Predictors for prolonged hospital stay solely to complete intravenous antifungal treatment in patients with candidemia: Results from the ECMM candida III multinational European observational cohort study

Background To date, azoles represent the only viable option for oral treatment of invasive Candida infections, while rates of azole resistance among non-albicans Candida spp. continue to increase. The objective of this sub-analysis of the European multicenter observational cohort study Candida III was to describe demographical and clinical characteristics of the cohort requiring prolonged hospitalization solely to complete intravenous (iv) antifungal treatment (AF Tx). Methods Each participating hospital (number of eligible hospitals per country determined by population size) included the first ~ 10 blood culture proven adult candidemia cases occurring consecutively after July 1st, 2018, and treating physicians answered the question on whether hospital stay was prolonged only for completion of intravenous antifungal therapy. Descriptive analyses as well as binary logistic regression was used to assess for predictors of prolonged hospitalization solely to complete iv AF Tx. Findings Hospital stay was prolonged solely for the completion of iv AF Tx in 16% (100/621) of candidemia cases by a median of 16 days (IQR 8 – 28). In the multivariable model, initial echinocandin treatment was a positive predictor for prolonged hospitalization to complete iv AF Tx (aOR 2.87, 95% CI 1.55 – 5.32, p < 0.001), while (i) neutropenia, (ii) intensive care unit admission, (iii) catheter related candidemia, (iv) total parenteral nutrition, and (v) C. parapsilosis as causative pathogen were found to be negative predictors (aOR 0.22 – 0.45; p < 0.03). Interpretation Hospital stays were prolonged due to need of iv AF Tx in 16% of patients with candidemia. Those patients were more likely to receive echinocandins as initial treatment and were less severely ill and less likely infected with C. parapsilosis

Echinocandin resistance and population structure of invasive Candida glabrata isolates from two university hospitals in Germany and Austria

In dieser Arbeit wurde die Empfindlichkeit gegenüber Antimykotika, die Anwesenheit von FKS-Mutationen und die genetische Struktur von Candida glabrata-Isolaten aus Blutkulturen von zwei unterschiedlichen Krankenhäusern in Deutschland und Österreich untersucht. Die Empfindlichkeitstestung der 64 C. glabrata-Isolate wurde mittels Bouillon-Mikrodilution nach EUCAST und dem Vitek®-2 System durchgeführt. Zusätzlich wurden alle Isolate auf das Vorhandensein einer FKS-Mutation untersucht. Molekulargenetische Untersuchungen wurden mittels Mikrosatelliten-PCR mit drei unterschiedlichen Oligonukleotiden und halbautomatisierter repetitiver sequenz-basierter PCR (rep-PCR) durchgeführt. Grundsätzlich sollte bei allen C. glabrata-Isolaten, welche aus Blutkulturen isoliert werden, eine Resistenztestung durchgeführt werden. Das Vitek®-2 System ist eine routinetaugliche Alternative für die MHK-Bestimmung von Fluconazol, Amphotericin B und Caspofungin (CAS). Die Resistenztestung für Voriconazol sollte über die Bouillon-Mikrodilution nach EUCAST erfolgen. Anhand der Ergebnisse kann eine adäquate Therapieentscheidung bei einer Candidämie mit C. glabrata getroffen werden. Eine Initialtherapie sollte mit einem Echinocandin begonnen werden. Nur ein C. glabrata-Isolat aus Deutschland zeigte eine Resistenz gegenüber CAS und eine entsprechende Mutation in der Hotspot 1 Region auf dem FKS2-Gen. Die Diskriminationsstärke der Mikrosatelliten-PCR war höher, als die der rep-PCR (Simpson-Index von 0,94 vs. 0,88). Es zeigten sich prominente Genotypen von Isolaten aus Deutschland und Österreich, allerdings ohne epidemiologische Hinweise nosokomialer Übertragungen. Wir konnten zeigen, dass die Populationsstruktur von C. glabrata hauptsächlich polyklonal mit spezifischen Genotypen und abhängig von der geografischen Lokalisation der Isolate ist. Obwohl wir eine geringe Inzidenz von Echinocandin-Resistenzen in C. glabrata-Isolaten gefunden haben, sind weitere Studien zur Überwachung epidemiologischer Trends in Europa notwendig. Die genetische Populationsstruktur der C. glabrata-Isolate demonstriert überrepräsentative geographische Genotypen.In this work, the susceptibility of antifungal agents, the presence of FKS-mutations and the population-genetics of Candida glabrata were investigated. C. glabrata isolates from blood cultures were taken from two different sampling sites, the University Hospital Essen and the University Hospital Vienna for analysis. The susceptibility testing of a total of 64 C. glabrata isolates were carried out after EUCAST, using the microdilution method as well as by the use of Vitek®-2 System. In addition, each isolate FKS-mutations were investigated. Genetics were investigated by molecular-biological tools, microsatellite PCR including three oligonucleotides and by half-automated repetitive sequenced-based PCR (rep-PCR). The aim of this study was to identify resistances in the tested isolates against fluconazole, amphotericin B, caspofungin and voriconazole. The above mentioned methods were tested on efficiency for this purpose. After comparison of all methods, the Vitek®-2 System is recommended for susceptibility testing including the determination of minimum inhibitory concentration (MIC) of fluconazole, amphotericin B and caspofungin. In contrast, the susceptibility against voriconazole should be tested in a microdilution, as recommended by EUCAST. On behalf of these results, an initial therapy could be started, mostly recommended using an echinocandin. Only one isolate (1.5 %) was detected with a hotspot mutation in the FKS2-gene, resulting in a resistance against caspofungin. The analysis of the molecular methods revealed higher discriminatory power for the microsatellite-PCR compared to the rep-PCR (Simpson-Index 0.94 vs. 0.88). Prominent genotypes were detected in isolates from both hospitals. However, epidemiological, no nosocomial route of transmission could be detected. We could show that the population structure of C. glabrata is mainly polyclonal with specific genotypes, depending on the geographical origin of the isolate. In conclusion, we found weak incidence of echinocandin resistance in C. glabrata. However, further epidemiological screening in Europe is recommended. The population structure of the C. glabrata isolates shows the overrepresented geographical genotypes

Evaluation of Three Commercial PCR Assays for the Detection of Azole-Resistant Aspergillus fumigatus from Respiratory Samples of Immunocompromised Patients

This is the first study comparing three commercially available PCR assays for the detection of Aspergillus DNA from respiratory specimen of immunocompromised patients and the presence of cyp51A gene mutations. Bronchoalveolar lavages (BALs, N = 103) from patients with haematological/oncological underlying diseases were retrospectively investigated. The performance of three PCR assays, namely MycoGENIE®Aspergillus fumigatus Real-Time PCR Kit (Adamtech), Fungiplex®Aspergillus Azole-R IVD Real-Time PCR Kit (Bruker Daltonik GmbH) and AsperGenius® (PathoNostics B.V.), were evaluated. All patients were categorised following current EORTC/MSG criteria, with exclusion of the PCR-results. From the 11 invasive pulmonary aspergillosis (IPA) probable samples, eight were detected with MycoGENIE®, resulting in a sensitivity of 80% and a specificity of 73%. Furthermore, Fungiplex® resulted in six positive BALs with a sensitivity of 60% and a specificity of 91% and AsperGenius® in seven positive BAL samples, with a sensitivity of 64% and a specificity of 97%. No proven IPA was detected. One isolate showed phenotypically an azole-resistance, which was also detected in each of the tested PCR assays with the mutation in TR34. The here tested PCR assays were capable of reliably detecting A. fumigatus DNA, as well as differentiation of the common cyp51A gene mutations. However, evaluation on the AsperGenius® assay revealed a low risk of false positive results

Microbiological Non-Culture-Based Methods for Diagnosing Invasive Pulmonary Aspergillosis in ICU Patients

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

Evaluation of Three Commercial PCR Assays for the Detection of Azole-Resistant <i>Aspergillus fumigatus</i> from Respiratory Samples of Immunocompromised Patients

This is the first study comparing three commercially available PCR assays for the detection of Aspergillus DNA from respiratory specimen of immunocompromised patients and the presence of cyp51A gene mutations. Bronchoalveolar lavages (BALs, N = 103) from patients with haematological/oncological underlying diseases were retrospectively investigated. The performance of three PCR assays, namely MycoGENIE®Aspergillus fumigatus Real-Time PCR Kit (Adamtech), Fungiplex®Aspergillus Azole-R IVD Real-Time PCR Kit (Bruker Daltonik GmbH) and AsperGenius® (PathoNostics B.V.), were evaluated. All patients were categorised following current EORTC/MSG criteria, with exclusion of the PCR-results. From the 11 invasive pulmonary aspergillosis (IPA) probable samples, eight were detected with MycoGENIE®, resulting in a sensitivity of 80% and a specificity of 73%. Furthermore, Fungiplex® resulted in six positive BALs with a sensitivity of 60% and a specificity of 91% and AsperGenius® in seven positive BAL samples, with a sensitivity of 64% and a specificity of 97%. No proven IPA was detected. One isolate showed phenotypically an azole-resistance, which was also detected in each of the tested PCR assays with the mutation in TR34. The here tested PCR assays were capable of reliably detecting A. fumigatus DNA, as well as differentiation of the common cyp51A gene mutations. However, evaluation on the AsperGenius® assay revealed a low risk of false positive results

Prevalence of COVID-19 Associated Mucormycosis in a German Tertiary Care Hospital

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

Comparison of genotyping methods for Cunninghamella bertholletiae

Background Invasive fungal infections caused by filamentous fungi of the order Mucorales are serious complications in immunocompromised patients and often associated with fatal outcome. As a member of this order, Cunninghamella bertholletiae is a saprophytic fungus with naturally exhibited high minimum inhibitory concentrations against common antifungal drugs and with the potential for outbreaks in clinical settings. Objectives and methods In a proof-of-principle study, we evaluated the performance of microsatellite markers for the discrimination of thirteen C. bertholletiae isolates from various sources in comparison with a repetitive sequence-based PCR (rep-PCR) and random amplification of polymorphic DNA (RAPD). Based on the higher discriminatory power of the microsatellite PCR with five separate primer pairs (Simpson's index of 1 vs 0 [RAPD] and 0 [rep-PCR]), the novel method was applied to eight additional isolates, including four well-characterised isolates from a cluster of infections in a next step. Results In total, microsatellite PCR identified 21 separate genotypes. A probable epidemiological association of the cluster isolates could be demonstrated by microsatellite genotyping. Conclusion In conclusion, our findings demonstrate the value of microsatellite PCR in genotyping Cunninghamella bertholletiae and its potential for future applications with other species of the order Mucorales

Variable Correlation between Bronchoalveolar Lavage Fluid Fungal Load and Serum-(1,3)-β-d-Glucan in Patients with Pneumocystosis-A Multicenter ECMM Excellence Center Study.

Pneumocystis jirovecii pneumonia is a difficult invasive infection to diagnose. Apart from microscopy of respiratory specimens, two diagnostic tests are increasingly used including real-time quantitative PCR (qPCR) of respiratory specimens, mainly in bronchoalveolar lavage fluids (BAL), and serum β-1,3-d-glucan (BDG). It is still unclear how these two biomarkers can be used and interpreted in various patient populations. Here we analyzed retrospectively and multicentrically the correlation between BAL qPCR and serum BDG in various patient population, including mainly non-HIV patients. It appeared that a good correlation can be obtained in HIV patients and solid organ transplant recipients but no correlation can be observed in patients with hematologic malignancies, solid cancer, and systemic diseases. This observation reinforces recent data suggesting that BDG is not the best marker of PCP in non-HIV patients, with potential false positives due to other IFI or bacterial infections and false-negatives due to low fungal load and low BDG release.status: Published onlin