Infectious keratitis in southern Brazil: a comparison culture negative and culture positive patients / Ceratite infecciosa no sul do Brasil: comparação entre pacientes com cultura negativa e positiva

Abstract Purpose: To compare clinical-epidemiological profile and treatment outcome between culture negative and culture positive keratitis patients. Methods: Patients with suspected infectious keratitis seen at two ophthalmic hospitals in Curitiba, Brazil, between June 2014 and April 2016, were prospectively studied. Ophthalmological exam with corneal scraping and microbiological tests were performed. Data regarding follow up, surgical interventions and treatment outcome were collected after 12 weeks of the first visit trough medical chart review. From the results of the culture, two groups were formed: culture negative keratitis (CNK) and culture positive keratitis (CPK). Results: According to inclusion criteria 21 patients were classified as culture negative keratitis and 20 patients as culture positive keratitis. The number of patients on antibiotic drops at the first visit was greater in CNK group (90.5% versus 60%; p=0.032). Surgical procedures were necessary in 3 patients (15%) in CNK group and in 7 patients (36,8%) in CPK group (p=0.155). Treatment success was achieved by 85% (17/20) of the patients in CNK group and by 61% (11/18) of the patients in CPK group (p=0.144). There was no significant difference between groups regarding age, gender, place of residence, presence of comorbidities, risk factors for infectious keratitis, duration of symptoms and characteristics of corneal ulcer. Conclusions: Previous treatment with antibiotics correlates with negative culture results. There was no significant difference in treatment outcome between culture negative and culture positive keratitis patients.

Resumo Objetivo: Comparar os perfis clinico-epidemiológicos e os desfechos entre pacientes com ceratite com cultura positiva e pacientes com ceratite com cultura negativa. Métodos: Pacientes com ceratite infecciosa, atendidos em dois hospitais oftalmológicos em Curitiba, Brasil, entre junho de 2014 e abril de 2016, foram estudados prospectivamente. Exame oftalmológico, raspado de córnea e exames microbiológicos foram realizados no primeiro atendimento. Os dados quanto a seguimento e desfecho foram coletados após 12 semanas do primeiro atendimento através de revisão de prontuário. A partir dos resultados das culturas, dois grupos foram formados: ceratite com cultura negativa e ceratite com cultura positiva. Resultados: Vinte e um pacientes foram classificados como ceratite com cultura negativa e 20 como ceratite com cultura positiva. O número de pacientes em uso de colírio antibiótico no primeiro atendimento foi maior no grupo de cultura negativa (90,5% versus 60%; p=0,032). Sete pacientes (37%) no grupo cultura positiva precisaram de procedimentos cirúrgicos no manejo da ceratite, versus 3 pacientes (15%) do grupo cultura negativa (p=0,155). Oitenta e cinco por cento (17/20) dos pacientes do grupo cultura negativa alcançaram sucesso no tratamento, contra 61% (11/18) dos pacientes no grupo cultura positiva (p=0,144). Não houve diferença entre os grupos quanto a idade, gênero, local de procedência, presença de comorbidades, fatores de risco, duração dos sintomas e características da úlcera de córnea. Conclusão: Tratamento prévio com colírio de antibiótico correlaciona-se com resultados negativos de cultura. Não houve diferença no desfecho após tratamento entre os pacientes com cultura negativa e cultura positiva.

Down-Regulation of Cellulose Synthase Inhibits the Formation of Endocysts in Acanthamoeba

Acanthamoeba cysts are resistant to unfavorable physiological conditions and various disinfectants. Acanthamoeba cysts have 2 walls containing various sugar moieties, and in particular, one third of the inner wall is composed of cellulose. In this study, it has been shown that down-regulation of cellulose synthase by small interfering RNA (siRNA) significantly inhibits the formation of mature Acanthamoeba castellanii cysts. Calcofluor white staining and transmission electron microscopy revealed that siRNA transfected amoeba failed to form an inner wall during encystation and thus are likely to be more vulnerable. In addition, the expression of xylose isomerase, which is involved in cyst wall formation, was not altered in cellulose synthase down-regulated amoeba, indicating that cellulose synthase is a crucial factor for inner wall formation by Acanthamoeba during encystation.

Phospholipase Activities in Clinical and Environmental Isolates of Acanthamoeba

The pathogenesis and pathophysiology of Acanthamoeba infections remain incompletely understood. Phos-pholipases are known to cleave phospholipids, suggesting their possible involvement in the host cell plasma membrane disruption leading to host cell penetration and lysis. The aims of the present study were to determine phospholipase activities in Acanthamoeba and to determine their roles in the pathogenesis of Acanthamoeba. Using an encephalitis isolate (T1 genotype), a keratitis isolate (T4 genotype), and an environmental isolate (T7 genotype), we demonstrated that Acanthamoeba exhibited phospholipase A2 (PLA2) and phospholipase D (PLD) activities in a spectrophotometry-based assay. Interestingly, the encephalitis isolates of Acanthamoeba exhibited higher phospholipase activities as compared with the keratitis isolates, but the environmental isolates exhibited the highest phospholipase activities. Moreover, Acanthamoeba isolates exhibited higher PLD activities compared with the PLA2. Acanthamoeba exhibited optimal phospholipase activities at 37degrees C and at neutral pH indicating their physiological relevance. The functional role of phospholipases was determined by in vitro assays using human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. We observed that a PLD-specific inhibitor, i.e., compound 48/80, partially inhibited Acanthamoeba encephalitis isolate cytotoxicity of the host cells, while PLA2-specific inhibitor, i.e., cytidine 5'-diphosphocholine, had no effect on parasite-mediated HBMEC cytotoxicity. Overall, the T7 exhibited higher phospholipase activities as compared to the T4. In contract, the T7 exhibited minimal binding to, or cytotoxicity of, HBMEC.

Analysis of the risk factors predisposing to fungal, bacterial & Acanthamoeba keratitis in south India.

Background & objectives: Infective keratitis is rare in the absence of predisposing factors. The pattern of risk factors predisposing to infective keratitis varies with geographical regions and also influences the type of infection that occurs. The present study was aimed to identify the specific risk factors that predispose the infective keratitis (non viral) and to determine the association between the risk factors identified and the microbial aetiology of infective keratitis in a geographic region (south India). Methods: A retrospective analysis of all patients clinically diagnosed infective keratitis (non viral) presenting between September 1999 and September 2002 was performed to identify risk factors. After diagnosing infective keratitis clinically, corneal scrapes were performed and subjected to microscopy and culture. Results: A total of 3295 patients with infective keratitis were evaluated, of whom, 1138 (34.5%) patients had fungal growth alone, 1066 (32.4%) had bacterial growth alone, 33 (1%) had Acanthamoeba growth alone, 83 (2.5%) had mixed microbial growth and the remaining 975 (29.6%) had no growth. Corneal injury was identified in 2356 (71.5%) patients and it accounted for 91.9 per cent in fungal keratitis (P<0.0001) (OR: 73.5; 95%CI: 61.3-98.5), 28.1 per cent in bacterial keratitis and 100 per cent in Acanthamoeba keratitis (P<0.0001). Injuries due to vegetative matter (61.2%) was identified as significant risk for fungal keratitis (P<0.0001) (OR: 15.73; 95%CI: 12.7-19.49) and mud (84.85%) for Acanthamoeba keratitis (P<0.0001) (OR: 16.52; 95%CI: 6.35-42.99). Co-existing ocular diseases predisposing to bacterial keratitis accounted for 724 (69%) patients (P<0.0001) (OR: 33.31; 95%CI: 26.98-41.12). Bacterial pathogens alone were recovered from all 35 patients with contact lens associated keratitis (100%). Co-existing ocular diseases (78.3%) were frequently documented among patients older than 50 yr (P<0.0001) (OR: 27.0; 95%CI: 25.0-28.0) and corneal injury (89.7%) was frequently recorded among patients younger than 51 yr (P<0.0001) (OR: 72.0; 95%CI: 70.0-73.0). Interpretation & conclusions: Corneal injury was found to be the principal risk factor for fungal and Acanthamoeba keratitis, while co-existing ocular diseases for bacterial keratitis. Corneal injury with vegetative matter was more often associated with fungal keratitis and injury with mud with Acanthamoeba keratitis.

Review of epidemiological features, microbiological diagnosis and treatment outcome of microbial keratitis: Experience of over a decade.

Purpose: To review the epidemiological characteristics, microbiological profile, and treatment outcome of patients with suspected microbial keratitis. Materials and Methods: Retrospective analysis of a non-comparative series from the database was done. All the patients presenting with corneal stromal infiltrate underwent standard microbiologic evaluation of their corneal scrapings, and smear and culture-guided antimicrobial therapy. Results: Out of 5897 suspected cases of microbial keratitis 3563 (60.4%) were culture-proven (bacterial – 1849, 51.9%; fungal – 1360, 38.2%; Acanthamoeba – 86, 2.4%; mixed – 268, 7.5%). Patients with agriculture-based activities were at 1.33 times (CI 1.16–1.51) greater risk of developing microbial keratitis and patients with ocular trauma were 5.33 times (CI 6.41–6.44) more likely to develop microbial keratitis. Potassium hydroxide with calcofluor white was most sensitive for detecting fungi (90.6%) and Acanthamoeba (84.0%) in corneal scrapings, however, Gram stain had a low sensitivity of 56.6% in detection of bacteria. Majority of the bacterial infections were caused by Staphylococcus epidermidis (42.3%) and Fusarium species (36.6%) was the leading cause of fungal infections. A significantly larger number of patients (691/1360, 50.8%) with fungal keratitis required surgical intervention compared to bacterial (799/1849, 43.2%) and Acanthamoeba (15/86, 17.4%) keratitis. Corneal healed scar was achieved in 75.5%, 64.8%, and 90.0% of patients with bacterial, fungal, and Acanthamoeba keratitis respectively. Conclusions: While diagnostic and treatment modalities are well in place the final outcome is suboptimal in fungal keratitis. With more effective treatment available for bacterial and Acanthamoeba keratitis, the treatment of fungal keratitis is truly a challenge.

Acometimento ocular em pacientes com mansonelose / Ocular manifestations in mansonelliasis

OBJETIVOS: Avaliar as alterações oculares observadas em indivíduos com mansonelose residentes na área rural do município de Coari, AM, na região do médio Amazonas. MÉTODOS: Foi conduzido um estudo de corte transversal envolvendo 543 moradores residentes na área rural do município de Coari, na margem direita do rio Solimões. Todos os indivíduos foram submetidos a exame de gota espessa para pesquisa de microfilária e os indivíduos positivos foram submetidos a exame oftalmológico. Lesões oculares suspeitas foram biopsiadas e enviadas para exame histopatológico. RESULTADOS: Mansonella ozzardi foi encontrada no sangue periférico em 103 (18,9 por cento) indivíduos. Noventa e cinco pacientes com mansonelose foram examinados, observando ceratite puntata em 12 (11,7 por cento) destes, ceratite numular em um indivíduo (1 por cento) e esclerosante em outro (1 por cento). Biópsia de pele foi negativa em todos os indivíduos. Biópsia de conjuntiva e limbo foi realizada em cinco pacientes com mansonelose e biópsia de córnea em outros três pacientes. Duas biópsias de conjuntiva e limbo e uma biópsia de córnea foram enviadas para exame histopatológico, não sendo observadas microfilárias ou alterações sugestivas da presença destas nas peças estudadas. O restante das biópsias foi enviado para pesquisa de DNA do parasita por PCR, não se encontrando microfilária. CONCLUSÕES: Associação entre infecção por Mansonella ozzardi e lesões corneanas sem etiologia aparente foi verificada neste estudo, mas a ausência de achados específicos no exame histopatológico e a não identificação de microfilárias por PCR em lesões suspeitas põem em dúvida a o acometimento corneano na mansonelose. Estudos mais amplos de populações com alta prevalência de M. ozzardi e a realização de outros testes para identificação de microfilárias em tecido ocular são necessários para investigar a patogenicidade da microfilária ao tecido ocular.

PURPOSE: To evaluate eye manifestations in mansonelliasis patients from Coari, AM, rural area on the middle Amazonas River. METHODS: Cross-sectional study consisted of evaluation of 543 subjects from Coari rural area on Solimões River right margin. All subjects were submitted to blood test for microfilaria and the subjects who had a positive test also were submitted to skin biopsies and ophthalmologic examination. Histopathology and PCR research for microfilaria were done on eye biopsies of suspect lesions. RESULTS: Mansonella ozzardi was found in peripheral blood of 103 (18.9 percent) subjects. Ninety-five mansonelliasis patients were examined. Punctate keratitis was observed in 12 of them, nummular keratitis in one subject and sclerosing keratitis in another one. Skin biopsies were negative in all subjects. Conjunctival and limbal biopsies were done in five mansonelliasis patients and corneal biopsy in another three patients. Conjunctival and limbal biopsies of two patients and corneal biopsy of one patient showed no microfilaria or alterations due to its presence on histopathology. PCR showed no evidence of microfilaria in the other biopsies. CONCLUSION: Association between Mansonella ozzardi infection and corneal lesions with no evident etiology was observed in this study, but the lack of conclusive findings on histopathology and PCR make us doubt the corneal involvement in mansonelliasis. Large studies of high mansonelliasis prevalence populations and extensive employment of microfilaria identification tests in ocular tissue are needed to evaluate the microfilaria corneal pathogenicity.

Queratitis infecciosa no viral: factores predisponentes, agentes etiológicos y diagnóstico de laboratorio / Non viral infectious keratitis: predisposing factors, etiologic agents and laboratory diagnosis

Las queratitis infecciosas poseen una elevada morbilidad, poniendo en riesgo la visión en casos graves. Dada la eficaz protección que brinda el epitelio corneal, para que ocurra una infección se requiere la presencia de factores condicionantes. El principal predisponente para las queratitis infecciosas es el uso de lentes de contacto, seguido por traumatismos y cirugías oculares y luego diversas afecciones locales o generales. Los agentes etiológicos abarcan una enorme diversidad de microorganismos, incluyendo bacterias, micobacterias, virus, hongos y parásitos. Para poder instaurar un tratamiento acotado se necesita un diagnóstico etiológico, lo que requiere una correcta toma de muestra y un exhaustivo análisis microbiológico.

Infectious keratitis cause significant morbidity and, if it is not promptly and appropriately treated, can lead to severe ocular disability. Almost all cases of keratitis are associated to predisposing conditions. In occident, the main risk factor is contact lens wear, but previous ocular surgery or trauma are also important, as well as various ocular surface diseases. An enormous diversity of etiologic agents for infectious keratitis exist, including virus, bacteria, mycobacteria, fungi and parasites. This review provides literature and personal based information about main predisposing factors, etiologic agents and pathophysiology of infectious keratitis, excluding those of viral origin. Focus is made on microbiologic procedures, describing stains and media that should be used, and highlighting their utility. A special mention on particular situations is made, including laboratory diagnosis of Acanthamoeba keratitis, utility of lens cases analysis, keratitis in patients with previous treatment, as well as molecular biology techniques described in ophthalmology.

Chronic microsporidial stromal keratitis in an immunocompetent, non-contact lens wearer.

An 82-year-old healthy man with unilateral chronic stromal keratitis, initially diagnosed to have viral keratitis and refractory to medical therapy, showed numerous oval, microsporidial organisms, measuring 4-5 m in length in the corneal biopsy. Penetrating keratoplasty, followed by treatment with systemic albendazole and topical propamidine isethionate resulted in resolution of the infection. Electron microscopy of the keratoplasty specimen demonstrated sporoblasts with diplokaryotic nuclei and multiple coils of the filament. The light and electron microscopic features were consistent with microsporidial keratitis.

Cytopathogenicity of Acanthamoeba isolates on rat glial C6 cell line.

The pathogenicity of Acanthamoeba isolates from keratitis patients (the Hamburg isolate from Germany, H-1 and a Philippine isolate, IB-1-7) as well as an environmental isolate, W4 was assayed in vitro using rat glial C6 cell line. Results indicate that both live amebae and cell-free supenatants from H-1 and IB-1-7 clones produced cytopathic effects (CPE) on rat glial C6 cells in a dose-and-time-dependent fashion. A dose of 10(5) cells/ml induced death and moderate areas of destruction of individual cells after 48 hours of incubation. Results of both free zone capillary electrophoresis and sodium dodecyl sulphate polyacrylamide gel electrophoresis suggest the release of amebic products to the culture medium that could at least partially explain the observed cytopathogenicity after 48 hours. Furthermore, results of SDS-PAGE indicate differences between the secretions of the isolates, with bands produced by the two ocular isolates that were not seen with the environmental isolates. That the secretions can produce a cytopathic effect (CPE) has been shown by the cytotoxicity assays using protein concentrations of the secretory products. Protein concentration of 0.30 microg/microl of culture supenatants from H-1 and IB-1-7 clones produced similar effects on the cell monolayers after 2 hours of incubation. This concentration caused the highest % cell death as measured by both trypan blue exclusion (TBE) and 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl-tetrazolium bromide (MTT) assays. In contrast, using W4 clone, corresponding concentrations of both trophozoites and culture supernatant did not cause significant cell death and cellular disintegration.

Acanthamoeba culbertsoni: sensibilidade aos diferentes métodos de desinfecçäo de lentes de contato hidrofílicas / Acanthamoeba culbertsoni: susceptibility to soft contact lens disinfection systems

Desde 1973, úlceras de córnea por Acanthamoeba spp têm sido descritas em número crescente e muitas vezes relacionados com o uso de lentes de contacto hidrofílicas (L.C.H.). Neste estudo foi testada a sensibilidade da Acanthamoeba culbertsoni aos diferentes métodos de desinfecçäo química (Hidrocare - Trietanol alquilamonio miramide e Flexcare - Clorixidine) e térmica (fervura simples por dez minutos com flutuador e aparelhos de asseptizaçäo - Contact Lens e Hydroset). Foram utilizadas 36 L.C.H. novas Soflens (Baush & Lomb) e 36 L.C.H. usadas Hydrosol (Solótica) que foram contaminadas com cistos e trofozoítos de Acanthamoeba culbertsoni por 12 horas. Após este período as lentes foram cortadas ao meio, ficando uma das metades semeada para controle e a outra submetida aos diferentes métodos de desinfecçäo, salientando-se que as lentes foram lavadas previamente com xampú apropriado por 20 segundos e enxaguadas com 5 ml de soluçäo fisiológica näo preservadas. As placas semeadas com as lentes eram observadas diariamente sob microspia óptica para detectar a passagem da forma cística para a trofozoítica, o que traduziria a viabilidade do parasita. Todos os resultados foram negativos. Todos os métodos testados se mostraram, eficientes. Concluímos que os métodos de desinfecçäo testados, desde que corretamente utilizados, säo eficazes na eliminaçäo da Acanthamoeba culbertsoni

case of onchocerciasis of the anterior segment

A 25 years old male patient from Uganda was referred from the United Nations Office in Cairo to the Ophthalmological Clinic of the Greek Community Hospital. The examination revealed a bilateral punctate keratitis with microfilariae in the cornea and the anterior chamber. Comments follow on the pathogenesis and the excellent results of 5% Hetrazan solution as eye drops for the treatment