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Detection of Pneumocystis carinii by in situ hybridization in the lungs of immunosuppressed rats
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Korean J Parasito > Volume 34(3):1996 > Article

Original Article
Korean J Parasitol. 1996 Sep;34(3):177-184. English.
Published online Sep 20, 1996.  http://dx.doi.org/10.3347/kjp.1996.34.3.177
Copyright © 1996 by The Korean Society for Parasitology
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).

Tables


Table 1
Procedure for in situ hybridization

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