Pneumocystis jirovecii Can Be Productively Cultured in Differentiated CuFi-8 Airway Cells

Abstract

Although Pneumocystis jirovecii is a well-known and serious pathogen, all previous attempts to isolate, cultivate, and propagate this fungus have failed. This serious challenge in microbiology was addressed in the present study. We examined whether P. jirovecii could be cultured in a permanent three-dimensional air-liquid interface culture system formed by CuFi-8 cells, a differentiated pseudostratified airway epithelial cell line. Cultured pseudostratified cells were inoculated with bronchoalveolar fluid that had been confirmed to be positive for P. jirovecii using PCR. Five days later, the cells and basal medium were harvested and tested for P. jirovecii using quantitative PCR (qPCR), commercially available immunofluorescence detection assays, and Grocott staining of formalin-fixed, paraffin-embedded thin sections of infected-cell cultures. We successfully productively cultivated and propagated P. jirovecii from these P. jirovecii-positive bronchoalveolar lavage fluid (BALF) samples. Furthermore, we provide evidence that P. jirovecii induced cytopathic effects on lung epithelial cells and was even invasive in cell culture. To the best of our knowledge, the cell culture system developed herein represents the first methodology to enable molecular analyses of this pathogen’s life cycle and further in vitro studies of P. jirovecii, such as assessments of drug sensitivity and resistance as well as investigations of the pathogen’s stability against environmental factors and disinfectants.

Epidemiological Outbreaks of Pneumocystis jirovecii Pneumonia Are Not Limited to Kidney Transplant Recipients: Genotyping Confirms Common Source of Transmission in a Liver Transplantation Unit

“…Pneumocystis jirovecii replication cannot be obtained routinely in culture (7), so PCP diagnosis relies on microscopic observation of respiratory samples, mostly bronchoalveolar lavage fluid (BALF) samples, or on molecular detection of P. jirovecii DNA by realtime quantitative PCR (qPCR) (1,8). These techniques are not suitable for investigating the transmission routes of P. jirovecii (1,9) or for epidemiological investigations of a PCP outbreak (10).…”

Diagnosis and management ofPneumocystis jiroveciiinfection

“…32 result. 48 Even when using higher positivity thresholds BDG specificity when testing BAL 236 fluid remained compromised (241pg/ml: 39%; 783pg/ml 79%).…”

“…Several methods using various co-culture cell lines were described but 141 failed to attain widespread use. 32 Most attempts have used rat-models and 142 subsequently P. carinii not P. jirovecii. In 1999, P. carinii initially isolated from rat lung 143 was cultured using continuous axenic cultivation.…”

Impact of HIV Infection Status on Interpretation of Quantitative PCR for Detection of Pneumocystis jirovecii

“…While an in vitro culture system for Pneumocystis has recently been described (5), laboratory diagnosis of PCP currently relies on the demonstration of cysts and/or trophic forms of P. jirovecii directly from respiratory samples, such as bronchoalveolar lavage (BAL) fluid samples or induced sputum (6). The fungal elements can be visualized using standard staining, such as Giemsa staining, silver staining, or toluidine blue staining, but a direct immunofluorescence assay, notably one targeting the cysts, significantly enhances the sensitivity of detection (7).…”