Clinical and molecular diagnosis of Acanthamoeba keratitis in contact lens wearers in southern Brazil reveals the presence of an endosymbiont.

Acanthamoeba keratitis (AK) is an infection that is mostly observed in contact lens wearers. It is often misdiagnosed causing delays in the administration of the correct treatment. The aim of this study was to report the outcome of clinical and molecular diagnosis of AK cases during the summer of 2019 in the southern region of Brazil. Three suspected cases of AK were discovered after an ophthalmic examination at a public hospital in the city of Porto Alegre. These cases were then confirmed through laboratory diagnosis (cell culture and molecular analysis by PCR and sequencing). In each of the three clinical sample cell cultures of corneal scraping and molecular analysis confirmed the presence of Acanthamoeba spp., all belonging to the morphological group II and to the genotype T4, which is the most common genotype associated with AK. In addition, Acanthamoeba spp. isolated from one of the clinical samples was found to harbor the Candidatus Paracaedibacter acanthamoeba, a bacterial endosymbiont. The presence of Ca. Paracaedibacter acanthamoeba in clinical isolates requires further research to reveal its possible role in the pathogenicity of Acanthamoeba infections.

Acanthamoeba keratitis in Porto Alegre (southern Brazil): 28 cases and risk factors.

The increasing use of contact lenses worldwide has led to an increase in cases of Acanthamoeba keratitis, which are often associated with inappropriate cleaning of contact lenses and lens cases. This study aimed to retrospectively review 28 cases of Acanthamoeba keratitis in Porto Alegre (southern Brazil) and identify the risk factors and clinical outcomes of affected patients. Most patients had higher education (66.6%), all were users of contact lenses, mostly women (67.9%). Most patients were soft contact lens wearers (66.7%) and 85.7% used multipurpose cleaning solutions. Sixteen patients (64.0%) used to wear contact lenses while swimming and/or bathing. Pain was the most common symptom (92.6%). For treatment, patients used polyhexamethylene biguanide drops (92.6%), propamidine isethionate drops (81.5%), chlorhexidine drops (55.6%), topical corticosteroids (63.0%), and systemic corticosteroids (37.0%). Herpes simplex keratitis was the most common misdiagnosis (72.7%). The majority of patients (76.0%) underwent a corneal transplant to control the disease.

Antifungal Susceptibility, Morphological and Molecular Characterization of Lasiodiplodia theobromae Isolated from a Patient with Keratitis.

Lasiodiplodia theobromae is a rare ocular pathogen. We report a patient with fungal keratitis caused by L. theobromae. The patient was a 75-year-old male, a farmer with diabetes type II, and no previous history of ocular trauma. Histopathology analysis revealed the presence fungi invading Descemet's membrane of the cornea. The fungus was characterized by septate, highly bulged fungal filaments involving full corneal thickness in the corresponding histopathology specimens. A dematiaceous mold was isolated and initally identified as L. theobromae by microscopic and macroscopic morphology, and further confirmed by PCR-based determination of internal transcribed spacer (ITS) regions of ribosomal DNA. Antifungal susceptibility tests showed sensitivity to amphotericin B (AMB) and voriconazole ( VRC), and resistance to other azoles, including itraconazole (ITC) and fluconazole (FLC). Corneal transplant was performed. Despite in vitro itraconazole resistance, the patient was successfully treated with oral itraconazole, topical voriconazole and natamycin, combined with ocular injections of amphotericin B and voriconazole.

Antifungal Efficacy and Safety of Cycloheximide as a Supplement in Optisol-GS.

PURPOSE: The incidence of fungal infection after corneal transplant has increased significantly in recent years, especially Candida spp. This study aimed to evaluate the efficacy and safety of the addition of cycloheximide in Optisol-GS media in decreasing the growth of Candida spp. strains. METHODS: This in vitro laboratory efficacy study measured fungal colony growth in 24 vials of Optisol-GS that were divided into 6 groups of 4 vials each, as follows: (1) MIC/2 cycloheximide, (2) MIC cycloheximide, (3) MICx5 cycloheximide, (4) MICx10 cycloheximide, from MIC values obtained for each strain, (5) unsupplemented optisol-GS as a positive control (added inoculum), and (6) unsupplemented optisol-GS as a negative control (no inoculum). In each group was added Candida albicans, C. glabrata and C. parapsilosis, except in the negative control. The evaluated variables were fungal colony growth from the Optisol-GS vials, corneal endothelial cell density and endothelial cell viability at different concentrations of cycloheximide. RESULTS: In the efficacy study, all strains showed a reduction in fungal cell growth from the second day at all evaluated concentrations of optisol-GS supplemented with cycloheximide, even at subinhibitory concentrations (MIC/2). For C. glabrata, the colony count was reduced to 99%. No evidence of corneal endothelial toxicity was found at any concentration, in the safety study, compared with the paired control. CONCLUSION: The addition of cycloheximide to optisol-GS decreased the fungal growth, demonstrating fungicide action against C. glabrata and fungistatic action against C. albicans and C. parapsilosis. This drug did not demonstrate toxicity to the corneal endothelium at different concentrations.

Fusarium solani keratitis: role of antifungal susceptibility testing and identification to the species level for proper management.

We report a patient with fungal keratitis caused by a multiresistant Fusarium solani in a tertiary care hospital located in southern Brazil. A 55-year-old man with a history of ocular trauma presented with keratitis in left eye. The patient has a complicated clinical course and failed to respond to local and systemic antifungal treatment, and required eye enucleation. Despite multiple topical, intraocular and systemic antifungal treatments, hyphal infiltration persisted in the corneal transplant causing continuous recurrences. The cultures of corneal biopsy scrapings were positive for Fusarium spp. The organism was identified to species level by multi-locus sequencing for translation elongation factor 1 alpha (EF-1α), and RNA polymerase II subunit (RPB2). In vitro antifungal susceptibility testing of the isolate by the broth microdilution method, according to CLSI M38-A2, disclosed susceptibility to natamycin and resistance to amphotericin B, voriconazole, itraconazole and fluconazole. Considering previous unsuccessful antifungal treatments due to multiple drug resistance, the eye was enucleated. Our case report illustrates that management of fungal keratitis remains a therapeutic challenge. Optimal treatment for F. solani infection has not yet been established and should include susceptibility testing for different antifungal agents.

Autologous serum eye drops diluted with 0.5% methylcellulose and 0.9% saline solution: a clinical comparative study / Colírio de soro autólogo diluído com metilcelulose 0,5% e solução salina 0,9%: um estudo clínico comparativo

ABSTRACT Purpose: This clinical study compared autologous serum eye drops diluted with 0.5% methylcellulose and 0.9% saline solution. The subjective criteria for symptom improvement and the objective clinical criteria for response to therapy were evaluated. Methods: This longitudinal prospective study enrolled 23 patients (42 eyes) with persistent epithelial defects or severe dry eye disease refractory to conventional therapy who had been using autologous serum 20% prepared with methylcellulose for > 6 months and started on autologous serum diluted in 0.9% saline solution. The control and intervention groups consisted of the same patients under alternate treatments. The subjective criteria for symptom relief were evaluated using the Salisbury Eye Evaluation Questionnaire. The objective clinical criteria were evaluated through a slit-lamp examination of the ocular surface, tear breakup time, corneal fluorescein staining, Schirmer's test, rose Bengal test, and tear meniscus height. These criteria were evaluated before the diluent was changed and after 30, 90, and 180 days. Results: In total, 42 eyes were analyzed before and after 6 months using autologous serum diluted with 0.9% saline. No significant differences were found in the subjective criteria, tear breakup time, tear meniscus, corneal fluorescein staining, or rose Bengal test. Schirmer's test scores significantly worsened at 30 and 90 days (p=0.008). No complications or adverse effects were observed. Conclusions: This study reinforces the use of autologous serum 20% as a successful treatment for severe dry eye disease resistant to conventional therapy. Autologous serum in 0.9% saline was not inferior to the methylcellulose formulation and is much more cost-effective.

RESUMO Objetivo: Este estudo comparou o colírio de soro au tólogo manipulado com metilcelulose a 0,5% com solução salina 0,9%. Critérios subjetivos de melhora dos sintomas e critérios clínicos objetivos para resposta à terapia foram avaliados. Métodos: Este estudo prospectivo longitudinal envolveu 23 pacientes (42 olhos) com defeitos epiteliais persistentes ou doença de olho seco grave refratária à terapia convencional que usavam colírio de soro autólogo 20% preparado com metilcelulose por mais de 6 meses e iniciaram soro autólogo diluído em solução salina 0,9%. Os grupos controle e intervenção consistiam dos mesmos pacientes sob tratamentos alternados. Os critérios subjetivos para o alívio dos sintomas foram avaliados usando o Salisbury Eye Evaluation Questionnaire. Os critérios objetivos foram avaliados por meio de exame em lâmpada de fenda incluindo: tempo de ruptura da lágrima, coloração da córnea com fluoresceína, teste de Schirmer, coloração com rosa bengala e altura do menisco lacrimal. Esses critérios foram avaliados antes da troca do diluente e após 30, 90 e 180 dias. Resultados: Um total de 42 olhos foram analisados antes e após 6 meses usando soro autólogo diluído com solução salina 0,9%. Nenhuma diferença significativa foi encontrada nos critérios subjetivos, tempo de ruptura da lágrima, menisco lacrimal, coloração com fluoresceína ou rosa bengala. Os resultados dos testes de Schirmer pioraram significativamente em 30 e 90 dias (p=0,008). Não foram observadas complicações ou efeitos adversos. Conclusões: Este estudo reforça o uso do colírio de soro autólogo 20% como um tratamento de sucesso para a doença do olho seco grave resistente à terapia convencional. O soro autólogo diluído em solução salina a 0,9% não foi inferior à formulação de metilcelulose.

[Positive corneoscleral rim culture in penetrating keratoplasty at the Porto Alegre Clinical Hospital]. / Contaminação de halos doadores córneo-esclerais em ceratoplastia penetrante no Hospital de Clínicas de Porto Alegre.

PURPOSE: To determine the incidence of positive corneoscleral rim cultures preserved in Optisol GS medium, to identify pathogens involved and possible recipient eye infection. METHODS: A hundred sixty-three corneoscleral rim cultures penetrating keratoplasties performed from January 2001 to January 2003 in the Hospital de Clínicas de Porto Alegre were reviewed. Enucleations and corneal storage were done as aseptic as possible and gentamicin 0.3% was instilled. Corneoscleral rim was divided into two segments, half was inoculated into Sabouraud broth and the other half into thioglycolate broth; inoculation into blood agar, chocolate agar and MacConkey agar was done later if necessary for pathogen identification. The receiver's eye data were reviewed. RESULTS: There were eleven positive cultures (6.7%) out of 163 evaluated corneoscleral rim cultures. Of these, four were Staphylococcus epidermidis, one was Staphylococcus aureus, one was Serratia sp., one was Pseudomonas aeruginosa and the other four were different subtypes of Candida (two Candida sp., one Candida albicans and one Candida parapapilosis). All pathogens were resistant to gentamicin. None of the eleven cases of positive corneoscleral rim cultures resulted in ocular infection at the receiver's eyes (six months follow-up). CONCLUSIONS: We found low rates of positive corneoscleral rim cultures after penetrating keratoplasty at the Porto Alegre Clinical Hospital. The most frequent involved pathogens were Staphylococcus sp and Candida sp. Although we did not identify any postoperative infection at the receiver's eyes, we recommend corneoscleral rim culture for guidance of postoperative infection, a rare but possible devastating ocular event.

Virulent T4 Acanthamoeba causing keratitis in a patient after swimming while wearing contact lenses in Southern Brazil.

Several strains of free-living amoebae belonging to the genus Acanthamoeba can cause a painful sight-threatening disease of the cornea known as Acanthamoeba keratitis (AK). The numbers of AK cases keep rising worldwide mainly due to an increase in contact lens wearers and lack of hygiene in the maintenance of contact lenses and their cases. We report a case of AK in a healthy young woman admitted to the Hospital de Clinicas in Porto Alegre, southern Brazil. Corneal scrapings were examined for the presence of Acanthamoeba strains. The initial isolate was characterized by morphological and genotypic properties. The isolate belonged to group III according to Pussard and Pons' cyst morphology. Analysis of its 18S rDNA sequence identified the isolate as genotype T4. The T4 genotype is the most commonly reported among keratitis isolates and the most common in environmental samples.

A Case-Control Study on the Oxidative Balance of 50% Autologous Serum Eye Drops.

Importance. Autologous serum (AS) eye drops are recommended for severe dry eye in patients with ocular surface disease. No description of the antioxidant balance of AS eye drops has been reported in the literature. Objective. This study sought to evaluate the total reactive antioxidant potential (TRAP) and concentration of reactive oxygen species (ROS) in samples of 50% AS eye drops and their correlations with the demographic characteristics and lifestyle habits of patients with ocular surface disease and healthy controls. Design. This was a case-control study with a 3-month follow-up period. Participants. 16 patients with severe dry eye disease of different etiologies and 17 healthy controls matched by age, gender, and race were included. Results. TRAP and ROS were detected at all evaluated times. There were no differences in the mean ROS (p = 0.429) or TRAP (p = 0.475) levels between cases and controls. No statistically significant differences in the concentrations of ROS or TRAPs were found at 0, 15, or 30 days (p for ROS = 0.087 and p for TRAP = 0.93). Neither the demographic characteristics nor the lifestyle habits were correlated with the oxidative balance of the 50% AS eye drops. Conclusions and Relevance. Both fresh and frozen 50% AS eye drops present antioxidant capacities and ROS in an apparently stable balance. Moreover, patients with ocular surface disease and normal controls produce equivalent AS eye drops in terms of oxidative properties.

Fusarium solani keratitis: role of antifungal susceptibility testing and identification to the species level for proper management

ABSTRACT We report a patient with fungal keratitis caused by a multiresistant Fusarium solani in a tertiary care hospital located in southern Brazil. A 55-year-old man with a history of ocular trauma presented with keratitis in left eye. The patient has a complicated clinical course and failed to respond to local and systemic antifungal treatment, and required eye enucleation. Despite multiple topical, intraocular and systemic antifungal treatments, hyphal infiltration persisted in the corneal transplant causing continuous recurrences. The cultures of corneal biopsy scrapings were positive for Fusarium spp. The organism was identified to species level by multi-locus sequencing for translation elongation factor 1 alpha (EF-1α), and RNA polymerase II subunit (RPB2). In vitro antifungal susceptibility testing of the isolate by the broth microdilution method, according to CLSI M38-A2, disclosed susceptibility to natamycin and resistance to amphotericin B, voriconazole, itraconazole and fluconazole. Considering previous unsuccessful antifungal treatments due to multiple drug resistance, the eye was enucleated. Our case report illustrates that management of fungal keratitis remains a therapeutic challenge. Optimal treatment for F. solani infection has not yet been established and should include susceptibility testing for different antifungal agents.

Contaminação de halos doadores córneo-esclerais em ceratoplastia penetrante no Hospital de Clínicas de Porto Alegre / Positive corneoscleral rim culture in penetrating keratoplasty at the Porto Alegre Clinical Hospital

OBJETIVOS: Avaliar a incidência de positividade de culturas de halos doadores córneo-esclerais preservados em Optisol GS, identificar os patógenos envolvidos, a sensibilidade dos mesmos à gentamicina e a ocorrência de infecções em olhos receptores. MÉTODOS: Foram analisadas retrospectivamente 163 culturas de halos córneo-esclerais cujos botões corneanos foram utilizados em transplantes de córnea no Hospital de Clínicas de Porto Alegre entre janeiro de 2001 e janeiro de 2003. Os halos foram divididos em dois segmentos, metade inoculada em meio Sabouroud e a outra metade em tioglicolato, com posterior semeadura em ágar-sangue, ágar-chocoloate e meio de MacConkey, conforme necessidade para identificação dos patógenos. Os prontuários dos pacientes receptores foram revisados. RESULTADOS: Dos 163 halos analisados, 11 apresentaram culturas positivas, correspondendo a 6,7 por cento do total. Destes, quatro foram por Staphylococcus epidermidis, um por Staphylococcus aureus, um por Serratia sp, um por Pseudomonas aeruginosa e os outros quatro por diferentes subtipos de Candida (dois por Candida sp, um por Candida albicans e um por Candida parapapilosis). No antibiograma, todas as bactérias apresentaram-se resistentes à gentamicina. Nenhum olho que recebeu córnea com cultura positiva apresentou infecção após a cirurgia. CONCLUSÕES: Baixos índices de positividade de cultura de halos utilizados em transplantes de córnea no Hospital de Clínicas de Porto Alegre foram encontrados. Os patógenos mais freqüentemente identificados não apresentam boa cobertura pelos antimicrobianos presentes nos meios de preservação. A cultura de halos corneanos é recomendada para auxílio no tratamento de possível infecção ocular pós-cirúrgica.

PURPOSE: To determine the incidence of positive corneoscleral rim cultures preserved in Optisol GS medium, to identify pathogens involved and possible recipient eye infection. METHODS: A hundred sixty-three corneoscleral rim cultures penetrating keratoplasties performed from January 2001 to January 2003 in the Hospital de Clínicas de Porto Alegre were reviewed. Enucleations and corneal storage were done as aseptic as possible and gentamicin 0.3 percent was instilled. Corneoscleral rim was divided into two segments, half was inoculated into Sabouraud broth and the other half into thioglycolate broth; inoculation into blood agar, chocolate agar and MacConkey agar was done later if necessary for pathogen identification. The receiver's eye data were reviewed. RESULTS: There were eleven positive cultures (6.7 percent) out of 163 evaluated corneoscleral rim cultures. Of these, four were Staphylococcus epidermidis, one was Staphylococcus aureus, one was Serratia sp., one was Pseudomonas aeruginosa and the other four were different subtypes of Candida (two Candida sp., one Candida albicans and one Candida parapapilosis). All pathogens were resistant to gentamicin. None of the eleven cases of positive corneoscleral rim cultures resulted in ocular infection at the receiver's eyes (six months follow-up). CONCLUSIONS: We found low rates of positive corneoscleral rim cultures after penetrating keratoplasty at the Porto Alegre Clinical Hospital. The most frequent involved pathogens were Staphylococcus sp and Candida sp. Although we did not identify any postoperative infection at the receiver's eyes, we recommend corneoscleral rim culture for guidance of postoperative infection, a rare but possible devastating ocular event.

Conjunctival endogenous microbiota in patients submitted to cataract surgery

A partir de material ocular coletado de 46 pacientes submetidos à cirurgia de catarata foram realizados isolamento, identificacão e teste de susceptibilidade de microrganismos frente a antimicrobianos, utilizando-se suabes e lentes intraoculares (LIO). Foram obtidos 76 isolados e identificados 7 tipos de microrganismos. Estafilococos coagulase-negativos (CNS) foram os microrganismos mais freqüentemente detectados de suabes (71,4 per center dos casos), lentes de PMMA (81,3 pree center) e lentes de silicone (77,8 per center). Isolados de CNS apresentaram elevada resistência à penicilina G, seguida por tetraciclina, cloranfenicol e aminoglicosídeos. No entanto, estes isolados mostraram grande sensibilidade à vancomicina, cefalotina e ofloxacina. Com excecão da penicilina G, os isolados de Staphylococcus aureus foram bastante sensíveis aos agentes antimicrobianos, incluindo a oxacilina. Entre as gram-negativas, Proteus mirabilis foi a bactéria mais freqüente e também se mostrou bastante resistente à tetraciclina e ao cloranfenicol. Os isolados de Enterococcus se mostraram sensíveis à vancomicina.

Aderência bacteriana in vitro a lentes intra-oculares de polimetilmetacrilato e de silicone / In vitro bacterial adherence to silicone and polymetylmethacrylate intraocular lenses

OBJETIVOS: Verificar a aderência bacteriana a lentes intra-oculares de silicone e de polimetilmetacrilato como possível fator de risco no desenvolvimento de endoftalmite pós-operatória, utilizando-se um modelo in vitro com três microrganismos potencialmente patogênicos. MÉTODOS: As análises foram realizadas com cepas de Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis (amostra clínica) e Pseudomonas aeruginosa ATCC 27853 incluindo a determinação de curvas de crescimento, testes para verificação de produção de cápsula, avaliação da hidrofobicidade, testes de aderência a diferentes materiais, microscopia óptica, microscopia eletrônica de varredura e microscopia de força atômica. RESULTADOS: A produção de cápsula e a aderência das três diferentes cepas não mostraram qualquer relação com a quantidade de microrganismos; em relação às lentes intra-oculares de polimetilmetacrilato e de silicone, não houve diferença estatisticamente significativa na aderência de S. aureus e S. epidermidis; P. aeruginosa foi o microrganismo mais aderente a ambos os materiais. A microscopia eletrônica de varredura confirmou estes achados em relação à aderência, ao peso que a microscopia de força atômica evidenciou a produção de biofilme pelas cepas de S. aureus, S. epidermidis e P. aeruginosa. CONCLUSÕES: Constatou-se, in vitro, que os materiais analisados não diferiram com relação à taxa de aderência bacteriana, porém, P. aeruginosa apresentou maior eficiência de adesão entre as bactérias testadas. Todas as cepas produziram biofilme. Silicone foi o material mais hidrofóbico, quando comparado ao polimetilmetacrilato.

Influência de fatores climáticos na toxoplasmose ocular em Guaporé - Brasil / Influence of climatic factors on ocular toxoplasmosis in Guaporé

Toxoplasmose ocular apresenta alta prevalência na regiäo de Guaporé-RS, sendo desconhecidas as causas deste fenômeno. Foram estudados, no período de dez anos, todos os pacientes com diagnóstico de toxoplasmose ocular que se consultaram numa clínica oftalmológica localizada na regiäo. Os fatores climáticos avaliados no mesmo período foram precipitaçäo, temperatura e umidade relativa do ar. A distribuiçäo máxima das lesöes ocorreu entre os 20 e 29 anos (43 por cento). Näo houve variaçäo significativa na distribuiçäo dos pacientes com lesöes únicas ou recidivadas ao longo dos meses através do teste do qui-quadrado para um alfa de 0,05. Também näo foi encontrada nenhuma correlaçäo entre o aparecimento da lesöes únicas ou recidivadas com os índices climáticos