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.

Estudo epidemiológico das queratites infeciosas internadas num centro hospitalar terciário - revisão de 5 anos / Epidemiological study of infeccious keratitis in inpatients of a terciary hospital center - revision of 5 years

Resumo Objetivo: A queratite infeciosa é uma doença de incidência relativamente elevada e é responsável por um número importante de internamentos. Neste estudo pretende-se estudar diversas características epidemiológicas e clínicas associadas às queratites infeciosas de alto risco num hospital terciário em Portugal. Métodos: Realizou-se um estudo retrospetivo, onde foram incluídos todos os doentes internados por abcesso da córnea no Centro Hospitalar Universitário do Porto (CHUP), entre Abril de 2013 a Março de 2018. Caracterizou-se a população em relação aos fatores de risco, apresentação clínica, tempo de internamento, resultados de culturas, resistência antibiótica in vitro, tratamento efetuado e resultado funcional. Resultados: O estudo incluiu 105 doentes. Os principais fatores de risco foram antecedentes de cirurgia de córnea, uso de lentes de contacto e história recente de trauma ocular. 74,3% dos doentes tiveram cultura positiva com 87,9% a corresponderem a cultura bacteriana pura, sendo a Pseudomonas aeruginosa e o Streptococcus pneumoniae os agentes etiológicos mais frequentes. 27,9% das culturas positivas eram resistentes a 3 ou mais classes de antibióticos. Todos os doentes iniciaram tratamento com colírios fortificados. 29,5% dos doentes necessitaram de realizar transplante de córnea. Ao final de 6 meses de seguimento, apenas 20,9% apresentavam AV>20/40. Conclusão: Na maioria dos casos, a etiologia foi bacteriana. Observou-se um número considerável de bactérias multirresistentes. Apesar do tratamento ter permitido uma melhoria da visão na maioria dos casos, um número considerável de doentes ficou com sequelas visuais importantes.

Abstract Objective: Infectious keratitis is a pathology with a high incidence and is responsible for a large number of prolonged stay hospital admissions. The purpose was to analyze the epidemiological and clinical data associated with high risk microbial keratitis at a central hospital in Portugal. Methods: A retrospective study of all inpatients presenting with corneal abscess in Centro Hospitalar Universitário do Porto, from April 2013 to March 2018 was performed. Target population was characterized by risk factors, clinical features, length of stay, culture results, in vitro antibiotic resistance, treatment and outcome. Results: This study included 105 patients. The main risk factors were previous corneal surgery, contact lenses wear and recent history of ocular trauma. 74.3% of patients had a positive culture, 87.9% of these corresponding to a pure bacterial culture, with Pseudomonas aeruginosa and Streptococcus pneumoniae being the most common pathogens. 27.9% of positive cultures were resistant to 3 or more classes of antibiotics. All patients began treatment with fortified drops. 29.5% of patients required a corneal transplant. After 6 months of follow-up, only 20.9% presented a VA>20/40. Conclusion: Most cases were caused by bacteria. A considerable number of multi-resistant bacteria was identified. Despite most cases having improved after treatment, a large number of patients had a significant visual acuity sequelae.

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.

Potentially pathogenic Acanthamoeba genotype T4 isolated from dental units and emergency combination showers

BACKGROUND Acanthamoeba is the genus of free-living amoebae that is most frequently isolated in nature. To date, 20 Acanthamoeba genotypes have been described. Genotype T4 is responsible for approximately 90% of encephalitis and keratitis cases. Due to the ubiquitous presence of amoebae, isolation from environmental sources is not uncommon; to determine the clinical importance of an isolation, it is necessary to have evidence of the pathogenic potential of amoebae. OBJECTIVE The aim of this study was to physiologically characterise 8 Acanthamoeba T4 isolates obtained from dental units and emergency combination showers and to determine their pathogenic potential by employing different laboratory techniques. METHODS Eight axenic cultures of Acanthamoeba genotype T4 were used in pathogenic potential assays. Osmotolerance, thermotolerance, determination and characterisation of extracellular proteases and evaluation of cytopathic effects in MDCK cells were performed. FINDINGS All of the isolates were osmotolerant, thermotolerant and had serine proteases from 44-122 kDa. Two isolates had cytopathic effects on the MDCK cell monolayer. MAIN CONCLUSION The presence of Acanthamoeba T4 with pathogenic potential in areas such as those tested in this study reaffirms the need for adequate cleaning and maintenance protocols to reduce the possibility of infection with free-living amoebae.

Presencia de Acanthamoeba spp. en agua para consumo ganadero en la provincia de La Pampa, Argentina / Presence of Acanthamoeba spp. in watering trough in La Pampa province, Argentina

Las amebas de vida libre del género Acanthamoeba incluyen cepas patógenas y no patógenas que actualmente se encuentran clasificadas en 18 genotipos diferentes (T1-T18). Se llevó a cabo un relevamiento para determinar la presencia de cepas de Acanthamoeba en agua de bebida para consumo ganadero en la provincia de La Pampa, Argentina. Las muestras fueron sembradas en agar no nutritivo. La identificación de cepas de Acanthamoeba se realizó mediante polymerase chain reaction con los primers JDP1/JDP2 específicos para género. De las 65 muestras tomadas, 13 fueron positivas a Acanthamoeba spp. Estas fueron caracterizadas a nivel de genotipo mediante la secuenciación del fragmento DF3. Los resultados de la secuenciación revelaron la presencia de los genotipos T4, T5 y T15 dentro de las muestras estudiadas, siendo la más frecuente la T4. Nuestro estudio revela importancia de la presencia de Acanthamoeba en el ambiente ganadero y la necesidad de realizar más estudios, para asociar la presencia de estos organismos y el papel que cumplen en patologías veterinarias. Este es el primer estudio en la provincia de La Pampa que demuestra la presencia de Acanthamoeba y el primero estudiado a nivel de genotipo de Argentina.

Free-living Amoebae of Acanthamoeba genus include non-pathogenic and pathogenic strains that are currently classified in 18 different genotypes, T1-T18. In this study, a survey was carried out to evaluate the presence of Acanthamoeba strains inwatering trough sample in La Pampa province, Argentina. Sample were inoculated onto non-nutrient agar plates and were checked for the presence of Acanthamoeba. Polymerase chain reaction was performed with genus-specific primers JDP1/JDP2, followed by direct sequencing of the polymerase chain reaction product for molecular identification. Sequencing results revealed the presence of T4, T5 and T15 genotypes within the studied samples. Sequencing revealed presence of T4, T5 and T15 in the samples studied genotypes, the most frequent T4. Our study reveals importance of the presence of Acanthamoeba in the livestock environment and the need for further studies to associate the presence of these organisms and the role in veterinary pathology. To the best of our knowledge, this is the first report demonstrating the presence Acanthamoeba in La Pampa province and the first study at the genotype level in Argentina.

Epidemiological profile of infectious keratitis / Perfil epidemiológico da ceratite infecciosa

Abstract Objective: To evaluate the epidemiologic aspects of cornea ulcers in a referred ophthalmology private practice center. Methods: Retrospective study over electronic files of patients treated for corneal ulcers during a period of 7 years by the same physician (RJMF) at Centro de Olhos São Francisco, between june 2007 and june 2014. These patients were evaluated for risk factors such as: trauma causes, co-morbidities and contact lenses use. They were also evaluated by the microorganism found at the smears, treatment and patient's outcome. Patients that didn't return with the smears results or that didn´t have a complete follow-up were excluded from the study. Results: A total of 242 patients were fully treated during this period. 55 patients were excluded and 187 patients were included. 28.88% (54/187) were positive for gram negative bacteria, 27.81% (52/187) for filamentous fungi, 16.04% (29/187) for gram positive bacteria and only 1.07% for acanthamoeba. Twenty-four patients had negative cultures and 21 patients had combined infections (bacteria + fungi or other combination). Corneal collagen Cross-linking was effective on melting arrest in 16 of the 16 patients that we have submitted to this procedure during 2008 and 2009. Among all the patients that had corneal infection and were previously using contact lenses, 81% were infected by pseudomonas or pseudomonas associated with other gram negative bacteria. Filamentous fungi had a strong association with physical trauma. While use of saline solution for contact lenses cleaning had a strong association with gram negative bacteria and biological trauma, a strong association with combined infection. Conclusion: Gram negative bacteria (Pseudomonas) followed by filamentous fungi (Fusarium sp) were the most frequent etiologic agent found in our study. Cross-link was effective on corneal melting arrest. Pseudomonas aeruginosa was the most frequent isolated agent on hydrophilic contact lenses wearers.

Resumo Objetivo: Avaliar os aspectos epidemiológicos de úlceras de córnea em um centro privado de referência oftalmológica. Métodos: Estudo retrospectivo a partir de arquivos eletrônicos de pacientes tratados por úlceras de córnea durante um período de 7 anos pelo mesmo médico (RJMF) no Centro de Olhos São Francisco, entre Junho de 2007 e Junho de 2014. Estes pacientes foram avaliados quanto a fatores de risco, trauma, co-morbidades, uso de lentes de contato, tipo de microrganismo encontrado, tratamento e evolução do doente. Os pacientes que não retornaram com os resultados das culturas ou que não possuíam um seguimento completo foram excluídos do estudo. Resultados: Um total de 242 pacientes foram tratados durante este período. 55 pacientes foram excluídos e 187 foram incluídos no estudo. 28,88% (54/187) foram positivos para as bactérias gram-negativas, 27,81% (52/187) para fungos filamentosos, 16,04% (29/187) para as bactérias gram-positivas e apenas 1,07% para acanthamoeba. Vinte e quatro pacientes tiveram culturas negativas e 21 pacientes tinham infecções combinadas (bactérias fungos + ou outra combinação). O Cross-link foi eficaz em interromper o melting em 16 dos 16 pacientes que se submeteram a este procedimento durante 2008 e 2009. Entre todos os pacientes que tiveram infecção de córnea e usavam previamente lentes de contato, 81% foram infectados por pseudomonas isolada ou pseudomonas associada ou não a outras bactérias gram-negativas. Os fungos filamentosos tiveram uma forte associação com trauma físico, uso de solução salina para a limpeza de lentes de contato teve uma forte associação com bactérias gram- negativas e trauma biológico, uma forte associação com a infecção combinada. Conclusão: Bactérias Gram negativas (em especial, Pseudomonas) seguido por fungos filamentosos (Fusarium sp) foram os agentes etiológicos mais freqüentemente encontrados em nosso estudo. O Cross-linking foi eficaz em interromper o melting de córnea. Pseudomonas aeruginosa foi o agente isolado mais frequente em usuários de lente de contato hidrofílica.

Microbiological and epidemiological study of infectious keratitis in children and adolescents / Estudo microbiológico e epidemiológico da ceratite infecciosa em crianças e adolescentes

ABSTRACT Purpose: To analyze epidemiological and microbiological aspects of microbial keratitis in children and adolescents. Methods: This retrospective cohort study was conducted at the Department of Ophthalmology and Visual Science, Escola Paulista de Medicina, Universidade Federal de São Paulo, between July 15, 1975, and December 31, 2010. We analyzed corneal samples from 859 patients with clinical suspicion of infectious keratitis, comparing epidemiological and microbiological characteristics of bacterial keratitis with those of non-bacterial and non-viral keratitis. We also compared Gram-positive and Gram-negative pathogens in patients with bacterial keratitis. We created a susceptibility profile of the bacterial microorganisms studied. Results: Of the 859 patients, 346 (40.3%) showed positive culture results for non-viral microorganisms. Teenagers (13-18 years) made up the group with the highest number of patients with keratitis (164, 47.4%). The most frequent risk factors for keratitis were trauma (33.5%) and previous ocular surgery (24.9%). Gram-positive bacteria (71.8%) were the most often isolated, with coagulase-negative Staphylococcus (23.8%) the most prevalent microorganism. Logistic regression analysis showed age (p=0.002), topical antimicrobial drug use (p=0.01), and trauma due to non-chemical burns (p=0.005) were risk factors for non-bacterial keratitis. Age (p=0.01) was also a risk factor for Gram-negative bacterial keratitis. Conclusion: Our study showed that in the age range studied, the prevalence of keratitis caused by Gram-negative bacteria or by the non-viral microorganisms evaluated increases with age. Previous use of topical antimicrobial drug and trauma due to non-chemical burns are associated with non-bacterial keratitis. Knowledge of the risk factors and the microorganisms involved may help improve treatment of keratitis in children and adolescents and minimize visual impairment.

RESUMO Objetivos: Descrever o perfil epidemiológico e microbiológico de ceratite microbiana em crianças e adolescentes. Métodos: Estudo retrospectivo tipo coorte, utilizando fichas laboratoriais de pacientes, atendidos no Departamento de Oftalmologia e Ciências Visuais - Escola Paulista de Medicina - Universidade Federal de São Paulo, entre 15 de julho de 1975 a 31 de dezembro de 2010. Foram comparados pacientes com ceratite bacteriana e não bacteriana (não viral). Entre os pacientes com ceratite bacteriana, foram comparados aqueles em que a ceratite foi causada por bactérias Gram positivas e Gram negativas. O perfil de sensibilidade dos microrganismos bacterianos aos antimicrobianos também foi estudado. Resultados: Foram analisadas amostras corneanas de 859 pacientes com suspeita clínica de ceratite infecciosa, na faixa etária estudada. Destes, 346 (40,3%) apresentaram resultados de culturas positivas para microrganismos não virais. Adolescentes (13 a 18 anos) compuseram o grupo com maior número de pacientes com ceratite (164-47,4%). Os principais fatores de risco foram trauma (33,5%) e cirurgias oculares prévias (24,9%). Bactérias Gram positivas foram isoladas com maior frequência (71,8%), sendo prevalente o patógeno Staphylococcus coagulase negativo (23,8%). De acordo com a análise de regressão logística, idade (p=0,002), uso tópico de drogas antimicrobianas (p=0,01) e trauma por queimadura não química (p=0,005) foram fatores predisponentes para ceratite não bacteriana. Idade (p=0,01) também foi fator de risco para ceratite causada por bactérias Gram negativas. Conclusões: Nosso estudo mostrou que quanto maior a idade, na faixa etária estudada, maior a probabilidade da ceratite ser causada por bactérias Gram negativas e/ou por outros microrganismos não virais avaliados. O uso tópico de drogas antimicrobianas prévias e trauma devido à queimadura não química predispõe à ceratite não bacteriana. O conhecimento dos fatores de risco e dos microrganismos envolvidos resultarão em tratamento específico da ceratite em crianças e adolescentes, com menores danos visuais.

Systemic acanthamoebiasis associated with canine distemper in dogs in the semiarid region of Paraíba, Brazil

Infections by free-living amoebae can cause systemic disease in animals and humans. We describe the epidemiological, clinical and pathological aspects of disseminated acanthamoebiasis associated with canine distemper in three dogs of the semiarid region of Paraíba, Northeastern Brazil. Affected dogs developed progressive neurological and respiratory signs that progressed to death within in two to 20 days. Gross lesions were irregular and with yellow-reddish nodules randomly distributed in the lungs, heart, kidneys, spleen, lymph nodes, adrenals, and intestine. One dog had foci of malacia in the parietal cortex and another one in nucleus of brain basis. Histologically, pyogranulomas with areas of necrosis and hemorrhage in all organs affected were observed, associated with myriads of intralesional amoebic trophozoites. All three cases were concomitant canine distemper, that possibly triggered immunosuppression in the dogs. The diagnosis was performed through microscopic findings of infection by free-living amoebae and confirmed Acanthamoeba sp. by immunohistochemistry.

Infecções por amebas de vida livre podem causar doença sistêmica nos animais e no homem. Descrevem-se os aspectos epidemiológicos, clínicos e patológicos de acanthamoebíase disseminada associada com cinomose em três cães na região semiárida da Paraíba, Nordeste do Brasil. Os cães afetados desenvolveram sinais respiratórios e neurológicos progressivos, que evoluíram para a morte em dois a 20 dias. Na necropsia havia áreas nodulares, irregulares e amarelo-avermelhadas distribuídas aleatoriamente em pulmões, coração, rins, fígado, baço, linfonodos, adrenal e intestino. Em um caso havia foco de malácia no córtex parietal e outro em núcleo da base encefálica. Histologicamente, foram observados piogranulomas com áreas de necrose e hemorragia em todos os órgãos afetados, associados a miríades de amebas intralesionais. Nos três casos havia cinomose concomitante, que possivelmente desencadeou imunossupressão nos cães. O diagnóstico foi realizado através dos achados microscópicos de infecção por amebas de vida livre e confirmado Acanthamoeba sp. pela imuno-histoquímica.

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.

Axenic cultivation and pathogenic assays of acanthamoeba strains using physical parameters

The main goal of the present study was to set up an axenic cultivation of Acanthamoeba and assess the pathogenic ability of T4 genotypes from different clinical and environmental strains of Acanthamoeba using two physical assays. Sixteen Acanthamoeba isolates including 10 environmental and 6 clinical strains were cultured axenically. Axenic cultivation was performed using Proteosepepton, yeast extract and glucose medium and TY-I-S33culture. Pathogenic survey was done using osmotolerance and thermotolerance assay. Briefly, differentosmolarity [0.5 M and 1 M] of non-nutrient agar plates were performed. One hundred fiftymicrol of axenic culture were collected and were inoculated in 1% agar medium. For thermotolerance assay 150 micro L of amoebas from axenic culture were divided into fresh culture mediums. Cultures were incubated at 37degreeC and 42degreeC. All plates were monitored for 24 h, 48 h and 72 h. Overall, 16 strains of Acanthamoeba isolates previously genotyped as T4 were cultivated axenically after several months. Thermotolerance and osmotolerance assay revealed that all of clinical strains, soil and animal feces strains were highly pathogenic isolates. Two dust and water strains did not grow at high temperature [42degreeC] and osmolarity [1.5 M] and thus they were classified as weak pathogens. Most of T4 genotypes are highly pathogenic organisms. This is an important finding since Acanthamoeba belonging to T4 type is the predominate genotype in environmental and clinical samples. The presence of highly pathogenic Acanthamoeba may pose a risk within susceptible people.

Caracterización molecular de aislados de Acanthamoeba spp / Molecular characterization of Acanthamoeba spp isolates

Las especies del género Acanthamoeba tiene una amplia distribución, lo cual junto a sus estrecha relación con el humano aumenta la probabilidad de actuar como agente etiológico de enfermedades de gravedad variable según el estado inmunológico del individuo . La presencia de estos microorganismos en pacientes con queratitis, dermatitis y encefalitis, es subestimada como agente causal. Debido a la subjetividad limitada sensibilidad y especificidad en la identificación de estos microorganismos solo por morfología, es importante el empleo de herramientas moleculares. El objetivo de esta investigación se centró en la caracterización molecular de 14 aislados mantenidos en medio Pagé modificado en el Laboratorio de Amibiasis de la Escuela de Bioanálisis de la UCV, procedentes de muestras biológicas, mediante el empleo de PCR-RFLP (HinfI, HhaI, HaeIII), previamente identificados morfológicamente como el género Acanthamoeba, según los parámetros propuestos por Pussard y Pons, 1997. Todos los aislados se excluyeron del Grupo I por tener quistes de menos de 18 µm. De los 14 aislados seleccionados , 50% presentó características morfológicas compatibles con el Grupo II mientras que el otro 50% fue compatible con el grupo III. Desde el punto de vista molecular, 86% de los aislados clasificados como Grupo II amplificaron productos de PCR de 900 pb, y el 100% de los aislados del Grupo III de 700 pb, así como el 14% restante del Grupo II. Se Observó una tendenciaque sugiere, que a mayor tiempo (años) de mantenimiento en medios de cultivo, se espera un producto de PCR de 700 pb y variaciones fenotípicas en estos microorgnismos. Los aislados del Grupo III no se pudieron caracterizar molecularmente por PCR-RFLP, debido a que el patrón de digestión con las enzimas de restricción no coincidió con patrones publicados por Kong y Chung, 1996. Respecto al Grupo II, 71% (5/6) de los aislados se identificó a nivel de especie por medio del RFLP. Se identificó a A13 y A14 como A...

Members of the genus Acanthamoeba, have a wide distribution of pathogenic species, it`s close relationship with humans increases the probality of life threatening or critical health conditions depending on the immne status of the patient. Usually these protozoans are not considered as the main cause of keratitis, dermatitis, and encephalitis in patients. As a consequence of the low sensibility, specificity, and high subjectivity that involves the morphologic identification of this microorganisms, has been considered the importance of the use of molecular analyses for its identification. By PCR-RFLP analyses, where characterized 14 isolates of Acanthamoeba spp. maintained in Page (modified) culture media in El Laboratorio de Amibiasis de la Escuela de Bioanálisis de la UCV, from clinical samples. These isolates where previously identified as Acanthamoeba using morphological methods as established for Pussard and Pons, 1997. 100% of the isolates where excluded from morphological Group I for showing sizes under 18 µm. However 50% of the isolates where classified as members of morphological Group II and the other 50% as members of morphological Group III. Furthermore, 86% of the isolates classified as Group II exhited PCR products of 900 pb as expected, while 100% of the isolated classified as Group III and 14% of the remaining isolates from Group II showed PCR products of 700 pb. In addition, it`s been observed a tendency suggesting that the longer (time in years) the microorganism are maintained in culture conditions, it is expected to obtain a PCR product of 700 pb and major phenotypic alteracions. Moreover, species of isolates from Group III were not identified, because they showed patterns of digestion with restriction enzymes HinfI, HhaI y HaeIII that differed from the reference ones published in 1996 by Kong y Chung. On the other hand, 71% of the isolates belonging to Group II showed patterns of identificatiob compatibles with the reference Kong & Chung...

Acanthamoeba spp. en piscinas cubiertas de la ciudad de Bahía Blanca, provincia de Buenos Aires, Argentina / Acanthamoeba spp. in indoor swimming pools in Bahía Blanca city, Buenos Aires province, Argentina

Acanthamoeba ha sido aislada de numerosos hábitats incluyendo piletas de natación. Estudiar su distribución es importante ya que algunas especies causan enfermedad en el hombre. El objetivo del presente trabajo fue la búsqueda, aislamiento y caracterización de protozoos del género Acanthamoeba en piscinas cubiertas de la ciudad de Bahía Blanca, Argentina, en las cuatro estaciones del año durante 2007-2008. Se estudiaron 7 piscinas y en cada una se tomaron cuatro muestras: fondo, superficie, raspado de pared y para análisis bacteriológico. Las muestras se analizaron por observación directa y por cultivo en agar no nutritivo a 37 °C y a 42 °C. La identificación genérica se realizó de acuerdo con las características morfológicas de quistes y trofozoítos y para identificar Naegleria se realizó la prueba de transformación ameboflagelar. En 5 de las 7 piscinas se aislaron amebas de vida libre al menos en una época del año. La prueba de transformación ameboflagelar resultó negativa, descartando al género Naegleria. Todos los aislamientos correspondieron al género Acanthamoeba Grupos II y III de Pussard y Pons. Si bien la eliminación de Acanthamoeba en las aguas de natatorios resulta muy difícil por tratarse de un protozoario ubicuo y sumamente resistente a los desinfectantes comúnmente utilizados, se recomienda una limpieza profunda de las piscinas que minimice los riesgos de infección.

Acanthamoeba spp. has been isolated from many habitats, including swimming pools. Investigations on its distribution are relevant because many of its species cause human diseases. The aim of the present work was to investigate, isolate and characterize protozoan of the genus Acanthamoeba from indoor swimming pools in Bahía Blanca, Argentina, in the four seasons, during the 2007-2008 period. Seven pools were studied and samples were collected from the bottom, surface and wall by scraping them. Besides, samples for bacteriological analysis were taken from each pool. The samples were analyzed by direct observation and by culturing on non nutritive agar at 37 °C and at 42 °C. The generic identification was performed according to the morphologic characteristics of cysts and trofozoites, while the amoebo-flagellate transformation test was carried out to identify Naegleria. Free-living amoebas were isolated from five of the seven swimming pools examinated, in at least one season. Naegleria genus was not found as the amoebo-flagellate transformation tests were negative in all samples. All the isolations corresponded to the genus Acanthamoeba belonging to Pussard & Pons morphological Groups II and III. Although elimination of Acanthamoeba from the water is difficult because it is a ubiquitous protozoan. which is highly resistant to the commonly used disinfectants, a thorough cleaning of the pools to minimize the risks of infection is recommended.

Acanthamoeba spp: ecoepidemiología, biología, ultraestructura, patogénesis y diagnóstico en el hombre / Acanthamoeba: ecoepidemiology, biology, ultrastructure, pathogenesis and diagnosis in humans

En el presente trabajo presentamos nuestros estudios referidos al protozoo Acanthamoeba, desde lo morfológico, diagnóstico de laboratorio, patología en el ser humano, aspectos epidemiológicos y resistencia al cloro. Se aisló Acanthamoeba en el 28.6% de los tanques domiciliarios de agua, en el 71% de piscinas cubiertas, en la totalidad de las muestras del arroyo Napostá, que cruza la ciudad de Bahía Blanca, y no fue detectada en el agua de la red. Con referencia a la acción quisticida del cloro, se logró inhibir el crecimiento recién a las tres horas de exposición en 60 ppm de cloro. Realizamos el primer aislamiento de un paciente con queralitis, en Bahía Blanca. Hasta el momento no aislamos Acanthamoeba de otras muestras biológicas. Con referencia a los aspectos morfológicos, documentamos la ultraestructura de las cepas regionales de Acanthamoeba y realizamos la tipificación molecular de la cepa aislada del paciente con queralitis, una afección que, si bien es de baja prevalencia y su diagnóstico etiológico es de fácil ejecución y bajo costo, no justifica no realizar el estudio oportuna y rápidamente. La sensibilidad de la observación directa por microscopia óptica fue del 11.43%, comparada con los cultivos. Finalmente efectuamos un listado de recomendaciones para usuarios de lentes de contacto.

Prevalence of potentially pathogenic free-living amoebae from Acanthamoeba and Naegleria genera in non-hospital, public, internal environments from the city of Santos, Brazil

Acanthamoeba and Naegleria species are free-living amoebae (FLA) found in a large variety of natural habitats. The prevalence of such amoebae was determined from dust samples taken from public non-hospital internal environments with good standards of cleanliness from two campuses of the same University in the city of Santos (SP), Brazil, and where young and apparently healthy people circulate. The frequency of free-living amoebae in both campuseswas 39 percent and 17 percent respectively, with predominance of the genus Acanthamoeba. On the campus with a much larger number of circulating individuals, the observed frequency of free-living amoebae was 2.29 times larger (P< 0.00005). Two trophozoite forms of Naegleria fowleri, are the only species of this genus known to cause primary amoebian meningoencephalitis, a rare and non-opportunistic infection. We assume that the high frequency of these organisms in different internal locations represents some kind of public health risk.

Acanthameba meningitis with successful outcome.

We report a rare cause of sub acute meningitis in a 15-yr-old immunocompetent female child with successful outcome. The etiological agent was Acanthameba. The child was sucessfully treat with combination of Ketoconazole. Rifampicin, cotrimoxa zole and for a period of 9 month.

Endosymbionts of Acanthamoeba Isolated from Domestic Tap Water in Korea

In a previous study, we reported our discovery of Acanthamoeba contamination in domestic tap water; in that study, we determined that some Acanthamoeba strains harbor endosymbiotic bacteria, via our molecular characterization by mitochondrial DNA restriction fragment length polymorphism (Mt DNA RFLP). Five (29.4%) among 17 Acanthamoeba isolates contained endosymbionts in their cytoplasm, as demonstrated via orcein staining. In order to estimate their pathogenicity, we conducted a genetic characterization of the endosymbionts in Acanthamoeba isolated from domestic tap water via 16S rDNA sequencing. The endosymbionts of Acanthamoeba sp. KA/WP3 and KA/WP4 evidenced the highest level of similarity, at 97% of the recently published 16S rDNA sequence of the bacterium, Candidatus Amoebophilus asiaticus. The endosymbionts of Acanthamoeba sp. KA/WP8 and KA/WP12 shared a 97% sequence similarity with each other, and were also highly similar to Candidatus Odyssella thessalonicensis, a member of the alpha-proteobacteria. The endosymbiont of Acanthamoeba sp. KA/WP9 exhibits a high degree of similarity (85-95%) with genus Methylophilus, which is not yet known to harbor any endosymbionts. This is the first report, to the best of our knowledge, to show that Methylophilus spp. can live in the cytoplasm of Acanthamoeba.

Free-living Acanthamoeba and Naegleria spp. amebae in water sources of León, Nicaragua

free-living amebae (FLA) are known to occur worldwide in water-related biotopes, but only limited information is available on these organisms in developing countries and so far no information on their presence is available from Nicaragua. The aims of this study were to evaluate the prevalence of potentially pathogenic Acanthamoeba spp. and Naegleria spp. in different water sources to which the population of León municipality is exposed. Since pathogenic amebae are thermotolerant, we were especially interested in the occurrence of FLA in geothermal areas. Water samples were collected from León area in Nicaragua: 88 samples were from rivers and springs, 111 from wells, 74 from water taps and 21 from water tanks in urban and suburban León and from three nearby geothermal areas of San Jacinto, Posoltega and Tipitapa. Amebae were identified using morphological and physiological criteria, immunohistochemical staining procedures and molecular methods. Indirect immunofluorescent test was performed on cysts and trophozoites fixed on microscopical slides and incubated for 30 min at room temperature in separate experiments with the following antibodies: rabbit-anti N. fowleri/N. lovanensis (Nf-Pab), mouse monoclonal antibody anti N. fowleri (Nf-5D12u), rabbit antibodies against Acanthamoeba spp. And fluorescent in situ hybridization (FISH) was performed using 18S rRNA-targeted fluorescent oligonucleotide probes. Probes: GSP for the detection of Acanthamoeba and NAEG1088 for the detection of Naegleria. Free-living amebae were recovered from approximately 43 % of the samples. Acanthamoeba spp was found in 21 % of samples from León municipality and in 2 % of samples from geothermal areas. Amoeboflagellates were found in 10 % of samples from León and in 19 % in geothermal areas. Fifty three percent of tested wells in the geothermal area contained thermotolerant amoeboflagellates. Naegleria spp. was identified in 24 out of 39 (61.5 %) of isolated amoeboflagellates. Twelve of them were assigned to N. lovanenesis while none of the isolates could be identified as N. fowleri. However, the common presence of thermotolerant Naegleria in water, specially N. lovanensis, which is an indicator species for N. fowleri, suggests that also this pathogenic amoeba may pose a risk to public health in the area. On the other side, direct pathogenicity, free-living amebae are receiving increasing attention as reservoirs and potential vehicles for the transmission of bacteria in the environment. Thus the information provided in this study may serve as base-line for future studies on the role of free-living amebae e.g. in waterborne-disease outbreaks in the region. Among such potentially important enteropathgens are Vibrio cholerae, E. coli 0157, and Helicobacter pylori. Rev. Biol. Trop. 56 (2): 439-446. Epub 2008 June 30.

Las amebas de vida libre (AVL) son un grupo de organismos de distribución mundial. Entre las AVL hay parásitos facultativos en humanos y otros animales, los cuales pertenecen a los géneros Acanthamoeba, Naegleria y Balamuthia que causan infecciones severas en el sistema nervioso central. Sin embargo no se tiene ninguna información de Nicaragua. El objetivo de este estudio fue evaluar la presencia de amebas de vida libre, en diferentes fuentes de agua de la parte urbana y rural del Departamento de León, y áreas geotérmicas de Nicaragua. Estas amebas fueron identificadas usando criterios morfológicos, fisiológicos, histoquímicos y moleculares. En los resultados se encontró amebas de vida libre en el 43% del total de las muestras. En la municipalidad de León, se encontraron 21% de Acanthamopeba sp. y en las áreas geotérmicas un 2%. Las amebaflagelados tipo Naegleria fueron 10% y 19% respectivamente. Del grupo amebaflagelados fueron 24, de las cuales 12 se dentificaron como N. lovanensis. En estas muestras no se aisló N. fowleri (ameba patógena), sin embargo, la presencia de N. lovanensis es in indicador de la presencia de N. fowleri, la cual puede ser un riesgo a la salud pública. Además, estas amebas pueden servir como vectores de bacterias enteropatógenas.

Acanthamoeba encephalitis.

Central nervous system infection with free-living amoebae is rare. We present a fatal case of Acanthamoeba encephalitis in a 63-year-old female from India where acanthamoebae were demonstrated and cultured from CSF. In spite of treatment with amphotericin B, fluconazole and rifampicin the patient did not survive. Amoebic infection should be suspected in a patient of encephalitis of unexplained aetiology as timely diagnosis can lead to a favourable outcome.

Acanthamoebae presenting as primary meningoencephalitis in AIDS.

A rare case of Acanthamoebae meningoencephalitis is diagnosed in cerebrospinal fluid (CSF) of a 24 years old male suffering from acquired immunodeficiency syndrome (AIDS) patient on the basis of bright field microscopy and culture growth on non-nutrient agar with Escherichia coli. This case illustrates that Acanthamoebae should be considered in the differential diagnosis of meningoencephalitis in AIDS in addition to tuberculosis and cryptococcus infection in tropical areas.