Parallel and cross-resistances of clinical yeast isolates determined by susceptibility pattern analysis.

For calculated initial antifungal therapy, knowledge on parallel and cross-resistances are vitally important particularly in the case of multiresistant isolates. Based on a strain collection of 1,062 yeast isolates from a German/Austrian multicentre study, susceptibility pattern analysis (SPA) was used to determine the proportion of parallel and cross-resistances to eight antifungal agents (AFAs) encompassing flucytosine, amphotericin B, azoles (fluconazole, voriconazole and posaconazole) and echinocandins (caspofungin, micafungin and anidulafungin). A total of 414 (39.0%) isolates were resistant for one or more of the AFAs. Resistance to one AFA was shown for 18.1% of all isolates. For 222 isolates (20.9%), resistance to two to seven AFAs was noted (7.7%; 7.7%; 3.6%; 1.0%; 0.7% and 0.2% to 2, 3, 4, 5, 6 and 7 antifungal compounds, respectively). Partial parallel resistances within the azole and echinocandin classes, respectively, were found for 81 (7.6%) and 70 (6.6%) isolates. Complete parallel resistances for azoles, echinocandins and combined for both classes were exhibited by 93 (8.8%), 18 (1.7%) and 6 (0.6%) isolates, respectively. Isolates displaying cross-resistances between azoles and echinocandins were infrequently found. Highly resistant isolates (resistance to ≥6 AFAs) were almost exclusively represented by Candida albicans. Highly standardized testing of AFAs in parallel and from the same inocula followed by SPA allows detailed insights in the prevalence and distribution of susceptibility patterns of microbial isolates.

Detection of azole susceptibility patterns in clinical yeast strains isolated from 1998 to 2008.

4,860 clinical yeast isolates (25 genera, 47 species) were tested in parallel to fluconazole, itraconazole, ketoconazole, and voriconazole. After re-evaluation of all species according to their current valid taxonomic denominations, the range of the top four of the dermatology, gynaecology and paediatrics associated species from superficial infections was similar to those isolated from other wards with mainly systemic/invasive infections. Candida albicans (44.7%) was the most frequent pathogen followed by C. glabrata, C. tropicalis, and C. parapsilosis. The MIC-assessment revealed for the ten-year test period an overall azole-susceptibility of about 75%, and ~80% for their associated ICUs. The overall susceptibility of the isolates from systemic and superficial infections to the four azoles was 79% and 80% respectively, and demonstrates a high in vitro activity. When two test periods (1998-2001 and 2002-2008) were compared by characteristic MIC values and multi-azole resistance, no significant increase could be detected in azole susceptibility/resistance over the two periods, respectively, over the total investigation period of ten years. This holds true when the characteristic MIC values were compared with those from different azole susceptibility studies from similar time periods and from different investigators around the world (1991 to 2010). With a new method, susceptibility pattern analysis for fungi, detailed information of multi-resistant microorganism populations could be obtained, and different characteristic resistance patterns in clinical yeast species detected. Although at a relatively low level, multi-resistance was seen in individual species populations demonstrating resistance to two (6.7%), three (4.4%), or all four (4%) azoles tested. A level of 4% and 2% fourfold parallel resistance was also determined in Candia spp. and non-Candida spp. derived of blood culture isolates.

Role for the fibrinogen-binding proteins coagulase and Efb in the Staphylococcus aureus-Candida interaction.

Staphylococcus aureus and Candida species are increasingly coisolated from implant-associated polymicrobial infections creating an incremental health care problem. Synergistic effects between both genera seem to facilitate the formation of mixed S. aureus-Candida biofilms, which is thought to play a critical role in coinfections with these microorganisms. To identify and characterize S. aureus factors involved in the interaction with Candida species, we affinity-panned an S. aureus phage display library against Candida biofilms in the presence or absence of fibrinogen. Repeatedly isolated clones contained DNA fragments encoding portions of the S. aureus fibrinogen-binding proteins coagulase or Efb. The coagulase binds to prothrombin in a 1:1 ratio thereby inducing a conformational change and non-proteolytic activation of prothrombin, which in turn cleaves fibrinogen to fibrin. Efb has been known to inhibit opsonization. To study the role of coagulase and Efb in the S. aureus-Candida cross-kingdom interaction, we performed flow-cytometric phagocytosis assays. Preincubation with coagulase reduced the phagocytosis of Candida yeasts by granulocytes significantly and dose-dependently. By using confocal laser scanning microscopy, we demonstrated that the coagulase mediated the formation of fibrin surrounding the candidal cells. Furthermore, the addition of Efb significantly protected the yeasts against phagocytosis by granulocytes in a dose-dependent and saturable fashion. In conclusion, the inhibition of phagocytosis of Candida cells by coagulase and Efb via two distinct mechanisms suggests that S. aureus might be beneficial for Candida to persist as it helps Candida to circumvent the host immune system.

Epidemiology and changes in patient-related factors from 1997 to 2009 in clinical yeast isolates related to dermatology, gynaecology, and paediatrics.

From 1997 to 2009, 1,862 dermatology, gynaecology, and paediatrics (DGP) associated clinical yeast isolates were analysed for species occurrence, specimen origin and type, (multi-) resistance pattern, and testing period. The top seven of the isolated DGP-associated species remained the same as compared to total medical wards, with Candida albicans (45%) as most frequent pathogen. However, the DGP wards and DGP ICUs showed species-specific profiles; that is, the species distribution is clinic-specific similar and however differs in their percentage from ward to ward. By applying the "one fungus one name" principle, respectively, the appropriate current taxonomic species denominations, it has been shown that no trend to emerging species from 1998 to 2008 could be detected. In particular the frequently isolated non-Candida albicans species isolated in the DGP departments have already been detected in or before 1997. As yeasts are part of the cutaneous microbiota and play an important role as opportunistic pathogens for superficial infections, proper identification of the isolates according to the new nomenclature deems to be essential for specific and calculated antifungal therapy for yeast-like DGP-related infectious agents.

Susceptibility screening of hyphae-forming fungi with a new, easy, and fast inoculum preparation method.

In vitro susceptibility testing of clinically important fungi becomes more and more essential due to the rising number of fungal infections in patients with impaired immune system. Existing standardized microbroth dilution methods for in vitro testing of molds (CLSI, EUCAST) are not intended for routine testing. These methods are very time-consuming and dependent on sporulating of hyphomycetes. In this multicentre study, a new (independent of sporulation) inoculum preparation method (containing a mixture of vegetative cells, hyphae, and conidia) was evaluated. Minimal inhibitory concentrations (MIC) of amphotericin B, posaconazole, and voriconazole of 180 molds were determined with two different culture media (YST and RPMI 1640) according to the DIN (Deutsches Institut für Normung) microdilution assay. 24 and 48 h MIC of quality control strains, tested per each test run, prepared with the new inoculum method were in the range of DIN. YST and RPMI 1640 media showed similar MIC distributions for all molds tested. MIC readings at 48 versus 24 h yield 1 log(2) higher MIC values and more than 90 % of the MICs read at 24 and 48 h were within ± 2 log(2) dilution. MIC end point reading (log(2 MIC-RPMI 1640)-log(2 MIC-YST)) of both media demonstrated a tendency to slightly lower MICs with RPMI 1640 medium. This study reports the results of a new, time-saving, and easy-to-perform method for inoculum preparation for routine susceptibility testing that can be applied for all types of spore-/non-spore and hyphae-forming fungi.

Intracerebral mass lesion diagnosed as cryptococcoma in a patient with sarcoidosis, a rare opportunistic manifestation induced by immunosuppression with corticosteroids.

Cryptococcal infection of the central nervous system (CNS) typically affects patients with HIV infection. In addition, opportunistic infections can also occur during immunosuppressive therapies. Some patients develop cryptococcal meningitis. Cryptococcomas, however, are rarely observed. A 42-year-old patient with sarcoidosis known for 2½ years presented with a cerebral mass lesion primarily thought to be CNS sarcoidosis. Stereotactic biopsy and extensive micro- and macrobiological investigations revealed a cryptococcoma which had emerged from cryptococcal meningitis despite 3 months of treatment. Differential diagnosis of cerebral cryptococcoma is difficult due to the unspecific findings in the CSF analysis if C. neoformans fails to be detected using Indian ink staining or PCR studies. In this case, stereotactic biopsy and pathohistological examination revealed C. neoformans causing intracerebral mass lesion. Intensive treatment with antifungal drugs was followed by remission of all symptoms. In conclusion, cryptococcoma of the CNS represents a very important indication of mass lesions in patients suffering from sarcoidosis and treated with corticosteroids.

Candida albicans and its metabolite gliotoxin inhibit platelet function via interaction with thiols.

Platelets bind to Candida albicans, the major cause of candidiasis. But in contrast to other microorganisms the fungus does not aggregate platelets. Gliotoxin (GT), which possesses immunosuppressive properties, is produced by various fungi, including the opportunistic pathogens Aspergillus fumigatus and C. albicans . Its mode of action involves the formation of mixed disulfides with host proteins. Disulfide exchanges play an important role in platelet activation. Therefore, the effect of C. albicans and GT on platelet function was tested. C. albicans yeast cells (5,000-10,000 cells/microl) and GT, in pathophysiologically relevant concentrations (0.05-0.5 microM), inhibited platelet fibrinogen binding, anti gp IIb/IIIa antibody PAC-1 binding, aggregation and procoagulant activity in a dose-dependent manner. Alpha granule release, measured via CD62P surface expression, was not affected. Addition of reduced glutathione partially counteracted the effect of C. albicans and GT on platelet fibrinogen binding and platelet aggregation. The C. albicans metabolite GT features antithrombotic properties in addition to its immunosuppressive functions. Since treatment with reduced glutathione partially counteracted the inhibitory effect of C. albicans yeast cells and GT on platelet fibrinogen binding, the antithrombotic activity is likely to depend on the disulfide bridge of this mycotoxin. GT production by C. albicans could contribute to its survival in the blood stream during vascular infections. The knowledge of the underlying mechanisms of the antithrombotic properties might help to treat fungal infections as well as thrombosis.

Safety, tolerance and outcome of treatment with liposomal amphotericin B in paediatric patients with cancer or undergoing haematopoietic stem cell transplantation.

OBJECTIVES: To assess safety, tolerance and efficacy of liposomal amphotericin B (LAMB) in a large unselected series of paediatric cancer/haematopoietic stem cell transplantation (HSCT) patients requiring LAMB therapy. PATIENTS AND METHODS: The study included 84 children and adolescents (median age: 11 years) who received 141 consecutive courses of LAMB for prophylaxis (32), empirical therapy (83), possible (19) or probable/proven (7) invasive infections. LAMB was administered until intolerance or maximum efficacy at dosages individually determined by the responsible physician. RESULTS: Fifty-nine courses were post-HSCT (42%, 49 allogeneic), and 92 courses were started during granulocytopenia (65%). The median duration of LAMB therapy was 13 days (range 1-79), and the median maximum dosage was 2.8 mg/kg (range 0.93-5.10). Mild-to-moderate adverse events were noted during 109 courses (77%; hepatic, 58.8%; electrolyte wasting, 52.5%; renal, 31.9%; infusion-related reactions, 8.5%); adverse events necessitating discontinuation of LAMB occurred in 6 courses (4.3%; renal, 3; anaphylaxis, 2; hepatic, 1). While median hepatic transaminase, alkaline phosphatase and blood urea nitrogen values were slightly (P < 0.01) higher at end of treatment (EOT), bilirubin and creatinine values were not different from baseline. Complete or partial responses were observed in 16/19 and 2/7 courses for possible and probable/proven invasive infections. Thirty-two of 33 courses of prophylaxis and 74 of 83 courses of empirical therapy were completed with success. Overall survival was 90.8% at 3 months post-EOT. CONCLUSIONS: LAMB had acceptable safety and tolerance and was useful in prevention and treatment in unselected, mostly granulocytopenic paediatric patients undergoing treatment for cancer or HSCT.

Prevalence, associations, and trends of biliary-tract candidiasis: a prospective observational study.

BACKGROUND: Biliary obstruction and cholangitis are common problems in gastroenterology. Infections of the biliary tract with Candida and other fungal species have increasingly been seen in the last few years. OBJECTIVE: To investigate the prevalence, associations, and trends of biliary-tract candidiasis. DESIGN: A prospective, observational, diagnostic study. SETTING: University Hospital, Muenster, Germany. PATIENTS: Consecutive patients undergoing ERCP for various indications. RESULTS: In 54 of 123 patients, we found Candida species in bile samples (44%). In only 7 patients, candidiasis was suspected on endoscopy before mycologic proof. Only 4 of these 7 patients were correctly diagnosed with biliary candidiasis by simple morphologic aspects. The fungus was mainly differentiated as Candida albicans or Candida glabrata and rarely as Candida parapsilosis, Candida tropicalis, or other subspecies. Immunosuppression for various reasons was significantly associated with bile-duct candidiasis (P < .02). No significant association was found between positive fungal cultures and prior endoscopic sphincterotomy (P = .0824) or prior ERCP (P = .1152). Biliary candidiasis was neither associated with positive fungal cultures of buccal smears (P = .0722) nor with positive findings in stool samples (P = .0860). LIMITATIONS: Highly selected patient population. Buccal smears and stool samples were not obtained from all patients. Contamination artifacts cannot totally be excluded with the ERCP procedure. CONCLUSIONS: Candida species very frequently can be detected in the bile. Positive fungal cultures of bile samples are not just contamination artifacts. This has to be taken into account when designing an anti-infectious treatment for recurrent cholangitis or even more cholangiosepsis. Especially in immunosuppressed patients or recipients of long-term antibiotic therapy, physicians should screen for biliary-tract candidiasis during endoscopic examination.

Systematic survey of nonspecific agglutination by Candida spp. in latex assays.

In order to demonstrate that cells of Candida spp. may show considerable nonspecific agglutination in latex agglutination tests, 150 clinical and reference isolates of 12 yeast species were systematically studied by applying various test parameters. In fact, 40 (26.7%) of these isolates revealed nonspecific results, significantly associated with the time allowed for agglutination.

Biliary tract candidiasis: diagnostic and therapeutic approaches in a case series.

BACKGROUND: Biliary obstruction with its wide range of potential causes is a common disorder in gastroenterology. Infections of the biliary tract with Candida and other fungal species leading to obstructive jaundice have increasingly been recognized in the last few years. Besides a few case reports, there are few data in the literature giving us an idea how to diagnose and treat these patients. METHODS: We report on a series of seven patients suffering from biliary tract candidiasis who were diagnosed and treated at our institution. Predisposition factors, reliability of various diagnostic modalities, and treatment options based on our own experience are presented and discussed. RESULTS: Besides the general diagnostic modalities such as laboratory findings or ultrasonography, we often observed mycelia in the bile duct system endoscopically. Typical morphological changes in peripheral bile ducts could be detected during endoscopic retrograde cholangiopancreatography (ERCP). Aspiration of bile and subsequent microbiological analysis in combination with ERCP findings revealed diagnosis of bile duct candidiasis in all cases. Treatment included both antiinfectious drugs and endoscopic therapy such as bile duct drainage, lavage, or débridement. With respect to fungal eradication, therapy was successful in 71% of cases as proven by microbiological analysis of bile aspirates. Since many of these patients suffer not only from biliary mycosis but also from disease necessitating immunosuppression, the prognosis was poor in some cases. CONCLUSION: Biliary tract candidiasis because of immunosuppression is an increasingly recognized disease and remains a major clinical challenge. Besides laboratory analysis and ultrasonography, diagnostic modalities should include aspiration of bile during ERCP and microbiological analysis. Antiinfectious drug treatment as the main therapeutic column for biliary candidiasis should be complemented by endoscopic intervention.

Fourier-transform infrared spectroscopic analysis is a powerful tool for studying the dynamic changes in Staphylococcus aureus small-colony variants.

Infections due to small-colony variants (SCVs) of Staphylococcus aureus in patients with chronic and recurrent infections are an emerging problem; however, studies with this subpopulation are hampered by the fact that SCVs may exhibit unstable phenotypes, making them difficult to study, particularly in broth media. In this study, two S. aureus sets comprising the (i) normal and the (ii) SCV phenotype (clonal with normal phenotype) recovered from clinical specimens, as well as (iii) corresponding site-directed mutants displaying the SCV phenotype (knockout of hemB) and (iv) their complemented mutants were examined by Fourier-transform infrared (FTIR) spectroscopy. Phenotypes were defined on solid and in broth media. Using first-derivative infrared spectra to calculate spectral distances, hierarchical clustering based on spectral information resulted in a dendrogram with clear discrimination between SCV and normal phenotypes. The SCVs gave an FTIR fingerprint that was easily recognizable and that was much closer to other SCVs than to their parent strains. This technique offers for the first time a noninvasive approach to investigate dynamic processes of reversion of SCVs to the normal phenotype and vice versa. Thus, FTIR spectroscopy allowed a rapid and reproducible tool for the examination of different subpopulations of S. aureus on solid and in broth media for diagnostic and research purposes.

Bacteria and granulation tissue associated with Montgomery T-tubes.

OBJECTIVES: Although complications (infection, development of granulation tissue) of silicone Montgomery T-tubes have been reported, the microbiological consequences and the origin of granulation tissue have not yet been evaluated. STUDY DESIGN: A prospective trial. METHODS: Twenty-three Montgomery T-tubes from 10 patients were analyzed with regard to the development of granulation tissue, bacterial growth (including genotyping with polymerase chain reaction), and results of sensitivity testing. Furthermore, stent sterilization (n = 6) was investigated. RESULTS: Granulation tissue occurred with 74% of the stents, and all specimens showed signs of infection but no foreign body reaction. The predominant organisms were Staphylococcus aureus (35%) and Pseudomonas aeruginosa (17%). The differences between groups with and without granulation tissue were significant for P aeruginosa. Polymerase chain reaction fingerprinting of the S aureus obtained from 15 stents (n = 3 patients) revealed a total of seven different genotypes. Whereas two of these patients harbored six different genotypes of S aureus, the third patient was persistently colonized by S aureus over a 15-month period with the identical genotype. Susceptibility testing showed most commonly (65%) sensitivity to a combination of amoxicillin-clavulanate and ofloxacin. After sterilization, 92% of analyzed stent segments showed no bacterial growth. CONCLUSIONS: Granulation tissue commonly occurred next to the silicone (subglottic area, stoma) where S aureus and P aeruginosa were commonly isolated. A combination of mechanical irritation and bacterial infection seems to account for the development of granulation tissue. Polymerase chain reaction fingerprinting showed both prolonged persistence and a change of colonizing strains after multiple stent replacements. A combination of amoxicillin-clavulanate and ofloxacin is the most effective antibiotic therapy. Sterilization of the cost-intensive silicone stents is feasible, and reuse in the same patient is justifiable from economic aspects.