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1.
Arch. Soc. Esp. Oftalmol ; 95(11): 569-572, nov. 2020. ilus
Artigo em Espanhol | IBECS | ID: ibc-197750

RESUMO

La queratoconjuntivitis por Microsporidium es un cuadro infrecuente. Se ha asociado a brotes epidémicos en Asia relacionados con la exposición a agua o tierra contaminada. Habitualmente estos cuadros son autolimitados y leves, pero pueden tener evoluciones prolongadas. Presentamos el caso de una paciente de 29 años que comenzó con dolor, enrojecimiento, visión borrosa en su ojo derecho tras su vuelta de un viaje a Singapur y que no mejoró tras un tratamiento convencional frente a conjuntivitis. Fue diagnosticada de queratoconjuntivitis por Microsporidium mediante PCR y tinción con PAS del epitelio corneal. El tratamiento inicial fue desbridamiento epitelial, albendazol oral y voriconazol, levofloxacino y propamidina, pero la enfermedad conjuntival y corneal no cedió hasta que si introdujeron corticoides tópicos 5 meses después para tratar la limbitis. Debemos tener la sospecha de queratitis por Microsporidium en casos de queratitis unilateral y conjuntivitis, sobre todo en pacientes que vuelvan de Asia


Microsporidium keratoconjunctivitis is an very rare disease. It is related to outbreaks in Asia due to exposure to contaminated water or soil. Microsporidium keratoconjunctivitis is a a self-limited disease, but it could have long term courses. We present the case of a 29 year old woman who started with pain, redness and blurred vision after a holiday in Singapore and did not respond to conjunctivitis treatment. PCR sequencing and PAS staining of corneal epithelial biopsy identified Vittaforma corneae as the causative organism. Treatment was initiated with corneal debridement, oral albendazol, and intensive topical voriconazole, levofloxacin and propamidine, but the conjunctival and corneal disease was only resolved 5 months later with the introduction of topical steroids to treat her severe limbitis. Suspicion of Microsporidium keratoconjunctivitis should be raised amongst ophthalmologists in unilateral keratitis with mild conjunctivitis in travelers from Asia


Assuntos
Humanos , Feminino , Adulto , Vittaforma/isolamento & purificação , Ceratoconjuntivite/microbiologia , Doença Relacionada a Viagens , Ceratoconjuntivite/patologia , Reação em Cadeia da Polimerase , Acuidade Visual , Singapura
2.
J Med Microbiol ; 69(3): 414-418, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32011230

RESUMO

Introduction. Ocular microsporidiosis is a significant emerging infectious disease reported in immunocompromised patients and immunocompetent persons throughout the world.Aim. To identify the pathogens responsible for human keratitis, via corneal scrapings.Methodology. Thirty-three hospitalized patients with epithelial keratitis were examined using staining and DNA sequencing. DNA was extracted from corneal samples and the small-subunit ribosomal RNA gene was amplified by polymerase chain reaction (PCR) and sequenced.Results. Twenty-one samples were positive by staining while PCR generated amplicons in 18 cases. Of the 18 sequences, 16 were identical with, or very similar to, those of Vittaforma corneae (99-100 % similarity) and the remaining two sequences were similar to that of unidentified Microsporidium species deposited in the GenBank.Conclusion. This study has reconfirmed that V. corneae causes epithelial keratitis in humans and that a newly detected Microsporidium species is also involved in microsporidial keratitis as one of the emerging pathogens in Thailand. Ophthalomologists should be aware of microsporidial keratitis in people from Thailand and those from neighbouring countries.


Assuntos
Ceratite/microbiologia , Microsporídios não Classificados/genética , Microsporidiose/microbiologia , Adolescente , Adulto , Idoso , Córnea/microbiologia , Feminino , Humanos , Ceratite/epidemiologia , Masculino , Microsporídios não Classificados/isolamento & purificação , Microsporidiose/epidemiologia , Pessoa de Meia-Idade , Epidemiologia Molecular , Tailândia/epidemiologia , Vittaforma/genética , Vittaforma/isolamento & purificação , Adulto Jovem
3.
Br J Ophthalmol ; 104(11): 1613-1620, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32051138

RESUMO

AIMS: To report the clinical manifestations, ultrastructure and evaluate the efficacy of therapeutic lamellar keratectomy (TLK) and penetrating keratoplasty (PK) for microsporidial stromal keratitis (MSK). METHODS: Fourteen MSK cases between 2009 and 2018 were recruited. Each patient's clinical presentation, light microscopy, histopathology, PCR and electron microscopy (EM) of corneal samples were reviewed. RESULTS: The patients were 70.0±4.7 years old (average follow-up, 4.5 years). Time from symptoms to presentation was 10.6±13.0 weeks. The corneal manifestations were highly variable. Corneal scrapings revealed Gram stain positivity in 12 cases (85.7%) and modified Ziehl-Neelsen stain positivity in 9 (64.3%). Histopathology revealed spores in all specimens, while sequencing of small subunit rRNA-based PCR products identified Vittaforma corneae in 82% of patients. EM demonstrated various forms of microsporidial sporoplasm in corneal keratocytes. All patients were treated with topical antimicrobial agents or combined with oral antiparasitic medications for >3 weeks. As all patients were refractory to medical therapy, they ultimately underwent surgical intervention (TLK in 7, PK in 6 and 1 received TLK first, followed by PK). Postoperatively, the infection was resolved in 78.6% of the patients. Nevertheless, a high recurrence rate (21.4%) was noted during 3-year follow-up, with only two patients retained a final visual acuity ≥20/100. CONCLUSION: MSK usually presents with a non-specific corneal infiltration refractory to antimicrobial therapy. The diagnosis relies on light microscopic examinations on corneal scrapings and histopathological analyses. Surgical intervention is warranted by limiting the infection; however, it was associated with an overall poor outcome.


Assuntos
Substância Própria/microbiologia , Substância Própria/ultraestrutura , Úlcera da Córnea , Infecções Oculares Fúngicas , Microsporidiose , Vittaforma/isolamento & purificação , Idoso , Transplante de Córnea , Úlcera da Córnea/diagnóstico , Úlcera da Córnea/patologia , Úlcera da Córnea/cirurgia , Infecções Oculares Fúngicas/diagnóstico , Infecções Oculares Fúngicas/patologia , Infecções Oculares Fúngicas/cirurgia , Feminino , Técnicas de Genotipagem , Humanos , Ceratoplastia Penetrante , Masculino , Microscopia Eletrônica , Microsporidiose/diagnóstico , Microsporidiose/patologia , Microsporidiose/cirurgia , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , RNA Fúngico/genética , RNA Ribossômico/genética , Estudos Retrospectivos , Resultado do Tratamento , Acuidade Visual/fisiologia
4.
Emerg Infect Dis ; 25(11): 2100-2103, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31625849

RESUMO

We analyzed 2 batches of environmental samples after a microsporidial keratoconjunctivitis outbreak in Taiwan. Results indicated a transmission route from a parking lot to a foot washing pool to a swimming pool and suggested that accumulation of mud in the foot washing pool during the rainy season might be a risk factor.


Assuntos
Ceratoconjuntivite Infecciosa/epidemiologia , Ceratoconjuntivite Infecciosa/microbiologia , Microsporidiose/epidemiologia , Microsporidiose/microbiologia , Piscinas , Vittaforma , Microbiologia da Água , Animais , Surtos de Doenças , Humanos , Vigilância em Saúde Pública , Taiwan/epidemiologia , Vittaforma/isolamento & purificação
5.
Ann Clin Microbiol Antimicrob ; 18(1): 17, 2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-31182106

RESUMO

BACKGROUND: The utility of formalin-fixed paraffin-embedded (FFPE) corneal tissue specimens for retrospective diagnosis of microsporidial keratitis was evaluated by transmission electron microscopy (TEM) analysis and the possible second case of microsporidial keratitis after Descemet stripping automated endothelial keratoplasty (DSAEK) was described. CASE PRESENTATION: A 68-year-old man presented with multiple crystalline opacities in the corneal stroma that progressed extremely slowly after DSAEK. Fungiflora Y staining of corneal scrapings from the affected regions revealed an oval microorganism. Topical voriconazole administration was ineffective and penetrating keratoplasty was performed. Histological and molecular analyses were carried out on the excised cornea. Ziehl-Neelsen staining revealed an acid-fast, oval organism that was visible by ultraviolet illumination after Fungiflora Y and Uvitex 2B staining, whereas periodic acid-Schiff and Grocott's staining did not yield any significant findings. Microsporidium was detected by TEM of FFPE tissue. Nosema or Vittaforma sp. was suspected as the causative microorganism by PCR of FFPE tissue and by the fact that those species are known to cause eye infection. The corneal graft has maintained transparency at 1 year and half postoperatively. CONCLUSIONS: This is the first known case of microsporidial keratitis diagnosed retrospectively by molecular and ultrastructural study of FFPE tissue, and the possible second case of microsporidial keratitis after DSAEK. Microsporidial keratitis should be considered when corneal opacity refractory to conventionally known therapy would occur after DSAEK. Our findings suggest that more microsporidial keratitis cases than have been reported to date can be identified by TEM or PCR examination of FFPE corneal specimens.


Assuntos
Córnea/patologia , Ceratite/patologia , Idoso , Córnea/microbiologia , Córnea/cirurgia , Córnea/ultraestrutura , Formaldeído , Humanos , Ceratite/diagnóstico , Ceratite/microbiologia , Ceratite/cirurgia , Ceratoplastia Penetrante , Masculino , Nosema/genética , Nosema/isolamento & purificação , Inclusão em Parafina , Estudos Retrospectivos , Vittaforma/genética , Vittaforma/isolamento & purificação
6.
Ocul Immunol Inflamm ; 27(5): 826-828, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-29672246

RESUMO

Purpose: To describe a case of microsporidial stromal keratitis with endophthalmitis in an immunocompetent patient.Methods: Case reportResults: A 58-year-old HIV-negative man presented with stromal keratitis in his right eye. The patient demonstrated subsequent vitritis, multifocal retinitis and arteritis, and macular edema with recurrent vitreous hemorrhage after therapeutic keratoplasty. Numerous microsporidial spores were detected in corneal tissues by modified trichrome stain. Both corneal tissues and vitreous sample of the affected eye showed positive results by polymerase chain reaction targeting the microsporidial small subunit rRNA gene whose sequences belonged to Vittaforma corneae. Post-keratoplasty and vitrectomy, his best-corrected visual acuity was hand motion due to pale optic disc.Conclusion: Endophthalmitis can be a consequence of microsporidial stromal keratitis in an immunocompetent host. Early recognition and prompt treatment should be considered in patients diagnosed with microsporidial keratitis presenting with mild vitritis, retinitis, and recurrent vitreous hemorrhage.


Assuntos
Endoftalmite/microbiologia , Infecções Oculares Fúngicas/microbiologia , Ceratite/microbiologia , Microsporidiose/diagnóstico , Vittaforma/isolamento & purificação , Substância Própria/patologia , Humanos , Masculino , Pessoa de Meia-Idade
7.
Parasitology ; 146(5): 569-579, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30486909

RESUMO

Obligately intracellular microsporidia regulate their host cell life cycles, including apoptosis, but this has not been evaluated in phagocytic host cells such as macrophages that can facilitate infection but also can be activated to kill microsporidia. We examined two biologically dissimilar human-infecting microsporidia species, Encephalitozoon cuniculi and Vittaforma corneae, for their effects on staurosporine-induced apoptosis in the human macrophage-differentiated cell line, THP1. Apoptosis was measured after exposure of THP-1 cells to live and dead mature organisms via direct fluorometric measurement of Caspase 3, colorimetric and fluorometric TUNEL assays, and mRNA gene expression profiles using Apoptosis RT2 Profiler PCR Array. Both species of microsporidia modulated the intrinsic apoptosis pathway. In particular, live E. cuniculi spores inhibited staurosporine-induced apoptosis as well as suppressed pro-apoptosis genes and upregulated anti-apoptosis genes more broadly than V. corneae. Exposure to dead spores induced an opposite effect. Vittaforma corneae, however, also induced inflammasome activation via Caspases 1 and 4. Of the 84 apoptosis-related genes assayed, 42 (i.e. 23 pro-apoptosis, nine anti-apoptosis, and 10 regulatory) genes were more affected including those encoding members of the Bcl2 family, caspases and their regulators, and members of the tumour necrosis factor (TNF)/TNF receptor R superfamily.


Assuntos
Apoptose/efeitos dos fármacos , Encephalitozoon cuniculi/fisiologia , Estaurosporina/farmacologia , Vittaforma/fisiologia , Apoptose/genética , Encefalitozoonose/microbiologia , Regulação da Expressão Gênica , Humanos , Microsporidiose/microbiologia , Células THP-1
8.
Environ Sci Pollut Res Int ; 25(19): 18823-18837, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29713979

RESUMO

Vittaforma corneae belongs to microsporidia, which include over 1500 species of opportunistic obligate intracellular fungi infecting almost all known animal taxa. Although outbreaks of ocular infections caused by waterborne V. corneae have been reported in recent years, little is known about the occurrence of this pathogen in aquatic environments. In this study, 50 water samples from rivers and reservoirs around Taiwan in two seasons were analyzed to explore the presence of this pathogen in natural aquatic environments. A high detection rate of Vittaforma-like amplicons (94%; 47/50) was observed in the water samples when examined by nested PCR with primer pairs specific to the small ribosomal subunit (SSU) rRNA gene. After electrophoresis, many lanes showed multiband patterns with expected molecular weights. After confirmation by DNA sequencing and by sequence alignment in the NCBI database, we identified a variety of Vittaforma-like microsporidia with weak sequence similarity, with approximately 85% identity to V. corneae, thus indicating high diversity of microsporidia in aquatic environments. Phylogenetic analysis showed clear-cut microsporidian clade classification and indicated that the most Vittaforma-like microsporidia in this study belong to clade IV and cluster into four major groups. The first group is similar to the microsporidia associated with ocular microsporidiosis. The second group is associated with the diarrheal pathogens, whereas the third and fourth groups are a novel group and a zoonotic group, respectively. This study provides abundant sequencing information, which will be useful for future molecular biological studies on microsporidia. Because microsporidia are important pathogens of animals and humans, it is urgently necessary to determine via a survey whether there are species with potential threats that have not yet been revealed.


Assuntos
Água Potável/microbiologia , Microsporídios/isolamento & purificação , Animais , Sequência de Bases , DNA Fúngico , Humanos , Microsporidiose/epidemiologia , Microsporidiose/microbiologia , Filogenia , Reação em Cadeia da Polimerase , Rios , Alinhamento de Sequência , Análise de Sequência de DNA , Taiwan/epidemiologia , Vittaforma/isolamento & purificação , Microbiologia da Água
9.
Am J Trop Med Hyg ; 99(1): 90-93, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29692301

RESUMO

Microsporidia are protists close to the kingdom of fungi that may cause eye infections. Most cases are reported in Asia and affect both immunocompromised and immunocompetent patients. Here, we report a rare case of microsporidial keratoconjunctivitis in an immunocompetent French patient 3 weeks after returning from India. In our patient, Weber trichrome staining of conjunctival scrapings revealed rounded elements approximately 1-3 µm in size. Conventional polymerase chain reaction analysis by ribosomal RNA subunit sequencing showed 100% identity with Vittaforma corneae. Treatment by corneal debridement combined with fluoroquinolone eye drops allowed complete resolution of the lesions. Although rare, ocular microsporidiosis should be investigated in a patient who is native to Asia or has returned from an endemic area and presents with keratoconjunctivitis of undetermined etiology.


Assuntos
Antifúngicos/uso terapêutico , Infecções Oculares Fúngicas/diagnóstico , Fluoroquinolonas/uso terapêutico , Ceratoconjuntivite/diagnóstico , Microsporidiose/diagnóstico , Córnea/efeitos dos fármacos , Córnea/microbiologia , Córnea/patologia , Córnea/cirurgia , Desbridamento/métodos , Infecções Oculares Fúngicas/tratamento farmacológico , Infecções Oculares Fúngicas/microbiologia , Infecções Oculares Fúngicas/cirurgia , França , Humanos , Índia , Ceratoconjuntivite/tratamento farmacológico , Ceratoconjuntivite/microbiologia , Ceratoconjuntivite/cirurgia , Masculino , Microsporidiose/tratamento farmacológico , Microsporidiose/microbiologia , Microsporidiose/cirurgia , Pessoa de Meia-Idade , Viagem , Vittaforma/efeitos dos fármacos , Vittaforma/crescimento & desenvolvimento , Vittaforma/patogenicidade
10.
Water Res ; 118: 208-216, 2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28432932

RESUMO

Vittaforma corneae is an obligate intracellular fungus and can cause human ocular microsporidiosis. Although accumulating reports of V. corneae causing keratoconjunctivitis in both healthy and immunocompromised persons have been published, little is known about the organism's occurrence in aquatic environments. Limitations in detection sensitivity have meant a large sampling volume is required to detect the pathogen up to now, which is problematic. A recent study in Taiwan has shown that some individuals suffering from microsporidial keratitis (MK) were infected after exposure to the pathogen at a hot spring. As a consequence of this, a survey and analysis of environmental V. corneae present in hot springs became an urgent need. In this study, sixty water samples from six hot spring recreation areas around Taiwan were analyzed. One liter of water from each sample site was filtered to harvest the fungi. The positive samples were detected using a modified nested PCR approach followed by sequencing using specific SSU rRNA gene primer pairs for V. corneae. In total fifteen V. corneae-like isolates were identified (25.0% of sites). Among them, six isolates, which were collected from recreational areas B, C and D, were highly similar to known V. corneae keratitis strains from Taiwan and other countries. Furthermore, five isolates, which were collected from recreation areas A, C, E and F, were very similar to Vittaforma-like diarrhea strains isolated in Portugal. Cold spring water tubs and public foot bath pools had the highest detection rate (50%), suggesting that hot springs might be contaminated via untreated water sources. Comparing the detection rate across different regions of Taiwan, Taitung, which is in the east of the island, gave the highest positive rate (37.5%). Statistical analysis showed that outdoor/soil exposure and a high heterotrophic plate count (HPC) were risk factors for the occurrence of V. corneae. Our findings provide empirical evidence supporting the need for proper control and regulations at hot spring recreational waters in order to avoid health risks from this pathogen. Finally, we have developed a small volume procedure for detecting V. corneae in water samples and this has proved to be very useful.


Assuntos
Monitoramento Ambiental , Fontes Termais , Vittaforma , Humanos , Microsporidiose/prevenção & controle , Portugal , Taiwan
11.
Sci Rep ; 6: 31430, 2016 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-27578396

RESUMO

Honeybee colony survival strongly relies on the queen to overcome worker losses exposed to combined stressors like pesticides and parasites. Queen's capacity to withstand these stressors is however very little known. The effects of the common neonicotinoid pesticide imidacloprid in a chronic and sublethal exposure together with the wide distributed parasite Nosema ceranae have therefore been investigated on queen's physiology and survivorship in laboratory and field conditions. Early physiological changes were observed on queens, particularly the increase of enzyme activities (catalase [CAT] and glutathione-S-transferase [GST] in the heads) related to protective responses to xenobiotics and oxidative stress against pesticide and parasite alone or combined. Stressors also alter the activity of two other enzymes (carboxylesterase alpha [CaE α] and carboxylesterase para [CaE p] in the midguts) involved in metabolic and detoxification functions. Furthermore, single and combined effects of pesticide and parasite decrease survivorship of queens introduced into mating hives for three months. Because colony demographic regulation relies on queen's fertility, the compromise of its physiology and life can seriously menace colony survival under pressure of combined stressors.


Assuntos
Abelhas/efeitos dos fármacos , Abelhas/microbiologia , Neonicotinoides/toxicidade , Nitrocompostos/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Praguicidas/toxicidade , Vittaforma/fisiologia , Animais , Abelhas/fisiologia , Encéfalo/enzimologia , Carboxilesterase/metabolismo , Catalase/metabolismo , Feminino , Glutationa Transferase/metabolismo , Proteínas de Insetos/metabolismo , Intestinos/enzimologia , Estimativa de Kaplan-Meier , Microsporidiose/mortalidade , Microsporidiose/patologia , Microsporidiose/veterinária
14.
J Med Microbiol ; 62(Pt 4): 553-559, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23319308

RESUMO

Microsporidia are obligate intracellular parasites that infect eukaryotic cells and have emerged as major opportunistic human pathogens. Due to the difficulties in definitive laboratory diagnosis and insufficient knowledge, ocular microsporidiosis is infrequently reported in India. To improve diagnostic facilities, we have developed a novel duplex PCR (dPCR) for the simultaneous identification of both genera and species of isolates with microsporidian aetiology that cause keratitis. The material scraped from the corneas of 12 clinically diagnosed microsporidial keratitis patients was subjected to routine microbiological examinations and molecular diagnosis using a novel dPCR that targeted the small-subunit rRNA gene (SSU-rRNA) of microsporidia and Vittaforma corneae using genus- and species-specific primers. Of the 12 corneal scrapes, 6 showed positive results in smears, while dPCR provided positive amplification with both pan-microsporidial and V. corneae species-specific primers for 9 corneal scrapes. The results were validated by sequencing and blast analysis. The sensitivity of this novel dPCR method was higher than that of conventional microscopy in the diagnosis of corneal microsporidial infection. dPCR with specific primers is potentially more sensitive, specific and depends less on more complicated methods for exact identification of the aetiology of microsporidial keratitis.


Assuntos
Técnicas de Laboratório Clínico/métodos , Ceratite/diagnóstico , Microsporidiose/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , Vittaforma/isolamento & purificação , Primers do DNA/genética , DNA Fúngico/química , DNA Fúngico/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Genes de RNAr , Humanos , Índia/epidemiologia , Ceratite/microbiologia , Microsporidiose/microbiologia , Técnicas de Diagnóstico Molecular/métodos , Dados de Sequência Molecular , Micologia/métodos , RNA Fúngico/genética , RNA Ribossômico 18S/genética , Sensibilidade e Especificidade , Análise de Sequência de DNA , Vittaforma/classificação , Vittaforma/genética
15.
J Invertebr Pathol ; 110(3): 408-10, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22546521

RESUMO

Queens are the primary female reproductive individuals in honey bee colonies and, while they are generally free from Nosema ceranae infection, they are nevertheless susceptible. We sought to determine whether queens are naturally infected by N. ceranae, as these infections could be a factor in the rapid spread of this parasite. Queens were analyzed using real-time PCR and included larval queens, newly emerged, and older mated queens. Overall, we found that all tissues we examined were infected with N. ceranae at low levels but no samples were infected with Nosema apis. The infection of the ovaries and spermatheca suggests the possibility of vertical transmission of N. ceranae.


Assuntos
Abelhas/microbiologia , Transmissão Vertical de Doenças Infecciosas/veterinária , Microsporidiose/veterinária , Vittaforma/isolamento & purificação , Animais , DNA Fúngico , Monitoramento Ambiental , Feminino , Larva/microbiologia , Microsporidiose/microbiologia , Ovário/microbiologia , Oviductos/microbiologia , Vittaforma/genética
16.
J Clin Microbiol ; 50(2): 414-8, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22116156

RESUMO

This retrospective study included 10 eyes of 9 patients diagnosed with microsporidial keratitis. All of them were known to contract this disease after taking baths in hot springs. The disease was diagnosed based on detecting microsporidia in corneal scrapings using Gram stain and the modified Kinyoun's acid-fast stain. The specimens from the last six patients were subjected to PCR and then sequencing. All of them revealed that the microorganism identified has a high similarity to Vittaforma corneae. Repeated debridement of the epithelial lesions successfully eradicated the microsporidial infection in all nine patients.


Assuntos
Ceratite/diagnóstico , Microsporidiose/diagnóstico , Vittaforma/isolamento & purificação , Adulto , Idoso , Técnicas de Laboratório Clínico/métodos , Córnea/patologia , DNA Fúngico/química , DNA Fúngico/genética , Feminino , Fontes Termais , Humanos , Ceratite/microbiologia , Ceratite/patologia , Masculino , Microscopia , Microsporidiose/microbiologia , Microsporidiose/patologia , Pessoa de Meia-Idade , Dados de Sequência Molecular , Micologia/métodos , Reação em Cadeia da Polimerase , Estudos Retrospectivos , Análise de Sequência de DNA , Vittaforma/citologia , Vittaforma/genética
17.
Indian J Med Microbiol ; 27(1): 35-9, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19172057

RESUMO

Being intracellular parasites, microsporidia can only be propagated in cell culture systems. This study evaluated three cell lines to determine the most suitable host-parasite In vitro system. Confluent monolayers of vero, SIRC, and HeLa cell lines, grown in 24-well tissue culture plates, were inoculated with varying concentrations (1 x 10(4) to 1 x 10(8) spores/mL) of Vittaforma corneae, Encephalitozoon hellem, Encephalitozoon cuniculi, and Encephalitozoon intestinalis spores. Growth was compared quantitatively at weekly intervals. Encephalitozoon species showed the highest amount of growth when cultured in vero cell line, while there was no significant difference in their growth in SIRC and HeLa cell lines. In comparison, V. corneae showed the highest growth in SIRC cells, followed by vero cells. The analytical sensitivity was found to be 1 x 10(4) spores/mL for vero cell line compared to 1 x 10(5) spores/mL for SIRC cell line and 1 x 10(7) spores/mL for HeLa cell line. HeLa cells also showed rapid disruption of cells, and the spores could not be easily distinguished from cell debris. This is the first report of the comparison of vero, SIRC, and HeLa for the propagation of microsporidial spores. Vero cell line was found to be more sensitive than SIRC and HeLa cells, and we believe that the inclusion of vero cell line in the routine culture protocols of ocular parasitology laboratories would result in a significant increase in the diagnostic yield.


Assuntos
Técnicas de Laboratório Clínico/métodos , Encephalitozoon/crescimento & desenvolvimento , Ceratite/microbiologia , Microsporidiose/microbiologia , Vittaforma/crescimento & desenvolvimento , Animais , Linhagem Celular , Chlorocebus aethiops , Contagem de Colônia Microbiana , Humanos , Coelhos , Sensibilidade e Especificidade
18.
Antimicrob Agents Chemother ; 52(2): 790-3, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18056284

RESUMO

We amplified, cloned, and sequenced the beta-tubulin gene of Vittaforma corneae, a microsporidium causing human infections. The beta-tubulin gene sequence has a substitution at Glu(198) (with glutamine), which is one of six amino acids reported to be associated with benzimidazole sensitivity. Benzimidazoles were assayed for antimicrosporidial activity and showed poor parasite inhibition.


Assuntos
Antifúngicos/farmacologia , Benzimidazóis/farmacologia , Farmacorresistência Fúngica/genética , Tubulina (Proteína)/genética , Vittaforma/efeitos dos fármacos , Sequência de Aminoácidos , Linhagem Celular , Clonagem Molecular , Fibroblastos/microbiologia , Humanos , Testes de Sensibilidade Microbiana , Microsporidiose/microbiologia , Dados de Sequência Molecular , Análise de Sequência de DNA , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Vittaforma/genética
19.
Curr Genet ; 51(3): 171-86, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17235519

RESUMO

Microsporidia are fungi-related obligate intracellular parasites that infect numerous animals, including man. Encephalitozoon cuniculi harbours a very small genome (2.9 Mbp) with about 2,000 coding sequences (CDSs). Most repeated CDSs are of unknown function and are distributed in subterminal regions that mark the transitions between subtelomeric rDNA units and chromosome cores. A potential multigenic family (interB) encoding proteins within a size range of 579-641 aa was investigated by PCR and RT-PCR. Thirty members were finally assigned to the E. cuniculi interB family and a predominant interB transcript was found to originate from a newly identified gene on chromosome III. Microsporidian species from eight different genera infecting insects, fishes or mammals, were tested for a possible intra-phylum conservation of interB genes. Only representatives of the Encephalitozoon, Vittaforma and Brachiola genera, differing in host range but all able to invade humans, were positive. Molecular karyotyping of Brachiola algerae showed a complex set of chromosome bands, providing a haploid genome size estimate of 15-20 Mbp. In spite of this large difference in genome complexity, B. algerae and E. cuniculi shared some similar interB gene copies and a common location of interB genes in near-rDNA subterminal regions.


Assuntos
Cromossomos Fúngicos/genética , Sequência Conservada , Encephalitozoon cuniculi/genética , Encephalitozoon cuniculi/patogenicidade , Proteínas Fúngicas/genética , Família Multigênica , Animais , Sequência de Bases , Linhagem Celular , Biologia Computacional , Cães , Encefalitozoonose/microbiologia , Proteínas Fúngicas/biossíntese , Regulação Fúngica da Expressão Gênica , Humanos , Dados de Sequência Molecular , Especificidade da Espécie , Vittaforma/genética
20.
Antimicrob Agents Chemother ; 50(6): 2146-55, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16723577

RESUMO

Therapies for microsporidiosis in humans are limited, and fumagillin, which appears to be the most broadly effective antimicrosporidial drug, is considered to be moderately toxic. The purpose of this study was to apply an in vitro drug screening assay for Encephalitozoon intestinalis and Vittaforma corneae and an in vivo athymic mouse model of V. corneae infection to assess the efficacy of TNP-470 (a semisynthetic analogue of fumagillin), ovalicin, and eight ovalicin derivatives. TNP-470, ovalicin, and three of the ovalicin derivatives inhibited both E. intestinalis and V. corneae replication by more than 70% in vitro. Another three of the ovalicin derivatives inhibited one of the two microsporidian species by more than 70%. None of the treated athymic mice survived the V. corneae infection, but they did survive statistically significantly longer than the untreated controls after daily treatment with fumagillin administered at 5, 10, and 20 mg/kg of body weight subcutaneously (s.c.), TNP-470 administered at 20 mg/kg intraperitoneally (i.p.), or ovalicin administered at 5 mg/kg s.c. Of two ovalicin derivatives that were assessed in vivo, NSC 9665 given at 10 mg/kg i.p. daily also statistically significantly prolonged survival of the mice. No lesions associated with drug toxicity were observed in the kidneys or livers of uninfected mice treated with these drugs at the highest dose of 20 mg/kg daily. These results thus support continued studies to identify more effective fumagillin-related drugs for treating microsporidiosis.


Assuntos
Ácidos Graxos Insaturados/farmacologia , Microsporídios/efeitos dos fármacos , Microsporidiose/tratamento farmacológico , Sesquiterpenos/farmacologia , Animais , Cicloexanos , Avaliação Pré-Clínica de Medicamentos , Encephalitozoon/efeitos dos fármacos , Encephalitozoon/crescimento & desenvolvimento , Técnicas In Vitro , Masculino , Camundongos , Camundongos Nus , O-(Cloroacetilcarbamoil)fumagilol , Fatores de Tempo , Vittaforma/efeitos dos fármacos , Vittaforma/crescimento & desenvolvimento
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