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.

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.

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.

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.

Environmental isolates of azole-resistant Aspergillus fumigatus in Germany.

Azole antifungal drug resistance in Aspergillus fumigatus is an emerging problem in several parts of the world. Here we investigated the distribution of such strains in soils from Germany. At a general positivity rate of 12%, most prevalently, we found strains with the TR34/L98H and TR46/Y121F/T289A alleles, dispersed along a corridor across northern Germany. Comparison of the distributions of resistance alleles and genotypes between environment and clinical samples suggests the presence of local clinical clusters.

Proteomic analysis of hyperadhesive Candida glabrata clinical isolates reveals a core wall proteome and differential incorporation of adhesins.

Attachment to human host tissues or abiotic medical devices is a key step in the development of infections by Candida glabrata. The genome of this pathogenic yeast codes for a large number of adhesins, but proteomic work using reference strains has shown incorporation of only few adhesins in the cell wall. By making inventories of the wall proteomes of hyperadhesive clinical isolates and reference strain CBS138 using mass spectrometry, we describe the cell wall proteome of C. glabrata and tested the hypothesis that hyperadhesive isolates display differential incorporation of adhesins. Two clinical strains (PEU382 and PEU427) were selected, which both were hyperadhesive to polystyrene and showed high surface hydrophobicity. Cell wall proteome analysis under biofilm-forming conditions identified a core proteome of about 20 proteins present in all C. glabrata strains. In addition, 12 adhesin-like wall proteins were identified in the hyperadherent strains, including six novel adhesins (Awp8-13) of which only Awp12 was also present in CBS138. We conclude that the hyperadhesive capacity of these two clinical C. glabrata isolates is correlated with increased and differential incorporation of cell wall adhesins. Future studies should elucidate the role of the identified proteins in the establishment of C. glabrata infections.

Molecular types of Cryptococcus gattii/Cryptococcus neoformans species complex from clinical and environmental sources in Nairobi, Kenya.

Cryptococcal meningitis infections cause high mortality rates among HIV-infected patients in Sub-Saharan Africa. The high incidences of cryptococcal infections may be attributed to common environmental sources which, if identified, could lead to institution of appropriate control strategies. To determine the genotypes of Cryptococcus gattii/C. neoformans- species complex from Nairobi, Kenya, 123 clinical and environmental isolates were characterised. Typing was done using orotidine monophosphate pyrophosphorylase (URA5) gene restriction fragment length polymorphism (URA5-RFLP). The majority of the isolates [105/123; 85.4%] were C. neoformans genotype (AFLPI/VNI) and 1.6% AFLP1A/VNB/VNII, whereas (13%) were C. gattii (AFLP4/VGI). This is the first report on the genotypes of C. gattii/C. neoformans species complex from clinical and environmental sources in Nairobi, Kenya and the isolation of C. gattii genotype AFLP4/VGI from the environment in Kenya.

Yeast on-target lysis (YOTL), a procedure for making auxiliary mass spectrum data sets for clinical routine identification of yeasts.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based species identification has become a reliable and fast tool for use in clinical diagnostics, including in mycology. To identify yeasts in the MALDI Biotyper system, a multistep extraction protocol, which is also used to generate the reference spectra, is recommended. Sample preparation by on-target lysis (OTL) requires significantly less hands-on time and is therefore highly desirable, but it results in too-low MALDI Biotyper log score values to allow automated species identification. To overcome this problem, we developed a procedure for generating and validating an OTL spectrum data set for the most relevant and frequently occurring yeast species in clinical specimens. The performance was evaluated against a set of OTL spectra derived during clinical routine procedures and from a set of closely related yeasts. In the diagnostic setting, the OTL procedure significantly decreased the workload but allowed species identification with high specificity and sensitivity. False identifications were not observed. The use of in-house-generated OTL reference spectra can highly accelerate MALDI-TOF MS-based yeast species identification using the MALDI Biotyper.

Adhesins in human fungal pathogens: glue with plenty of stick.

Understanding the pathogenesis of an infectious disease is critical for developing new methods to prevent infection and diagnose or cure disease. Adherence of microorganisms to host tissue is a prerequisite for tissue invasion and infection. Fungal cell wall adhesins involved in adherence to host tissue or abiotic medical devices are critical for colonization leading to invasion and damage of host tissue. Here, with a main focus on pathogenic Candida species, we summarize recent progress made in the field of adhesins in human fungal pathogens and underscore the importance of these proteins in establishment of fungal diseases.

[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.

cyp51A-Based mechanisms of Aspergillus fumigatus azole drug resistance present in clinical samples from Germany.

Since the mid-1990s, a steady increase in the occurrence of itraconazole-resistant Aspergillus fumigatus isolates has been observed in clinical contexts, leading to therapeutic failure in the treatment of aspergillosis. This increase has been predominantly linked to a single allele of the cyp51A gene, termed TR/L98H, which is thought to have arisen through the use of agricultural azoles. Here, we investigated the current epidemiology of triazole-resistant A. fumigatus and underlying cyp51A mutations in clinical samples in Germany. From a total of 527 samples, 17 (3.2%) showed elevated MIC0 values (the lowest concentrations with no visible growth) for at least one of the three substances (itraconazole, voriconazole, and posaconazole) tested. The highest prevalence of resistant isolates was observed in cystic fibrosis patients (5.2%). Among resistant isolates, the TR/L98H mutation in cyp51A was the most prevalent, but isolates with the G54W and M220I substitutions and the novel F219C substitution were also found. The isolate with the G54W substitution was highly resistant to both itraconazole and posaconazole, while all others showed high-level resistance only to itraconazole. For the remaining six isolates, no mutations in cyp51A were found, indicating the presence of other mechanisms. With the exception of the strains carrying the F219C and M220I substitutions, many itraconazole-resistant strains also showed cross-resistance to voriconazole and posaconazole with moderately increased MIC0 values. In conclusion, the prevalence of azole-resistant A. fumigatus in our clinical test set is lower than that previously reported for other countries. Although the TR/L98H mutation frequently occurs among triazole-resistant strains in Germany, it is not the only resistance mechanism present.

Discrimination of multilocus sequence typing-based Campylobacter jejuni subgroups by MALDI-TOF mass spectrometry.

BACKGROUND: Campylobacter jejuni, the most common bacterial pathogen causing gastroenteritis, shows a wide genetic diversity. Previously, we demonstrated by the combination of multi locus sequence typing (MLST)-based UPGMA-clustering and analysis of 16 genetic markers that twelve different C. jejuni subgroups can be distinguished. Among these are two prominent subgroups. The first subgroup contains the majority of hyperinvasive strains and is characterized by a dimeric form of the chemotaxis-receptor Tlp7(m+c). The second has an extended amino acid metabolism and is characterized by the presence of a periplasmic asparaginase (ansB) and gamma-glutamyl-transpeptidase (ggt). RESULTS: Phyloproteomic principal component analysis (PCA) hierarchical clustering of MALDI-TOF based intact cell mass spectrometry (ICMS) spectra was able to group particular C. jejuni subgroups of phylogenetic related isolates in distinct clusters. Especially the aforementioned Tlp7(m+c)(+) and ansB+/ ggt+ subgroups could be discriminated by PCA. Overlay of ICMS spectra of all isolates led to the identification of characteristic biomarker ions for these specific C. jejuni subgroups. Thus, mass peak shifts can be used to identify the C. jejuni subgroup with an extended amino acid metabolism. CONCLUSIONS: Although the PCA hierarchical clustering of ICMS-spectra groups the tested isolates into a different order as compared to MLST-based UPGMA-clustering, the isolates of the indicator-groups form predominantly coherent clusters. These clusters reflect phenotypic aspects better than phylogenetic clustering, indicating that the genes corresponding to the biomarker ions are phylogenetically coupled to the tested marker genes. Thus, PCA clustering could be an additional tool for analyzing the relatedness of bacterial isolates.

Two clinical isolates of Candida glabrata exhibiting reduced sensitivity to amphotericin B both harbor mutations in ERG2.

Two novel isolates of Candida glabrata exhibiting reduced sensitivity to amphotericin B (MIC, 8 µg ml(-1)) were found to be ERG2 mutants, wherein Δ(8)-sterol intermediates comprised >90% of the total cellular sterol fraction. Both harbored an alteration at Thr(121) in ERG2; the corresponding residue (Thr(119)) in Saccharomyces cerevisiae is essential for sterol Δ8-Δ7 isomerization. This constitutes the first report of C. glabrata harboring mutations in ERG2 and exhibiting reduced sensitivity to amphotericin B.

Facultative sterol uptake in an ergosterol-deficient clinical isolate of Candida glabrata harboring a missense mutation in ERG11 and exhibiting cross-resistance to azoles and amphotericin B.

We identified a clinical isolate of Candida glabrata (CG156) exhibiting flocculent growth and cross-resistance to fluconazole (FLC), voriconazole (VRC), and amphotericin B (AMB), with MICs of >256, >256, and 32 µg ml(-1), respectively. Sterol analysis using gas chromatography-mass spectrometry (GC-MS) revealed that CG156 was a sterol 14α-demethylase (Erg11p) mutant, wherein 14α-methylated intermediates (lanosterol was >80% of the total) were the only detectable sterols. ERG11 sequencing indicated that CG156 harbored a single-amino-acid substitution (G315D) which nullified the function of native Erg11p. In heterologous expression studies using a doxycycline-regulatable Saccharomyces cerevisiae erg11 strain, wild-type C. glabrata Erg11p fully complemented the function of S. cerevisiae sterol 14α-demethylase, restoring growth and ergosterol synthesis in recombinant yeast; mutated CG156 Erg11p did not. CG156 was culturable using sterol-free, glucose-containing yeast minimal medium ((glc)YM). However, when grown on sterol-supplemented (glc)YM (with ergosta 7,22-dienol, ergosterol, cholestanol, cholesterol, Δ(7)-cholestenol, or desmosterol), CG156 cultures exhibited shorter lag phases, reached higher cell densities, and showed alterations in cellular sterol composition. Unlike comparator isolates (harboring wild-type ERG11) that became less sensitive to FLC and VRC when cultured on sterol-supplemented (glc)YM, facultative sterol uptake by CG156 did not affect its azole-resistant phenotype. Conversely, CG156 grown using (glc)YM with ergosterol (or with ergosta 7,22-dienol) showed increased sensitivity to AMB; CG156 grown using (glc)YM with cholesterol (or with cholestanol) became more resistant (MICs of 2 and >64 µg AMB ml(-1), respectively). Our results provide insights into the consequences of sterol uptake and metabolism on growth and antifungal resistance in C. glabrata.

Interlaboratory comparison of PCR-based identification of Candida and Aspergillus DNA in spiked blood samples.

Despite PCR per se being a powerful and sensitive technique, regarding the detection of fungi in patients' blood, no consensus for a standardised PCR protocol yet exists. To complement other ongoing or accomplished studies which tackle this problem, the German Reference Center for Systemic Mycoses conducted an interlaboratory comparison starting with blood samples spiked with fungal cell elements. Altogether, six laboratories using in-house PCR-protocols from Germany and Austria participated in the trial. Blood samples were spiked with vital cells of Candida albicans or Aspergillus fumigatus. Candida was used in the yeast form, whereas Aspergillus cells were either spiked as conidia or as very young germlings, also known as smoo cells. Spiked blood samples contained between 10 and 10 000 cells ml(-1). Depending on the techniques used for fungal cell disruption and DNA-amplification, detection quality was variable between laboratories, but also differed within single laboratories in different trials particularly for samples spiked with less than 100 cells ml(-1). Altogether, at least regarding the detection of A. fumigatus, two of six laboratories showed constant reliable test results also with low fungal cell number spiked samples. Protocols used by these labs do not differ substantially from others. However, as particularities, one protocol included a conventional phenol chloroform extraction during the DNA preparation process and the other included a real time PCR-protocol based on FRET probes. Other laboratory comparisons on the basis of clinical samples should follow to further evaluate the procedures. The difficulties and problems of such trials in general are discussed.

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.

Mucormycosis in paediatric patients: demographics, risk factors and outcome of 12 contemporary cases.

Mucormycosis is associated with high morbidity and mortality and is perceived as an emerging fungal infection. However, contemporary paediatric data are limited. We present a series of paediatric cases of mucormycosis reported from Germany and Austria collected within a voluntary epidemiological survey through standardised, anonymized case report forms. Twelve cases were reported between January 2004 and December 2008 (six men; mean age: 12.6 years, range: 0.1-17 years). Mucormycosis was proven in nine, and probable in three cases. Isolates included Lichtheimia (syn. Absidia pro parte, Mycocladus) (five), Rhizopus (three) and Mucor (one) species. Infection was limited to soft tissue in three cases, the lung in two cases, and an infected thrombus in one case; rhinocerebral disease was found in three cases, and pulmonary-mediastinal, pulmonary-cerebral and soft tissue-cerebral involvement in one case each. All three patients with isolated soft tissue infection were cured, whereas seven of the remaining patients died (one patient without follow-up). The overall mortality rate was 67%. While these data cannot provide conclusive data on incidence and disease burden of mucormycosis in paediatric patients, they reflect the continuing threat of these infections to immunocompromised patients and the need for improved diagnosis and management.

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.