carinii oligoprobe, negative (× 100). Fig. 8. Candidiasis in human esophagus. A. PAS stain; B.In situ hybridization with P. carimii oligoprobe, negative (× 200). Fig. 9. Toxoplasmosis in mouse brain. A. Hematoxylin and eosin stain; B.In situ hybridization with P. carinii oligoprobe, negative (× 200). Fig. 10. Amebiasis in human colon. A. Hematoxylin and eosin stain (× 200); B.In situ hybridization with P. carinii oligoprobe, negative (× 400).
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990;215(3):403–410.
2.
Bashir MS, Lewis FA, Quirke P, Lee A, Dixon MF. In situ hybridisation for the identification of Helicobacter pylori in paraffin wax embedded tissue. J Clin Pathol 1994;47(9):862–864.
3.
Brigati DJ, et al. J Histotechnol 1988;11:165–183.
4.
Brigati DJ, Myerson D, Leary JJ, Spalholz B, Travis SZ, Fong CK, Hsiung GD, Ward DC. Detection of viral genomes in cultured cells and paraffin-embedded tissue sections using biotin-labeled hybridization probes. Virology 1983;126(1):32–50.
6.
Gigliotti F, Stokes DC, Cheatham AB, Davis DS, Hughes WT. Development of murine monoclonal antibodies to Pneumocystis carinii. J Infect Dis 1986;154(2):315–322.
7.
Hayashi Y, Watanabe J, Nakata K, Fukayama M, Ikeda H. A novel diagnostic method of Pneumocystis carinii. In situ hybridization of ribosomal ribonucleic acid with biotinylated oligonucleotide probes. Lab Invest 1990;63(4):576–580.
8.
Hong ST, Park KH, Lee SH. Susceptibility of various animals to Pneumocystis carinii infection. Korean J Parasitol 1992;30(4):277–281.
9.
Hong ST, Yu JS, Lee M. Relation of cyst counts with numbers of total nuclei of Pneumocystis carinii in rats. Korean J Parasitol 1994;32(3):171–176.
10.
John HA, Birnstiel ML, Jones KW. RNA-DNA hybrids at the cytological level. Nature 1969;223(5206):582–587.
11.
Kessler C. The digoxigenin:anti-digoxigenin (DIG) technology--a survey on the concept and realization of a novel bioanalytical indicator system. Mol Cell Probes 1991;5(3):161–205.
12.
Kovacs JA, Halpern JL, Lundgren B, Swan JC, Parrillo JE, Masur H. Monoclonal antibodies to Pneumocystis carinii: identification of specific antigens and characterization of antigenic differences between rat and human isolates. J Infect Dis 1989;159(1):60–70.
13.
Langer PR, Waldrop AA, Ward DC. Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes. Proc Natl Acad Sci U S A 1981;78(11):6633–6637.
14.
Leibovitz E, Pollack H, Moore T, Papellas J, Gallo L, Krasinski K, Borkowsky W. Comparison of PCR and standard cytological staining for detection of Pneumocystis carinii from respiratory specimens from patients with or at high risk for infection by human immunodeficiency virus. J Clin Microbiol 1995;33(11):3004–3007.
15.
Lu JJ, Chen CH, Bartlett MS, Smith JW, Lee CH. Comparison of six different PCR methods for detection of Pneumocystis carinii. J Clin Microbiol 1995;33(10):2785–2788.
16.
Montone KT. Acta Histochemi Cytochem 1994;27:601–606.
17.
Montone KT, et al. J Clin Pathol 1995;103:48–51.
18.
Pardue ML, Gall JG. Molecular hybridization of radioactive DNA to the DNA of cytological preparations. Proc Natl Acad Sci U S A 1969;64(2):600–604.
19.
Park CS, et al. J Histotechnol 1991;14:219–229.
20.
Tanabe K, Fuchimoto M, Egawa K, Nakamura Y. Use of Pneumocystis carinii genomic DNA clones for DNA hybridization analysis of infected human lungs. J Infect Dis 1988;157(3):593–596.
21.
Wakefield AE, Hopkin JM, Burns J, Hipkiss JB, Stewart TJ, Moxon ER. Cloning of DNA from Pneumocystis carinii. J Infect Dis 1988;158(4):859–862.
22.
Wazir JF, Brown I, Martin-Bates E, Coleman DV. EB9, a new antibody for the detection of trophozoites of Pneumocystis carinii in bronchoalveolar lavage specimens in AIDS. J Clin Pathol 1994;47(12):1108–1111.
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