Laboratory Diagnosis Of Rat Model Of Pneumocystis Carinii Pneumonia

Pneumocystis carinii(PC) is an opportunistic pathogen causing serious and even life-threatening Pneumocystis carinii pneumonia(PCP) in immunosuppressed patients, especially those with AIDS, malignancies, organ transplantation or CD4~+T cell functional deficit. For years, diagnosis of Pneumocystis carinii pneumonia has relied on microscopic visualization of PC in specimens obtained from the lung either by bronchoalveolar lavage or by induction of sputum in our country. But the sensitivity of conventionally stained specimens is variable. Hence, convenient, stable, sensitivity and specificity diagnostic procedures are needed for patients with PCP. In this research, we studied the possible application of improved conventionally stained and molecular biology assay in the detection of PCP in infected rats.Objective: To research the sensitivity and possible application ofWright-Giemsa's stained, nested PCR asssy(PC-ITS-PCR) and RNA in situ hybridization to detect the specimens of PCP in infected rats.Methods: Wistar rat model of PCP was established by subcutaneousinjection with Dexanethasone. Wright-Giemsa's stained was used to detect the organism in lung impression preparation and bronchoalveolar lavage fluid(BALF). In situ hybridization detection was performed with Dig-labelled oligonucleotide probe complement to small subunit mRNA of Pneumocystis carinii. PCR detection amplified internal transcribed spacers of the rRNA genes of Pneumocystis carinii in serum, lung and BALF by nested PCR method using PC-ITS primers.Result: In this study, samples from 43 rats in experimental group weretested. The sensitivity of Wright-Giemsa's stained is 74.2% in lung impression preparation and 67.44% in BALF. RNA in situ hybridization successfully demonstrate PC cyst and tachyzoite in infected rat lung without background staining .Specific positive results were observed on sections 3 weeks after injection with Dexanethasone in experimental group. Many cysts were observed in 7-8 weeks, a large number of trophozoites were observed in 9-10 weeks, and empty cysts were observed in 8-9 weeks. The sensitivity of in situ hybridization is 88.37% in lung sections. Usingnested PC-ITS-PCR, Pneumocystis carinii DNA was amplified at 550bp. The sensitivity of PCR is 86.05% in lung tissue , 83.72% in BALF and 76.74% in serum. Positive rate of detection by in situ hybridization is much higher than Wright-Giemsa's stained (p<0.01) ,but has no significant difference compared with nested PC-ITS-PCR (p>0.05) . Positive rate of detection by PCR is much higher than Wright-Giemsa's stained (p<0.01) . No positive signal was observed in the rat tissue of the contrast group.Conclusion: RNA in situ hybridization is a simple and precise tool inpathological detection of PC. Positive rate of detection by RNA in situ hybridization and the nested PC-ITS-PCR method d is much higher than that by conventionally stained. With the improved Wright-Giemsa's stained to detect PC, the specificity is high. The improved Wright-Giemsa's stained is simple ,but the sensitivity is low.