In this study a commercially available multiplex real-time PCR (AsperGenius®) was evaluated for its efficacy in detecting Aspergillus fumigatus and azole resistance markers in comparison with conventional culture methods and galactomannan (GM) testing from BAL fluids in allogeneic HSCT recipients. Between January 2015 and May 2017 100 allogeneic HSCT recipients with pulmonary infiltrates and suspicion of invasive fungal infection were recruited to the study from a tertiary care center in Germany. BAL fluid was routinely assessed using the following diagnostic tests: AsperGenius® PCR assay, GM testing (cut-off: 1.0) and conventional culture. Susceptibility testing of azoles was performed by using Etest and, in case presenting elevated MICs, PCR for mutations in the cyp51A gene was carried out. Criteria of EORTC/MSG were used to classify the patients for invasive fungal disease. According to the EORTC/MSG criteria 23 patients presented with probable invasive aspergillosis (IA). Aspergillus PCR showed a sensitivity of 65% for probable IA cases. A combination of PCR and GM results in BAL displayed a sensitivity of 96% (22/23) and 100% specificity. Mutations in the cyp51A gene were detected by PCR in three cases (3/23; 13%) which were also found resistant with the culture method. In one case a Y121F/T289A mutation and in two cases a L98H were found. The combination of a commercial Aspergillus PCR assay and GM testing from BAL demonstrated a high sensitivity and specificity for diagnosing IA in allogeneic HSCT recipients. The Aspergillus PCR assay was not superior in detecting azole resistant A. fumigatus compared to culture.

Objectives Aspergillus fumigatus is the most prevalent filamentous fungus in the respiratory tract of patients with cystic fibrosis (CF). The aim of this prospective multicentre study was to investigate the prevalence of azole-resistant A. fumigatus (ARAF) in respiratory secretions from CF patients across Germany and to characterize ARAF isolates by phenotypic and molecular methods. Methods Twelve tertiary care centres from Germany participated in the study. In total, 2888 A. fumigatus isolates from 961 CF patients were screened for ARAF by using azole-containing agar plates. Antifungal susceptibility testing of isolates was performed by broth microdilution according to EUCAST guidelines. Analysis of mutations mediating resistance was performed using PCR and sequencing of the cyp51A gene. Furthermore, genotyping by microsatellite PCR was performed. Results Of a total of 2888 A. fumigatus isolates, 101 isolates from 51 CF patients were found to be azole resistant (prevalence per patient 5.3%). The Essen centre had the highest prevalence (9.1%) followed by Munich (7.8%), Münster (6.0%) and Hannover (5.2%). Most ARAF isolates (n = 89) carried the TR34/L98H mutation followed by eight G54E/R, one TR46/Y121F/T289A and one F219S mutation. In two isolates no mutation was found. Genotyping results showed no major clustering. Forty-five percent of CF patients with ARAF had previously received azole therapy. Conclusions This is the first multicentre study analysing the prevalence of ARAF isolates in German CF patients. Because of a resistance rate of up to 9%, susceptibility testing of A. fumigatus isolates from CF patients receiving antifungal treatment should be part of standard diagnostic work-up.

BACKROUND:Pneumocystis jirovecii pneumonia (PCP) is an opportunistic fungal infection that is associated with a high morbidity and mortality in immunocompromised individuals. In this study, we analysed the microbiome of the lower respiratory tract from critically ill intensive care unit patients with and without pneumocystosis.METHODS:Broncho-alveolar fluids from 65 intubated and mechanically ventilated intensive care unit patients (34 PCP+ and 31 PCP- patients) were collected. Sequence analysis of bacterial 16S rRNA gene V3/V4 regions was performed to study the composition of the respiratory microbiome using the Illumina MiSeq platform.RESULTS:Differences in the microbial composition detected between PCP+ and PCP- patients were not statistically significant on class, order, family and genus level. In addition, alpha and beta diversity metrics did not reveal significant differences between PCP+ and PCP- patients. The composition of the lung microbiota was highly variable between PCP+ patients and comparable in its variety with the microbiota composition of the heterogeneous collective of PCP- patients.CONCLUSIONS:The lower respiratory tract microbiome in patients with pneumocystosis does not appear to be determined by a specific microbial composition or to be dominated by a single bacterial species.

Members of the recently introduced fungal genus Rasamsonia (formerly included in the Geosmithia genus) have been described as emerging pathogens in immunosuppressed hosts or patients with cystic fibrosis (CF). Rasamsonia species have often been misidentified as Penicillium or Paecilomyces because of similar morphological characteristics. We validated a commercially available real-time PCR assay (Primerdesign™, UK) for accurate detection of species from the Rasamsonia argillacea complex. First, we tested this assay with a collection of 74 reference strains and clinical isolates and then compared the PCR with cultures of 234 respiratory samples from 152 patients with CF from two University Hospitals in Germany and France. The assay reliably detected the three main species within the Rasamsonia argillacea species complex (R. argillacea,R. piperina,R. aegroticola), which are typically encountered in CF patients. The limit of DNA detection was between 0.01 and 1 pg/μL. Analysis of the DNA extracts from respiratory specimens of CF patients revealed that four out of the 153 patients studied (2.6%) were colonized with R. argillacea species complex. Two species from the R. argillacea complex grew in the parallel cultures from the same patients. In one patient the PCR was positive 5 months before culture. The real-time PCR assay is a sensitive and specific method for detecting the three most important species of the R. argillacea species complex encountered in the CF context. Detection of these emerging pathogens in respiratory secretions from CF patients by this novel assay may increase our understanding of the occurrence and epidemiology of the R. argillacea species complex.

Background To describe a simple quantitative immunofluorescence assay (IFA) for the detection of specific Stenotrophomonas maltophilia antibodies in serum of CF patients. Methods A total of 100 sera (64 CF patients and 36 healthy subjects) were collected over a period of 2years at the University Hospital Essen, Germany. Sputum culture status classified CF patients into groups. Serologic response was determined after Pseudomonas aeruginosa absorption by indirect IFA to Sm whole cell. Results CF patients with “chronic S. maltophilia” showed significantly higher S. maltophilia antibody levels compared with healthy individuals (P<0.0001) and CF patients with “intermittent” (P=0.0315) or “never S. maltophilia/P. aeruginosa” (P=0.0002). A discriminant cut-off value of >1:120 titre was established to differentiate “CF chronic S. maltophilia” from the other groups. For “CF chronic S. maltophilia”, the IFA showed sensitivity and specificity values of 70.7% and 84.7%, respectively. Conclusion Our data demonstrated that quantitative IFA is a simple serological assay for the detection of specific S. maltophilia antibodies, which could be useful as a diagnostic tool for monitoring immune response of CF patients to S. maltophilia.

The laboratory identification of Pseudallescheria and Scedosporium isolates at the species level is important for clinical and epidemiological purposes. This study used semiautomated repetitive sequence-based polymerase chain reaction (rep-PCR) to identify Pseudallescheria/Scedosporium. Reference strains of Pseudallescheria boydii (n = 12), Scedosporium prolificans (n = 8), Scedosporium apiospermum (n = 9), and clinical/environmental isolates (P. boydii, 7; S. prolificans, 7; S. apiospermum, 7) were analyzed by rep-PCR. All clinical isolates were identified by morphological and phenotypic characteristics and by sequence analysis. Species identification of reference strains was based on the results of available databases. Rep-PCR studies were also conducted with various molds to differentiate Pseudallescheria/Scedosporium spp. from other commonly encountered filamentous fungi. All tested Pseudallescheria/Scedosporium isolates were distinguishable from the other filamentous fungi. All Scedosporium prolificans strains clustered within the cutoff of 85%, and species identification by rep-PCR showed an agreement of 100% with sequence analysis. However, several isolates of P. boydii and S. apiospermum did not cluster within the 85% cutoff with the same species by rep-PCR. Although the identification of P. boydii and S. apiospermum was not correct, the semiautomated rep-PCR system is a promising tool for the identification of S. prolificans isolates.