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1.
Artigo em Inglês | MEDLINE | ID: mdl-30104280

RESUMO

VT-1129 is a novel fungal enzyme-specific Cyp51 inhibitor with potent cryptococcal activity. Because of its long half-life (>6 days in mice) and our desire to quickly reach potent efficacy, we evaluated a VT-1129 loading dose-maintenance dose strategy against cryptococcal meningitis. VT-1129 plasma and brain pharmacokinetics were first studied in healthy mice, and these data were used to model loading dose-maintenance dose regimens to generate different steady-state concentrations. Mice were inoculated intracranially with Cryptococcus neoformans, and oral treatment began 1 day later. Treatment consisted of placebo or one of three VT-1129 loading dose-maintenance dose regimens, i.e., loading dose of 1, 3, or 30 mg/kg on day 1, followed by once-daily maintenance doses of 0.15, 0.5, or 5 mg/kg, respectively. In the fungal burden arm, therapy continued for 14 days and brains were collected on day 15 for fungal burden assessments. In the survival arm, treatment continued for 10 days, after which mice were monitored without therapy until day 30. VT-1129 plasma and brain concentrations were also measured. All VT-1129 doses significantly improved survival and reduced fungal burdens, compared to placebo. VT-1129 plasma and brain levels correlated with fungal burden reductions (R 2 = 0.72 and R 2 = 0.67, respectively), with a plasma concentration of 1 µg/ml yielding a reduction of ∼5 log10 CFU/g. With the highest loading dose-maintenance dose regimen, fungal burdens were undetectable in one-half of the mice in the fungal burden arm and in one-fourth of the mice in the survival arm, 20 days after the final dose. These data support a loading dose-maintenance dose strategy for quickly reaching highly efficacious VT-1129 concentrations for treating cryptococcal meningitis.


Assuntos
Antifúngicos/farmacologia , Meningite Criptocócica/tratamento farmacológico , Piridinas/farmacologia , Tetrazóis/farmacologia , Animais , Encéfalo/microbiologia , Cryptococcus neoformans/efeitos dos fármacos , Masculino , Meningite Criptocócica/microbiologia , Camundongos , Camundongos Endogâmicos ICR , Testes de Sensibilidade Microbiana/métodos
2.
Artigo em Inglês | MEDLINE | ID: mdl-29987152

RESUMO

Cryptococcal meningitis is a significant cause of morbidity and mortality in immunocompromised patients. VT-1129 is a novel fungus-specific Cyp51 inhibitor with potent in vitro activity against Cryptococcus species. Our objective was to evaluate the in vivo efficacy of VT-1129 against cryptococcal meningitis. Mice were inoculated intracranially with Cryptococcus neoformans Oral treatment with VT-1129, fluconazole, or placebo began 1 day later and continued for either 7 or 14 days, and brains and plasma were collected on day 8 or 15, 1 day after therapy ended, and the fungal burden was assessed. In the survival study, treatment continued until day 10 or day 28, after which mice were monitored off therapy until day 30 or day 60, respectively, to assess survival. The fungal burden was also assessed in the survival arm. VT-1129 plasma and brain concentrations were also measured. VT-1129 reached a significant maximal survival benefit (100%) at a dose of 20 mg/kg of body weight once daily. VT-1129 at doses of ≥0.3 mg/kg/day and each dose of fluconazole significantly reduced the brain tissue fungal burden compared to that in the control after both 7 and 14 days of dosing. The fungal burden was also undetectable in most mice treated with a dose of ≥3 mg/kg/day, even ≥20 days after dosing had stopped, in the survival arm. In contrast, rebounds in fungal burden were observed with fluconazole. These results are consistent with the VT-1129 concentrations, which remained elevated long after dosing had stopped. These data demonstrate the potential utility of VT-1129 to have a marked impact in the treatment of cryptococcal meningitis.


Assuntos
Inibidores de 14-alfa Desmetilase/farmacologia , Cryptococcus neoformans/efeitos dos fármacos , Meningite Criptocócica/tratamento farmacológico , Piridinas/farmacologia , Esterol 14-Desmetilase/metabolismo , Tetrazóis/farmacologia , Animais , Antifúngicos/farmacologia , Criptococose/tratamento farmacológico , Fluconazol/farmacologia , Camundongos , Testes de Sensibilidade Microbiana/métodos , Modelos Teóricos
4.
Drug Dev Ind Pharm ; 43(2): 264-274, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27645428

RESUMO

Previously, modified release itraconazole in the form of a melt-extruded amorphous solid dispersion based on a pH dependent enteric polymer combined with hydrophilic additives (HME-ITZ), exhibited improved in vitro dissolution properties. These properties agreed with pharmacokinetic results in rats showing high and sustained itraconazole (ITZ) systemic levels. The objective of the present study was to better understand the best choice of rodent model for evaluating the pharmacokinetic and efficacy of this orally administered modified release ITZ dosage form against invasive Aspergillus fumigatus. A mouse model and a guinea pig model were investigated and compared to results previously published. In the mouse model, despite similar levels as previously reported values, plasma and lung levels were variable and fungal burden was not statistically different for placebo controls, HME-ITZ and Sporanox® (ITZ oral solution). This study demonstrated that the mouse model is a poor choice for studying modified release ITZ dosage forms based on pH dependent enteric polymers due to low fluid volume available for dissolution and low intestinal pH. To the contrary, guinea pig was a suitable model to evaluate modified release ITZ dosage forms. Indeed, a significant decrease in lung fungal burden as a result of high and sustained ITZ tissue levels was measured. Sufficiently high intestinal pH and fluids available for dissolution likely facilitated the dissolution process. Despite high ITZ tissue level, the primary therapeutic agent voriconazole exhibited an even more pronounced decrease in fungal burden due to its reported higher clinical efficacy specifically against Aspergillus fumigatus.


Assuntos
Antifúngicos/administração & dosagem , Aspergillus fumigatus/efeitos dos fármacos , Itraconazol/química , Itraconazol/farmacologia , Administração Oral , Animais , Antifúngicos/química , Antifúngicos/farmacologia , Aspergillus fumigatus/química , Varredura Diferencial de Calorimetria , Modelos Animais de Doenças , Cobaias , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Itraconazol/farmacocinética , Camundongos , Ratos , Solubilidade
5.
Antimicrob Agents Chemother ; 60(11): 6703-6708, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27572393

RESUMO

Candida albicans, normally found as a commensal in the gut, is a major human fungal pathogen responsible for both mucosal and systemic infections in a wide variety of immunocompromised individuals, including cancer patients and organ transplant recipients. The gastrointestinal tract represents a major portal of entry for the establishment of disseminated candidiasis in many of these individuals. Here we report the development of a diet-based mouse model for disseminated candidiasis acquired via the gastrointestinal tract. Using this model, as well as an appropriate immunosuppression regimen, we demonstrate that dissemination of C. albicans from the gastrointestinal tract can result in mortality within 30 days postinfection. We also show a significant increase in fungal burden in systemic organs, but not gastrointestinal tract organs, upon immunosuppression. Importantly, we demonstrate that the administration of two widely used antifungals, fluconazole and caspofungin, either pre- or postimmunosuppression, significantly reduces fungal burdens. This model should prove to be of significant value for testing the ability of both established and experimental therapeutics to inhibit C. albicans dissemination from the gastrointestinal tract in an immunocompromised host as well as the subsequent mortality that can result from disseminated candidiasis.


Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candidíase/tratamento farmacológico , Dieta/efeitos adversos , Equinocandinas/farmacologia , Fluconazol/farmacologia , Hospedeiro Imunocomprometido , Lipopeptídeos/farmacologia , Animais , Candida albicans/crescimento & desenvolvimento , Candida albicans/patogenicidade , Candidíase/etiologia , Candidíase/imunologia , Candidíase/mortalidade , Caspofungina , Contagem de Colônia Microbiana , Ciclofosfamida/efeitos adversos , Modelos Animais de Doenças , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/microbiologia , Humanos , Imunossupressores/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Prednisolona/efeitos adversos , Prednisolona/análogos & derivados , Análise de Sobrevida
6.
Antimicrob Agents Chemother ; 60(9): 5600-3, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27324761

RESUMO

We evaluated the efficacy of isavuconazole against cryptococcal meningitis. Treatment with either oral isavuconazole (120 mg/kg and 240 mg/kg twice a day [BID]) or fluconazole as the positive control significantly improved survival in mice infected intracranially with either Cryptococcus neoformans USC1597 or H99 and significantly reduced brain fungal burdens for both isolates. Concentrations of isavuconazole in plasma and brain tissue also demonstrated that the greatest improvements in survival and fungal burden were associated with elevated exposures.


Assuntos
Antifúngicos/farmacologia , Meningite Criptocócica/tratamento farmacológico , Nitrilas/farmacologia , Piridinas/farmacologia , Triazóis/farmacologia , Animais , Encéfalo/microbiologia , Cryptococcus neoformans/efeitos dos fármacos , Modelos Animais de Doenças , Meningite Criptocócica/microbiologia , Camundongos , Testes de Sensibilidade Microbiana
7.
J Clin Microbiol ; 54(4): 960-6, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26791366

RESUMO

The EuropeanAspergillusPCR Initiative (EAPCRI) has provided recommendations for the PCR testing of whole blood (WB) and serum/plasma. It is important to test these recommended protocols on nonsimulated "in vivo" specimens before full clinical evaluation. The testing of an animal model of invasive aspergillosis (IA) overcomes the low incidence of disease and provides experimental design and control that is not possible in the clinical setting. Inadequate performance of the recommended protocols at this stage would require reassessment of methods before clinical trials are performed and utility assessed. The manuscript describes the performance of EAPCRI protocols in an animal model of invasive aspergillosis. Blood samples taken from a guinea pig model of IA were used for WB and serum PCR. Galactomannan and ß-d-glucan detection were evaluated, with particular focus on the timing of positivity and on the interpretation of combination testing. The overall sensitivities for WB PCR, serum PCR, galactomannan, and ß-d-glucan were 73%, 65%, 68%, and 46%, respectively. The corresponding specificities were 92%, 79%, 80%, and 100%, respectively. PCR provided the earliest indicator of IA, and increasing galactomannan and ß-d-glucan values were indicators of disease progression. The combination of WB PCR with galactomannan and ß-d-glucan proved optimal (area under the curve [AUC], 0.95), and IA was confidently diagnosed or excluded. The EAPRCI-recommended PCR protocols provide performance comparable to commercial antigen tests, and clinical trials are warranted. By combining multiple tests, IA can be excluded or confirmed, highlighting the need for a combined diagnostic strategy. However, this approach must be balanced against the practicality and cost of using multiple tests.


Assuntos
Testes Diagnósticos de Rotina/métodos , Aspergilose Pulmonar Invasiva/diagnóstico , Mananas/análise , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase/métodos , beta-Glucanas/análise , Animais , Sangue/microbiologia , Modelos Animais de Doenças , Cobaias , Masculino , Sensibilidade e Especificidade , Fatores de Tempo
8.
mBio ; 6(3): e00647, 2015 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-26106079

RESUMO

UNLABELLED: Recent estimates suggest that >300 million people are afflicted by serious fungal infections worldwide. Current antifungal drugs are static and toxic and/or have a narrow spectrum of activity. Thus, there is an urgent need for the development of new antifungal drugs. The fungal sphingolipid glucosylceramide (GlcCer) is critical in promoting virulence of a variety of human-pathogenic fungi. In this study, we screened a synthetic drug library for compounds that target the synthesis of fungal, but not mammalian, GlcCer and found two compounds [N'-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) and its derivative, 3-bromo-N'-(3-bromo-4-hydroxybenzylidene) benzohydrazide (D0)] that were highly effective in vitro and in vivo against several pathogenic fungi. BHBM and D0 were well tolerated in animals and are highly synergistic or additive to current antifungals. BHBM and D0 significantly affected fungal cell morphology and resulted in the accumulation of intracellular vesicles. Deep-sequencing analysis of drug-resistant mutants revealed that four protein products, encoded by genes APL5, COS111, MKK1, and STE2, which are involved in vesicular transport and cell cycle progression, are targeted by BHBM. IMPORTANCE: Fungal infections are a significant cause of morbidity and mortality worldwide. Current antifungal drugs suffer from various drawbacks, including toxicity, drug resistance, and narrow spectrum of activity. In this study, we have demonstrated that pharmaceutical inhibition of fungal glucosylceramide presents a new opportunity to treat cryptococcosis and various other fungal infections. In addition to being effective against pathogenic fungi, the compounds discovered in this study were well tolerated by animals and additive to current antifungals. These findings suggest that these drugs might pave the way for the development of a new class of antifungals.


Assuntos
Antifúngicos/isolamento & purificação , Antifúngicos/farmacologia , Compostos de Benzil/isolamento & purificação , Compostos de Benzil/farmacologia , Vias Biossintéticas/efeitos dos fármacos , Fungos/efeitos dos fármacos , Esfingolipídeos/biossíntese , Animais , Antifúngicos/efeitos adversos , Antifúngicos/toxicidade , Compostos de Benzil/efeitos adversos , Compostos de Benzil/toxicidade , Candidíase/tratamento farmacológico , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Contagem de Colônia Microbiana , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Sinergismo Farmacológico , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Fungos/citologia , Fungos/metabolismo , Fungos/fisiologia , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Camundongos , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Microscopia Eletrônica de Transmissão , Estrutura Molecular , Esfingolipídeos/antagonistas & inibidores , Resultado do Tratamento
9.
Antimicrob Agents Chemother ; 59(5): 2875-81, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25753643

RESUMO

ASP9726 is an investigational echinocandin with in vitro activity against Aspergillus species. We evaluated the pharmacokinetics and efficacy of this agent in an established guinea pig model of invasive pulmonary aspergillosis. ASP9726 plasma concentrations were measured in guinea pigs administered either a single dose or multiple doses of this agent at 2.5, 5, and 10 mg/kg of body weight/day by subcutaneous injection. Immunosuppressed guinea pigs were inoculated with A. fumigatus AF293, and ASP9726 (2.5, 5, and 10 mg/kg/day), voriconazole (10 mg/kg by oral gavage twice daily), or caspofungin (3 mg/kg/day by intraperitoneal injection) was administered for 8 days. Changes in fungal burden were measured by enumerating CFU and by quantitative PCR of specimens from within the lungs, as well as by analysis of serum (1 → 3)-ß-D-glucan and galactomannan. Lung histopathology was also evaluated. ASP9726 plasma concentrations increased in a dose-proportional manner, and the drug was well tolerated at each dose. Each dose of ASP9726, voriconazole, and caspofungin significantly reduced pulmonary fungal burden as measured by quantitative PCR and by determining (1 → 3)-ß-D-glucan and galactomannan levels, but only voriconazole significantly reduced numbers of CFU. ASP9726 at 5 mg/kg also significantly improved survival. Histopathology demonstrated morphological changes in hyphae in animals exposed to ASP9726 and caspofungin, consistent with the activities of the echinocandins. These results suggest that ASP9726 may be efficacious for the treatment of invasive pulmonary aspergillosis.


Assuntos
Equinocandinas/uso terapêutico , Aspergilose Pulmonar Invasiva/tratamento farmacológico , Animais , Modelos Animais de Doenças , Equinocandinas/farmacocinética , Cobaias , Pulmão/microbiologia , Masculino
10.
Antimicrob Agents Chemother ; 59(1): 690-2, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25331706

RESUMO

The in vitro and in vivo activity of the inositol acyltransferase inhibitor E1210 was evaluated against echinocandin-resistant Candida albicans. E1210 demonstrated potent in vitro activity, and in mice with invasive candidiasis caused by echinocandin-resistant C. albicans, oral doses of 10 and 40 mg E1210/kg of body weight twice daily significantly improved survival and reduced fungal burden compared to those of controls and mice treated with caspofungin (10 mg/kg/day). These results demonstrate the potential use of E1210 against resistant C. albicans infections.


Assuntos
Aminopiridinas/farmacologia , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candidíase Invasiva/tratamento farmacológico , Isoxazóis/farmacologia , Aminopiridinas/uso terapêutico , Animais , Antifúngicos/uso terapêutico , Caspofungina , Relação Dose-Resposta a Droga , Farmacorresistência Fúngica , Equinocandinas/farmacologia , Equinocandinas/uso terapêutico , Fluconazol/farmacologia , Fluconazol/uso terapêutico , Isoxazóis/uso terapêutico , Lipopeptídeos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Testes de Sensibilidade Microbiana
11.
Antimicrob Agents Chemother ; 59(2): 1341-3, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25451054

RESUMO

We evaluated the in vitro and in vivo activities of the investigational arylamidine T-2307 against echinocandin-resistant Candida albicans. T-2307 demonstrated potent in vitro activity, and daily subcutaneous doses between 0.75 and 6 mg/kg of body weight significantly improved survival and reduced fungal burden compared to placebo control and caspofungin (10 mg/kg/day) in mice with invasive candidiasis caused by an echinocandin-resistant strain. Thus, T-2307 may have potential use in the treatment of echinocandin-resistant C. albicans infections.


Assuntos
Amidinas/farmacologia , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Candida albicans/patogenicidade , Equinocandinas/farmacologia , Equinocandinas/uso terapêutico , Animais , Candida albicans/efeitos dos fármacos , Candidíase/tratamento farmacológico , Farmacorresistência Fúngica , Masculino , Camundongos , Camundongos Endogâmicos ICR , Testes de Sensibilidade Microbiana
12.
Artigo em Inglês | MEDLINE | ID: mdl-23312542

RESUMO

OBJECTIVE: We monitored the epidemiology and microbiology of oral yeast colonization in patients undergoing hemopoietic progenitor cell transplantation (HPCT) to examine associations between yeast colonization and oral mucositis. STUDY DESIGN: One hundred twenty-one consecutive HPCT patients were sampled for oral yeasts prior to fluconazole (FLC) prophylaxis, at transplantation, and weekly until discharge. Clinical oral mucositis screenings were performed triweekly. RESULTS: Yeast colonization was evident at 216 of 510 total visits. Candida albicans and Candida glabrata were the predominant organisms. Eight patients showed elevated minimal inhibitory concentrations to FLC. One patient developed fungal septicemia. Patients with oral mucositis assessment scale scores <20 had higher colonization rates than those with higher scores. CONCLUSIONS: FLC is effective in controlling a variety of oral yeasts in HPCT recipients. FLC-resistant yeasts do emerge and can be the source of fungal sepsis. A positive association was not shown between yeast colonization and the presence or severity of oral mucositis.


Assuntos
Candida/isolamento & purificação , Transplante de Células-Tronco Hematopoéticas , Boca/microbiologia , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Candida/classificação , Candida albicans/isolamento & purificação , Candida glabrata/isolamento & purificação , Candida tropicalis/classificação , Candidíase Bucal/prevenção & controle , Quimioprevenção , Estudos de Coortes , Farmacorresistência Fúngica , Feminino , Fluconazol/uso terapêutico , Seguimentos , Fungemia/etiologia , Neoplasias Hematológicas/cirurgia , Humanos , Estudos Longitudinais , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Estudos Prospectivos , Saccharomyces cerevisiae/isolamento & purificação , Estomatite/microbiologia , Condicionamento Pré-Transplante/métodos , Adulto Jovem
13.
Med Mycol ; 51(6): 592-602, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23356446

RESUMO

Improved diagnostics are needed to detect invasive pulmonary aspergillosis, a life-threatening infection caused by the pathogenic fungus Aspergillus fumigatus. We are investigating secreted fungal proteases as novel biomarkers for the diagnosis of this disease. Although the A. fumigatus genome encodes a multitude of secreted proteases, few have been experimentally characterized. Here, we employed an unbiased combinatorial library of internally quenched fluorogenic probes to detect infection-associated proteolysis in the lungs of guinea pigs experimentally infected with A. fumigatus. Comparative protease activity profiling revealed a prolyl endopeptidase activity that is reproducibly induced during infection but is not observed in healthy animals. This proteolytic activity was found in four independent animal experiments involving two A. fumigatus isolates. We synthesized a small, focused fluorogenic probe library to define the substrate specificity of the prolyl endopeptidase substrate motif and to identify optimal Probe sequences. These efforts resulted in the identification of a panel of six individual substrate-based fluorescent probes capable of detecting infection in guinea pigs with high statistical significance (P<0.005 in most cases). Receiver operating characteristic analyses demonstrated that this fluorogenic assay could detect A. fumigatus infection-associated proteolysis with comparable sensitivity and specificity as existing diagnostic procedures, suggesting that further optimization of the methodology may lead to improved diagnostics options for invasive pulmonary aspergillosis.


Assuntos
Aspergillus fumigatus/enzimologia , Biomarcadores/análise , Líquido da Lavagem Broncoalveolar/química , Aspergilose Pulmonar Invasiva/diagnóstico , Serina Endopeptidases/análise , Animais , Modelos Animais de Doenças , Corantes Fluorescentes/metabolismo , Cobaias , Prolil Oligopeptidases , Curva ROC , Sensibilidade e Especificidade
14.
Antimicrob Agents Chemother ; 57(2): 745-50, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23165465

RESUMO

Miltefosine is an alkyl phosphocholine with good oral bioavailability and in vitro activity against Cryptococcus species that has gained interest as an additional agent for cryptococcal infections. Our objective was to further evaluate the in vivo efficacy of miltefosine in experimental in vivo models of cryptococcal meningoencephalitis and disseminated cryptococcosis. Mice were infected intracranially or intravenously with either C. neoformans USC1597 or H99. Miltefosine treatment (1.8 to 45 mg/kg of body weight orally once daily) began at either 1 h or 1 day postinoculation. Fluconazole (10 mg/kg orally twice daily) or amphotericin B deoxycholate (3 mg/kg intraperitoneally once daily) served as positive controls. In our standard models, miltefosine did not result in significant improvements in survival or reductions in fungal burden against either C. neoformans isolate. There was a trend toward improved survival with miltefosine at 7.2 mg/kg against disseminated cryptococcosis with the H99 strain but only at a low infecting inoculum. In contrast, both fluconazole and amphotericin B significantly improved survival in mice with cryptococcal meningoencephalitis and disseminated cryptococcosis due to USC1597. Amphotericin B also improved survival against both cryptococcal infections caused by H99. Combination therapy with miltefosine demonstrated neither synergy nor antagonism in both models. These results demonstrate limited efficacy of miltefosine and suggest caution with the potential use of this agent for the treatment of C. neoformans infections.


Assuntos
Antifúngicos/uso terapêutico , Criptococose/tratamento farmacológico , Cryptococcus neoformans/efeitos dos fármacos , Meningite Criptocócica/tratamento farmacológico , Meningoencefalite/tratamento farmacológico , Fosforilcolina/análogos & derivados , Anfotericina B/administração & dosagem , Animais , Criptococose/microbiologia , Cryptococcus neoformans/isolamento & purificação , Ácido Desoxicólico/administração & dosagem , Combinação de Medicamentos , Fluconazol/administração & dosagem , Meningite Criptocócica/microbiologia , Meningoencefalite/microbiologia , Camundongos , Testes de Sensibilidade Microbiana , Fosforilcolina/uso terapêutico , Sobrevida
15.
J Clin Microbiol ; 51(2): 459-65, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23175252

RESUMO

Interest in lateral-flow devices (LFDs) as potential point-of-care assays for the diagnosis of infectious diseases has increased. Our objective was to evaluate the interlaboratory and interstudy reproducibility and the effects of antifungal therapy on an LFD developed for invasive pulmonary aspergillosis (IPA) detection. An established neutropenic guinea pig model of IPA caused by Aspergillus fumigatus was used. At predetermined time points (1 h and 3, 5, and 7 days postinoculation), blood and bronchoalveolar lavage (BAL) fluid were collected from infected and uninfected animals. In a separate experiment, guinea pigs were treated with posaconazole (10 mg/kg of body weight orally [p.o.] twice a day [BID]), voriconazole (10 mg/kg p.o. BID), liposomal amphotericin B (10 mg/kg intraperitoneally [i.p.] once a day [QD]), or caspofungin (2 mg/kg i.p. QD), and samples were collected on days 7 and 11. Each laboratory independently evaluated the IgG monoclonal antibody-based LFD. Galactomannan and (1 → 3)-ß-D-glucan were also measured using commercially available kits. Good interlaboratory agreement was observed with the LFD, as the results for 97% (32/33) of the serum and 78.8% (26/33) of the BAL fluid samples from infected animals were in agreement. Good interstudy agreement was also observed. The serum sensitivity of each surrogate-marker assay was reduced in animals treated with antifungals. In contrast, these markers remained elevated within the BAL fluids of treated animals, which was consistent with the fungal burden and histopathology results. These results demonstrate that the LFD assay is reproducible between different laboratories and studies. However, the sensitivity of this assay and other markers of IPA may be reduced with serum in the presence of antifungal therapy.


Assuntos
Aspergilose Pulmonar Invasiva/diagnóstico , Sistemas Automatizados de Assistência Junto ao Leito , Animais , Antifúngicos/administração & dosagem , Antifúngicos/farmacologia , Aspergillus/efeitos dos fármacos , Aspergillus/imunologia , Aspergillus/isolamento & purificação , Líquido da Lavagem Broncoalveolar/imunologia , Líquido da Lavagem Broncoalveolar/microbiologia , Contagem de Colônia Microbiana , Modelos Animais de Doenças , Glucanos/metabolismo , Cobaias , Humanos , Aspergilose Pulmonar Invasiva/tratamento farmacológico , Aspergilose Pulmonar Invasiva/microbiologia , Masculino , Mananas/metabolismo , Sistemas Automatizados de Assistência Junto ao Leito/normas , Reprodutibilidade dos Testes , Fatores de Tempo
16.
AIDS Res Treat ; 2012: 262471, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22970352

RESUMO

The impact of antiretroviral therapy (ART) on opportunistic conditions in HIV patients continues to evolve. We specifically studied the changing epidemiology of oropharyngeal candidiasis (OPC) in 215 HIV/AIDS patients. Status of yeast colonization was assessed from oral rinse samples, and preliminary yeast identification was made using CHROMagar Candida and confirmed with standard microbiological techniques and/or molecular sequencing. Susceptibility to fluconazole was determined by CHROMagar Candida agar dilution screening and CLSI broth microdilution. 176 (82%) patients were colonized and 59 (27%) patients had symptomatic OPC. Candida albicans was the most prevalent species, though C. glabrata and C. dubliniensis were detected in 29% of isolates. Decreased fluconazole susceptibility occurred in 10% of isolates. Previous ART reduced the risk of OPC, while smoking increased the risk of colonization. Oral yeast colonization and symptomatic infection remain common even with advances in HIV therapy. C. albicans is the most common species, but other yeasts are prevalent and may have decreased susceptibility to fluconazole.

17.
J Antimicrob Chemother ; 67(6): 1432-8, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22378683

RESUMO

OBJECTIVES: Meningoencephalitis caused by Cryptococcus gattii is associated with significant morbidity and the need for aggressive therapy, and often necessitates neurosurgical intervention. We adapted a previously described murine model of cryptococcal meningoencephalitis due to Cryptococcus neoformans to that caused by C. gattii. METHODS: Mice were inoculated intracranially with either C. gattii (genotype VGIIa) or C. neoformans. In virulence studies, different C. gattii infecting inocula were compared with a fixed inoculum of C. neoformans, and differences were assessed by survival, brain tissue fungal burden, serum antigen titres and histopathological changes within brain tissue. For treatment, fluconazole or posaconazole (10 mg/kg orally twice daily) was initiated 24 h post-inoculation. RESULTS: C. gattii was more virulent than C. neoformans, as evident by shorter median survival, earlier histopathological changes and higher serum antigen titres. However, no differences in fungal burden or dissemination to other organs were observed among the various groups. In treatment studies, both fluconazole and posaconazole improved the median survival of mice infected with either species. However, neither regimen improved the percentage of animals surviving to the predetermined study endpoint. CONCLUSIONS: These results demonstrate the virulence of C. gattii meningoencephalitis and the potential of this model for the assessment of new treatment strategies.


Assuntos
Criptococose/microbiologia , Criptococose/patologia , Cryptococcus gattii/patogenicidade , Modelos Animais de Doenças , Meningoencefalite/microbiologia , Meningoencefalite/patologia , Animais , Antifúngicos/administração & dosagem , Antígenos de Fungos/sangue , Encéfalo/microbiologia , Contagem de Colônia Microbiana , Criptococose/tratamento farmacológico , Cryptococcus neoformans/patogenicidade , Fluconazol/administração & dosagem , Histocitoquímica , Meningoencefalite/tratamento farmacológico , Camundongos , Camundongos Endogâmicos ICR , Análise de Sobrevida , Triazóis/administração & dosagem
18.
Int J Mol Sci ; 13(1): 726-36, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22312282

RESUMO

In this study we pursued a diagnostic target in Aspergillus fumigatus (AF) by using qualitative Realtime PCR combined with proprietary DNA primers and a hydrolysis probe specific for this fungal target. Qualitative Realtime PCR is a diagnostic tool that utilizes Realtime PCR technology and detects the presence or absence target specific DNA within a predetermined detection range. Respiratory tissue and fluids from experimentally infected guinea pigs were tested by extracting DNA from the samples which were amplified and detected using AF specific DNA primers and probe. This study included qualitative evaluations of all specimens for the presence of the DNA of AF. The findings in the tissues after AF infection were compared to the numbers of spores in aerosolized samples used to inoculate the animals. Results demonstrated that the specific probe and primer set could detect the presence or absence of AF DNA in the sample. The qualitative detection limit of the assay ranged from 6 × 10(4) copies to 6 copies. Since blood cultures are rarely positive for Aspergillosis, our data indicate that qualitative Realtime PCR, in combination with the appropriate DNA primers and probe can serve as an effective diagnostic tool in the early detection of fungal infections.


Assuntos
Aspergillus fumigatus/genética , Líquido da Lavagem Broncoalveolar/microbiologia , Pulmão/microbiologia , Animais , Aspergillus fumigatus/isolamento & purificação , Primers do DNA/genética , Primers do DNA/metabolismo , DNA Fúngico/análise , Cobaias , Pulmão/patologia , Masculino , Reação em Cadeia da Polimerase em Tempo Real
19.
J Antimicrob Chemother ; 67(4): 970-6, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22240402

RESUMO

OBJECTIVES: Amphotericin B inhalation powder (ABIP) is a novel dry-powder amphotericin B formulation that is directly delivered to the lung, resulting in elevated lung tissue drug concentrations of this polyene. We evaluated the prophylactic efficacy of single dose administration of ABIP in a guinea pig model of invasive pulmonary aspergillosis. METHODS: Guinea pigs were immunosuppressed with cyclophosphamide and cortisone acetate and challenged with Aspergillus fumigatus conidia in an aerosol chamber. Guinea pigs received prophylaxis with a single inhaled dose of ABIP at 0.05, 0.5, 4 or 10 mg/kg administered 24 h prior to infection. Treatment with oral voriconazole at doses of 5 or 10 mg/kg twice daily beginning 24 h post-challenge served as the positive control. RESULTS: Improvements in survival were observed with ABIP prophylaxis. A single inhaled dose of 4 mg/kg ABIP and treatment with 5 mg/kg voriconazole both improved median and percentage survival compared with untreated controls. In addition, pulmonary fungal burden, as assessed by cfu, quantitative PCR and galactomannan, was also reduced in a dose-dependent fashion with ABIP prophylaxis as well as with both doses of voriconazole treatment. CONCLUSIONS: Single-dose prophylaxis with inhaled ABIP as prophylaxis demonstrated a significant survival advantage and reductions in pulmonary fungal burden in this model of invasive pulmonary aspergillosis. Optimization of the dose and dosing frequency of ABIP dose may help to further enhance the anti-Aspergillus activity of this novel amphotericin B formulation.


Assuntos
Anfotericina B/administração & dosagem , Antifúngicos/administração & dosagem , Aspergillus fumigatus/patogenicidade , Quimioprevenção/métodos , Aspergilose Pulmonar Invasiva/prevenção & controle , Administração por Inalação , Animais , Aspergillus fumigatus/efeitos dos fármacos , Contagem de Colônia Microbiana , Cortisona/administração & dosagem , Cortisona/análogos & derivados , Ciclofosfamida/administração & dosagem , Modelos Animais de Doenças , Cobaias , Imunossupressores/administração & dosagem , Pulmão/microbiologia , Masculino , Pirimidinas/administração & dosagem , Análise de Sobrevida , Triazóis/administração & dosagem , Voriconazol
20.
J Clin Microbiol ; 50(3): 602-8, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22189110

RESUMO

We developed and assessed the diagnostic value of a novel quantitative nested real-time (QNRT) PCR assay targeting the internal transcribed spacer region of ribosomal DNA (rDNA) in a guinea pig model of invasive pulmonary aspergillosis. Groups of 5 immunosuppressed animals that were infected using an aerosol chamber with Aspergillus fumigatus conidia were humanely terminated 1 h postinoculation and at days 3, 5, 7, and 11 postchallenge, and lung tissue, bronchoalveolar lavage (BAL) fluid, whole blood, and serum samples were collected. The QNRT PCR results obtained with the serum and BAL fluid were compared to those achieved with galactomannan and (1→3)-ß-d-glucan assays. High fungal burden levels were detected by QNRT PCR in both lung tissue and BAL fluid in all infected animals at each time point, and the sensitivity of each assay in BAL fluid was 100% by day 3 and remained so through the remainder of the study. The sensitivity of detection of fungi in whole blood and serum samples was significantly lower, and some samples remained negative by all three assays despite the advanced stage of the infection. From these data, we can conclude that this novel QNRT PCR method was highly sensitive for the detection of A. fumigatus from different types of samples in this model. In addition, BAL fluid samples appeared to be the most suitable for the early diagnosis of invasive pulmonary aspergillosis. When testing serum, the use of a combination of available assays may increase the possibility of early detection of this opportunistic mycosis.


Assuntos
Aspergillus fumigatus/isolamento & purificação , Aspergilose Pulmonar Invasiva/microbiologia , Mananas/análise , Micologia/métodos , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , beta-Glucanas/análise , Animais , Líquido da Lavagem Broncoalveolar/microbiologia , Contagem de Colônia Microbiana/métodos , DNA Fúngico/genética , DNA Espaçador Ribossômico/genética , Modelos Animais de Doenças , Cobaias , Aspergilose Pulmonar Invasiva/patologia , Pulmão/microbiologia , Masculino , Sensibilidade e Especificidade
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