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Members of the recently introduced fungal genus Rasamsonia (formerly included in the Geosmithia genus) have been described as emerging pathogens in immunosuppressed hosts or patients with cystic fibrosis (CF). Rasamsonia species have often been misidentified as Penicillium or Paecilomyces because of similar morphological characteristics. We validated a commercially available real-time PCR assay (Primerdesign™, UK) for accurate detection of species from the Rasamsonia argillacea complex. First, we tested this assay with a collection of 74 reference strains and clinical isolates and then compared the PCR with cultures of 234 respiratory samples from 152 patients with CF from two University Hospitals in Germany and France. The assay reliably detected the three main species within the Rasamsonia argillacea species complex (R. argillacea, R. piperina, R. aegroticola), which are typically encountered in CF patients. The limit of DNA detection was between 0.01 and 1 pg/μL. Analysis of the DNA extracts from respiratory specimens of CF patients revealed that four out of the 153 patients studied (2.6%) were colonized with R. argillacea species complex. Two species from the R. argillacea complex grew in the parallel cultures from the same patients. In one patient the PCR was positive 5 months before culture. The real-time PCR assay is a sensitive and specific method for detecting the three most important species of the R. argillacea species complex encountered in the CF context. Detection of these emerging pathogens in respiratory secretions from CF patients by this novel assay may increase our understanding of the occurrence and epidemiology of the R. argillacea species complex.

We investigated the in vitro and in vivo activities of epigallocatechin-3-gallate (EGCg), a green tea component, against Stenotrophomonas maltophilia (Sm) isolates from cystic fibrosis (CF) patients. In vitro effects of EGCg and the antibiotic colistin (COL) on growth inhibition, survival, and also against young and mature biofilms of S. maltophilia were determined. Qualitative and quantitative changes on the biofilms were assessed by confocal laser scanning microscopy (CLSM). Further, in vivo effects of nebulized EGCg in C57BL/6 and Cftr mutant mice during acute Sm lung infection were evaluated. Subinhibitory concentrations of EGCg significantly reduced not only biofilm formation, but also the quantity of viable cells in young and mature biofilms. CLSM showed that EGCg-exposed biofilms exhibited either a change in total biofilm biovolume or an increase of the fraction of dead cells contained within the biofilm in a dose depended manner. Sm infected wild-type and Cftr mutant mice treated with 1,024 mg/L EGCg by inhalation exhibited significantly lower bacterial counts than those undergoing no treatment or treated with COL. EGCg displayed promising inhibitory and anti-biofilm properties against CF Sm isolates in vitro and significantly reduced Sm bacterial counts in an acute infection model with wild type and CF mice. This natural compound may represent a novel therapeutic agent against Sm infection in CF.

Background: The toxin Panton-Valentine leukocidin (PVL) produced by S. aureus is known as a virulence factor that leads to severe infections of skin and soft tissue. However the effect of PVL on wound healing is not known yet. Therefore we examined the detection rate of PVL in patients with chronic wounds., Patients and Methods: The study included 100 patients with chronic wounds of the lower limb. We determined in all S. aureus isolates the presence of the PVL gene using a PCR technique., Results: Altogether 94% of the patients had a leg ulcer, while 6% had a foot ulcer; 65% were women. PVL was found in two patients. One of the strains was methicillin-resistant (MRSA) and the other was methicillin-sensitive (MSSA)., Conclusion: In our investigation there was detection rate for PVL of 2% of all S. aureus isolates in patients with chronic wounds of the lower extremities. Although the role of PVL as a virulence factor of S. aureus in wound healing remains unclear, the detection of PVL should be taken as a cause for a consequent topical antimicrobial wound therapy because of the increased risk of serious infections.

Background: To describe a simple quantitative immunofluorescence assay (IFA) for the detection of specific Stenotrophomonas maltophilia antibodies in serum of CF patients., Methods: A total of 100 sera (64 CF patients and 36 healthy subjects) were collected over a period of 2 years at the University Hospital Essen, Germany. Sputum culture status classified CF patients into groups. Serologic response was determined after Pseudomonas aeruginosa absorption by indirect IFA to Sm whole cell., Results: CF patients with "chronic S. maltophilia" showed significantly higher S. maltophilia antibody levels compared with healthy individuals (P<0.0001) and CF patients with "intermittent" (P=0.0315) or "never S. maltophilia/P. aeruginosa" (P=0.0002). A discriminant cut-off value of >1:120 titre was established to differentiate "CF chronic S. maltophilia" from the other groups. For "CF chronic S. maltophilia", the IFA showed sensitivity and specificity values of 70.7% and 84.7%, respectively., Conclusion: Our data demonstrated that quantitative IFA is a simple serological assay for the detection of specific S. maltophilia antibodies, which could be useful as a diagnostic tool for monitoring immune response of CF patients to S. maltophilia., (Copyright © 2013 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Because published reports indicate that the antibiotic colistin (COL) has antifungal properties, this study investigated the antifungal in vitro activity of COL as single agent and in combination with the antifungal compounds voriconazole (VRC), caspofungin (CAS) and amphotericin B (AMB) against Scedosporium/Pseudallescheria spp., Exophiala dermatitidis and Geosmithia argillacea. In total, susceptibility was determined for 77 Scedosporium/Pseudallescheria spp., 82 E. dermatitidis and 17 G. argillacea isolates. The minimal inhibitory concentrations (MICs) of COL and the antifungals as single compound and in combination were determined with MIC test strips. Drug interactions were detected by crossing the MIC test strips at a 90º angle. The fractional inhibitory concentration index was used to categorise the drugs' interaction. The MIC50 value of COL was 12 μg ml(-1) for S. prolificans, 16 μg ml(-1) for P. apiosperma, 16 μg ml(-1) for P. boydii, 12 μg ml(-1) for E. dermatiditis and 6 μg ml(-1) for G. argillacea. VRC was the most active drug in combination without any antagonism with the exception of few P. boydii isolates. COL as single agent and in most combinations with antifungals exhibits in vitro antifungal activity against filamentous ascomycetes occurring in cystic fibrosis patients and may offer a novel therapeutic option, especially for multidrug-resistant S. prolificans., (© 2012 Blackwell Verlag GmbH.)

This prospective study evaluated the utility of the SeptiFast (SF) test in detecting 25 clinically important pathogens in 225 blood samples from 170 intensive care unit (ICU) patients with suspected sepsis after liver transplantation (LTX) or after other major abdominal surgery (non-LTX). SF yielded a significantly higher positivity rate in the LTX group (52.3%) than in the non-LTX group (30.5%; P = 0.0009). SF may be a powerful tool for the early diagnosis of bloodstream infections in LTX patients.

Background: Sepsis is a common cause of death in children. Early detection of bloodstream pathogens is crucial for the appropriate antibio­tic treatment. Blood cultures (BC) are the gold standard test used for detection. Recently, additional molecular detection methods of microbial DNA by multiplex PCR (SeptiFast, SF) have become available., Aim: Our retrospective study was aimed to compare results of BC to those of SF regarding results and therapeutic relevance., Method: We identified a total of 110 SF samples in 75 patients with suspected systemic infection by retrospective chart review. Each patient underwent SF and BC testing simultaneously., Results: The initial analysis displayed no statistical significant difference in positive SF results compared to BC (p=0.19): in 26 of 110 samples (24%) microbial DNA was found. 19 BC (17%) showed microbial growth. 14 samples were positive in SF but negative in BC (13%). In patients who were pretreated with antibiotics (n=97) pathogens were identified in 24 samples by SF (25%) but only in 11 samples by BC (11%). Based on the clinical presentation and the spectrum of bacterial isolates 3 BC were considered contaminated. Considering this, SF yielded pathogens significantly more often than BC in the overall study population (p=0.04). SF results were available at least 31 h before BC results. Based on SF result antibiotic therapy was adjusted in 14 patients (13%)., Conclusion: Molecular detection of pathogens by SF was faster and more frequently positive than BC. We have therefore demonstrated that SF might be superior to BC in testing for bloodstream pathogens. Prospective multicentric studies are required to determine whether this hypothesis can be maintained., (© Georg Thieme Verlag KG Stuttgart · New York.)

Background: We implemented a selective medium for improved detection of Stenotrophomonas maltophilia in sputum samples from CF patients. We also performed antimicrobial susceptibility testing with eight antibiotics., Methods: A total of 623 consecutive sputum samples from 165 CF patients in a German CF center were cultured onto conventional media and onto Steno medium agar (SMA). All isolates confirmed as S. maltophilia by biochemical and molecular methods were subjected to antimicrobial susceptibility testing. The following agents were tested by Etest: ceftazidime, levofloxacin, moxifloxacin, tigecycline, trimethoprim-sulfamethoxazole, fosfomycin, colistin, and ticarcillin-clavulanate acid., Results: Conventional media supported the growth of S. maltophilia in 7.1% of samples, whereas SMA supported its growth in 11.6%, increasing the detection rate to 64%. Trimethoprim-sulfamethoxazole and tigecycline exhibited the highest in vitro activity, whereas ceftazidime, colistin, and ticarcillin-clavulanate acid exhibited higher resistance rates., Conclusions: SMA is a promising medium allowing improved isolation of S. maltophilia from sputum samples from CF patients. Trimethoprim-sulfamethoxazole and tigecycline demonstrated excellent inhibitory effects against S. maltophilia, which may suggest a potential clinical effect., (Copyright © 2011 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

The aim of this study was to develop and validate a novel bioassay for determining serum voriconazole (VRC) concentrations and to compare its routine clinical performance with that of high-performance liquid chromatography (HPLC). The biological activity of VRC was measured by a plate diffusion assay using a VRC-hypersusceptible Candida kefyr strain. The bioassay's utility was tested by measuring steady-state VRC concentrations in 100 serum probes from VRC-treated patients. The HPLC system used solvent extraction with hexane:dichloromethane followed by reversed-phase HPLC with ultraviolet detection. The intra-day and inter-day accuracy of the bioassay was <5%, while that of HPLC was <1%. The precision (mean coefficient of variation, 3.5%) was equal for both the methods. The limit of quantification was lower for HPLC (0.2 mg l(-1)) than for the bioassay (0.5 mg l(-1)). The result of linear regression analysis was HPLC = 1.0178 (bioassay) + 0.328; R(2) = 0.88; n = 100. Results of the serum panel ranged from 0.5 to more than 8.0 mg l(-1) for the bioassay and from 0.26 to 10.1 mg l(-1) for HPLC. Especially in laboratories without access to HPLC, the bioassay may be a clinically useful tool for therapeutic drug monitoring., (© 2010 Blackwell Verlag GmbH.)

We describe the epidemiology and characteristics of the pathogen and patients (n=7) associated with an outbreak of a carbapenem-resistant Klebsiella pneumoniae (CRKP) strain in a German university hospital from July 2010 to January 2011. Species identification and detection of carbapenem resistance were carried out using standard microbiological procedures. Carbapenemases were detected by phenotypic methods and specific polymerase chain reactions (PCRs). DNA fingerprinting profiles were performed with repetitive sequence-based PCR. Medical records of colonised or infected patients were retrospectively reviewed. Antibiotic resistance profiles, PCR-specific amplification products and genotyping demonstrated that the outbreak occurred because of the spread of a single CRKP clone harbouring both KPC-2 and VIM-1. Five of the seven patients had invasive infections with the CRKP strain; the deaths of four of them were directly related to the infection. Early implementation of infection control interventions brought about efficient containment of further cross-transmission. Rapid dissemination of carbapenemase-producing Enterobacteriaceae is a serious concern in patient care and is a problem that has emerged in western Europe.

The laboratory identification of Pseudallescheria and Scedosporium isolates at the species level is important for clinical and epidemiological purposes. This study used semiautomated repetitive sequence-based polymerase chain reaction (rep-PCR) to identify Pseudallescheria/Scedosporium. Reference strains of Pseudallescheria boydii (n = 12), Scedosporium prolificans (n = 8), Scedosporium apiospermum (n = 9), and clinical/environmental isolates (P. boydii, 7; S. prolificans, 7; S. apiospermum, 7) were analyzed by rep-PCR. All clinical isolates were identified by morphological and phenotypic characteristics and by sequence analysis. Species identification of reference strains was based on the results of available databases. Rep-PCR studies were also conducted with various molds to differentiate Pseudallescheria/Scedosporium spp. from other commonly encountered filamentous fungi. All tested Pseudallescheria/Scedosporium isolates were distinguishable from the other filamentous fungi. All Scedosporium prolificans strains clustered within the cutoff of 85%, and species identification by rep-PCR showed an agreement of 100% with sequence analysis. However, several isolates of P. boydii and S. apiospermum did not cluster within the 85% cutoff with the same species by rep-PCR. Although the identification of P. boydii and S. apiospermum was not correct, the semiautomated rep-PCR system is a promising tool for the identification of S. prolificans isolates.

The aim of this study was to assess the vancomycin MIC distribution for MRSA blood culture isolates over a period of six years in Germany. The study examined 287 MRSA isolates from blood cultures collected at several hospitals in two German cities between 2004 and 2009. The vancomycin MIC was determined by Etest. Genotypic features of the MRSA strains with vancomycin MIC ≥ 1 mg/L were determined by semiautomated repetitive-sequence-based polymerase chain reaction. The range of vancomycin MIC as determined by Etest was 0.25 to 2.0 mg/L. The geometric mean MIC increased by 1.34-fold in city A over the study period (p < 0.05), but there was no meaningful change in city B (a 1.09-fold increase, p > 0.05). Furthermore, in city A a shift in vancomycin MICs occurred as an increase in the percentage of isolates with MIC ≥ 1 mg/L from period one (2004-2006) to period two (2007-2009) (p < 0.0001). Typing results showed that in city A a single clone was predominant (55% of the creep isolates). In this study, the creep phenomenon seems to be a regional problem. We suggest that all hospitals should monitor their local status of elevated vancomycin MICs in invasive MRSA isolates.

A female patient with end-stage chronic obstructive pulmonary disease received a double-lung transplant from a donor with serologic evidence of past syphilis. The recipient showed a Treponema pallidum-specific IgG seroconversion 8 days after transplantation with increasing titers at follow-up. In Month 3 post-transplantation the immunoglobulin G (IgG) antibody titer decreased. Retrospectively, we argue that the reactive syphilis serology in the recipient was due to a transmission of immunocompetent B cells in the graft rather than a transmission of T pallidum.

Infective endocarditis is a rare complication affecting solid organ transplant recipients. Staphylococcus aureus is a common cause of infective endocarditis accounting for about 30% of cases. We present a case of nosocomial methicillin-resistant S. aureus endocarditis with persistent bacteremia, in a patient following orthotopic liver transplantation. We were unable to eradicate this infection with primary linezolid therapy or with secondary treatment with combined vancomycin and rifampicin, but successfully treated it with daptomycin, in addition to tricuspid and aortic valve replacement.

We report the clinical features, diagnosis, and monitoring findings of invasive aspergillosis (IA) in 2 liver transplant recipients. Blood/serum samples and bronchoalveolar lavage (BAL) fluids were analyzed by a novel commercial polymerase chain reaction (PCR) assay (SeptiFast) and an Aspergillus galactomannan (GM) enzyme-linked immunoassay (EIA). The diagnosis of IA could be performed in <6 h with the detection of Aspergillus fumigatus DNA in blood and BAL fluid. High GM values (mean: 9.1, range: 7.3-10.8) in serum and BAL fluid confirmed the SeptiFast result. Follow-up of the SeptiFast findings and GM index correlated with the clinical course. The molecular detection of A. fumigatus-specific DNA and GM test in blood/serum and BAL samples appears to be a useful tool for prompt diagnosis of IA. Further prospective clinical trials are necessary to evaluate the accuracy of SeptiFast and the GM test in diagnosing IA.

Background: Infectious complications occur in approximately 50% of cadaveric liver transplant (CDLT) recipients. Living-donor liver transplantation (LDLT) is an established alternative to shorten the waiting time. Currently, the incidence of pulmonary infections after LDLT and the microbiologic causes are unknown. In the present cohort study, we compared the incidence and profiles of pulmonary and blood stream infections (BSI) between LDLT and CDLT recipients. We hypothesized a lower incidence in LDLT recipients., Methods: The clinical course of 55 LDLT recipients consecutively transplanted between January 2003 and December 2006 was analyzed. The 173 CDLT recipients who were transplanted in the same period served as a control group. Patients were treated in a single Intensive Care Unit, applying standardized postoperative care., Results: Mean model for end-stage liver disease score did not differ between LDLT and CDLT recipients (14.2 vs. 13.3). The overall incidence of pulmonary and BSI for both groups was 8% and 24%, respectively. Pulmonary infections were experienced by 18% of LDLT versus 5% of CDLT recipients (P=0.005) and BSI occurred in 33% of LDLT versus 21% of CDLT recipients (P=0.1)., Conclusions: In contrast to our hypothesis, LDLT recipients experienced significantly more pulmonary infections and a trend toward increased higher incidence of BSI. These findings emphasize the need for future research on the causative agents and prevention of infection in LDLT recipients. The observation that patients with pulmonary infection had a significantly reduced 1-year survival rate underscores the importance of our observations.

Pulmonary infections are a significant cause of morbidity and mortality after liver transplantation. Infections with methicillin-resistant Staphylococcus aureus (MRSA) have increased in the last 10 years. Mortality may exceed 80% in liver transplant recipients who develop MRSA pneumonia. A 57-year-old male following living-donor liver transplantation developed a right-sided MRSA pneumonia 6 weeks after transplantation, which required artificial ventilation for 14 weeks. Initially, pneumonia was treated with linezolid. However, after 12 days under current therapy, the infection spread out to both lungs. At that time. we initiated the treatment with tigecycline. Under this therapy, the patient could be cured from MRSA pneumonia and was extubated. We detected no tigecycline related hepatotoxic effect. In conclusion, this case suggests that tigecycline may be useful in the salvage therapy of pneumonia due to MRSA after linezolid failure., ((c) 2006 AASLD)

Background: NOD2 and TLR-4 genes belong to the innate immune system that detects invading pathogens through several pattern-recognition receptors. Here we analyzed 403 patients for NOD2 gene mutations and 307 patients for TLR-4 gene mutations (Thr399Ile) with their respective donors and correlated the results with the incidence of acute graft-versus-host disease (aGVHD), severe acute GVHD (saGVHD), the risk for transplant-related mortality (TRM), overall survival (OS) and incidence of infectious complications., Methods: We performed a retrospective single-center study. Genotyping of TLR-4 and NOD2 were evaluated by real-time polymerase chain reaction., Results: Surprisingly, we found a significant reduced incidence of aGVHD, saGVHD, and intestinal GVHD for patients with NOD2 gene mutations on the donor side with 50%, 0% and 2% compared to patients with the wild-type NOD2 gene with 65%, 17%, and 26%, respectively (P<0.02). However, the incidence of saGVHD increased in patients with NOD2 mutations on the patient and donor (P/D) side with 44% versus 17% compared to patients with the wild-type gene (P<0.03). TLR-4 gene mutations at P/D side had an increased risk for saGVHD with 42% versus 15% of patients with wild-type gene (P<0.04). OS, TRM, and incidence of infectious complications were not influenced by the mutated genes. Multivariate analysis confirmed that NOD2 gene mutations on the donor side had a reduced risk for saGVHD (P<0.001), whereas mutations of the NOD2 gene on P/D side had an increased risk for saGVHD (P<0.01) in our analysis., Conclusions: These results suggest that NOD2 mutations have influence on the occurrence of acute GVHD after transplantation.

Caspofungin (CAS) is the first of a new class of antifungal agents, the echinocandins, that interfere with fungal cell wall synthesis by inhibition of glucan synthesis. Here, we report the results of 31 patients treated with CAS following allogeneic SCT. CAS was administered as a second-line agent to patients with invasive fungal infection (IFI) (n=15) or fever of unknown origin (n=16) who were recalcitrant to or intolerant of prior antifungal therapy. Unsuccessful first-line regimes included amphotericin B (n=17), liposomal amphotericin B (n=5), fluconazole (n=3), itraconazole (n=1), and voriconazole (n=2). All patients received concomitant immunosuppressive therapy for graft-versus-host disease. In 23 patients, cyclosporin A (CSA) and CAS were administered concurrently without any major side effects detected. Observed increases in GPT were not clinically significant. Normalization of serum creatinine and significant reductions in C-reactive protein were observed in response to CAS. Favorable outcome to CAS were documented in eight of 15 patients with IFI and in 15 of 16 patients with fever of unknown origin. CAS is a promising alternative in patients with IFI and fever of unknown origin in the setting of allogeneic SCT.

Using gellan gum as a substitute for agar-agar in a mycological medium and sequencing of the ITS 1 and 2 regions resulted in an accurate identification of Aspergillus fumigatus, Aspergillus nidulans, Aspergillus terreus and Aspergillus ustus within 24 h of subculture.

Cellular fatty acids of Helicobacter pylori have taxonomic, physiological, and pathogenic implications. However, little is known about the fatty acid composition under various culture conditions. H. pylori is usually grown on blood-supplemented complex media, and the fatty acids in the blood may affect the fatty acids in the cells. In addition, frequently subcultivated laboratory-adapted strains may have properties different from those of fresh clinical isolates, which are culturable only for a limited number of passages. Therefore, the cellular fatty acid profiles of laboratory-adapted strains (LAS) and freshly isolated strains (FIS) were compared after growth on agar that was fatty acid free and growth on blood agar that contained fatty acids. LAS ATCC 43504, 51932, and 700392 and the FIS IMMi 88, 89, and 92, each with <10 subcultures, were cultured in parallel on a fatty acid-free agar (ISAF) and on 5% sheep blood agar (SBA), which contained oleic acid (18:1 9c), hexadecanoic acid (16:0), and octadecanoic acid (18:0). ISAF-grown cultures showed no 18:1 9c and no appreciable differences between the profiles of FIS and LAS. After culture on SBA, the strains showed 18:1 9c and increased 16:0 and 18:0 content combined with decreased tetradecanoic acid (14:0) content compared to ISAF-grown cells. The changes in the fatty acid profiles were much more pronounced in FIS than in LAS. LAS are obviously characterized by a lower uptake of the fatty acids from the growth medium than FIS. Furthermore, it could be shown that this LAS behavior is most likely a primary strain attribute that is favored under laboratory conditions. The pronounced uptake of fatty acids by strains with FIS behavior may be associated with the expression of virulence properties.

The antiseptic Lavasept (LS), containing the polymeric biguanide polihexanide (CAS 28757-48-4), possesses microbicidal activity against a broad spectrum of bacteria including Staphylococcus aureus. It is used for antiseptic wound care in concentrations corresponding to 0.2-0.4 mg polihexanide per ml. To obtain basic data on its ability to eradicate S. aureus colonizing the nasal mucosa, the influence of mucin on the anti-staphylococcal activity was investigated. A disk agar-diffusion method was applied. Two reference strains of S. aureus (methicillin-sensitive S. aureus ATCC 29213 and methicillin-resistant S. aureus ATCC 33591) and 20 fresh clinical isolates were used. In the absence of mucin, the growth of all strains was inhibited by polihexanide concentrations of 0.1 mg/ml. In the presence of 0.25% mucin in the test medium, a concentration of 0.4 mg/ml was necessary to inhibit all strains. Mucin concentrations of 0.5% and 1%, that are even lower than the mucin concentrations in healthy nasal secretions, abolished the activity of the therapeutic concentrations of polihexanide. It is concluded that the inactivation of LS by mucin obstructs a reliable clearance of nasal S. aureus carriage.

Methane-forming microbes of the phylogenetic domain Archaea are part of the strictly anaerobic microflora of the human intestine. In bone marrow transplant (BMT) recipients, the regimen of intestinal decontamination with metronidazole is targeted to anaerobic bacteria. The effect on the anaerobic methanoarchaea, however, is unknown. Therefore, the faeces of patients undergoing BMT were investigated for methane production. The anoxic Hungate technique and an archaeal growth medium were used to culture faecal specimens. Methane production was measured in the head space of the culture bottles by gas chromatography using a thermal conductivity detector. In a testing serial specimen of 100 patients, 13 patients were found to bear methanogens, and 11 of these patients received metronidazole. The methane-producing faecal specimens occurred before metronidazole use in three patients, during the first week in five patients, and after cessation in three patients. No specimen of the 11 patients that was obtained during the 2nd-5th week of gut decontamination showed methane production. It is concluded that use of metronidazole directed against faecal anaerobic bacteria also suppresses or eliminates faecal methanogenic Archaea.

Background: Lavasept, containing the polymeric biguanide polyhexanide, may be effective against Staphylococcus aureus colonizing the nasal mucosa. To obtain basic data on its suitability for that purpose, its antimicrobial activity was examined in the presence of mucin., Methods: A disk diffusion test was applied using Mueller-Hinton agar, and disks soaked with a therapeutic and a 10-fold higher concentration of Lavasept. Commercially available mucin preparations from porcine stomach were added to the test system. Reference strains and 20 clinically isolates of S. aureus, and reference strains of Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, and Candida albicans were tested., Results: Supplementation of the test medium with 1% mucin completely abolished the activity of disks loaded with the therapeutically used concentration of the antiseptic. The neutralizing effect of mucin occurred with all test strains., Conclusions: The inactivation of Lavasept by mucin may hamper a reliable clearance of nasal S. aureus carriage., (Copyright 2002 S. Karger AG, Basel)

The BacT/Alert blood culture system was evaluated for the MIC determination of amphotericin B for 27 strains of Aspergillus from five different species. An inoculum of c. 10(8) cfu/mL and an incubation time of 48 h resulted in MICs for 26 of 27 strains that corresponded with published MICs obtained in a microdilution assay. Twelve of 13 strains with MIC(48) values >/=2 mg/L showed growth within 24 h in bottles containing 0.25 mg/L amphotericin B, indicating that results can be obtained the day following inoculation. It is concluded that the BacT/Alert blood culture system can be used for susceptibility testing of Aspergillus spp. with amphotericin B.

The sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of water-soluble proteins and the randomly amplified polymorphic DNA (RAPD) patterns of whole-cell lysates from 21 Aspergillus ustus isolates, including 11 reference strains and 10 patient and environmental strains from one hospital, were investigated. All isolates showed identical protein patterns. The RAPD assay discriminated between all reference strains. Comparison of hospital isolates showed identical RAPD patterns in some of the patient and environmental isolates. The data indicate that the RAPD technique is useful for fingerprinting A. ustus.

Twenty-five culture collection strains from four Aspergillus species (A. fumigatus n = 8, A. flavus n = 8, A. niger n = 4, A. nidulans n = 5) were characterized by four methods: (i) determination of patterns in an assimilation assay; (ii) protein pattern of whole mycelial cell lysates in the sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE); (iii) reactivity of a pool serum obtained from cystic fibrosis patients with mycelial lysates in the immunoblot; and (iv) random amplification of polymorphic DNA (RAPD) with eight primers having arbitrary or repetitive sequences. In the assimilation assay the A. fumigatus strains showed identical patterns in contrast to the strains of the species A. flavus, A. niger, and A. nidulans, which each showed four patterns. In the SDS-PAGE no differences in the band patterns in the A. fumigatus strains were found, in contrast to the A. flavus (three patterns), A. nidulans (five patterns) and A. niger strains (two patterns). The immunoblot patterns were characteristic for each species with bands at 62 and 17/18 kDa in the A. fumigatus strains, at 51 and 18 kDa in the A. flavus strains, at 51 kDa in the A. niger strains, and at 51, 40 and 17/18 kDa in the A. nidulans strains allowing, however, no intraspecies typing. In the RAPD assay four out of eight primers gave interpretable patterns with 3-20 bands. None of the primers showed sufficient discriminatory power when used alone. However, when combining the results of two of the primers (5'-GTA TTG CCC T-3' and 5'-GAT AGA TAG ATA GAT A-3') all strains except two A. fumigatus strains could be clearly separated from each other. It is concluded that the the RAPD assay showed the most discriminatory power in all Aspergillus species investigated. In contrast to the phenotypically similar A. fumigatus strains, the strains of the species A. flavus, A. nidulans and A. niger differed in their phenotypic characteristics. The presented data of strains from international culture collections may serve as basis for interlaboratory standardization of typing methods.

The amplified 5.8S RNA coding DNA with the neighbouring internal transcribed spacers ITS I and ITS II (ITS I--5.8S rDNA--ITS II) of 27 culture collection strains of Aspergillus fumigatus, Aspergillus flavus, Aspergillus nidulans, Aspergillus niger, and Aspergillus terreus were investigated by single strand conformational polymorphism (SSCP) analysis. All strains showed a polymerase gel electrophoresis (PCR) product of 0.6 kb. Separation of DNA single strands of the PCR product in an acrylamide-bisacrylamide gel containing formamide SSCP resulted in individual patterns for each of the species. A minor variability within the species A. fumigatus and A. flavus did not affect the correct species identification. The results were confirmed when investigating 55 wild strains from patients and the environment. It is concluded that the analysis of the amplified ITS I--5.8S rDNA--ITS II region by SSCP allows the differentiation of the medically most relevant aspergilli. As the method does not require morphologically fully developed fungal colonies, it yields species diagnosis faster than the conventional macroscopic and microscopic identification.

A case of invasive pulmonary aspergillosis in an allogeneic bone marrow transplant recipient caused by Aspergillus ustus is presented. A. ustus was also recovered from the hospital environment, which may indicate that the infection was nosocomially acquired. A literature review revealed seven cases of invasive infections caused by A. ustus, and three of these were primarily cutaneous infections. In vitro susceptibility testing of 12 A. ustus isolates showed that amphotericin B and terbinafine had fungicidal activity and that itraconazole and voriconazole had fungistatic activity.

To evaluate different methods for strain differentiation, 10 isolates of Aspergillus terreus from Germany and two epidemiologically unrelated strains were investigated. The sources of the isolates were patients with cystic fibrosis (4), immunosuppression (2), otitis externa (2), sinusitis (1) and endocarditis (1). Environmental isolates were obtained from a contaminated cell culture and from soil. The isolates did not differ in their macroscopic and microscopic morphology, in their protein patterns analysed by SDS-PAGE and in their susceptibility to amphotericin B and itraconazole. The RFLP analysis of total genomic DNA digested by EcoRI resulted in patterns that were too faint for interpretation. However, after hybridisation of the digested DNA with a short DNA probe of repetitive sequence, six different patterns were found. Based on the patterns of the randomly amplified polymorphic DNA (RAPD) with three primers, nine different genotypes were discriminate. RAPD patterns discriminated the epidemiologically unrelated reference strains (endocarditis isolate from Thailand, soil isolate from the USA) and the isolates from Germany. It is concluded that, in contrast to the phenotypic methods, the analysis of RAPD patterns is useful for strain differentiation of A. terreus.

Susceptibility testing of 27 Aspergillus reference strains belonging to five species was performed using the microdilution broth method with yeast nitrogen broth and RPMI-1640. Similar results were found using the two media. The strains of Aspergillus fumigatus (n = 8) and Aspergillus niger (n = 4) had MICs of amphotericin B in the range 0.125-0.5 mg/L. In contrast, nine out of 13 strains of Aspergillus flavus and Aspergillus nidulans had MICs in the range 2-16 mg/L. All strains had MICs of itraconazole in the range 0.03-0.5 mg/L. It is concluded that both media can be used for testing. Susceptibility testing to amphotericin B is recommended in clinical settings.

The specificity of the Pastorex Aspergillus latex agglutination test for the diagnosis of manifest aspergillosis is hampered by the occurrence of false-positive results. In order to prove whether or not the false-positive reactions may be caused by the uptake of the soluble galactomannan antigen from the environment, the presence of the antigen was tested in foods, air samples, antibiotics for therapeutic use and faeces. Reactions of the Aspergillus latex agglutination test were found in 15 (79%) out of 19 samples of meals prepared in a hospital kitchen, in five out of six canned vegetables from a supermarket, in all of six samples of pasta and rice bought in health shops, in the faeces of four bone marrow transplant (BMT) recipients and of four healthy subjects and in one and two batches of the antibiotics co-amoxyclav and piperacillin respectively. The concentration of the antigen in faecal material was calculated to be in the range of 1.2-38.4 micrograms g-1. It is concluded that the faecal galactomannan antigen may reach the circulation in patients with dysfunction of the intestinal mucosal barrier, e.g. BMT recipients, thus leading to diagnostically false-positive antigenaemia.

Phenotypic and genotypic characteristics of 11 strains of Exophiala dermatitidis were investigated. Ten strains (including three reference strains) were isolated from sputum samples of six patients with cystic fibrosis (CF) in Germany, and one reference strain was isolated from a patient with phaeohyphomycosis in Japan. The strains showed differences in their ability to assimilate sorbitol, palatinose, rhamnose, gluconate and melezitose, leading to the differentiation of seven auxotypes. The IC30 of amphotericin B, and ketoconazole and itraconazole, respectively, indicated susceptibility, whereas the IC30 of fluconazole and 5-fluorocytosine indicated resistance in all strains. Protein patterns in SDS-PAGE revealed no major differences. The glycoconjugate patterns distinguished the Japanese strain from the other strains. Cluster analysis of whole-cell fatty acid methyl ester (FAME) profiles with the Microbial Identification System (MIS) revealed two major clusters separating a reference strain and the Japanese strain from the other strains. Analysis of patterns resulting from random amplification of polymorphic DNA (RAPD) with two arbitrary primers showed four genotypes. Comparison of the results revealed no agreement between the different fingerprinting methods, except the separation of the Japanese strain from the European CF strains. As the results of assimilation tests seem to vary between different laboratories, the analysis of FAME profiles and RAPD analysis are recommended for typing E. dermatitidis.

In order to investigate the risk of hospital-acquired infections due to environmental Aspergillus, air sampling outside and inside the bone marrow transplantation (BMT) clinic of the University Hospital, Essen, Germany, was performed prospectively for one year. The spore concentration in the air was matched with meteorological data. Two BMT-patients, who were hospitalized during the sampling period, suffered from aspergillosis after discharge. The patients' isolates obtained at re-admission were compared with environmental isolates obtained during the first hospitalization. Analysis by randomly amplified polymorphic DNA showed that the two BMT-patients were infected with Aspergillus strains that were different from the environmental strains. It is concluded that it is not possible to predict the environmental Aspergillus spore concentration by analysis of meteorological data. Since the concentration of specific strains may fluctuate rapidly, a hospital-acquired Aspergillus infection cannot be excluded even if the infecting strain is not found in the hospital environment.

Strains of Aspergillus fumigatus (n = 24) were isolated from the sputa of six patients with cystic fibrosis during periods from 3 to 11 months. The genetic polymorphisms of the strains were studied using the random amplified polymorphic DNA (RAPD) assay with three single oligonucleotides and pairwise combined primers. The analysis of RAPD patterns resulted in 15 different RAPD types. In four patients, the colonizing type changed, whereas in two others the same types were detected over periods between 3 and 11 months. The genetic diversity as well as the shift of the colonizing strains found in some patients might be important for the epidemiology of Aspergillus infections in patients with cystic fibrosis.

A commercially available antigen assay (Pastorex Aspergillus) was used to detect Aspergillus antigen in serial bronchoalveolar lavage (BAL) fluids and sera of patients undergoing bone marrow transplantation (six patients with autopsyproven aspergillosis and 10 control patients without evidence of fungal infection). Aspergillus antigen was not detected in 17 BAL fluids of the six patients with proven aspergillosis. In two of the six patients the assay gave positive results in serum specimens. Three of the 10 control patients showed reactive BAL fluids. It is concluded that the latex agglutination assay of BAL fluids has no value in the diagnosis of invasive (pulmonary) aspergillosis in patients undergoing bone marrow transplantation.

During the course of extensive building activity in the vicinity of bone marrow transplantation wards, the patients were routinely screened for the occurrence of Aspergillus galactomannan antigen in serum. In 19 (6.7%) out of 285 patients, an antigenemia was detected. Eleven (58%) of the 19 antigenemic patients suffered from autopsy-proven or clinically suspected invasive aspergillosis. The yearly incidence of antigenemic patients differed significantly, ranging from 0% in the year without building activities to 20.9% in the year with major activities, particularly interior completion works and landscaping. It is concluded that Aspergillus antigen monitoring of bone marrow transplant recipients has a limited value for the diagnosis of manifest invasive aspergillosis. However, it it epidemiologically useful to assess the extent of intensive contact with aspergilli and to control the effectivity of preventive measures.

In 1992 blood samples were taken from 630 forestry workers in the state of Brandenburg, Germany, and an inquiry about tick bites and possible symptoms of Lyme borreliosis carried out in order to determine the seroprevalence of the disease. To estimate the rate of seroconversion within six months, 406 of the individuals were investigated a second time. IgG and IgM antibodies against Borrelia burgdorferi were detected in serum using an indirect immunofluorescence assay (IFA) and an immunoblot assay (IBA). Fifty-three percent of the forestry workers reported suffering a tick bite, 8% of whom recalled an erythema after the bite. Positive results were found more frequently in the forestry workers than in a control group of 200 healthy blood donors in both the IgG-IFA (8% vs. 4%, p < 0.05) and the IgG-IBA (18% vs. 5%, p < 0.05). The detection of IgG antibodies correlated with a tick bite and erythema history. There was a tendency of lower seropositivity by the IgG-IBA in individuals who treated the ticks before removal with chemicals or other agents compared to those without such treatment (16.8% vs. 23.9%, 0.05 > p < 0.1). Likewise, there was a tendency of lower seropositivity by the IgG-IFA in individuals being treated with antibiotics for other reasons compared to untreated individuals (3.15% vs. 8.9%, p < 0.05), although the two groups did not differ in the IgG-IBA (13.8% vs. 18.5%, p > 0.1). The rate of seroconversion within six months ranged from 5 to 7%. It is concluded that forestry workers in Brandenburg, Germany, are at risk for infection with Borrelia burgdorferi, but clinical signs of infection are rare.

Aspergillus fumigatus isolates (n = 6) from a lung transplant recipient, one isolate from a patient who had been on the same ward and a reference strain (NCPF 2140) were compared using three typing methods: SDS-PAGE, immunoblotting with serum from the transplant patient and random amplified polymorphic DNA (RAPD) assay. Neither the SDS-PAGE, immunoblot nor RAPD assay with single primers revealed differences between the eight isolates. Digestion of one primer product with the endonuclease EcoRI discriminated between the six patient isolates and the other two strains. The RAPD assay using pairwise combined primers showed identical patterns for the patient's strains but differentiated between the two other strains. It is concluded that any single technique may fail to detect strain differences and that a spectrum of typing methods is necessary in order to reveal or to exclude cross-infections with Aspergillus fumigatus.

A decrease of the susceptibility of Aspergillus fumigatus strains to amphotericin B was found when tested in combination with high concentrations (128-2048 mg/l) of the antibacterial agent imipenem by checkerboard titration and agar diffusion assay. Only bacteriologically active imipenem showed the antagonism. The mechanism of action is unknown. However, imipenem and amphotericin B did not react directly, as shown by checkerboard titration with bacterial strains as well as by HPLC analysis. It is concluded that imipenem medication may influence the efficacy of amphotericin B treatment in aspergillosis.

The detection of galactomannan antigen in urine was investigated in 26 bone marrow transplant recipients using an Aspergillus latex agglutination test (Pastorex). After modification of the method, which was originally devised for serum testing, the detection limit in native urine was approximately 20 ng/ml. Antigen was found in 79 (36.4%) of 217 serial urine samples, compared to 40 (11.8%) of 340 serum samples. As a rule, antigenuria preceded antigenemia and was more persistent. The sensitivity, specificity, positive predictive value and negative predictive value of antigenuria for autopsy-proven aspergillosis and clinically suspected Aspergillus infection were 57%, 53%, 31% and 77%, respectively, while those of antigenemia were 43%, 53%, 25% and 71%. It is concluded that urine testing is more reliable than serum testing for the detection of Aspergillus galactomannan. The detection of antigen, however, whether in serum or in urine, allows no clear distinction between Aspergillus infection and exposure to non-infectious Aspergillus antigens.

The serological distinction of an immune response to the agent of syphilis, Treponema (T.) pallidum, and Lyme disease borreliae is difficult due to the existence of cross-reacting antibodies. In this study, the immunoblot technique is used to compare the immune response of sera from patients with syphilis or Lyme borreliosis to either T. pallidum or Borrelia (B.) burgdorferi. Patients with syphilis showed an IgG response to the 17 and 15 kDa antigens of T. pallidum, whereas sera from patients with Lyme borreliosis-especially in late stages of the disease-reacted with the 95 and 19-17 kDa antigens of B. burgdorferi. Thus, IgG antibodies against low molecular weight antigens of T. pallidum (17, 15 kDa) or B. burgdorferi (19-17 kDa) may represent useful markers for the serological diagnosis of syphilis or Lyme borreliosis.