Biomedical subjects
Publications and source records attributed to R Rüchel.
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.
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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.
The influence of the heterologous acid secretory Candida proteinases on the adherence of the non-proteinase secreting strain of C. tropicalis DSM 4959 to epitheloid cells (vero line) was examined. The proteinases of the following Candida strains were used: C. albicans ATCC 10261 (serotype A), C. albicans ATCC 48867 (serotype B), C. tropicalis DSM 4238. The assays were performed with the previously described in-vitro-adherence test [1] using the following principle steps: Candida proteinases and C. tropicalis blastoconidia were incubated with verocells in microtest plates in phosphate-buffer in the range of pH 4.0 to pH 7.0. Adherent Candida cells were detected according to Filler et al. [2] with anti-Candida-mannoprotein antibodies and a secondary anti-rabbit-peroxidase conjugate. Compared to controls with denaturated proteinases, the photometric evaluation of adherent C. tropicalis cells showed, under optimal conditions, an augmentation of the adherence due to the Candida proteinases of about 50%. The optimum of this adherence augmentation was in the range of pH 5.5 which is outside the general activity optimum of Candida proteinases (pH 3). The degree of purity of these proteinases had no marked influence on the adherence. The specificity of the proteinase dependent adherence augmentation could be demonstrated with the enzyme inhibitor Pepstatin A. C. tropicalis blastoconidia supplemented by pepstatin A and active Candida proteinase adhered in the same range as with denaturated proteinases in control tests. Our results suggest a function of Candida proteinases in the adherence process of blastoconidia to epithelia.
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Oligonucleotide primers based on conserved regions of the aspergillopepsins (EC 3.4.23.18) were used to PCR amplify a 650 bp segment of the gene encoding the extracellular aspartic proteinase (PEP) from Aspergillus Fumigatus. The segment was used as a probe for isolating and sequencing the gene from a genomic library of the fungus. Likewise the cDNA was amplified by reverse PCR, cloned and sequenced. The pep gene was found to consist of four exons encoding for 395 aa. The pre-proenzyme deduced has an N-terminal leader sequence of 70 aa preceding the sequence of the mature enzyme consisting of 325 aa with a calculated molecular mass of 34.4 kDa and an isoelectric point of 3.95. The N-terminal sequence of the mature enzyme matched the N-terminal aa sequence of PEP exactly. The nucleotide and the aa sequences of the pre-proenzyme were 70% and 71% homologous to the corresponding sequences of the aspergillopepsin from A. niger var. awamori. Southern analysis of digested genomic A. fumigatus DNA with a specific PCR probe suggested the presence of a single copy of the pep gene.
Domestic invasive mycoses are typically present as secondary diseases in patients definitely immunocompromised. This truth should not obscure the fact that a transient overload of the immune system, e.g. in the polytrauma patients, may likewise favour the development of mycoses. The two groups of patients show a comparable course of infection and, to some extent, diagnostic signs: surveillance cultures and monitoring of antibodies are more helpful with trauma patients and less reliable in the typically immunocompromised patients. In the latter, however, antigen tests may yield more reliable results than in the trauma patients. The different functional capacities of the immune system in the two groups of patients may also affect the appearance of fungal elements, particularly of aspergilli, in secretions and biopsies.
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The analysis of genomic DNA fragment patterns has revealed as a powerful tool for strain discrimination in Staphylococcus aureus; for use as an epidemiological marker, stability during the course of an outbreak is an essential prerequisite. Genomic DNA fragment patterns (SmaI restriction, pulsed-field electrophoresis) of four different epidemic MRSA strains were compared along with intra- and interhospital and country-wide spread over more than 12 months in Germany. Strain I was isolated from infections in 8 hospitals. In one hospital a subclone arised which differed from the original strain by 4 fragments. Strain II was spread among 4 hospitals, isolates from three of these hospitals exhibited a variability of one to three fragments in the 150-200 kb range. Two hospitals in the Hannover-area were affected by strain III; in 17 isolates of this strain a variability up to three fragments was found in the 170-200 kb range. Strain IV was isolated from 19 cases of infections in 3 hospitals in Berlin. The fragment patterns were completely stable. When S. aureus strains are typed by genomic DNA fragment patterns, a variability in a definite range of molecular masses during the course of an epidemic should be taken into consideration.
Fournier's gangrene is a necrotising soft-tissue infection of the scrotum and perineal region caused by gram-negative and gram-positive Enterobacteriaceae. The disease is characterised by its unique appearance, its speed of onset, and its high mortality. CASE REPORT. A 26-year-old male presented to the emergency room complaining of a painful, tremendously swollen scrotum and penis (Fig. 1) that had developed within the past 24 h. Later, slurred speech, pallor, and hypotension were recognised, leading to the patient's admission to the intensive care unit. Suspecting a severe internal haemorrhage, vigorous volume therapy was started using crystalloids and colloids until blood and fresh frozen plasma were available. One hour later, septic shock was presumed and therapy augmented by IV antibiotics, tracheal intubation, and mechanical ventilation. Despite all efforts, the patients condition deteriorated rapidly and he died a few hours later due to multiple organ failure in septic shock. Postmortem, a perforated external hemorrhoidal node was found to be the primary focus of sepsis. Microbiologic cultures revealed Escherichia coli in blood and tissue samples. DISCUSSION. Fournier's gangrene is a rare disease; nevertheless, its clinical picture has to be recognised immediately in order to provide appropriate treatment in time. It occurs predominantly in males after minor trauma, colorectal or urological disease, and perineal or abdominal surgery. Fournier's gangrene usually begins with itching and pain in the scrotal region followed by swelling and dark-blueish discolouration of the scrotum and penis, occasionally including the lower abdominal wall. Fever and chills are usually present. The illness progresses to severe prostation and septic shock with a mortality of 20%-50%. Tissue cultures mostly reveal E. coli, gram-positive enterococci, Pseudomonas, Proteus, and various anaerobes. The treatment should include immediate radical surgical debridement, i.v. administration of broad-spectrum antibiotics, and cardiopulmonary support. CONCLUSION. The dramatic course of Fournier's gangrene requires early recognition, extensive surgical debridement, as well as intensive care treatment in order to prevent irreversible septic shock.
An aspartic proteinase (PEP) from the culture supernatant of a clinical isolate of Aspergillus fumigatus was purified to virtual homogeneity at a yield of 24%. The procedure involved affinity chromatography on pepstatin agarose, the interaction requiring a chaotropic salt (sodium trifluoroacetate) for complete elution of the enzyme. Among 11 amino acids of the N-terminal region, nine were identical with the corresponding sequence of the aspartic proteinase aspergillopepsin A from Aspergillus niger var. awamori (previously called Aspergillus awamori). Thus PEP belongs to the aspergillopepsins, a family of closely related aspartic proteinases produced by aspergilli. Specific antibodies against PEP were detected by dot blot assay in sera of two patients with aspergillosis. In addition, PEP antigen was demonstrated by immunofluorescence in mycotic human lung, using specifically elicited antibodies from guinea-pigs. PEP had an estimated molecular mass of 38 kDa and the pI was determined at pH 4.2. PEP is therefore likely to be closely related to an acid proteinase of A. fumigatus which was originally described in 1981.
In compromised patients, invasive mycoses are being observed at an increasing frequency and with a broadening spectrum of causative fungi. The rapid and definitive diagnosis of such opportunistic mycoses requires the synoptical consideration of predisposing clinical conditions, of culture isolates, and of the results of nonculture techniques. At present, the latter comprise the monitoring of specific antibodies and of fungal antigens, and the microscopical examination of suspected biopsies. The validity of the individual techniques (e.g., the monitoring of antibodies or antigens) varies with individual mycoses. Alternative methods for monitoring fungal metabolites or nucleic acids are still in the developmental stage. Particularly the amplification of DNA by the polymerase chain reaction (PCR) has a high diagnostic potential. However, at present, it is uncertain whether PCR allows the necessary distinction between colonization and truly invasive infection, and whether PCR can be simplified sufficiently to allow the continuous surveillance of high-risk patients.
We report on four cases of aspergillosis which developed during therapy with fluconazole in patients who were immunosuppressed or granulocytopenic. Fluconazole may obscure the onset of aspergillosis. We feel that it should neither be given prophylactically nor for fever of unknown origin in patients who are at high risk of contracting aspergillosis.