[Surgical therapy and pathogen detection in endogenous endophthalmitis : Analysis of data from five German eye hospitals]. / Operative Therapie und Keimnachweis bei endogener Endophthalmitis : Datenauswertung aus 5 Augenkliniken.

BACKGROUND: Endogenous endophthalmitis results from hematogenous spread of bacterial or fungal infection in severely diseased patients. Specific systemic and intraocular therapy is required. The basis for this treatment is causal pathogen detection in blood culture or vitreous sample. However, functional results are limited. OBJECTIVE: The current article provides practical hints for surgical therapy and pathogen detection in patients with endogenous endophthalmitis. METHODS: A retrospective analysis of anonymous data of 68 male and female patients from 2018-2023 from five ophthalmology clinics in Germany was performed. RESULTS: Mean age of affected patients was 71.4 years (31-96 years). Surgical therapy included pars plana vitrectomy (ppV) and intravitreal injection (IVOM). In 44 of 68 patients (65%), 1-3 surgeries were performed, 4-6 surgeries were required in 14/68 (21%) of patients, and 10 or more surgeries were required in 4/68 patients (6%). Pathogen detection was possible in 34% of vitreous specimens and in 11% of anterior chamber samples. Mean initial visual acuity was logMAR 1.5. After treatment and a mean follow-up of 2.5 months, mean visual acuity was logMAR 1.3. Preanalytical methods for specimen collection like the Freiburg endophthalmitis set to optimize pathogen detection are presented. CONCLUSION: Severe inflammatory intraocular reactions in endogenous endophthalmitis necessitate a combination of ppV and repeated IVOM. In addition to providing a vitreous sample, ppV also serves to remove inflammatory fibrin membranes. Early pars plana vitrectomy with specific antibiotic or antifungal therapy should be sought in addition to the focus search and systemic therapy.

CHROMAgar™ LIN-R as an efficient screening tool to assess the prevalence of linezolid-resistant enterococci in German hospital patients-a multicentre study approach, 2021-2022.

BACKGROUND: In recent years, an increasing number of linezolid-resistant enterococci (LRE) was recognized at the German National Reference Centre (NRC) for Enterococci. National guidelines on infection prevention recommend screening for LRE in epidemiologically linked hospital settings without referring to a reliable and rapid diagnostic method. Since 2020, CHROMAgar™ provide a chromogenic linezolid screening agar, LIN-R, suitable to simultaneously screen for linezolid-resistant staphylococci and enterococci. OBJECTIVES: To assess the applicability of CHROMAgar™ LIN-R in clinical settings for detecting LRE directly from patient material and to infer prevalence rates of LRE amongst German hospital patients. METHODS: During the 3-month trial period, clinical samples were plated on CHROMAgar™ LIN-R. Antimicrobial susceptibility testing was performed using VITEK2 or disc diffusion. At the NRC, linezolid resistance was determined by broth microdilution, multiplex-PCR for cfr/optrA/poxtA and by a restriction-based assay for 23S rDNA mutations. RESULTS: The 12 participating study sites used 13 963 CHROMAgar™ LIN-R plates during the study period. Of 442 presumptive LRE, 192 were confirmed by phenotypic methods. Of these, 161 were received by the NRC and 121 (75%) were verified as LRE. Most of LR-E. faecium 53/81 (65%) exhibited a 23S rRNA gene mutation as the sole resistance-mediating mechanism, whereas optrA constituted the dominant resistance trait in LR-E. faecalis [39/40 (98%)]. Prevalence of LRE across sites was estimated as 1% (ranging 0.18%-3.7% between sites). CONCLUSIONS: CHROMAgar™ LIN-R represents a simple and efficient LRE screening tool in hospital settings. A high proportion of false-positive results demands validation of linezolid resistance by a reference method.

Comparison and Harmonization of Different Semi-Automated and Automated qRT-PCR Assays in the Assessment of SARS-CoV-2.

In SARS-CoV-2 diagnostics, cycle threshold (Ct) values from qRT-PCRs semi-quantitatively estimate a patient's viral load. However, relevant analytical differences between qRT-PCR assays are often neglected. This study was designed (i) to identify such differences between five commonly used assays and (ii) to demonstrate a straightforward strategy to harmonize them. QRT-PCRs for SARS-CoV-2 were carried out in 85 oropharyngeal swab samples using three fully automated (Alinity m, cobas®6800 and GeneXpert) and two semi-automated (genesig® and RIDA®GENE) assays. Qualitative results (positive/negative) showed excellent comparability between the fully automated assays, but not between the Alinity m and semi-automated methods. Ct values significantly varied between all the methods, with the median values ranging from 22.76 (Alinity m) to 30.89 (RIDA®GENE) and 31.50 (genesig®), indicating the lowest sensitivity for semi-automated methods. Passing-Bablok analysis further revealed systemic biases. Assay-specific viral load concentration calculations-based on generated individual standard curves-resulted in much better comparability between the assays. Applying these calculations, significant differences were no longer detectable. This study highlights relevant analytical differences between SARS-CoV-2 qRT-PCR assays, leading to divergent decisions about the mandatory isolation of infected individuals. Secondly, we propose a strategy to harmonize qRT-PCR assays to achieve better comparability. Our findings are of particular interest for laboratories utilizing different assays.

Epidemiology and Prevalence of Oral Candidiasis in HIV Patients From Chad in the Post-HAART Era.

Oral candidiasis remains a common problem in HIV-infected individuals, especially in sub-Saharan Africa. Here, we performed the first study in Chad on the prevalence of oral yeasts carriage and oral candidiasis in HIV-positive subjects from southern Chad and analyzed the influence of HAART, CD4+ T-cell numbers, and antimycotics in 589 patients. These patients were recruited from a specialized medical center for HIV patients in Sarh and from a rural medical health dispensary in the vicinity, including a total of 384 HIV-positive and 205 HIV-negative individuals. Yeasts obtained from oral specimen were identified by MALDI-TOF MS and their antifungal susceptibility profiles determined. The overall prevalence of yeast colonization and symptomatic oral candidiasis in HIV-infected patients was 25.1%. The prevalence of oral candidiasis was higher in untreated than in HAART-treated HIV-positive patients (16% vs. 2%; p < 0.01). Oral candidiasis was furthermore associated with high fungal burdens of Candida albicans and a CD4+ T-cell number <200/µl. A shift toward non-albicans Candida species was observed under nucleoside-based HAART therapy. Azole antifungal drug resistance was only observed for the intrinsically resistant species Candida krusei and Candida glabrata. Prevalence of oral candidiasis in the studied area was very low. The species distribution was similar to other countries around the world, with C. albicans being dominant. Candida dubliniensis was not isolated. Nucleoside-based HAART therapy significantly reduced oral colonization as well as occurrence of oral candidiasis caused by C. albicans and led to a species shift toward non-albicans species. Antifungal resistance was not yet a concern in Chad.

Ultra-deep long-read sequencing detects IS-mediated gene duplications as a potential trigger to generate arrays of resistance genes and a mechanism to induce novel gene variants such as blaCTX-M-243.

BACKGROUND: Extended-spectrum ß-lactamases (ESBLs) are enzymes that can render their hosts resistant to various ß-lactam antibiotics. CTX-M-type enzymes are the most prevalent ESBLs and the main cause of resistance to third-generation cephalosporins in Enterobacteriaceae. The number of described CTX-M types is continuously rising, currently comprising over 240 variants. During routine screening we identified a novel blaCTX-M gene. OBJECTIVES: To characterize a novel blaCTX-M variant harboured by a multidrug-resistant Escherichia coli isolate of sequence type ST354. METHODS: Antibiotic susceptibilities were determined using broth microdilution. Genome and plasmid sequences were reconstructed using short- and long-read sequencing. The novel blaCTX-M locus was analysed using long-read and Sanger sequencing. Plasmid polymorphisms were determined in silico on a single plasmid molecule level. RESULTS: The novel blaCTX-M-243 allele was discovered alongside a nearly identical blaCTX-M-104-containing gene array on a 219 kbp IncHI2A plasmid. CTX-M-243 differed from CTX-M-104 by only one amino acid substitution (N109S). Ultra-deep (2300-fold coverage) long-read sequencing revealed dynamic scaling of the blaCTX-M genetic contexts from one to five copies. Further antibiotic resistance genes such as blaTEM-1 also exhibited sequence heterogeneity but were stable in copy number. CONCLUSIONS: We identified the novel ESBL gene blaCTX-M-243 and illustrate a dynamic system of varying blaCTX-M copy numbers. Our results highlight the constant emergence of new CTX-M family enzymes and demonstrate a potential evolutionary platform to generate novel ESBL variants and possibly other antibiotic resistance genes.

Proteogenomics analysis of CUG codon translation in the human pathogen Candida albicans.

BACKGROUND: Yeasts of the CTG-clade lineage, which includes the human-infecting Candida albicans, Candida parapsilosis and Candida tropicalis species, are characterized by an altered genetic code. Instead of translating CUG codons as leucine, as happens in most eukaryotes, these yeasts, whose ancestors are thought to have lost the relevant leucine-tRNA gene, translate CUG codons as serine using a serine-tRNA with a mutated anticodon, [Formula: see text]. Previously reported experiments have suggested that 3-5% of the CTG-clade CUG codons are mistranslated as leucine due to mischarging of the [Formula: see text]. The mistranslation was suggested to result in variable surface proteins explaining fast host adaptation and pathogenicity. RESULTS: In this study, we reassess this potential mistranslation by high-resolution mass spectrometry-based proteogenomics of multiple CTG-clade yeasts, including various C. albicans strains, isolated from colonized and from infected human body sites, and C. albicans grown in yeast and hyphal forms. Our data do not support a bias towards CUG codon mistranslation as leucine. Instead, our data suggest that (i) CUG codons are mistranslated at a frequency corresponding to the normal extent of ribosomal mistranslation with no preference for specific amino acids, (ii) CUG codons are as unambiguous (or ambiguous) as the related CUU leucine and UCC serine codons, (iii) tRNA anticodon loop variation across the CTG-clade yeasts does not result in any difference of the mistranslation level, and (iv) CUG codon unambiguity is independent of C. albicans' strain pathogenicity or growth form. CONCLUSIONS: Our findings imply that C. albicans does not decode CUG ambiguously. This suggests that the proposed misleucylation of the [Formula: see text] might be as prevalent as every other misacylation or mistranslation event and, if at all, be just one of many reasons causing phenotypic diversity.

Host Age and Denture Wearing Jointly Contribute to Oral Colonization with Intrinsically Azole-Resistant Yeasts in the Elderly.

In elderly patients, several morbidities or medical treatments predisposing for fungal infections occur at a higher frequency, leading to high mortality and morbidity in this vulnerable patient group. Often, this is linked to an innately azole-resistant yeast species such as Candida glabrata or C. krusei. Additionally, host age per se and the wearing of dentures have been determined to influence the mix of colonizing species and, consequently, the species distribution of invasive fungal infections. Since both old age and the wearing of dentures are two tightly connected parameters, it is still unclear which of them is the main contributor. Here, we performed a cross-sectional study on a cohort (N = 274) derived from three groups of healthy elderly, diseased elderly, and healthy young controls. With increasing host age, the frequency of oral colonization by a non-albicans Candida species, mainly by C. glabrata, also increased, and the wearing of dentures predisposed for colonization by C. glabrata irrespectively of host age. Physically diseased hosts, on the other hand, were more frequently orally colonized by C. albicans than by other yeasts. For both C. albicans and C. glabrata, isolates from the oral cavity did not generally display an elevated biofilm formation capacity. In conclusion, intrinsically azole-drug-resistant, non-albicans Candida yeasts are more frequent in the oral cavities of the elderly, and fungal cells not contained in biofilms may predispose for subsequent systemic infection with these organisms. This warrants further exploration of diagnostic procedures, e.g., before undergoing elective abdominal surgery or when using indwelling devices on this patient group.

Mass Spectrometry-Based Proteomic and Immunoproteomic Analyses of the Candida albicans Hyphal Secretome Reveal Diagnostic Biomarker Candidates for Invasive Candidiasis.

Invasive candidiasis (IC) is associated with high morbidity and mortality in hospitalized patients if not diagnosed early. Long-term use of central venous catheters is a predisposing factor for IC. Hyphal forms of Candida albicans (the major etiological agent of IC) are related to invasion of host tissues. The secreted proteins of hyphae are involved in virulence, host interaction, immune response, and immune evasion. To identify IC diagnostic biomarker candidates, we characterized the C. albicans hyphal secretome by gel-free proteomic analysis, and further assessed the antibody-reactivity patterns to this subproteome in serum pools from 12 patients with non-catheter-associated IC (ncIC), 11 patients with catheter-associated IC (cIC), and 11 non-IC patients. We identified 301 secreted hyphal proteins stratified to stem from the extracellular region, cell wall, cell surface, or intracellular compartments. ncIC and cIC patients had higher antibody levels to the hyphal secretome than non-IC patients. Seven secreted hyphal proteins were identified to be immunogenic (Bgl2, Eno1, Pgk1, Glx3, Sap5, Pra1 and Tdh3). Antibody-reactivity patterns to Bgl2, Eno1, Pgk1 and Glx3 discriminated IC patients from non-IC patients, while those to Sap5, Pra1 and Tdh3 differentiated between cIC and non-IC patients. These proteins may be useful for development of future IC diagnostic tests.

Diagnosing SARS-CoV-2 with Antigen Testing, Transcription-Mediated Amplification and Real-Time PCR.

This study was performed as a head-to-head comparison of the performance characteristics of (1) two SARS-CoV-2-specific rapid antigen assays with real-time PCR as gold standard as well as (2) a fully automated high-throughput transcription-mediated amplification (TMA) assay and real-time PCR in a latent class analysis-based test comparison without a gold standard with several hundred samples in a low prevalence "real world" setting. Recorded sensitivity and specificity of the NADAL and the LumiraDx antigen assays and the Hologic Aptima SARS-CoV-2 TMA assay were 0.1429 (0.0194, 0.5835), 0.7644 (0.7016, 0.8174), and 0.7157 (0, 1) as well as 0.4545 (0.2022, 0.7326), 0.9954 (0.9817, 0.9988), and 0.9997 (not estimable), respectively. Agreement kappa between the positive results of the two antigen-based assays was 0.060 (0.002, 0.167) and 0.659 (0.492, 0.825) for TMA and real-time PCR. Samples with low viral load as indicated by cycle threshold (Ct) values > 30 were generally missed by both antigen assays, while 1:10 pooling suggested higher sensitivity of TMA compared to real-time PCR. In conclusion, both sensitivity and specificity speak in favor of the use of the LumiraDx rather than the NADAL antigen assay, while TMA results are comparably as accurate as PCR, when applied in a low prevalence setting.

CryptoType - Public Datasets for MALDI-TOF-MS Based Differentiation of Cryptococcus neoformans/gattii Complexes.

Yeasts of the Cryptococcus neoformans/gattii species complexes are human pathogens mostly in immune compromised individuals, and can cause infections from dermal lesions to fungal meningitis. Differences in virulence and antifungal drug susceptibility of species in these complexes indicate the value of full differentiation to species level in diagnostic procedures. MALDI-TOF MS has been reported to sufficiently discriminate these species. Here, we sought to re-evaluate sample pre-processing procedures and create a set of publicly available references for use with the MALDI Biotyper system. Peak content using four different pre-processing protocols was assessed, and database entries for 13 reference strains created. These were evaluated against a collection of 153 clinical isolates, typed by conventional means. The use of decapsulating protocols or mechanical disruption did not sufficiently increase the information content to justify the extra hands-on-time. Using the set of 13 reference entries created with the standard formic acid extraction, we were able to correctly classify 143/153 (93.5%) of our test isolates. The majority of the remaining ten isolates still gave correct top matches; only two isolates did not give reproducible identifications. This indicates that the log score cut-off can be lowered also in this context. Ease to identify cryptococcal isolates to the species level is improved by the workflow evaluated here. The database references are freely available from https://github.com/oliverbader/BioTyper-libraries for incorporation into local diagnostic systems.

Candida parapsilosis Colony Morphotype Forecasts Biofilm Formation of Clinical Isolates.

Candida parapsilosis is a frequent cause of fungal bloodstream infections, especially in critically ill neonates or immunocompromised patients. Due to the formation of biofilms, the use of indwelling catheters and other medical devices increases the risk of infection and complicates treatment, as cells embedded in biofilms display reduced drug susceptibility. Therefore, biofilm formation may be a significant clinical parameter, guiding downstream therapeutic choices. Here, we phenotypically characterized 120 selected isolates out of a prospective collection of 215 clinical C. parapsilosis isolates, determining biofilm formation, major emerging colony morphotype, and antifungal drug susceptibility of the isolates and their biofilms. In our isolate set, increased biofilm formation capacity was independent of body site of isolation and not predictable using standard or modified European Committee on Antimicrobial Susceptibility Testing (EUCAST) drug susceptibility testing protocols. In contrast, biofilm formation was strongly correlated with the appearance of non-smooth colony morphotypes and invasiveness into agar plates. Our data suggest that the observation of non-smooth colony morphotypes in cultures of C. parapsilosis may help as an indicator to consider the initiation of anti-biofilm-active therapy, such as the switch from azole- to echinocandin- or polyene-based strategies, especially in case of infections by potent biofilm-forming strains.

Rapid direct detection of pathogens for diagnosis of joint infections by MALDI-TOF MS after liquid enrichment in the BacT/Alert blood culture system.

Pathogen identification is a critical step during diagnosis of infectious diseases. Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight mass spectrometry (MALDI-TOF-MS) has become the gold standard for identification of microorganisms cultured on solid media in microbiology laboratories. Direct identification of microbes from liquid specimen, circumventing the need for the additional overnight cultivation step, has been successfully established for blood culture, urine and liquor. Here, we evaluate the ability of MALDI-TOF MS for direct identification of pathogens in synovial fluid after liquid enrichment in BacT/Alert blood culture bottles. Influence of synovial specimen quality on direct species identification with the MALDI BioTyper/Sepsityper was tested with samples inoculated from pretested native synovia with concomitant inoculation of blood or pus, or highly viscous fluid. Here, we achieved >90% concordance with culture on solid medium, and only mixed-species samples posed significant problems. Performance in routine diagnostics was tested prospectively on bottles inoculated by treating physicians on ward. There, we achieved >70% concordance with culture on solid media. The major contributors to test failure were the absence of a measurable mass signal and mixed-specimen samples. The Sepsityper workflow worked well on samples derived from BacT/Alert blood culture bottles inoculated with synovial fluid, giving concordant results to identification from solid media. Host remnant material in the inoculum, such as blood or pus, had no detrimental effect on identification score values of the BioTyper system after processing with the Sepsityper workflow, and neither had the initial viscosity of the synovial sample.

Phenotypic Variability in a Coinfection With Three Independent Candida parapsilosis Lineages.

The human pathogenic yeast Candida parapsilosis has gained significant importance over the past decades as one of the principal causes of fungal bloodstream infections. Isolates of C. parapsilosis are known to be able to switch between several different colony morphologies in vitro, which are correlated with different cell shapes, altered cell surface properties, and thus different capacities to form biofilms on indwelling medical devices. In a set of six clinical specimens from a single surgery patient yielding stable smooth- as well as crepe-morphology isolates, we investigated the differences between five of them on a phenotypic and genomic level. In contrast to the initial assumption that they were switched forms of a clonal strain, karyotyping and genome sequencing showed that the patient was colonized by at least three distinct linages. Statistical analysis placed these groups distantly across the population of C. parapsilosis. Interestingly the single blood culture isolate was of smooth morphology and matched with an isolate from the patient's nose of similar morphology. Strong variation between the isolates was seen in adhesin-encoding genes, where repeat regions showed significant variation in length and repeat-numbers, most strikingly in HWP1 of the smooth isolates. Although no differences in drug susceptibility were evident, the high phylogenetic distance separating the individual strains highlights the need for testing of multiple colonies in routine practice. The absence of biofilm formation in the blood stream isolate indicates a lack of respective adhesins in the cell wall, in turn pointing toward lack of adhesion as a positively contributing factor for dissemination.

There is no intraocular affection on a SARS-CoV-2 - Infected ocular surface.

PURPOSE: The presence of SARS-CoV-2 RNA in anterior chamber fluid and/or the vitreous in patients with SARS-CoV-2 RNA on the ocular surface is unclear. Knowledge about the infectious state of intraocular structures could influence the daily work of ophthalmic surgeons. OBSERVATIONS: We analyzed ocular samples from a patient who had succumbed to COVID-19 pneumonia for the prevalence of SARS-CoV-2 RNA. We detected viral RNA in the ocular-surface samples on one swab and in one excidate from the conjunctiva. Samples from the anterior chamber and vitreous revealed no SARS-CoV-2 RNA. CONCLUSIONS: SARS-CoV-2 can effectively be inactivated with standard disinfection agents. The now proven absence of SARS-CoV-2 in intraocular fluids could influence how ophthalmic surgeons work. Without having to account for the risk of a contagion via the anterior chamber and/or vitreous body, the surgical staff would require no additional, more elaborate protection.

Molecular Tools for the Detection and Deduction of Azole Antifungal Drug Resistance Phenotypes in Aspergillus Species.

The incidence of azole resistance in Aspergillus species has increased over the past years, most importantly for Aspergillus fumigatus. This is partially attributable to the global spread of only a few resistance alleles through the environment. Secondary resistance is a significant clinical concern, as invasive aspergillosis with drug-susceptible strains is already difficult to treat, and exclusion of azole-based antifungals from prophylaxis or first-line treatment of invasive aspergillosis in high-risk patients would dramatically limit drug choices, thus increasing mortality rates for immunocompromised patients. Management options for invasive aspergillosis caused by azole-resistant A. fumigatus strains were recently reevaluated by an international expert panel, which concluded that drug resistance testing of cultured isolates is highly indicated when antifungal therapy is intended. In geographical regions with a high environmental prevalence of azole-resistant strains, initial therapy should be guided by such analyses. More environmental and clinical screening studies are therefore needed to generate the local epidemiologic data if such measures are to be implemented on a sound basis. Here we propose a first workflow for evaluating isolates from screening studies, and we compile the MIC values correlating with individual amino acid substitutions in the products of cyp51 genes for interpretation of DNA sequencing data, especially in the absence of cultured isolates.

Oral findings and dental behaviour before and after liver transplantation - a single-centre cross-sectional study.

OBJECTIVE: The aim of this single-centre, cross-sectional study was to evaluate dental, periodontal and mycological findings, as well as oral behaviour, in patients before (pre-LTx) and after (post-LTx) liver transplantation. METHODS: A total of 47 patients pre-LTx and 119 patients post-LTx were asked to participate. Oral health behaviour was assessed using a standardised questionnaire. Oral examinations included dental [decayed, missing and filled teeth (DMFT) index] and periodontal [papillary bleeding index (PBI), periodontal probing depth (PPD) and clinical attachment loss (CAL)] findings. For Candida screening, swabs from the oral mucosa were cultured. Statistical analysis was performed using the Student's t-test or the Mann-Whitney U-test, depending on whether or not the data followed a normal distribution; Fisher's exact test was also performed. The significance level was α = 5%. RESULTS: A total of 110 patients were included (pre-LTx, n = 35; post-LTx, n = 75). Different patients were investigated in the post-LTx and pre-LTx groups. Lack of use of supplemental oral-hygiene aids was noted. Between-group comparisons failed to find significant overall differences in DMFT and periodontal status. The post-LTx group showed fewer decayed teeth (P = 0.03). A total of 86% of patients pre-LTx and 84% of patients post-LTx were found to need dental treatment, and 60% of patients pre-LTx and 55% of patients post-LTx showed a need for periodontal treatment. The prevalence of Candida albicans was high; however, there were no statistically significant differences between the groups in regard to fungal infection. CONCLUSION: Improved dental care pre- and post-transplant, including screening for fungal infections, is recommended to avoid systemic infections in LTx patients. Increased attention to oral health care, and interdisciplinary collaboration to provide guidance, is needed to improve the oral health of patients before and after LTx.

Diversity and Antifungal Drug Susceptibility of Cryptococcus Isolates in Thailand.

Yeasts of the Cryptococcus species complex are the causative agent of cryptococcosis, especially in human immunodeficiency virus (HIV) positive individuals. Cerebral or disseminated cryptococcosis has a very high mortality rate worldwide, including in Thailand. Additionally, an increasing rate of antifungal drug resistant cryptococcal isolates has been reported in several neighboring countries, complicating therapeutic approaches. To understand the situation of this infection in Thailand, we retrospectively investigated the molecular epidemiology and antifungal drug resistance in a collection of 74 clinical, 52 environmental and two veterinary isolates using the URA5-RFLP for typing and the EUCAST guideline for susceptibility testing. Where no EUCAST breakpoints (AMB and 5FC) were available, CLSI epidemiologic cutoff values were used for interpretation. Cryptococcal molecular type diversity showed most isolates were C. grubii, molecular type VNI. One clinical isolate was C. deuterogattii (mol. type VGII) and another C. grubii (mol. type VNII). One strain from environment was classified as C. grubii (mol. type VNII). No resistant strains were detected in this retrospective study for either of the antimycotics tested; however, monitoring of the epidemiology of Cryptococcus species in infected patients in Thailand needs to be continued to detect emergence of resistance.

Subtyping of Campylobacter jejuni ssp. doylei Isolates Using Mass Spectrometry-based PhyloProteomics (MSPP).

MALDI-TOF MS offers the possibility to differentiate some bacteria not only at the species and subspecies level but even below, at the strain level. Allelic isoforms of the detectable biomarker ions result in isolate-specific mass shifts. Mass spectrometry-based phyloproteomics (MSPP) is a novel technique that combines the mass spectrometric detectable biomarker masses in a scheme that allows deduction of phyloproteomic relations from isolate specific mass shifts compared to a genome sequenced reference strain. The deduced amino acid sequences are then used to calculate MSPP-based dendrograms. Here we describe the workflow of MSPP by typing a Campylobacter jejuni ssp. doylei isolate collection of seven strains. All seven strains were of human origin and multilocus sequence typing (MLST) demonstrated their genetic diversity. MSPP-typing resulted in seven different MSPP sequence types, sufficiently reflecting their phylogenetic relations. The C. jejuni ssp. doylei MSPP scheme includes 14 different biomarker ions, mostly ribosomal proteins in the mass range of 2 to 11 kDa. MSPP can in principle, be adapted to other mass spectrometric platforms with an extended mass range. Therefore, this technique has the potential to become a useful tool for strain level microbial typing.

Towards proteomic species barcoding of fungi - An example using Scedosporium/Pseudallescheria complex isolates.

MALDI-ToF mass spectrometry offers fast and reliable species identification for bacteria and yeasts under clinical routine conditions. Here, we produced mass spectra for identification of clinically important species of the Pseudallescheria/Scedosporium complex using the recently suggested new nomenclature and use this example to discuss to what extent the principle of DNA barcoding might be transferred to mass spectrometry.

Aspergillus oerlinghausenensis, a new mould species closely related to A. fumigatus.

Two isolates belonging to Aspergillus section Fumigati were recovered from German soil on itraconazole containing agar media. Phylogenetic analysis and phenotypic characterization of both isolates show that they represent a novel species named Aspergillus oerlinghausenensis (holotype CBS H-22119(HT), ex-type CBS 139183(T) = IBT 33878 = DTO 316-A3). The species is phylogenetically related to A. fischeri and A. fumigatus. Aspergillus oerlinghausenensis can be differentiated from A. fischeri by its higher growth rate at 50°C. Furthermore, A. oerlinghausenensis is protoheterothallic as only the MAT1-1 idiomorph was detected, while A. fischeri is homothallic. The species differs from A. fumigatus by a weak sporulation on malt extract agar at 25°C, a floccose colony texture on Czapek yeast extract agar and malt extract agar and subglobose instead of subclavate vesicles. The cyp51A promoter region of A. oerlinghausenensis deviates from the previously reported cyp51A promoter regions in A. fumigatus and potentially presents a novel azole resistance conferring modification. Due to the close relationship of A. oerlinghausenensis with A. fischeri and A. fumigatus, this species is placed in a good position for comparative studies involving these species.