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.

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%.

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.

[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.

An outbreak of candidemia due to Candida tropicalis in a neonatal intensive care unit.

An outbreak of candidemia due to Candida tropicalis involving 16 neonates (gestational age 28-36 weeks) is reported. All infants had received hyperalimentation and at least one course of antibiotics. The commonest clinical manifestations included episodes of acute respiratory distress and lack of response to antibacterial antibiotic therapy. Candida tropicalis was recovered from blood in all the 16 infants and urine cultures were positive in 14 infants. Environmental sampling yielded C. tropicalis from one each of the blankets and mattresses used for neonates. Four of five urinary tract isolates and both environmental isolates genotyped by arbitrarily primed-PCR with several random primers were shown to belong to the same genotype.

Oropharyngeal carriage of Candida species in HIV-infected patients in India.

The present investigation represents the first study of oropharyngeal carriage of Candida and other yeasts in HIV-infected patients in India. One hundred and fifty HIV-positive patients were investigated by culturing their swish samples on plates of CHROMagar Candida. Ninety-eight patients (65.3%) were positive for Candida and four (2.7%) were positive for other yeasts. Among them, the first Indian C. dubliniensis isolate has been recovered. Molecular typing of selected C. albicans isolates by AP-PCR revealed two major genotypes based on the banding patterns. The susceptibilities of 30 Candida isolates to five antifungal agents including the new triazole voriconazole were determined in a micro-dilution test, according to the NCCLS protocol M 27. All the 22 C. albicans isolates were susceptible to five antimycotic agents (flucytosine, amphotericin B, fluconazole, voriconazole and itraconazole) except one isolate (VPCI-122), which was resistant to flucytosine (MIC > or = 64 mg l-1). The azole-resistant isolates reported here endorse the role of antifungal susceptibility testing whenever antifungal treatment with azoles is planned.

Treatment of severe Candida infections in high-risk patients in Germany: consensus formed by a panel of interdisciplinary investigators.

Now that modern medicine can provide increasing chances of cure to patients with formerly incurable disorders, therapy-related complications play the key role in outcome. Thus, among opportunistic infections, severe candidiasis remains a challenge. A multidisciplinary panel of 20 investigators was formed to find a consensus on antifungal strategies for various underlying conditions in neutropenic and non-neutropenic patients. To record their preferences, the investigators used an anonymous voting system. Among antifungal agents, fluconazole emerged as the major alternative to the classic amphotericin B, being therapeutically at least equivalent but clearly less toxic. Factors that restrict the use of fluconazole include pretreatment with azoles, involvement of resistant species like Candida krusei, and an inability to exclude aspergillosis. Flucytosine can be reasonably combined with both amphotericin B and fluconazole. Within the limited antifungal armamentarium, amphotericin B lipid formulations and itraconazole also appear useful and require further investigation. The general consensus of the group is that antifungal agents should be administered at sufficient dosages, rather early, and often empirically.

Isolation and characterization of a species-specific DNA fragment for identification of Candida (Torulopsis) glabrata by PCR.

A PCR specific for Candida glabrata that amplifies a mitochondrial rRNA gene fragment was developed by analysis of C. glabrata-specific agarose gel bands, which were generated by arbitrarily primed PCR. The expected PCR product was successfully amplified with genomic DNA from 95 C. glabrata isolates but not from a number of other fungal isolates.

Common bile duct obstruction due to candidiasis.

Biliary obstruction with its wide range of potential causes (e.g. neoplastic lesions, gallstones and inflammatory processes) is a common disease in gastroenterology. Although infections with Candida and other fungal species have increasingly been recognized in patients with certain predispositions, fungal involvement of the biliary tract is extremely rare. We report the case of a male patient with a past history of long-time mechanical ventilation and who was referred to our department with cholangitis. Endoscopic retrograde cholangio-pancreatography (ERCP) of the septic patient revealed a high-degree stenosis of the distal common bile duct with a prestenotic dilation which was strongly suspicious of an underlying malignancy. Control ERCP revealed a beads-like deformation of the intra- and extrahepatic bile duct system which was compatible with chronic secondary sclerosing cholangitis. Examining the bile duct system with a balloon catheter, a long tubular. filamentous structure with several branches at its sides could be extracted and was assessed histologically to be a Candida conglomerate. Candida colonization of the bile duct was confirmed by microbiological analysis of aspirated bile.

Liposomal amphotericin B for prophylaxis of invasive fungal infections in high-risk paediatric patients with chemotherapy-related neutropenia: interim analysis of a prospective study.

Invasive fungal infections (IFI) are a major cause of morbidity and mortality in patients with cancer. A retrospective analysis of children with cancer at high risk for IFI treated at Münster University Hospital showed that the incidence (7.4% vs. 1.8%) and lethality (28.1% vs. 0) of documented IFI were lower in patients receiving systemic antifungal prophylaxis with liposomal amphotericin B (l-AmB) in comparison to a historical control group. To determine whether this decline in incidence and lethality was due to antifungal prophylaxis or was produced by advances in diagnostic procedures and early empirical antifungal therapy, a prospective study was initiated. Patients in the prophylaxis arm received thrice-weekly 1 mg kg(-1) body weight l-AmB, whilst patients in the early intervention arm received no prophylaxis. Diagnostic procedures and antifungal therapy for suspected or proven IFI were initiated as clinically indicated for all patients. The primary endpoint of the study was the incidence of IFI. Secondary endpoints were the use of therapeutic doses of l-AmB, the safety of prophylactic l-AmB, and the total consumption of l-AmB for antifungal therapy. The interim analysis after 1 year showed no differences between the two approaches with respect to the incidence of IFI and to safety issues.

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.

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.

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 occurred 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.

[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.

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.

[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.

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.

Pulmonary fungal infections in immunocompromised patients: incidence and risk factors.

In a prospective study, 178 patients with fever > 38.4 degrees C and newly diagnosed pulmonary infiltrates underwent bronchoscopy with bronchoalveolar lavage (BAL), aspiration of bronchial secretions (BS) and, in 71 cases, protected specimen brushing (PSB). In 42/143 immunocompromised patients (haematological malignancies, n = 92; AIDS, n = 22; immunosuppressant therapy, n = 29) and in 4/35 patients with no defined underlying disease fungal pneumonia was present (candidosis, n = 35; aspergillosis, n = 8 mixed fungal infection, n = 3). Candidosis was combined, in 17 cases, with Aspergillus (n = 3), bacterial (n = 15) or cytomegalovirus (n = 2) infection. Aspergillosis was combined in eight cases with infection with Candida (n = 3), Pneumocystis carinii (n = 1) or bacteria (n = 5). The sensitivity of BAL and PSB in Candida pneumonia was 48% and 50%, respectively; specificity was 75% and 74% respectively. Bronchial secretions were more sensitive in detecting Candida pneumonia, but specificity was only 55%. In aspergillosis, the specificity of BAL, BS and PSB in each case was 100%; the sensitivity of BAL, BS and PSB was 38%, 64% and 100%. Twenty-four fungal infections were fatal. Unfavourable prognostic factors were respiratory failure needing mechanical ventilation, diffuse bilateral pulmonary infiltrates, mixed fungal infections and start of i.v. antifungal treatment > 14 days after fever onset, which were associated with a mortality rate of 74%, 67%, 67% and 63% respectively.