Abstract title 
Long-term pathology of the naturally-acquired Pneumocystis primary infection in rats.

Author: Prof Vargas MD, FIDSA, SL, Biomedical Sciences Institute, University of Chile School of Medicine, Santiago, Chile

Co-author(s): Ponce C.A (presenting author) Bustamante R., Iturra P.A., Rojas D.A., Méndez A. Biomedical Sciences Institute, University of Chile School of Medicine, Santiago, Chile

Objectives
To study whether the Pneumocystis primary infection induces long-lasting pathology is relevant and may suggest a role of this fungal infection in chronic airway disease. Pneumocystis is the most common pulmonary infection of healthy infants with a peak incidence between 2 and 5 months of age that goes undiagnosed. We have recently reported an animal model of this primary infection documenting that this infection induces lung damage consisting of increased mucus, airway epithelial thickening, perivascular and peribronchial infiltrates, and increased collagen suggesting chronic changes secondary to Pneumocystis in immunocompetent rats evaluated up to day 75 post-infection. This work extended our evaluations in the same model up to the age of 1 year.

Methods
120 newborn rats were kept in HEPA-filtered air containers throughout the duration of the experiment. They were randomized to exposure at birth to rats with Pneumocystis pneumonia by co-habitation for 48 hours, or to no-exposure and anti-Pneumocystis prophylaxis with TMP/SMZ during the duration of the experiment. Twelve rats per group were sacrificed each time at 40, 60, 80, 258, and 361 days of age under deep anaesthesia with Ketamine-Xylazine. Six of them to be used for histologic determinations were sacrificed by vascular perfusion of 3% buffered formalin via inferior cava vein at a pressure of 25 water cm, and their fixed lungs longitudinally sectioned to separate the proximal and distal lung zones for central and distal airway histologic determinations using Alcian Blue, H&E, and IHQ stains respectively, with Image Pro-plus software. The other 6 rats were used for Pneumocystis and collagen determinations. They were exsanguinated and their lungs removed, placed in RNA-later, and fresh frozen at -80°C until processing. Pneumocystis was diagnosed using nPCR and P. carinii-specific primers amplifying the mtLSUrRNA, and collagen levels were biochemically determined.

Results
Pneumocystis-DNA was amplified in none (0%) of the non-exposed rats throughout the experiment, and in 6/6(100%), 6/6(100%), 6/6(100%), 2/6(33%), 1/6(17%) of exposed rats at 40, 60, 81, 258, and 361 days respectively. Inflammatory cuffs significantly increased from day 81 and from day 60 up to day 361, respectively around bronchi and vessels. Infiltrates were more prominent and showed sustained increase around vessels. The area of airway epithelium occupied by mucins was 18 and 5 times higher on days 281 and 361 post infection, respectively, than measurements before day 80 in the Pneumocystis-exposed rats. Cuffing inflammation and the area of epithelium occupied by mucins did not vary along the experiment in the non-exposed rats. Collagen levels were not different in both groups throughout the experiment.

Conclusion
The primary infection by Pneumocystis induces long-term changes consisting of sustained peribronchial and perivascular inflammation and severely increased mucin secretion being consistent with long-term epithelium changes of mucous cells. These changes support a role of Pneumocystis in chronic airway disease.