Early detection of Pneumocystis carinii in experimental rats with nested PCR

Abstract

Objective To investigate the sensitivity and significance of nested PCR with ITS1-5.8S rRNA-ITS2 for early detection of Pneumocystis carinii (P. carinii) in rats. Methods Infection of P. carinii in rats was established by subcutaneous injection of an immunodepressant, dexamethasone. The study was carried out in 7 groups, 10 rats each, including one uninfected group as control. Samples of bronchoalveolar lavage fluid (BALF) and lung tissue (LT) from the rats were collected and assayed weekly after the 3 rd week of immunedepression by the nested PCR. The primer for ITS1-5.8S rRNA-ITS2 of P. carinii was used for the nested PCR. Meanwhile LT and BALF smears stained with Gomori(s) methenamine silver (GMS) were examined under microscope for P. carinii. Results Aspecific marker site of ITS1-5.8SrRNA-ITS2 of P. carinii from the samples of LT and BALF in the infected rats were amplified by the nested PCR, and it was found that the positive rates during 3-8 weeks were 20% and 0, 70% and 10%, 90% and 30%, 90% and 80%, 100% and 80% ,63% and 63%, respectively. In comparison, P. carinii was observed by the microscopic examination only at the 5th week post-infection, while the number of cysts reached to the highest at the 7th and 8th week.The experimental rats showed no obvious pneumonia symptoms before the 4th week, but becoming apparent at the 5th week and there after. Conclusion The nested PCR to identify specific marker site ITS1-5.8S rRNA-ITS2 is a sensitive method and could detect P. carinii3 weeks after infection while no obvious pneumonia symptoms in rats, 2 weeks earlier than the traditional microscopic method. Key words: Pneumocystis carinii; Internal transcribed spacer; 5.8S rRNA; Nested PCR; Early detection