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.

[Species differentiation of yeasts of the genus Malassezia with Fourier transform infrared spectroscopy]. / Speziesdifferenzierung von Hefen der Gattung Malassezia mittels Fourier-Transform-Infrarot-Spektroskopie.

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.

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.

[IWhich are the conditions for Cryptococcus neoformans var. neoformans-strains from avian excrements as a cause for human infections?]. / Unter welchen Voraussetzungen sind Cryptococcus neoformans var. neoformans-Stämme aus Vogelexkrementen als Ursache menschlicher Infektionen anzusehen?

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.

Candida africana sp. nov., a new human pathogen or a variant of Candida albicans?

Atypical Candida strains were isolated from patients in Madagascar, Angola and Germany. These isolates were slow growing and were unable to produce chlamydospores. They had atypical carbohydrate assimilation profiles. All strains were unable to assimilate the amino sugars N-acteylglucosamine and glucosamine as well as the disaccharide trehalose and the organic acid DL-lactate. They were germ-tube-positive in serum, but only some of these organisms produced pseudohyphae after a long incubation. As shown by Fourier transform infrared spectroscopy the atypical Candida isolates clustered as a monophyletic group different from C. albicans and C. dubliniensis. All strains belonged to C. albicans serotype B. Considering all data presented here, this group of Candida strains differs from any other known member of the genus Candida. Therefore, it is suggested to represent a new species within the genus Candida for which the name Candida africana is proposed.

[Evidence of Cryptococcus neoformans in domestic and sports pigeons in Thyringia]. / Nachweis von Cryptococcus neoformans bei Haus- und Sporttauben in Thüringen.

19 strains of Cryptococcus neoformans var. neoformans were isolated from 17 (= 40.5%) of 42 investigated pigeon breeder flocks in Thuringia.

[Fourier transform infrared spectroscopy, molecular biologic methods and antimyocotic susceptibility patterns for identification and differentiation of cryptococcus species]. / Fourier-Transform-Infrarot-Spektroskopie, molekularbiologische Methoden und Antimykotika-Empfindlichkeitsmuster zur Identifizierung und Differenzierung von Cryptococcus-Spezies.

Molecular biological methods as well as the FTIR method allows the rapid, reliable and reproducible determination and identification of Cryptococcus species from human, veterinary and environmental origin and their serovars. The results obtained by FTIR could be verified by the molecular methods. In addition, with the PCR and FTIR fingerprinting methods it is possible to distinctly group the serovars and differentiate the different Cryptococcus strains.

Occurrence of Cryptococcus spp. in excreta of pigeons and pet birds.

In pooled samples of faeces from 25 pet bird flocks in Thuringia, a high rate of contamination with Cryptococcus neoformans var. neoformans was found. The prevalence of Cr. neoformans in the bird-breeding establishments correlated with the numbers of the different pet bird species in these flocks. The differentiation between varieties of Cr. neoformans by means of proline assimilation and canavanine resistance detection as well as with the aid of Cr. neoformans factor sera, polymerase chain reaction (PCR) fingerprinting, sequencing of PCR products as well as Fourier transform infrared spectroscopy showed uniform results which also corresponded to the serological differentiation between serovars A and D. A predominance of serovar A could be observed among the pet bird breeding flocks. This corresponded to the frequency distribution of serovars A and D in cases of human diseases in Germany. In 50% of the samples of pigeon excreta examined (n = 30) in Innsbruck (Austria), Cryptococcus albidus could be isolated but not Cr. neoformans. However, this Cryptococcus species is of minor pathogenetic importance for man. Cryptococcus albidus may be clearly distinguished from Cr. neoformans by means of microbiological methods, PCR and Fourier transform infrared spectroscopy.

[Antifungal susceptibility testing in chronically recurrent vaginal candidosis as basis for effective therapy]. / Resistenzbestimmungen bei Erregern chronisch rezidivierender Vaginalcandidosen als Voraussetzung für eine effektive Therapie.

The chronically recidivist vulvo-vaginal candidiasis is one of the most stubborn problematic diagnosis in the dermatology and gynaecology ward. Prognosis and therapy are primarily determined by the causative micro-organism and the interaction of the fungal species with the currently available antifungal agents. Objective of the study was the investigation of vaginal yeast isolates from patients with chronically recidivist vaginal candidiasis against 8 antifungal agents with the aim of optimising the standard therapy with azole antifungal agents and assessment of alternative therapy schemes. 55 clinical isolates (Dermatology, Charité) of 40 patients were tested by microdilution according to DIN 58940-84. Species differentiation and identification was performed by Fourier-Transform Infrared Spectroscopy (FTIR). In the result Candida glabrata was the predominant causative agent for the recidivist vaginal candidiasis. MIC-mode values for C. glabrata were: fluconacole 32 micrograms/ml, itraconacole 1 microgram/ml, ketoconacole 1 microgram/ml, amphotericine B, voriconacole 0.03 microgram/ml, amphotericin B 0.5 microgram/ml, terbinafine 128 micrograms/ml, cicloproxolamine 4 micrograms/ml, 5-fluorocytosine 0.03 microgram/ml. Some strains of Patients with suboptimal introductory low doses of fluconacole showed increasing of MIC in course of therapy. Parallel resistance with itraconacole was observed in all these cases. Consecutively isolated strains could be clearly and reliably identified by FTIR. In conclusion of most importance is the initial dose adapatation of the drug used, e.g. for fluconacole 800/d p.o., when C. glabrata is the causative agent. Low dose fluconacole therapy is always unsuccessful in recurrent vaginal candidiasis and induces secondary resistance. Demonstrated high susceptibility of voriconacole, amphotericine B an 5-fluorocytosine particularly for C. glabrata may indicate of an anitmycotic therapy potential unconsidered regarding to dermatological indication up to now.

Identification of dermatophytes by Fourier transform infrared spectroscopy (FT-IR).

Fourier transform infrared spectroscopy is an established method in the routine diagnosis of various micro-organisms, including bacteria and yeasts, on a species level. Its possible value in the diagnostics of dermatophytes was analysed using three clinical isolates each of the three most frequently found species, namely Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum canis. The results encourage further work to establish a library which would allow the use of this method in the clinical setting. This might help to make repeated subcultures, which are money- and time-consuming, redundant.

[Differentiation and characterization of yeasts pathogenic for humans (Candida albicans, Exophiala dermatitidis) and algae pathogenic for animals (Prototheca spp.) using Fourier transform infrared spectroscopy (FTIR) in comparison with conventional methods]. / Differenzierung und Charakterisierung von humanpathogenen Hefen (Candida albicans, Exophiala dermatitidis) und tierpathogenen Algen (Prototheca spp.) mittels Fourier-Transform-Infrarot-Spektroskopie (FT-IR) im Vergleich zu konventionellen Methoden.

Due to the Fourier-Transform Infrared Spectroscopy (FT-IR) of strain specific traits demonstrated to be a suitable and efficient method for diagnostic and epidemiological determinations for the yeasts Candida albicans, Exophiala dermatitidis and the chlorophylless algae of the genus Prototheca. FT-IR leads in a rapid and economical way to reproducible results according to the spectral differences of intact cells (IR-fingerprints). Different genera, species and sub-species respectively, different strains can be recognized and grouped into different clusters and subclusters. The FT-IR analysis of Candida albicans isolates (n = 150) of 22 newborns-at-risk of an intensive care unit showed, that 86% of the children were colonised with several (2-4) different strains in the oral cavities and faeces. Stationary cross-infections could definitely be determined. Exophiala dermatitidis isolates (n = 31), mostly isolated repetitively within a period of 3 years from sputa of patients suffering from cystic fibrosis could be characterized and grouped patient-specifically over the total sampling period. Of 6 from 8 patients (75%) their individual strains remain the same and could be tracked over the three years. Cross-infections during the stationary treatment could be clearly identified by FT-IR. The Prototheca isolate (n = 43) from live-stock and farm environment showed clear distinguishable clusters differentiating the species P. wickerhamii, P. zopfii and P. stagnora. In addition, the biotypes of P. zopfii could be distinguished, especially the subclusters of variants II and III. It could be demonstrated, that FT-IR is suitable for the routine identification and differentiation of yeasts and algae. However, in spite of the gain of knowledge by using FT-IR for the characterization of microorganisms, the conventional phenotyping and/or genetic analysis of yeast or algae strains cannot be replaced completely. For a final taxonomic classification a combination of conventional methods on FT-IR together with more sophisticated molecular genetic procedures is necessary.

Susceptibility testing of macrolide and lincosamide antibiotics according to DIN guidelines. Deutsches Institut für Normung.

The in-vitro activity of azithromycin, clarithromycin, erythromycin, josamycin, midekamycin, roxithromycin and clindamycin against 674 Gram-negative and Gram-positive clinical isolates, including methicillin-resistant Staphylococcus aureus, was determined by agar dilution, microdilution and agar diffusion with Mueller-Hinton medium according to the Deutsches Institut für Normung (DIN) 58940 guidelines. The results obtained by regression analysis and the error-rate-bounded method of Metzler-DeHaan indicate that common interpretative criteria (breakpoints) for test discs may be assigned to susceptible/resistant Gram-positive strains for all antibiotics tested. The following tentative DIN values are suggested for 15 microg macrolide discs: for susceptible Gram-positive and Gram-negative strains, an inhibition zone diameter (IZD) of > or = 26 mm at a corresponding MIC of < or = 2 mg/L; for resistant Gram-positive strains, an IZD of < or = 21 mm; for resistant Gram-negative strains, an IZD of < or = 19 mm at a corresponding MIC of > or = 8 mg/L. For Haemophilus influenzae only, breakpoints for azithromycin are suggested with IZDs of > or = 21 mm for susceptible and < or = 18 mm for resistant.

Fluconazole: comparison of pharmacokinetics, therapy and in vitro susceptibility.

Fluconazole shows good penetration into the tissues and body fluids examined and a rapid equilibrium is achieved between the concentrations in the various compartments. The pharmacokinetics of fluconazole after intravenous or oral administration are proportional to the dose. This finding, together with the slow elimination of the triazole (t1/2 30 h), makes it easier to forecast the therapeutically effective dosage. Measurements of fluconazole concentration in blood can be used to predict levels in some tissues (lung, brain, gynaecological samples), body fluids (sputum, saliva, vaginal secretions) or exudates. Concentrations in cerebrospinal fluid and vitreous humour of the eye reach approximately 80% of the levels found in blood. A very high proportion of fluconazole is excreted unchanged in the urine, where concentrations of the drug are 10-20-fold higher than in blood. Whilst this pharmacokinetic profile is valuable in the treatment of fungal infections of the urinary tract, it also means that the dosage may need to be decreased in patients with renal impairment. The susceptibility of fungi to fluconazole in vitro and in vivo correlates well with the concentrations of the drug measured in various compartments of the body.

[Prerequisites for effective therapy of chronic recurrent vaginal candidiasis]. / Voraussetzungen für eine effektive Therapie chronisch rezidivierender Vaginalcandidosen.

67 women with chronic recurrent or persistent vaginal candidosis between 5-79 years of age were seen in our outdoor department. In 34 cases, yeasts could be isolated in a vaginal swab taken at the first consultation. On average the patients reported 5 episodes per year during the last years. Typical symptoms consisted of pruritus vulvae, local inflammation and a curdy vaginal discharge. Nearly all of the women had received local or systemic antimycotic treatment for several times. In 53% (18 patients), C. albicans had been isolated, in 29% (10 patients) C. glabrata and in 9% (3 patients) C. krusei. While candidosis due to C. albicans and C. krusei was frequently associated with distressing complaints, infections with C. glabrata caused only very few symptoms. Independent of the species, severe and persistent infections were characterized by long term persisting specific IgM-antibody-titers and remarkable lack of IgG-antibodies. The laboratory parameters of WBC, CRP and immunelectrophoresis were normal. The minimum inhibitory concentrations (MIC) of 60 Candida strains against fluconacole were determined by microdilution assay. The MIC for C. albicans (n = 35) were between 0.78 and 3.125 micrograms/ml, for C. glabrata (n = 20) between 8 and 32 micrograms/ml and for C. krusei (n = 5) between 25 and 128 micrograms/ml. In 7 cases, local antimycotic treatment was sufficient. Correlating to the sensitivity, 18 women were treated with 100-800 mg fluconacole/d for 10-20 days. In 13 of them, clearance of symptoms and yeasts was achieved. The treatment of fluconacole-resistant strains with itraconazole (100-200 ml/d for 10-20 days) together with local application of nystatin (2 x 1 Mio. IE for 10 days) was without any effect. Three women with C. albicans, C. glabrata and C. krusei infection received a candidin-vaccination (0.005 BE/ml-500 BE/ml). In all of these cases, production of IgM-antibodies was induced. However, the clinical symptoms could not be influenced. Only in two cases it was not possible to reach a clearance of symptoms and yeasts. The results show the benefit of a precise differentiation before therapy. Serologic controls of antibody titers seem to be useful tools to control the efficacy of treatment.

[Susceptibility testing of fluconazole: evaluation of a multicenter study of the working group "Clinical Mycology" of the German Speaking Mycological Society]. / Empfindlichkeitsprüfung von Fluconazol: Auswertung einer Multizenter-Studie der Arbeitsgemeinschaft "Klinische Mykologie" der Deutschsprachigen Mykologischen Gesellschaft.

In a collaborative study for in vitro testing of fluconazole against clinical yeast isolates participated 21 laboratories of the working group "Clinical Mycology" of the German Speaking Mycological Society. In these centers, according to a standard test protocol 1181 clinical isolates from 1033 patients were tested to their susceptibility against fluconazole by microdilution, agar diffusion and partly by agar dilution. Approximately 600 strains (59.1%) of the collective of 1106 isolates sent to a reference center underwent retesting in one laboratory (center 13). These strains demonstrated almost the same species distribution as the total collective. For 80% of all isolates a MIC of < or = 4 micrograms/ml and for 90% of the Candida albicans strains a MIC of < or = 2 micrograms/ml has been determined. Only approx, 9% of all isolates (4% with Candida albicans) showed a MIC of < or = 25 micrograms/ml. By parallel testing of 10 control strains issued by the reference center to the laboratories, the inter- and intra-laboratory comparability of the susceptibility testing of fluconazole was checked. The results demonstrated that under appropriate technical prerequisites and standardised test conditions, the methods used routinely in bacteriology microdilution, agar dilution and agar diffusion may also be applied in a reproducible way in the routine mycological laboratory for the susceptibility testing of yeasts.

[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.

[Susceptibility testing of yeasts against fluconazole: proposal for a standardized agar diffusion method with 25 microgram fluconazole paper discs]. / Empfindlichkeitsprüfung von Hefen gegenüber Fluconazol: Vorschlag für eine standardisierte Agardiffusions-Methode mit 25-microgram-Fluconazol-Testblättchen.

This paper gives a proposal for a standardised agar diffusion susceptibility testing method with 25 micrograms fluconazole discs. The methodology compiles the results of several years of work to develop a reliable and reproducible routine-method for the microbiology laboratory. In this proposal, in addition, the critics and experiences of a collaborative study for susceptibility testing of fluconazole with 21 laboratories from Germany and Austria are included.

[Susceptibility testing of yeasts against fluconazole: comparison of the Etest method with microdilution and agar dilution]. / Empfindlichkeitsprüfung von Hefen gegenüber Fluconazol: Vergleich der Etest-Methode mit der Mikrodilution und der Agardilution.

In bacteriology, the Etest has a broad field of application in bacteriology and is recently available for the antimycotics fluconazole and itraconazole. By means of the presence of gradient concentrations of the active substance on the carrier material, it is possible to obtain reproducible MICs of the antimycotic substances. The results of susceptibility testing of 326 clinical yeast isolates with the Etest were compared to those MICs obtained by microdilution and agar dilution. A 100% concordance of the MIC markers (mode-, MIC50- and MIC90-value, standard deviation of the mean log2-MIC-dilution steps) was given when compared by a +/- 1 MIC-dilution step range with microdilution and by +/- 2 MIC-dilution steps with agar dilution; species dependent all strains were within 2 x standard deviation of the individual MIC-mean of the species. By comparison of the individual MIC-values maximum differences of +/- 6 MIC-dilution steps were obtained, where 70% of all results were within +/- 2 MIC-dilution steps, and more than 92% of all strains were within +/- 3 MIC-dilution steps. The Pearson's correlation coefficients show a good agreement of the Etest with microdilution (r = 0.92) resp., agar dilution (r = 0.88) demonstrate, however, clearly insufficient correlations (r < 0.65) to the reference methods, for species with difficult to read Etest inhibition zones (e.g., Candida glabrata, Candida krusei, Candida parapsilosis). The differences between the proposed test methods recommended by the NCCLS and the working group "Clinical Mycology" of the German Speaking Mycological Society (AG-KMYK) are tabled.