Reduced Clostridioides difficile infections in hospitalised older people through multiple quality improvement strategies.

OBJECTIVES: To reduce infections with Clostridioides difficile (CDI) in geriatric patients by interventions easily implementable in standard clinical care. METHODS: Prevalence and incidence of CDI between January 2015 and February 2020 were analysed (n = 25,311 patients). Pre-intervention status was assessed from April 2016 to March 2017 (n = 4,922). Between May 2017 and August 2019, a monocentric interventional crossover study (n = 4,655) was conducted including standard care and three interventions: (A) sporicidal cleaning of hospital wards, (B) probiotics and (C) improvement in personal hygiene for CDI patients. This was followed by a multicentric comparison of the interventional bundle (A + B + C) between September 2019 and February 2020 (n = 2,593) with the pre-intervention phase. In 98 CDI cases and matched controls individual risk factors for the development of CDI were compared. RESULTS: Time series analyses of CDI cases revealed a reduction in the prevalence of CDI in all three participating centres prior to the multicentric intervention phase. In the monocentric phase, no effect of individual interventions on CDI prevalence was identified. However, an aggregated analysis of CDI cases comparing the pre-intervention and the multicentric phase revealed a significant reduction in CDI prevalence. Risk factors for the development of CDI included use of antibiotics, anticoagulants, previous stay in long-term care facilities, prior hospital admissions, cardiac and renal failure, malnutrition and anaemia. CONCLUSIONS: The observed reduction in CDI may be attributed to heightened awareness of the study objectives and specific staff training. Individual interventions did not appear to reduce CDI prevalence. A further randomised trial would be necessary to confirm whether the bundle of interventions is truly effective.

Proteome Analysis Reveals the Conidial Surface Protein CcpA Essential for Virulence of the Pathogenic Fungus Aspergillus fumigatus.

Aspergillus fumigatus is a common airborne fungal pathogen of humans and a significant source of mortality in immunocompromised individuals. Here, we provide the most extensive cell wall proteome profiling to date of A. fumigatus resting conidia, the fungal morphotype pertinent to first contact with the host. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified proteins within the conidial cell wall by hydrogen-fluoride (HF)-pyridine extraction and proteins exposed on the surface using a trypsin-shaving approach. One protein, designated conidial cell wall protein A (CcpA), was identified by both methods and was found to be nearly as abundant as hydrophobic rodlet layer-forming protein RodA. CcpA, an amphiphilic protein, like RodA, peaks in expression during sporulation on resting conidia. Despite high cell wall abundance, the cell surface structure of ΔccpA resting conidia appeared normal. However, trypsin shaving of ΔccpA conidia revealed novel surface-exposed proteins not detected on conidia of the wild-type strain. Interestingly, the presence of swollen ΔccpA conidia led to higher activation of neutrophils and dendritic cells than was seen with wild-type conidia and caused significantly less damage to epithelial cells in vitro In addition, virulence was highly attenuated when cortisone-treated, immunosuppressed mice were infected with ΔccpA conidia. CcpA-specific memory T cell responses were detectable in healthy human donors naturally exposed to A. fumigatus conidia, suggesting a role for CcpA as a structural protein impacting conidial immunogenicity rather than possessing a protein-intrinsic immunosuppressive effect. Together, these data suggest that CcpA serves as a conidial stealth protein by altering the conidial surface structure to minimize innate immune recognition.IMPORTANCE The mammalian immune system relies on recognition of pathogen surface antigens for targeting and clearance. In the absence of immune evasion strategies, pathogen clearance is rapid. In the case of Aspergillus fumigatus, the successful fungus must avoid phagocytosis in the lung to establish invasive infection. In healthy individuals, fungal spores are cleared by immune cells; however, in immunocompromised patients, clearance mechanisms are impaired. Here, using proteome analyses, we identified CcpA as an important fungal spore protein involved in pathogenesis. A. fumigatus lacking CcpA was more susceptible to immune recognition and prompt eradication and, consequently, exhibited drastically attenuated virulence. In infection studies, CcpA was required for virulence in infected immunocompromised mice, suggesting that it could be used as a possible immunotherapeutic or diagnostic target in the future. In summary, our report adds a protein to the list of those known to be critical to the complex fungal spore surface environment and, more importantly, identifies a protein important for conidial immunogenicity during infection.

Monitoring microevolution of OXA-48-producing Klebsiella pneumoniae ST147 in a hospital setting by SMRT sequencing.

Objectives: Carbapenemase-producing Klebsiella pneumoniae pose an increasing risk for healthcare facilities worldwide. A continuous monitoring of ST distribution and its association with resistance and virulence genes is required for early detection of successful K. pneumoniae lineages. In this study, we used WGS to characterize MDR blaOXA-48-positive K. pneumoniae isolated from inpatients at the University Medical Center Göttingen, Germany, between March 2013 and August 2014. Methods: Closed genomes for 16 isolates of carbapenemase-producing K. pneumoniae were generated by single molecule real-time technology using the PacBio RSII platform. Results: Eight of the 16 isolates showed identical XbaI macrorestriction patterns and shared the same MLST, ST147. The eight ST147 isolates differed by only 1-25 SNPs of their core genome, indicating a clonal origin. Most of the eight ST147 isolates carried four plasmids with sizes of 246.8, 96.1, 63.6 and 61.0 kb and a novel linear plasmid prophage, named pKO2, of 54.6 kb. The blaOXA-48 gene was located on a 63.6 kb IncL plasmid and is part of composite transposon Tn1999.2. The ST147 isolates expressed the yersinabactin system as a major virulence factor. The comparative whole-genome analysis revealed several rearrangements of mobile genetic elements and losses of chromosomal and plasmidic regions in the ST147 isolates. Conclusions: Single molecule real-time sequencing allowed monitoring of the genetic and epigenetic microevolution of MDR OXA-48-producing K. pneumoniae and revealed in addition to SNPs, complex rearrangements of genetic elements.

[Not Available]. / Multiresistente Erreger - Prävention und Diagnostik.

Multiresistant bacteria play an increasingly important role in everyday clinical practice. This is particularly the case in intensive care units and wards with critically ill patients. Often there is insufficient knowledge concerning diagnostic screening indications and strategies to avoid cross-transmission via infection control strategies. Hereby, we provide an orienting overview and assessment about current guidelines and recommendations with special focus on methicillin-resistant Staphylococcus aureus (MRSA) and multiresistantgramnegative bacteria (MRGN).

Vaccination approaches against opportunistic fungal infections caused by Aspergillus fumigatus.

Although innate immunity primarily combats systemic infections of opportunistic fungi such as Aspergillus and Candida spp., acquired and protective immunoreactions were observed long ago in animal trials following sublethal systemic infections caused by viable fungi or after challenging animals with inactivated fungal cells. Based on these observations, fungal antigens should exist which mediate such protective immunoreactions and have in part already been identified. In this context, this review focuses primarily on the various approaches that have been used to identify protection-mediating Aspergillus-antigens and their rationale. Emphasis is placed on screening methods that have exploited genetic or proteomic approaches on the basis of the corresponding fungal genome projects. Thereby, a survey and description is given of the antigens so far known to be capable of inducing immune responses that protect animals against acquiring lethal systemic aspergillosis.

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.

Sensing of mammalian IL-17A regulates fungal adaptation and virulence.

Infections by opportunistic fungi have traditionally been viewed as the gross result of a pathogenic automatism, which makes a weakened host more vulnerable to microbial insults. However, fungal sensing of a host's immune environment might render this process more elaborate than previously appreciated. Here we show that interleukin (IL)-17A binds fungal cells, thus tackling both sides of the host-pathogen interaction in experimental settings of host colonization and/or chronic infection. Global transcriptional profiling reveals that IL-17A induces artificial nutrient starvation conditions in Candida albicans, resulting in a downregulation of the target of rapamycin signalling pathway and in an increase in autophagic responses and intracellular cAMP. The augmented adhesion and filamentous growth, also observed with Aspergillus fumigatus, eventually translates into enhanced biofilm formation and resistance to local antifungal defenses. This might exemplify a mechanism whereby fungi have evolved a means of sensing host immunity to ensure their own persistence in an immunologically dynamic environment.

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.

Secreted glutamic protease rescues aspartic protease Pep deficiency in Aspergillus fumigatus during growth in acidic protein medium.

In an acidic protein medium Aspergillus fumigatus secretes an aspartic endoprotease (Pep) as well as tripeptidyl-peptidases, a prolyl-peptidase and carboxypeptidases. In addition, LC-MS/MS revealed a novel glutamic protease, AfuGprA, homologous to Aspergillus niger aspergillopepsin II. The importance of AfuGprA in protein digestion was evaluated by deletion of its encoding gene in A. fumigatus wild-type D141 and in a pepΔ mutant. Either A. fumigatus Pep or AfuGprA was shown to be necessary for fungal growth in protein medium at low pH. Exoproteolytic activity is therefore not sufficient for complete protein hydrolysis and fungal growth in a medium containing proteins as the sole nitrogen source. Pep and AfuGprA constitute a pair of endoproteases active at low pH, in analogy to A. fumigatus alkaline protease (Alp) and metalloprotease I (Mep), where at least one of these enzymes is necessary for fungal growth in protein medium at neutral pH. Heterologous expression of AfuGprA in Pichia pastoris showed that the enzyme is synthesized as a preproprotein and that the propeptide is removed through an autoproteolytic reaction at low pH to generate the mature protease. In contrast to A. niger aspergillopepsin II, AfuGprA is a single-chain protein and is structurally more similar to G1 proteases characterized in other non-Aspergillus fungi.

Novel cytosolic allergens of Aspergillus fumigatus identified from germinating conidia.

Aspergillus fumigatus is the common cause of allergic broncho-pulmonary aspergillosis (ABPA) and most of the allergens have been described from its secreted fraction. In the present investigation, germinating conidial cytosolic proteins of A. fumigatus were extracted from a 16 h culture. The proteome from this fraction was developed, and immuno-blots were generated using pooled ABPA patients' sera. Well separated Immunoglobulin-E (IgE) and Immunoglobulin-G (IgG) reactive spots were picked from corresponding 2DE gels and subjected to mass spectrometric analysis. As a result, 66 immuno-reactive proteins were identified from two geographically different strains (190/96 and DAYA) of A. fumigatus. Only 3 out of 66 proteins reacted with IgG, and the remaining 63 proteins were found to be IgE reactive. These 63 IgE-reactive cytosolic proteins from germinating conidia included 2 already known (Asp f12 and Asp f22) and 4 predicted allergens (Hsp88, Hsp70, malate dehydrogenase, and alcohol dehydrogenase) based on their homology with other known fungal allergens. In view of this, the panel of presently identified IgE-reactive novel proteins holds the potential of providing a basis for the wider diagnostic application in assay for allergic aspergillosis. We could demonstrate that recombinantly expressed proteins from this panel showed consistent reactivity with IgE of individual sera of ABPA patients. The recombinantly expressed proteins may also be useful in desensitization therapy of allergic disorders including ABPA.

Immuno-reactive molecules identified from the secreted proteome of Aspergillus fumigatus.

The secreted proteomes of a three week old culture of an Indian (190/96) and a German (DAYA) Aspergillus fumigatus isolate were investigated for reactivity with IgG and/or IgE antibodies derived from pooled allergic broncho-pulmonary aspergillosis (ABPA) patients' sera. Two dimensional Western blotting followed by mass spectrometric analysis of the reactive protein spots revealed 35 proteins from the two A. fumigatus strains. There were seven known A. fumigatus allergens among them (Asp f1-4, Asp f9, Asp f10, and Asp f13/15), whereas three proteins displaying significant sequence similarity to known fungal allergens have been assigned as predicted allergens (Dipeptidyl-peptidase-V precursor, Nuclear transport factor 2, and Malate dehydrogenase, NAD-dependent). Eight IgG and IgE reactive proteins were common in both strains; however, 12 proteins specifically reacted in 190/96 and 15 in DAYA. Further testing with sera of 5 individual ABPA patients demonstrated that 12 out of 20 immunoreactive proteins of 190/96 strain of A. fumigatus had consistent reactivity with IgE. Seven of these proteins reacted with IgG also. The 25 of 35 identified proteins are novel with respect to immuno-reactivity with ABPA patients' sera and could form a panel of molecules to improve the currently existing less-sensitive diagnostic methods. Through expressing recombinantly, these proteins may also serve as a tool in desensibilization strategies.

Analysis of the cellular Aspergillus fumigatus proteome that reacts with sera from rabbits developing an acquired immunity after experimental aspergillosis.

Invasive aspergillosis caused by the mould Aspergillus fumigatus is a life-threatening lung or systemic infection in the immunocompromised host. In this study, a protective immune response against the disease was achieved in two infected rabbits, and the cellular fungal antigenic proteome that mediated such a response was investigated against the background of vaccine development efforts. Altogether, 59 different Aspergillus proteins were found becoming reactive in the course of the developing immunity, many of which are described in this context for the first time. These included proteins related to oxidative stress management, glycolysis and other metabolic pathways. As oxidative stress is suspected to be one of the major defense mechanisms, the results may indicate at least in part a continuous response of the pathogen to evade the host's immune system. In addition, proteins with suspected cell surface association or crucial function for fungal cell development were identified. As these antigens are newly recognized during the process of the developing immunoprotection, they may not only represent valuable infection markers but also importantly broaden the list of possible vaccine candidates.

Aspergillus protein degradation pathways with different secreted protease sets at neutral and acidic pH.

Aspergillus fumigatus grows well at neutral and acidic pH in a medium containing protein as the sole nitrogen source by secreting two different sets of proteases. Neutral pH favors the secretion of neutral and alkaline endoproteases, leucine aminopeptidases (Laps) which are nonspecific monoaminopeptidases, and an X-prolyl dipeptidase (DppIV). Acidic pH environment promotes the secretion of an aspartic endoprotease of pepsin family (Pep1) and tripeptidyl-peptidases of the sedolisin family (SedB and SedD). A novel prolyl peptidase, AfuS28, was found to be secreted in both alkaline and acidic conditions. In previous studies, Laps were shown to degrade peptides from their N-terminus until an X-Pro sequence acts as a stop signal. X-Pro sequences can be then removed by DppIV, which allows Laps access to the following residues. We have shown that at acidic pH Seds degrade large peptides from their N-terminus into tripeptides until Pro in P1 or P'1 position acts as a stop for these exopeptidases. However, X-X-Pro and X-X-X-Pro sequences can be removed by AfuS28 thus allowing Seds further sequential proteolysis. In conclusion, both alkaline and acidic sets of proteases contain exoprotease activity capable of cleaving after proline residues that cannot be removed during sequential digestion by nonspecific exopeptidases.

Comparison between pp65 antigenemia assay and quantitative real-time polymerase chain reaction for detection of active cytomegalovirus infection in routine diagnostics.

Real-time polymerase chain reaction (PCR) and pp65 antigenemia assay for the detection of active cytomegalovirus infection in immunocompromised patients experiencing neutropenia after bone marrow or kidney transplantation have been compared with a special focus on evaluability and embedment in daily routine diagnostics. Investigating 334 specimens from 97 patients, real-time PCR was shown to be the superior assay with regard to the parameters focused on.

Characterization and kinetics of the major purine transporters in Aspergillus fumigatus.

Three genes encoding putative purine transporters have been identified in silico in the genome of Aspergillus fumigatus by their very close similarity of their translation products to well-studied homologues in A. nidulans. Two of these transporters, called AfUapC and AfAzgA, were found responsible for bulk uptake of purines and studied in detail herein. Genetic knock-out analysis, regulation of transcription, direct purine uptake assays and heterologous expression in A. nidulans have unequivocally shown that AfUapC and AfAzgA are high-affinity, high-capacity, purine/H(+) symporters, the first being specific for xanthine, uric acid and oxypurinol, whereas the second for adenine, hypoxanthine, guanine and purine. The expression of these transporters is primarily controlled at the level of transcription. Transcription of both genes is purine-inducible, albeit with different efficiencies, whereas AfuapC is also ammonium-repressible. We characterised in detail the kinetics of the AfUapC and AfAzgA transporters, both in A. fumigatus and in A. nidulans, using a plethora of possible purine substrates. This analysis led us to propose kinetic models describing the molecular interactions of AfUapC and AfAzgA with purines. These models are discussed comparatively with analogous models from other purine transporters from fungi, bacteria and humans, and within the frame of a systematic development of novel purine-related antifungals.

Epidemiology and antifungal susceptibilities of Candida spp. to six antifungal agents: results from a surveillance study on fungaemia in Germany from July 2004 to August 2005.

OBJECTIVES: Data on fungal infections occurring in Germany are rare to date. The aim of the present study was to survey the epidemiological situation in Germany, to provide data on the susceptibility of the fungal isolates to antifungals. METHODS: Five hundred and sixty-one Candida isolates were collected from primarily sterile sites of patients from July 2004 to August 2005 with the aid of a nationwide established laboratory network, MykolabNet-D. The MICs of amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole and caspofungin were determined using the microdilution reference procedure M27-A2 of the CLSI. RESULTS: Candida albicans was the most frequently isolated species (58.5%), followed by Candida glabrata (19.1%), Candida parapsilosis (8.0%) and Candida tropicalis (7.5%). In contrast, the isolation rate of Candida krusei (1.4%) was low. Candida kefyr appeared as a new pathogen in this profile. Amphotericin B revealed excellent activity, with only three resistant isolates (0.5%). A total of 25 isolates (4.5%) showed resistance against flucytosine. All 25 isolates were identified as C. tropicalis indicating a peculiarity within German isolates. The resistance rate of all tested isolates to fluconazole and to itraconazole was 3.7% and 17.6%, respectively. According to the provisional breakpoints, two isolates (0.4%) were tested as resistant to voriconazole. Caspofungin was active against the majority of isolates where an intrinsic resistance is unknown. CONCLUSIONS: This latest German survey of isolates from patients with fungaemia demonstrates a favourable situation with respect to antifungal susceptibilities for the antifungal substances tested.

Sulphite efflux pumps in Aspergillus fumigatus and dermatophytes.

Dermatophytes and other filamentous fungi excrete sulphite as a reducing agent during keratin degradation. In the presence of sulphite, cystine in keratin is directly cleaved to cysteine and S-sulphocysteine, and thereby, reduced proteins become accessible to hydrolysis by a variety of secreted endo- and exoproteases. A gene encoding a sulphite transporter in Aspergillus fumigatus (AfuSSU1), and orthologues in the dermatophytes Trichophyton rubrum and Arthroderma benhamiae (TruSSU1 and AbeSSU1, respectively), were identified by functional expression in Saccharomyces cerevisiae. Like the S. cerevisiae sulphite efflux pump Ssu1p, AfuSsu1p, TruSsu1p and AbeSsu1p belong to the tellurite-resistance/dicarboxylate transporter (TDT) family which includes the Escherichia coli tellurite transporter TehAp and the Schizosaccharomyces pombe malate transporter Mae1p. Seven genes in the A. fumigatus genome encode transporters of the TDT family. However, gene disruption of AfuSSU1 and of the two more closely related paralogues revealed that only AfuSSU1 encodes a sulphite efflux pump. TruSsulp and AbeSsulp are believed to be the first members of the TDT family identified in dermatophytes. The relatively high expression of TruSSU1 and AbeSSU1 in dermatophytes compared to that of AfuSSU1 in A. fumigatus likely reflects a property of dermatophytes which renders these fungi pathogenic. Sulphite transporters could be a new target for antifungal drugs in dermatology, since proteolytic digestion of hard keratin would not be possible without prior reduction of disulphide bridges.

Characterization of an extracellular subtilisin protease of Rhizopus microsporus and evidence for its expression during invasive rhinoorbital mycosis.

An endoprotease Arp (alkaline Rhizopus protease) was identified and purified to virtual homogeneity from the culture supernatant of an isolate of Rhizopus microsporus var. rhizopodiformis recovered from a non-fatal case of rhinoorbital mucormycosis. N-terminal sequencing of the mature native enzyme was obtained for the first 20 amino acids and revealed high homology to serine proteases of the subtilisin subfamily. Arp migrated in SDS-PAGE with an estimated molecular mass of 33 kDa and had a pI determined to be at pH 8.8. Arp is proteolytically active against various substrates, including elastin, over a broad pH range between 6 and 12 with an optimum at pH 10.5. After invasive mucormycosis, specific antibodies against Arp were detected in stored serum samples taken from the patient from whom the R. microsporus strain of this study had been isolated. Furthermore, in search of factors involved in thrombosis as a typical complication of mucormycosis, a procoagulatory effect of the enzyme has recently been shown. Altogether, these data substantiate the expression of Arp during human rhinoorbital mucormycosis and suggest a role of the enzyme in pathogenesis.

Proteome of conidial surface associated proteins of Aspergillus fumigatus reflecting potential vaccine candidates and allergens.

Aspergillus fumigatus is a mold causing most of the invasive fungal lung infections in the immunocompromised host. In addition, the species is the causative agent of certain allergic diseases. Both in invasive and in allergic diseases, the conidial surface mediates the first contact with the human immune system. Thus, conidial surface proteins may be reasonable vaccine candidates as well as important allergens. To broaden the list of those antigens, intact viable Aspergillus conidia were extracted with mild alkaline buffer at pH 8.5 in the presence of a 1,3-beta-glucanase. The proteome of this fraction was separated by two- dimensional gel electrophoresis (2-DE) and analyzed by liquid chromatography coupled with tandem mass spectrometry. Altogether 26 different A. fumigatus proteins were identified, twelve of which contain a signal for secretion. Among these were the known major conidial surface protein rodlet A, one acid protease PEP2, one lipase, a putative disulfide isomerase and a putative fructose-1,6-biphosphatase. The known allergen Aspf 3 was identified among the proteins without a signal for secretion. On the basis of the recently annotated A. fumigatus genome (Nature 2005, 438, 1151-1156), proteome analysis is now a powerful tool to confirm expression of hypothetical proteins and, thereby to identify additional vaccine candidates and possible new allergens of this important fungal pathogen.