Cultivo de Acanthamoeba spp. en agua apirógena / Acanthamoeba spp. culture in pyrogen-free water / Cultura de Acanthamoeba spp. em água apirogênica

Resumen El objetivo de este estudio fue evaluar el cultivo de cepas de Acanthamoeba spp. en agua destilada estéril apirógena de uso farmacéutico. Se utilizaron dos cepas de genotipo T4 [un aislamiento de encefalitis granulomatosa amebiana (EGA) y una ambiental] y cepas correspondientes a los genotipos T5 y T15. Los quistes de cada una de las cepas se sembraron en placas de Petri con agar no nutritivo con diferentes soluciones (agua destilada estéril apirógena uso médico para preparaciones inyectables, agua destilada filtrada, medio Page) y combinados con suspensiones de Escherichia coli. Las placas se incubaron a 37 °C y se monitorearon diariamente durante 15 días para la detección de trofozoítos. El crecimiento se evaluó mediante examen microscópico directo. Cada cultivo contó con cuatro repeticiones para cada una de las cepas (n=96). En conclusión, se hallaron diferencias estadísticamente significativas en el crecimiento de las cepas por día. Las cepas T5 y T4 (encefalitis amebiana granulomatosa) desarrollaron mayor cantidad de trofozoítos en el primer día respecto de la cepa T15 (H=16,42; p=0,001). En el agua apirógena con E. coli se obtuvo un crecimiento igual a la solución de Page con E. coli (H=24,64; p=0,0001). No se hallaron diferencias estadísticamente significativas en la cantidad de trofozoítos obtenidos en agua apirógena con E. coli y solución de Page con E. coli en la cepa T4 (EGA) (U=4; p<0,05) pero sí en la cepa T4 ambiental (U=0; p<0,05).

Abstract The objective of this study was to evaluate the culture of strains of Acanthamoeba spp. in sterile apyrogenic distilled water for pharmaceutical use. Two T4 genotype strains [one isolate of granulomatous amebic encephalitis (GAE) and one environmental], a T5 and T15 genotype strains were used. The cysts of each of the strains were seeded in Petri dishes with non-nutritive agar with different solutions (pyrogenic sterile distilled water for medical use for injectable preparations, filtered distilled water, Page medium) and combined with Escherichia coli suspensions. Plates were incubated at 37 °C and monitored daily for 15 days for the detection of trophozoites. Growth was assessed by direct microscopic examination. Each medium culture counted four replicates for each of the strains (n=96). Concluding, statistically significant differences were found in the growth of the strains per day. Strains T5 and T4 (granulomatous amebic encephalitis) developed a greater number of trophozoites on the first day compared to strain T15 (H=16.42; p=0.001). In apyrogenic water with E. coli, a growth equal to Page's solution with E. coli was obtained (H=24.64; p=0.0001). No statistically significant differences were found in the amount of trophozoites obtained in apyrogenic water with E. coli and Page's solution with E. coli in strain T4 (GAE) (U=4; p<0.05), but significant differences were found in the environmental T4 strain (U=0; p<0.05).

Resumo O objetivo deste trabalho foi avaliar o cultivo de cepas Acanthamoeba spp. em água destilada apirogênica estéril para uso farmacêutico. Foram utilizadas duas cepas de genótipo T4 [um isolamento de encefalite granulomatosa amebiana (EGA) e uma ambiental], e cepas correspondentes aos genótipos T5 e T15. Os cistos de cada uma das cepas foram semeados em placas de Petri com ágar não nutritivo com diferentes soluções (água destilada estéril apirogênica para uso médico para preparações injetáveis, água destilada filtrada, meio Page) e combinados com suspensões de Escherichia coli. As placas foram incubadas a 37 °C e monitoradas diariamente durante 15 dias para detecção de trofozoítos. O crescimento foi avaliado através de exame microscópico direto. Cada cultura contou com quatro réplicas para cada uma das cepas (n=96). Em conclusão, diferenças estatisticamente significativas foram encontradas no crescimento das cepas por dia. As cepas T5 e T4 (encefalite amebiana granulomatosa) desenvolveram maior número de trofozoítos no primeiro dia em relação à cepa T15 (H=16,42; p=0,001). Em água apirogênica com E. coli, foi obtido crescimento igual ao da solução de Page com E. coli (H=24,64, p=0,0001). Não foram encontradas diferenças estatisticamente significativas na quantidade de trofozoítos obtidos em água apirogênica com E. coli e solução de Page com E. coli na cepa T4 (EGA) (U=4; p%<0,05), mas sim na cepa T4 ambiental (U=0; p<0,05).

Encefalitis por ameba de vida libre: reporte de caso / Free-living amoeba encephalitis: case report

La encefalitis amebiana granulomatosa es una enfermedad considerada atípica debido a la baja incidencia desde que fue descubierta. Sin embargo, conforme han avanzando los métodos diagnósticos, en retrospectiva, se ha descrito la presencia de esta enfermedad desde principios del siglo XX, identificando a Acanthamoeba spp y Balamuthia mandrillaris como agentes más comunes.Es una enfermedad de difícil diagnóstico debido al amplio abanico clínico que puede presentar, siendo el sitio anatómico dentro del sistema nervioso central quien determinará estas características. El conocimiento y sospecha de la misma permitirá un abordaje terapéutico oportuno aunque, en la mayoría de los casos, con un desenlace fatal para el paciente.A continuación se presenta el caso clínico de un paciente masculino de 26 años de edad originario de la ciudad de Durango, México, sin antecedentes de importancia, de ocupación ladrillero, quien sufre golpe con ladrillo en región naso-orbitaria, ingresando al hospital al tercer día del evento con diagnóstico de celulitis periorbitaria izquierda y nasal, cefalea de leve intensidad, rinorrea abundante, hemiparesia fasciocorporal izquierda, parestesia de extremidad superior izquierda, diplopía y vértigo postural. Los estudios imagenológicos revelaron lesiones heterogéneas en el lóbulo occipital, hemisferio cerebeloso izquierdo y tallo cerebral. Se realizó escisión de lesión tumoral cerebelar, con estudio anatomopatológico, reportando encefalitis granulomatosa por presencia de ameba de vida libre. El paciente evolucionó hacia deterioro rostro-caudal declarándose su fallecimiento a los 16 días de su estancia intrahospitalaria.En el Estado de Durango, México, no se cuenta con casos reportados por infección de ameba de vida libre

Granulomatous amoebic encephalitis is a disease considered atypical due to the low incidence since it was discovered. However, as diagnostic methods have progressed, in retrospect, the presence of this disease has been described since the beginning of the 20th century, identifying Acanthamoeba spp. and Balamuthia mandrillaris as the most common agents.A disease that is difficult to diagnose due to the wide clinical range that it can present, being the anatomical site within the central nervous system that will determine these characteristics. The knowledge and suspicion of it will allow a timely therapeutic approach although, in most cases, with a fatal outcome for the patient.The clinical case is presented below of a 26 year old male patient from the city of Durang, México, with no significant history, of brick occupation, who suffers a blow with a brick in the naso-orbital region, follows, entering our hospital on the third day of the event with a diagnosis of left periorbital and nasal cellulitis, mild headache, profuse rhinorrhea, left fasciocorporeal hemiparesis, left upper limb parestesia, diplopia, and postural vertigo.Imaging studies were performed observing heterogeneous lesions in the occipital lobe, left cerebellar hemisphere and brain stem; excision of the cerebellar tumor lesion being carried out, with pathological study, reporting granulomatous encephalitis due to the presence of free-living amoeba. The patient progresses to face-caudal deterioration, declaring his death after 16 days of hospital stay.Within the state of Durango, Mexico, there are no reported cases of free-living amoeba infection

Encefalitis amebiana granulomatosa por Amebas de Vida Libre en un paciente pediátrico / Granulomatous amoebic encephalitis due to free-living amebas in a pediatric patient

Introducción: Las amebas de vida libre (AVL) Naegleria fowleri, Acanthamoeba spp., Balamuthia mandrillaris, son protozoos ampliamente distribuidos en la naturaleza. Son microorganismos oportunistas, que afectan preferentemente al sistema nervioso central causando cuadros de meningoencefalitis amebiana primaria o de encefalitis granulomatosa amebiana (EGA), ambas patologías de alta mortalidad. Descripsión del Caso: Paciente femenina de 10 años, que ingresa a nuestro hospital por presentar hemiparesia braquiocrural izquierda, vómitos y cefalea de 72 horas de evolución. Se realiza tomografía computada y Resonancia Magnética en la que se identifica una lesión expansiva, sólido-quística ubicada en la región fronto derecha. Se realiza exéresis completa de la misma y se implementa tratamiento farmacológico lográndose la restitución ad-integrum de la función motora y la resolución de la EGA a 30 meses de seguimiento. Discusión: La encefalitis granulomatosa amebiana es una patología causada por amebas de vida libre. Su presentacíon clínica puede ser indistinguible de otras causas de leptomeningitis o encefalitis, como las de origen bacteriano, viral o por micobacterias lo que impide conocer su real incidencia. Su tratamiento antiparasitario es muy complejo y su evolución es habitualmente fatal. Conclusión: Reportamos un caso de Encefalitis Amebiana Granulomatosa en una paciente pediátrica inmunocompetente, con excelente evolución.

Introduction: Free-living amoebas (FLA) Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris, are protist widely distributed in nature. Are opportunistic microorganisms, preferentially affect the central nervous system causing primary amoebic meningoencephalitis or amoebic granulomatous encephalitis (AGE), both with high mortality. Case report: A 10 year-old female patient was admitted with a three-day history of left hemiparesis accompanied with headaches and vomiting. Computed tomography and magnetic resonance were performed, in which an expansive solid cystic mass was observed in the right fronto-parietal region. Complete resection was performed and pharmacological treatment was started, achieving complete restitution of motor function and resolution of AGE after 30 months of follow-up. Discussion: AGE is a desease caused by free-living amoebas. Its clinical presentation is similar to other leptomeningitis or encephalitis of different etiology such as bacterial, viral or by mycobacterial, which cannot know its real incidence. Its pharmacological treatment is complex and its evolution is usually fatal. Conclusion: We report a case of Amoebic Granulomatous Encephalitis in an immunocompetent pediatric patient with good outcome.

Occurrence of Acanthamoeba in hospitals: a literature review / Ocorrência de Acanthamoeba em hospitais: uma revisão da literatura / Ocurrencia de Acanthamoeba en hospitales: una revisión de la literature

Background and Objectives: Acanthamoeba are among the most prevalent environmental protozoans and have a cosmopolitan distribution. The main concern in public health is that they can also be isolated from contact lenses, storage cases and cleaning solutions, ventilation and air conditioning systems, dental treatment units, dialysis units, emergency showers and eyewash stations. As these genera include a diversity of pathogenic microorganisms that leads to infectious diseases inside the hospital environment, it is of the utmost importance to carry out the surveillance, considering mainly the immunocompromised patients, who are more susceptible to these diseases. The level of human health risk and its associations in the hospital environment are unknown, and part of this problem is potentially the lack of correlation between protozoan exposure and the onset of symptoms that can occur in each patient at different periods in time. Thus, this review offers a current overview of the presence of Acanthamoeba spp. in hospital environments with the aim to detect its presence in these environments. Methods: This is a review of the literature on Lilacs, Scielo, Medline and Bdenf databases to gather and synthesize publications and search for effective ways of controlling the presence of Acanthamoeba through disinfection and monitoring measures. Results: We found that Acanthamoeba is present in different hospital environments, namely in water, dust, biofilm, cooling waters and air conditioners. Conclusion: Studies on the ecology and distribution of non-enteric pathogens in the hospital environment are necessary for understanding their potential threat to human health.(AU)

Justificativa e objetivos: Acanthamoeba estão entre os protozoários ambientais mais prevalentes e possuem distribuição cosmopolita. A principal preocupação para fins de saúde pública é que eles também podem ser isolados de lentes de contato, caixas de armazenamento e soluções de limpeza, sistemas de ventilação e ar condicionado, unidades de tratamento odontológico, unidades de diálise, chuveiros de emergência e lava-olhos. O gênero Acanthamoeba compreende várias espécies que podem determinar doenças infecciosas dentro do ambiente hospitalar, e é de suma importância realizar a vigilância, considerando principalmente os pacientes imunocomprometidos, que são mais suscetíveis a essas doenças. O nível de risco para a saúde humana e suas associações no ambiente hospitalar é desconhecido, e parte desse problema ocorre pela falta de correlação entre a exposição ao protozoário e o início dos sintomas, que cada paciente pode desenvolver em diferentes períodos. Assim, esta revisão apresenta uma visão geral atual de Acanthamoeba spp. em ambientes hospitalares, com o objetivo de verificar sua presença nesses ambientes. Métodos: Trata-se de uma revisão da literatura nas bases Lilacs, Scielo, Medline e Bdenf para reunir e sintetizar publicações e buscar formas eficazes de controle de sua presença por meio de medidas de desinfecção e monitoramento. Resultados: Verificamos que Acanthamoeba está presente em diferentes ambientes hospitalares, estando presente em água, poeira, biofilme, águas de refrigeração e ar condicionados. Conclusão: Estudos sobre a ecologia e distribuição de patógenos não-entéricos no ambiente hospitalar são necessários para entender sua ameaça potencial à saúde humana.(AU)

Justificativa y Objetivos: Acanthamoeba están entre los protozoos ambientales más prevalentes y poseen una distribución cosmopolita. La principal preocupación por la salud pública es que también pueden aislarse de lentes de contacto, cajas de almacenamiento y soluciones de limpieza, sistemas de ventilación y aire acondicionado, unidades de tratamiento odontológico, unidades de diálisis, duchas de emergencia y lava- los ojos. Estos géneros incluyen una diversidad de microorganismos patógenos que conducen a enfermedades infecciosas dentro del ambiente hospitalario, y es de suma importancia realizar la vigilancia, considerando principalmente a los pacientes inmunocomprometidos, que son más susceptibles a esas enfermedades. El nivel de riesgo para la salud humana y sus asociaciones en el ambiente hospitalario es desconocido, y parte de este problema es potencialmente la falta de correlación entre la exposición al protozoario y el inicio de los síntomas, que cada paciente puede desarrollar en diferentes períodos en el tiempo. Así, esta revisión presenta una visión general actual de la presencia de Acanthamoeba spp. en ambientes hospitalarios, con el objetivo de verificar su presencia en esos ambientes. Métodos: Se trata de una revisión de la literatura en las bases Lilacs, Scielo, Medline y Bdenf para reunir y sintetizar publicaciones y buscar formas eficaces de control de su presencia por medio de medidas de desinfección y monitoreo. Resultados: En el presente trabajo se analizan los resultados de la evaluación de la calidad de los alimentos y de los productos alimenticios en el medio ambiente hospitalario, que se encuentran en el agua, polvo, biofilm, aguas de refrigeración y aire acondicionados. Conclusión: Estudios sobre la ecología y distribución de patógenos no entéricos en el ambiente hospitalario son necesarios para entender su amenaza potencial salud humana.(AU)

Anti-encystment and amoebicidal activity of Lonicera japonica Thunb. and its major constituent chlorogenic acid in vitro

OBJECTIVE@#To examine the acanthamoebicidal effects of ethyl acetate, aqueous and butanol fractions of dried flower buds of Lonicera japonica (L. japonica) Thunb. (Flos Lonicerae) in vitro.@*METHODS@#Acanthamoeba triangularis isolates were obtained from environmental water samples and identified by PCR. They were exposed to ethyl acetate, water and butanol fractions of L. japonica Thunb. at concentrations ranging from 0.5 mg/mL to 1.5 mg/mL. The extracts were evaluated for growth inhibition at 24, 48 and 72 h, respectively. Chlorogenic acid at a concentration of 1 mg/mL was examined for inhibition of encystment.@*RESULTS@#Ethyl acetate fraction at a concentration of 1.5 mg/mL evoked a significant reduction of trophozoite viability by 48.9% after 24 h, 49.2% after 48 h and 33.7% after 72 h chlorogenic acid, the major active constituent of L. japonica Thunb. at the concentration of 1 mg/mL reduced the cysts/trophozoite ratio by 100% after 24 h, 84.0% after 48 h and 72.3% after 72 h. This phenolic compound at concentration of 1 mg/mL concurrent with 0.6% hydrogen peroxide inhibited hydrogen peroxide-induced encystment by 92.8% at 72 h.@*CONCLUSIONS@#Results obtained from this study show that ethyl acetate fraction at 1.5 mg/mL is the most potent fraction of L. japonica Thunb. and its major constituent chlorogenic acid showed the remarkable inhibition of encystment at a concentration of 1 mg/mL.

Anti-encystment and amoebicidal activity of Lonicera japonica Thunb. and its major constituent chlorogenic acid in vitro

Objective To examine the acanthamoebicidal effects of ethyl acetate, aqueous and butanol fractions of dried flower buds of Lonicera japonica (L. japonica) Thunb. (Flos Lonicerae) in vitro. Methods Acanthamoeba triangularis isolates were obtained from environmental water samples and identified by PCR. They were exposed to ethyl acetate, water and butanol fractions of L. japonica Thunb. at concentrations ranging from 0.5 mg/mL to 1.5 mg/mL. The extracts were evaluated for growth inhibition at 24, 48 and 72 h, respectively. Chlorogenic acid at a concentration of 1 mg/mL was examined for inhibition of encystment. Results Ethyl acetate fraction at a concentration of 1.5 mg/mL evoked a significant reduction of trophozoite viability by 48.9% after 24 h, 49.2% after 48 h and 33.7% after 72 h chlorogenic acid, the major active constituent of L. japonica Thunb. at the concentration of 1 mg/mL reduced the cysts/trophozoite ratio by 100% after 24 h, 84.0% after 48 h and 72.3% after 72 h. This phenolic compound at concentration of 1 mg/mL concurrent with 0.6% hydrogen peroxide inhibited hydrogen peroxide-induced encystment by 92.8% at 72 h. Conclusions Results obtained from this study show that ethyl acetate fraction at 1.5 mg/mL is the most potent fraction of L. japonica Thunb. and its major constituent chlorogenic acid showed the remarkable inhibition of encystment at a concentration of 1 mg/mL.

Light and electron microscopic studies of the structural damages of acanthamoeba cysts by pouzolzia indica methanolic extract fraction 4 solution.

Objective : This study was to investigate the morphological changes and ultrastructural damages of the Acanthamoeba cysts after treated with the minimal cysticydal concentration (MCC) of Pouzolzia indica methanolic extract fraction 4 solution by light and electron microscopies. Materials and Methods : Acanthamoeba cyst which prepared followed the method of Roongruangchai K15,16 were adjusted to the final concentration of 104 cysts/ml and treated with 1:4 soltion of fraction 4 Pouzolzia indica methanolic extract, the Thai medicinal plant. which was the minimal cysticidal concentration (MCC) 16. The cysts then, were centrifuged and the pellets were prepared for light, scanning and transmission electron microscopies. Result : Light micrographs showed the cytoplasmic clumping and some empty cyst wall. Transmission electron microscopy showed the ruptures of both ectocyst and the endocyst wall with some opercula damages. The cytoplasm aggregated and clumped. Scanning electron microscopy show steps of damage which started from shrinkage and collapsed of the cyst walls, then the cysts began to bulge and swell resulted in decreasing in the wrinkles of the cyst walls. The cytoplasmic contents drain out from the cyst wall and finally the cyst walls were ripped and torn into small pieces. Conclusion : Pouzolzia indica methanolic extract fraction 4 at the MCC of 1:4 caused structural damages to the Acanthamoeba cyst including shrinking of the cyst walls with the ruptured of the ectocyst, endocyst and operculum, edema and break out into pieces. The active ingredients of this Thai medicinal plant should be further studied as this can eventually be one of the regimen for the treatment of the Acanthamoeba keratitis or as a solution for cleaning the contact lens or contact lens case solution.

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...

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.

Several fractions of pouzolzia indica methanolic extract were lethal to the acanthamoeba cyst: in vitro study.

Objective: The purpose of this study was to compare the least concentrations of 5 fractions of Pouzolzia indica methanolic extract which can be lethal to the cyst form of the Acanthamoeba spp. Methods: Acanthamoeba spp. was isolated from a keratitis patient and was cultured using nonnutrient agar plates enriched with heat-killed E.coli for seven days at room temperature for the production of mature cysts. The cysts were harvested, washed in normal saline solution and adjusted to the final concentration of 104 cysts/ml. They were mixed with several dilutions of each fraction of Pouzolzia indica methanolic extract. After incubation for 1 hour, they were washed and centrifuged to remove the herbal extract supernatants. The cysts were recultured in the same medium for 7 days to confirm that they were all dead. Results: Pouzolzia indica methanolic extract fraction No. 1 which was eluted by water could not kill the cyst, while the crude extract (Fraction C) could at the concentration of 1:2. The fraction No.2 which was eluted by water: methanol had the minimal cysticidal concentration of 1:4, fraction No.3 which was eluted by methanol had the minimal cysticidal concentration of 1:8 and the fraction No.4 which was eluted by ethyl acetone had the minimal cysticidal concentration of 1:4. Concluison: Our results demonstrated that the Pouzolzia indica methanolic extract of several fractions can be cysticidal to an Acanthamoeba cyst, and this can modified to be a better disinfecting solution for contact lens cases.