Identification of extrapulmonary Pneumocystis carinii in immunocompromised rats by PCR

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Abstract
Pneumocystis carinii has been shown to cause extra-alveolar infections in humans, but the lack of a reproducible animal model has hindered the elucidation of mechanisms of P. carinii dissemination. In the present study, PCR and the immunosuppressed rat model were used to gain further insight into the dissemination of P. carinii organisms in extrapulmonary (EP) tissues. Primer sequences specific to major surface glycoprotein (MSG) and dihydrofolate reductase (DHFR) were used to detect P. carinii in lungs and EP tissues. Sprague-Dawley rats were grouped into two classes: one group included rats that had primary episodes of pneumocystosis and the other group included rats that had undergone treatment for P. carinii infection and that had second episodes of pneumocystosis. PCR analysis with MSG primers with tissues obtained from both groups of rats showed the presence of P. carinii DNA in adrenal tissue, bone marrow, blood, and heart, kidney, liver, lymph node, spleen, and thyroid tissues. Reverse transcriptase-PCR (RT-PCR) analysis was carried out with DHFR primers with lung, spleen, heart, kidney, and liver tissues from both groups of rats. Only those tissues that showed a positive PCR result and hybridization signal for the MSG gene were used for the RT-PCR experiments. RT-PCR analysis showed that the P. carinii DHFR gene is actively transcribed in these tissues, thereby indicating the presence of viable P. carinii organisms in EP tissues. Our observations suggest that P. carinii dissemination is influenced by factors other than P. carinii chemotherapy and that heavy organism load and destruction of lung tissue may contribute to the dissemination of P. carinii. The study provides an animal model that can be used for further investigations of the causes of EP pneumocystosis.