COVID-19 Co-infection with Legionella pneumophila in 2 Tertiary-Care Hospitals, Germany

Background: COVID‐19‐associated invasive pulmonary aspergillosis (CAPA) is associated with increased mortality. Cases of CAPA caused by azole‐resistant Aspergillus fumigatus strains have been reported.Objectives: To analyse the twelve‐month CAPA prevalence in a German tertiary care hospital and to characterise clinical A. fumigatus isolates from two German hospitals by antifungal susceptibility testing and microsatellite genotyping.Patients/Methods.Retrospective observational study in critically ill adults from intensive care units with COVID‐19 from 17 February 2020 until 16 February 2021 and collection of A. fumigatus isolates from two German centres. EUCAST broth microdilution for four azole compounds and microsatellite PCR with nine markers were performed for each collected isolate (N = 27) and additional for three non‐COVID A. fumigatus isolates.Results: welve‐month CAPA prevalence was 7.2% (30/414), and the rate of azole‐resistant A. fumigatus isolates from patients with CAPA was 3.7% with detection of one TR34/L98H mutation. The microsatellite analysis revealed no major clustering of the isolates. Sequential isolates mainly showed the same genotype over time.Conclusions: Our findings demonstrate similar CAPA prevalence to other reports and a low azole‐resistance rate. Genotyping of A. fumigatus showed polyclonal distribution except for sequential isolates.

The diagnosis of invasive pulmonary aspergillosis (IPA) in intensive care unit (ICU) patients is crucial since most clinical signs are not specific to invasive fungal infections. To detect an IPA, different criteria should be considered. Next to host factors and radiological signs, microbiological criteria should be fulfilled. For microbiological diagnostics, different methods are available. Next to the conventional culture-based approaches like staining and culture, non-culture-based methods can increase sensitivity and improve time-to-result. Besides fungal biomarkers, like galactomannan and (1→3)-β-D-glucan as nonspecific tools, molecular-based methods can also offer detection of resistance determinants. The detection of novel biomarkers or targets is promising. In this review, we evaluate and discuss the value of non-culture-based microbiological methods (galactomannan, (1→3)-β-D-glucan, Aspergillus PCR, new biomarker/targets) for diagnosing IPA in ICU patients.

Numbers of infections with azole-resistant Aspergillus fumigatus (ARAf) were rising in the last decades. We assessed ARAf susceptibility trends towards five antifungal agents (amphotericin B (AMB), itraconazole (ITR), voriconazole (VCZ), olorofim (OLO) and manogepix (MGX)) over twelve years in a German Excellence Center for Medical Mycology (ECMM). In addition, underlying mutations were studied and correlated with trends in minimum inhibitory concentration (MIC). Broth microdilution (BMD) was performed following EUCAST guidelines for 143 clinical ARAf isolates collected between the years 2011 and 2022 in a West German tertiary care centre. BMD was carried out for all antifungal agents in the following concentration ranges: 0.016–8 mg/L for AMB, ITR and VCZ as well as 0.001–0.5 mg/L for OLO and 0.004–2 mg/L for MGX. Molecular assays on mutations associated with antifungal resistance were performed for all 143 isolates (AsperGenius® 1.0, Pathonostics, Maastricht, The Netherlands) and for a total of ten non TR₃₄/L98H and TR₄₆/Y121F/T289A mutated ARAf isolates additional cyp51A sequencing was carried out. For all isolates, microdilution revealed a MIC₅₀ of > 8 mg/L for ITR, 4 mg/L for VCZ, 0.03 mg/L for OLO, 0.016 mg/L for MGX, and 0.5 mg/L for AMB. Considering EUCAST breakpoints, 97.9% of the strains (n = 140) were resistant to VCZ, 1.4% (n = 2) towards AMB and 92.3% towards ITR (n = 132). Molecular assays revealed 123 (86%) isolates with the azole resistance underlying mutation TR₃₄/L98H, 10 (7%) with a TR₄₆/Y121F/T289A mutation and 10 (7%) with other cyp51A mutations. A comparison of triazole MICs of isolates collected from 2011 to 2019 with the MICs of isolates collected between 2020 and 2022 revealed no significant differences for itraconazole (p = 0.543) and for voriconazole (p = 0.148),with a trend of increased geometric mean for ITR and VCZ MICs over time. MICs for OLO and MGX did not significantly differ between isolates with the distinct azole-resistance underlying mutations. Before 2016, the azole resistance underlying mutations were mainly TR₃₄/L98H, but the portion of isolates with TR₄₆/Y121F/T289A and other Cyp51A mutated isolates increased afterwards. We showed almost stable MICs for ITR and VCZ over twelve years in ARAf isolates from West Germany while occurring azole resistance underlying mutations varied with an increase in the proportion of TR₄₆/Y121F/T289A and other Cyp51A mutations after 2016.

Cystic fibrosis (CF) is an autosomal recessive disorder caused by the deficiency of the cystic fibrosis transmembrane conductance regulator (CFTR) and often leads to pulmonary infections caused by various pathogens, including Staphylococcus aureus, Pseudomonas aeruginosa, and nontuberculous mycobacteria, particularly Mycobacterium abscessus. Unfortunately, M. abscessus infections are increasing in prevalence and are associated with the rapid deterioration of CF patients. The treatment options for M. abscessus infections are limited, requiring the urgent need to comprehend infectious pathogenesis and develop new therapeutic interventions targeting affected CF patients. Here, we show that the deficiency of CFTR reduces sphingosine levels in bronchial and alveolar epithelial cells and macrophages from CF mice and humans. Decreased sphingosine contributes to the susceptibility of CF tissues to M. abscessus infection, resulting in a higher incidence of infections in CF mice. Notably, treatment of M. abscessus with sphingosine demonstrated potent bactericidal activity against the pathogen. Most importantly, restoration of sphingosine levels in CF cells, whether human or mouse, and in the lungs of CF mice, provided protection against M. abscessus infections. Our findings demonstrate that pulmonary sphingosine levels are important in controlling M. abscessus infection. These results offer a promising therapeutic avenue for CF patients with pulmonary M. abscessus infections.

Due to Coronavirus disease (COVID-19) a new group of patients at risk emerged with COVID-19-associated mucormycosis (CAM). Systematic studies, evaluating the prevalence of CAM are missing. To assess CAM prevalence in a tertiary care hospital in Germany, we applied direct microscopy, fungal culture and quantitative realtime in-house PCR targeting Mucorales-specific fragments of 18S and 28S rRNA on respiratory specimens of 100 critically ill COVID-19 patients. Overall, one Mucorales-PCR positive bronchoalevolar lavage was found whereas direct microscopy and fungal culture were negative in all cases. We conclude that a routine screening for CAM in Germany is not indicated.