Identification of Fungal Pathogens in Tissue Samples from Patients with Proven Invasive Infection by Fluorescence In Situ Hybridization.

Identification of fungal pathogens in clinical samples by hybridization with short oligonucleotide probes is increasingly used in the diagnosis of invasive fungal infections. Rapid and specific fungal identification has been documented in different diagnostic settings allowing for specific patient management. Identification of fungal pathogens in formalin-fixed, paraffin-embedded tissue samples appears to be rewarding as these materials are stored in pathology archives offering an insight into the etiology of deep fungal infections that is often not achieved by non-molecular tests. In contrast to PCR based methods, amplification of target sequences is unnecessary limiting the potential for contamination and localization within infected tissue is possible helping to distinguish between colonization and infection.

Cryptococcosis due to Cryptococcus gattii in Germany from 2004-2013.

The fungal pathogen Cryptococcus gattii was considered to be restricted to tropic and sub- tropic regions. A recent outbreak in North America due to isolates belonging to molecular type VG II, affecting mostly non-immunocompromised hosts, documented the potential public health impact of this fungal pathogen also in temperate regions. Surveillance of these infections in Germany is challenging, as cryptococcosis is not notifiable and often C. gattii is diagnostically not distinguished from the more prevalent Cryptococcus neoformans. We used hospital discharge data and identified cryptococcal isolates received by the German cryptococcosis reference laboratory at the species level to gain insights into the epidemiology of C. gattii-infections in Germany between 2004 and 2013. Between 49 and 60 (Median 57) hospitalizations for cryptococcosis are documented per year. Between 5 and 28 (Median 14) isolates were received at the reference laboratory per year. Among 155 single patient isolates, four C. gattii (3%) of the molecular types VGI and VG III were identified from patients with meningoencephalitis, including one interspecies hybrid. Patient histories and molecular typing suggest that half of the infections were acquired abroad. Only one patient survived the infection. C. gattii remains rarely identified as agent of cryptococcosis in Germany but underestimation is likely. Definition of environmental niches occupied by C. gattii in Germany may help to assess the associated risk of infection and prevent this deadly fungal infection.

Molecular typing of clinical Cryptococcus neoformans isolates collected in Germany from 2004 to 2010.

Cryptococcosis is a fungal infection mostly caused by Cryptococcus neoformans. We identified agents of cryptococcosis diagnosed in Germany from 2004 to 2010. We used multi-locus sequence typing (MLST) to understand the molecular epidemiology of cryptococcosis. Sero- and mating types of individual patient isolates were determined by PCR. MLST was performed using the seven-locus scheme. Allele and nucleotide diversity was calculated for each locus of C. neoformans var. grubii and C. neoformans var. neoformans. Phylogenetic relations were assessed by dendrograms. Clinical data were compared between infections caused by the two variants. We studied 101 isolates. Eight were identified as hybrids (8%). All non-hybrids were of the α mating type. Among 78 C. neoformans var. grubii (77%), 16 sequence types (STs) were identified including three novel STs. They clustered in four groups, previously isolated in Asia, Europe or worldwide. Among 15 C. neoformans var. neoformans (15%), 10 STs were identified, without clustering. These isolates showed higher allele, and nucleotide diversity compared with C. neoformans var. grubii. C. neoformans var. neoformans was more likely to cause soft-tissue infections (3/9, 33 vs. 1/63, 2%, p = 0.005) and to affect non-AIDS patients (7/14, 50 vs. 15/76, 20%, p = 0.036). C. neoformans var. grubii is the predominant agent of cryptococcosis in Germany. MLST suggests that a part of these cases are acquired abroad by immigrants or tourists. C. neoformans var. neoformans isolates represent a greater genetic diversity and are associated with more variable clinical presentations.