Evaluation of clinical and environmental of Aspergillus spp. isolates : morphological and molecular identification; susceptibility profile and search for azoles resistance related mutations

Orientador: Angélica Zaninelli SchreiberTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: Membros do gênero Aspergillus são patógenos fúngicos oportunistas responsáveis por altas taxas de mortalidade, principalmente em pacientes imunocomprometidos, que se infectam após inalação dos conídios presentes no ar. O derivado azólico voriconazol (VRZ) é a primeira linha na terapia e o surgimento de isolados de A. fumigatus resistentes a este e outros azólicos está sendo investigado em diversos países. Isolados resistentes apresentam mutações no gene cyp51A, que codifica a 14?-demetilase, enzima alvo para estes compostos. No Hospital de Clínicas da Unicamp (HC-Unicamp) muitos pacientes de alto risco estão suscetíveis a agentes infecciosos oportunistas como os do gênero Aspergillus. Estudos recentes demonstraram que os pacientes podem estar colonizados ou infectados com mais de um genótipo de A. fumigatus e ainda que esses podem apresentar diferentes perfis frente aos antifúngicos. Análise de microssatélites (STRAF) é útil para estudar a epidemiologia molecular de A. fumigatus devido ao seu alto poder discriminatório. Desta forma, o acompanhamento do possível surgimento de isolados resistentes e a análise dos isolados sequenciais de um mesmo paciente quanto a sua genotipagem se tornam importantes. Neste contexto, o presente trabalho buscou estudar a ocorrência das espécies e o perfil de suscetibilidade de Aspergillus ambientais e clínicos frente aos antifúngicos, além de analisar a presença de mutações no gene cyp51A dos isolados de A. fumigatus resistentes aos azólicos seguida da genotipagem de A. fumigaus clínicos e ambientais. Duzentos e vinte e nove isolados clínicos e 123 isolados ambientais foram estudados. Para a identificação de espécies foi realizado sequenciamento de DNA das regiões ITS e ?-tubulina. O perfil de suscetibilidade aos antifúngicos foi avaliado de acordo com o documento M38-A2 do Clinical and Laboratory Standards Institute (CLSI, 2008) para determinação da concentração efetiva mínima (CEM) para as equinocandinas e concentração inibitória mínima (CIM) para os demais. Os antifúngicos avaliados foram anfotericina B (AMB) itraconazol (ITZ), voriconazol (VRZ), posaconazol (PSZ), micafungina (MCF) e caspofungina (CPF). Isolados clínicos resistentes aos azólicos e os isolados ambientais de A. fumigatus foram submetidos ao sequenciamento do gene cyp51A. A tipagem de A. fumigatus por microssatélites foi realizada por sequenciamento dos pares de oligonucleotídeos iniciadores: 2AB, 3AB, 4AB, BDA, BDB, BDC e BDD e analisada com auxílio do programa e-Burst v.3. Dez isolados de A. fumigatus apresentaram valores elevados de CIM para pelo menos um azólico, mas mutações no gene cyp51A relacionadas com resistência não foram observadas. A observação mais preocupante foi que valores de CIM de AMB foram elevados (?2 mg L-1) em 87% dos pacientes com isolados de A. flavus e 43% com isolados de A. fumigatus. Não foi observada resistência às equinocandinas. Quanto a genotipagem, os isolados de A. fumigatus apresentaram elevada diversidade. Seis isolados de 3 pacientes foram englobados como parte de um complexo clonal. Estes 6 isolados possuem as já conhecidas mutações de ponto F46Y, M127V, N248T, D225E e E427K no gene cyp51A. Fazer parte desse complexo clonal significa que os isolados têm a mesma origem clonal, mas com 1 a 3 ST de diferença. Os resultados confirmam estudos anteriores que mostraram o alto polimorfismo das populações de A. fumigatus. A heterogeneidade dos genótipos confirma que um paciente pode estar contaminado ou infectado com diferentes isolados e diferentes pacientes podem ser infectados com genótipos diferentes ou, por outro lado, com genótipos muito próximos sugerindo, neste caso, a possibilidade de infecção durante internação hospitalar. Dado que a AMB é usada para tratar infecções resistentes aos azólicos, os dados obtidos no presente trabalho quanto a este antifúngico destacam a necessidade de vigilância contínua em Aspergillus para toda a resistência antifúngica a fim de permitir a implementação de estratégias de tratamento corretas para o manejo desses patógenos. Nenhuma mutação no gene cyp51A que tenha sido relacionada com resistência foi observada, nem mesmo em isolados resistentes aos azólicos, indicando que outros mecanismos podem estar envolvidos. Estudos adicionais serão necessários para um maior entendimento da epidemiologia e a heterogeneidade desses patógenos, além dos mecanismos envolvidos na resistência não apenas aos antifúngicos azólicos mas também frente a AMB e às equinocandinasAbstract: Members of the Aspergillus genus are opportunistic fungal pathogens responsible for high mortality rates, especially in immunocompromised patients, who become infected after inhalation of conidia present in the air. The azole voriconazole (VRZ) is the first line in therapy and the emergence of resistant A. fumigatus isolates to this and other azole is being investigated in several countries. Resistant isolates have mutations in the cyp51A gene, which encodes 14?-demethylase, the target enzyme for these compounds. In the Hospital de Clínicas of Campinas University (HC-Unicamp) many high-risk patients are susceptible to opportunistic infectious agents like those of the genus Aspergillus. Recent studies have shown that patients may be colonized or infected with more than one A. fumigatus genotype and that they may present different susceptibility profiles. Microsatellite analysis (STRAF) is useful for studying the molecular epidemiology of A. fumigatus due to its high discriminatory power. In this way, monitoring the possible emergence of resistant isolates and analyzing the sequential isolates from the same patient regarding their genotyping become important. In this context, the present work aimed to study the occurrence of the species and the antifungal susceptibility profile of environmental and clinical Aspergillus, as well as to analyze the presence of cyp51A gene mutations in A. fumigatus azoles resistant isolates, followed by genotyping clinical and environmental A. fumigatus. Two hundred twenty-nine clinical isolates and 123 environmental isolates were studied. For the identification of species, DNA sequencing of the ITS and ?-tubulin regions was performed. The antifungal susceptibility profile was evaluated according to the M38-A2 of the Clinical and Laboratory Standards Institute (CLSI, 2008) to determine the minimum effective concentration (MEC) for the echinocandins and minimum inhibitory concentration (MIC) for the others. The antifungal agents evaluated were amphotericin B (AMB) itraconazole (ITZ), voriconazole (VRZ), posaconazole (PSZ), micafungin (MCF) and caspofungin (CPF). Clinical azoles resistant A. fumigatus and environmental A. fumigatus were submitted to the cyp51A gene sequencing. A. fumigatus isolates were submitted to microsatelites typing (STRAfs 2AB, 3AB, 4AB, BDA, BDB, BDC and BDD) to assess if the serially isolates were genetically related and then they were analyzed with e-Burst v.3. Ten isolates of A. fumigatus showed high MIC values for at least one azole, but mutations in the cyp51A gene related resistance were not observed. The most disturbing observation was that the MIC values of AMB were high (?2 mg L-1) in 87% of the patients with A. flavus isolates and 43% with A. fumigatus isolates. No resistance to echinocandins was observed. Regarding to genotyping, A. fumigatus showed high diversity. Six isolates of three patients were part of a clonal complex. These 6 isolates have the already known point mutations F46Y, M127V, N248T, D225E and E427K in the cyp51A gene. Being part of this clonal complex means that the isolates have the same clonal origin, but with 1 to 3 STs of difference. Our results confirm previous studies showing the high polymorphism of A. fumigatus populations. The heterogeneity of the genotypes confirms that a patient may be colonized or infected with different isolates and different patients may be infected with different or, on the other hand, infected with very close genotypes suggesting, in this case, the possibility of infection during hospitalization. Since AMB is used to treat azole-resistant infections, the data obtained in the present study highlight the need for continuous resistance surveillance in Aspergillus for all important antifungal to implement correct treatment strategies for the management of these pathogens. No related to resistance mutation in cyp51A gene was observed, not even in those azole-resistant isolates, suggesting that other mechanisms may be involved. Further studies will be necessary to better understand the epidemiology and heterogeneity of these pathogens, as well as the resistance mechanisms involved not only to azole antifungals but to AMB and echinocandinsDoutoradoCiencias BiomedicasDoutora em Ciências Médicas870369/1997-088881.134235/2016-01CNPQCAPE

Cryptococcus species obtained from clinical and environmental isolates from the city of Campinas, SP : genotyping and assessment of in vitro susceptibility of antifungal agents alone and in different combinations

Orientador: Angélica Zaninelli SchreiberDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: O gênero Cryptococcus engloba duas espécies consideradas patogênicas: C.neoformans e C.gattii. Apesar dos avanços na área médica, a criptococose permanece uma das infecções fúngicas sistêmicas mais importantes no Brasil. Anfotericina B (AMB) associada a flucitosina (5FC) é a terapia de indução indicada porém, no Brasil, 5FC não está disponível e o tratamento segue somente com AMB ou em associação com fluconazol (FCL). Este trabalho avaliou a ocorrência de espécies e os genótipos de isolados clínicos de pacientes atendidos no Hospital de Clínicas da UNICAMP em um período de 5 anos; isolados ambientais coletados na cidade de Campinas-SP e o perfil de suscetibilidade aos antifúngicos sozinhos contra isolados clínicos e ambientais e o efeito de combinações de antifúngicos frente a isolados clínicos de Cryptococcus spp.. A identificação de espécies e genótipos foi realizada por testes bioquímicos, Restriction Fragment Length Polymorphism do gene URA5 (URA5-RFLP) e sequenciamento da região Internal Transcribed Spacer (ITS) do DNA ribossomal. Testes de suscetibilidade para AMB, 5FC, FCL, voriconazol (VRC), itraconazol (ITC) e terbinafina (TRB) isolados foram realizados conforme CLSI M27-A3 (2008). Os testes com antifúngicos combinados (AMB+5FC; AMB+FCL; AMB+TRB; FCL+TRB), foram realizados pelo método do "tabuleiro de xadrez" para determinação do Coeficiente de Inibição Fracional (CIF) para avaliar o tipo de interação entre as substâncias (sinergismo, indiferença ou antagonismo). Dentre 75 isolados clínicos reativados, foram identificados 66 C.neoformans e 9 C.gattii. Todos C.gattii pertenceram ao genótipo VGII enquanto que 62 isolados de C.neoformans ao genótipo VNI e apenas 4 ao genótipo VNII. Foram obtidos 92 isolados ambientais de Cryptococcus, pertencentes às espécies C.neoformans (genótipo VNI), C.laurentii, C.albidus, C.flavescens e Cryptococcus spp.. Os valores dos intervalos de CIM para C.neoformans clínicos foram AMB: ? 0,125-1 µg/mL; 5FC: ? 0,125-2 µg/mL; FCL: 0,25-8 µg/mL; VRC: ? 0,015-0,125 µg/mL; ITC: 0,03-0,25 µg/mL e TRB: 0,125-2 µg/mL. Intervalos de CIM para C.gattii variaram de 0,25-1 µg/mL para AMB; 0,5-4 µg/mL para 5FC; 2-16 µg/mL para FCL; 0,06-0,25µg/mL para VRC; 0,06-0,5 µg/mL para ITC e 0,5-4 µg/mL para TRB. Foram observados elevados valores de CIM para os antifúngicos 5FC e FCL frente aos isolados ambientais de C.albidus e C.laurentii. O genótipo VNI de C.neoformans clínico mostrou 75,80% de interação sinérgica para AMB+5FC; 79,03% para AMB+FCL; 77,42% para AMB+TRB e 95,16% para FCL+TRB. O genótipo VNII apresentou 100% de sinergismo em todas as combinações. C.gattii (VGII) apresentou 88,9% de sinergismo nas combinações AMB+5FC e AMB+FCL; 100% para AMB+TRB e FCL+TRB. Não foi observado efeito antagônico nas combinações de antifúngicos. Foi observado bom desempenho nas combinações realizadas, especialmente naquelas envolvendo a TRB para ambas as espécies C.neoformans e C.gattii. O genótipo VNI foi o predominante entre os genótipos que afetam os pacientes com criptococose na região de Campinas. Em infecções de difícil tratamento ou que não respondem aos antifúngicos convencionais, a combinação de diferentes antifúngicos como AMB+TRB ou FCL+TRB podem vir a ser uma alternativa em países onde 5FC não está disponível, como no Brasil. Mais estudos são necessários para avaliar o papel dos genótipos na sensibilidade aos antifúngicos, assim como estudos de combinações de antifúngicos in vitro e in vivo para que novas estratégias possam ser empregadas no tratamento da criptococoseAbstract: Cryptococcus genus comprises two species considered pathogenic: C.neoformans and C.gattii. Despite advances in the medical field, cryptococcosis remains one of the most important systemic fungal infections in Brazil. Amphotericin B (AMB) associated with flucytosine (5FC) induction therapy is indicated but, in Brazil, 5FC is not available and treatment follows only with AMB or in combination with fluconazole (FCL). This study evaluated the prevalence of species and molecular subtypes of clinical isolates from patients treated at the Hospital of UNICAMP in a period of 5 years; environmental isolates collected in Campinas ¿ SP and in vitro antifungal susceptibility profile of antifungal agents alone against clinical and enviromental isolates of Cryptococcus spp. and the effect of combinations of antifungal agents against clinical isolates of Cryptococcus spp.. The species identification and subtyping was performed by biochemical tests, Restriction Fragment Length Polymorphism of URA5 gene (RFLP - URA5) and sequencing of the Internal Transcribed Spacer region (ITS) of ribosomal DNA. Susceptibility tests for AMB, 5FC, FCL, voriconazole (VRC), itraconazole (ITC) and terbinafine (TRB) isolated were performed according to CLSI M27-A3 (2008). Combined antifungals tests (AMB +5FC; AMB+FCL; AMB +TRB, FCL+TRB), were performed by the "Checkerboard" method to determine the Fractional Inhibitory Coefficient index (FIC) to assess the type of interaction between substances (synergism, indifference or antagonism). Among the 75 viable clinical isolates, 66 were identified as C.neoformans and 9 as C.gattii. All C.gattii belonged to subtype VGII while 62 isolates belonged C.neoformans VNI genotype and only 4 VNII. 92 environmental isolates of Cryptococcus were obtained. The species C.neoformans (subtype VNI) C.laurentii, C.albidus, C.flavescens and Cryptococcus spp.were identified. The MIC range values of clinical C.neoformans for AMB were: ? 0.125 -1 µg/mL; 5FC: ? 0.125 to 2 µg/mL; FCL: 0.25-8 µg/mL; VRC: ? 0.015 to 0.125 µg/mL; ITC: 0.03 to 0.25 µg/mL and TRB: 0.125 to 2 µg/mL. MIC for C.gattii ranged from 0.25-1 µg/mL for AMB; 0.5-4 µg/mL for 5FC; 2-16 µg/mL for FCL; 0.06 to 0.25 µg/mL for VRC; 0.06 to 0.5 µg/mL for ITC and 0.5-4 µg/mL for TRB. High MIC values were observed for FCL and 5FC against environmental isolates of C.albidus and C.laurentii. The VNI C.neoformans genotype showed 75.80 % of synergistic interaction for AMB+5FC; 79.03 % for AMB + FCL; 77.42 % for AMB + TRB and 95.16 % for FCL+TRB. The VNII genotype showed 100% synergism in all combinations. C.gattii (VGII) showed 88.9 % of synergism in combinations AMB+5 FC and AMB+FCL; 100 % for AMB+TRB and TRB+FCL. No antagonistic effect was observed in all evaluated antifungal combinations. Good performance was observed in all combinations performed, especially those involving TRB for both C.neoformans and C.gattii species. The VNI was the predominant genotype among genotypes affecting patients with cryptococcosis in Campinas region. In difficult to treat infections or unresponsive to conventional antifungal agents, the combination of different antifungals so as AMB+FCL or TRB+TRB may become an alternative in countries where 5FC is not available, as in Brazil. More studies are needed to evaluate the role of genotypes in sensitivity to antifungal agents, as well as antifungal agents combination studies in vitro and in vivo so that new strategies can be employed in the treatment of cryptococcosisMestradoCiencias BiomedicasMestra em Ciências Médica

Evaluation Of Antifungal Combination Against Cryptococcus Spp.

The second cause of death among systemic mycoses, cryptococcosis treatment represents a challenge since that 5-flucytosine is not currently available in Brazil. Looking for alternatives, this study evaluated antifungal agents, alone and combined, correlating susceptibility to genotypes. Eighty Cryptococcus clinical isolates were genotyped by URA5 gene restriction fragment length polymorphism. Antifungal susceptibility was assessed following CLSI-M27A3 for amphotericin (AMB), 5-flucytosine (5FC), fluconazole (FCZ), voriconazole (VRZ), itraconazole (ITZ) and terbinafine (TRB). Drug interaction chequerboard assay evaluated: AMB + 5FC, AMB + FCZ, AMB + TRB and FCZ + TRB. Molecular typing divided isolates into 14 C. deuterogattii (VGII) and C. neoformans isolates were found to belong to genotype VNI (n = 62) and VNII (n = 4). C. neoformans VNII was significantly less susceptible than VNI (P = 0.0407) to AMB; C. deuterogattii was significantly less susceptible than VNI and VNII to VRZ (P < 0.0001). C. deuterogattii was less susceptible than C. neoformans VNI for FCZ (P = 0.0170), ITZ (P < 0.0001) and TRB (P = 0.0090). The combination FCZ + TRB showed 95.16% of synergistic effect against C. neoformans genotype VNI isolates and all combinations showed 100% of synergism against genotype VNII isolates, suggesting the relevance of cryptococcal genotyping as it is widely known that the various genotypes (now species) have significant impact in antifungal susceptibilities and clinical outcome. In difficult-to-treat cryptococcosis, terbinafine and different antifungal combinations might be alternatives to 5FC.599585593JICA (Japan International Cooperation Agency)SATREPS (Science and Technology Research Partnership for Sustainable Development) [02P-29548-09

Lysine acetylation as drug target in fungi: an underexplored potential in Aspergillus spp.

In recent years, the intensification of the use of immunosuppressive therapies has increased the incidence of invasive infections caused by opportunistic fungi. Considering that, the spread of azole resistance and amphotericin B (AmB) inefficiency against some clinical and environmental isolates has been described. Thus, to avoid a global problem when controlling fungal infections and critical failures in medicine, and food security, new approaches for drug target identification and for the development of new treatments that are more effective against pathogenic fungi are desired. Recent studies indicate that protein acetylation is present in hundreds of proteins of different cellular compartments and is involved in several biological processes, i.e., metabolism, translation, gene expression regulation, and oxidative stress response, from prokaryotes and eukaryotes, including fungi, dem- onstrating that lysine acetylation plays an important role in essential mechanisms. Lysine acetyltransferases (KATs) and lysine deacetylases (KDACs), the two enzyme families responsible for regulating protein acetylation levels, have been explored as drug targets for the treatment of several human diseases and infections. Aspergilli have on average 8 KAT genes and 11 KDAC genes in their genomes. This review aims to summarize the available knowledge about Aspergillus spp. azole resistance mechanisms and the role of lysine acetylation in the control of biological processes in fungi. We also want to discuss the lysine acetylation as a potential target for fungal infection treatment and drug target discovery.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP: 2018/09948-0CNPq: 424729/201

Surveillance for azoles resistance in Aspergillus spp. highlights a high number of amphotericin B-resistant isolates

Aspergillus spp. are the most common invasive mould infection and are responsible for high mortality. Aspergillus fumigatus is currently of interest because resistance to azole antifungals has emerged. The Campinas University Hospital (HC-UNICAMP) receives high-risk patients susceptible to opportunistic infections but there have been no reports of resistant A.fumigatus. This study aimed to assess the susceptibility profile of Aspergillus isolates, specifically looking for azole resistance. ITS and -tubulin DNA sequencing was performed on 228 sequential clinical isolates. Broth microdilution susceptibility testing was performed for all isolates. A.fumigatus represented 74% of the isolates followed by Aspergillus flavus (12%). Nine A.fumigatus isolates from 9 different patients showed high MIC values to at least 1 azole, but cyp51A polymorphisms were detected in only 6 isolates and none correlated with known resistance mutations. The most troubling observation was that the minimum inhibitory concentration for amphotericin B was elevated (2mgL(-1)) in 87% of patients with A.flavus isolates and 43% with Aspergillusfumigatus isolates. Given that amphotericin B is used to treat azole-resistant infections, these data highlight the need for continuous surveillance in Aspergillus for all antifungal resistance to implement correct treatment strategies for the management of these pathogens616360365CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQ157884/2014-7; 246701/2013-

Evaluation of antifungal combination against Cryptococcus spp

The second cause of death among systemic mycoses, cryptococcosis treatment represents a challenge since that 5-flucytosine is not currently available in Brazil. Looking for alternatives, this study evaluated antifungal agents, alone and combined, correlating susceptibility to genotypes. Eighty Cryptococcus clinical isolates were genotyped by URA5 gene restriction fragment length polymorphism. Antifungal susceptibility was assessed following CLSI-M27A3 for amphotericin (AMB), 5-flucytosine (5FC), fluconazole (FCZ), voriconazole (VRZ), itraconazole (ITZ) and terbinafine (TRB). Drug interaction chequerboard assay evaluated: AMB + 5FC, AMB + FCZ, AMB + TRB and FCZ + TRB. Molecular typing divided isolates into 14 C. deuterogattii (VGII) and C. neoformans isolates were found to belong to genotype VNI (n = 62) and VNII (n = 4). C. neoformans VNII was significantly less susceptible than VNI (P = 0.0407) to AMB; C. deuterogattii was significantly less susceptible than VNI and VNII to VRZ (P < 0.0001). C. deuterogattii was less susceptible than C. neoformans VNI for FCZ (P = 0.0170), ITZ (P < 0.0001) and TRB (P = 0.0090). The combination FCZ + TRB showed 95.16% of synergistic effect against C. neoformans genotype VNI isolates and all combinations showed 100% of synergism against genotype VNII isolates, suggesting the relevance of cryptococcal genotyping as it is widely known that the various genotypes (now species) have significant impact in antifungal susceptibilities and clinical outcome. In difficult-to-treat cryptococcosis, terbinafine and different antifungal combinations might be alternatives to 5FC599585593JICA (Japan International Cooperation Agency); SATREPS (Science and Technology Research Partnership for Sustainable Development

MIC range (μg/mL) of LIF 12560 and LIF-E10 against amphotericin B, 5-flucytosine, fluconazole, itraconazole and voriconazole.

<p>MIC range (μg/mL) of LIF 12560 and LIF-E10 against amphotericin B, 5-flucytosine, fluconazole, itraconazole and voriconazole.</p

MICs Distribution (μg/mL) of 143 <i>Candida albicans</i> bloodstream isolates against amphotericin B, 5-flucytosine, fluconazole, itraconazole, voriconazole and caspofungin.

<p>MICs Distribution (μg/mL) of 143 <i>Candida albicans</i> bloodstream isolates against amphotericin B, 5-flucytosine, fluconazole, itraconazole, voriconazole and caspofungin.</p

Nucleotide mutations and amino acid substitutions.

<p>Nucleotide mutations and amino acid substitutions.</p