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Objectives: Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is a widely used method for bacterial species identification. Incomplete databases and mass spectral quality (MSQ) still represent major challenges. Important proxies for MSQ are the number of detected marker masses, reproducibility, and measurement precision. We aimed to assess MSQs across diagnostic laboratories and the potential of simple workflow adaptations to improve it.Methods: For baseline MSQ assessment, 47 diverse bacterial strains, which are challenging to identify by MALDI-TOF MS, were routinely measured in 36 laboratories from 12 countries, and well-defined MSQ features were used. After an intervention consisting of detailed reported feedback and instructions on how to acquire MALDI-TOF mass spectra, measurements were repeated and MSQs were compared.Results: At baseline, we observed heterogeneous MSQ between the devices, considering the median number of marker masses detected (range = [2–25]), reproducibility between technical replicates (range = [55%–86%]), and measurement error (range = [147 parts per million (ppm)–588 ppm]). As a general trend, the spectral quality was improved after the intervention for devices, which yielded low MSQs in the baseline assessment as follows: for four out of five devices with a high measurement error, the measurement precision was improved (p-values Discussion: We have identified simple workflow adaptations, which, to some extent, improve MSQ of poorly performing devices and should be considered by laboratories yielding a low MSQ. Improving MALDI-TOF MSQ in routine diagnostics is essential for increasing the resolution of bacterial identification by MALDI-TOF MS, which is dependent on the reproducible detection of marker masses. The heterogeneity identified in this external quality assessment (EQA) requires further study.

The objective of this study was to evaluate the performance of the Copan Colibrí™ against the manual preparation of the MALDI targets. We analyzed 416 (31 different species) non-duplicate strains covering the most important species identified in clinical routine. We also assessed the intra-strain repeatability between the comparable methods. We then analyzed the performance of this new method after implementation in routine on 12,253 aerobic bacterial isolates and yeasts, encompassing a total of 42 different species. Among the 416 strains analyzed, 6.3% (26/416) and 10.8% (45/416) had a score value

Keratitis is a public health issue in developing countries and a potentially sight-threatening condition. Collagen fibrils in the corneal stroma are parallels to each other. Fundamental substance maintains the same space between collagen fibrils. That is how corneal transparency can be achieved. Any damage which can modify this structure will lead to corneal opacity and loss of vision. Fungal keratitis might appear in up to one-third of cases. Nevertheless, fungal keratitis remains poorly described and understood. Herein, we present the first ever reported case of corneal infection due to Phaeoacremonium parasiticum in a young patient. We describe the clinical and microbial characteristics, and we also discuss the use of confocal microscopy in early diagnosis of this infection.

Relying on a biomarker to diagnose or follow up the treatment of a Candida albicans meningitis would have an impact on patient management. The biomarker 1,3, β-D-glucan (BDG), developed for serum testing, shows inconsistent values when applied on cerebrospinal fluid (CSF), and its use with the current protocol on CSF samples warrants caution.

Among 400 Aspergillus species from respiratory samples in Switzerland, Aspergillus fumigatus was the most frequent species. Non-fumigatus Aspergillus spp were more prevalent among solid organ transplant recipients and after azole exposure. Azole resistance was detected in 4 A fumigatus isolates, 3 of them with the “environmental” mutation TR34/L98H in the cyp51A gene.

Background Despite progress in diagnostic, prevention, and treatment strategies, invasive mold infections (IMIs) remain the leading cause of mortality in allogeneic hematopoietic cell transplant (allo-HCT) recipients. Methods We describe the incidence, risk factors, and mortality of allo-HCT recipients with proven/probable IMI in a retrospective single-center 10-year (01/01/2010–01/01/2020) cohort study. Results Among 515 allo-HCT recipients, 48 (9.3%) patients developed 51 proven/probable IMI: invasive aspergillosis (IA; 34/51, 67%), mucormycosis (9/51, 18%), and other molds (8/51, 15%). Overall, 35/51 (68.6%) breakthrough IMIs (bIMIs) were identified: 22/35 (62.8%) IA and 13/35 (37.1%) non-IA IMI. One-year IMI cumulative incidence was 7%: 4.9% and 2.1% for IA and non-IA IMI, respectively. Fourteen (29.2 %), 10 (20.8%), and 24 (50.0%) patients were diagnosed during the first 30, 31–180, and >180 days post-HCT, respectively. Risk factors for IMI included prior allo-HCT (sub hazard ratio [SHR], 4.06; P = .004) and grade ≥2 acute graft-vs-host disease (aGvHD; SHR, 3.52; P Conclusions IA mortality has remained relatively unchanged during the last 2 decades. More than two-thirds of allo-HCT recipients with IMI die by 1 year post–IMI diagnosis. Dedicated intensified research efforts are required to further improve clinical outcomes.

We determined the susceptibility of 182 Fusarium species isolates to five antifungal drugs (amphotericin B, voriconazole, posaconazole, isavuconazole, and terbinafine) by the EUCAST method. Based on the latest taxonomic insights, isolates collected from 20 European centers were distributed into seven complexes and 27 species. The susceptibility was variable, depending on the species. Comparison with the gradient concentration strip method, which was used for 77 isolates, showed essential agreement values for voriconazole, posaconazole, isavuconazole, and amphotericin B of 17%, 91%, 83%, and 70%, respectively.

Background The increasing incidence of candidemia and emergence of drug-resistant Candida species are major concerns worldwide. Long-term surveillance studies are needed. Methods The Fungal Infection Network of Switzerland (FUNGINOS) conducted a 15-year (2004–2018), nationwide, epidemiological study of candidemia. Hospital-based incidence of candidemia, Candida species distribution, antifungal susceptibility, and consumption were stratified in 3 periods (2004–2008, 2009–2013, 2014–2018). Population-based incidence over the period 2009–2018 derived from the Swiss Antibiotic Resistance Surveillance System (ANRESIS). Results A total of 2273 Candida blood isolates were studied. Population and hospital-based annual incidence of candidemia increased from 2.96 to 4.20/100 000 inhabitants (P = .022) and 0.86 to 0.99/10 000 patient-days (P = .124), respectively. The proportion of Candida albicans decreased significantly from 60% to 53% (P = .0023), whereas Candida glabrata increased from 18% to 27% (P < .0001). Other non-albicans Candida species remained stable. Candida glabrata bloodstream infections occurred predominantly in the age group 18–40 and above 65 years. A higher proportional increase of C glabrata was recorded in wards (18% to 29%, P < .0001) versus intensive care units (19% to 24%, P = .22). According to Clinical and Laboratory Standards Institute, nonsusceptibility to fluconazole in C albicans was observed in 1% of isolates, and anidulafungin and micafungin nonsusceptibility was observed in 2% of C albicans and C glabrata. Fluconazole consumption, the most frequently used antifungal, remained stable, whereas use of mold-active triazoles and echinocandins increased significantly in the last decade (P < .0001). Conclusions Over the 15-year period, the incidence of candidemia increased. A species shift toward C glabrata was recently observed, concurring with increased consumption of mold-active triazoles., The incidence of candidemia increased in Switzerland from 2004 to 2018. A species shift toward C glabrata was observed after 2013, now accounting for one fourth of all candidemia, concurring with increased consumption of mold-active triazoles.

Fusarium spp. are widespread environmental fungi as well as pathogens that can affect plants, animals and humans. Yet the epidemiology of human fusariosis is still cloudy due to the rapidly evolving taxonomy. The Mass Spectrometry Identification database (MSI) has been developed since 2017 in order to allow a fast, accurate and free-access identification of fungi by matrix-assisted laser desorption ionization—time of flight (MALDI-TOF) mass spectrometry. Taking advantage of the MSI database user network, we aim to study the species distribution of Fusarium spp. isolates in an international multicenter prospective study. This study also allowed the assessment of the abilities of miscellaneous techniques to identify Fusarium isolates at the species level. The identification was performed by PCR-sequencing and phylogenic-tree approach. Both methods are used as gold standard for the evaluation of mass spectrometry. Identification at the species complex was satisfactory for all the tested methods. However, identification at the species level was more challenging and only 32% of the isolates were correctly identified with the National Center for Biotechnology Information (NCBI) DNA database, 20% with the Bruker MS database and 43% with the two MSI databases. Improvement of the mass spectrometry database is still needed to enable precise identification at the species level of any Fusarium isolates encountered either in human pathology or in the environment.

BACKGROUND Cryptococcosis is a frequent complication in immunosuppressed patients, causing mainly central nervous system and lung infection, and leading to increased mortality risk. CASE PRESENTATION We present the first documented case in Switzerland of Cryptococcus gattii pneumonia in a kidney-pancreas transplant patient, with a concomitant Pneumocystis jirovecii infection mimicking an immune reconstitution syndrome. Diagnosis of cryptococcal pneumonia was based on a positive serum cryptococcal antigen and confirmed by Grocott’s methenamine silver and periodic acid-Schiff stains on bronchoalveolar lavage fliud. C. gattii was identified with mass spectrometry and antifungal susceptibility testing by microdilution was performed. After an initial successful treatment with liposomal amphotericin-B, flucytosine and tapering of immunosuppression, the patient clinically deteriorated, developing bilateral diffuse ground-glass opacities with consolidations on chest computed tomography. A diagnosis of probable P. jirovecii pneumonia versus an immune reconstitution syndrome was considered. Because of a high titre of Pneumocystis on polymerase chain-reaction testing of bronchoalveloar lavage fluid and high serum b-D-glucan, a diagnosis of probable P. jirovecii pneumonia was made. CONCLUSION This case illustrates the potential complications of a cryptococcal infection in immunosuppressed hosts, despite timely diagnosis and appropriate antifungal therapy.

Echinocandins represent the first-line therapy of candidemia. Echinocandin resistance among Candida spp. is mainly due to acquired FKS mutations. In this study, we report the emergence of FKS-mutant Candida albicans/glabrata in Switzerland and provide the microbiological and clinical characteristics of 9 candidemic episodes. All patients were previously exposed to echinocandins (median 26 days; range 15–77). Five patients received initial echinocandin therapy with persistent candidemia in 4 of them. Overall mortality was 33%.

Background Aspergillus spp. of section Usti (A. ustus) represent a rare cause of invasive aspergillosis (IA). This multicenter study describes the epidemiology and outcome of A. ustus infections. Methods Patients with A. ustus isolated from any clinical specimen were retrospectively identified in 22 hospitals from 8 countries. When available, isolates were sent for species identification (BenA/CaM sequencing) and antifungal susceptibility testing. Additional cases were identified by review of the literature. Cases were classified as proven/probable IA or no infection, according to standard international criteria. Results Clinical report forms were obtained for 90 patients, of whom 27 had proven/probable IA. An additional 45 cases were identified from literature review for a total of 72 cases of proven/probable IA. Hematopoietic cell and solid-organ transplant recipients accounted for 47% and 33% cases, respectively. Only 8% patients were neutropenic at time of diagnosis. Ongoing antimold prophylaxis was present in 47% of cases. Pulmonary IA represented 67% of cases. Primary or secondary extrapulmonary sites of infection were observed in 46% of cases, with skin being affected in 28% of cases. Multiple antifungal drugs were used (consecutively or in combination) in 67% of cases. The 24-week mortality rate was 58%. A. calidoustus was the most frequent causal agent. Minimal inhibitory concentrations encompassing 90% isolates (MIC90) were 1, 8, >16, and 4 µg/mL for amphotericin B, voriconazole, posaconazole, and isavuconazole, respectively. Conclusions Aspergillus ustus IA mainly occurred in nonneutropenic transplant patients and was frequently associated with extrapulmonary sites of infection. Mortality rate was high and optimal antifungal therapy remains to be defined.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) is routinely used in mycology laboratories to rapidly identify pathogenic yeasts. Various methods have been proposed to perform routine MS-based identification of clinically relevant species. In this study, we focused on Bruker technology and assessed the identification performance of three protocols: two pretreatment methods (rapid formic acid extraction directly performed on targets and full extraction using formic acid/acetonitrile in tubes) and a direct deposit protocol that omits the extraction step. We also examined identification performance using three target types (ground-steel, polished-steel, and biotargets) and two databases (Bruker and online MSI [biological-mass-spectrometry-identification application]) in a multicenter manner. Ten European centers participated in the study, in which a total of 1511 yeast isolates were analyzed. The 10 centers prospectively performed the three protocols on approximately 150 yeast isolates each, and the corresponding spectra were then assessed against two reference spectra databases (MSI and Bruker), with appropriate thresholds. Three centers evaluated the impact of the targets. Scores were compared between the various combinations, and identification accuracy was assessed. The protocol omitting the extraction step was inappropriate for yeast identification, while the full extraction method yielded far better results. Rapid formic acid extraction yielded variable results depending on the target, database and threshold. Selecting the optimal extraction method in combination with the appropriate target, database and threshold may enable simple and accurate identification of clinically relevant yeast samples. Concerning the widely used polished-steel targets, the full extraction method still ensured better scores and better identification rates.

Aspergillus fumigatus is a ubiquitous opportunistic pathogen. This fungus can acquire resistance to azole antifungals due to mutations in the azole target ( cyp51A ). Recently, cyp51A mutations typical for environmental azole resistance acquisition (for example, TR 34 /L98H) have been reported. These mutations can also be found in isolates recovered from patients. Environmental azole resistance acquisition has been reported on several continents. Here we describe, for the first time, the occurrence of azole-resistant A. fumigatus isolates of environmental origin in Switzerland with cyp51A mutations, and we show that these isolates can also be recovered from a few patients. While the TR 34 /L98H mutation was dominant, a single azole-resistant isolate exhibited a cyp51A mutation (G54R) that was reported only for clinical isolates. In conclusion, our study demonstrates that azole resistance with an environmental signature is present in environments and patients of Swiss origin and that mutations believed to be unique to clinical settings are now also observed in the environment.

Dermatophytes cause human infections limited to keratinised tissues. We showed that the direct transfer method allows reliable identification of non-dermatophytes mould and yeast by MALDI-TOF/MS. We aimed at assessing whether the direct transfer method can be used for dermatophytes and whether an own mass spectra library would be superior to the Bruker library. We used the Bruker Biotyper to build a dermatophyte mass spectra library and assessed its performance by 1/testing a panel of mass spectrum produced with strains genotypically identified and, 2/comparing MALDI-TOF/MS identification to morphology-based methods. Identification of dermatophytes using the Bruker library is poor. Our library provided 97% concordance between ITS sequencing and MALDI-TOF/MS analysis with a panel of 1104 spectra corresponding to 276 strains. Direct transfer method using unpolished target plates allowed proper identification of 85% of dermatophytes clinical isolates most of which were common dermatophytes. A homemade dermatophyte MSP library is a prerequisite for accurate identification of species absent in the Bruker library but it also improves identification of species already listed in the database. The direct deposit method can be used to identify the most commonly found dermatophytes such as T. rubrum and T. interdigitale/mentagrophytes by MALDI-TOF/MS.

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) identification (ID) of Candida yeast isolates is fast and reliable, but the optimal workflow is debated ([1][1], [2][2]). Two different steel target plates are available for the Microflex system (Bruker

The identification of moulds by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF MS) has been evaluated repeatedly for use in clinical diagnostic settings. However, recommended protocols are based on the analysis of time-consuming subcultures rather than primary growth obtained on solid isolation media. In order to compare the identification performance of MALDITOF MS applied to primary colonies (protocol A) versus subculture samples (protocol B), 111 reference specimens representing 30 clinically relevant species were analyzed. Each of these species was identified, at least once, by internal transcribed spacer (ITS) sequence analysis, which was considered the gold standard in this study (Table 1). In addition, each of the 111 strains was identified by phenotype testing. In protocol A, primary growth on Sabouraud agar (Difco Sabouraud Agar, Modified) after 24 to 72 h of incubation (time required for the first visible mycelium growth) was collected carefully, avoiding agar, by means of a wooden toothpick. The biomass was deposited on an unpolished target plate, air-dried, and overlaid with 1 ml alpha-cyano-4-hydroxycinnamic acid. In protocol B, and in accordance with the Bruker protocol, the filamentous fungi were inoculated in 8 ml of Sabouraud liquid broth (Difco Sabouraud Maltose Broth), which was rotated overnight at room temperature. The sediments of overnight subcultures were washed thoroughly, extracted with acetonitrile–formic acid, and then 1 ml of supernatant was placed onto an unpolished target plate and processed further, as above.

PURPOSE: To report the use of accelerated photoactivated chromophore for keratitis–corneal collagen cross-linking (PACK-CXL) as a first-line treatment in a patient with an atypical fungal keratitis. METHODS: Case report and literature review. RESULTS: A patient who presented with a painful peripheral corneal infiltrate underwent PACK-CXL with a local limited abrasion and accelerated ultraviolet-A irradiation at 365 μm and 9 mW/cm 2 for 10 minutes. Cultures grew Aureobasidium pullulans . The corneal epithelium closed completely within 3 days and the infiltrate was completely eradicated without administration of antibiotics. CONCLUSIONS: Accelerated PACK-CXL was successfully used as a first-line and sole treatment in a case of early fungal keratitis caused by Aureobasidium pullulans . Further characterization of the antifungal effect of PACK-CXL is needed in prospective studies. [ J Refract Surg . 2014;30(12):855–857.]