The susceptibility/resistance profile of bifonazole (BFZ) in 170 dermatophyte strains including azole parallel-resistance in 324 clinical yeast isolates was determined, additionally with impact on patient-relevant factors. Overall susceptibility to four azoles tested in parallel was 70%, with differences to both, the azoles, and species-specific for isolates from patients with superficial or invasive/systemic infections. 86% of the C. glabrata (n=166) isolates were susceptible to bifonazole, 76% were BFZ-susceptible to fluconazole-resistant C. glabrata (n=184) isolates, whereas 45% of the bifonazole-resistant strains (n=82) were susceptible to FLC. However, compared to voriconazole most of the other non-C. albicans Candida, and non-Candida species were less susceptible (< 50%) to bifonazole. As the other azoles tested, BFZ showed bimodular MIC-distribution. Susceptibility pattern analysis (SPA) demonstrated that isolates from antifungal agent pretreated patients had zero to significant less complete susceptible isolates (SP: SSSS) compared to non-treated patients. Furthermore, SPA revealed zero to fourfold parallel-resistance, species-specifically distributed, most prominently in C. glabrata and C. parapsilosis. Evaluation of azole susceptibility and two-way hierarchical clustering revealed a high grade of diversity and heterogeneity among the clinical C. glabrata isolates. A modified MIC assessment system was introduced to achieve a more realistic, well-arranged, and therapy oriented reporting of MIC in vitro data.

377 clinical isolates and seven dermatophyte culture collection strains were tested in vitro by microdilution against a panel of 18 antifungal agents. A newly introduced method for rapid and reliable inoculum preparation for moulds using the fragmented mycelia of freshly grown isolates as inoculum was firstly applied to dermatophytes. The performance of the method was tested with two different culture media which are recommended by different standardized testing methods for moulds. The standardized fragmented mycelia provided both, countable single colonies (viable units) on solid culture media and distinct readable endpoints (minimum inhibitory concentrations) in the microdilution wells for all tested topical and systemic drugs, including the echinocandins. As with moulds, and as shown by the culture collection strains, reproducible minimum inhibitory concentrations were obtained with an essential agreement (± 1 log2-dilution) of 97% to 100%, by a significant reduction of the overall testing time. As exemplarily shown for the microdilution technique, this inoculum method should also provide a solid basis for improved agar-based susceptibility testing methods such as disc-, tablet-, or strip-tests.

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 log2 higher MIC values and more than 90 % of the MICs read at 24 and 48 h were within ±2 log2 dilution. MIC end point reading (log2 MIC-RPMI 1640−log2 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.

Fluconazole is an azole antifungal agent active against Candida spp. and Cryptococcus spp. It can be administered orally or intravenously. It has been used for treating Candida infections, and is effective in treating infections caused by strains of Candida albicans, Candida tropicalis and Candida parapsilosis without acquired resistance mechanisms. The drug is ineffective for treating infections caused by Candida krusei, which is naturally resistant. The response of infections caused by Candida glabrata is variable, as the wild-type MIC distribution straddles most reasonable MIC breakpoints. Every attempt should be made to identify Candida isolates to the species level before or in conjunction with antimicrobial susceptibility testing. The EUCAST-AFST (European Committee on Antimicrobial Susceptibility Testing—Subcommittee on Antifungal Susceptibility Testing) has determined breakpoints of fluconazole for Candida spp. This Technical Note is based on the EUCAST fluconazole rationale document (available on the EUCAST website: http://www. eucast.org). The rationale document includes more detail and published references related to the selection of EUCAST-AFST breakpoints. D O S A G E

Fluconazole is an azole antifungal agent active against Candida spp. and Cryptococcus spp. It can be administered orally or intravenously. It has been used for treating Candida infections, and is effective in treating infections caused by strains of Candida albicans, Candida tropicalis and Candida parapsilosis without acquired resistance mechanisms. The drug is ineffective for treating infections caused by Candida krusei, which is naturally resistant. The response of infections caused by Candida glabrata is variable, as the wild-type MIC distribution straddles most reasonable MIC breakpoints.

Yeasts of the genus Malassezia are part of the normal flora of human skin. Under appropriate conditions they may cause skin infections such as pityriasis versicolor, Malassezia folliculitis, se-borrhoeic dermatitis, and dandruff. It has been reported that Malassezia may be a trigger factor for atopic dermatitis. The in vitro antifungal susceptibility testing of Malassezia still remains a problem. There is no recommended method of antifungal susceptibility testing designed for lipophilic genus Malassezia. 81 strains of Malassezia were examined for their in vitro susceptibility to antifungal substances (17 were reference or pre-identified strains, 64 were obtained from clinical specimens). Identification of the clinical strains based on biochemical features and Fourier-Transform-Infrared-Spectroscopy. All strains were maintained 7 days on modified Dixon agar. In vitro susceptibility testing was performed on microtitration plates with laid out of 6 dehydrated antifungals (ketoconazole, voriconazole, itraconazole, fluconazole, amphotericin B, and 5-flucytosin) in 12 different concentrations. The yeast cell inoculum was adjusted to 5" 103 cfu (colony forming unit) suspended in modified Dixon without peptone. All Malassezia strains were very susceptible to the azole drugs. Especially, voriconazole (MIC - minimum inhibitory concentration -0.007 - 1 mg/ml), itraconazole (MIC 0.007 - 0.05 mg/ml), and ketoconazole (MIC 0.03 - 0.5 mg/ml) were the most effective agents. MIC values among Malassezia sympodialis, Malassezia globosa and Malassezia obtusa were comparable, whereas MIC values for M. furfur were significantly higher (p < 0.05). All values ranged among those accepted according the NCCLS standards. Amphotericin B was also effective against Malassezia in vitro (MIC 0.03125 - 8 mg/ml). 5-Flucytosin was unlikely to inhibit the growth of Malassezia. In some cases it could prevent the growth, however high concentrations were needed. There were variations in susceptibility of different Malassezia species to azole antifungal substances. Malassezia furfur was found to be the less susceptible species when compared to Malassezia obtusa, Malassezia globosa and Malassezia sympodialis.

Species Specific Susceptibility of Malassezia against Antifungal Agents in vitro

BACKGROUND:83 Malassezia strains (65 wild isolates and 18 reference strains) were differentiated to the species level using conventional methods including morphological and biochemical features. These strains were further analyzed by Fourier transform infrared spectroscopy (FT-IRS).RESULTS:FT-IRS analysis allowed a clear separation of Malassezia strains according to species-specific cluster formation. The main differences were found between Malassezia furfur and other Malassezia species. In addition, within the species Malassezia furfur, a separation in two similar groups could be demonstrated. A disadvantage of FT-IRS is the relatively expensive apparatus. A great advantage is the speed and simplicity of the procedure, producing results within minutes.CONCLUSION:In pityriasis versicolor, Malassezia globosa was the dominant species found in 62% of cases. In addition, Malassezia furfur was found in 60% of dandruff cases.

Detection of antigen factors of Cryptococcus with factor sera in slide agglutination confirms diagnosis of species and varieties of Cryptococcus neoformans (Cr. n). This method is important in investigations of sources of infections. Serotype D strains of Cr. neoformans were detected in pigeon breedings from Thuringia exclusively. Because of that an essential difference exists in comparison to human isolates in Germany and strains from breeding stocks of companion birds in Thuringia where serotype A strains are predominant in pet birds and in human infections. Using different primers in PCR fingerprinting Cr. neoformans isolates can be assigned to serotypes A, B, C and D and to varieties Cr. neoformans neoformans and Cr. neoformans gattii (primer FM 1). On the other hand, genetic heterogeneity of Cr. neoformans strains is detectable within the serotypes A and D (primer 60-26). This genetic heterogeneity can be demonstrated in investigations by Fourier Transform Infrared (FTIR) spectroscopy, too. Isolated Cr. neoformans strains from pigeons (serotype D) could be divided into 3 and from pet birds (serotype A) into 2 different clusters by FTIR spectroscopy. It is important to take into account heterogeneity of strains within serotypes for determination of infection chains of human disease.

MycosesVolume 45, Issue S2 p. 16-16 INFLUENCE OF CULTURE MEDIA ON THE SUSCEPTIBILITY OF CANDIDA SPECIES TO VORICONAZOLE W. Fegeler, W. Fegeler Institute of Med. Microbiology, University of Muenster, GermanySearch for more papers by this authorK. Becker, K. Becker Institute of Med. Microbiology, University of Muenster, GermanySearch for more papers by this authorA. Schmalreck, A. Schmalreck MBS, Munic, GermanySearch for more papers by this authorWorking Group, Working Group Members of the Working Group “Clinical Mycology” of German Speaking Mycological Society (DMykG)Search for more papers by this author W. Fegeler, W. Fegeler Institute of Med. Microbiology, University of Muenster, GermanySearch for more papers by this authorK. Becker, K. Becker Institute of Med. Microbiology, University of Muenster, GermanySearch for more papers by this authorA. Schmalreck, A. Schmalreck MBS, Munic, GermanySearch for more papers by this authorWorking Group, Working Group Members of the Working Group “Clinical Mycology” of German Speaking Mycological Society (DMykG)Search for more papers by this author First published: 14 April 2011 https://doi.org/10.1111/j.1439-0507.2002.tb04594.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume45, IssueS2August 2002Pages 16-16 RelatedInformation

. 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.

Detection of antigen factors of Cryptococcus with factor sera in slide agglutination confirms diagnosis of species and varieties of Cryptococcus neoformans (Cr. n). This method is important in investigations of sources of infections. Serotype D strains of Cr neoformans were detected in pigeon breedings from Thuringia exclusively. Because of that an essential difference exists in comparison to human isolates in Germany and strains from breeding stocks of companion birds in Thuringia where serotype A strains are predominant in pet birds and in human infections.Using different primers in PCR fingerprinting Cr neoformans isolates can be assigned to serotypes A, B, C and D and to varieties Cr neoformans neoformans and Cr neoformans gathi (primer FM 1). On the other hand, genetic heterogeneity of Cr neoformans strains is detectable within the serotypes A and D (primer 60-26). This genetic heterogeneity can be demonstrated in investigations by Fourier Transform Infrared (FTIR) spectroscopy, too. Isolated Cr neoformans strains from pigeons (serotype D) could be divided into 3 and from pet birds (serotype A) into 2 different clusters by FTIR spectroscopy. It is important to take into account heterogeneity of strains within serotypes for determination of infection chains of human disease.

The ubiquitous encapsulated yeast Cryptococcus neoformans causes serious, life-threatening infections of the central nervous system in AIDS patients [1]. Fluconazole is the drug of choice for long-term suppressive therapy because it is effective and can be given by mouth. Anecdotal reports suggest that the use of fluconazole for long-term maintenance therapy in AIDS patients may be associated with the selection of less susceptible cryptococcal isolates to fluconazole. However, the impact of the development of resistance on the clinical management of recurrent cryptococcosis has not been particularly addressed. This case report relates the clinical course and treatment response in a patient with AIDS, who relapsed four times with severe cryptococcal meningitis during anti-fungal maintenance therapy, to in-vitro anti-fungal drug resistance profiles of serial C. neoformans isolates. In May 1998, a 33-year-old man with an 11 year history of HIV infection [CD4 cell count 9 cells/μl, 1.6%; HIV-RNA (nucleic acid sequence-based amplification) 20 000 copies/ml] presented with fever, non-productive cough, and clinical signs of meningitis. Chest X-ray showed diffuse bilateral reticular infiltration, whereas cranial computerized tomography was unremarkable. C. neoformans was found in cytospins obtained from both bronchoalveolar lavage and cerebrospinal fluid (CSF). Cryptococci grown from both sources were highly sensitive to fluconazole [minimum inhibitory concentration (MIC) < 8 μg/ml], intraconoazole (MIC < 0.5 μg/ml), flucytosine (MIC < 2 μg/ml) and amphotericin B (MIC < 2 μg/ml) when tested by broth microdilution technique in vitro. After intravenous triple therapy with amphotericin B (0.7 mg/kg a day), flucytosine (150 mg/kg a day) and fluconazole (400 mg twice a day) for 6 weeks cryptococcus meningitis and pneumonia resolved; maintenance therapy with fluconazole (200 mg twice a day) was started, together with antiretroviral therapy. After two meningitis relapses despite ongoing fluconazole maintenance therapy in March and August 1999, both again responsive to intravenous triple agent anti-fungal therapy, maintenance therapy was intensified to 400 mg fluconazole twice a day. This did not prevent a further cryptococcal meningitis relapse in November 1999, complicated by ocular involvement with chorioretinitis. At this time Cryptococci grown from the CSF were found to be resistant to fluconazole (MIC 64 μg/ml), itraconazole (MIC 0.5 μg/ml) and flucytosine (8 μg/ml) in microdilution assays, but remained sensitive to amphotericin B (MIC 0.125 μg/ml) and voriconazole (MIC 0.5 μg/ml). After treatment with amphotericin B (0.7 mg/kg a day) for 6 weeks, supplemented by prednisolone (40 mg a day) to control severe ocular disease, meningitis and chorioretinitis resolved. Oral maintenance therapy with voriconazole (200 mg twice a day after a 400 mg twice a day loading dose) in an approval study was initiated. However, this maintenance regimen did not prevent a fourth cryptococcal menigitis relapse in March 2000. Surprisingly, C. neoformans grown from the CSF during that relapse was still completely sensitive to voriconazole in vitro (MIC 1.0 μg/ml). After daily intravenous amphotericin B treatment for 6 weeks, an intravenous maintenance regimen with 50 mg amphotericin B three times a week was started, which effectively prevented further cryptococcal meningitis episodes. Antiretroviral therapy achieved only partial suppression of viral replication within the first 12 months. After modification in March 1999, plasma HIV levels were consistently below a detection limit of 50 copies/ml. In parallel, there was a continuous increase of CD4 cells to 197/mm3 in April 2001. The presented patient with severe disseminated cryptococcosis showed an excellent clinical response to established triple antifungal therapy with amphotericin B, flucytosine and fluconazole, but repeatedly relapsed despite fluconazole maintenance therapy initially dosed as recommended [2,3] and subsequently intensified. At the time of relapse, the patient showed no clinical evidence of non-compliance, maldigestion or malabsorption; no drugs potentially affecting triazole pharmacokinetics were taken. The occurrence of C. neoformans strains less susceptible to fluconazole after primary and secondary azole antifungal prophylaxis has been reported previously [4,5]. In line with the present observation, this report may well explain the relapses of cryptococcal disease. Interestingly, the loss of azole susceptibility of C. neoformans, which developed during fluconazole maintenance therapy, apparently affects various azole compounds differentially, as susceptibility to voriconazole in vitro was consistently found even after clinical failure. Therefore, the relapse observed during voriconazole maintenance therapy was probably caused by insufficient bioavailability of this particular compound within the CSF with the present dosing regimen. In summary, this report demonstrates that: (i) C. neoformans resistance to triazoles (fluconazole, itraconazole) may emerge during fluconazole maintenance therapy; (ii) fluconazole/itraconazole-resistant strains remain sensitive to voriconazole in vitro; (iii) despite its preserved in-vitro activity, voriconazole may not be potent enough to prevent cryptococcal meningitis relapse with the standard dose used; and (iv) after the development of azole resistance, intravenous amphotericin B maintenance therapy remains an option for the long-term control of cryptococcal meningitis in AIDS patients. Georg Friesea Thomas Dischera Roswitha Füssleb Arno Schmalreckc Jürgen Lohmeyera