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.

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)-ß-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)-ß-D-glucan, Aspergillus PCR, new biomarker/targets) for diagnosing IPA in ICU patients.

European candidaemia is characterised by notable differential epidemiology and susceptibility pattern: Results from the ECMM Candida III study.

The objectives of this study were to assess Candida spp. distribution and antifungal resistance of candidaemia across Europe. Isolates were collected as part of the third ECMM Candida European multicentre observational study, conducted from 01 to 07-07-2018 to 31-03-2022. Each centre (maximum number/country determined by population size) included ∼10 consecutive cases. Isolates were referred to central laboratories and identified by morphology and MALDI-TOF, supplemented by ITS-sequencing when needed. EUCAST MICs were determined for five antifungals. fks sequencing was performed for echinocandin resistant isolates. The 399 isolates from 41 centres in 17 countries included C. albicans (47.1%), C. glabrata (22.3%), C. parapsilosis (15.0%), C. tropicalis (6.3%), C. dubliniensis and C. krusei (2.3% each) and other species (4.8%). Austria had the highest C. albicans proportion (77%), Czech Republic, France and UK the highest C. glabrata proportions (25-33%) while Italy and Turkey had the highest C. parapsilosis proportions (24-26%). All isolates were amphotericin B susceptible. Fluconazole resistance was found in 4% C. tropicalis, 12% C. glabrata (from six countries across Europe), 17% C. parapsilosis (from Greece, Italy, and Turkey) and 20% other Candida spp. Four isolates were anidulafungin and micafungin resistant/non-wild-type and five resistant to micafungin only. Three/3 and 2/5 of these were sequenced and harboured fks-alterations including a novel L657W in C. parapsilosis. The epidemiology varied among centres and countries. Acquired echinocandin resistance was rare but included differential susceptibility to anidulafungin and micafungin, and resistant C. parapsilosis. Fluconazole and voriconazole cross-resistance was common in C. glabrata and C. parapsilosis but with different geographical prevalence.

In Vitro and In Vivo Effect of the Imidazole Luliconazole against Lomentospora prolificans and Scedosporium spp.

Infections with Scedosporium spp. and Lomentospora prolificans have become a serious threat in clinical settings. The high mortality rates associated with these infections can be correlated with their multidrug resistance. The development of alternative treatment strategies has become crucial. Here, we investigate the in vitro and in vivo activity of luliconazole (LLCZ) against Scedosporium apiospermum (including its teleomorph Pseudallescheria boydii) and Lomentospora prolificans. The LLCZ MICs were determined for a total of 37 isolates (31 L. prolificans isolates, 6 Scedosporium apiospermum/P. boydii strains) according to EUCAST. Furthermore, the LLCZ antifungal activity was tested in vitro, using an XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt] growth kinetics assay and biofilm assays (crystal violet and XTT assay). In addition, a Galleria mellonella infection model was used for in vivo treatment assays. The MIC90 of LLCZ was determined to be 0.25 mg/L for all tested pathogens. Growth was inhibited within 6 to 48 h of the start of incubation. LLCZ inhibited biofilm formation in both preadhesion stages and late-stage adhesion. In vivo, a single dose of LLCZ increased the survival rate of the larvae by 40% and 20% for L. prolificans and Scedosporium spp., respectively. This is the first study demonstrating LLCZ activity against Lomentospora prolificans in vitro and in vivo and the first study showing the antibiofilm effect of LLCZ in Scedosporium spp. IMPORTANCE Lomentospora prolificans and S. apiospermum/P. boydii are opportunistic, multidrug-resistant pathogens causing invasive infections in immunosuppressed patients and sometimes in healthy persons. Lomentospora prolificans is panresistant against the currently available antifungals, and both species are associated with high mortality rates. Thus, the discovery of novel antifungal drugs exhibiting an effect against these resistant fungi is crucial. Our study shows the effect of luliconazole (LLCZ) against L. prolificans and Scedosporium spp. in vitro, as well as in an in vivo infection model. These data reveal the previously unknown inhibitory effect of LLCZ against L. prolificans and its antibiofilm effect in Scedosporium spp. It represents an extension of the literature regarding azole-resistant fungi and could potentially lead to the development of future treatment strategies against these opportunistic fungal pathogens.

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.

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.

Variable Correlation between Bronchoalveolar Lavage Fluid Fungal Load and Serum-(1,3)-ß-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 ß-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.

Evaluation of two lateral flow assays in BAL fluids for the detection of invasive pulmonary aspergillosis: A retrospective two-centre study.

BACKGROUND: Commonly, the application of radiological and clinical criteria and the determination of galactomannan (GM) in respiratory samples are used as a diagnostic tool for the detection of invasive pulmonary aspergillosis (IPA). MATERIALS/METHODS: In this study, two lateral flow assays, OLM Aspergillus lateral flow device (LFD) and IMMY sona Aspergillus Galactomannan lateral flow assay (LFA), were evaluated at two tertiary hospitals in Germany. A total of 200 bronchoalveolar lavage (BAL) samples from patients with suspicion of IPA were analysed retrospectively. LFD and LFA were evaluated against four different criteria: Blot, EORTC/MSG, Schauwvlieghe and extended Blot criteria and additionally against GM. RESULTS: The evaluation of four algorithms for the diagnosis of IPA showed that there exist good diagnostic tools to rule out an IPA even before results of Aspergillus culture are available. Sensitivities and negative predictive values are generally higher for the LFA than for the LFD in all four criteria. Specificity and positive predictive values varied depending on the classification criteria. The total agreement between the GM and the LFA cube reader (cut-off = 1) was 84%. The correlation between the GM and LFA was calculated with r = 0.8. CONCLUSION: The here presented data indicate that a negative LFA result in BAL fluid can reliable rule out an IPA in a heterogeneous group of ICU patients based on the original Blot criteria. LFA seems to be a promising immunochromatographic test exhibiting a good agreement with positive GM values.

Evaluation of a commercial Loop-mediated Isothermal Amplification (LAMP) assay for rapid detection of Pneumocystis jirovecii.

BACKGROUND: Various tools are obtainable for the detection of Pneumocystis jirovecii, among them qPCR promising highest sensitivity. A novel molecular method is commercially available, the loop-mediated isothermal amplification (LAMP) assay. OBJECTIVES: We compared the performance of the LAMP eazyplex® Pneumocystis jirovecii with the RealStar Pneumocystis jirovecii PCR 1.0 qPCR. MATERIAL/METHODS: Overall, 162 lower respiratory tract specimens from 146 critically ill patients were investigated. LAMP assay and qPCR were carried out according to the manufacturer's recommendations. Positive results of the LAMP were described as time to positivity (TTP). The limit of detection (LOD) of the LAMP was analysed using 10-fold serial dilutions of a high positive P jirovecii respiratory sample. For each serial dilution, TTP of the LAMP was plotted against cycle threshold (Ct) values of the qPCR. RESULTS: The LOD of the LAMP was determined to be approximately 4 × 103 copies/mL. While the LAMP revealed 28 (17%) positive signals from 20 patients, by using qPCR 41 (25%) positive samples from 28 patients were identified. Overall agreement with qPCR was 92%. Five false-negative, one false-positive and nine invalid results were detected by the LAMP. Positive and negative predictive values were 96% each, and sensitivity and specificity were 84% and 99%, respectively. There was a low correlation between the TTP and the fungal load. CONCLUSION: The LAMP is a time-saving and easy-to-perform method. It can be used as an alternative diagnostic method. However, for quantification purposes the qPCR is still the gold standard.

Comparison of four commercially available chromogenic media to identify Candida albicans and other medically relevant Candida species.

BACKGROUND: The number of invasive Candida infections has significantly increased in recent decades. For the successful treatment of fungal infections, rapid identification at the species level, particularly in polyfungal infections, is a key factor. In this study, four commercially available chromogenic media, CandiSelect™ 4 (CS4), chromID™ Candida Agar (CCA), BBL™ CHROMagar™ Candida Medium (BBL) and Brilliance™ Candida Agar (BCA) were evaluated for Candida identification. MATERIAL/METHODS: Overall, 181 bronchial secretion samples from intensive care patients were analysed prospectively. In addition, 18 primarily sterile materials, previously tested positive for Candida, were investigated retrospectively. All samples were cultured as recommended by the manufacturer and visually inspected after 24 and 48 hours by three independent investigators. As a control, colonies were identified by MALDI-TOF MS. Specificity and sensitivity were determined for C albicans identification prospectively. RESULTS: CS4 and BCA showed the best overall consensus with the identification results reached by MALDI-TOF MS for Candida albicans and species. A clear differentiation between the species could be ascertained via easily identifiable, species-specific coloration in contrast to BBL and CCA. Sensitivity for C albicans (n = 73) identification varied between 32% (BCA) and 69% (CS4 and CCA) after 24 hours and 68% (BBL) and 82% (BCA) after 48 hours incubation, while specificity ranged between 62% (BBL) and 81% (CCA) after 24 hours and 82% (BBL) and 85% (CS4) after 48 hours. CONCLUSION: CS4 and BCA are recommended for routine identification of Candida species in human samples.

Phenotypical Characteristics of the Black Yeast Exophiala dermatitidis Are Affected by Pseudomonas aeruginosa in an Artificial Sputum Medium Mimicking Cystic Fibrosis-Like Conditions.

Research into the cooperative pathogenicity of microbes in cystic fibrosis (CF) lungs is crucial for an understanding of the pathophysiology of infections and the development of novel treatment strategies. This study investigated the impact of the common CF-associated bacterial pathogen Pseudomonas aeruginosa on the black yeast Exophiala dermatitidis. It evaluated the planktonic growth, biofilm formation, morphology, and virulence of the fungus in the presence or absence of P. aeruginosa. It also determined the role of P. aeruginosa quorum-sensing (QS) molecules within these interactions, e.g., by using sterile culture filtrate and QS-deficient mutants. P. aeruginosa is known to inhibit the planktonic growth of E. dermatitidis. We found that fungal biofilm formation increased in the presence of P. aeruginosa after 24 h but is decreased significantly after 48 h. This effect was reversed when, instead of QS wild-type strains, ΔlasR, and ΔrhlR mutants were added to E. dermatitidis biofilm formation. The number and length of hyphae were substantially reduced when E. dermatitidis was co-cultivated with P. aeruginosa, but not when it was co-cultivated with the mutants. Experiments testing the virulence of E. dermatitidis in the greater wax moth Galleria mellonella showed a synergetic effect on larval killing when E. dermatitidis was injected together with P. aeruginosa culture filtrate. Survival rates were decreased when biofilm culture filtrate was added but not when planktonic culture filtrate was added. In summary, P. aeruginosa affects the growth, morphology, biofilm formation, and virulence of E. dermatitidis. N-acyl-L-homoserine lactone (AHL) QS molecules regulated factors that have been shown to contribute to the inhibition of the ability of E. dermatitidis to form filaments and biofilm.

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.