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Though not overtly immunosuppressed, an elderly female patient suffered from chronic pleurisy due to Aspergillus fumigatus. In spite of nearly six months of itraconzole-therapy, she eventually succumbed to CNPA. Pleurisy may be a typical manifestation of this rare mycosis which is often ill fated.

Fluorescent staining using optical brighteners (diaminostilbenes) affords the semispecific and rapid detection of fungal elements in clinical specimens. After yielding a first hint of mycosis, the identification of the involved fungal genus is often desirable in cases when culture proves unsuccessful. In such cases, immunohistochemistry or in situ hybridisation may further the diagnosis with respect to establish an appropriate therapy.

Invasive zygomycoses (syn. mucormycoses) are rather rare but life-threatening diseases which often take a peracute course. Particularly endangered are diabetics and patients suffering from siderophilia. Zygomycosis is regularly complicated by thrombosis and subsequent necrosis. Usually it evolves from sinusitis in a rhinocerebral form. With the use of a clinical isolate (Rhizopus microsporus) and sera of the same female survivor, we investigated possible sources of the typical blood clotting. The results suggest that coagulation is probably initiated in a bimodal manner by an extracellular serine proteinase of the fungus and by elastase from the patients' leukocytes. The former causes a partial hydrolysis of fibrinogen, while the latter activates coagulation factor XIII (fibrin stabilizing factor). Both proteinases were present in the patient at the site of infection, and in vitro they jointly bring about regular clotting of fibrinogen.

In the case of a 53-year-old woman with acute myeloid leukaemia and fever in late aplasia after chemotherapy, invasive mycosis with characteristic involvement of liver and spleen was diagnosed. For the serological identification of Candida in the early phase of the infection, methods for the detection of antibodies against Candida antigens were compared. By ELISA-based detection of IgM and IgG antibodies against a mixture of Candida antigens (ESR 117G and 117M, Virion-Serion, Wurzburg, Germany) evidence for invasive candidosis was obtained significantly earlier (22 days) when compared with the immunofluorescence detection of IgG antibodies against Candida albicans germ tube antigens (Vircell, Granada, Spain). In the case of this patient, the detection of a humoral response against Candida germ tube antigens was of little diagnostic value.

Activation of blood coagulation to a varying extent affect the course of domestic invasive mycoses. Upon invasion of blood vessels by Candida or aspergilli, occasionally thrombi are formed, which may cause septic embolism. In the course of mucormycosis (syn. zygomycosis) thrombotic occlusion of afflicted blood vessels and subsequent necrosis of dependent tissue regularly occurs. Coagulation during candidosis or aspergillosis may be triggered by secreted aspartic proteinases which are able to activate factor X as has been shown previously [1, 2]. During mucormycosis, severe blood coagulation apparently is due to paracoagulation of fibrinogen which is triggered by low concentrations of extracellular fungal subtilisin-like proteinase (Arp). The enzyme is also able to inactivate the major inhibitor of blood coagulation (antithrombin III). Recent findings on the action of Arp are discussed.

The occurrence of community-acquired pneumonia due to yeast-like fungi of the genus Candida in patients without manifest immunodeficiency has previously been discounted. However, such pneumonias may indeed occur in patients with chronic parenchymal lung damage, e.g. from nicotine. Candida pneumonia can be triggered in these patients for example by trivial viral infections. Three corresponding cases are discussed.

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.

The present knowledge on the pathogenetic relevance of extracellular and cell wall-attached proteinases from fungal pathogens is briefly reviewed.

Small pre-term neonates are susceptible to cutaneous aspergillosis because of their immature immune system and because of the vulnerability of their skin. In addition, the common therapy with broad-spectrum antibiotic drugs and corticoids creates a favourable milieu for fungal superinfections. We present four pre-term neonates who succumbed to cutaneous aspergillosis that subsequently developed into a systemic infection. The source of the infection proved to be contaminated latex finger stalls. Three of the four patients died. The poor prognosis for systemic aspergillosis can only be improved by an early therapy with amphotericin B, possibly in liposomal form.

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.

Sporulating colonies of Aspergillus fumigatus, A. flavus, and A. niger were subjected to immunofluorescence using specific polyclonal antibodies against the aspergillopepsin PEP (EC 3.4.23.18), a secretory aspartic proteinase produced by A. fumigatus. The proteinase antigen was found mainly in developing conidiophores of aspergilli, in submerged mycelia and on the tips of growing aerial mycelia. Mature aerial hyphae and spores showed no immunofluorescence at all. Sporulating conidiophores revealed only weak activity in A. fumigatus and A. flavus. The distinct pattern of expression of the aspartic proteinase antigens suggests a role for such enzymes in the growth of hyphae and the development of conidiophores and thus for the sporulation process in aspergilli.

Scedosporium apiospermum, an anamorph of Pseudallescheria boydii, is a hyalohyphomycete which can be mistaken, histologically, for Aspergillus. S. apiospermum is clinically distinguished by resistance to most antifungals and its ability to cause invasive mycoses in immunocompetent patients. Here we present a typical case of lethal cerebral Pseudallescheria mycosis in a child after near-drowning.

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.

In the University Hospital in Göttingen, the spectra of fungal species in clinical specimens of respiratory secretions, bronchial secretions and urine were compared over periods of 15 months (10/87 to 12/88 and 1/91 to 3/92) before and after the introduction of fluconazole. The following changes could be demonstrated: 1. In all specimens analysed the number of Candida albicans isolates decreased, while the number of Candida tropicalis isolates remained almost unchanged. 2. During the observation period the number of Candida glabrata isolates doubled. In 1991 C. glabrata was second to C. albicans as the most common of all fungal isolates, appearing in 8.6% of all specimens. 3. The total number of Candida krusei isolates increased only slightly, but the rise in the number of isolates in bronchial secretions was statistically significant. 4. The prevalence of rarely isolated Candida yeasts, such as Candida guilliermondii, Candida lipolytica and Candida kefyr, and Candida isolates which were not further differentiated increased. 5. During the observation period the number of mixed cultures showed a fourfold increase. C. glabrata and C. krusei were associated in more than 75% of all isolates with C. albicans or C. tropicalis respectively. 6. The number of mould isolates increased. These changes in the spectra of fungal isolates are discussed with respect to the broad therapeutic and prophylactic usage of fluconazole in the University Hospital of Göttingen.

A fluorescent staining procedure, which used to be recommended for the detection of fungi in skin and nail scrapings, can be adapted for the rapid detection of fungal elements in non-sectioned biopsies or respiratory secretions. The specimens are directly stained with an optical brightener (Blankophor P flüssig, Cellufluor, Uvitex 2B) preferentially after exposure to 20% aqueous potassium hydroxide solution. The procedure can also be applied to Gram-stained microscopic mounts. The high intensity of the fluorescence elicited allows rapid microscopic screening at low magnifications.

Clinical specimens of respiratory secretions, urine, and vaginal secretions were continuously monitored for Candida species and accompanying bacteria. The distribution of the major bacterial species was analysed with respect to the presence or absence of Candida co-isolates. Statistically significant differences were observed particularly between the distributions of certain aerobic gram-positive cocci, which tended to be associated with the yeast-like fungi, and certain aerobic gram-negative rods, which were found more often in the absence of Candida. Likewise, the profile of bacterial isolates as a whole correlated significantly with the presence of yeast-like fungi.

By repeated testing of sera after heating (56 degrees C, 15 min) certain false-positives in the Cand-Tec test can be identified from the thermo-resistance of the reaction. True titers are drastically reduced by such pretreatment of sera. The thermostable antigen giving rise to falsely positive agglutination is not identical with mannan.

The diagnosis of deep-seated candidosis still poses severe problems to the physician. While laboratory tests for a long time have been mainly based on the detection of antibodies to Candida and in particular Candida albicans the detection of Candida antigen in the blood gains more and more interest. When it comes to the evaluation of the various laboratory tests the correlation with autopsy data is most rewarding. The more specimens are analysed for the presence of hints at deep-seated candidosis in a given hospital setting the least frequent death due to deep-seated candidosis occurs.