A 57-year-old woman was hospitalized due to dyspnoea, cough, and bilateral interstitial infiltrates on chest X-ray (Figure 1a). She moved from Venezuela to Germany a year prior to presentation. A chest computed tomography (CT) showed diffuse interstitial changes (Figure 1b). Flexible bronchoscopy revealed a black exophytic mass migrating through the left main bronchus wall. Subsequently, a peripheral lung biopsy was obtained using video-assisted thoracoscopy (VATS). Histological analysis of the peripheral lung biopsy by local pathology showed large interstitial granulomas, giant cells, and no necrosis or signs of vasculitis, compatible with sarcoidosis. In combination with elevated serum interleukin-2-receptor levels (1654 U/ml, normal 220-710 U/ml), pulmonary sarcoidosis was diagnosed and systemic corticosteroid therapy (1 mg per kg bodyweight) was initiated; 3 months later clinic and chest CT remained unchanged (Figure 1c), as did the lesion in the left main bronchus under bronchoscopy. A lesional biopsy revealed necrosis and inflammation. Viridans streptococci were isolated from the broncheoalveolar lavage (BAL), as well as low levels of unspecified yeast after 7 days of incubation. The latter was not found on microscopic analysis of the BAL or, at the time of testing, in the lesional biopsy. Meropenem was initiated but the patient developed progressive respiratory failure requiring noninvasive ventilation (NIV) and was transferred to our university clinic.
Subsequent re-examination of the initial (VATS) lung biopsy by our pathologists led to the detection of intracellular Grocott positive particles of 2-5 microm, accompanied by giant cell pneumonia, suggestive of a yeast infection. While initial stains showed small yeasts more in line with Histoplasmosis, more in depth examination of the samples showed multi-budded yeasts (Figure 1d) more closely resembling Paracoccidioides, albeit not matching the textbook description of a multipolar 'pilot's wheel' configuration. Due to this ambiguity, we were unable to differentiate between these fungi by histology alone.
Due to the histologic finding, and since the patient had recently moved from a region endemic for dimorphic fungal pathogens, serologic testing was performed for histoplasmosis using immunodiffusion (ID) and complement fixing (CF) methods, as well as for paracoccidioidomycosis (ID) and coccidioidomycosis (ID, CF). Histoplasma and Paracoccidioides antibodies were detected by ID. However, a lack of antibodies against Histoplasma in the CF-test was interpreted as a likely cross-reaction in Histoplasma ID and evidence for paracoccidioidomycosis. CF testing for PCM was not available at our laboratory. In addition, a specific qPCR assay targeting the ITS-1 region of Histoplasma and broad range PCR assays targeting the 28S and ITS-2 region were performed from the formalin-fixed, paraffin-embedded (FFPE) lung biopsy as described previously. While correct DNA extraction was shown by amplification of the human 18S rRNA gene, and an internal amplification control excluded PCR inhibition, both broadrange fungal PCRs amplified DNA. Negative Mastermix-, and extraction controls ruled out contamination with fungal DNA. Sequencing of both broad range PCR amplicons showed 99.7% pairwise identity with sequences of Paracoccidioides brasiliensis as the causative pathogen. The specific Histoplasma PCR did not amplify DNA.
In conclusion, the patient had paracoccidioidomycosis as suggested by the detection of antibodies, histology (Figure 1d,e) and PCR from FFPE tissue. Cerebral or abdominal involvement was ruled out by cMRI and abdominal ultrasound. Sarcoidosis was excluded after histopathological re-evaluation. We initiated a therapy using liposomal amphotericin B 150 mg once per day. Within 3 days, the respiratory situation improved and noninvasive ventilation was discontinued. After 3 weeks, therapy was switched to oral itraconazole 200 mg twice daily, and, after 8 weeks of antifungal treatment, oxygenation had returned to baseline and no additional oxygen supplementation was required. Chest CT showed decreasing pulmonary infiltrates (Figure 1f) which resolved completely after 6 months (Figure 1g). To monitor the lesion in the left main bronchus, another flexible bronchoscopy was performed after 6 months, showing a decrease in size of the fungal lesion (Figure 1h). However, in the BAL, yeasts were still detected by microscopy but fungal culture and PCR remained negative. Treatment with itraconazole 200 mg twice daily was continued.