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1.
Infect Immun ; 88(2)2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31767773

RESUMO

Aspergillus fumigatus is a ubiquitous fungal pathogen capable of causing multiple pulmonary diseases, including invasive aspergillosis, chronic necrotizing aspergillosis, fungal colonization, and allergic bronchopulmonary aspergillosis. Intact mucociliary barrier function and early airway neutrophil responses are critical for clearing fungal conidia from the host airways prior to establishing disease. Following inhalation, Aspergillus conidia deposit in the small airways, where they are likely to make their initial host encounter with epithelial cells. Challenges in airway infection models have limited the ability to explore early steps in the interactions between A. fumigatus and the human airway epithelium. Here, we use inverted air-liquid interface cultures to demonstrate that the human airway epithelium responds to apical stimulation by A. fumigatus to promote the transepithelial migration of neutrophils from the basolateral membrane surface to the apical airway surface. Promoting epithelial transmigration with Aspergillus required prolonged exposure with live resting conidia. Swollen conidia did not expedite epithelial transmigration. Using A. fumigatus strains containing deletions of genes for cell wall components, we identified that deletion of the hydrophobic rodlet layer or dihydroxynaphthalene-melanin in the conidial cell wall amplified the epithelial transmigration of neutrophils, using primary human airway epithelium. Ultimately, we show that an as-yet-unidentified nonsecreted cell wall protein is required to promote the early epithelial transmigration of human neutrophils into the airspace in response to A. fumigatus Together, these data provide critical insight into the initial epithelial host response to Aspergillus.


Assuntos
Aspergilose/imunologia , Aspergillus fumigatus/imunologia , Parede Celular/imunologia , Células Epiteliais/imunologia , Neutrófilos/imunologia , Aspergilose/microbiologia , Linhagem Celular Tumoral , Células Epiteliais/microbiologia , Humanos , Pulmão/imunologia , Pulmão/microbiologia , Melaninas/imunologia , Naftóis/imunologia , Esporos Fúngicos/imunologia
2.
Eur J Ophthalmol ; 30(1): 88-93, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30474397

RESUMO

PURPOSE: To identify the causative microorganism of fungal endogenous endophthalmitis in our tertiary referral uveitis center and review the therapeutic role of pars plana vitrectomy in patients with fungal endogenous endophthalmitis. METHODS: Seven eyes of six cases were identified as fungal endogenous endophthalmitis through positive cultures of ocular fluids and clinical presentations. The final anatomical and functional results were evaluated. RESULTS: Four women (66.7%) and two men (33.3%) underwent vitrectomy. Control of infection was achieved early on in all cases. Candida (71.4%) and Aspergillus (28.6%) species were identified as causative fungi in patients with fungal endogenous endophthalmitis. Two patients were reoperated due to reinfection and retinal detachment, respectively. Visual acuity improved in six eyes (85.7%) and worsened in one eye (14.3%). At the final examination, the retina was flat in all cases. No eye developed phthisis bulbi. CONCLUSION: Candida species are the most common causative organisms of fungal endogenous endophthalmitis in this study. Pars plana vitrectomy in fungal endogenous endophthalmitis may enhance the treatment of infection by removing fungal elements in the vitreous and aid in diagnosis. Vitrectomy may also be an important tool in the management of vision-threatening post-infectious sequelae such as retinal detachment and reinfections.


Assuntos
Aspergilose/cirurgia , Candidíase/cirurgia , Endoftalmite/cirurgia , Infecções Oculares Fúngicas/cirurgia , Vitrectomia/métodos , Adulto , Idoso , Aspergilose/microbiologia , Aspergilose/fisiopatologia , Candidíase/microbiologia , Candidíase/fisiopatologia , Endoftalmite/microbiologia , Endoftalmite/fisiopatologia , Infecções Oculares Fúngicas/microbiologia , Infecções Oculares Fúngicas/fisiopatologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reoperação , Descolamento Retiniano/cirurgia , Estudos Retrospectivos , Centros de Atenção Terciária , Acuidade Visual/fisiologia , Corpo Vítreo/microbiologia , Adulto Jovem
3.
Vet Clin North Am Small Anim Pract ; 50(2): 331-357, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31866094

RESUMO

Fungal rhinosinusitis, including sinonasal aspergillosis (SNA) and sino-orbital aspergillosis (SOA), is the most common type of aspergillosis encountered in cats. Other focal forms of aspergillosis including disseminated invasive aspergillosis occur less frequently. SOA is an invasive mycosis that is increasingly recognized and is most commonly caused by Aspergillus felis, a close relative of Aspergillus fumigatus. SNA can be invasive or noninvasive and is most commonly caused by A fumigatus and Aspergillus niger. Molecular methods are required to correctly identify the fungi that cause SNA and SOA. SNA has a favorable prognosis with treatment, whereas the prognosis for SOA remains poor.


Assuntos
Aspergilose/veterinária , Doenças do Gato/microbiologia , Sinusite/veterinária , Animais , Antifúngicos/uso terapêutico , Aspergilose/diagnóstico , Aspergilose/tratamento farmacológico , Aspergilose/microbiologia , Aspergillus fumigatus/isolamento & purificação , Aspergillus niger/isolamento & purificação , Doenças do Gato/diagnóstico , Doenças do Gato/tratamento farmacológico , Gatos , Diagnóstico Diferencial , Sinusite/diagnóstico , Sinusite/tratamento farmacológico , Sinusite/microbiologia
4.
Nat Commun ; 10(1): 4275, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31537789

RESUMO

Calcineurin is important for fungal virulence and a potential antifungal target, but compounds targeting calcineurin, such as FK506, are immunosuppressive. Here we report the crystal structures of calcineurin catalytic (CnA) and regulatory (CnB) subunits complexed with FK506 and the FK506-binding protein (FKBP12) from human fungal pathogens (Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans and Coccidioides immitis). Fungal calcineurin complexes are similar to the mammalian complex, but comparison of fungal and human FKBP12 (hFKBP12) reveals conformational differences in the 40s and 80s loops. NMR analysis, molecular dynamic simulations, and mutations of the A. fumigatus CnA/CnB-FK506-FKBP12-complex identify a Phe88 residue, not conserved in hFKBP12, as critical for binding and inhibition of fungal calcineurin. These differences enable us to develop a less immunosuppressive FK506 analog, APX879, with an acetohydrazine substitution of the C22-carbonyl of FK506. APX879 exhibits reduced immunosuppressive activity and retains broad-spectrum antifungal activity and efficacy in a murine model of invasive fungal infection.


Assuntos
Antifúngicos/farmacologia , Aspergillus fumigatus/metabolismo , Inibidores de Calcineurina/farmacologia , Calcineurina/metabolismo , Cryptococcus neoformans/metabolismo , Proteína 1A de Ligação a Tacrolimo/metabolismo , Tacrolimo/farmacologia , Animais , Aspergilose/tratamento farmacológico , Aspergilose/microbiologia , Aspergillus fumigatus/efeitos dos fármacos , Sítios de Ligação , Candida albicans/efeitos dos fármacos , Candida albicans/metabolismo , Células Cultivadas , Coccidioides/efeitos dos fármacos , Coccidioides/metabolismo , Criptococose/tratamento farmacológico , Criptococose/microbiologia , Cryptococcus neoformans/efeitos dos fármacos , Cristalografia por Raios X , Descoberta de Drogas/métodos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos C57BL , Simulação de Dinâmica Molecular , Tacrolimo/metabolismo
5.
mSphere ; 4(4)2019 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-31391280

RESUMO

The genetic stability of every living organism depends on accurate DNA replication and repair systems. Here, we investigated the Aspergillus fumigatus MSH2 mismatch repair (MMR) gene MshA and how it impacts virulence and the evolution of azole resistance. We examined mshA gene variation in 62 environmental and clinical A. fumigatus strains. We have observed 12 strains with variants (18.2%), and 8 strains among them showed missense variants. We demonstrated that A. fumigatus mshA null mutants are haploid and have conserved karyotypes with discrete gross chromosomal rearrangements. The ΔmshA strains are not sensitive to several DNA-damaging agents. The lack of mshA caused a significant reduction of virulence of A. fumigatus in a neutropenic murine model of invasive pulmonary aspergillosis and in the invertebrate alternative model Galleria mellonella Wild-type and ΔmshA populations did not show any significant changes in drug resistance acquisition after they were transferred 10 times in minimal medium in the absence of any stress. However, these populations rapidly acquired virulence in the ΔmshA background and high levels of resistance to posaconazole in the presence of this drug (at least 200-fold-higher levels of resistance than those derived from the wild-type strain). Taken together, these results suggest that genetic instability caused by ΔmshA mutations can confer an adaptive advantage, mainly increasing posaconazole resistance and virulence acquisition.IMPORTANCE Invasive aspergillosis (IA) has emerged as one of the most common life-threatening fungal diseases in immunocompromised patients, with mortality rates as high as 90%. Systemic fungal infections such as IA are usually treated with triazoles; however, epidemiological research has shown that the prevalence of azole-resistant Aspergillus fumigatus isolates has increased significantly over the last decade. There is very little information about the importance of genomic stability for A. fumigatus population structure, azole resistance, and virulence. Here, we decided to investigate whether the mismatch repair system could influence A. fumigatus azole resistance and virulence, focusing on one of the components of this system, MSH2 Although the mutation frequency of mshA (the A. fumigatus MSH2 homologue) is low in environmental and clinical isolates, our results indicate that loss of mshA function can provide increased azole resistance and virulence when selected for. These results demonstrate the importance of genetic instability in A. fumigatus as a possible mechanism of evolving azole resistance and establishing fitness in the host.


Assuntos
Antifúngicos/farmacologia , Aspergillus fumigatus/genética , Aspergillus fumigatus/patogenicidade , Azóis/farmacologia , Farmacorresistência Fúngica , Proteína 2 Homóloga a MutS/genética , Animais , Aspergilose/microbiologia , Aspergillus fumigatus/efeitos dos fármacos , Reparo de Erro de Pareamento de DNA , Feminino , Proteínas Fúngicas/genética , Larva/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Mariposas/microbiologia , Neutropenia , Homologia de Sequência , Virulência
6.
mSphere ; 4(4)2019 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-31434750

RESUMO

Jorge Amich studies several aspects of sulfur and nitrogen metabolism in Aspergillus fumigatus, with the ultimate aim of identifying targets for the development of novel antifungals. In this mSphere of Influence article, he reflects on how "Sub-Telomere Directed Gene Expression during Initiation of Invasive Aspergillosis" (A. McDonagh, N. D. Fedorova, J. Crabtree, Y. Yu, S. Kim, et al., PLoS Pathog 4:e1000154, 2008, https://doi.org/10.1371/journal.ppat.1000154) impacted his thinking about in vivo metabolism and how to investigate it.


Assuntos
Adaptação Fisiológica , Aspergillus fumigatus/metabolismo , Aspergillus fumigatus/patogenicidade , Interações Hospedeiro-Patógeno , Antifúngicos/farmacologia , Aspergilose/microbiologia , Metabolismo Secundário , Virulência
8.
J Med Life ; 12(2): 128-132, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31406513

RESUMO

Aspergillus species (sp.) that causes opportunistic infections have been increasingly found in human mainly immunosuppressive patients around the world every year. The main objective was to use a rapid and cheap molecular method for monitoring Aspergillus infections and epidemiological approaches. In order to identity Aspergilli species (spp.), a number of molecular methods including restriction fragment length polymorphism (RFLP) have been employed in accordance with ribosomal RNA amplification. The focus of this study - a group of hospitalized patients with clinical and subclinical signs of infection. All of the collected clinical specimens were transported to the medical mycology lab and examined for Aspergillus identification. The environmental specimens were collected from air and surfaces inspected for the Aspergillus within the hospital sources. At first, growth characteristics and microscopic features on mycological media for the identification of Aspergillus sp. were performed. For the confirmation of Aspergillus isolates which similarly found in clinical and environmental sources, molecular method polymerase chain reaction/restriction fragment length polymorphism was carried out. From the mentioned specimens, 102 fungal isolates included Candida spp., Aspergillus spp. and other fungi. Aspergillus flavus (47%), Aspergillus fumigatus (29.4%) and Aspergillus niger (23.5%) all were found as the most common clinical isolates. In addition, Aspergillus isolates from environmental were Aspergillus niger (43.7%), Aspergillus flavus (41.7%), Aspergillus fumigatus (14.6%). Therefore, polymerase chain reaction-restriction fragment length polymorphism with a single restriction enzyme can be very useful in the identification of Aspergillus spp., because of its facility in use, speed, robust, and high sensitivity of diagnosis.


Assuntos
Aspergilose/microbiologia , Aspergillus/fisiologia , Infecção Hospitalar/microbiologia , Meio Ambiente , Hospitais Universitários , Aspergillus/isolamento & purificação , Humanos , Polimorfismo de Fragmento de Restrição
9.
BMJ Case Rep ; 12(8)2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31420430

RESUMO

A male patient in his mid-60s presented with a severe pneumonia following return to the UK after travel to Crete. He was diagnosed with Legionnaire's disease (caused by an uncommon serogroup of Legionella pneumophila). He was pancytopenic on admission, and during a long stay on critical care he was diagnosed with a disseminated Aspergillus infection. Bone marrow aspiration revealed an underlying hairy cell leukaemia that undoubtedly contributed to his acute presentation and subsequent invasive fungal infection.


Assuntos
Aspergillus , Legionella pneumophila , Doença dos Legionários/microbiologia , Pneumonia/microbiologia , Doença Relacionada a Viagens , Aspergilose/microbiologia , Grécia , Humanos , Leucemia de Células Pilosas/microbiologia , Masculino , Pessoa de Meia-Idade , Reino Unido
10.
Mycoses ; 62(11): 1035-1042, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31402465

RESUMO

Invasive aspergillosis (IA) is a severe complication in immunocompromised patients. Early diagnosis is crucial to decrease its high mortality, yet the diagnostic gold standard (histopathology and culture) is time-consuming and cannot offer early confirmation of IA. Detection of IA by polymerase chain reaction (PCR) shows promising potential. Various studies have analysed its diagnostic performance in different clinical settings, especially addressing optimal specimen selection. However, direct comparison of different types of specimens in individual patients though essential, is rarely reported. We systematically assessed the diagnostic performance of an Aspergillus-specific nested PCR by investigating specimens from the site of infection and comparing it with concurrent blood samples in individual patients (pts) with IA. In a retrospective multicenter analysis PCR was performed on clinical specimens (n = 138) of immunocompromised high-risk pts (n = 133) from the site of infection together with concurrent blood samples. 38 pts were classified as proven/probable, 67 as possible and 28 as no IA according to 2008 European Organization for Research and Treatment of Cancer/Mycoses Study Group consensus definitions. A considerably superior performance of PCR from the site of infection was observed particularly in pts during antifungal prophylaxis (AFP)/antifungal therapy (AFT). Besides a specificity of 85%, sensitivity varied markedly in BAL (64%), CSF (100%), tissue samples (67%) as opposed to concurrent blood samples (8%). Our results further emphasise the need for investigating clinical samples from the site of infection in case of suspected IA to further establish or rule out the diagnosis.


Assuntos
Aspergilose/diagnóstico , Hospedeiro Imunocomprometido , Infecções Fúngicas Invasivas/diagnóstico , Técnicas de Diagnóstico Molecular/normas , Reação em Cadeia da Polimerase/normas , Adolescente , Adulto , Idoso , Aspergilose/sangue , Aspergilose/microbiologia , Aspergillus/isolamento & purificação , Criança , Pré-Escolar , Feminino , Humanos , Infecções Fúngicas Invasivas/sangue , Infecções Fúngicas Invasivas/microbiologia , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular/métodos , Estudos Retrospectivos , Sensibilidade e Especificidade , Adulto Jovem
11.
ACS Chem Biol ; 14(7): 1643-1651, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31265232

RESUMO

Fungal secondary metabolites (SMs) include medically valuable compounds as well as compounds that are toxic, carcinogenic, and/or contributors to fungal pathogenesis. It is consequently important to understand the regulation of fungal secondary metabolism. McrA is a recently discovered transcription factor that negatively regulates fungal secondary metabolism. Deletion of mcrA (mcrAΔ), the gene encoding McrA, results in upregulation of many SMs and alters the expression of more than 1000 genes. One gene strongly upregulated by the deletion of mcrA is llmG, a putative methyl transferase related to LaeA, a major regulator of secondary metabolism. We artificially upregulated llmG by replacing its promoter with strong constitutive promoters in strains carrying either wild-type mcrA or mcrAΔ. Upregulation of llmG on various media resulted in increased production of the important toxin sterigmatocystin and compounds from at least six major SM pathways. llmG is, thus, a master SM regulator. mcrAΔ generally resulted in greater upregulation of SMs than upregulation of llmG, indicating that the full effects of mcrA on secondary metabolism involve genes in addition to llmG. However, the combination of mcrAΔ and upregulation of llmG generally resulted in greater compound production than mcrAΔ alone (in one case more than 460 times greater than the control). This result indicates that deletion of mcrA and/or upregulation of llmG can likely be combined with other strategies for eliciting SM production to greater levels than can be obtained with any single strategy.


Assuntos
Aspergillus nidulans/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Metiltransferases/genética , Aspergilose/microbiologia , Aspergillus nidulans/metabolismo , Proteínas Fúngicas/metabolismo , Humanos , Metiltransferases/metabolismo , Metabolismo Secundário , Esterigmatocistina/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para Cima
12.
Eur J Clin Microbiol Infect Dis ; 38(11): 2021-2027, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31332609

RESUMO

To investigate the expression of AFB1 gene in isolates obtained from corneal scrapping samples from keratitis patients and to correlate the quantity of AFB1 to the severity of keratitis. An observational study was undertaken in Medical Microbiology and Immunology department, Mansoura University, Egypt, over corneal scrapping samples that were cultured aiming to isolate fungal causes of infective keratitis followed by AFB1 gene detection in Aspergillus flavus isolates by nested PCR then quantitation of the toxin by TLC. Out of 843 corneal scrapping samples collected from patients with infective keratitis, positive fungal growth was identified in 277 cases (32.9%). A. flavus was the commonest fungal agent isolated in 93 cases (33.6%). The AFB1 toxin-encoding gene was detected in 63.4% of A. flavus isolates. There was a positive correlation between the quantity of produced AFB1 toxin and the degree of severity of keratitis (P value < 0.0001*). Aspergillus flavus was the most common cause of fungal keratitis, with the AFB1 toxin-encoding gene detected in more than half of the isolates. A significant correlation between the degree of severity of keratitis and the quantity of produced AFB1 toxin was detected. Therefore, exploring presence or absence of AFB1 toxin is important for the clinicians in their diagnostic assessment and selection of proper treatment choices.


Assuntos
Aflatoxina B1/metabolismo , Aspergilose/patologia , Aspergillus flavus/metabolismo , Ceratite/patologia , Adolescente , Adulto , Aflatoxina B1/genética , Aspergilose/microbiologia , Aspergillus flavus/genética , Aspergillus flavus/isolamento & purificação , Biomarcadores/metabolismo , Criança , Córnea/microbiologia , Córnea/patologia , Egito , Feminino , Humanos , Ceratite/microbiologia , Masculino , Pessoa de Meia-Idade , Centros de Atenção Terciária , Adulto Jovem
13.
J Microbiol Immunol Infect ; 52(5): 728-735, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31302087

RESUMO

BACKGOUND: Conventional diagnosis of invasive fungal disease from blood cultures is often notoriously delayed and inadequately sensitive. We aimed to develop a universal primers-based polymerase chain reaction (PCR) assay and restriction fragment length polymorphisms (RFLP) for rapid identification of invasive fungal disease (IFD). METHODS: We evaluated 16 clinical fungal species using a combination of PCR assays with 3 different restriction endonucleases targeting various internal transcribed spacer (ITS) regions and high resolution melting analysis (HRMA). Serial samples from 75 patients suspected to have IFD were analyzed for clinical verification. RESULTS: We have designed a universal PCR capable of amplifying a portion of the 18S rRNA gene of 16 clinically important fungal species. The restriction patterns of most PCR products generated by EcoRI or double digested by ClaI and AvaI were different, except Aspergillus niger and Aspergillus flavus had a similar pattern, and Aspergillus fumigatus and Aspergillus terreus had a similar pattern. All these species had a unique melting curve shape using the HRMA. Both HRMA and universal PCR had adequate sensitivity, and all sixteen reference fungal species can be clearly distinguished by the universal PCR-RFLP-HRMA assay. With a reference library of 176 clinically relevant fungal strains, and 75 clinical samples from patients with suspicious IFD were tested, our assay identified 100% and 61.1% of isolates from the reference library and clinical samples, respectively. CONCLUSIONS: Universal PCR and RFLP coupled with HRMA could be a highly discriminative and useful molecular diagnostic that could enhance the current diagnostic, treatment, and surveillance methods of invasive fungal disease.


Assuntos
Primers do DNA , Enzimas de Restrição do DNA , Fungos/isolamento & purificação , Espécies Introduzidas , Técnicas de Tipagem Micológica/métodos , Micoses/microbiologia , Reação em Cadeia da Polimerase/métodos , Aspergilose/diagnóstico , Aspergilose/microbiologia , Aspergillus/classificação , Aspergillus/genética , Aspergillus/isolamento & purificação , Candida/classificação , Candida/genética , Candida/isolamento & purificação , DNA Fúngico/genética , Fungos/classificação , Fungos/genética , Humanos , Micoses/diagnóstico , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 18S/genética , Sensibilidade e Especificidade
14.
Diagn Microbiol Infect Dis ; 95(2): 171-178, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31239090

RESUMO

The study evaluated the change in the clinical antifungal sensitivity profile of A. flavus strains after exposure to azole and benzimidazole fungicide. Exposure to fungicide altered the sensitivity profile for the antifungal itraconazole, voriconazole and posaconazole. This change was characterized by an increase in the minimum inhibitory concentration (MIC) from 16 to 32 times, evidencing the development of resistance phenotypes. The most significant changes were found after exposure to a pool of the fungicide with MIC of up to 256 times, which is considered, to the best of our knowledge, the first case report of such a high level of resistance induced by azole fungicide exposure. This observation probably indicates a synergistic action among azole compounds that potentiates the development of resistance phenotypes. In addition, exposure to fungicide changed the pigmentation of the colonies from green to white. The development of resistance to fungicides represents risks to human health, since azole fungicides are used widely in the agriculture, and a single agricultural fungicide spray often includes more than one azole compound.


Assuntos
Aspergillus flavus/efeitos dos fármacos , Azóis/farmacologia , Benzimidazóis/farmacologia , Farmacorresistência Fúngica , Fungicidas Industriais/farmacologia , Antifúngicos/farmacologia , Aspergilose/microbiologia , Aspergillus flavus/crescimento & desenvolvimento , Aspergillus flavus/isolamento & purificação , Farmacorresistência Fúngica/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Testes de Sensibilidade Microbiana
15.
Exp Eye Res ; 186: 107700, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31233730

RESUMO

Corneal mycotic ulceration is predominantly due to Aspergillus and Fusarium solani infection in tropical countries. In this study, we examined the proteome profile of tear samples from A. flavus keratitis patients at various stages of infection. The proteome was profiled using 2D PAGE and the protein levels were quantified using 2D DIGE. Alpha-1-antitrypsin, apolipoprotein, haptoglobin, lactoferrin and albumin were up regulated while cystatin SA III precursor, lacrimal lipocalin precursor, lacritin precursor and Zinc alpha-2 glycoprotein (ZAG) were down regulated in tear fluid. In the case of ZAG all proteoforms were down regulated as the disease progressed from early to late stage of infection. Western blot analysis confirmed the results observed using DIGE. Further, there were no gender specific differences in the levels of ZAG expression in keratitis patient tear film. Published results show up regulation of ZAG in Fusarium keratitis patient tear indicating subtle changes in the early events of host response to these two fungal pathogens. We conclude that ZAG level could be used as an indicator of A. flavus or F. solani infection, even during the early stage of the disease.


Assuntos
Aspergilose , Aspergillus flavus , Infecções Oculares Fúngicas , Ceratite , Proteoma/metabolismo , Proteínas de Plasma Seminal/metabolismo , Lágrimas/metabolismo , Adolescente , Adulto , Idoso , Aspergilose/metabolismo , Aspergilose/microbiologia , Regulação para Baixo , Infecções Oculares Fúngicas/metabolismo , Infecções Oculares Fúngicas/microbiologia , Feminino , Humanos , Ceratite/metabolismo , Ceratite/microbiologia , Masculino , Pessoa de Meia-Idade , Adulto Jovem
16.
Emerg Infect Dis ; 25(7): 1347-1353, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31211684

RESUMO

Azole resistance is a major concern for treatment of infections with Aspergillus fumigatus. Environmental resistance selection is a main route for Aspergillus spp. to acquire azole resistance. We investigated the presence of environmental hotspots for resistance selection in the Netherlands on the basis of the ability of A. fumigatus to grow and reproduce in the presence of azole fungicide residues. We identified 3 hotspots: flower bulb waste, green waste material, and wood chippings. We recovered azole-resistant A. fumigatus from these sites; all fungi contained cyp51A tandem repeat-mediated resistance mechanisms identical to those found in clinical isolates. Tebuconazole, epoxiconazole, and prothioconazole were the most frequently found fungicide residues. Stockpiles of plant waste contained the highest levels of azole-resistant A. fumigatus, and active aerobic composting reduced Aspergillus colony counts. Preventing plant waste stockpiling or creating unfavorable conditions for A. fumigatus to grow in stockpiles might reduce environmental resistance burden.


Assuntos
Antifúngicos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Azóis/farmacologia , Farmacorresistência Fúngica , Microbiologia Ambiental , Antifúngicos/uso terapêutico , Aspergilose/tratamento farmacológico , Aspergilose/epidemiologia , Aspergilose/microbiologia , Aspergillus fumigatus/classificação , Aspergillus fumigatus/genética , Aspergillus fumigatus/isolamento & purificação , Azóis/uso terapêutico , Genes Bacterianos , Humanos , Testes de Sensibilidade Microbiana , Países Baixos/epidemiologia
17.
mBio ; 10(3)2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-31164462

RESUMO

Aspergillus fumigatus is a leading cause of invasive fungal infections. Resistance to first-line triazole antifungals has led to therapy with echinocandin drugs. Recently, we identified several high-minimum-effective-concentration (MEC) A. fumigatus clinical isolates from patients failing echinocandin therapy. Echinocandin resistance is known to arise from amino acid substitutions in ß-(1,3)-d-glucan synthase encoded by the fks1 gene. Yet these clinical isolates did not contain mutations in fks1, indicating an undefined resistance mechanism. To explore this new mechanism, we used a laboratory-derived strain, RG101, with a nearly identical caspofungin (CAS) susceptibility phenotype that also does not contain fks1 mutations. Glucan synthase isolated from RG101 was fully sensitive to echinocandins. Yet exposure of RG101 to CAS during growth yielded a modified enzyme that was drug insensitive (4 log orders) in kinetic inhibition assays, and this insensitivity was also observed for enzymes isolated from clinical isolates. To understand this alteration, we analyzed whole-enzyme posttranslational modifications (PTMs) but found none linked to resistance. However, analysis of the lipid microenvironment of the enzyme with resistance induced by CAS revealed a prominent increase in the abundances of dihydrosphingosine (DhSph) and phytosphingosine (PhSph). Exogenous addition of DhSph and PhSph to the sensitive enzyme recapitulated the drug insensitivity of the CAS-derived enzyme. Further analysis demonstrated that CAS induces mitochondrion-derived reactive oxygen species (ROS) and that dampening ROS formation by antimycin A or thiourea eliminated drug-induced resistance. We conclude that CAS induces cellular stress, promoting formation of ROS and triggering an alteration in the composition of plasma membrane lipids surrounding glucan synthase, rendering it insensitive to echinocandins.IMPORTANCE Resistance to first-line triazole antifungal agents among Aspergillus species has prompted the use of second-line therapy with echinocandins. As the number of Aspergillus-infected patients treated with echinocandins is rising, clinical observations of drug resistance are also increasing, indicating an emerging global health threat. Our knowledge regarding the development of clinical echinocandin resistance is largely derived from Candida spp., while little is known about resistance in Aspergillus. Therefore, it is important to understand the specific cellular responses raised by A. fumigatus against echinocandins. We discovered a new mechanism of resistance in A. fumigatus that is independent of the well-characterized FKS mutation mechanism observed in Candida This study identified an off-target effect of CAS, i.e., ROS production, and integrated oxidative stress and sphingolipid alterations into a novel mechanism of resistance. This stress-induced response has implications for drug resistance and/or tolerance mechanisms in other fungal pathogens.


Assuntos
Antifúngicos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Farmacorresistência Fúngica/genética , Equinocandinas/farmacologia , Glucosiltransferases/genética , Estresse Fisiológico , Aspergilose/microbiologia , Aspergillus fumigatus/enzimologia , Proteínas Fúngicas/genética , Humanos , Testes de Sensibilidade Microbiana , Estresse Oxidativo , Processamento de Proteína Pós-Traducional , Espécies Reativas de Oxigênio/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo
19.
J Vet Diagn Invest ; 31(4): 652-655, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31113331

RESUMO

An adult female Cape porcupine (Hystrix africaeaustralis) was presented because of marked abdominal distension, acute dyspnea, and lethargy. Physical examination and standard radiographs revealed marked and diffuse gaseous dilation of the stomach and intestines of undetermined origin. The porcupine died despite medical therapy and surgical intervention. Postmortem examination revealed chronic fungal (Aspergillus flavus) rhinitis. Given that rodents are obligate nasal breathers, the rhinitis likely resulted in substantial upper airway obstruction leading to aerophagia.


Assuntos
Aspergilose/veterinária , Aspergillus flavus , Porcos-Espinhos , Rinite/veterinária , Doenças dos Roedores/microbiologia , Animais , Animais de Zoológico , Aspergilose/microbiologia , Aspergilose/patologia , Evolução Fatal , Feminino , Gases , Rinite/microbiologia , Doenças dos Roedores/patologia
20.
mBio ; 10(3)2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-31113894

RESUMO

The emergence of azole resistance in the pathogenic fungus Aspergillus fumigatus has continued to increase, with the dominant resistance mechanisms, consisting of a 34-nucleotide tandem repeat (TR34)/L98H and TR46/Y121F/T289A, now showing a structured global distribution. Using hierarchical clustering and multivariate analysis of 4,049 A. fumigatus isolates collected worldwide and genotyped at nine microsatellite loci using analysis of short tandem repeats of A. fumigatus (STRAf), we show that A. fumigatus can be subdivided into two broad clades and that cyp51A alleles TR34/L98H and TR46/Y121F/T289A are unevenly distributed across these two populations. Diversity indices show that azole-resistant isolates are genetically depauperate compared to their wild-type counterparts, compatible with selective sweeps accompanying the selection of beneficial mutations. Strikingly, we found that azole-resistant clones with identical microsatellite profiles were globally distributed and sourced from both clinical and environmental locations, confirming that azole resistance is an international public health concern. Our work provides a framework for the analysis of A. fumigatus isolates based on their microsatellite profile, which we have incorporated into a freely available, user-friendly R Shiny application (AfumID) that provides clinicians and researchers with a method for the fast, automated characterization of A. fumigatus genetic relatedness. Our study highlights the effect that azole drug resistance is having on the genetic diversity of A. fumigatus and emphasizes its global importance upon this medically important pathogenic fungus.IMPORTANCE Azole drug resistance in the human-pathogenic fungus Aspergillus fumigatus continues to emerge, potentially leading to untreatable aspergillosis in immunosuppressed hosts. Two dominant, environmentally associated resistance mechanisms, which are thought to have evolved through selection by the agricultural application of azole fungicides, are now distributed globally. Understanding the effect that azole resistance is having on the genetic diversity and global population of A. fumigatus will help mitigate drug-resistant aspergillosis and maintain the azole class of fungicides for future use in both medicine and crop protection.


Assuntos
Antifúngicos/farmacologia , Aspergilose/microbiologia , Aspergillus fumigatus/efeitos dos fármacos , Azóis/farmacologia , Farmacorresistência Fúngica , Microbiologia Ambiental , Aspergillus fumigatus/classificação , Aspergillus fumigatus/genética , Aspergillus fumigatus/isolamento & purificação , Análise por Conglomerados , Variação Genética , Genótipo , Humanos , Repetições de Microssatélites , Tipagem Molecular , Técnicas de Tipagem Micológica , Filogenia , Sequências de Repetição em Tandem
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