Search

Long-term continuous flow culture allows the investigation of dynamic biofilms under microaerophilic or aerobic conditions. We studied the biofilm formation and changes of susceptibility in 30 blood culture isolates (48 experiments) of different Candida species exposed to anidulafungin in 0.16 ml or 7.7 ml flow chambers. The flow rate (F) was adjusted to a very low rate of 1.3 ml h(-1) resulting in an exchange rate of up to 180 and 6.25 times chamber volumes per 24 hours in the small and large chambers, respectively. The results of culture at a very low flow rate were markedly different from cultures in micro well plates. Low flow rates may better mimic the in vivo situation and thus may be of higher relevance for the clinical setting. Under these conditions, a general resistance of fungal biofilms against anidulafungin cannot be confirmed. Strains of C. albicans and C. glabrata showed very uniform results whereas the C. parapsilosis group and C. lusitaniae varied from high susceptibility to resistance. Species differentiation of the C. parapsilosis group appears to be appropriate in clinical microbiological diagnostics. For the majority of the tested Candida species, anidualafungin was more effective than voriconazole. For the species C. lusitaniae and C. guilliermondii susceptibility testing should be considered prior to clinical use of echinocandin antifungals., (© 2011 Blackwell Verlag GmbH.)

The autopsy rate in Germany has drastically diminished in the last decades and is below 10% nowadays. Possible reasons for this development are discussed. Pressure of cost is a quoted cause, although it is not so high. There is a large discrepancy between the clinically supposed cause of death and the by autopsy confirmed diagnosis (40-60%). This especially applies to mycoses. Every year in Germany 1200 crimes of causing death and 11.000 non-natural deaths are not found because of missing autopsy. Another important aspect for a sufficient number of autopsies is their value for the quality assurance in diagnosis and therapy and also in education and further training of physicians and students.

In a miniature continuous flow cell system we investigated the influence of voriconazole (VCZ) (27 trials) or anidulafungin (ANID) (five trials) on the antimycotic resistance of C. albicans (type strain SC5314 and three strains from blood cultures). The duration of the trials was up to 10 d. After continuous addition of VCZ or ANID to the media, biomass decreased and glucose levels and pH values increased. After exposure to VCZ, the dry weight of biofilm dropped by 90%. There was no evidence of resistance. No resistance also developed against ANID after 1 day or 3 days of exposure. No viable cells were detected in the biofilm after 5 days of exposure to ANID. Therefore, VCZ and ANID effectively inhibited biofilm formation of different C.albicans strains. Our experiments show that biofilm production is not necessarily associated with development of resistance.

The autopsy rates in Germany became low like in other European, American and Asian countries. Main reasons for this development are the lack of acceptance of autopsy in the society as well as in the medical profession, the introduction of a requirement for consent, unclear legal position, the public health system, pressure of costs and a change in the field of activity in pathology with much more diagnostics of surgical and biopsy material. The autopsy is missing with respect to the reliability of causes of death and morbidity statistics and other epidemiological studies. Published data indicate that up to 20-30% of patients who die in hospitals have important diseases/lesions that remain undetected before death but that are found at autopsy. For infectious diseases, the data are similar. Therefore, a higher incidence of invasive fungal infections was found. Some rare fungal disorders are diagnosed by autopsy. Only exact death statistics makes specific health care possible and is cost saving in a public health system in the long term. Autopsy remains an important tool for quality control in medical diagnostic and therapeutic activity. It is also essential for fundamental medical education and further training.

In November 1839 the young university lecturer Bernhard (von) Langenbeck (1810-1887) published the first description of a fungus as aetiological agent of an oro-pharyngeal and oesophageal candidosis. We present his in English translated paper entitled 'Finding of fungi on the mucous membrane of the gullet of a typhoid fever corpse' (Germ.) in 'Neue Notizen aus dem Gebiete der Natur- und Heilkunde' (Froriep). There are interesting particulars in Langenbeck's description and discussion. The publication happened at the same time as that by Johann Lukas Schoenlein about fungi as the cause of the favus lesions. In this time Langenbeck lived at Göttingen. Later he went to Kiel and Berlin and became one of the most prominent surgeons of the 19th century. He sponsored the founding of the German Surgical Society, the Berlin Medical Society and the 'Archiv für Klinische Chirurgie'. In 1864 he was ennobled. Some of his assistants were Th. Billroth, R.U. Kroenlein, F. von Esmarch, and F. Trendelenburg.

We analysed the autopsy records of the Greifswald University Institute of Pathology (located in Eastern Germany) in respect of findings of candidosis and aspergillosis from 1994 to 2003. We also present eight immature aborted fetuses and premature infants with a mycosis. In a total of 2027 autopsies we found 164 cases of invasive candidosis and aspergillosis (8.1%) including a combination of both on four occasions. Other authors cited between 0.7 and 7.3%. In these 10 years in our material mycoses and in particular candidosis increased in spite of slightly decreased numbers of autopsies. The differences comparing the 5-years periods (1994-98 and 1999-2003) are highly significant for both mycoses and candidosis. They are not significant for aspergillosis. A similar relationship was observed in the distribution of mycotic organs and causative origin for candidosis alone. In the last 5 years the gastrointestinal and respiratory tracts, including the peritoneum, were more frequently infected by Candida. Non-haematological neoplasia and pneumonia as basic diseases more often appeared in cases of candidosis. All eight immature aborted fetuses and premature infants suffered from candidosis. The survey confirms the importance of autopsy as a tool for education and quality control in medical diagnostic and therapeutic activity in the field of mycoses, too.

Reconstituted multilayered oesophageal epithelium appears to be a good basis to test the efficacy of voriconazole (VOR) and fluconazole (FLU) in the tissue. The resulting model of a Candida oesophagitis was approaching the in vivo situation. We infected the tissue with 2 x 10(6) cfu of the Candida albicans strain SC5314. In the trials with FLU we also used clinical strains. Four hours after infection a good growth of C. albicans appeared mainly with hyphae on the surface of the tissue and a tendency to invasion. The destruction of the tissue began after 36 h. VOR (2 and 16 microg ml-1, respectively) prevented the penetration of hyphae into the tissue, when it was given 4-8 h after infection. It was less effective in reduction of Candida growth on the tissue surface. When VOR was given 16-24 h postinfection, the Candida infiltration stopped more slowly. Thirty-six hours after infection VOR application could not stop the destruction of the tissue despite reducing the fungi. The results with FLU (32 microg ml-1) were in principle the same, but not so distinct. FLU seems to be more effective against clinical strains of C. albicans than against the type strain.

The titres of indirect anti-Candida haemagglutination test (C-HAT) and specific immunoglobulins C-IgM, C-IgG and C-IgA in 328 intensive care (IT) patients and 166 non-intensive care (NIT) patients were compared by statistical test methods. Positive correlations were found between values of C-HAT and all three Candida-specific immunoglobulins. At an interval of < or =7 days in the first patient group only, the changes in the course of titres were statistically highly significant. For C-IgM the short-term increase of titres can be interpreted in the sense of happened mycotic infection in connection with the clinical picture. In IT patients we found an average increase of C-IgM at 259.3, C-IgG at 174.7, C-IgA at 59.8 U ml(-1) and of three titre steps in C-HAT. In our experience of diagnostics of Candida infections in intensive care area with non-neutropenic patients the short-term determination of the anti-Candida immunoglobulin subclasses C-IgM and C-IgG and of C-HAT as screening test should not be neglected.

Clinical applications and in vitro studies show that all antimycotics are most effective against infection when applied as early as possible. An early diagnosis is, therefore, essential. At this time for aspergillosis and particularly candidosis, a statement is only possible by combining several diagnostic methods. Inflammatory parameters like procalcitonin, C-reactive protein and proinflammatory cytokines are most important evidence of infection. Antigen tests are more significant by higher sensitivity. Attention should be focused on the detection of mannan by ELISA test, beta-glucan and D-arabinitol. Given the present research level in the field of proteomics, the diagnostic importance of transmembranal receptor proteins or other regulatory proteins seems promising.

The aim was the investigation of fungal colonization and morphological alterations under the influence of voriconazole in an in vitro system. Voriconazole stopped growth and colonization of Candida albicans (wild type SC5314) on cover slips in microtiter plates dependent on drug concentration, the time of Candida growth before the input of voriconazole and oxygen concentration. The direct microscopy by fluorescence staining with the optical brightener Blancophor showed short bizarrely deformed mycelia looking swollen. The colonization on cover glass was diminished. Microcolonies or starting of biofilm formation as in the control was not observed. The metabolic activity was demonstrated by vital staining with FUN 1 resulting in red fluorescent structures in the yeast forms and mycelia in the controls. Under voriconazole influence the remaining cells only showed a green or pale yellow fluorescence. Most of the cells lost their metabolic activity.

In 1847 the student of medicine Theodor Sluyter (1817-1895) from Greifswald published his thesis in Berlin including the first well-documented case of human pulmonary aspergillosis. In 1856 Rudolf Virchow (1821-1902) classified the depicted fungi as an Aspergillus species. Possibly Carl Ferdinand Eichstedt (1816-1892) carried out the autopsy. He is known by the first description of a fungus as the cause of pityriasis versicolor in 1846. Further involved scientists from Greifswald were Wilhelm Baum (1799-1883), Theodor Litzmann (1815-1890) and the botanist Johann Konrad Schauer (1813-1848). Their curricula vitae are given in further details as well the curriculum of T. Sluyter.

We investigated the influence of voriconazole and fluconazole in a long term trial of continuous flow culture (cfc) up to 9 days. The effects of these azoles were different in dependence on the growth circumstances. Under anaerobic conditions a fungicidal effect of voriconazole was detectable, defined by an inhibition of 99.9%. This also applied to fluconazole for the majority of tested strains of C. albicans. Under aerobic conditions with an otherwise similar situation we found only a fungistatic reaction (inhibition of 90%). Fluorescence microscopy comparing fungal morphology in biofilms on glass surfaces in the cfc revealed a differentiation into blastospores, germ tubes, pseudomycelia and mycelia in the control trial after a cultivation of 8 days. Under anaerobic conditions with azoles only some single cells could be found, sometimes in cell detritus. The adhesion was clearly reduced. Under aerobic conditions more blastospores but no differentiated mycelia were to be seen.

Unlabelled: Funguria, indirect anti-Candida haemagglutination test (C-IHT) and Candida-specific immunoglobulins C-IgM, C-IgG and C-IgA were investigated under suspicion of systemic candidosis in critically ill patients. A total of 143 urine cultures were studied for Candida from 74 adults and a median count of log 3.0 CFU ml-1 was found. Most isolated Candida species were Candida albicans and Candida glabrata. In 14 cases of candidaemia there was no regular agreement between the finding of Candida species in blood and urine. In cases with candiduria > = or log 3.0 CFU ml-1 a stronger increase of C-IHT titres and all three Candida-specific immunoglobulins after 5-7 days was observed. Some statistically significant correlations were found between the levels of urinary yeast counts and immunological parameters concerning C-IHT, C-IgA and C-IgG on the first day and after 5-7 days. Clinical findings in some cases coincided well with funguria and courses of titres before and after treatment., Conclusion: In critically ill patients suspected of having systemic candidosis not only blood cultures should be made. Cultural studies with specimens taken from different sites including funguria are essential for a complete specific serological investigation.

We conducted upper intestinoscopies in 124 intensive care patients, six of whom had oesophageal candidosis. Of these, two also had Candida plaque in the stomach. The patients at the intensive care unit (ICU) had a mean Apache-II score of 26.7; whereas the score was 29.5 in patients with Candida oesophagitis. A significant increase of Candida antibodies was found in 59 of 124 patients (47.6%), including all patients with oesophageal candidosis. Presumably, mycotic infections of other sites were present. The severity by which mucous membranes were affected correlated well with microscopically evident invasiveness.

We used the model of continuous flow culture (cfc) to study the growth of Candida species. This model allows special test conditions: a long generation time of 15-20 hs, controlled limitation of nitrogen sources and carbohydrates, comparison of the growth under aerobic and anaerobic conditions simultaneously. These conditions were used to study the effect of antimycotic drugs, mainly during a long time of 7 to 10 days. Germ tube formation as a virulence factor was more abundant and faster in cfc of strains with a stronger adherence to buccal epithelium cells. Co-cultivation of C. albicans and C. glabrata allowed conclusions for their colonization in vivo. A biofilm on the glass wall of the culture vessel led to mycelium formation by C. albicans. Concomitantly the growth of C. glabrata was favoured. Growth of C. albicans in the gastrointestinal flora was reduced by masses of bacteria and their multiple metabolic activities. A remarkable growth of C. albicans was only to be seen if the ecosystem was destroyed e.g. by antibacterial antibiotics. The influence of fluconazole in a long-term follow up study under anaerobic conditions showed an inhibition of C. albicans in 99.9 %. This means fungicidal efficacy., (1999 Blackwell Science Ltd.)

Besides the activation of phagocytes, the release of cytokines is the most important immunological defence mechanism of an organism against infection with Candida albicans. On the other hand cytokines induced in the organism by the yeast itself are able to modulate the immune responses of the host. We investigated whether eight clinically isolated strains of C. albicans inactivated by formalin as well as a laboratory strain were able to induce proinflammatory and regulatory cytokines in peripheral blood mononuclear cells (PBMC) of four different donors. Under our assay conditions the yeast strains induced the cytokines interleukin-1 beta (IL-1 beta), interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) in PBMC to varying extents, but not the cytokine interleukin-4 (IL-4). We observed a difference in the reaction of the individual donors to the stimulus C. albicans but on the other hand the extent of the cytokine signal seemed to be dependent on the yeast strain as well. No correlation was found between the ability of the individual C. albicans strains to induce cytokines in PBMC and their ability to adhere to buccal epithelial cells. Determination of the cytokine induction potential of C. albicans strains possibly may contribute to the detection of new virulence factors of this yeast.

We used the model of continuous flow culture (cfc) to study the growth of Candida species. This model allows special test conditions: a long generation time of 15-20 hs, controlled limitation of nitrogen sources and carbohydrates, comparison of the growth under aerobic and anaerobic conditions simultaneously. These conditions were used to study the effect of antimycotic drugs, mainly during a long time of 7 to 10 days. Germ tube formation as a virulence factor was more abundant and faster in cfc of strains with a stronger adherence to buccal epithelium cells. Co-cultivation of C. albicans and C. glabrata allowed conclusions for their colonization in vivo. A biofilm on the glass wall of the culture vessel led to mycelium formation by C. albicans. Concomitantly the growth of C. glabrata was favoured. Growth of C. albicans in the gastrointestinal flora was reduced by masses of bacteria and their multiple metabolic activities. A remarkable growth of C. albicans was only to be seen if the ecosystem was destroyed e.g. by antibacterial antibiotics. The influence of fluconazole in a long-term follow up study under anaerobic conditions showed an inhibition of C. albicans in 99.9%. This means fungicidal efficacy.

Mucosal adherence and germ tube formation have been considered as important virulence factors of Candida albicans. We investigated 11 clinical isolates (among them six isolates from oesophageal thrush) for quantification of adherence to buccal epithelial cells and germ tube formation in the continuous flow culture in vitro, and correlated the results with the clinical data of the patients. Adherence varied considerably between the different C. albicans strains. Strains recovered from clinically, culturally and serologically confirmed oesophageal thrush adhered stronger to buccal epithelial cells. Isolates from cases with heavy colonisation but clinically without candidosis were less adherent. Only after 30 min germ tube formation was observed in the continuous flow culture. Strains with stronger adherence also showed significantly faster and increased germ tube formation. The patients with oesophageal thrush did not suffer any particular immunosuppression such as HIV infection, although in most cases chronic alcoholism was apparent. We conclude, that in cases with minor immunosuppression the expression of the virulence factors adherence and germ tube formation plays an important role in the pathogenesis of candidosis, whereas it may be of less importance in cases with severe immunosuppression. In the latter they may, however, influence outcome.

In the period 1992-95 there was a significant shift in the spectrum of Candida species in the University Hospital in Greifswald. During this time, the annual number of specimens taken for mycological investigations of adults increased threefold (total n = 11,568). The isolation rate of Candida species was 50.5%. The percentage of C. albicans isolates decreased from 76% to 54.4% with the lowest level in 1994. The opposite trend in the occurrence of non-C. albicans species was seen, for example the occurrence of C. glabrata, from 11.7% to 28.4%. We found only 98 strains of C. parapsilosis (1.4%) during the 4 years. The occurrence of Candida species in a variety of habitats was different. During the 4 years, the same annual percentages of C. albicans (mean 87.9%) were isolated from endoscopic oesophageal smears, whereas the distribution of Candida yeasts from the oral cavity, the respiratory tract, faeces and urine had changed. Over the years, at these locations, C. albicans was less frequently isolated and non-C. albicans species clearly increased. The highest occurrence of C. glabrata was found in urine, in which the isolation percentage almost doubled from 23.1% to 40.9% in 1994. In contrast to adults, in all specimens originating from a paediatric clinic that included neonatology the occurrence of C. albicans was high (83.5% in 1995), but the isolation rate of Candida species was low (12.3%). These results are important because of the differences in yeast susceptibility, of non-C. albicans species in particular, against antifungal drugs.

We report about our experiences obtained with the Vitek system (bioMerieux, Nürtingen) for identification of 1160 clinical Candida isolates. The Vitek system correctly identified 1005 (86.6%) Candida isolates (probability > or = 85%): 776 (66.9%) strains were identified after 24 h of incubation, 229 (19.7%) required 48 h of incubation. In 4.4% (n = 51) we found results with a lower probability (< 85%). After 24 h of incubation 38 (14.6%) of the 260 C. tropicalis isolates tested were misidentified as C. parapsilosis; we obtained the correct results after 48 h of incubation. 104 (9%) isolates were wrongly identified after 48 h of incubation. The majority of these strains (n = 82) were biochemically minimally reactive Candida species. Overall, the Yeast Biochemical Card system provides a reliable method for the rapid, automated identification of medically important Candida species.

The investigation of susceptibility of Candida species to fluconazole was performed in microdilution to a supplemented HR-medium. The sufficient reproducibility of the test was verified using special control isolates and isolates of patients. The excellent applicability of the method in routine diagnostics was evaluated by in vitro testing of susceptibility of 279 Candida isolates from patients being colonised or suffering from endomycoses. The Candida species showed different susceptibility against fluconazole: 96% of the C. albicans isolates were sensitive, 55% of the C. glabrata isolates had a reduced sensitivity, and 26% were resistant against fluconazole (MIC > 25 micrograms/ml). C. krusei isolates were highly resistant (9 of 11 strains).

Within the microbial ecosystem of the orointestinal tract Candida spp. are common but not always detectable organisms. Therefore they belong to the transient flora. Candida spp. are found at various sites in healthy subjects in quantities between 10(2)-10(4)/ml or g. In different diseases an increased growth of Candida can occur depending on the presence of typical risk factors. In any case the yeasts only account for a minimal part of the bacterial flora. In this regard the contribution to the formation of metabolic products-in particular carbon dioxide, hydrogen and alcohols-is very small. Growth and development of Candida are dependent on nutrient and oxygen supply. In the colon under anaerobic conditions the flora competes for limited nutrients and fermentation is the dominant metabolic pathway in Candida. There is only minimal growth. Under aerobic conditions as can be demonstrated in experimental settings a marked growth with the formation of mycelium takes place. The generation time in vivo is much longer than under laboratory conditions and takes several hours. The detection of Candida in stool specimens can be interpreted in the first line only as a colonization. This is not equivalent with an infection, which, unfortunately, often is wrongly suggested in positive stool specimens.

We used the model of faecal microflora under continuous flow culture (CFC) conditions to study the growth of Candida albicans in mixed cultures. The development of Candida is usually limited by the high germ counts of aerobic and anaerobic bacteria. Candida growth in faecal flora could be obtained neither by the continuous inflow of C. albicans CFC monocultures nor by intermittent input of highly concentrated suspensions of C. albicans into the system. If the system was run under aerobic conditions development of C. albicans could be observed. After some days, however, the faecal flora again suppressed further development of the yeasts. Only marked destruction of the aerobic and anaerobic microflora by antibiotics resulted in growth or overgrowth of Candida albicans.

Unlabelled: We studied 15 patients with mostly alcoholic liver diseases and 25 patients with acute or chronic pancreatitis with regard to occurrence of yeasts in different microbiological samples and corresponding serological findings. In about a half of the patients with liver diseases yeast counts and serological titres were already raised in the first mycological investigation. Patients with pancreatitis, however, showed only little or negative cultural and serological results. This changed during the course of disease, where they developed significantly higher yeast counts and serotitres. Finally two case reports are presented: two patients with infected pancreatic pseudocysts (including a case of aspergillosis)., Conclusions: in patients with decompensated chronic liver diseases an early search for mycological complications is recommended. In pancreatic diseases these complications are rather seen later in the course of the disease, especially under intensive care conditions. Therefore, we encourage surveillance cultures and control of serotitres in these patients.

We used the model of fecal microflora under continuous flow culture (cfc) conditions to study the growth of Candida albicans in mixed cultures. The development of Candida is usually limited by the high germ counts of aerobic and anaerobic bacteria. Neither by the continuous inflow of C. albicans-cfc-monocultures nor by intermittent input of highly concentrated suspensions of C. albicans into the system a growth in fecal flora could be obtained. If the system was run under aerobic conditions a development of C. albicans with few hyphal growth could be observed. After some days however, the fecal flora again suppressed a further development of the yeasts. Only a marked destruction of the aerobic and anaerobic microflora by antibiotics resulted in a growth or overgrowth of Candida albicans.

We investigated the opportunistic pathogen Candida albicans in a continuous flow culture with regard to particular growth conditions. Germ counts of only 10(7) cfu/ml were obtained in a steady state of an anaerobic culture. In a parallel experiment with aerobic conditions excessive growth occurred and the invasive forms of Candida, i.e. germ tubes and pseudomycelium, also developed. Neither by different carbohydrates and concentration of nutrients in the culture medium nor by changing the flow rate the growth of Candida could be influenced. Excessive overgrowth was triggered by a sufficient oxygen supply.

The api-Zym system offers a useful method for biotyping of Candida albicans. We assessed 19 hydrolytic enzymes with this method. Our 53 strains of human origin showed up to 10 different enzymatic activities.

Three different Candida antigen tests were studied in internal-surgical patients: two tests for Candida mannan antigen (C-Mannan-Dresden, Pastorex-Candida and Cand-Tec test for cytoplasmatic antigen. Most positive findings could be seen by both tests for mannan. In cases with increased occurrence of fungi in feces, sputum, urine, and mouth smears the tests differently became negative. Some clinical cases are discussed. With increased positivity of titer courses of all 3 antigen tests the probability of detection of an endomycosis also increased.