Loading...
Publication
Avaliação da resistência de Trichophyton rubrum ao antifúngico terbinafina e caracterização fenotípica e genotípica de fungos dermatófitos
| Name: | Description: | Size: | Format: | |
|---|---|---|---|---|
| 2.59 MB | Adobe PDF |
Authors
Advisor(s)
Abstract(s)
Os dermatófitos são um grupo de fungos filamentosos com uma distribuição cosmopolita, que têm a capacidade de invadir tecidos queratinizados (pele, cabelo e unhas) de humanos e outros animais e provocar uma infeção superficial denominada de dermatofitose. As dermatofitoses são responsáveis por 3 - 4% dos casos afeções dermatológicas, sendo a infeção superficial mais comum em humanos (com uma prevalência entre os 20-25%). No entanto, espera-se que a sua prevalência continue a aumentar devido ao aparecimento de novos fatores de risco. As espécies do género Trichophyton são os principais agentes causadores de patologias como pé de atleta e onicomicose nas mãos e pés e estima-se que afete ~1.000.000.000 de pessoas em todo o mundo. T. rubrum e T. interdigitale são os principais agentes etiológicos com prevalências de 80% e 20%, respetivamente. Em Portugal, é estimado que 1.510.391 de portugueses sofram de dermatofitoses, correspondendo a uma incidência de 14.300 infeções por dermatófitos por cada 1.000.000 de habitantes. Para o tratamento das dermatofitoses, a primeira terapia a ser prescrita é, na grande maioria dos casos, a terbinafina. Desde 2017 que os casos de resistência a este antifúngico por parte de T. rubrum e T. interdigitale têm vindo a aumentar gradualmente. Os mecanismos de resistência à terbinafina são, essencialmente, descritos como mutações pontuais no gene que codifica a esqualeno epoxidase.
Assim, neste estudo, procurámos perceber se a problemática de resistência ao antifúngico terbinafina em T. rubrum e T. interdigitale estaria ou não negligenciada em Portugal. Cento e sessenta e quatro isolados de T. rubrum e T. interdigitale foram identificados até à espécie através de tecnologias moleculares e também com recurso a espectoctrometria de massa (MALDI-TOF). De seguida, verificámos a presença da resistência ao antifúngico terbinafina nestes isolados e pesquisámos as mutações associadas a essa mesma resistência. Para tal, procedemos à otimização e implementação de metodologias que permitiram efetuar a avaliação da resistência de isolados de T. rubrum e T. interdigitale, quer por método cultural quer por metodologias moleculares com sequenciação do gene da esqualeno epoxidase (SQLE) seguida de deteção de mutações neste gene que conferem resistência a este composto.
A caracterização dos padrões de suscetibilidade ao antifúngico terbinafina foi feita recorrendo a meios de screening (agar suplementado com terbinafina) e ao método das microdiluições em caldo. Numa primeira abordagem, de 102 isolados de T. rubrum, apenas 1 isolado (nº126) revelou ser resistente á terbinafina por meio de screening e dos 17 isolados T. interdigitale, apenas 3 demonstraram ser resistentes à terbinafina por meio de screening em ambas as concentrações utilizadas definidas pela norma EUCAST (0.06 mg/L e 0.125 mg/L). De acordo com a metodologia das microdiluições, 2,44% dos isolados apresentaram menor suscetibilidade à terbinafina. Os nossos resultados demonstram que o isolado nº126 (T. rubrum) revelou ser bastante resistente à terbinafina com uma CMI de 8 mg/L, os isolados nº 63 e nº94 (T. interdigitale) apresentaram CMIs superiores a 8 mg/L o que representa uma baixa suscetibilidade à terbinafina. O isolado T. interdigitale nº 59 apresentou uma CMI igual a 1 mg/L o que revela uma resistência moderada à terbinafina. A otimização da PCR de amplificação e sequenciação do gene da SQLE de T. rubrum e T. interdigitale permitiu a deteção de mutações pontuais apenas num isolado de cada espécie num total de quatro isolados classificados como resistentes. O isolado T. rubrum nº126, após análise da sequência do gene da SQLE, apresentou uma mutação pontual na posição 1189 na ORF do gene da SQLE o que corresponde a uma substituição de uma fenilalanina na posição 397 da sequência de aminoácidos da proteína por uma isoleucina. Apesar de as substituições mais comuns e que levam a uma maior resistência à terbinafina serem L393F e F397L, a substituição F397I detetada no isolado T. rubrum deste estudo, sugere causar uma elevada resistência à terbinafina. Relativamente aos isolados resistentes T. interdigitale, apenas o isolado nº 94 apresentou uma mutação pontual na posição 1189 na ORF do gene da SQLE, ocorrendo uma substituição de uma fenilalanina na posição 397 da sequência de aminoácidos da proteína por uma leucina, o que confirma a sua CMI superior a 8 mg/L. Nos restantes isolados resistentes pertencentes à espécie T. interdigitale, não foram encontradas quaisquer mutações no gene que codifica a SQLE.
Este estudo, permitiu-nos efetuar uma caracterização da população amostrada, das amostras biológicas e dos isolados do género Trichophyton obtidos por cultura destas amostras. Foi também possível detetar, pela primeira vez, isolados do género Trichophyton resistentes à terbinafina em Portugal. Foi ainda possível estimar, dentro dos isolados estudados, uma frequência de resistência de 2,44%. A resistência à terbinafina por parte de T. rubrum e T. interdigitale é um problema real em todo o mundo e estima-se que os casos continuem em aumentar e por isso acreditamos que, a aplicação do método de screening e das microdiluições bem como a implementação da reação de PCR para pesquisa de mutações que conferem essa mesma resistência será uma mais valia e um contributo importante no que toca ao diagnóstico e posterior terapia das dermatofitoses reportadas em Portugal.
Dermatophytes are a group of filamentous fungi with a cosmopolitan distribution, which have the ability to invade keratinized tissues (skin, hair and nails) of humans and other animals and cause a superficial infection called dermatophytosis. Dermatophytosis are responsible for 3 - 4% of cases of dermatological disorders, being the most common superficial infection in humans (with a prevalence of 20-25%). However, it is expected that this prevalence will continue to increase, due to the appearance of new risk factors. Species of the genus Trichophyton are the main causative agents of pathologies such as athlete's foot and onychomycosis in the hands and feet and are estimated to affect ~ 1,000,000,000 people worldwide. T. rubrum and T. interdigitale are the main etiological agents with a prevalence of 80% and 20%, respectively. In Portugal, it is estimated that 1,510,391 of Portuguese suffer from dermatophytosis, corresponding to an incidence of 14,300 infections by dermatophytes per 1,000,000 inhabitants. For the treatment of dermatophytoses, the first therapy to be prescribed is, in the majority of the cases, terbinafine. Since 2017, cases of resistance to this antifungal by T. rubrum and T. interdigitale have been gradually increasing. The mechanisms of resistance to terbinafine are essentially described as point mutations in the gene encoding squalene epoxidase. Thus, in this study, we sought to understand whether the problem of resistance to the terbinafine antifungal in T. rubrum and T. interdigitale would be neglected or not in Portugal. One hundred and sixty-four isolates of T. rubrum and T. interdigitale were identified up to the species using molecular technologies and also using mass spectrometry (MALDI-TOF). Then, we verified the presence of resistance to the antifungal agent terbinafine in these isolates and investigated the mutations associated with that same resistance. To this end, we proceeded to the optimization and implementation of methodologies that made it possible to evaluate the resistance of T. rubrum and T. interdigitale isolates, either by cultural method or by molecular methodologies with sequencing of the squalene epoxidase gene (SQLE) followed by detection of mutations in this gene that confer resistance to this compound. The characterization of the susceptibility patterns to the terbinafine antifungal was performed using agar screening medium (agar supplemented with terbinafine) and the broth microdilution method. In a first approach, of 102 isolates of T. rubrum, only one isolate (nº 126) showed to be resistant to terbinafine and of the 17 isolates of T. interdigitale, only 3 demonstrated to be resistant to terbinafine by screening agar medium in both concentrations used defined by EUCAST (0.06 mg/L and 0.125 mg/L). According to the microdilution method, 2,44% of the isolates were less susceptible to terbinafine. Our results demonstrate that isolate nº 126 (T. rubrum) proved to be quite resistant to terbinafine with a MIC of 8 mg/L, isolates nº 63 and 94 (T. interdigitale) had MICs greater than 8 mg/L, which represents a low susceptibility to terbinafine. The isolate T. interdigitale nº 59 presented a MIC equal to 1 mg / L, which reveals a moderate resistance to terbinafine. The optimization of the PCR for amplification and sequencing of the SQLE gene of T. rubrum and T. interdigitale allowed the detection of point mutations in only one isolate of each species in a total of four isolates classified as resistant. The isolate T. rubrum nº126, after sequence analysis of the SQLE gene, showed a point mutation at the position 1189 in the ORF of the SQLE gene, which corresponds to a substitution of a phenylalanine at the position 397 of the amino acid sequence of the protein with an isoleucine. Although the most common substitutions that lead to greater resistance to terbinafine are L393F and F397L, the F397I substitution detected in the isolate T. rubrum in this study, suggests causing a high resistance to terbinafine. Regarding resistant isolates T. interdigitale, only isolate 94 showed a point mutation at position 1189 in the SQLE gene ORF, with a substitution of a phenylalanine at position 397 of the protein's amino acid sequence for a leucine, which confirms the its MIC greater than 8 mg / L. In the remaining resistant isolates belonging to the species T. interdigitale, no mutations were found in the gene encoding SQLE. This study allowed us to characterize the sampled population, the obtained biological samples and characterize the isolates of the genus Trichophyton obtained by culture. It was also possible to detect, for the first time in Portugal, terbinafine resistant isolates belonging to the genus Trichophyton to . It was also possible to estimate, within the studied isolates, a resistance frequency of 2.44%. Resistance to terbinafine in T. rubrum and T. interdigitale is a real problem worldwide and it is estimated that cases continue to increase and that is why we believe that the application of the screening method and microdilutions, as well as the implementation of the PCR reaction to search for mutations that confer this same resistance will be an important contribution with regard to the diagnosis and subsequent therapy of the dermatophytosis reported in Portugal.
Dermatophytes are a group of filamentous fungi with a cosmopolitan distribution, which have the ability to invade keratinized tissues (skin, hair and nails) of humans and other animals and cause a superficial infection called dermatophytosis. Dermatophytosis are responsible for 3 - 4% of cases of dermatological disorders, being the most common superficial infection in humans (with a prevalence of 20-25%). However, it is expected that this prevalence will continue to increase, due to the appearance of new risk factors. Species of the genus Trichophyton are the main causative agents of pathologies such as athlete's foot and onychomycosis in the hands and feet and are estimated to affect ~ 1,000,000,000 people worldwide. T. rubrum and T. interdigitale are the main etiological agents with a prevalence of 80% and 20%, respectively. In Portugal, it is estimated that 1,510,391 of Portuguese suffer from dermatophytosis, corresponding to an incidence of 14,300 infections by dermatophytes per 1,000,000 inhabitants. For the treatment of dermatophytoses, the first therapy to be prescribed is, in the majority of the cases, terbinafine. Since 2017, cases of resistance to this antifungal by T. rubrum and T. interdigitale have been gradually increasing. The mechanisms of resistance to terbinafine are essentially described as point mutations in the gene encoding squalene epoxidase. Thus, in this study, we sought to understand whether the problem of resistance to the terbinafine antifungal in T. rubrum and T. interdigitale would be neglected or not in Portugal. One hundred and sixty-four isolates of T. rubrum and T. interdigitale were identified up to the species using molecular technologies and also using mass spectrometry (MALDI-TOF). Then, we verified the presence of resistance to the antifungal agent terbinafine in these isolates and investigated the mutations associated with that same resistance. To this end, we proceeded to the optimization and implementation of methodologies that made it possible to evaluate the resistance of T. rubrum and T. interdigitale isolates, either by cultural method or by molecular methodologies with sequencing of the squalene epoxidase gene (SQLE) followed by detection of mutations in this gene that confer resistance to this compound. The characterization of the susceptibility patterns to the terbinafine antifungal was performed using agar screening medium (agar supplemented with terbinafine) and the broth microdilution method. In a first approach, of 102 isolates of T. rubrum, only one isolate (nº 126) showed to be resistant to terbinafine and of the 17 isolates of T. interdigitale, only 3 demonstrated to be resistant to terbinafine by screening agar medium in both concentrations used defined by EUCAST (0.06 mg/L and 0.125 mg/L). According to the microdilution method, 2,44% of the isolates were less susceptible to terbinafine. Our results demonstrate that isolate nº 126 (T. rubrum) proved to be quite resistant to terbinafine with a MIC of 8 mg/L, isolates nº 63 and 94 (T. interdigitale) had MICs greater than 8 mg/L, which represents a low susceptibility to terbinafine. The isolate T. interdigitale nº 59 presented a MIC equal to 1 mg / L, which reveals a moderate resistance to terbinafine. The optimization of the PCR for amplification and sequencing of the SQLE gene of T. rubrum and T. interdigitale allowed the detection of point mutations in only one isolate of each species in a total of four isolates classified as resistant. The isolate T. rubrum nº126, after sequence analysis of the SQLE gene, showed a point mutation at the position 1189 in the ORF of the SQLE gene, which corresponds to a substitution of a phenylalanine at the position 397 of the amino acid sequence of the protein with an isoleucine. Although the most common substitutions that lead to greater resistance to terbinafine are L393F and F397L, the F397I substitution detected in the isolate T. rubrum in this study, suggests causing a high resistance to terbinafine. Regarding resistant isolates T. interdigitale, only isolate 94 showed a point mutation at position 1189 in the SQLE gene ORF, with a substitution of a phenylalanine at position 397 of the protein's amino acid sequence for a leucine, which confirms the its MIC greater than 8 mg / L. In the remaining resistant isolates belonging to the species T. interdigitale, no mutations were found in the gene encoding SQLE. This study allowed us to characterize the sampled population, the obtained biological samples and characterize the isolates of the genus Trichophyton obtained by culture. It was also possible to detect, for the first time in Portugal, terbinafine resistant isolates belonging to the genus Trichophyton to . It was also possible to estimate, within the studied isolates, a resistance frequency of 2.44%. Resistance to terbinafine in T. rubrum and T. interdigitale is a real problem worldwide and it is estimated that cases continue to increase and that is why we believe that the application of the screening method and microdilutions, as well as the implementation of the PCR reaction to search for mutations that confer this same resistance will be an important contribution with regard to the diagnosis and subsequent therapy of the dermatophytosis reported in Portugal.
Description
Dissertação de mestrado em Biologia Humana e Ambiente apresentada à Faculdade de Ciências da Universidade de Lisboa, 2021.
Orientadora Doutora Raquel Filipa Pinheiro Sabino (INSA).
Discussão pública a 24 de novembro de 2021.
Orientadora Doutora Raquel Filipa Pinheiro Sabino (INSA).
Discussão pública a 24 de novembro de 2021.
Keywords
Trichophyton Terbinafina Resistência T. rubrum Infecções Sistémicas e Zoonoses Portugal