Biomedical subjects
Publications and source records attributed to R Rüchel.
We report on the recurrence of Candida albicans among yeast isolates from our university hospital. After a decline in occurrence which coincided with the onset of the use of fluconazole, the fraction of C. albicans recovered and at present has reached the pre-fluconazole level. No permanent rise of C. glabrata or C. krusei has been observed.
An extracellular aspartic proteinase (Rmap) from Rhizopus microsporus var. rhizopodiformis was detected in the culture supernatant of a fungal isolate from a case of rhinocerebral mucormycosis (case HA). The proteinase was purified to near homogeneity by ion exchange and affinity chromatography on pepstatin agarose. Based on its N-terminus the RMAP gene was cloned and found to code for 388 amino acids. The preproenzyme has an aminoterminal leader sequence of 65 amino acids, whereas the mature enzyme consists of 323 amino acids. The deduced amino-acid sequence of the preproenzyme was 82% homologous to an extracellular aspartic proteinase of Rhizopus niveus. Low stringency Southern blot analysis of R. microsporus DNA suggested the presence of other homologous genes. Expression of Rmap in Pichia pastoris was achieved, and the recombinant enzyme was active in the yeast culture supernatant. Both enzyme preparations exhibited a similar optimum of activity in the pH 2.5 region. Furthermore, Rmap was shown to activate bovine blood coagulation factor X at slightly acidic pH in vitro. Expression of the proteinase during mycosis was proven by a specific immune response of patient HA.
A novel subtilisin-related serine proteinase (ALP2) [EC 3.4.21.48] with a broad range of activity between pH 4.5 and 11.0 was released from a cell wall fraction of Aspergillus fumigatus by an alkaline pH shift. The enzyme which was not detected in the culture supernatant was partially purified by phenylbutylamine agarose chromatography. The N-terminal sequence revealed that ALP2 is the same protein identified as the major allergen of A. fumigatus in patients suffering from extrinsic bronchial asthma (Shen et al. 1999, Int. Arch. Allergy Immunol. 119, 259-264). Based on this N-terminal sequence and on a conserved region of fungal subtilisins, a specific PCR probe was generated and the ALP2 genomic and cDNA were isolated from corresponding phage libraries. ALP2 shares a 49% identity with the vacuolar proteinase B (PrB) of Saccharomyces cerevisiae. In addition there is a 78% identity with PEPC, a serine proteinase which has been described in Aspergillus niger. Targeted disruption of the ALP2-encoding gene resulted in a slightly decreased speed of vegetative growth and in a more than 80% reduction of sporulation in the alp2-negative mutants, correlated with an approximately 50% reduction of the median diameter of conidiophore vesicles. The requirement of ALP2 for regular sporulation, in addition to its role in allergic asthma, raises further interest in cellular proteinases in respect to morphogenesis and pathogenesis in A. fumigatus.
An aspartic proteinase PEP2 [EC 3.4.23.25] was purified from a cell wall fraction of Aspergillus fumigatus. The enzyme, which showed a broad range of activity from pH 2.0 to 7.0 and migrated as a single band of 39 kDa in SDS-PAGE, was not detected in the culture supernatant. A specific gene probe was designed on the basis of the N-terminal sequence of the native protein, and the PEP2 genomic and cDNA were isolated from corresponding libraries. The deduced amino acid sequence of PEP2 consists of 398 amino acids. A signal sequence of 18 amino acids and a proregion of another 52 amino acids were identified. The mature protein consists of 328 amino acids which include the two DTG-motifs of the active site common to almost all pepsin-like enzymes. PEP2 showed a 64% identity with the vacuolar proteinase A (PrA), of Saccharomyces cerevisiae, and an 88% identity with PEPE, an aspartic proteinase of Aspergillus niger. Recombinant PEP2 was overexpressed in Pichia pastoris and the active enzyme was secreted into the culture supernatant. Targeted deletion of PEP2 did not affect vegetative growth or cell and colony morphology. Identification of proteinases, such as PEP2, which are apparently associated with the Aspergillus cell wall raises new interest in these molecules with respect to their possible function in the pathogenesis of invasive aspergillosis.
Invasive pulmonary aspergillosis (IPA) is increasingly recognized especially in immunocompromised patients, but early diagnosis remains a problem. Fungal elements in clinical specimens can be directly stained with an optical brightener. The high intensity of the elicited fluorescence allows for rapid and reliable microscopic screening. In the present study the authors aimed to validate this method. All specimens from bronchial secretions or pleural fluid suspected of mycosis (n=94) and all bronchoalveolar lavages (n=439) were prospectively evaluated by culture and staining with the optical brightener Blankophor-P-Flüssig. IPA was diagnosed for 17 specimens from 13 patients, using a combination of clinical, culture and radiological data, and by biopsy (n=3) or autopsy (n=3). The overall incidence of IPA was 3.3%. Nine of the 13 patients with IPA died (mortality=69%). Staining with the optical brightener and consecutive microscopic screening took 9+/-3 min. For the diagnosis of invasive aspergillosis, sensitivity was 0.88 and specificity was 0.99. Using culture, sensitivity was 0.76 and specificity was 0.99. In conclusion, direct examination of clinical specimens using the optical brightener has a high diagnostic potential for the diagnosis of invasive pulmonary aspergillosis. The reliability, simplicity and speed of the method render it suitable for routine diagnostic work.
Fungal infections represent an increasing problem in immunocompromised patients. The majority of cases are caused by one single fungal pathogen and infections with more than one fungus are very rare. Here we describe a case of combined infection with Aspergillus and a zygomycete species, involving the lungs, spleen and the brain and leading to fatal outcome in spite of early antimycotic therapy.
Optical brighteners of the diaminostilbene type are fluorescent dyes which are popular diagnostic tools in the mycology laboratory. While these dyes are conventionally used for the in vitro diagnosis of mycoses, their low toxicity and chemical reactivity have led us to investigate their potential use for in vivo staining of fungal elements in mycotic tissue. In mice we have established deep-seated candidiasis, cryptococcosis, aspergillosis and zygomycosis, as well as coccidioidomycosis, histoplasmosis and blastomycosis. After establishment of infection, which mostly required immunosuppression, a single dose of 100 microl of an aqueous solution (2.2 x 10(-4) M) of the optical brightener Blankophor P fluessig (4,4'-Bis [(4-anilino-6-substituted-1,3,5-triazine-2-yl) amino] stilbene-2,2'-disulfonic acid) was injected by the tail vein and the animals were sacrificed 1 h later. Sections of freshly prepared target organs were directly subjected to epifluorescence microscopy using an appropriate filter kit. In most cases, fluorescent fungal elements could be detected in the murine tissue. There was little evidence for uptake of the dye by non-infected tissues. It is suggested that radioactive labeling may render parenteral Blankophor suitable for radiographic localization of deep-seated mycotic foci in the host.
The ever increasing numbers of immunosuppressed individuals has led to a significant increase in the incidence of opportunistic infections, particularly those caused by fungi. The epidemiology of infections caused by the common fungal pathogens such as Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus has been well documented. However, in addition to these, a number of species which have previously been unrecognized (e.g., C. dubliniensis) or have previously been assumed to be non-pathogenic (e.g., Saccharomyces cerevisiae, Scedosporium spp. and Fusarium spp.) have emerged as agents of human disease. Since these species have only been identified recently as human pathogens, their role in disease is poorly understood. In most cases, identification of these species is problematic and therefore their epidemiology has yet to be elucidated adequately. In addition, several of these species fail to respond to conventional antifungal therapies. In this article, we describe the emergence of two separate yeast species (C. dubliniensis and S. cerevisiae) and two separate groups of moulds (Scedosporium prolificans and Fusarium spp.), as human pathogens. It is apparent from what we already know, that much work has yet to be performed before we have a clear understanding of how these species cause disease and most importantly how they can be controlled.
We investigated the prophylactical administration of liposomal amphotericin B (Ambisome) in the early phase after liver transplantation (LTx). Fifty-eight patients received Ambisome prophylactically after LTx. Ambisome (1 mg kg-1 day-1) was given intravenously for 7 days after LTx. Immunosuppressive prophylaxis was cyclosporin A (CsA) based in 11 patients. Forty-seven patients had a tacrolimus-based immunosuppressive regimen. CsA and tacrolimus dosages were adjusted to trough levels of 150-250 ng ml-1 (EMIT) and 5-15 ng ml-1 (MEIA II) respectively. Three patients died from sepsis due to Aspergillus fumigatus infection. Reasons for a fatal outcome were foudroyant Aspergillus pneumonia in a patient transplanted for fulminant hepatic failure on post-operative day (pod) 8; Aspergillus sepsis with severe endocardidtis in a patient with two retransplantations for graft non/dysfunction on pod 24; and disseminated aspergillosis due to Aspergillus fumigatus in a patient retransplanted for primary non-function (pod 19). All three patients underwent haemofiltration for renal failure. One patient with Candida albicans sepsis (pod 4) recovered under increased dosage of Ambisome (3 mg kg-1 per day). Ambisome (1 mg kg-1 per day) seems to be beneficial against systemic Candida infections. However, the onset of systemic Aspergillus infections could not be prevented. Obviously, higher Ambisome doses appear to be necessary against Aspergillus. We recommend the use of Ambisome (3 mg kg-1 per day) for patients with risk factors such as graft dys-/non-function, retransplantation, haemofiltration and complicated acute liver failure to prevent invasive aspergillosis.
From 1986 to 1988, in the prefluconazole era, 67,765 clinical specimens from the Göttingen University Hospital were investigated for bacteria and fungi in our institution. Oral and throat swabs, respiratory secretions, gastric juices, faeces, urine, genital swabs, blood, wound secretions and skin swabs were analysed for yeast-like fungi, and opportunistic or pathogenic bacteria. A total of 5195 specimens (7.7%) yielded Candida spp. alone or in combination with bacteria (fungal (F-) group) and 62,570 specimens yielded bacteria only or remained sterile (non-fungal group, N-group). Elevated rates of accompanying bacteria were detected with Candida spp. colonizing blood, urine, and skin. Among the dominant bacterial isolates, the distribution of staphylococci and enterococci did not reflect a distinct association pattern. Among the enterobacterial isolates from patients in intensive care, colonization patterns of the throat, gastric juices, and faeces reflected the use of a selective decontamination of the digestive tract (SDD). A statistically significant association between Candida and enterobacteria of the genus Enterobacter which was unaffected by SDD, was observed throughout this study. Such an association pattern was also observed, to a lesser extent, with the related genera Klebsiella and Serratia, but not with Escherichia coli.
A biphasic outbreak of methicillin-resistant Staphylococcus aureus in intensive-care units of a German tertiary-care hospital afflicted 89 patients within 4 years. The spread of the outbreak most likely was facilitated by the contamination of mobile radiograph equipment. The outbreak was controlled by measures of hospital hygiene.
The present knowledge on the pathogenetic relevance of extracellular and cell wall-attached proteinases from fungal pathogens is briefly reviewed.
Fluorescent staining of fungi in clinical specimens with the optical brightener Blankophor can be performed concomitantly with maceration of surrounding tissue and may be accelerated by heating. The procedure is suitable for disclosing fungi in gram-stained microscopical mounts and can be used for screening of tissue sections prior to immunofluorescence.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
A method for the detection of Candida albicans from up to 15 ml of blood by polymerase chain reaction (PCR), based on the differential resistance of mammalian and fungal cells towards detergent was developed. The procedure essentially involved removal of the blood cells by sodium dodecyl sulfate (SDS) induced lysis, followed by DNA extraction after degradation of fungal cell walls by a recombinant beta-1,3-glucanase. The genes of two different aspartic proteinases from C. albicans, SAP1 and SAP2, with an overall homology of 77% in their nucleotide sequences, were chosen as targets for PCR. The oligonucleotide primers used were directed to strictly conserved regions similar in both genes. As the number of base pairs between the primers are different in the two genes, amplification products of 220 bp and 238 bp in length were obtained. This led to a characteristic double band in subsequent agarose gel electrophoresis. The detection limit for a nested PCR was less than 10 C. albicans cells ml-1 of seeded blood. The detection limit of conventional PCR from a blood volume in the 10 ml range was less than 100 yeasts ml-1. Preliminary trials with clinical blood specimens suggested, that conventional PCR from large blood samples, being less laborious and prone to contamination than nested PCR, could be suited for the detection of deepseated C. albicans mycosis.