RESUMO
BACKGROUND: Biofilm formation by Candida species is an influential virulence factor in candidemia pathogenesis. We investigated the relationship between biofilm formation of Candida tropicalis isolates with the clinical characteristics and mortality outcomes in patients with candidemia. MATERIALS AND METHODS: Thirty-nine C. tropicalis isolates were recovered from patients with candidemia admitted to two university hospitals in Tehran, Iran. Biofilm mass and metabolic activity of C. tropicalis biofilms were assessed in vitro with two colorimetric methods. The sessile minimum inhibitory concentrations (SMICs) were evaluated in vitro by treating preformed biofilms with diluted concentrations of azoles according to CLSI-M27 A3/S4 protocol, followed by metabolic activity quantification. The expressions of ERG11, UPC2, MDR1, and CDR1 genes were also evaluated. RESULTS: All C. tropicalis isolates produced biofilm. Respectively, higher <7-day and ≥7-day mortality rates were found among cases with high metabolic activity (46.7% vs. 13%, P = 0.03) and high biofilm mass (31.8% vs. 0, P = 0.029). Sessile cells had high resistance to fluconazole, voriconazole, and itraconazole. The azole minimum inhibitory concentrations (MICs) of C. tropicalis sessile were significantly greater than the planktonic minimum inhibitory concentrations (PMICs). In fluconazole-treated biofilms, the expression of ERG11 and UPC2 genes was increased. CONCLUSION: Our findings highlight the importance of C. tropicalis biofilm formation as an important factor in candidemia pathogenesis and the clinical outcome of patients with candidemia.
Assuntos
Candida tropicalis , Candidemia , Antifúngicos/farmacologia , Biofilmes , Candida tropicalis/genética , Farmacorresistência Fúngica , Fluconazol/farmacologia , Humanos , Irã (Geográfico) , Testes de Sensibilidade MicrobianaRESUMO
This study aimed to determine the prevalence, the causative agents, clinical features, and the risk factors associated with the fungal rhinosinusitis in a tertiary health center with a view to providing valid grounds that may guide healthcare professionals to effectively prevent, control, and treat fungal infections. All patients were subjected to diagnostic nasal endoscopy and CT scan of paranasal sinuses and FRS were confirmed by routine and complementary mycological and molecular methods. The inclusion criteria for invasive FRS were: confirmed diagnosis of IFRS according to the guidelines of the EORTC/MSG criteria (i.e., clinical, microbiological, and histological evidence of invasive fungal infection). From a total of 512 suspected patients, FRS was confirmed in 108 cases (21.1%). Our results showed FB (38/108; 35.2%) is the most common form of FRS followed by AIFRS (33/108; 30.6%), AFS (32/108; 29.6%), and CIFRS (5/108; 4.6%). A. flavus and Rhizopus oryzae were the most common causes of infection in AFS, FB, CIFRS, and AIFRS, respectively. Univariate analysis of variables predictive of AIFRS revealed 3 variables significantly associated with AIFRS. These included mucosal abnormalities of the middle turbinate and septum, and specifically, necrosis of the middle turbinate (P < .0001). Microbiological cultures, although useful for mycological speciation, are less sensitive. Furthermore, we used molecular methods to confirm the identity of some isolates that were not detectable using routine methods. Our data showed that the molecular methods and histologic diagnosis in all patients were more sensitive than the unenhanced sinus CT scan, and conventional microbiological methods.
Assuntos
Micoses , Sinusite , Fungos/genética , Humanos , Micoses/diagnóstico por imagem , Micoses/epidemiologia , Nariz , Sinusite/diagnóstico por imagem , Tomografia Computadorizada por Raios XRESUMO
Acute respiratory distress syndrome is a common complication of severe viral pneumonia, such as influenza and COVID-19, that requires critical care including ventilatory support, use of corticosteroids and other adjunctive therapies to arrest the attendant massive airways inflammation. Although recommended for the treatment of viral pneumonia, steroid therapy appears to be a double-edged sword, predisposing patients to secondary bacterial and invasive fungal infections (IFIs) whereby impacting morbidity and mortality. Mucormycosis is a fungal emergency with a highly aggressive tendency for contiguous spread, associated with a poor prognosis if not promptly diagnosed and managed. Classically, uncontrolled diabetes mellitus (DM) and other immunosuppressive conditions including corticosteroid therapy are known risk factors for mucormycosis. Upon the background lung pathology, immune dysfunction and corticosteroid therapy, patients with severe viral pneumonia are likely to develop IFIs like aspergillosis and mucormycosis. Notably, the combination of steroid therapy and DM can augment immunosuppression and hyperglycaemia, increasing the risk of mucormycosis in a susceptible individual. Here, we report a case of sinonasal mucormycosis in a 44-year-old woman with hyperglycaemia secondary to poorly controlled diabetes following dexamethasone therapy on a background of influenza pneumonia and review 15 available literatures on reported cases of influenza and COVID-19 associated mucormycosis.
Assuntos
Corticosteroides/uso terapêutico , COVID-19/complicações , Influenza Humana/complicações , Mucormicose/tratamento farmacológico , Mucormicose/etiologia , Pneumonia Viral/tratamento farmacológico , Adulto , Anfotericina B/uso terapêutico , Antifúngicos/uso terapêutico , Complicações do Diabetes , Feminino , Humanos , Lipossomos/uso terapêutico , Triazóis/uso terapêuticoRESUMO
BACKGROUND: Limited knowledge exists on the virulence factors of Candida tropicalis and the mechanisms of azole resistance that lead to an intensified pathogenicity and treatment failure. We aimed to evaluate the virulence factors and molecular mechanisms of azole resistance among C. tropicalis isolated from patients with candidemia. MATERIALS AND METHODS: Several virulence factors, including extracellular enzymatic activities, cell surface hydrophobicity (CSH), and biofilm formation, were evaluated. Antifungal susceptibility pattern and expression level of ERG11, UPC2, MDR1, and CDR1 genes of eight (4 fluconazole resistance and 4 fluconazole susceptible) clinical C. tropicalis isolates were assessed. The correlation between the virulence factors and antifungal susceptibility patterns was analyzed. RESULTS: During a 4 year study, forty-five C. tropicalis isolates were recovered from candidemia patients. The isolates expressed different frequencies of virulence determinants as follows: coagulase 4 (8.9%), phospholipase 5 (11.1%), proteinase 31 (68.9%), esterase 43 (95.6%), hemolysin 44 (97.8%), biofilm formation 45 (100%) and CSH 45(100%). All the isolates were susceptible to amphotericin B and showed the highest resistance to voriconazole. There was a significant positive correlation between micafungin minimum inhibitory concentrations (MICs) and hemolysin production (rs = 0.316). However, we found a negative correlation between fluconazole MICs and esterase production (rs = -0.383). We observed the high expression of ERG11 and UPC2 genes in fluconazole-resistant C. tropicalis isolates. CONCLUSION: C. tropicalis isolated from candidemia patients extensively displayed capacities for biofilm formation, hemolysis, esterase activity, and hydrophobicity. In addition, the overexpression of ERG11 and UPC2 genes was considered one of the possible mechanisms of azole resistance.
Assuntos
Candida tropicalis , Candidemia , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Azóis , Candida tropicalis/genética , Candidemia/tratamento farmacológico , Farmacorresistência Fúngica , Fluconazol/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Fatores de Virulência/genéticaRESUMO
Otomycosis is a fungal infection of the external auditory canal caused mainly by the genus Aspergillus. Aspergillus luchuensis, an industrially important fungus, is a member of Aspergillus section Nigri. In this report, we present a case of otomycosis due to Aspergillus luchuensis in a 43-year-old female patient. We performed a partial PCR-sequencing of ß-tubulin and calmodulin genes to identify the isolate to the species level. Further, we determined the in vitro susceptibility of the isolate to nystatin, clotrimazole and itraconazole according to the Clinical & Laboratory Standards Institute (CLSI) M38-A2 protocol. Accordingly, the minimum inhibitory concentrations of clotrimazole, nystatin and itraconazole were 0.25µg/mL, 0.5µg/mL and 1µg/mL, respectively. This is the first report of clinically relevant isolation of Aspergillus luchuensis identified by a molecular technique as a causative agent of otomycosis.
Assuntos
Otomicose , Adulto , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Aspergillus/genética , Feminino , Humanos , Testes de Sensibilidade Microbiana , Otomicose/diagnóstico , Otomicose/tratamento farmacológico , Reação em Cadeia da PolimeraseRESUMO
The mutation at codon L98 accompanied by a tandem repeat of 34 base pairs (TR34/L98H) in the 5´upstream region of cyp51A is the principal mechanism of triazole resistance of Aspergillus fumigatus. We aimed to evaluate a simple and low-cost tetra-primer amplification refractory mutation system (ARMS)-PCR technique for detection of TR34/L98H mutations in the cyp51A gene of azole-resistant A. fumigatus. The tetra-primer ARMS-PCR assay optimized by four primers in one reaction consists of external primers for detection of tandem repeats in the promoter region and internal primers for detection of a point mutation in codon 98 (L98H) in the cyp51A gene of azole-resistant A. fumigatus. The specificity of TR34/L98H mutation detection was assessed by testing 36 clinical and environmental A. fumigatus strains. The tetra-primer ARMS-PCR assay from A. fumigatus, containing wild-type sequence (T allele) and L98H mutation at cyp51A (A allele), yielded two DNA fragments of 908 bp and 740 bp and two of 942 bp and 212 bp, respectively. None of the A. fumigatus isolates without the TR34/L98H mutation yielded false-positive results. The ARMS-PCR assay was 100% concordant with DNA sequencing results. Prevalence and screening of the TR34/L98H mutation in the cyp51A gene in A. fumigatus isolates may now be determined by a fast, low-cost, and simple method in resource-poor settings.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Reação em Cadeia da Polimerase/métodos , Alelos , Aspergillus fumigatus/genética , Primers do DNA/genética , Testes de Sensibilidade Microbiana , Mutação , Análise de Sequência de DNA , Sequências de Repetição em Tandem , Triazóis/farmacologiaRESUMO
Free-living amoebae belong to the genus Acanthamoeba; can feed on microbial population by phagocytosis, and with the capability to act as a reservoir and a vehicle of microorganisms to susceptible host. Therefore, the role of endosymbiosis in the pathogenesis of Acanthamoeba is complex and not fully understood. The aim of the present study was to identify bacterial, fungal, and human adenovirus (HADV) endosymbionts as well as evaluating the endosymbionts role of such organisms in the pathogenesis of Acanthamoeba in keratitis patients living in Iran. Fifteen Acanthamoeba (T4 genotype) isolates were recovered from corneal scrapes and contact lenses of patients with keratitis. Cloning and purification was performed for all isolate. Gram staining was performed to identify bacterial endosymbionts. DNA extraction, PCR, and nested PCR was set up to identify endosymbiont of amoeba. Evaluation of pathogenicity was conducted by osmo-tolerance and thermo-tolerance assays and cell culture, and then CPE (cytopathic effect) was survey. Statistical analysis was used between Acanthamoeba associated endosymbionts and Acanthamoeba without endosymbiont at 24, 48, 72, and 96â¯h. A p valueâ¯<â¯0.05 was considered as significant, statistically. A total of 9 (60%) Acanthamoeba (T4 genotypes) isolates were successfully cloned for detecting microorganism endosymbionts. The only isolate negative for the presence of endosymbiont was ICS9. ICS7 (Pseudomonas aeruginosa, Aspergillus sp., and human adenovirus endosymbionts) and ICS2 (Escherichia coli endosymbiont) isolates were considered as Acanthamoeba associated endosymbionts. ICS7 and ICS2 isolates were highly pathogen whereas ICS9 isolate showed low pathogenicity in pathogenicity evaluated. Positive CPE for ICS7 and ICS2 isolates and negative CPE for ICS9 isolate were observed in cell culture. The average number of cells, trophozoites, and cysts among ICS7, ICS2, and ICS9 isolates at 24, 48, 72, and 96â¯h was significant. This is the first survey on microbial endosymbionts of Acanthamoeba in keratitis patients of Iran, and also the first report of Aspergillus sp, Achromobacter sp., Microbacterium sp., Brevibacillus sp, Brevundimonas sp and Mastadenovirus sp in Acanthamoeba as endosymbionts. Our study demonstrated that microbial endosymbionts can affect the pathogenicity of Acanthamoeba; however, further research is required to clarify the exact pattern of symbiosis, in order to modify treatment protocol.
Assuntos
Ceratite por Acanthamoeba/complicações , Acanthamoeba/fisiologia , Adenovírus Humanos/isolamento & purificação , Bactérias/isolamento & purificação , Fungos/isolamento & purificação , Simbiose , Acanthamoeba/isolamento & purificação , Acanthamoeba/microbiologia , Acanthamoeba/patogenicidade , Adenovírus Humanos/genética , Adenovírus Humanos/fisiologia , Animais , Bactérias/genética , Chlorocebus aethiops , Clonagem Molecular , Doenças Transmissíveis/microbiologia , Doenças Transmissíveis/transmissão , Lentes de Contato/parasitologia , Córnea/parasitologia , Reservatórios de Doenças , Fungos/genética , Humanos , Irã (Geográfico) , Reação em Cadeia da Polimerase , Células Vero , VirulênciaRESUMO
The aim of this study was to determine the in vitro interactions of geldanamycin (Hsp90-inhibitor) with triazoles and echinocandins against common and emerging Candida species. Twenty clinically important Candida strains comprising C. auris, C. albicans, C. parapsilosis, and C. glabrata (each five strains) were included. In vitro interactions of geldanamycin with fluconazole, itraconazole, caspofungin and anidulafungin were determined using a checkerboard method. The results were interpreted as synergistic, indifferent and antagonistic based on the fractional inhibitory concentration index (FICI). In vitro combination of fluconazole with geldanamycin resulted in synergistic effect against C. albicans (100%), C. glabrata (80%) and C. parapsilosis (80%) (FICI range 0.009-0.5), while indifferent interactions were obtained against C. auris (FICI range 1.5-2). The overall minimum inhibitory concentration (MIC) range of fluconazole against C. albicans, C. glabrata and C. parapsilosis reduced from 16-256 to 0.25-64 mg/L when combined with geldanamycin. Regarding the synergistic effect of geldanamycin with itraconazole against all strains of C. albicans, C. glabrata and C. parapsilosis (FICI range 0.009-0.375), the MIC range of this antifungal was reduced from 0.125-32 mg/L when tested alone, to 0.03-1 mg/L. Combinations of geldanamycin with fluconazole and itraconazole against C. auris, as well as combination of geldanamycin with caspofungin and anidulafungin against all studied Candida species, resulted in indifferent effects. No antagonism was observed. Simultaneous targeting of Hsp90 and lanosterol 14-α demethylase seems an effective approach against C. albicans, C. glabrata and C. parapsilosis. However, this combination is ineffective against the emerging pathogen C. auris.
Assuntos
Antifúngicos/farmacologia , Benzoquinonas/farmacologia , Candida/efeitos dos fármacos , Interações Medicamentosas , Equinocandinas/farmacologia , Lactamas Macrocíclicas/farmacologia , Triazóis/farmacologia , Testes de Sensibilidade MicrobianaRESUMO
Mycotic keratitis or keratomycosis is a fungal infection with global distribution. The dominant aetiology of this disease varies based on geographical origin, socioeconomic status, and climatic condition. Generally, Aspergillus spp. and Fusarium spp. are common in tropical and subtropical regions and Candida spp. are dominant in temperate areas. Demonstration of fungal elements in microscopic examination besides the isolation of fungi in culture is the gold standard of laboratory diagnosis. As the culture is a time-consuming procedure, other approaches such as in vivo confocal microscopy which produces real-time imaging of corneal tissue and molecular techniques have been developed to facilitate rapid diagnosis of fungal keratitis. The first choice of treatment is topical natamycin, although topical amphotericin B is the best choice for Aspergillus and Candida keratitis. Regarding the diversity of fungal aetiology and the emergence of drug resistance in some genera and species, proper identification using molecular methods and antifungal susceptibility testing could provide useful data. Furthermore, as the better efficacy of combination therapy in comparison to monotherapy is reported, in vitro determination of interactions between various drugs seem informative. This review aims to provide a general and updated view on the aetiology, risk factors, epidemiology, clinical and laboratory diagnosis, and management of fungal keratitis.
Assuntos
Infecções Oculares Fúngicas/diagnóstico , Infecções Oculares Fúngicas/epidemiologia , Fungos/isolamento & purificação , Ceratite/diagnóstico , Ceratite/epidemiologia , Técnicas Microbiológicas/métodos , Microscopia/métodos , Administração Tópica , Anfotericina B/administração & dosagem , Antifúngicos/administração & dosagem , Clima , Quimioterapia Combinada/métodos , Infecções Oculares Fúngicas/tratamento farmacológico , Infecções Oculares Fúngicas/microbiologia , Fungos/classificação , Saúde Global , Humanos , Ceratite/tratamento farmacológico , Ceratite/microbiologia , Técnicas de Diagnóstico Molecular/métodos , Natamicina/administração & dosagem , Fatores de RiscoRESUMO
Dihydroxyacid dehydratase (DHAD) is a key enzyme in biosynthetic pathway of isoleucine and valine. This pathway is absent in human but exists in various organisms such as fungi. Using RNA-seq analysis in this study, we identified MGL_3741gene which encodes DHAD protein in Malassezia globosa (M. globosa). Furthermore, we found that mentioned gene is homologous to the Ustilago maydis, Saccharomyces cerevisiae, Aspergillus flavus, and Aspergillus fumigatus ILV3P. For understanding the probable role of this gene in pathogenicity of M. globosa, we applied Real-time PCR to investigate the differentially expressed of the MGL_3741 gene in healthy and pathogenic states. Our results indicate a significant difference between two mentioned stats. These results revealed that ILV3-like gene in M. globosa can be related to the pathogenicity of this yeast.
Assuntos
Hidroliases/genética , Malassezia/enzimologia , Malassezia/patogenicidade , Tinha Versicolor/patologia , Fatores de Virulência/genética , Perfilação da Expressão Gênica , Humanos , Hidroliases/metabolismo , Malassezia/genética , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA , Homologia de Sequência , Tinha Versicolor/microbiologia , Fatores de Virulência/metabolismoRESUMO
Aspergillus flavus is the second most important Aspergillus species causing human infections in tropical countries. Despite an increasing number of infections of A. flavus in Iran, the molecular epidemiology of clinical and environmental strains has not been well studied. We used a panel of nine microsatellite markers to analyse the genetic relatedness of A. flavus. Microsatellite typing of 143 (n = 119 clinical and n = 24 environmental) isolates demonstrated 118 different genotypes. A possible outbreak at a pulmonary ward was discovered. The discriminatory power for the individual markers ranged from 0.4812 to 0.9457 and the panel of all nine markers combined yielded a diversity index of 0.9948. This high-resolution typing method assists in better understanding of the molecular epidemiology of A. flavus.
Assuntos
Aspergilose/microbiologia , Aspergillus flavus/classificação , Aspergillus flavus/genética , Microbiologia Ambiental , Técnicas de Genotipagem/métodos , Repetições de Microssatélites , Técnicas de Tipagem Micológica/métodos , Aspergillus flavus/isolamento & purificação , Humanos , Irã (Geográfico) , Epidemiologia Molecular/métodosRESUMO
BACKGROUND: Global reports have highlighted the increasing prevalence of Candida tropicalis infections as well as organism(') s drug resistance. This study aimed at identifying azole resistance markers in clinical isolates of C. tropicalis, which will be a great resource for developing new drugs. METHODS: Two susceptible and resistant isolates of C. tropicalis were recovered from an epidemiological investigation of candidiasis in immunocompromised patients. C. tropicalis ATCC 750 was used as reference strain. Antifungal susceptibility to fluconazole and itraconazole was determined using Clinical and Laboratory Standards Institute (CLSI) method. Complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) technology and real-time reverse-transcriptase (RT) PCR were used for identification of potential genes involved in azole resistance of C. tropicalis clinical isolates. RESULTS: Five genes encoding the following enzymes were identified as superoxide dismutase (SOD) implicated in antioxidant defense, ornithine aminotransferase (OAT), acetyl ornithine aminotransferase (ACOAT), adenosylmethionine-8-amino-7-oxononanoate aminotransferase (DAPA AT), and 4-aminobutyrate aminotransferase (ABAT)-belonging to pyridoxal phosphate (PLP) dependent enzymes and acting in an important physiological role in many fungal-cell cycles. Real-time RT-PCR confirmed mRNA level of the aforementioned genes. CONCLUSION: Our findings showed that factors such as PLP-dependent enzymes and SOD might be implicated in drug resistance in C. tropicalis clinical isolate. Therefore, further studies are required to explore the accurate biological functions of the mentioned genes that would be helpful for effective drug development.
Assuntos
Análise do Polimorfismo de Comprimento de Fragmentos Amplificados/métodos , Azóis/farmacologia , Candida tropicalis/efeitos dos fármacos , Candida tropicalis/isolamento & purificação , DNA Complementar/genética , Farmacorresistência Fúngica/efeitos dos fármacos , Candida tropicalis/genética , Eletroforese em Gel de Poliacrilamida , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Marcadores Genéticos , Humanos , Testes de Sensibilidade MicrobianaRESUMO
Incidence of fungal sinusitis due to the genus Fusarium has increased during the last two decades. We report a case of fungus ball sinusitis with multiple sinuses involvement in an Iranian 21-year-old woman. The patient was diagnosed as having a fungus ball-type sinusitis in computed tomography scan. The sinus biopsy revealed fungal structures on histopathological and direct microscopic examinations and a Fusarium species arose in culture. Partial sequencing of the translation elongation factor 1-alpha identified the isolate as F. proliferatum. Removal of all lesions by endoscopic surgery resulted in a favorable outcome. To the best of our knowledge, this is the first case of F. proliferatum-associated fungus ball which involved multi-sinus and highlights the efficiency of molecular methods for discrimination of fungal agents involved.
Assuntos
Fusariose/diagnóstico , Fusariose/patologia , Fusarium/isolamento & purificação , Sinusite/diagnóstico , Sinusite/patologia , Endoscopia , Feminino , Fusariose/microbiologia , Fusariose/cirurgia , Histocitoquímica , Humanos , Irã (Geográfico) , Técnicas Microbiológicas , Microscopia , Seios Paranasais/diagnóstico por imagem , Seios Paranasais/patologia , Sinusite/microbiologia , Sinusite/cirurgia , Tomografia Computadorizada por Raios X , Resultado do Tratamento , Adulto JovemRESUMO
OBJECTIVE: To investigate manufacturing smooth muscle cells (SMCs) for regenerative bladder reconstruction from differentiation of endometrial stem cells (EnSCs), as the recent discovery of EnSCs from the lining of women's uteri, opens up the possibility of using these cells for tissue engineering applications, such as building up natural tissue to repair prolapsed pelvic floors as well as building urinary bladder wall. MATERIALS AND METHODS: Human EnSCs that were positive for cluster of differentiation 146 (CD146), CD105 and CD90 were isolated and cultured in Dulbecco's modified Eagle/F12 medium supplemented with myogenic growth factors. The myogenic factors included: transforming growth factor ß, platelet-derived growth factor, hepatocyte growth factor and vascular endothelial growth factor. Differentiated SMCs on bioabsorbable polyethylene-glycol and collagen hydrogels were checked for SMC markers by real-time reverse-transcriptase polymerase chain reaction (RT-PCR), western blot (WB) and immunocytochemistry (ICC) analyses. RESULTS: Histology confirmed the growth of SMCs in the hydrogel matrices. The myogenic growth factors decreased the proliferation rate of EnSCs, but they differentiated the human EnSCs into SMCs more efficiently on hydrogel matrices and expressed specific SMC markers including α-smooth muscle actin, desmin, vinculin and calponin in RT-PCR, WB and ICC experiments. The survival rate of cultures on the hydrogel-coated matrices was significantly higher than uncoated cultures. CONCLUSIONS: Human EnSCs were successfully differentiated into SMCs, using hydrogels as scaffold. EnSCs may be used for autologous bladder wall regeneration without any immunological complications in women. Currently work is in progress using bioabsorbable nanocomposite materials as EnSC scaffolds for developing urinary bladder wall tissue.
Assuntos
Endométrio/ultraestrutura , Miócitos de Músculo Liso/ultraestrutura , Células-Tronco/citologia , Engenharia Tecidual/métodos , Actinas/biossíntese , Actinas/genética , Adulto , Biópsia , Western Blotting , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Endométrio/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Microscopia Eletrônica de Varredura , Miócitos de Músculo Liso/metabolismo , Fator de Crescimento Derivado de Plaquetas/biossíntese , Fator de Crescimento Derivado de Plaquetas/genética , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/metabolismo , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/genética , Bexiga Urinária/citologiaRESUMO
Pentavalent antimonial compounds have been the first line therapy for leishmaniasis; unfortunately the rate of treatment failure of anthroponotic cutaneous leishmaniasis (ACL) is increasing due to emerging of drug resistance. Elucidation of the molecular mechanisms operating in antimony resistance is critical for development of new strategies for treatment. Here, we used a cDNA-AFLP approach to identify gene(s) which are differentially expressed in resistant and sensitive Leishmania tropica field isolates. We identified five genes, aquaglyceroporin (AQP1) acts in drug uptake, ATP-binding cassette (ABC) transporter (MRPA) involved in sequestration of drug, phosphoglycerate kinase (PGK) implicated in glycolysis metabolism, mitogen activated protein kinase (MAPK) and protein tyrosine phosphatase (PTP) responsible for phosphorylation pathway. The results were confirmed using real time RT-PCR which revealed an upregulation of MRPA, PTP and PGK genes and downregulation of AQP1 and MAPK genes in resistant isolate. To our knowledge, this is the first report of identification of PTP and PGK genes potentially implicated in resistance to antimonials. Our findings support the idea that distinct biomolecules might be involved in antimony resistance in L. tropica field isolates.
Assuntos
Antiprotozoários/farmacologia , Resistência a Medicamentos/genética , Leishmania tropica/efeitos dos fármacos , Meglumina/farmacologia , Compostos Organometálicos/farmacologia , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Antimônio/farmacologia , Linhagem Celular , Fragmentação do DNA , DNA Complementar/química , DNA Complementar/metabolismo , DNA de Protozoário/química , DNA de Protozoário/metabolismo , Genes de Protozoários , Concentração Inibidora 50 , Leishmania tropica/genética , Antimoniato de Meglumina , Testes de Sensibilidade Parasitária , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The mainstay therapy against leishmaniasis is still pentavalent antimonial drugs; however, the rate of antimony resistance is increasing in endemic regions such as Iran. Understanding the molecular basis of resistance to antimonials could be helpful to improve treatment strategies. This study aimed to recognize genes involved in antimony resistance of Leishmania tropica field isolates. Sensitive and resistant L. tropica parasites were isolated from anthroponotic cutaneous leishmaniasis patients and drug susceptibility of parasites to meglumine antimoniate (Glucantime®) was confirmed using in vitro assay. Then, complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) and real-time reverse transcriptase-PCR (RT-PCR) approaches were utilized on mRNAs from resistant and sensitive L. tropica isolates. We identified 2 known genes, ubiquitin implicated in protein degradation and amino acid permease (AAP3) involved in arginine uptake. Also, we identified 1 gene encoding hypothetical protein. Real-time RT-PCR revealed a significant upregulation of ubiquitin (2.54-fold), and AAP3 (2.86-fold) (P<0.05) in a resistant isolate compared to a sensitive one. Our results suggest that overexpression of ubiquitin and AAP3 could potentially implicated in natural antimony resistance.
Assuntos
Sistemas de Transporte de Aminoácidos/genética , Antimônio/farmacologia , Antipruriginosos/farmacologia , Resistência a Medicamentos , Leishmania tropica/genética , Leishmaniose Cutânea/parasitologia , Proteínas de Protozoários/genética , Ubiquitina/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Humanos , Leishmania tropica/efeitos dos fármacos , Leishmania tropica/enzimologia , Leishmania tropica/isolamento & purificação , Proteínas de Protozoários/metabolismo , Ubiquitina/metabolismoRESUMO
BACKGROUND: Candida glabrata causes significant medical problems in immunocompromised patients. Many strains of this yeast are intrinsically resistant to azole antifungal agents, and treatment is problematic, leading to high morbidity and mortality rates in immunosuppressed individuals. The primary goal of this study was to investigate the genes involved in the drug resistance of clinical isolates of C. glabrata. METHODS: The clinical isolates of C. glabrata were collected in an epidemiological survey of candidal infection in immunocompromised patients and consisted of four fluconazole and itraconazole resistant isolates, two fluconazole and itraconazole sensitive isolates, and C. glabrata CBS 138 as reference strain. Antifungal susceptibility patterns of the organisms were determined beforehand by the Clinical and Laboratory Standards Institute (CLSI). The potential gene(s) implicated in antifungal resistance were investigated using complementary DNA- Amplified Fragment Length Polymorphism (cDNA-AFLP). Semi-quantitative RT-PCR was carried out to evaluate the expression of gene(s) in resistant isolates as compared to sensitive and reference strains. RESULTS AND CONCLUSIONS: The aldo-keto-reductase superfamily (AKR gene) was upregulated in the resistant clinical isolates as assessed by cDNA-AFLP. Semi-quantitative RT-PCR revealed AKR mRNA expression approximately twice that seen in the sensitive isolates. Overexpression of the AKR gene was associated with increased fluconazole and itraconazole resistance in C. glabrata. The data suggest that upregulation of the AKR gene might give a new insight into the mechanism of azole resistance.
RESUMO
The introduction of RNA silencing machinery in fungi has led to the promising application of RNAi methodology to knock down essential vital factor or virulence factor genes in the microorganisms. Efg1p is required for development of a true hyphal growth form which is known to be essential for interactions with human host cells and for the yeast's pathogenesis. In this paper, we describe the development of a system for presenting and studying the RNAi function on the EFG1 gene in C. albicans. The 19-nucleotide siRNA was designed on the basis of the cDNA sequence of the EFG1 gene in C. albicans and transfection was performed by use of a modified-PEG/LiAc method. To investigate EFG1 gene silencing in siRNA-treated cells, the yeasts were grown in human serum; to induce germ tubes a solid medium was used with the serum. Quantitative changes in expression of the EFG1 gene were analyzed by measuring the cognate EFG1 mRNA level by use of a quantitative real-time RT-PCR assay. Compared with the positive control, true hyphae formation was significantly reduced by siRNA at concentrations of 1 µM, 500 nM, and 100 nM (P < 0.05). In addition, siRNA at a concentration of 1 µM was revealed to inhibit expression of the EFG1 gene effectively (P < 0.05). On the basis of the potential of post-transcriptional gene silencing to control the expression of specific genes, these techniques may be regarded as promising means of drug discovery, with applications in biomedicine and functional genomics analysis.
Assuntos
Candida albicans/crescimento & desenvolvimento , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas Fúngicas/antagonistas & inibidores , Hifas/crescimento & desenvolvimento , Interferência de RNA , RNA Fúngico/metabolismo , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Antifúngicos/farmacologia , Produtos Biológicos/farmacologia , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica , Humanos , Hifas/efeitos dos fármacos , Hifas/genética , RNA Fúngico/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição/genéticaRESUMO
Dermatophytosis is caused by a group of pathogenic fungi namely, dermatophytes, is among the most prevalent infectious diseases worldwide. Azole drugs are widely used in the treatment of dermatomycosis, but can cause various side effects and drug resistance to the patients. Hence, for solving this problem can be used from the plant extract as alternative for chemical drugs. Allicin is a pure bioactive compound isolated from garlic was tested for its potential as a treatment of dermatomycosis in this study. This study evaluated the in vitro efficacy of pure allicin against ten isolates of Trichophyton rubrum and the MIC50 and MIC90 ranged from 0.78-12.5 µg/ml for allicin. The results revealed that the order of efficacy based on the MICs values, all isolates showed almost comparable response to allicin and ketoconazole except for some isolates, at 28 °C for both 7 and 10 days incubation. Mann-Whitney test indicate that MICs at 7 days incubation was not observed a significant difference between the effects of allicin and ketoconazole (p > 0.05), but MICs at 10 days incubation, a significant difference was observed (p ≤ 0.05). On the other side, time kill studies revealed that allicin used its fungicidal activity within 12-24 h of management in vitro as well as ketoconazole. In conclusion, allicin showed very good potential as an antifungal compound against mycoses-causing dermatophytes, almost the same as the synthetic drug ketoconazole. Therefore, this antifungal agent appears to be effective, safe and suitable alternative for the treatment of dermatomycosis.
RESUMO
[This corrects the article DOI: 10.1007/s40201-021-00734-6.].