Background: Voriconazole is a first line agent for the treatment for invasive pulmonary aspergillosis. There are increasing reports of Aspergillus fumigatus isolates with reduced susceptibility to voriconazole. I investigated the pharmacodynamics of voriconazole against drug susceptible and drug resistant strains of Aspergillus fumigatus through the development of a novel dynamic in vitro model of the human alveolus. I then investigated whether combination therapy with voriconazole and anidulafungin would be beneficial in the treatment of infection with these isolates. Methods: An in vitro dynamic model of IPA was developed that enabled simulation of human-like voriconazole pharmacokinetics. Galactomannan was used as a biomarker. The pharmacodynamics of voriconazole against wild-type and three resistant strains of A. fumigatus were defined. The results were bridged to humans to provide decision support for setting breakpoints for voriconazole using Clinical Laboratory Standards Institute (CLSI) and European Committee of Antimicrobial Susceptibility Testing (EUCAST) methodology. The interaction of voriconazole and anidulafungin in an in vitro static model was described using the Greco model. Results: Isolates with higher MICs required higher area under the concentration time curve (AUCs) to achieve suppression of galactomannan. An AUC:MIC using CLSI and EUCAST methodology that achieved suppression of galactomannan was 55 and 32.1, respectively. A trough concentration:MIC using CLSI and EUCAST methodology that achieved suppression of galactomannan was 1.68 and 1, respectively. Potential CLSI breakpoints for voriconazole are: susceptible ≤ 0.5 mg/L; resistant > 1 mg/L. Potential EUCAST breakpoints for voriconazole are: susceptible ≤ 1 mg/L; resistant > 2 mg/L. Galactomannan concentrations were only marginally reduced by anidulafungin monotherapy in the static model. An additive effect between voriconazole and anidulafungin was apparent. Conclusions: Voriconazole resistance mechanisms can be overcome with higher drug exposures, but this may require concentrations likely to cause toxicity in humans. The addition of anidulafungin does not markedly alter the exposure-response relationship of voriconazole. A rise in serum galactomannan during combination therapy with voriconazole and anidulafungin should be interpreted as treatment failure and not attributed to a paradoxical reaction related to echinocandin treatment. The dynamic in vitro model is a useful tool to address many remaining questions related to treatment of invasive fungal infection.

Candida glabrata, geralmente comensal, surgiu como uma causa comum de infecções fúngicas graves com risco de morte. Dada a resistência crescente aos azóis, a orientação recente é utilizar as equinocandinas como a primeira escolha para o tratamento de infecções sistêmicas por C. glabrata. No entanto, esta é a primeira espécie de Candida que foi detectada com resistência significativa às equinocandinas. Esta levedura é capaz de colonizar tecidos do hospedeiro, bem como superfícies abióticas (catéteres, próteses) onde desenvolve um crescimento em multicamadas caracterizado como biofilme. A natureza da estrutura do biofilme e os atributos fisiológicos a ele conferidos resultam em uma resistência inerente a agentes antimicrobianos, impactando negativamente na saúde do paciente. Este estudo teve como objetivo a indução in vitro de resistência à anidulafungina em células planctônicas e sésseis de sete cepas sensíveis de C. glabrata, além da verificação do desenvolvimento de resistência cruzada com fluconazol. A indução de resistência foi realizada submetendo as cepas a concentrações sub-inibitórias do antifúngico. A determinação de concentração inibitória mínima através de microdiluição em caldo foi realizada previamente e posteriormente à indução de resistência e, também, para a verificação de resistência cruzada com fluconazol. O método de indução de resistência resultou em cepas fortemente resistentes à anidulafungina, com concentrações inibitórias mínimas variando de 1 a 2 μg/mL. Antes da indução da resistência, as formas planctônica e séssil das cepas eram todas sensíveis ou sensíveis dose-dependente ao fluconazol. Após a indução de resistência à anidulafungina esta sensibilidade ao fluconazol não foi mantida, tornando as cepas resistentes a este antifúngico. Clinicamente, esta resistência cruzada poderia implicar em falha terapêutica ao utilizar o fluconazol em pacientes previamente expostos a concentrações sub-inibitórias de anidulafungina por longos períodos. / Candida glabrata, usually commensal, has emerged as a common cause of serious life threatening fungal infections. Given the increasing resistance to azoles, the recent guidance is to use echinocandins as the first choice for the treatment of systemic infections by C. glabrata. However, C. glabrata is the first species of Candida that has been detected with significant resistance to echinocandins. This yeast is able to colonize host tissues as well as abiotic surfaces (catheters, prostheses) where it develops a multi-layer growth characterized as biofilm. The nature of the biofilm structure and the physiological attributes conferred on it, result in an inherent resistance to antimicrobial agents, negatively impacting the patient's health. This study aimed to induce in vitro resistance to anidulafungin in planktonic and sessile cells of seven sensitive C. glabrata strains, as well as to verify the development of cross-resistance with fluconazole. The induction of resistance was performed by subjecting the isolates to sub-inhibitory concentrations of the antifungal. The determination of minimum inhibitory concentration by broth microdilution was performed before and after induction of resistance and also for fluconazole cross-resistance verification. The resistance induction test resulted in strains strongly resistant to anidulafungin, with minimum inibitory concentrations ranging from 1 to 2 μg mL-1. Prior to induction of resistance, the planktonic and sessile forms of the strains were all sensitive or sensitive dose-dependent to fluconazole. However, after the induction of resistance to anidulafungin, this sensitivity to fluconazole was not maintained, making the strains resistant to this antifungal. Clinically, this cross-resistance could lead to therapeutic failure when using fluconazole in patients previously exposed to sub-inhibitory concentrations of anidulafungin for long periods.

Candida glabrata, geralmente comensal, surgiu como uma causa comum de infecções fúngicas graves com risco de morte. Dada a resistência crescente aos azóis, a orientação recente é utilizar as equinocandinas como a primeira escolha para o tratamento de infecções sistêmicas por C. glabrata. No entanto, esta é a primeira espécie de Candida que foi detectada com resistência significativa às equinocandinas. Esta levedura é capaz de colonizar tecidos do hospedeiro, bem como superfícies abióticas (catéteres, próteses) onde desenvolve um crescimento em multicamadas caracterizado como biofilme. A natureza da estrutura do biofilme e os atributos fisiológicos a ele conferidos resultam em uma resistência inerente a agentes antimicrobianos, impactando negativamente na saúde do paciente. Este estudo teve como objetivo a indução in vitro de resistência à anidulafungina em células planctônicas e sésseis de sete cepas sensíveis de C. glabrata, além da verificação do desenvolvimento de resistência cruzada com fluconazol. A indução de resistência foi realizada submetendo as cepas a concentrações sub-inibitórias do antifúngico. A determinação de concentração inibitória mínima através de microdiluição em caldo foi realizada previamente e posteriormente à indução de resistência e, também, para a verificação de resistência cruzada com fluconazol. O método de indução de resistência resultou em cepas fortemente resistentes à anidulafungina, com concentrações inibitórias mínimas variando de 1 a 2 μg/mL. Antes da indução da resistência, as formas planctônica e séssil das cepas eram todas sensíveis ou sensíveis dose-dependente ao fluconazol. Após a indução de resistência à anidulafungina esta sensibilidade ao fluconazol não foi mantida, tornando as cepas resistentes a este antifúngico. Clinicamente, esta resistência cruzada poderia implicar em falha terapêutica ao utilizar o fluconazol em pacientes previamente expostos a concentrações sub-inibitórias de anidulafungina por longos períodos. / Candida glabrata, usually commensal, has emerged as a common cause of serious life threatening fungal infections. Given the increasing resistance to azoles, the recent guidance is to use echinocandins as the first choice for the treatment of systemic infections by C. glabrata. However, C. glabrata is the first species of Candida that has been detected with significant resistance to echinocandins. This yeast is able to colonize host tissues as well as abiotic surfaces (catheters, prostheses) where it develops a multi-layer growth characterized as biofilm. The nature of the biofilm structure and the physiological attributes conferred on it, result in an inherent resistance to antimicrobial agents, negatively impacting the patient's health. This study aimed to induce in vitro resistance to anidulafungin in planktonic and sessile cells of seven sensitive C. glabrata strains, as well as to verify the development of cross-resistance with fluconazole. The induction of resistance was performed by subjecting the isolates to sub-inhibitory concentrations of the antifungal. The determination of minimum inhibitory concentration by broth microdilution was performed before and after induction of resistance and also for fluconazole cross-resistance verification. The resistance induction test resulted in strains strongly resistant to anidulafungin, with minimum inibitory concentrations ranging from 1 to 2 μg mL-1. Prior to induction of resistance, the planktonic and sessile forms of the strains were all sensitive or sensitive dose-dependent to fluconazole. However, after the induction of resistance to anidulafungin, this sensitivity to fluconazole was not maintained, making the strains resistant to this antifungal. Clinically, this cross-resistance could lead to therapeutic failure when using fluconazole in patients previously exposed to sub-inhibitory concentrations of anidulafungin for long periods.

Candida glabrata, geralmente comensal, surgiu como uma causa comum de infecções fúngicas graves com risco de morte. Dada a resistência crescente aos azóis, a orientação recente é utilizar as equinocandinas como a primeira escolha para o tratamento de infecções sistêmicas por C. glabrata. No entanto, esta é a primeira espécie de Candida que foi detectada com resistência significativa às equinocandinas. Esta levedura é capaz de colonizar tecidos do hospedeiro, bem como superfícies abióticas (catéteres, próteses) onde desenvolve um crescimento em multicamadas caracterizado como biofilme. A natureza da estrutura do biofilme e os atributos fisiológicos a ele conferidos resultam em uma resistência inerente a agentes antimicrobianos, impactando negativamente na saúde do paciente. Este estudo teve como objetivo a indução in vitro de resistência à anidulafungina em células planctônicas e sésseis de sete cepas sensíveis de C. glabrata, além da verificação do desenvolvimento de resistência cruzada com fluconazol. A indução de resistência foi realizada submetendo as cepas a concentrações sub-inibitórias do antifúngico. A determinação de concentração inibitória mínima através de microdiluição em caldo foi realizada previamente e posteriormente à indução de resistência e, também, para a verificação de resistência cruzada com fluconazol. O método de indução de resistência resultou em cepas fortemente resistentes à anidulafungina, com concentrações inibitórias mínimas variando de 1 a 2 μg/mL. Antes da indução da resistência, as formas planctônica e séssil das cepas eram todas sensíveis ou sensíveis dose-dependente ao fluconazol. Após a indução de resistência à anidulafungina esta sensibilidade ao fluconazol não foi mantida, tornando as cepas resistentes a este antifúngico. Clinicamente, esta resistência cruzada poderia implicar em falha terapêutica ao utilizar o fluconazol em pacientes previamente expostos a concentrações sub-inibitórias de anidulafungina por longos períodos. / Candida glabrata, usually commensal, has emerged as a common cause of serious life threatening fungal infections. Given the increasing resistance to azoles, the recent guidance is to use echinocandins as the first choice for the treatment of systemic infections by C. glabrata. However, C. glabrata is the first species of Candida that has been detected with significant resistance to echinocandins. This yeast is able to colonize host tissues as well as abiotic surfaces (catheters, prostheses) where it develops a multi-layer growth characterized as biofilm. The nature of the biofilm structure and the physiological attributes conferred on it, result in an inherent resistance to antimicrobial agents, negatively impacting the patient's health. This study aimed to induce in vitro resistance to anidulafungin in planktonic and sessile cells of seven sensitive C. glabrata strains, as well as to verify the development of cross-resistance with fluconazole. The induction of resistance was performed by subjecting the isolates to sub-inhibitory concentrations of the antifungal. The determination of minimum inhibitory concentration by broth microdilution was performed before and after induction of resistance and also for fluconazole cross-resistance verification. The resistance induction test resulted in strains strongly resistant to anidulafungin, with minimum inibitory concentrations ranging from 1 to 2 μg mL-1. Prior to induction of resistance, the planktonic and sessile forms of the strains were all sensitive or sensitive dose-dependent to fluconazole. However, after the induction of resistance to anidulafungin, this sensitivity to fluconazole was not maintained, making the strains resistant to this antifungal. Clinically, this cross-resistance could lead to therapeutic failure when using fluconazole in patients previously exposed to sub-inhibitory concentrations of anidulafungin for long periods.

Candida glabrata ist die zweithäufigste Ursache von Candidämien und invasiven Hefepilzinfektionen in Europa. Im Gegensatz zu C. albicans zeigt C. glabrata eine reduzierte Empfindlichkeit gegen bestimmte Antimykotika und kann unter Therapie rasch Resistenzen entwickeln. Diese Arbeit umfasst eine systematische geno- und phänotypische Resistenzanalyse einer der größten europäischen - durch das NRZMyk in 5 Jahren zusammengetragenen - C. glabrata Stammsammlungen bestehend aus 176 klinisch relevanter Isolate. 84 der Stämme wurden anhand Referenztestung nach EUCAST zunächst als Anidulafungin (AND) resistent eingestuft. 71 wiesen konkordante Mutationen in den für die Glucan-Synthetase kodierenden FKS-Genen auf (13 % in FKS1, 87 % in FKS2). Vor allem die Position Ser-663 (FKS2-HS1) imponierte mit signifikant erhöhten AND MHK-Werten. 11 FKS-Wildtyp-Isolate, die ursprünglich als AND resistent klassifiziert wurden, wiesen in multiplen Nachtestungen um den Breakpoint undulierende AND MHK-Werte auf. 2 FKS-Wildtyp Isolate zeigten durchgängig hohe AND MHK-Werte und mussten daher - trotz fehlender Zielgenmutationen - als resistent eingestuft werden. Diese extremen Phänotypen wurden durch einen verblindeten nationalen Ringversuch bestätigt. Über ein Drittel der Isolate war multiresistent. Stämme aus Blutstrominfektionen und Ser-663 Mutation waren mit einer erhöhten Mortalität assoziiert. Ein weiteres Kernelement war die Detektion von Azol-resistenten C. glabrata petite-Phänotypen in der Routinediagnostik. Hier wurden innerhalb von 8 Monaten 20 relevante Isolate identifiziert. Die Ergebnisse belegen das regelmäßige Auftreten single- / multidrug-resistenter C. glabrata Isolate in Deutschland. Phänotypische Resistenztestungen können zu Fehlklassifizierung von sensiblen Isolaten führen. FKS-Genotypisierungen hingegen sind ein nützliches Tool zur Identifizierung relevanter Resistenzen. In seltenen Fällen scheint jedoch eine Echinocandin-Resistenz ohne genotypisches Korrelat möglich zu sein. / Candida glabrata is the second most common cause of candidaemia and invasive yeast infections in Europe. In contrast to C. albicans, C. glabrata shows reduced susceptibility to certain antifungal agents and can rapidly acquire resistance under therapy. This work comprises a systematic geno- and phenotypic resistance analysis of one of the largest European C. glabrata strain collections - compiled by NRZMyk in 5 years - consisting of 176 clinically relevant isolates. 84 of the strains were initially classified as anidulafungin (AND) resistant by reference testing according to EUCAST. 71 showed concordant mutations in FKS genes encrypting the glucan synthetase (13 % in FKS1, 87 % in FKS2). In particular, the position Ser-663 (FKS2-HS1) impressed with significantly increased AND MIC-values. 11 FKS wild-type isolates, originally classified as AND resistant, showed fluctuating AND MIC-values near the clinical breakpoint after retests with multiple assays. Two FKS wild-type isolates showed consistently high AND MIC values and therefore had to be classified as resistant - despite the absence of target gene mutations. These extreme phenotypes were confirmed in a blinded national ring trial. More than one third of echinocandin-resistant isolates showed concordant fluconazole resistance. Strains from bloodstream infections and Ser-663 mutation were associated with high mortality. Another core element was the detection of azole-resistant C. glabrata petite phenotypes in routine diagnostics. Here, 20 relevant isolates were identified within 8 months, which could be assigned to 8 patients. These results demonstrate the regular occurrence of single- / multidrug-resistant C. glabrata isolates in Germany. Phenotypic resistance testing can lead to misclassification of susceptible isolates. FKS genotyping, on the other hand, is a useful tool for identifying resistant strains. However, in rare cases, echinocandin resistance without a genotypic correlate seems to be possible.

Die Zahl invasiver Pilzinfektionen ausgelöst durch C. glabrata steigt zunehmend und auch die Ausbildung multipler Resistenzen wird immer häufiger registriert. In dieser Arbeit wurden zwei klinische MDR-C. glabrata-Stämme systematisch analysiert, um den Ursprung der Mehrfachresistenz zu finden. Aufgefallen waren jene Isolate in vorhergehenden Untersuchungen von Aldejohann et. al., die 176 Stämme, die dem Referenzzentrum NRZ-Myk zugesandt wurden, auf ihr Resistenzverhalten gegen Echinocandine analysierten und auf FKS-Mutationen untersuchten. Die Isolate CG22 und CG56 zeigten ein Resistenzverhalten gegen Anidulafungin ohne eine FKS-Mutation aufzuweisen. In Mehrfachtestungen wurde das einheitliche Verhalten von CG56 in zehn Einzelkolonien verifiziert, um Mischkulturen oder heterogenes Verhalten innerhalb des Isolates ausschließen zu können. Nach Analyse der gesamten Genomsequenz von CG56 zeigte sich eine Mutation kurz vor der HS-Region von FKS2, die eine Erklärung für das Resistenzverhalten zu liefern scheint. Neben der Mutation in FKS2 wurde ebenfalls eine Mutation in FKS1 und in ERG3 bestätigt. Die Mutation in ERG3 führt zu einer Verschiebung im Sterolsynthesepathway und zu einer Neuverteilung der Zellmembranbestandteile. Das klinische Isolat CG22 fällt mit Resistenzen gegen Azole, Echinocandine und Amphotericin B auf und zeigte ebenfalls eine Mutationen in ERG3. Zusätzlich dazu ergab sich eine Loss-of- Function-Mutation in ERG4 und damit verbunden einen massiv reduzierten Ergosterolgehalt der Zellmembran. Die seltene Kombination aus ERG3 und ERG4 Mutation scheint die Erklärung für die außergewöhnliche Amphotericin B-Resistenz von CG22 zu liefern und wird hier als erstmals bei einem C. glabrata Isolat beschrieben. Dieser besondere Stamm, der sogar als panresistent bezeichnet werden kann, sollte Bestandteil weiterer Forschung werden. Der Sterolsynthesepathway dient als Angriffspunkt vieler Antimykotika und kann durch seine vielen Intermediate und abweichenden Abläufen zu unterschiedlichen Stoffwechselendprodukten führen. Der Ergosterolgehalt der Zellmembran eines C. glabrata-Stammes kann weitere Rückschlüsse auf die Empfindlichkeit des Isolates geben und somit die Chancen des Therapieerfolges der Antimykotikagabe besser vorhersagen und könnte somit einen vielversprechenden Beitrag zur Behandlung lebensbedrohlicher Candidosen leisten. / The number of invasive fungal infections caused by C. glabrata is increasing and the development of resistance to multiple drug classes is also being recorded more frequently. In this study, two clinical MDR C. glabrata strains were systematically analyzed to find the origin of their Multidrug Resistance. These isolates had attracted attention in previous studies by Aldejohann et. al. who studied 176 strains that have been sent to the NRZ-Myk reference center. The strains have been analyzed for their resistance to echinocandins and have been examined for mutations in FKS1 and FKS2 hotspots. The isolates CG22 and CG56 showed resistance to anidulafungin without showing mutation in the FKS hotspots. In multiple testing, the uniform behavior of CG56 was verified in ten individual colonies in order to exclude mixed cultures or heterogeneous behavior within the isolate. Analysis of the entire genome sequence of CG56 revealed a mutation just upstream of the HS region of FKS2, which appears to provide an explanation for the resistance behavior. In addition to the mutation in FKS2, a mutation in FKS1 and ERG3 was also confirmed. The mutation in ERG3 leads to a shift in the sterol synthesis pathway and to a redistribution of the cell membrane components. The clinical isolate CG22 was found to be resistant to azoles, echinocandins and amphotericin B. CG22 showed a mutation in ERG3 and additionally a loss-of-function mutation in ERG4. This leads to a massively reduced ergosterol content of the cell membrane. The rare combination of ERG3 and ERG4 mutation seems to explain the extraordinary amphotericin B resistance of CG22 and is described for the first time in a C. glabrata isolate. This particular strain, which can even be described as panresistant, should become part of further research. The sterol synthesis pathway serves as a target for many antimycotics and can lead to different metabolic products due to its many intermediates and divergent processes. The ergosterol content of the cell membrane of a C. glabrata strain can provide further information on the susceptibility of the isolate. This could possibly predict the chances of therapeutic success of antimycotic treatment better and could therefore make a promising contribution to the treatment of life-threatening candidiasis.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED, APS-I) is exceptionally common in Finland. Most patients have chronic oral candidiasis since childhood. Thus, most patients receive repeated courses of antifungals throughout their life. Eleven of our patients (31.4%) have become colonized with Candida albicans with decreased sensitivity to fluconazole. A total of 43 isolates of C. albicans from 23 APECED patients isolated during the years 1994 to 2004 were divided into 2 groups: fluconazole-susceptible dose-dependent (MIC, 16-32 μg/mL, 18 isolates) and fluconazole-susceptible (MIC ≤8 μg/mL, 25 isolates) groups. Antifungal activity of amphotericin B, echinocandins, and azoles was determined by the Clinical and Laboratory Standards Institute M27-A2 methodology. All isolates were highly susceptible to amphotericin B and echinocandins. Posaconazole and voriconazole were active against all isolates. Our data suggest that topical amphotericin B could continue to be a safe and active drug for daily administration for APECED patients. Posaconazole, voriconazole, and echinocandins may be useful in some complicated cases.