A conceptual framework for nomenclatural stability and validity of medically important fungi: a proposed global consensus guideline for fungal name changes supported by ABP, ASM, CLSI, ECMM, ESCMID-EFISG, EUCAST-AFST, FDLC, IDSA, ISHAM, MMSA, and MSGERC.

The rapid pace of name changes of medically important fungi is creating challenges for clinical laboratories and clinicians involved in patient care. We describe two sources of name change which have different drivers, at the species versus the genus level. Some suggestions are made here to reduce the number of name changes. We urge taxonomists to provide diagnostic markers of taxonomic novelties. Given the instability of phylogenetic trees due to variable taxon sampling, we advocate to maintain genera at the largest possible size. Reporting of identified species in complexes or series should where possible comprise both the name of the overarching species and that of the molecular sibling, often cryptic species. Because the use of different names for the same species will be unavoidable for many years to come, an open access online database of the names of all medically important fungi, with proper nomenclatural designation and synonymy, is essential. We further recommend that while taxonomic discovery continues, the adaptation of new name changes by clinical laboratories and clinicians be reviewed routinely by a standing committee for validation and stability over time, with reference to an open access database, wherein reasons for changes are listed in a transparent way.

Large inter-individual variability of cellular and humoral immunological responses to mRNA-1273 (Moderna) vaccination against SARS-CoV-2 in health care workers.

PURPOSE: Studies on the immune responses to severe acute respiratory syndrome coronavirus 2 vaccines are necessary to evaluate the ongoing vaccination programs by correlating serological response data and clinical effectiveness data. We performed a longitudinal immunological profiling of health care workers vaccinated with mRNA-1273 (Moderna, Cambridge, MA, USA). Half of these vaccinees had experienced a mild coronavirus disease 2019 (COVID-19) infection in the spring of 2020 ("COVID-recovered" cohort), whereas the other half of the vaccinees had no previous COVID-19 infection ("COVID-naive" cohort). MATERIALS AND METHODS: Serum was drawn at multiple time points and subjected to assays measuring anti-Spike immunoglobulin G (IgG), avidity of anti-Spike IgG, avidity of anti-receptor binding domain (RBD) IgG, virus neutralizing activity, and interferon-γ release from stimulated lymphocytes. RESULTS: Between both cohorts and within each cohort, we found remarkable inter-individual differences regarding cellular and humoral immune responses to the Moderna mRNA-1273 vaccine. CONCLUSION: First, our study indicates that the success of mRNA-1273 vaccinations should be verified by serological assays in order to identify "low-responders" to vaccination. Second, the kinetics of anti-S IgG and neutralizing activity correlate well with clinical effectiveness data, thus explaining incipient protection against infection 2 weeks after the first dose of mRNA-1273 in COVID-naive vaccinees. Third, our IgG-avidity data indicate that this incipient protection is mediated by low-avidity anti-RBD IgG and low-avidity anti-S IgG.

Evaluation of the QuantiFERON SARS-CoV-2 interferon-É£ release assay in mRNA-1273 vaccinated health care workers.

Current studies focus on cellular and humoral immunity induced by novel SARS-CoV-2 vaccines. Non-responders to vaccinations are not uncommonly encountered in clinical medicine (e.g. in the field of hepatitis B). Whereas vaccine-induced humoral immunity against SARS-CoV-2 is compromised by emerging Variants of Concern (VOCs), cellular immunity against SARS-CoV-2 is emerging as resilient against VOCs. Thus commercially available test kits for diagnostic laboratories designed to evaluate cellular immune responses to SARS-CoV-2 are urgently needed. Here we evaluated the novel QuantiFERON SARS-CoV-2 assay (Qiagen) measuring INF-É£ release induced by two spike-derived peptide pools (Ag1 and Ag2) in a cohort of health care workers vaccinated with the mRNA-1273 vaccine and confirmed humoral response. Our study indicates the usefulness of this novel assay for routine laboratories to evaluate cellular immunity against SARS-CoV-2 in response to mRNA-1273 vaccination.

Heterogeneity of Streptococcus anginosus ß-hemolysis in relation to CRISPR/Cas.

Streptococcus anginosus is a commensal of the oral mucosa that can cause severe invasive infections. A considerable proportion of Streptococcus anginosus strains are ß-hemolytic due to the presence of an SLS-like gene cluster. However, the majority of strains do not display ß-hemolysis. To investigate ß-hemolysin heterogeneity in S. anginosus, we determined the presence of sag genes and correlated it with the presence of CRISPR/Cas genes in a collection of ß-hemolytic and non-ß-hemolytic strains. All of the ß-hemolytic strains carried the sag gene cluster. In contrast to other streptococci, clinical S. anginosus strains that do not display ß-hemolysis do not harbor sag genes. Phylogenetic analysis of the ß-hemolytic strains revealed that they belong to two previously defined clusters within S. anginosus. Correlation with CRISPR/Cas genes showed a significant difference for the presence of CRISPR/Cas in ß-hemolytic versus non-ß-hemolytic isolates. The presence of the CRISPR/Cas type IIA or type IIC locus is associated with the absence of sag genes; in 65% of the non-ß-hemolytic strains a CRISPR/Cas locus was found, while only 24% of ß-hemolytic strains carry CRISPR/Cas genes. Further analysis of the spacer content of the CRISPR systems revealed the presence of multiple self-targeting sequences directed against S. anginosus genes. These results support the hypothesis that horizontal gene transfer is involved in the acquisition of ß-hemolysin genes and that CRISPR/Cas may limit DNA uptake in S. anginosus.

Disseminated Emergomycosis in a Person with HIV Infection, Uganda.

We describe emergomycosis in a patient in Uganda with HIV infection. We tested a formalin-fixed, paraffin-embedded skin biopsy to identify Emergomyces pasteurianus or a closely related pathogen by sequencing broad-range fungal PCR amplicons. Results suggest that emergomycosis is more widespread and genetically diverse than previously documented. PCR on tissue blocks may help clarify emergomycosis epidemiology.

Biofilm formation of the black yeast-like fungus Exophiala dermatitidis and its susceptibility to antiinfective agents.

Various fungi have the ability to colonize surfaces and to form biofilms. Fungal biofilm-associated infections are frequently refractory to targeted treatment because of resistance to antifungal drugs. One fungus that frequently colonises the respiratory tract of cystic fibrosis (CF) patients is the opportunistic black yeast-like fungus Exophiala dermatitidis. We investigated the biofilm-forming ability of E. dermatitidis and its susceptibility to various antiinfective agents and natural compounds. We tested 58 E. dermatitidis isolates with a biofilm assay based on crystal violet staining. In addition, we used three isolates to examine the antibiofilm activity of voriconazole, micafungin, colistin, farnesol, and the plant derivatives 1,2,3,4,6-penta-O-galloyl-b-D-glucopyranose (PGG) and epigallocatechin-3-gallate (EGCG) with an XTT reduction assay. We analysed the effect of the agents on cell to surface adhesion, biofilm formation, and the mature biofilm. The biofilms were also investigated by confocal laser scan microscopy. We found that E. dermatitidis builds biofilm in a strain-specific manner. Invasive E. dermatitidis isolates form most biomass in biofilm. The antiinfective agents and the natural compounds exhibited poor antibiofilm activity. The greatest impact of the compounds was detected when they were added prior cell adhesion. These findings suggest that prevention may be more effective than treatment of biofilm-associated E. dermatitidis infections.

Epidemiology of invasive aspergillosis and azole resistance in patients with acute leukaemia: the SEPIA Study.

Invasive aspergillosis (IA) is a serious hazard to high-risk haematological patients. There are increasing reports of azole-resistant Aspergillus spp. This study assessed the epidemiology of IA and azole-resistant Aspergillus spp. in patients with acute leukaemia in Germany. A prospective multicentre cohort study was performed in German haematology/oncology centres. The incidence of probable and proven aspergillosis according to the revised EORTC/MSG criteria was assessed for all patients with acute leukaemia [acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL)]. Cases were documented into a web-based case report form, and centres provided data on standards regarding prophylactic and diagnostic measures. Clinical isolates were screened centrally for azole resistance and, if applicable, underlying resistance mechanisms were analysed. Between September 2011 and December 2013, 179 cases of IA [6 proven (3.4%) and 173 probable (96.6%)] were diagnosed in 3067 patients with acute leukaemia. The incidence of IA was 6.4% among 2440 AML patients and 3.8% among 627 ALL patients. Mortality at Day 84 was 33.8% (49/145) and attributable mortality was 26.9% (39/145). At Day 84, 53 patients (29.6%) showed a complete response, 25 (14.0%) a partial response and 17 (9.5%) a deterioration or failure. A total of 77 clinical Aspergillus fumigatus isolates were collected during the study period. Two episodes of azole-resistant IA (1.1%) were caused by a TR/L98H mutation in the cyp51A gene. With only two cases of IA due to azole-resistant A. fumigatus, a change of antifungal treatment practices in Germany does not appear warranted currently.

Name changes in medically important fungi and their implications for clinical practice.

Recent changes in the Fungal Code of Nomenclature and developments in molecular phylogeny are about to lead to dramatic changes in the naming of medically important molds and yeasts. In this article, we present a widely supported and simple proposal to prevent unnecessary nomenclatural instability.

Analysis of black fungal biofilms occurring at domestic water taps. I: compositional analysis using Tag-Encoded FLX Amplicon Pyrosequencing.

Mass growth of dark fungal biofilms on water taps and associated habitats was observed in various German drinking water distribution systems recently. Customers of affected drinking water systems are anxious about potential and unknown health risks. These environments are known to harbour a fungal flora also comprising a variety of fungal opportunists that are well known to cause superficial mycoses in humans (Exophiala equina, Exophiala lecanii-corni) but are not known to establish dark biofilms so far. To gain profound insight on composition of respective biofilms, a metagenomic approach using Tag-Encoded FLX Amplicon Pyrosequencing (TEFAP) of the ribosomal internal transcribed spacer 2 region in comparison with a classical cultivation approach using Sabouraud agar with chloramphenicol and erythritol-chloramphenicol-agar was performed. E. lecanii-corni was found to be the major component in 10 of 13 biofilms analysed independently of the method used. Alternaria sp., E. equina, Fusarium spp. and Ochroconis spp. were also relatively abundant. As expected, TEFAP usually revealed a higher diversity than the cultivation approaches. For example, opportunistic species like Candida albicans or Exophiala dermatitidis were detected in very low amounts. In conclusion, TEFAP turned out to be a promising and powerful tool for the semi-quantitative analysis of fungal biofilms. Referring to relevant literature, potential biological hazards caused by fungi of the dark biofilms can be regarded as low.

Analysis of black fungal biofilms occurring at domestic water taps. II: potential routes of entry.

Formation of tenacious and massive black biofilms was occasionally observed at the water-air interphase of water taps and in associated habitats at several locations in Germany. Exophiala lecanii-corni was proven to be the dominant component of these biofilms. Water utility companies were interested to understand by which route fungi building these black biofilms enter their habitat at affected sites in domestic sanitary. A wide variety of fungi is known to be common in wet indoor environments, as well as in the drinking water resources. Two possible routes of entry are therefore considered as follows: (a) distribution by the drinking water system or (b) a retrograde route of colonisation. Previous compositional analysis revealed that the black constituents of biofilms primarily belong to the herpotrichiellaceous black yeast and relatives. Therefore, a systematic search for black fungi in the drinking water system was performed using Sabouraud's glucose agar medium with chloramphenicol and erythritol-chloramphenicol agar as isolation media. Cadophora malorum was the dominant fungus in the investigated drinking water systems, and samples taken from the house connections (n = 50; 74 %, <200 cfu/L), followed by a so far undescribed Alternaria sp. (28 %; <10 cfu/L) and E. castellanii (26 %; <10 cfu/L). Of note, C. malorum was not present in any previously analysed biofilm. Since E. lecanii-corni was not found in any water sample from the distribution system tested, but represented the most abundant species in dark biofilms previously analysed, a retrograde route of contamination in case of E. lecanii-corni can be assumed.

Barcode identifiers as a practical tool for reliable species assignment of medically important black yeast species.

Herpotrichiellaceous black yeasts and relatives comprise severe pathogens flanked by nonpathogenic environmental siblings. Reliable identification by conventional methods is notoriously difficult. Molecular identification is hampered by the sequence variability in the internal transcribed spacer (ITS) domain caused by difficult-to-sequence homopolymeric regions and by poor taxonomic attribution of sequences deposited in GenBank. Here, we present a potential solution using short barcode identifiers (27 to 50 bp) based on ITS2 ribosomal DNA (rDNA), which allows unambiguous definition of species-specific fragments. Starting from proven sequences of ex-type and authentic strains, we were able to describe 103 identifiers. Multiple BLAST searches of these proposed barcode identifiers in GenBank revealed uniqueness for 100 taxonomic entities, whereas the three remaining identifiers each matched with two entities, but the species of these identifiers could easily be discriminated by differences in the remaining ITS regions. Using the proposed barcode identifiers, a 4.1-fold increase of 100% matches in GenBank was achieved in comparison to the classical approach using the complete ITS sequences. The proposed barcode identifiers will be made accessible for the diagnostic laboratory in a permanently updated online database, thereby providing a highly practical, reliable, and cost-effective tool for identification of clinically important black yeasts and relatives.

Study on the association of Helicobacter species with viral hepatitis-induced hepatocellular carcinoma.

Helicobacter species are important pathogens and previous studies in mice suggested a link between colonization by Helicobacter hepaticus (H. hepaticus) and hepatocellular carcinoma (HCC). This study aimed at corroborating this potential link in human patients. We used a sensitive and specific Helicobacter ssp PCR assay to screen stool samples from a collective of patients with viral-induced HCC (hepatitis B or hepatitis C) and a control group for presence of Helicobacter ssp DNA. Although retrieving DNA of H. pylori and H. canadensis from stool samples of non-HCC patients, we found no evidence indicating the presence of H. hepaticus in HCC-patients with chronic hepatitis B or hepatitis C. Interestingly we found H. canadensis in a stool sample of a patient presenting with diarrhea. Taken together, our data argue against a pathogenic role of H. hepaticus in viral-induced HCC. Yet, our results do not exclude a role of H. hepaticus in those HCC cases caused by other carcinogens, such as aflatoxin. Moreover, we speculate that H. canadensis might be a novel gastrointestinal pathogen.

Isolation of Streptococcus urinalis from a human blood culture.

Streptococcus urinalis was isolated from a blood culture of a 60-year-old man with a history of urethral stricture. This species has been recently described as a new member of the pyogenic subgroup of streptococci that cause urinary tract infections.

Unusual Aspergillus species in patients with cystic fibrosis.

Poorly sporulating Aspergillus isolates from patients with cystic fibrosis (CF) are generally identified in routine procedures as Aspergillus spp. In this study, we identified and characterized 11 isolates belonging to two unusual Aspergillus species of the section Fumigati (A. lentulus and Neosartorya pseudofischeri) recovered from four different patients. Aspergillus lentulus was found occasionally during a 10-year follow-up study of one CF patient colonized by A. fumigatus. Neosartorya pseudofischeri was isolated from three patients followed in different European hospitals. This species was recovered from two sputum samples of one patient, and from four successive samples of the two other patients, suggesting that it may be responsible for chronic colonization. Both species were isolated together with A. fumigatus. Isolates from both species did not grow at 50°C, and DNA sequence analysis, together with further morphological observations permitted identification at the species level. Growth at different temperatures and antifungal susceptibility were also investigated. All the isolates of N. pseudofischeri exhibited a very low susceptibility to voriconazole (VRZ) whereas a very low susceptibility to VRZ and amphotericin B was seen with the A. lentulus isolates.

Association between respiratory and herpes viruses on pulmonary exacerbations in cystic fibrosis patients.

Respiratory viruses discovered in the 21st century and human herpes viruses (N=13) were seldom (4/50) detected in our cystic fibrosis patients although exacerbation frequency (7.75+/-2.9/a versus 4.45+/-2.1/a; p=0.03) and colonization with Aspergillus fumigatus (RR: 2.6; CI95: 1.8-3.7), Pseudomonas aeruginosa (RR: 1.84; CI95: 1.4-2.4), and Staphylococcus aureus (RR: 1.5; CI95: 1.2-1.9) including MRSA (RR: 4.6; CI95: 1.3-16.6) were associated with virus positivity. Further studies should clarify whether this finding reflects non-specific colonization (human Bocavirus) or reactivation (Epstein-Barr virus) or rather an acceleration of lung tissue inflammation.

Rapid detection of Streptococcus agalactiae from swabs by peptide nucleic acid fluorescence in situ hybridization.

The applicability of the PNA FISH (peptide nucleic acid fluorescence in situ hybridization) method for detection of Streptococcus agalactiae [group B streptococci (GBS)] from swab samples was evaluated. Three swab-sample-processing protocols with different time-to-result (TTR) values were compared: (i) direct smearing of fresh swabs onto microscope slides (n=153, TTR 2.5 h), (ii) further extraction and concentration of cells from these same swabs (n=153, TTR 2.7 h), and (iii) short-term LIM broth enrichment culture incubation (7 h, 37 degrees C) of fresh swabs (n=120, TTR 9.5 h). The sensitivity, specificity, positive predictive value and negative predictive value for GBS PNA FISH for sample processing procedures, with TTR values of 2.5, 2.7 and 9.5 h, were 68, 100, 100 and 95 %; 91, 100, 100 and 98 %; and 100, 100, 100 and 100 %; respectively. Improved test results were achieved by subjecting swabs to an extraction procedure or abbreviated LIM broth enrichment culture incubation prior to performing GBS PNA FISH.

Comparison of two chromogenic media for selective isolation of vancomycin-resistant enterococci from stool specimens.

Two chromogenic media (Chromagar VRE and chromID VRE [C-ID]) performed equally well in the direct detection of vancomycin-resistant enterococci (VRE) in stool specimens after an overnight enrichment step and a 48-h incubation period, with a sensitivity of 98.2% (56/57) for both and specificities of 96.5% (195/202) and 97.5% (197/202), respectively. However, assigning discriminatory colony color was sometimes difficult, especially on C-ID. In order to facilitate simple species identification, biochemical key reactions were implemented.

Molecular typing and colonization patterns of Aspergillus fumigatus in patients with cystic fibrosis.

Aspergillus fumigatus is a chronic colonizer of the respiratory tract of patients with cystic fibrosis (CF). A total of 204 A. fumigatus isolates from 36 CF patients from three different medical centers, collected over a period of four months till 9.5 years, were genotyped using the short tandem repeat panel for A. fumigatus (STRAf assay). Four different colonization patterns were observed. Colonization patterns with only unique genotypes were found in 36% of the patients. In contrast 17% of the patients were chronically colonized with a single genotype. The remaining patients showed a predominant genotype or genotypes that succeed each other. In this collection no relation was found between colonization patterns and allergic bronchopulmonary aspergillosis.