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)

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.

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.

Citologia de impressão no diagnóstico de infecção corneana por Acanthamoeba: relato de caso / Diagnosis of Acanthamoeba corneal infection by impression cytology: case report

Relatamos três casos de infecção corneana por Acanthamoeba sp em que foi possível detectar cistos do microorganismo com a técnica de citologia de impressão. Três pacientes encaminhados ao Laboratório de Doenças Externas Oculares em 2004 com alterações superficiais da córnea foram submetidos ao exame de citologia de impressão para investigação da presença de cistos de Acanthamoeba sp. Duas amostras foram obtidas da córnea de cada paciente e coradas com PAS, hematoxilina e Papanicolaou. Investigação microbiológica de rotina e cultura também foram realizadas após raspado da córnea. O cultivo das amostras e a citologia de impressão foram positivas para Acanthamoeba sp em todos os pacientes, ao passo que os raspados corados com Giemsa foram positivos em dois casos. A citologia de impressão revelou cistos de Acanthamoeba sp entre feixe de células epiteliais corneanas e como células isoladas. Foram observados cistos no epitélio de um dos pacientes com a citologia de impressão após três meses de tratamento, enquanto o raspado foi negativo. No exame anatomopatológico observaram-se cistos no epitélio e estroma de uma córnea receptora de um dos pacientes após transplante. Neste estudo, a citologia de impressão detectou com sucesso cistos de Acanthamoeba sp em pacientes com acometimento epitelial. Por tratar-se de método não invasivo, a técnica pode ser usada para facilitar o diagnóstico mais precoce da infecção por Acanthamoeba, sendo útil também no acompanhamento do tratamento da doença.

To describe three cases of corneal infection due to Acanthamoeba sp in which was possible to detect Acanthamoeba sp cysts by the corneal impression cytology technique. Three patients referred to the External Eye Disease Laboratory in 2004 with superficial corneal alterations were submitted to corneal specimen collection by impression cytology filter paper to investigate the presence of Acanthamoeba sp cysts. Two impression cytology samples were obtained from each patient and were stained by PAS, hematoxylin and Papanicolaou. Routine microbiological investigation and culture were also performed using corneal scraping. Positive culture and impression cytology for Acanthamoeba sp was observed in all patients while smears with Giemsa stain were positive in two. Impression cytology Acanthamoeba sp cysts were observed among sheets of corneal epithelial cells and as isolated cells. Cysts were also found in the superficial epithelium in one of these patients after treatment while corneal scraping did not reveal any cyst. Histopathology revealed cysts in the epithelium and stroma in a transplanted cornea in one of these patients. The first description of impression cytology as a diagnostic method for Acanthamoeba keratitis occurred recently. In this study corneal impression cytology detected Acanthamoeba sp cysts successfully in these patients with only superficial involvement. Impression cytology as a non invasive technique can be used to facilitate early recognition of Acanthamoeba infection playing a useful role in the follow-up of the disease.

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.

Ceratite por Acanthamoeba: relato de casos no Rio Grande do Sul / Acanthamoeba keratitis: report of cases in Rio Grande do Sul

Relato de três primeiros casos comprovados de ceratite por Acanthamoeba sp no Rio Grande do Sul. Todos os pacientes eram usuários de lentes rígidas gás-permeáveis, usando água corrente para higiene das mesmas. Um paciente teve resoluçäo do quadro clínico após 4 meses de tratamento intensivo com neomicina, biguanida, metronidazol e dexametasona tópicos. Os outros pacientes necessitaram de ceratoplastia penetrante, um após 2 meses e o outro após 14 meses de tratamento clínico intensivo (neomicina, biguanida, propamidine e dexametasona tópicos) sem sucesso. Näo houve recidiva da infestaçäo por Acanthamoeba em nenhum dos casos durante o período de seguimento (16,5 e 22 meses). O diagnóstico etiológico foi realizado pela identificaçäo de cistos e trofozoítos de Acanthamoeba no exame direto do raspado da úlcera de córnea em dois pacientes e no exame anátomo-patológico dos botöes corneanos dos que se submeteram ao transplante de córnea

Amebas patógenas de vida libre / Pathogenic free living amebas

Las pequeñas amebas de vida libre están ampliamente distribuidas en todo el mundo en continuo contacto con el hombre y animales; sus formas quísticas son capaces de sobrevivir en el suelo, aire y agua. Las infecciones causadas por las mismas han tomado en los últimos treinta años notable importancia médica ya que muchos casos fatales no fueron diagnosticados clínicamente ni por el laboratorio, debido al desconocimiento de la potencial capacidad patogénica de estas amebas. Hoy se sabe que la meningoencefalitis amebiana primaria (MAP) causada por Naegleria fowleri y la encefalitits amebiana granulomatosa (EAG) originada por especies de Acanthamoeba spp se han incrementado en el mundo tanto en sujetos sanos como en inmunocomprometidos, incluyendo muchos individuos con SIDA. El grupo más reciente de infecciones causadas por especies del género Acanthamoeba es la queratitis amebiana relacionada principalmente con la falta de cuidado en el mantenimiento de las lentes de contacto. La terapia de la queratitis es problemática debido a la presencia de quistes en los tejidos, y aunque se han informado algunas curas de pacientes, la terapéutica médica aún constituye un capítulo no resuelto

Changing patterns of infectious keratitis: overview of clinical and histopathologic features of keratitis due to acanthamoeba or atypical mycobacteria, and of infectious crystalline keratopathy.

Acanthamoeba keratitis, infectious crystalline keratopathy and atypical mycobacterial keratitis have recently emerged as important types of infectious keratitis. These corneal infections have been associated with contact lens wear and with corneal surgical procedures such as radial keratotomy and penetrating keratoplasty, and the clinical setting of each of these infections is important in alerting the clinician to the possible diagnosis. There have been improvements in rapid diagnostic techniques for such infections in the last several years. Treatment has also improved, but remains a difficult problem, especially for Acanthamoeba. An overview of recent developments in the clinical and histopathologic methods for diagnosis and treatment options of these three corneal infections is provided.