Phylogenetic relationships matter: antifungal susceptibility among clinically relevant yeasts.

The objective of this study was 2-fold: to evaluate whether phylogenetically closely related yeasts share common antifungal susceptibility profiles (ASPs) and whether these ASPs can be predicted from phylogeny. To address this question, 9,627 yeast strains were collected and tested for their antifungal susceptibility. Isolates were reidentified by considering recent changes in taxonomy and nomenclature. A phylogenetic (PHYLO) code based on the results of multilocus sequence analyses (large-subunit rRNA, small-subunit rRNA, translation elongation factor 1α, RNA polymerase II subunits 1 and 2) and the classification of the cellular neutral sugar composition of coenzyme Q and 18S ribosomal DNA was created to group related yeasts into PHYLO groups. The ASPs were determined for fluconazole, itraconazole, and voriconazole in each PHYLO group. The majority (95%) of the yeast strains were Ascomycetes. After reclassification, a total of 23 genera and 54 species were identified, resulting in an increase of 64% of genera and a decrease of 5% of species compared with the initial identification. These taxa were assigned to 17 distinct PHYLO groups (Ascomycota, n=13; Basidiomycota, n=4). ASPs for azoles were similar among members of the same PHYLO group and different between the various PHYLO groups. Yeast phylogeny may be an additional tool to significantly enhance the assessment of MIC values and to predict antifungal susceptibility, thereby more rapidly initiating appropriate patient management.

Species and susceptibility distribution of 1062 clinical yeast isolates to azoles, echinocandins, flucytosine and amphotericin B from a multi-centre study.

Descriptive values were determined for eight antifungal agents within the course of a multi-centre study encompassing 1062 German and Austrian clinical yeast isolates. Candida albicans (54%) was the predominant species isolated followed by Candida glabrata (22%), Candida parapsilosis (6%), Candida tropicalis (5.7%), Candida krusei (4.3%), as well as eleven further candidal and four non-Candida yeast species. While 519 (48.9%) isolates were tested susceptible to all antifungals tested, no isolate was found to exhibit complete cross resistance. For C. albicans, the proportions of susceptible isolates were 93.2% (amphotericin B), 95.6% (flucytosine), 84.3% (fluconazole), 83.8% (posaconazole), 91.8% (voriconazole), 96.5% (anidulafungin), 96.2% (caspofungin) and 97.6% (micafungin). Patterns of complete parallel resistances were observed within azoles (8.8%) and echinocandins (1.7%). While a decreased susceptibility was found infrequently for echinocandins and flucytosine, it was more common for azoles with highest proportions for isolates of C. glabrata (fluconazole, 40.6%; posaconazole, 37.2%), Candida guilliermondii (fluconazole and posaconazole, each 25.0%), C. krusei (posaconazole, 28.3%; voriconazole, 60%), C. parapsilosis (fluconazole, 70.3%) and C. tropicalis (fluconazole, 62.3%). The descriptive values obtained in this study represent a valid basis for the comparison of recent and future epidemiological surveys to analyse the susceptibility of yeast isolates towards major antifungal substances.

Catheter-associated aspergillosis of the chest wall following allogeneic hematopoietic stem cell transplantation.

Invasive aspergillosis (IA) at the insertion site of central venous catheters is a rare event. Here we report the occurrence of chest wall aspergillosis at the insertion site of a Broviac catheter in a 5-year-old child undergoing allogeneic hematopoietic stem cell transplantation. The infection arose during profound granulocytopenia under conditions of reverse isolation with laminar air flow and high efficiency particulate air filtration and was successfully managed with repeat surgical debridement, voriconazole/caspofungin combination therapy guided by therapeutic drug monitoring, and adjunctive use of granulocyte colony-stimulating factor. The case reflects the occurrence of IA despite reverse isolation and air decontamination, the principles of treatment of Aspergillus soft tissue infections in granulocytopenic patients, and the need for therapeutic drug monitoring of voriconazole particularly in young children.

Molecular genotyping of Candida species with special respect to Candida (Torulopsis) glabrata strains by arbitrarily primed PCR.

A set of 46 epidemiologically related or unrelated Candida (Torulopsis) glabrata isolates from four different medical centres in Germany and Hungary, and the type strain of this species, were genetically typed by arbitrarily primed PCR (AP-PCR). The resulting band patterns of C. glabrata strains were compared with those of other species of the genus Candida including C. albicans, C. guilliermondii, C. kefyr, C. parapsilosis, C. tropicalis and C. krusei. After preliminary trials of various reaction parameters and control experiments to test the reproducibility of this method, it was found that consistently reproducible amplification patterns were obtained only when rigorously optimised and standardised reaction conditions were employed. Discriminatory abilities were studied with 29 generated 10-mer oligonucleotides of different G+C content. Typing of clinical isolates with the optimised AP-PCR protocol was then performed with the primer 50-1, with a G+C content of 50%. Sufficiently discriminatory polymorphisms were observed among the band patterns of the Candida species included. The gel electrophoresis patterns of each species showed an adequate similarity. Variations in minor bands were characteristic for comparison at the isolate level. Only three AP-PCR genotypes were identified among the clinical isolates of C. glabrata tested. Two of these genotypes were closely related and appeared to be widespread within German and Hungarian isolates. The third genotype of C. glabrata showed a distinct band pattern. With optimised, validated and standardised assay conditions, the feasibility, sensitivity and rapidity of AP-PCR may offer a discriminatory method for genotyping of yeasts in epidemiological studies, as well as in the control of nosocomial infections.

Hospital-acquired candida and aspergillus pneumonia--diagnostic approaches and clinical findings.

Bronchoscopy with bronchoalveolar lavage (BAL), collection of bronchial secretions (BS) and/or high resolution computed tomography (CT) of the lungs was performed in 70 patients with candida and/or aspergillus pneumonia. The sensitivity of bronchoscopy in detecting histologically proven fungal disease was 59%. Characteristic CT signs were found in 11 of 14 patients with candida pneumonia and 16 of 19 patients with aspergillosis. The more frequent use of bronchoscopy and CT scans between 1990 and 1992 compared with 1986-1989 for the differential diagnosis of new pulmonary infiltrates in immunocompromised patients resulted in earlier antifungal treatment (14 vs. nine days; P < 0 center dot 025). In the second treatment period survival was improved from 36 to 50% (not significant). Bronchoscopy and high resolution CT scans are mutually complementary diagnostic tools and should be performed as early as possible in the course of pneumonia in patients at high risk of fungal diseases.

A double-blind clinical trial of fenticonazole (2%) spray versus naftifine (1%) spray in patients with cutaneous mycoses.

A multi-centre, double-blind trial was carried out in 100 patients with cutaneous mycotic infections, confirmed by direct microscopy and/or culture, to compare the efficacy and tolerability of spray formulations of 2% fenticonazole and 1% naftifine. On entry, patients were allocated at random to receive once daily topical applications of one or other drug over a period of 2 to 4 weeks, treatment being stopped when patients had recovered or substantially improved. Clinical and mycological assessments were made before (baseline), at weekly intervals during treatment and, if possible, 2 to 3 weeks after the end of treatment (drug-free period). Treatment was continued for 19.25 days with fenticonazole and 19.62 days with naftifine. All patients had positive mycological findings on entry. The most frequently isolated pathogens were dermatophytes, mainly Trichophyton rubrum; however, Candida albicans was present in 33.3% of patients in the fenticonazole group and in 20.8% of those treated with naftifine. At the end of treatment, only 3 (6.3%) and 5 (10.4%) patients, respectively, of the 48 patients assessed in each group still had positive mycological findings. Assessments of symptoms indicated comparable, significant improvement in both groups, and at the end of treatment the overall opinion of doctors and patients was that about 90% of patients were cured or greatly improved. The end of the drug-free period evaluation showed that, of the patients assessed as cured or greatly improved at the end of treatment, only 1 (3.2%) patient who had received fenticonazole and 2 (6.3%) who had received naftifine were confirmed mycologically as having relapsed.(ABSTRACT TRUNCATED AT 250 WORDS)

Early empiric antifungal therapy of infections in neutropenic patients comparing fluconazole with amphotericin B/flucytosine.

We compared the efficacy and tolerability of fluconazole (FCA) with amphotericin B/flucytosine (ABF) in neutropenic patients with haematological malignancies. Antifungal therapy started on day 4 when fever was unresponsive to antibiotics or on day 1 together with the antibiotics, if there was evidence of mycosis. If patients did not respond to FCA after 7 days they switched to ABF. 98 patients, 51 FCA and 47 ABF were included in the study. Response to fever was achieved in 28/51 FCA patients and in another 16 after switching to ABF. So in overall 44/51 (86.2%) of the FCA and 37/47 (78.8%) of the ABF group defervescence was observed. 46 patients (21 FCA, 25 ABF) developed radiological signs of pneumonia. Resolution of infiltrates was achieved in 5/21 FCA and 20/25 ABF patients, and another 10 of 15 initially not responding patients showed regression when switched to ABF, 5 of these had highly suspected aspergillosis. Adverse events occured in 19.6% of FCA and 97.9% of ABF patients. In conclusion fluconazole and amphotericin B/flucytosine seem to be equally effective. In view of its lower toxicity fluconazole may be preferred as first line empiric antifungal agent, but in case of nonresponse, pneumonia or aspergillosis it may be replaced by amphotericin B combined with flucytosine.

[Current status of determining bacteria and resistance in urinary tract infections]. / Aktueller Erreger-Nachweis und Resistenzsituation bei Harnwegsinfektionen.

A total of 5.584 bacterial strains of different species from urine samples of various University clinics were isolated in 1983. These were tested with regard to their susceptability towards antibacterial substances. E. coli was the most common rod bacteria isolated, followed by Proteus species, Klebsiella und Pseudomonas. Staphylococci and Enterococci were found relatively frequently. A list was set up concerning the resistance pattern of the most important gram-negative rod bacteria toward four penicillin- and three cephalosporin derivatives as well as towards gentamicin and three chemotherapeutics in a stricter sense. For the first substance group mezlocillin supplied the best results. In the second group cefotaxime was most effective. More unfavorable on the whole were the results of the three chemotherapeutics. Enterococci were inhibited most by azlocillin, mezlocillin, ampicillin and nalidixic-acid. Cefsulodin as a special Pseudomonas cephalosporin showed strong activity towards this bacterial species.

Candidaemia in a European Paediatric University Hospital: a 10-year observational study.

In this retrospective observational study covering 1998 to 2008, 32 patients (mean age: 7.50 years) were identified that had 35 episodes of candidaemia (0.47 cases/1000 hospital discharges). Cancer/allogeneic haematopoietic stem cell transplantation (43%) and congenital malformations/syndromes (21%) were the predominant underlying conditions. Central venous catheterization (90%), a history of antibacterial therapy (69%) and previous bacteraemia (54%) were frequent comorbidities. Candida albicans (46%) was most common, followed by Candida parapsilosis (17%) and Candida glabrata (14%). Resistance was infrequent and limited to non-albicans Candida spp. The 30-day and 100-day mortality rates were 11.4%.

[Aspects in the diagnosis of deep-seated opportunistic mycoses]. / Aspekte zur Diagnostik tieflokalisierter, opportunistischer Mykosen.

Possibilities for the mycological, mycoserological and clinical diagnostics of deep-seated opportunistic mycoses--cryptococcosis, aspergillosis and candidosis--are shown. Improvements of the diagnostics and interpretation by consideration of pathogenesis and clinical situation are discussed.

[Evaluation of a breakpoint test for determination of fluconazole susceptibility of yeasts]. / Evaluierung eines Breakpoint-Testes zur Empfindlichkeitsbestimmung an Hefen gegenüber Fluconazol.

Using the breakpoint test at 1 g/ml and 4 g/ml fluconazole, a minimal inhibitory concentration (MIC) of < or = 4 g/ml fluconazole was determined against 78.5% of the 1254 clinical yeast isolates. When compared with the micro broth dilution test, none of a subset of 128/1254 strains had a higher MIC in the dilution test than in the breakpoint test, however, in 43.0% of the 128 strains the MIC was lower in the micro broth dilution test when compared to the MIC of the breakpoint test. In a subset of 94 strains with an MIC of > 4 g/ml fluconazole determined in the breakpoint test, the elevated MIC could be confirmed only in 45.7% of the strains when using the micro broth dilution test. The percentage of breakpoint test confirmation as well as the number of strains with decreased susceptibility towards fluconazole (> 4 g/ml) were species dependent, thus, the number of decreased-susceptible Candida albicans strains was smaller than that of C. glabrata or other Candida species such as C. krusei, C. inconspicua and some C. tropicalis strains. The breakpoint test allows to identify susceptible strains with a high accuracy. Strains with an MIC > 4 g/ml fluconazole should be tested in the micro dilution test to confirm decreased susceptibility and thus to indicate the need for higher dosage of fluconazole or a change of the antifungal therapy. The breakpoint test proved to be a rapid and reliable screening test.

[Candida- and Aspergillus-antibody-Elisa--expected positive antibody results in sera of the clinical-mycological routine diagnostics]. / Candida- und Aspergillus-Antikorper-ELISA--Erwartungswerte positiver Antikorpernachweise aus Seren der klinisch-mykologischen Routinediagnostik.

Based on the ELISA results of more than 15000 serum samples of clinical mycological routine diagnostics, the expected frequency of positive antibody ELISA results within the immunoglobulin classes IgM, IgG and IgA was determined, to optimize the diagnostic assessment of first or single result of Candida or Aspergillus antibody ELISA. In general diagnostics the expected frequency of positive antibody ELISA results of the first sample within the immunoglobulin classes were as follows: Candida antibody IgM 6.1%; IgG 6.0%; IgA 2.1% and Aspergillus antibody IgM 11.4%; IgG 22.1% and IgA 5.1%, respectively. Using the Candida antibody ELISA as confirmation test only, percentages of positive antibody results in the first sample were 2.5 to 3 times higher than in general diagnostics. In follow-up examinations the Candida antibodies showed different kinetics within the immunoglobulin classes compared to those of the Aspergillus antibodies.

Methodological aspects of a microidentification technique for the differentiation of coagulase-negative staphylococci to species level.

The increasing number of coagulase-negative staphylococci (CNS) obtained from clinical material requires a biochemical identification on a broad basis in order to get a better determination of the clinical relevance of the single species. The method of differentiation presented is based on an inoculated microtiter plate provided with reagents and indicators which is evaluated by means of a coding system including most variations of the typical enzyme patterns. As a result of 7040 test series with 55 defined strains, a high degree of plausibility and reproducibility of the single reactions was found in 1320 tests under the recommended conditions. The biochemical patterns of 1380 strains taken from the diagnostic routine were established by means of this method. Comparisons with a commercially available system showed a large congruence of the single reactions and the results of identification.

[Criteria for a microdilution susceptibility testing method of fluconazole: proposal of a standardized testing method for yeasts]. / Kriterien zur Empfindlichkeitsprüfung von Fluconazol im Mikrodilutionstest: Vorschlag für eine standardisierte Methode zur Testung von Sprosspilzen.

For the proposal of a standardised susceptibility testing method of yeasts against fluconazole the laboratory experiences of the last years and the results of a collaborative study of 21 laboratories from Germany and Austria were compiled. The present paper reflects the work flow and gives advice for performing the microdilution method.

[Experiences with an antimycotic tampon in the treatment of saccharomycetic vaginal infection]. / Erfahrungen mit antimykotisch präparierten Tampons bei der Behandlung des vaginalen Hefepilz-Befalls.

Antimycotic (miconazole) medicated tampons were used for treatment in 53 patients with vaginal saccharomycetic infections as evidenced by hanging drop and on culture. A short term 2 1/2 day treatment with 5 tampons showed a success rate of 92.5%. The fast relief of symptoms like discharge, itching and burning is an improvement of the medicated tampon treatment. The new medicated tampons are considered as improvement of local vaginal treatment.

[Cultural and serologic examination for saccharomycetic infection in pregnant women (author's transl)]. / Kulturelle und serologische Untersuchungen auf Hefepilzbefall bei Schwangeren.

Cultures from retrolingual, vaginal, and anal smears were used to determine the presence of saccharomycetic infection in 177 pregnant women. The retrolingual smear was saccharomyces-positive in 29.1% of the cases; the vaginal smear, in 18.1%; the anal smear, in 7.3%. The fungus differentiation revealed the pathogen Candida albicans 85 times and the pathogen Candida krusei, 1 time. The Candida HA Test and the Candida JF Test (Roche) were carried out in 160 of the patients. A relationship could be determined between the clinical findings and the serologic results. The danger to the newborn when the mother has a saccharomycetic infection as well as the fundamentals for therapy were discussed.

Resistance-pattern-analysis--a step toward predictable differentiated antibiotic therapy.

The Resistance-Pattern-Analysis (RPA)-the procedure will be described- makes possible a comparison of susceptibility test results of different antibiotics independent of patient and test related factors. Using two examples, the comparative assessment of a more recent antibiotic with well-tried older antibiotics and the selection of antibiotics for an interventive therapy, whereby the pathogen being unknown the therapy is based upon the probable pathogen, the expected susceptibility and the localization of the infection the practicability will be illustrated. A RPA of 1526 bacterial isolates was carried out using aztreonam, gentamicin and amikacin. Aztreonam was superior to amikacin against all species tested. In comparison to gentamicin, aztreonam showed the best results against Pseudomonadaceae. Using common combinations of antibiotics for the initial interventive therapy the possible use of RPA for a cost-risk-analysis from a medical microbiological viewpoint will be demonstrated.

Glucocorticoid-induced alterations of monocyte defense mechanisms against Candida albicans.

The mechanisms of glucocorticoid-induced immunosuppression of human monocytes for the defense of Candida albicans were examined in this in vitro study. Dexamethasone at pharmacological concentrations (10(-7)-10(-5) M) dose-dependently attenuated growth inhibition of C. albicans by resident monocytes. In interferon-gamma (IFN-gamma)-primed monocytes, fungal growth inhibition was maximal and not altered by dexamethasone. Similarly, phagocytosis and killing of Candida by monocytes were not affected by steroids. To study the underlying mechanisms, we found that Candida-induced synthesis of tumor necrosis factor-alpha (TNF-alpha) by monocytes was completely abrogated by dexamethasone. Substitution of TNF-alpha to dexamethasone-treated monocytes fully reversed the alterations of growth inhibition of C. albicans induced by steroids. These findings suggest that glucocorticoids affect the growth control of Candida by monocytes indirectly via suppression of the formation of TNF-alpha, which is required as an autocrine cofactor for full monocyte activation.

[Bronchoscopic diagnosis of lung infiltrates in immunocompromised patients]. / Bronchoskopische Diagnostik von Lungeninfiltraten bei Patienten mit zellulärer Abwehrschwäche.

Bronchoscopy with bronchoalveolar lavage was undertaken in 21 immunocompromised patients for microbiological and cytological diagnosis. In all of these patients radiologically confirmed pneumonic lesions had developed which, under empirical antibiotic treatment, had progressed or not clearly regressed. In four patients each the causative organisms of the pneumonia were identified as Pneumocystis carinii and Legionella, respectively, and Aspergilla in a further three. Proof of the causative organism in these patients resulted in a change of the antibiotic treatment. The pneumonia was healed in 11 patients. Ten patients died. Early bronchoscopy to identify the causative organism is recommended in immunocompromised patients so that, by administering microorganism-specific treatment, the at present high mortality in this group may be reduced.

Infection surveillance and selective decontamination of the digestive tract (SDD) in critically ill patients--results of a controlled study.

All ICU patients were continuously monitored for infections according to a standard protocol by the physician in charge and an infection control nurse during the first quarter of five consecutive years (1980-1984). The number of patients surveyed was 1,009. The average age was 45.5 years and the average period of stay about four days. 733 patients (72.6%) were intubated and artificially ventilated for three days. A fatal outcome resulted in 13.2% of all patients, 1,129 nosocomial infections were registered in 331 patients, which means an infection rate of 32.8%. The most frequent nosocomial infections were those of the respiratory tract (24.3%). Wound infections developed in 16.6%. The urinary tract was affected in 8.8%. Nosocomial septicaemias were observed in 8.7%. Catheter-associated infections were found in 6.7% of the patients. A fatal outcome resulted in 26% of the patients with nosocomial infections and in 6.9% of the non-infected patients, respectively. There was no significant reduction in nosocomial infections over the five-year period in our ICU. Therefore, a study was designed to evaluate the concept of selective decontamination of the digestive tract (SDD) in critically ill patients in our two surgical/traumatological ICUs. A prospective, consecutive, placebo-controlled study in two ICUs was carried out during four six-month periods. 200 patients who were intubated for at least three days, required intensive care for a minimum of five days, and belonged to either class III or IV according to the "Therapeutic Intervention Scoring System" were included in the study. They received either placebo or a prophylaxis regimen, consisting of polymyxin E, tobramycin and amphotericin B.(ABSTRACT TRUNCATED AT 250 WORDS)