Molecular typing and in vitro resistance of Cryptococcus neoformans clinical isolates obtained in Germany between 2011 and 2017.

Cryptococcosis is a fungal infection of the central nervous system predominantly caused by Cryptococcus neoformans in immunocompromised patients. In several countries worldwide, up to 50% of isolates show in vitro resistance to clinically used antifungals including fluconazole. No prospective data on susceptibility to antifungal drugs are available for Germany. In this study, we characterised all C. neoformans isolates collected from individual patients' samples at the German reference laboratory for cryptococcosis 2011 and 2017 (n = 133) by multi-locus sequence typing and phenotypic drug susceptibility testing. We identified serotype A/genotype VNI isolates belonging to clonal complexes previously described from Europe, Africa, Asia and South America as the most prevalent agents of cryptococcosis in Germany. Overall, we observed minimal inhibitory concentrations (MICs) above the epidemiological cut-offs (ECVs) in 1.6% of isolates regarding fluconazole and 2.3% of isolates regarding 5-flucytosine. Here, two C. neoformans var. grubii isolates displayed decreased drug susceptibility to fluconazole, one of them additionally to 5-flucytosine. We also found 5-flucytosine MICs above the ECV for two C. neoformans var. neoformans isolates. We identified a novel mutation in the ERG11 gene which might be associated with the elevated fluconazole MIC in one of the isolates. The clinical importance of the detected in vitro resistance is documented by patient histories showing relapsed infection or primary fatal disease. Of note, sertraline demonstrated antifungal activity comparable to previous reports. Systematic collection of susceptibility data in combination with molecular typing of C. neoformans is important to comprehensively assess the spread of isolates and to understand their drug resistance patterns.

Pitfalls in Serological Diagnosis of Cryptococcus gattii Infections.

The detection of cryptococcal antigen by latex agglutination tests (LATs), enzyme-linked immunoassays (ELISA), or lateral flow assay (LFA) is an important tool for diagnosis of a Cryptococcus infection. Cerebrospinal fluid and/or serum samples of 10 patients with cryptococcosis due to Cryptococcus gattii or a hybrid of Cryptococcus neoformans and C. gattii were examined by three LATs (the IMMY Latex-Crypto(®) test, the Pastorex(TM) Crypto Plus, and the Remel Cryptococcus Antigen Test Kit) and the LFA made by Immuno-Mycologics. LATs based on monoclonal antibodies (mAbs) like the Pastorex(TM) Crypto Plus or the Remel Cryptococcus Antigen Test Kit turned out to have an insufficient sensitivity to detect four out of 10 C. gattii infections, including one infection by a hybrid between C. gattii and C. neoformans. Reflecting the ongoing expansion of C. gattii in geographical zones outside of tropical and subtropical areas like Mediterranean countries, Vancouver Island (British Columbia, Canada) and the Pacific Northwest region (USA), these findings are alarming because of the risk of delayed diagnosis of infections caused by C. gattii. Therefore, the preliminary serological screening for cryptococcal antigen in the case of a suspected Cryptococcus infection should be performed by using an assay with a broad range specificity and sensitivity for C. neoformans and C. gattii, including their hybrids.

Comparison of susceptibility and transcription profile of the new antifungal hassallidin A with caspofungin.

This is the first report on the antifungal effects of the new glycolipopeptide hassallidin A. Due to related molecular structure moieties between hassallidin A and the established antifungal drug caspofungin we assumed parallels in the effects on cell viability. Therefore we compared hassallidin A with caspofungin by antifungal susceptibility testing and by analysing the genome-wide transcriptional profile of Candida albicans. Furthermore, we examined modifications in ultracellular structure due to hassallidin A treatment by electron microscopy. Hassallidin A was found to be fungicidal against all tested Candida species and Cryptococcus neoformans isolates. MICs ranged from 4 to 8 microg/ml, independently from the species. Electron microscopy revealed noticeable ultrastructural changes in C. albicans cells exposed to hassallidin A. Comparing the transcriptional profile of C. albicans cells treated with hassallidin A to that of cells exposed to caspofungin, only 20 genes were found to be similarly up- or down-regulated in both assays, while 227 genes were up- or down-regulated induced by hassallidin A specifically. Genes up-regulated in cells exposed to hassallidin A included metabolic and mitotic genes, while genes involved in DNA repair, vesicle docking, and membrane fusion were down-regulated. In summary, our data suggest that, although hassallidin A and caspofungin have similar structures, however, the effects on susceptibility and transcriptional response to yeasts seem to be different.