Imagenología diferencial por microscopia confocal in vivo entre la epiteliopatía por acanthamoeba y la queratitis por herpes simple / In vivo confocal microscopy imaging for the differential diagnosis of Acanthamoeba epitheliopathy and herpes simplex keratitis

La queratitis por Acanthamoeba es una infección corneal de baja incidencia, condicionada por varios factores, pero con manifestación clínica variada y sintomatología típica. En su fase epitelial debe ser diferenciada de otras queratitis, específicamente de la queratitis por herpes simple, por la similitud entre ambas en cuanto a las características de la lesión corneal. La microscopia confocal in vivo constituye una alternativa diagnóstica. Es una biopsia fotográfica en cuyas imágenes podemos describir los quistes y trofozoítos de Acanthamoeba desde etapas iniciales, que nos ayudan a diferenciarla de otros tipos de queratitis e iniciar el tratamiento precoz. Se realizó una búsqueda de artículos publicados, con el objetivo de mostrar las imágenes por microscopia confocal de la fase epitelial de la infección corneal por Acanthamoeba y herpes simple. Se utilizó la plataforma Infomed, específicamente la Biblioteca Virtual de Salud(AU)

Acanthamoeba keratitis is a low-incidence corneal infection caused by several factors and characterized by a variety of clinical manifestations and typical symptoms. In its epithelial phase, it should be differentiated from other keratitis, particularly from herpes simplex keratitis, due to the similar characteristics of the corneal lesion. In vivo confocal microscopy is a diagnostic alternative consisting in a photographic biopsy showing images of Acanthamoeba cysts and trophozoites since their initial stages, thus allowing differentiation from other types of keratitis and the initiation of early treatment. A search was conducted of published papers with the purpose of showing confocal microscopy images of the epithelial phase of Acanthamoeba and herpes simplex corneal infection. Use was made of the platform Infomed, specifically the Virtual Health Library(AU)

Cromoforo fotoactivado con crosslinking como tratamiento para la queratitis por Acanthamoeba / Photoactivated chromophore with crosslinking as treatment for Acanthamoeba keratitis

RESUMEN Se reporta el uso del crosslinking como tratamiento de la queratitis por Acanthamoeba en una serie de 7 pacientes quienes acudieron al Servicio de Córnea por queratitis multitratadas. Se les realizó biopsia corneal, la cual se cultivó en solución de Page. Los pacientes fueron tratados con un protocolo de PACK-CXL durante más de 5 minutos y fueron sometidos a la exposición a la luz UV-A. El edema del nuevo epitelio era de 2 cruces a las 24 horas, y desapareció a las dos semanas del procedimiento en todos los casos. El porcentaje de desepitelización basal al momento del diagnóstico fue de 75,7 por ciento. La agudeza visual mejor corregida fue de entre 20/20 y 20/30. Se concluye que el uso de crosslinking en pacientes con Acanthamoeba en fases inicales pudiera ser una opción terapéutica segura y efectiva(AU)

ABSTRACT A report is presented of the use of crosslinking as treatment for Acanthamoeba keratitis in a series of 7 patients attending the Cornea Service for multitreated keratitis. Corneal biopsy was performed, which was cultured in Page solution. The patients were treated with a PACK-CXL protocol for more than 5 minutes and subjected to UV-A light exposure. Edema of the new epithelium was 2 crosses at 24 hours and disappeared 2 weeks after the procedure in all cases. Basal de-epithelialization percentage at diagnosis was 75.7 percent. Best corrected visual acuity ranged between 20/20 and 20/30. It is concluded that the use of crosslinking in patients with Acanthamoeba keratitis in its initial stages could be a safe and effective therapeutic option(AU)

Detección de Acanthamoeba por reacción en cadena de la polimerasa en tiempo real en líquidos de conservación de lentes de contacto / Detection of Acanthamoeba sp. by real-time polymerase chain reaction in contact lens solutions

RESUMEN Objetivo: Estandarizar una técnica de reacción en cadena de la polimerasa en tiempo real para la detección del parásito e identificar Acanthamoeba en líquidos conservantes de lentes de contacto. Métodos: Se realizó un estudio descriptivo observacional de corte transversal sobre la técnica de reacción en cadena de la polimerasa en tiempo real para la detección de Acanthamoeba, en el Instituto de Investigaciones en Ciencias de la Salud de la ciudad de Asunción, en Paraguay. Se analizaron 110 líquidos conservantes aportados por usuarios sanos de lentes de contacto, mediante reacción en cadena de la polimerasa en tiempo real y cultivo en medio PAGE - SDS. Resultados: Se estandarizó con éxito la técnica de reacción en cadena de la polimerasa en tiempo real con límite de sensibilidad de 1 pg/µL. Se aisló Acanthamoeba a partir de una muestra (1 por ciento) por método de cultivo, mientras que la carga parasitaria en el líquido conservante fue inferior al límite de detección de la reacción en cadena de la polimerasa en tiempo real. El ADN obtenido del cultivo de dicha muestra fue positivo para Acanthamoeba por este método. Conclusión: El sistema estandarizado presenta buena sensibilidad y podrá ser incorporado en los laboratorios que cuentan con acceso a equipos de reacción en cadena de la polimerasa en tiempo real para un diagnóstico rápido y más eficiente en casos de sospechas de queratitis amebiana. Recomendamos el uso combinado de métodos moleculares y cultivo para aumentar la potencia del diagnóstico, sobre todo en muestras donde la carga parasitaria es muy baja(AU)

ABSTRACT Objective: Standardize a real-time polymerase chain reaction technique for detection of the parasite and identify Acanthamoeba in contact lens solutions. Methods: A cross-sectional observational descriptive study was conducted about a real-time polymerase chain reaction technique for detection of Acanthamoeba at the Institute of Health Sciences Research in the city of Asunción, Paraguay. A total 110 solutions were analyzed, which were provided by healthy contact lens users, by real-time polymerase chain reaction and culture in SDS-PAGE medium. Results: Successful standardization was achieved of the real-time polymerase chain reaction technique with a sensitivity limit of 1 pg/µl. Acanthamoeba was isolated from one sample (1 percent) by culture, whereas the parasite load in the contact lens solution was below the detection limit of the real-time polymerase chain reaction technique. The DNA obtained from the culture of that sample was positive for Acanthamoeba by the real-time polymerase chain reaction technique method. Conclusion: The system standardized exhibits good sensitivity and may be incorporated into laboratories with real-time polymerase chain reaction technique equipment for a rapid and more efficient diagnosis of suspected amoebic keratitis. We recommend the combined use of molecular methods and culture to enhance diagnostic power, mainly in samples where the parasite load is very low(AU)

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.

Situación del diagnóstico de Queratitis Amebiana en Paraguay / Status of the diagnosis of Amebic Keratitis in Paraguay

Las amebas de vida libre (AVL) existen ampliamente distribuidas en la naturaleza, donde el género Acanthamoeba es la más frecuentemente aislada en diversos ambientes del suelo, aire y agua y está asociada a enfermedades en humanos. Su capacidad para vivir en ambientes adversos se debe a su baja demanda de alimentación y a que en su ciclo biológico tiene un estadio en forma de quiste, lo que la hace muy resistente. El hombre, como hospedero puede desarrollar infecciones en el sistema nervioso central, en la piel y los pulmones. A nivel ocular es capaz de afectar la córnea y producir queratitis. Por lo tanto es considerado un importante agente etiológico de patologías humanas. En esta revisión se aborda la biología, patogénesis y los mecanismos de defensa del ser humano frente a la infección por Acanthamoeba. Además a esto, de los factores de riesgo por el uso de lentes de contacto y malos hábitos de higiene del usuario y por último el abordaje diagnóstico microbiológico y molecular. Esta herramienta ha ido mejorando con el avance de la tecnología, indispensable para la temprana identificación y el logro de una oportuna y eficaz intervención clínica y terapéutica. Finalmente exponemos la situación actual en relación a los métodos de diagnóstico con que se cuentan y los escasos reportes clínicos existentes en Paraguay. Palabras clave: Acanthamoeba sp., Lentes de contacto, Queratitis.

Free-living amoeba (FLA) exist widely in nature, where the genus Acanthamoeba is the most frequently isolated in various environments of soil, air and water. It is associated with diseases in human. Its ability to live in adverse environments is due to the low food demand and that in its biological cycle it has a cyst-shaped stage, which makes it very resistant. Man, as a host, can acquire infections in the central nervous system and in the skin and lungs. On the ocular level, it can affect the cornea and cause keratitis. Therefore, it is considered an important etiological agent of human pathologies. This review addresses the biology, pathogenesis and human defense mechanisms against Acanthamoeba infection. In addition to this, the review explores the risk factors related to the use of contact lenses and poor hygienic behavior of the users, and finally the microbiological and molecular diagnosis. This particular tool has been improved with the advancement of technology and is essential for timely identification and the achievement of a good clinical and therapeutic intervention. Finally, we present the current situation in Paraguay regarding the few existing clinical reports and the diagnostic methods available. Key words: Acanthamoeba sp., Contact lenses, Keratitis.

Parasitosis ocular por Acanthamoeba / Acanthamoeba parasitic eye infection

RESUMEN El parasitismo es uno de los fenómenos más sorprendentes de los observados en los animales. El número de organismos conocidos de vida parasitaria es muy elevado. Existe una gran cantidad de especies cuya supervivencia está relacionada con la de otras y dependen, en distinta medida, de ellas. Esta dependencia no implica que los animales parásitos sean organismos degenerados o deficientes; al contrario, a la vida parasitaria se ha llegado tras largo tiempo de evolución, en el que los parásitos han ido superando barreras y adaptándose a vivir en, o sobre sus hospedadores. Las queratitis por Acanthamoeba son infrecuentes; pero se describe un aumento a nivel mundial relacionado con el uso creciente de lentes de contacto. El diagnóstico precoz y el tratamiento adecuado deben realizarse para evitar la pérdida de la visión. Se realizó una búsqueda de artículos publicados, con el objetivo de conocer sobre la Acanthamoeba como parásito y su afectación ocular. Se utilizó la plataforma Infomed, específicamente la Biblioteca Virtual de Salud(AU)

ABSTRACT Parasitism is one of the most surprising phenomena among those occurring in animals. The number of known parasitic organisms is very high. There is a large number of species whose survival is related to that of others and depend on them to a greater or lesser degree. Such dependence does not imply that parasitic animals are either degenerate or deficient. On the contrary, parasitic life has been the result of a long process of evolution along which parasites have gradually overcome hurdles and have adapted to live in or on their hosts. Acanthamoeba keratitis is a rare disease, but a worldwide increase has been reported due to the growing use of contact lenses. Early diagnosis and appropriate treatment are required to prevent sight loss. A search was conducted for published papers with the purpose of learning about Acanthamoeba as a parasite and the related eye conditions. Use was made of the platform Infomed, specifically the Virtual Health Library(AU)

Accelerated collagen cross-linking in the management of advanced Acanthamoeba keratitis / Cross-linking acelerado de colágeno no controle da ceratite por Acanthamoeba avançada

ABSTRACT Purpose: To report our initial experience in the treatment of Acanthamoeba keratitis with accelerated corneal collagen cross-linking. Methods: Retrospective chart review of patients diagnosed with Acanthamoeba keratitis with progressive corneal melting who were treated with accelerated collagen cross-linking. Results: A total of 6 eyes (5 patients) were reviewed. All the patients received adjuvant therapy with moxifloxacin and chlorhexidine. In 4 cases, the ulcer healed with a mean interval to epithelialization of 108.8 days (range 59-217). In 2 eyes, there was a persistent neurotrophic ulcer. The melting was not progressive in any case, nor did any eye required emergency penetrating keratoplasy. Conclusion: This study suggests a beneficial effect of accelerated collagen cross-linking in cases of Acanthamoeba keratitis with corneal melting. Thus, collagen cross-linking may be considered as adjuvant treatment for Acanthamoeba keratitis.

RESUMO Objetivo: Relatar nossa experiência inicial no tra tamento da ceratite por Acanthamoeba com reticulação acelerada de colágeno corneano. Métodos: Revisão retrospectiva de prontuários de pacientes diagnosticados com ceratite por Acanthamoeba, com deformação progressiva da córnea, tratados com reticulação acelerada de colágeno. Resultados: Seis olhos (5 pacientes) foram incluídos. Todos os pacientes receberam terapia adjuvante com moxifloxacina e clorexidina. Em 4 casos, a úlcera cicatrizou com uma média de epitelização de 108,8 dias (amplitude de 59-217 dias). Em dois pacientes, a úlcera apresentou um comportamento neurotrófico. A deformação não foi progressiva em nenhum dos pacientes e nenhum dos olhos exigiu ceratoplastia penetrante de emergência. Conclusão: Este estudo sugeriu um efeito benéfico da reticulação acelerada de colágeno em casos de ceratite por Acanthamoeba infecciosa com deformação corneal. A reticulação de colágeno parece ser uma alternativa coadjuvante possível para casos de ceratite por Acanthamoeba.

The First Acanthamoeba keratitis Case of Non-Contact Lens Wearer with HIV Infection in Thailand

Acanthamoeba keratitis (AK) is a rare sight-threatening corneal infection, often reporting from contact lens wearers. An asymptomatic human immunodeficiency virus (HIV)-infected Thai male without history of contact lens use complained foreign body sensation at his left eye during motorbike riding. He had neither specific keratitis symptoms nor common drugs responding, which contributed to delayed diagnosis. By corneal re-scraping, Acanthamoeba-like cysts were detected by calcofluor white staining and agar culture. The etiological agent obtained from the culture was molecularly confirmed by Acanthamoeba spp.-specific PCR, followed by DNA sequencing. The results from BLAST and phylogenetic analysis based on the DNA sequences, revealed that the pathogen was Acanthamoeba T4, the major genotype most frequently reported from clinical isolates. The infection was successfully treated with polyhexamethylene biguanide resulting in corneal scar. This appears the first reported AK case from a non-contact lens wearer with HIV infection in Thailand. Although AK is sporadic in developing countries, a role of free-living Acanthamoeba as an opportunistic pathogen should not be neglected. The report would increase awareness of AK, especially in the case presenting unspecific keratitis symptoms without clinical response to empirical antimicrobial therapy.

The first Acanthamoeba keratitis case in the Midwest region of Brazil: diagnosis, genotyping of the parasite and disease outcome

Abstract We report an Acanthamoeba keratitis case associated with the use of contact lens in a 28-year-old female from Brasília, Brazil. Samples from corneal scraping and contact lens case were used for culture establishment, PCR amplification, and partial sequencing (fragments of ~400kb) of small subunit rDNA; both culture and PCR were positive. The sequence analyses of the cornea and of isolates from the contact lens case showed similarity with the T4 genotype. To the best of our knowledge, this is the first report of T4 Acanthamoeba keratitis case from the Midwest region of Brazil.

Comparison of Proteins Secreted into Extracellular Space of Pathogenic and Non-pathogenic Acanthamoeba castellanii

Pathogenic Acanthamoeba spp. cause granulomatous amoebic encephalitis and keratitis. Acanthamoeba keratitis (AK) is a rare but serious ocular infection that can result in permanent visual impairment or blindness. However, pathogenic factors of AK remain unclear and treatment for AK is arduous. Expression levels of proteins secreted into extracellular space were compared between A. castellanii pathogenic (ACP) and non-pathogenic strains. Two-dimensional polyacrylamide gel electrophoresis revealed 123 differentially expressed proteins, including 34 increased proteins, 7 qualitative increased proteins, 65 decreased proteins, and 17 qualitative decreased proteins in ACP strain. Twenty protein spots with greater than 5-fold increase in ACP strain were analyzed by liquid chromatography triple quadrupole mass spectrometry. These proteins showed similarity each to inosine-uridine preferring nucleoside hydrolase, carboxylesterase, oxygen-dependent choline dehydrogenase, periplasmic-binding protein proteinases and hypothetical proteins. These proteins expressed higher in ACP may provide some information to understand pathogenicity of Acanthamoeba.

Efficacy of Korean Multipurpose Contact Lens Disinfecting Solutions against Acanthamoeba castellanii

Acanthamoeba keratitis has been increasing in recent years. Main risk factors are contact lens wear and their cleaning solutions. Most contact lens wearers use multipurpose disinfecting solutions (MPDS) for cleansing and disinfecting microorganisms because of its convenience. We determined amoebicidal effects of MPDS made in Korea and their cytotoxicity on human corneal epithelium cells. Fifteen commercial MPDS (A to O) were tested for their amoebicidal effects on Acanthamoeba castellanii trophozoites and cysts by using a most probable number (MPN) technique. Among them, 7 kinds of MPDS showed little or no amoebicidal effects for 24 hr exposure. Solutions A, B, G, H, L, and O showed positive amoebicidal effects, and solutions M and N killed almost all trophozoites and cysts after 24 hr exposure. However, 50%-N solution showed 56% cytotoxicity on human corneal epithelial cells within 4 hr exposure, and 50%-O solution also showed 62% cytotoxicity on human cells within 4 hr exposure. Solution A did not show any cytotoxicity on human cells. These results revealed that most MPDS made in Korea were ineffective to kill Acanthamoeba. The solutions having amoebicidal activity also showed high levels of cytotoxicity on human corneal epithelial cells. New formulations for improved MPDS that are amoebicidal but safe for host cells are needed to prevent Acanthamoeba keratitis.

Aportes al diagnóstico de las infecciones por Acanthamoeba spp / Inputs for diagnosing Acanthamoeba spp. infections

Acanthamoeba spp., tiene capacidad para comportarse como parásito oportunista y ocasionar encefalitis granulomatosa amebiana (EGA), sinusitis y lesiones cutáneas, especialmente en pacientes inmunodeprimidos...

Two Cases of Corneal Toxicity in Acanthamoeba Keratitis by Combined Topical Anti-Acanthamoeba Keratitis Eye Solution

PURPOSE: To present a case of corneal toxicity caused by therapeutic duplication during treatment of acanthamoeba keratitis as a complication of contact lens use. CASE SUMMARY: A 12-year-old girl with a history of wearing orthokeratology contact lenses and a 14-year-old girl with a history of wearing cosmetic contact lenses presented with ocular pain, injection, and decreased visual acuity. They were diagnosed as having acanthamoeba keratitis based on slit lamp examination, confocal microscopy and culture. After the patients were treated with polyhexamethylene biguanide (PHMB) and chlorhexidine, corneal epithelial defect and erosion occurred. Use of chlorhexidine was stopped, and PHMB was used to treat patients and recovery of the corneal epithelium with improvement in symptoms of acanthamoeba keratitis was found. CONCLUSIONS: Using PHMB and chlorhexidine together in treating acanthamoeba keratitis increases the risk of corneal toxicity. Therefore, these drugs should be avoided in combination.

Sensibilidad antibiótica de bacterias obtenidas de queratitis e infecciones intraoculares en la Fundación Oftalmológica de Santander (FOSCAL), Floridablanca, Colombia / Antibiotic susceptibility patterns of bacteria isolated from keratitis and intraocular infections at Fundación Oftalmológica de Santander (FOSCAL), Floridablanca, Colombia

Introducción. La resistencia bacteriana es crítica para la selección de los antibióticos en el tratamiento de las infecciones, por ello es vital conocer su estado actual en nuestro medio. Objetivo. Determinar la sensibilidad antibiótica bacteriana in vitro obtenida de los cultivos de queratitis e infecciones intraoculares. Materiales y métodos. Se llevó a cabo un estudio retrospectivo en la Fundación Oftalmológica de Santander (FOSCAL), entre junio de 2011 y enero de 2012. Resultados. Se examinaron 92 muestras. Se identificaron 110 bacterias, 27 hongos y 12 amebas de vida libre. Del total de bacterias Gram positivas, 1,1 %, 0 %, 1,1 %, 16,9 %, 29,3 % y 85 % fue resistente a imipenem, moxifloxacina, gatifloxacina, levofloxacina, ciprofloxacina y tobramicina, respectivamente, mientras que la resistencia a estos mismos fármacos se presentó, respectivamente, en 0 %, 8,3 %, 0 %, 0 %, 18,2 % y 27,3 % de las bacterias Gram negativas. Los porcentajes de resistencia de los estafilococos positivos para coagulasa resistentes a la meticilina fueron 0 %, 0 %, 0 %, 7 %, 17 % y 100 %, respectivamente, y los porcentajes de los estafilococos negativos para coagulasa resistentes a la meticilina fueron 3 %, 0 %, 0 %, 24 %, 44 % y 100 %, respectivamente. Los porcentajes de resistencia bacteriana globales (tanto para bacterias Gram positivas como para Gram negativas) a imipenem, moxifloxacina, gatifloxacina, levofloxacina, ciprofloxacina y tobramicina fueron 1 %, 1 %, 1 %, 15,1 %, 28 % y 64,5 %, respectivamente. Conclusiones. Los niveles de resistencia bacteriana para imipenem, moxifloxacina y gatifloxacina fueron menores que para levofloxacina, ciprofloxacina y tobramicina. Los niveles de resistencia para la tobramicina fueron muy altos, lo que pone en duda su utilidad clínica en las infecciones oculares en nuestro medio.

Introduction: Bacterial resistance is critical for the selection of antibiotics in the treatment of infections, so it is vital to know its current status in our geographical area. Objective: To determine in vitro antibiotic susceptibility of bacterial isolates obtained from keratitis and intraocular infections. Materials and methods: A retrospective study of microbiological tests in Fundación Oftalmológica de Santander (FOSCAL) was carried out between June, 2011, and January, 2012. Results: A total of 92 samples were examined and 110 bacteria, 27 fungi and 12 free-living amoebae were identified. Polymicrobial infections constituted 50% of the total; 1.1%, 0%, 1.1%, 16.9%, 29.3% and 85% of Gram-positive bacteria were resistant to imipenem, moxifloxacin, gatifloxacin, levofloxacin, ciprofloxacin and tobramycin, respectively, while 0%, 8.3%, 0%, 0%, 18.2% and 27.3% of Gram-negative bacteria were resistant to imipenem, moxifloxacin, gatifloxacin, levofloxacin, ciprofloxacin and tobramycin, respectively. For methicillin-resistant coagulase-positive staphylococci, resistance percentages to imipenem, moxifloxacin, gatifloxacin, levofloxacin, ciprofloxacin and tobramycin were 0%, 0%, 0%, 7%, 17% and 100%, respectively. For methicillin-resistant coagulase-negative staphylococci, resistance percentages to imipenem, moxifloxacin, gatifloxacin, levofloxacin, ciprofloxacin and tobramycin were 3%, 0%, 0%, 24%, 44% and 100%, respectively. Overall bacterial resistance to imipenem, moxifloxacin, gatifloxacin, levofloxacin, ciprofloxacin and tobramycin, for both Gram-positive and Gram-negative, was 1%, 1%, 1%, 15.1%, 28% and 64.5%, respectively. Conclusions: The levels of bacterial resistance to imipenem, moxifloxacin and gatifloxacin were lower than for levofloxacin, ciprofloxacin and tobramycin. The levels of resistance to tobramycin were very high, which calls into question its usefulness in this region of our country.

Current advances in diagnostic methods of Acanthamoeba keratitis / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>The objective of this article was to review the current advances in diagnostic methods for Acanthamoeba keratitis (AK).</p><p><b>DATA SOURCES</b>Data used in this review were retrieved from PubMed (1970-2013). The terms "Acanthamoeba keratitis" and "diagnosis" were used for the literature search.</p><p><b>STUDY SELECTION</b>Data from published articles regarding AK and diagnosis in clinical trials were identified and reviewed.</p><p><b>RESULTS</b>The diagnostic methods for the eight species implicated in AK were reviewed. Among all diagnostic procedures, corneal scraping and smear examination was an essential diagnostic method. Polymerase chain reaction was the most sensitive and accurate detection method. Culturing of Acanthamoeba was a reliable method for final diagnosis of AK. Confocal microscopy to detect Acanthamoeba was also effective, without any invasive procedure, and was helpful in the early diagnosis of AK.</p><p><b>CONCLUSION</b>Clinically, conjunction of various diagnostic methods to diagnose AK was necessary.</p>

A Case of Serratia Marcescens Keratitis Presenting as Radial Keratoneuritis

PURPOSE: To report a case of Serratia marcescens keratitis presenting as radial keratoneuritis. CASE SUMMARY: A 15-year-old female who wore cosmetic and orthokeratology contact lenses but performed inadequate lens care visited our clinic with severe pain and visual disturbance in her left eye. On slit lamp examination, central corneal epithelial defect and stromal infiltration with radial keratoneuritis were observed. Based on clinical findings and past history, Acanthamoeba keratitis was highly suspected. The patient was treated with topical chlorhexidine 0.02% (Sigma-Aldrich Co., MO, USA) and moxifloxacin 0.5% (Vigamox(R), Alcon, TX, USA) per hour with 200 mg of oral itraconazol (Sporaone(R), LG, Seoul, Korea) once a day. Symptoms and corneal lesions did not improve after three days. After Serratia marsenscens was isolated from her contact lenses and solution, topical chlorhexidine 0.02% was discontinued, and intravenous ceftazidime (Tazime(R), Hanmi, Seoul, Korea) and fortified ceftazidime (50 mg/mL) eye drop was added. The corneal lesion dramatically improved, and after six months of follow-up, best-corrected visual acuity was 20/20 in the affected eye. CONCLUSIONS: Radial keratoneuritis can present not only in Acanthamoeba keratitis, but also in Serratia marsenscens keratitis. Confirmation of the isolated organism is useful when treating radial keratoneuritis.

Genetic Characterization of Clinical Acanthamoeba Isolates from Japan using Nuclear and Mitochondrial Small Subunit Ribosomal RNA

Because of an increased number of Acanthamoeba keratitis (AK) along with associated disease burdens, medical professionals have become more aware of this pathogen in recent years. In this study, by analyzing both the nuclear 18S small subunit ribosomal RNA (18S rRNA) and mitochondrial 16S rRNA gene loci, 27 clinical Acanthamoeba strains that caused AK in Japan were classified into 3 genotypes, T3 (3 strains), T4 (23 strains), and T5 (one strain). Most haplotypes were identical to the reference haplotypes reported from all over the world, and thus no specificity of the haplotype distribution in Japan was found. The T4 sub-genotype analysis using the 16S rRNA gene locus also revealed a clear sub-conformation within the T4 cluster, and lead to the recognition of a new sub-genotype T4i, in addition to the previously reported sub-genotypes T4a-T4h. Furthermore, 9 out of 23 strains in the T4 genotype were identified to a specific haplotype (AF479533), which seems to be a causal haplotype of AK. While heterozygous nuclear haplotypes were observed from 2 strains, the mitochondrial haplotypes were homozygous as T4 genotype in the both strains, and suggested a possibility of nuclear hybridization (mating reproduction) between different strains in Acanthamoeba. The nuclear 18S rRNA gene and mitochondrial 16S rRNA gene loci of Acanthamoeba spp. possess different unique characteristics usable for the genotyping analyses, and those specific features could contribute to the establishment of molecular taxonomy for the species complex of Acanthamoeba.

Les keratites amibiennes

L'objectif de cette revue generale est de decrire les caracteristiques des agents responsables des keratites amibiennes; les principes de leur diagnostic positif ainsi que les modalites de leur traitement.Les keratites amibiennes surviennent dans la majorite des cas chez des porteurs de lentilles de contact. Les manifestations cliniques peuvent etre atypiques. Les prelevements; en particulier corneens; permettent de rechercher les amibes par des examens directs et des cultures ou d'identifier leur acide desoxyribo- nucleique par reaction de polymerisation en chaine; cette derniere technique etant plus sensible. La microscopie confocale; permet par ailleurs; de rechercher in vivo des kystes amibiens. Le traitement medical; peut etre institue en cas de forte suspicion clinique du diagnostic. Les protocoles therapeutiques de premiere intention se basent sur l'association de collyres presentant une activite kysticide; le plus souvent un biguanide et une diamidine; pendant plusieurs semaines. Le pronostic visuel est lie a la severite du tableau initial et au delai de mise en route d'un traitement efficace. Les sequelles visuelles peuvent etre invalidantes d'ou l'interet de porter un diagnostic precoce

Evaluation of Three Different Methods to Establish Animal Models of Acanthamoeba Keratitis

PURPOSE: To produce animal models of Acanthamoeba keratitis and to evaluate the advantages and adaptation range of each of the three methods employed. MATERIALS AND METHODS: Mice and Wistar rats in three groups of 15 rats and 15 mice each were used to establish the models. Right corneas in group A were scratched and challenged with Acanthamoeba. Those in group B were scratched and covered with contact lenses incubated with Acanthamoeba. Those in group C received an intrastromal injection of Acanthamoeba. Five rats and 5 mice in each group were used for histopathological investigations and the other 10 in each group were used for clinical evaluation. The models were evaluated by slit lamp examination, microscopic examination and culture of corneal scrapings, HE staining of corneal sections, and pathological scoring of the infections. RESULTS: Four rats and 6 mice in group A, 7 rats and 8 mice in group B, and 10 rats and 10 mice in group C developed typical Acanthamoeba keratitis. CONCLUSION: Corneal scratching alone has the lowest infection rate, while scratching and then covering with contaminated contact lenses has a moderate rate of infection and most closely mimics what happens in most human infections. Intrastromal injection of Acanthamoeba gives a much higher infection rate and more severe Acanthamoeba keratitis.

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.