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1.
J Med Microbiol ; 69(1): 87-95, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31846414

RESUMO

Introduction. Acanthamoeba keratitis is a sight-threatening corneal infection that is commonly reported among contact lens users and those suffering from corneal trauma. The prevalence of Acanthamoeba species or genotypes in causing keratitis infection is not well known.Aim. This study was conducted to identify and genotype Acanthamoeba isolates from keratitis patients, targeting the ribosomal nuclear subunit (Rns) region, and describe the associated clinical presentation and treatment outcome.Methodology. Thirty culture-confirmed patients with Acanthamoeba keratitis, identified in a tertiary eye care centre in South India during the period from December 2016 to December 2018, were included in this study. The data collected from patient records include demographic details, history of illness, mode of trauma, treatment history and follow-up status. The genotype and species were identified based on the Rns sequence and phylogenetic tree analysis.Results. Acanthamoeba culbertsoni was the most predominant keratitis-causing species, followed by Acanthamoeba quina, Acanthamoeba castellanii, Acanthamoeba healyi, Acanthamoeba hatchetti, Acanthamoeba polyphaga and Acanthamoeba stevensoni. Three major genotypes were identified (T4, T11 and T12), with the T4 genotype being the most predominant, with four subclusters, i.e. T4A, T4B, T4D and T4E. This is the first report on corneal infection by the A. stevensoni T11 genotype and the A. healyi T12 genotype. No significant correlation was observed between the clinical outcomes of corneal disease and the genotypes or species.Conclusion. Rns genotyping is very effective in identifying the Acanthamoeba species and genotype in keratitis. Genotyping of Acanthamoeba spp. will help to advance our understanding of genotype-specific pathogenesis and geographical distribution.


Assuntos
Ceratite por Acanthamoeba/patologia , Ceratite por Acanthamoeba/parasitologia , Acanthamoeba castellanii/classificação , Acanthamoeba castellanii/genética , Genótipo , Filogenia , Acanthamoeba castellanii/isolamento & purificação , Genes de RNAr , Técnicas de Genotipagem , Humanos , Índia , Centros de Atenção Terciária
2.
Parasitol Res ; 119(2): 659-666, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31848745

RESUMO

Free-living amoebae of the genus Acanthamoeba are causative agents of Acanthamoeba keratitis and amoebic encephalitis in humans, both of which are serious infections. The ability to produce proteases is one of the factors involved in the pathogenesis of Acanthamoeba infections. The aim of this study was to evaluate the secreted proteases of six Acanthamoeba strains from distinct genotypes (T1, T2, T4 and T11) maintained in prolonged axenic culture and following three successive passages in Madin-Darby Canine Kidney (MDCK) cells. Conditioned medium was obtained from cultures before and after interaction with the MDCK monolayers, resolved in SDS-PAGE containing gelatine, then subjected to quantitative azocasein assays. Zymography profiles varied between the strains, with the predominant proteases found to be serine-type proteases from 49 to 128 kDa. A T1 genotype strain isolated from dust showed quantitatively higher protease secretion compared to the other strains. No changes were detected in the zymography profiles of MDCK-interacted cultures compared to long-term axenic cultures. Two strains presented lower proteolytic activity post-MDCK interaction, while the remaining strains presented similar values before and after MDCK passages. In conclusion, this study confirms the predominance of serine-type protease secretion by Acanthamoeba, with distinct profiles presented by the different strains and genotypes studied. Also, interaction of trophozoites with MDCK cells did not alter the zymography pattern.


Assuntos
Acanthamoeba/enzimologia , Acanthamoeba/metabolismo , Serina Proteases/metabolismo , Acanthamoeba/genética , Ceratite por Acanthamoeba/parasitologia , Animais , Cultura Axênica , Caseínas/análise , Linhagem Celular , Cães , Genótipo , Humanos , Células Madin Darby de Rim Canino , Trofozoítos/metabolismo
3.
Parasit Vectors ; 12(1): 467, 2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31597577

RESUMO

BACKGROUND: Pathogenic protozoans use extracellular vesicles (EVs) for intercellular communication and host manipulation. Acanthamoeba castellanii is a free-living protozoan that may cause severe keratitis and fatal granulomatous encephalitis. Although several secreted molecules have been shown to play crucial roles in the pathogenesis of Acanthamoeba, the functions and components of parasite-derived EVs are far from understood. METHODS: Purified EVs from A. castellanii were confirmed by electron microscopy and nanoparticle tracking analysis. The functional roles of parasite-derived EVs in the cytotoxicity to and immune response of host cells were examined. The protein composition in EVs from A. castellanii was identified and quantified by LC-MS/MS analysis. RESULTS: EVs from A. castellanii fused with rat glioma C6 cells. The parasite-derived EVs induced an immune response from human THP-1 cells and a cytotoxic effect in C6 cells. Quantitative proteomic analysis identified a total of 130 proteins in EVs. Among the identified proteins, hydrolases (50.2%) and oxidoreductases (31.7%) were the largest protein families in EVs. Furthermore, aminopeptidase activities were confirmed in EVs from A. castellanii. CONCLUSIONS: The proteomic profiling and functional characterization of EVs from A. castellanii provide an in-depth understanding of the molecules packaged into EVs and their potential mechanisms mediating the pathogenesis of this parasite.


Assuntos
Acanthamoeba castellanii/fisiologia , Exossomos/química , Exossomos/fisiologia , Proteômica , Ceratite por Acanthamoeba/parasitologia , Acanthamoeba castellanii/patogenicidade , Acanthamoeba castellanii/ultraestrutura , Aminopeptidases/análise , Animais , Infecções Protozoárias do Sistema Nervoso Central/parasitologia , Meios de Cultura , DNA Complementar/biossíntese , Exossomos/imunologia , Exossomos/ultraestrutura , Humanos , Microscopia Eletrônica de Transmissão , Neuroglia/parasitologia , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células THP-1/imunologia , Células THP-1/parasitologia
4.
Eye Contact Lens ; 45(5): 301-305, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31442212

RESUMO

PURPOSE: To compare the infectious contact lens-related corneal ulcer (CLRU) and non-CLRU cases at Saint Louis University. METHODS: Retrospective review of corneal ulcer cases identified by search of the ophthalmology and microbiology department databases between 1999 and 2016. RESULTS: Six hundred seventy-seven cases of corneal ulcers were identified, of which 46% were CLRU. CLRU cases were seen more commonly in younger patients (P<0.001) and women (P=0.03) than non-CLRU cases. Many of the infections were vision-threatening as defined by central/paracentral location (73% CLRU and 71% non-CLRU [P=0.60]) and large size of ulcer >2 mm in 36% CLRU and 51% non-CLRU (P=0.002). Causative pathogen in cultured CLRU was predominately Pseudomonas species (44%, P<0.001 vs. the non-CLRU group), other gram-negative (6%), gram-positive (33%), fungi (13%), and Acanthamoeba (5%). Comparatively, cultured non-CLRU was predominately gram-positive (64%, P<0.001 vs. the CLRU group), gram-negative (26%), and fungi (11%). The combined oxacillin-resistant Staphylococcus aureus and coagulase-negative Staphylococcus isolates were 35% and 34%, respectively. Despite the progressive increase in the number of corneal ulcers seen, the annual trend for any one particular organism for either CLRU cases or non-CLRU cases did not change significantly. CONCLUSIONS: Most of the cases were non-CLRU. CLRU was disproportionately associated with Pseudomonas species and non-CLRU with Staphylococcal species. Fungal infections were predominately caused by filamentous organisms in both groups. Acanthamoeba keratitis was exclusively associated with CL use.


Assuntos
Ceratite por Acanthamoeba/parasitologia , Lentes de Contato/microbiologia , Lentes de Contato/parasitologia , Úlcera da Córnea/microbiologia , Infecções Oculares Bacterianas/microbiologia , Infecções Oculares Fúngicas/microbiologia , Infecções Oculares Parasitárias/parasitologia , Centros Médicos Acadêmicos , Ceratite por Acanthamoeba/diagnóstico , Ceratite por Acanthamoeba/tratamento farmacológico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/uso terapêutico , Antiprotozoários/uso terapêutico , Criança , Pré-Escolar , Úlcera da Córnea/diagnóstico , Úlcera da Córnea/tratamento farmacológico , Infecções Oculares Bacterianas/diagnóstico , Infecções Oculares Bacterianas/tratamento farmacológico , Infecções Oculares Fúngicas/diagnóstico , Infecções Oculares Fúngicas/tratamento farmacológico , Infecções Oculares Parasitárias/diagnóstico , Infecções Oculares Parasitárias/tratamento farmacológico , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos
5.
Cornea ; 38(10): 1309-1313, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31306283

RESUMO

PURPOSE: To determine whether combinations of commonly used antiamoebic agents display synergy in their ability to kill Acanthamoeba cysts in vitro. METHODS: Synergy testing was performed with a microdilution checkerboard assay on 10 clinical Acanthamoeba keratitis isolates collected at the Proctor Foundation from 2008 to 2012. Each isolate was exposed to pairwise combinations of chlorhexidine, propamidine, and voriconazole. The minimum cysticidal concentration (MCC) for each drug pair was estimated for each isolate, and the summed fractional cysticidal concentration (ΣFCC) was calculated for each drug combination in the checkerboard, with synergy defined as a lack of growth at a ΣFCC ≤ 0.5 and antagonism as growth at a ΣFCC > 4. RESULTS: Chlorhexidine and propamidine were cysticidal, with median MCCs of 12.5 (range 1.5-50) and 11.7 (range 0.2-250), respectively. Voriconazole was not cysticidal, with a median MCC of >10,000 µg/mL. The combination of chlorhexidine and propamidine did not markedly change the cysticidal activity compared with either drug alone. By contrast, voriconazole antagonized the cysticidal activity of both chlorhexidine and propamidine, with Acanthamoeba growth observed at antagonistic ΣFCCs in 27 of 49 (55.1%, 95% confidence interval 35.7%-78.6%) checkerboard combinations of voriconazole and chlorhexidine and in 58 of 147 (39.5%, 95% confidence interval 14.3%-50.3%) combinations of voriconazole and propamidine. CONCLUSIONS: In an in vitro assay, voriconazole reduced the cysticidal activity of 2 commonly used antiamoebic drugs. Although the in vivo drug interactions could be different, these observations may be useful in cases of nonhealing Acanthamoeba keratitis being treated with combination therapies that include voriconazole.


Assuntos
Ceratite por Acanthamoeba/tratamento farmacológico , Acanthamoeba/isolamento & purificação , Amebicidas/farmacologia , Infecções Oculares Parasitárias/tratamento farmacológico , Voriconazol/farmacologia , Acanthamoeba/efeitos dos fármacos , Ceratite por Acanthamoeba/diagnóstico , Ceratite por Acanthamoeba/parasitologia , Animais , Antifúngicos/farmacologia , Sinergismo Farmacológico , Infecções Oculares Parasitárias/parasitologia , Humanos , Testes de Sensibilidade Parasitária
6.
Korean J Parasitol ; 57(3): 217-223, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31284343

RESUMO

Acanthamoeba castellanii has ubiquitous distribution and causes primary acanthamoebic keratitis (AK). AK is a common disease in contact lens wearers and results in permanent visual impairment or blindness. In this study, we observed the cytopathic effect, in vitro cytotoxicity, and secretion pattern of cytokines in human corneal epithelial cells (HCECs) induced by A. castellanii trophozoites and/or cysts. Morphological observation revealed that panked dendritic HCECs co-cultured with amoeba cysts had changed into round shape and gradually died. Such changes were more severe in co-culture with cyst than those of co-cultivation with trophozoites. In vitro cytotoxicity assay revealed the highest cytotoxicity to HCECs in the co-culture system with amoeba cysts. A. castellanii induced the expression of IL-1α, IL-6, IL-8, and CXCL1 in HCECs. Secreted levels of IL-1α, IL-6, and IL-8 in HCECs co-cultured with both trophozoites and cysts were increased at an early incubation time (3 and 6 hr). These results suggested that cytopathic changes and pro-inflammatory cytokines release of HCECs in response to A. castellanii, especially amoebic cysts, are an important mechanism for AK development.


Assuntos
Ceratite por Acanthamoeba/imunologia , Acanthamoeba castellanii/fisiologia , Córnea/citologia , Células Epiteliais/imunologia , Trofozoítos/fisiologia , Ceratite por Acanthamoeba/parasitologia , Acanthamoeba castellanii/crescimento & desenvolvimento , Células Cultivadas , Córnea/imunologia , Córnea/parasitologia , Células Epiteliais/parasitologia , Humanos , Interleucina-1/genética , Interleucina-1/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Interleucina-8/genética , Interleucina-8/imunologia , Trofozoítos/crescimento & desenvolvimento
7.
Parasitol Res ; 118(7): 2295-2304, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31093751

RESUMO

Acanthamoeba castellanii belonging to the T4 genotype is an opportunistic pathogen which is associated with blinding eye keratitis and rare but fatal central nervous system infection. A. castellanii pose serious challenges in antimicrobial chemotherapy due to its ability to convert into resistant, hardy shell-protected cyst form that leads to infection recurrence. The fatty acid composition of A. castellanii trophozoites is known to be most abundant in oleic acid which chemically is an unsaturated cis-9-Octadecanoic acid and naturally found in animal and vegetable fats and oils. This study was designed to evaluate antiacanthamoebic effects of oleic acid against trophozoites, cysts as well as parasite-mediated host cell cytotoxicity. Moreover, oleic acid-conjugated silver nanoparticles (AgNPs) were also synthesized and tested against A. castellanii. Oleic acid-AgNPs were synthesized by chemical reduction method and characterized by ultraviolet-visible spectrophotometry, atomic force microscopy, dynamic light scattering analysis, and Fourier transform infrared spectroscopy. Viability, growth inhibition, encystation, and excystation assays were performed with 10 and 5 µM concentration of oleic acid alone and oleic acid-conjugated AgNPs. Bioassays revealed that oleic acid alone and oleic acid-conjugated AgNPs exhibited significant antiamoebic properties, whereas nanoparticle conjugation further enhanced the efficacy of oleic acid. Phenotype differentiation assays also showed significant inhibition of encystation and excystation at 5 µM. Furthermore, oleic acid and oleic acid-conjugated AgNPs also inhibited amoebae-mediated host cell cytotoxicity as determined by lactate dehydrogenase release. These findings for the first time suggest that oleic acid-conjugated AgNPs exhibit antiacanthamoebic activity that hold potential for therapeutic applications against A. castellanii.


Assuntos
Ceratite por Acanthamoeba/tratamento farmacológico , Acanthamoeba castellanii/efeitos dos fármacos , Amebicidas/farmacologia , Nanopartículas Metálicas/química , Ácido Oleico/farmacologia , Ceratite por Acanthamoeba/parasitologia , Amebicidas/química , Animais , Olho/parasitologia , Humanos , Microscopia de Força Atômica , Ácido Oleico/química , Prata/química , Espectrofotometria Ultravioleta , Trofozoítos/efeitos dos fármacos
8.
Parasitol Res ; 118(8): 2443-2454, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31144032

RESUMO

Acanthamoeba keratitis (AK) is a devastating, painful corneal infection, which may lead to loss of vision. The development of resistance and failure of the currently used drugs represent a therapeutic predicament. Thus, novel therapies with lethal effects on resistant Acanthamoeba are necessary to combat AK. In the present study, the curative effect of Nigella sativa aqueous extract (N. sativa) and chitosan nanoparticles (nCs) and both agents combined were assessed in experimentally induced AK. All inoculated corneas developed varying grades of AK. The study medications were applied on the 5th day postinoculation and were evaluated by clinical examination of the cornea and cultivation of corneal scraps. On the 10th day posttreatment, a 100% cure of AK was obtained with nCs (100 µg/ml) in grades 1 and 2 of corneal opacity as well as with N. sativa 60 mg/ml-nCs 100 µg/ml in grades 1, 2, and 3 of corneal opacity, highlighting a possible synergistic effect. On the 15th day posttreatment, a 100% cure was reached with N. sativa aqueous extract (60 mg/ml). Moreover, on the 20th day posttreatment, N. sativa (30 mg/ml) provided a cure rate of 87.5%, while nCs (50 µg/ml) as well as N. sativa 30 mg/ml-nCs 50 µg/ml yielded a cure rate of 75%; the lowest percentage of cure (25%) was obtained with chlorhexidine (0.02%), showing a non-significant difference compared to the parasite control. The clinical outcomes were in agreement with the results of corneal scrap cultivation. The results of the present study demonstrate the effectiveness of N. sativa aqueous extract and nCs (singly or combined) when used against AK, and these agents show potential for the development of new, effective, and safe therapeutic alternatives.


Assuntos
Ceratite por Acanthamoeba/tratamento farmacológico , Antiprotozoários/administração & dosagem , Nigella sativa/química , Extratos Vegetais/administração & dosagem , Acanthamoeba/efeitos dos fármacos , Acanthamoeba/fisiologia , Ceratite por Acanthamoeba/parasitologia , Adulto , Animais , Antiprotozoários/química , Quitosana/química , Quitosana/farmacologia , Quitosana/uso terapêutico , Clorexidina/farmacologia , Córnea/parasitologia , Feminino , Humanos , Masculino , Nanopartículas/química , Extratos Vegetais/química , Ratos , Resultado do Tratamento
9.
Ann Parasitol ; 65(1): 19-25, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31095904

RESUMO

Acanthamoeba keratitis, the vision-threatening corneal disease reported with increasing frequency in Poland is difficult to treat due to extremely high resistance of the amoeba cysts to chemicals. The agents of possible anti-amoebic activity are still tested. Pathogenic Acanthamoeba samples/isolates acquired from severe cases of keratitis examined by molecular techniques to determine genotypes, compared to one another as well as to the environmental Acanthamoeba castellanii Neff strain were included in the studies. These strains were in vitro examined in terms of their sensitivity/resistance to selected chemicals and tolerance to temperature changes. Samples of the strains cultivated in vitro under bacteria-free conditions were monitored during different growth phases. Higher amoebic population dynamics was observed in both pathogenic Acanthamoeba strains during transfer to 37°C. Agents tested influenced population dynamics in different degree; they showed amoebostatic or amoebicidal effects, however a tendency toward induction of encystment also appeared. Because activation of the dormant cysts can lead to repeated development of amoebae, very important is cysticidal efficacy of chemicals. Further in vitro investigations on various Acanthamoeba strains with different chemicals are still necessary.


Assuntos
Acanthamoeba , Amebicidas , Temperatura , Acanthamoeba/efeitos dos fármacos , Acanthamoeba/genética , Ceratite por Acanthamoeba/parasitologia , Amebicidas/farmacologia , Genótipo , Humanos , Polônia , Saúde Pública
10.
Acta Microbiol Immunol Hung ; 66(3): 289-300, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-30835133

RESUMO

Genus Acanthamoeba is an opportunistic protozoan that is widely distributed in the environment. Within this genus, numerous species are recognized as human pathogens, potentially causing Acanthamoeba keratitis (AK). AK is a corneal disease, associated predominantly with contact lens (CL) wear; its epidemiology is related to the specific Acanthamoeba genotypes. This study reports seven CL wearer, Acanthamoeba PCR-positive patients with AK, diagnosed between January 2015 and 2018. Patients had the diagnosis of AK 1.36 months after first symptoms. Genotyping allowed the identification of six isolates of the T4 and one of the T8 genotypes. At first presentation, pseudendritiformic epithelopathy/dirty epithelium (four eyes, 57.1%), multifocal stromal infiltrates (five eyes, 71.4%), ring infiltrate (three eyes, 42.8%), and perineuritis (one eye, 14.3%) were observed. AK was healed without later recurrence in two eyes (28.5%) using triple-topical therapy, in three eyes (42.8%) following additional penetrating keratoplasty. In one patient (14.3%), AK recurred following successful application of triple-therapy and was treated successfully with repeated triple-topical therapy and in one patient (14.3%), no follow-up data were available after diagnosis. We could not observe correlation of genotype and clinical course or the necessity of corneal transplantation in our case series.


Assuntos
Ceratite por Acanthamoeba/patologia , Ceratite por Acanthamoeba/parasitologia , Acanthamoeba/classificação , Acanthamoeba/genética , Genótipo , Acanthamoeba/isolamento & purificação , Ceratite por Acanthamoeba/terapia , Administração Tópica , Adolescente , Adulto , Antiprotozoários/uso terapêutico , Transplante de Córnea , Feminino , Técnicas de Genotipagem , Humanos , Hungria , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento , Adulto Jovem
11.
Exp Parasitol ; 197: 29-35, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30648558

RESUMO

Free-living amoebae of the genus Acanthamoeba are the etiological agents of cutaneous lesions, granulomatous amoebic encephalitis (GAE) and amoebic keratitis (AK), which are chronic infections with poor prognosis if not diagnosed promptly. Currently, there is no optimal therapeutic scheme to eradicate the pathologies these protozoa cause. In this study we report the morphological and molecular identification of three species of the genus Acanthamoeba, belonging to T4 group; A. polyphaga isolated from the corneal ulcer of a patient sample of AK case; A. castellanii isolated from the contact lens of an AK patient and A. palestinensis obtained from a soil sample. The in vitro activity of chlorhexidine, itraconazole and voriconazole drugs against trophic stage was also evaluated through a colorimetric assay based on the oxidation-reduction of alamar blue. The strains in the study were sensitive to the evaluated drugs; although when determining the 50% inhibitory concentration (IC50) statistically significant differences were observed. A. castellanii showed to be highly sensitive to voriconazole (0.66 ±â€¯0.13 µM) but the least sensitive to chlorhexidine and itraconazole (8.61 ±â€¯1.63 and 20.14 ±â€¯4.93 µM, respectively), A. palestinensis showed the highest sensitivity to itraconazole (0.502 ±â€¯0.11 µM) and A. polyphaga expressed moderate sensitivity to chlorhexidine and itraconazole and lower sensitivity to voriconazole (10.10 ±â€¯2.21 µM). These results showed that species of the genus Acanthamoeba express different sensitivity to the tested drugs, which could explain the problems surrounding the establishment of a treatment of choice in the infections caused by these amoebae. We consider that although chlorhexidine and itraconazole show good activity on these amoebae and have been used in cases of AK in Mexico with acceptable results, voriconazole should be considered as the first therapeutic option of future Acanthamoeba infections that will be diagnosed in our country.


Assuntos
Acanthamoeba/efeitos dos fármacos , Amebíase/parasitologia , Anti-Infecciosos/farmacologia , Clorexidina/farmacologia , Itraconazol/farmacologia , Voriconazol/farmacologia , Acanthamoeba/classificação , Acanthamoeba/genética , Ceratite por Acanthamoeba/parasitologia , Amebíase/tratamento farmacológico , Lentes de Contato/parasitologia , Úlcera da Córnea/parasitologia , DNA de Protozoário/isolamento & purificação , Genótipo , Humanos , Concentração Inibidora 50 , México , Solo/parasitologia
12.
Parasit Vectors ; 12(1): 58, 2019 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-30683157

RESUMO

BACKGROUND: Acanthamoeba spp. are the causative agents of a severe keratitis occurring mainly in contact lens wearers. The genus comprises more than 24 species that are currently divided into 20 different genotypes (T1-T20) according to sequence variations in the 18S rRNA gene. The objective of this study was to identify the genotypes and sub-genotypes of Acanthamoeba isolates collected at the Parasitology Laboratory of the Public Health Institute of Chile, the only laboratory in the country where Acanthamoeba screening is performed. This is the first report of genotypic identification of clinical isolates of Acanthamoeba in Chile and one of the few in South America. RESULTS: In this study, 114 Acanthamoeba isolates from 76 Acanthamoeba keratitis patients, obtained between 2005-2016, were genotyped. T4 was the predominant genotype; T2 and T11 genotypes, which are scarcely reported worldwide, were also identified in Chilean patients (one and two patients, respectively). This is the first report of T2 and T11 genotypes isolated from Acanthamoeba keratitis patients in South America. It is also the first report of the T2 genotype circulating in this continent. Analysis of the diagnostic fragment 3 region of the 18S rRNA gene showed 24 T4 variants, with a predominance of the sub-genotype T4/A, followed by T4/B, T4/G, T4/C and T4/D. Bayesian analysis revealed three groups among the T4 variants: two well supported groups that included 12 and 7 sub-genotypes, respectively, and a weakly supported group that included 5 sub-genotypes. Most of the predominant T4 sub-genotypes belonged to the same group, which included 71.3% of the patients, while some minority variants lied mainly in the other two clusters. CONCLUSIONS: T2, T4 and T11 genotypes were predominantly isolated from the Acanthamoeba keratitis patients in Chile. Chilean predominant T4 sub-genotypes, which have also been reported worldwide, formed a separate cluster of the minority T4 variants. This study provides useful information about the predominant genotypes and subgenotypes that would be useful in selecting suitable strains to develop immunological and/or molecular diagnostic assays in Chile.


Assuntos
Ceratite por Acanthamoeba/parasitologia , Acanthamoeba/genética , Variação Genética , Acanthamoeba/classificação , Acanthamoeba/isolamento & purificação , Chile , Feminino , Genótipo , Humanos , Masculino , Filogenia
13.
Colloids Surf B Biointerfaces ; 173: 725-732, 2019 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-30384269

RESUMO

Acanthamoeba keratitis is an ophthalmic disease with no specific treatment that specially affects contact lens users. The silencing of serine phosphatase (SP) and glycogen phosphorylase (GP) proteins produced by Acanthamoeba has been shown to significantly reduce the cytopathic effect, although no vehicle was proposed yet to deliver the siRNA sequences to the trophozoites. In this study, PEGylated cationic liposomes were proposed and optimized using Box-Behnken design. The influence of DOTAP:DOPE ratio, DSPE-PEG concentration, and siRNA/DOTAP charge ratio were evaluated over both biological response and physicochemical properties of liposomes. The ratio of DOTAP:DOPE had an effect in the trophozoite activity whereas the charge ratio influenced both size and protease activity. The predicted values were very close to the observed values, yielding a formulation with good activity and toxicity profile, which was used in the following experiments. A murine model of ocular keratitis was treated with siGP + siSP-loaded liposomes, as well as their respective controls, and combined treatment of liposomes and chlorhexidine. After 15 days of eight daily administrations, the liposomal complex combined with chlorhexidine was the only treatment able to reverse the more severe lesions associated with keratitis. There was 60% complete regression in corneal damage, with histological sections demonstrating the presence of an integral epithelium, without lymphocytic infiltrate. The set of results demonstrate the efficacy of a combined therapy based on siRNA with classical drugs for a better prognosis of keratitis caused by Acanthamoeba.


Assuntos
Ceratite por Acanthamoeba/terapia , Acanthamoeba/efeitos dos fármacos , Clorexidina/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Lipossomos/química , Proteínas de Protozoários/antagonistas & inibidores , Trofozoítos/efeitos dos fármacos , Acanthamoeba/enzimologia , Acanthamoeba/patogenicidade , Ceratite por Acanthamoeba/parasitologia , Ceratite por Acanthamoeba/patologia , Animais , Córnea/efeitos dos fármacos , Córnea/parasitologia , Córnea/patologia , Modelos Animais de Doenças , Esquema de Medicação , Composição de Medicamentos/métodos , Quimioterapia Combinada , Análise Fatorial , Ácidos Graxos Monoinsaturados/química , Regulação da Expressão Gênica , Glicogênio Fosforilase/antagonistas & inibidores , Glicogênio Fosforilase/genética , Glicogênio Fosforilase/metabolismo , Humanos , Lipossomos/metabolismo , Fosfatidiletanolaminas/química , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Polietilenoglicóis/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Compostos de Amônio Quaternário/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Wistar , Trofozoítos/enzimologia , Trofozoítos/patogenicidade
14.
Exp Parasitol ; 196: 22-27, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30472333

RESUMO

Acanthamoeba keratitis (AK) is a sight-threatening corneal infection. The early symptoms include redness, pain, photophobia and intense tearing. Chronic infection usually progresses to stromal inflammation, ring ulcers, corneal opacification and hypopyon. Here we document an AK case in a high myopic 38-year-old woman from Mexico City, with a history of wearing contact lenses while swimming. Corneal scrapes cultures were positive only for amoebae, consequently a treatment including netilmicin 0.3% and oral itraconazole 100 mg/12 h was prescribed. The infection was resolved after 8 months, leaving a slight leucoma outside the visual axis, with a visual acuity of 20/150. In the laboratory, the amoebic isolate was axenized in PYG medium, with an optimal growth at 30 °C, and was identified morphologically as Acanthamoeba polyphaga according to the taxonomic criteria of Page (1988) and placed in the T4 group by genotyping. The virulence of this strain (40%) was determined by intranasal inoculation of 1 × 106/20 µl trophozoites in BALB/c mice recovering from brain, proving their invasion ability and by the interaction with monolayers of epithelial cells of the established MDCK line of canine kidney origin (1:2 ratio of interaction), at 1, 3, 6, 8 and 24 h; trophozoites migrated to cell junctions inducing few lytic zones. In addition to the biological characterization, in vitro drug sensitivity tests were performed using chlorhexidine, itraconazole, netilmicin and voriconazole. Results revealed that voriconazole was the most effective compound. A. polyphaga remains as one of the most frequently isolated species producing AK. The treatment of AK case using netilmicin and oral itraconazole solved the disease, but the healing process was wide-ranging (8 months). The use of voriconazole and chlorhexidine may be an alternative treatment of future AK cases in Mexico.


Assuntos
Ceratite por Acanthamoeba/parasitologia , Acanthamoeba/efeitos dos fármacos , Anti-Infecciosos/administração & dosagem , Acanthamoeba/isolamento & purificação , Ceratite por Acanthamoeba/tratamento farmacológico , Adulto , Animais , Antibacterianos/administração & dosagem , Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Antifúngicos/administração & dosagem , Antifúngicos/farmacologia , Clorexidina/farmacologia , Lentes de Contato/efeitos adversos , Lentes de Contato/parasitologia , Cães , Feminino , Humanos , Concentração Inibidora 50 , Itraconazol/administração & dosagem , Itraconazol/farmacologia , Células Madin Darby de Rim Canino , México , Camundongos , Camundongos Endogâmicos BALB C , Midriáticos/administração & dosagem , Netilmicina/administração & dosagem , Netilmicina/farmacologia , Testes de Sensibilidade Parasitária , Fenilefrina/administração & dosagem , Tropicamida/administração & dosagem , Voriconazol/farmacologia
16.
Ann Agric Environ Med ; 25(4): 725-731, 2018 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-30586959

RESUMO

INTRODUCTION: Free-living amoebae, ubiquitous in outer environments, in predisposing circumstances may exist as parasites, infectious agents of Acanthamoeba keratitis. In recent decades, the vision-threatening corneal infection is a growing human health threat worldwide, including Poland. The applied therapy is often ineffective due to diagnostic mistakes, various pathogenicity of Acanthamoeba strains and high resistance of cysts to drugs; many agents with possible anti-amoebic activity are still being tested. In the presented study, selected chemicals are investigated in terms of their in vitro effect on corneal and environmental Acanthamoeba strains. MATERIAL AND METHODS: Samples of a corneal isolate from a patient with severe Acanthamoeba keratitis,of assessed on the basis of genotype associations of 18S rRNA and the type strain, Acanthamoeba castellanii Neff cultivated in bacteria-free condition, were exposed to povidone iodine, chlorhexidine digluconate or toyocamycin. In vitro population dynamics of the strains were monitored and compared to those of control cultures. RESULTS: All chemicals showed anti-amoebic effects with different degrees of effectiveness. Significant differences were observed in the in vitro population dynamics, and the morpho-physiological status of A. castellanii Neff T4 and corneal strains determined as A. polyphaga T4 genotype, exposed to povidone iodine or toyocamycin, in comparison with chlorhexidine taken as reference. CONCLUSIONS: Time-dependent amoebstatic in vitro effects were demonstrated for all agents, in particular, the results of assays with povidone iodine are promising. No significant stimulation of encystation appeared; however, as cysticidal efficacy of chemicals is expected, complementary research is needed on different Acanthamoeba strains with modified agent concentrations and method application.


Assuntos
Ceratite por Acanthamoeba/parasitologia , Acanthamoeba/efeitos dos fármacos , Antiprotozoários/farmacologia , Clorexidina/análogos & derivados , Povidona-Iodo/farmacologia , Toiocamicina/farmacologia , Acanthamoeba/classificação , Acanthamoeba/genética , Acanthamoeba/isolamento & purificação , Ceratite por Acanthamoeba/tratamento farmacológico , Ceratite por Acanthamoeba/epidemiologia , Clorexidina/farmacologia , Genótipo , Humanos , Polônia/epidemiologia
18.
Exp Parasitol ; 195: 34-37, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30336121

RESUMO

Free-living amoebae (FLA) are widely spread in the environment and also known to cause rare but often serious infections. The present work focuses on a local survey on FLA. It is essential to know the prevalence and distribution of these microorganisms in order to get infections caused by them under control. In this study, FLA isolated from domestic tap water samples from homes of contact lens wearers were identified by morphology and by 18S rRNA gene sequence analysis. Morphological analysis and partial sequencing of the 18S rDNA revealed the presence of Acanthamoeba genotype T4 and Vermamoeba vermiformis in the investigated tap water samples. Naegleria fowleri, Balamuthia mandrillaris, and Sappinia spp. were not detected during this study. It was shown that species of FLA known to cause eye infections in humans are widely distributed in tap water in Istanbul, Turkey. Contact lens wearers should be aware of the risk of contamination from tap water and strictly apply stringent contact lens hygiene. With this study, we established Acanthamoeba genotype T4 and Vermamoeba vermiformis as contaminants of tap water in Istanbul.


Assuntos
Acanthamoeba/isolamento & purificação , Amoeba/isolamento & purificação , Água Potável/parasitologia , Acanthamoeba/classificação , Acanthamoeba/genética , Acanthamoeba/ultraestrutura , Ceratite por Acanthamoeba/etiologia , Ceratite por Acanthamoeba/parasitologia , Ceratite por Acanthamoeba/prevenção & controle , Amebíase/etiologia , Amebíase/parasitologia , Amebíase/prevenção & controle , Amoeba/classificação , Amoeba/genética , Amoeba/ultraestrutura , Análise por Conglomerados , Sequência Consenso , Soluções para Lentes de Contato/efeitos adversos , Criopreservação , DNA de Protozoário/química , Genótipo , Microscopia de Contraste de Fase , Reação em Cadeia da Polimerase , RNA Ribossômico 18S/genética , Alinhamento de Sequência , Trofozoítos/classificação , Trofozoítos/genética , Trofozoítos/isolamento & purificação , Trofozoítos/ultraestrutura , Turquia , Abastecimento de Água/normas
19.
Arch. Soc. Esp. Oftalmol ; 93(10): 497-502, oct. 2018. tab, ilus
Artigo em Espanhol | IBECS | ID: ibc-175125

RESUMO

Casos clínicos: Presentamos 10 casos de queratitis por Acanthamoeba tratados en nuestro hospital entre 2008 y 2017. Todos eran portadores de lentes de contacto. Como tratamiento todos recibieron una biguanida junto a una diamidina. En 3 casos la infección no superaba el estroma superficial, respondiendo al tratamiento tópico. En 7 alcanzaba el estroma profundo, precisando 6 de ellos una queratoplastia penetrante, 3 «en caliente» por riesgo de perforación o extensión ocular. La agudeza visual mejoró en todos los casos. Conclusión: La profundidad de la infección al diagnóstico aparece como el principal factor de riesgo para necesitar una queratoplastia penetrante


Clinical cases: Cases are presented of 10 patients with Acanthamoeba keratitis treated between 2008 and 2017. All were contact lens wearers. All of them received treatment with a biguanide combined with a diamidine. In 3 cases the infestation did not exceed the superficial stroma, responding to topical treatment. In 7, the infection reached the deep stroma, with 6 of these cases requiring penetrating keratoplasty (PKP), 3 of them therapeutic PKP because of perforation risk or ocular spreading. The visual acuity improved in all the cases. Conclusion: The infestation depth at the time of diagnosis appears to be the main risk factor for requiring a PKP


Assuntos
Humanos , Masculino , Feminino , Criança , Adolescente , Adulto Jovem , Adulto , Pessoa de Meia-Idade , Ceratite por Acanthamoeba/diagnóstico , Ceratite por Acanthamoeba/tratamento farmacológico , Biguanidas/uso terapêutico , Diaminas/uso terapêutico , Infecções Oculares/diagnóstico , Diagnóstico Precoce , Biópsia , Ceratite por Acanthamoeba/parasitologia , Acuidade Visual , Lentes de Contato/parasitologia , Infecções Oculares/tratamento farmacológico , Infecções Oculares/parasitologia
20.
Br J Ophthalmol ; 102(12): 1621-1628, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30232172

RESUMO

BACKGROUND/AIMS: Acanthamoeba keratitis (AK) is a chronic debilitating corneal infection principally affecting contact lens (CL) users. Studies were designed to test claims that the UK incidence may have increased in 2012-2014 and to evaluate potential causes. METHODS: Annualised incidence data were collected from January 1984 to December 2016. Case-control study subjects were recruited between 14 April 2011 and 05 June 2017. Reusable CL users with AK were recruited retrospectively and prospectively. Controls were reusable CL users, recruited prospectively, with any disorder other than AK. Multivariable analysis of questionnaire data measured independent risk factors for AK. RESULTS: The current outbreak of AK started in 2010-2011 with an incidence threefold higher than in 2004-2009. Risk factors for AK were: Oxipol disinfection, CLs made of group IV CL materials, poor CL hygiene, deficient hand hygiene, use of CLs while swimming or bathing, being white British, and for those in social classes 4-9. CONCLUSION: AK is a largely preventable disease. The current outbreak is unlikely to be due to any one of the identified risk factors in isolation. Improving CL and hand hygiene, avoiding CLs contamination with water and use of effective CL disinfection solutions, or daily disposable CLs, will reduce the incidence of AK. In the longer-term, water avoidance publicity for CL users can be expected to reduce the incidence further. Ongoing surveillance of AK numbers will identify changes in incidence earlier. Evaluation of Acanthamoeba contamination in end-user drinking water would contribute to our understanding of regional variations in the risk of exposure.


Assuntos
Ceratite por Acanthamoeba/epidemiologia , Lentes de Contato/parasitologia , Surtos de Doenças/estatística & dados numéricos , Higiene/normas , Ceratite por Acanthamoeba/parasitologia , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Soluções para Lentes de Contato , Grupos Étnicos , Feminino , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Fatores de Risco , Classe Social , Reino Unido/epidemiologia , Adulto Jovem
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