Central nervous system infections caused by pathogenic free-living amoebae: An Indian perspective

@#Pathogenic free-living amoebae (FLA), namely Acanthamoeba sp., Naegleria fowleri and Balamuthia mandrillaris are distributed worldwide. These neurotropic amoebae can cause fatal central nervous system (CNS) infections in humans. This review deals with the demographic characteristics, symptoms, diagnosis, and treatment outcomes of patients with CNS infections caused by FLA documented in India. There have been 42, 25, and 4 case reports of Acanthamoeba granulomatous amoebic encephalitis (GAE), N. fowleri primary amoebic meningoencephalitis (PAM), and B. mandrillaris meningoencephalitis (BAE), respectively. Overall, 17% of Acanthamoeba GAE patients and one of the four BAE patients had some form of immunosuppression, and more than half of the N. fowleri PAM cases had history of exposure to freshwater. Acanthamoeba GAE, PAM, and BAE were most commonly seen in males. Fever, headache, vomiting, seizures, and altered sensorium appear to be common symptoms in these patients. Some patients showed multiple lesions with edema, exudates or hydrocephalus in their brain CT/MRI. The cerebrospinal fluid (CSF) of these patients showed elevated protein and WBC levels. Direct microscopy of CSF was positive for amoebic trophozoites in 69% of Acanthamoeba GAE and 96% of PAM patients. One-fourth of the Acanthamoeba GAE and all the BAE patients were diagnosed only by histopathology following autopsy/biopsy samples. Twenty-one Acanthamoeba GAE survivors were treated with cotrimoxazole, rifampicin, and ketoconazole/amphotericin B, and all eleven PAM survivors were treated with amphotericin B alongside other drugs. A thorough search for these organisms in CNS samples is necessary to develop optimum treatment strategies.

Presencia de Acanthamoeba spp. en piscinas recreativas al aire libre de la ciudad de Córdoba, Argentina / Presence of Acanthamoeba spp. in outdoor recreational swimming pools in Córdoba city, Argentina / Presença de Acanthamoeba spp. em piscinas recreativas ao ar livre da cidade de Córdoba, Argentina

Resumen El objetivo del estudio fue determinar la presencia de Acanthamoeba spp. en piscinas de uso recreacional, al aire libre, de la ciudad de Córdoba, Argentina. Se recolectaron 30 muestras de agua correspondientes a un total de 10 piscinas. Estas se sembraron en agar no nutritivo en presencia de Escherichia coli en solución de Page. Luego de 72 horas de incubación a 37 °C, la identificación genérica se realizó mediante criterios morfológicos. La prueba de transformación amebo-flagelar se realizó para diferenciar amebas de vida libre que desarrollan trofozoítos flagelados, como Naegleria. Se midieron parámetros fisicoquímicos en cada una de las piscinas. Se identificó Acanthamoeba spp. en el 20% del total de las muestras. Las piscinas en las que se aisló este parásito presentaron niveles no detectables de cloro residual, pH moderadamente alcalino y temperatura templada. Este estudio demuestra la presencia de Acanthamoeba spp. en piscinas recreativas de Córdoba, lo que puede representar un potencial riesgo para la salud pública.

Abstract The aim of the study was to determine the presence of Acanthamoeba spp. in outdoor recreational pools, in Córdoba city, Argentina. Thirty water samples corresponding to a total of 10 pools were collected. These samples were sown on a non-nutritive agar in the presence of Escherichia coli in Pages's solution. After 72 hours of incubation at 37 °C, the generic identification was done based on morphological criteria. The amoebo-flagellate transformation test was performed to differentiate from genera that develop flagellated trophozoites, such as Naegleria. Physicochemical parameters were measured in each of the pools. Acanthamoeba spp. was identified in 20% of the samples. The pools, where this parasite was isolated, presented undetectable levels of residual chlorine, moderately alkaline pH and warm temperature. This study demonstrates the presence of Acanthamoeba spp. in recreational pools in Córdoba, which may represent a potential risk to public health.

Resumo O objetivo do estudo foi determinar a presença de Acanthamoeba spp. em piscinas para uso recreativo, ao ar livre, na cidade de Córdoba, Argentina. Foram coletadas 30 amostras de água correspondentes a um total de 10 piscinas. Elas foram semeadas em ágar não nutritivo na presença de Escherichia coli em solução de Page. Após 72 horas de incubação a 37 °C, a identificação genérica foi realizada utilizando critérios morfológicos. O teste de transformação amebo-flagelar foi realizado para diferenciar amebas de vida livre que desenvolvem trofozoítos flagelados, como Naegleria. Parâmetros físico-químicos foram medidos em cada uma das piscinas. Acanthamoeba spp. foi identificada em 20% do total das amostras. As piscinas onde este parasita foi isolado apresentaram níveis indetectáveis de cloro residual, pH moderadamente alcalino e temperatura temperada. Esse estudo demonstra a presença de Acanthamoeba spp. em piscinas recreativas de Córdoba, o que pode representar um risco potencial para a saúde pública.

Various brain-eating amoebae: the protozoa, the pathogenesis, and the disease / 医学前沿

Among various genera of free-living amoebae prevalent in nature, some members are identified as causative agents of human encephalitis, in which Naegleria fowleri followed by Acanthamoeba spp. and Balamuthia mandrillaris have been successively discovered. As the three dominant genera responsible for infections, Acanthamoeba and Balamuthia work as opportunistic pathogens of granulomatous amoebic encephalitis in immunocompetent and immunocompromised individuals, whereas Naegleria induces primary amoebic meningoencephalitis mostly in healthy children and young adults as a more violent and deadly disease. Due to the lack of typical symptoms and laboratory findings, all these amoebic encephalitic diseases are difficult to diagnose. Considering that subsequent therapies are also affected, all these brain infections cause significant mortality worldwide, with more than 90% of the cases being fatal. Along with global warming and population explosion, expanding areas of human and amoebae activity in some regions lead to increased contact, resulting in more serious infections and drawing increased public attention. In this review, we summarize the present information of these pathogenic free-living amoebae, including their phylogeny, classification, biology, and ecology. The mechanisms of pathogenesis, immunology, pathophysiology, clinical manifestations, epidemiology, diagnosis, and therapies are also discussed.

Free living amoebae in water sources of critical units in a tertiary care hospital in India.

Background: Isolation of free‑living amoebae (FLA) is reported sparsely from water taps, ventilators, air conditioners, haemodialysis units and dental irrigation systems of hospitals worldwide. Their prevalence in hospital environment especially in wards having immunocompromised patients may pose a risk to this group of susceptible population as they may cause disease themselves or may carry pathogens inside them. No study from India has performed such surveillance. Objective: To evaluate extent of FLA contamination in water sources of bone marrow transplant (BMT) intensive care unit (ICU), transplant ICU, haemodialysis unit and high dependency unit in a tertiary care hospital in India. Materials and Methods: A total of hundred samples including fifty each of tap water samples and swabs from mouth of taps used for drinking, bathing and hand washing purposes in these units were collected according to standard procedure. Samples were inoculated onto non‑nutrient agar plates at room temperature followed by morphological confirmation. Molecular identification including polymerase chain reaction (PCR) and sequencing was performed in culture positive samples. Results: Four tap water samples and ten swab samples showed growth of trophozoites and cyst formation. Morphologically, four amoebae resembled Acanthamoeba spp. which was further confirmed by PCR and sequencing showed them to be of T3 and T4 genotypes. Conclusion: The presence of these FLA in hospital water sources emphasises the urgent need of implementing effective preventive measures. Further studies are required to estimate the true prevalence of FLA in Indian hospitals by taking larger number of samples.

Potentially patoogenic free-living amoebae in sme flood-affected areas during 2011 chiang mai flood / Amebas potencialmente patogenicas de vida livre em algumas areas afetadas durante a inundacao de 2011 em Chiang Mai

SUMMARY The survey was carried out to investigate the presence of potentially pathogenic free-living amoebae (FLA) during flood in Chiang Mai, Thailand in 2011. From different crisis flood areas, seven water samples were collected and tested for the presence of amoebae using culture and molecular methods. By monoxenic culture, FLA were detected from all samples at 37 °C incubation. The FLA growing at 37 °C were morphologically identified as Acanthamoeba spp., Naegleria spp. and some unidentified amoebae. Only three samples (42.8%), defined as thermotolerant FLA, continued to grow at 42 °C. By molecular methods, two non-thermotolerant FlA were shown to have 99% identity to Acanthamoeba sp. and 98% identity to Hartmannella vermiformis while the two thermotolerant FLA were identified as Echinamoeba exundans (100% identity) and Hartmannella sp. (99% identity). This first report of the occurrence of FLA in water during the flood disaster will provide information to the public to be aware of potentially pathogenic FLA. .

RESUMO A pesquisa foi feita para investigar a presença de amebas de vida livre (FLA) durante a inundação em Chiang Mai, Tailândia, ano de 2011. A partir de diferentes áreas de inundação sete amostras de água foram coletadas e testadas para a presença de amebas usando métodos moleculares e de cultura. Através da cultura monoxênica, FLA foi detectada em todas as amostras após incubação a 37 °C. As FLA crescendo a 37 °C foram identificadas morfologicamente como Acanthamoeba spp, Naegleria spp e algumas amebas não determinadas. Somente três amostras (42,8%) definidas como FLA termotolerantes continuaram a crescer a 42 °C. Por métodos moleculares duas FLA termotolerantes tiveram 99% de identidade com a Acanthamoeba sp e 98% de identidade com Hartmannella vermiformis enquanto as duas FLA termotolerantes foram identificadas como Echinamoeba exundans (100% de identidade) e Hartmannella sp (99% de identidade). Este primeiro relato da ocorrência de FLA em águas durante inundações informa ao público que ele deve estar atento de FLA potencialmente patogênica. .

Reporte de caso y revisión de la literatura de caso de paciente con meningoencefalitis por amebas de vida libre / Case report and literature review of a patient with meningoencephalitis from free-living amoebae

Paciente de 14 años, proveniente de Apartadó (Antioquia), remitido al Hospital Pablo Tobón Uribe de Medellín por un cuadro febril agudo asociado a deterioro rápido del estado de conciencia, decantándose posteriormente como causa de su deterioro una ameba de vida libre (Naegleria fowleri) . Las amebas de vida libre son una causa etiológica poco común de meningitis, tienen un curso fulminante y requieren de un alto índice de sospecha para hacer un tratamiento oportuno.

A 14-year-old patient from Apartado (Antioquia, Colombia) was referred to Hospital Pablo Tobon Uribe in Medellin for acute febrile illness associated with headache and rapid loss of consciousness. The etiologic agent causing this deterioration was found to be a free-living amoeba ( Naegleria fowleri ). Free-living amoebae are a rare etiology for meningoencephalitis, have a fulminant course and require a high index of suspicion for early diagnosis.

Amibas de vida libre Acanthamoeba, Balamuthia, Naegleria, Sappinia y Paravahlkampfia: agentes de infección en humano y transmisoras de microorganismos / Free-living amoebae Acanthamoeba, Balamuthia, Naegleria, and Paravahlkampfia Sappinia: agents of human infection and transmission of microorganisms

Se han reportado como agentes causales de enfermedad en humanos: Naegleria fowleri, agente causal de meningoencefalitis amibiana primaria en individuos inmunocompetentes: varias especies de género Acanthamoeba que pueden producir encefalitis granulomatosa amibiana en individuos inmunosuprimidos, úlceras corneales, lesiones en piel y otros órganos en personas inmunocompetentes; Ballamuthia mandrillares agente causal de encefalitis y lesiones en piel en personas inmunocompetentes, Sappinia pedata responsable de un caso de encefalitis en un individuo inmunocompetente y Paravahlkamphfia sp en un caso de queratitis y Paravahlkamphfia francinae en un caso de meningoencefalitis aminiana primera, en paciente en paciente inmunocompetente. No se conoce totalmente los factores de los que depende que las enfermedades producidas por amibas de vida libre se presenten, influyen la presencia de algunas enfermedades, contacto con aguas en las cuales se encuentran estas amibas, y la presencia de enzimas capaces de ejercer efecto citopático. Además el uso de lentes de contacto, algunas costumbres y necesidades humanos rompen el equilibrio ecológico, aumentando así la probabilidad de contacto del hombre con estas amibas. Se han evidenciado en ellas microorganismo en un proceso de endosimbiosis lo que por lo que amibas de vida libre intervienen en la transmisión de ciertos microorganismos patógenos y las amibas se transforman en un incubador potencial y transmisorasde agentes infecciosos. Debe investigarse sobre aspectos biológicos y la relación con el humano.

Have been reported as causative agents of disease in humans: Naegleria fowleri, the causative agent of primary amoebic meningoencephalitis in immunocompetent individuals, several species of Acanthamoeba that con cause granulomatous amoebic encephalitis in immunosuppressed individuals, corneal ulcers, skin lesions and other organs in people immunocompetent; Ballamuthia madrillares causative agent of encephalitis and skin lesions in immunocompetent persons, Sappinia pedata, responsible for a case of encephalitis in an immunocompetent individual Paravahlkamphfia sp in a case of keratitis and Paravanhlkamphfia francinae in a case of primary amoebic meningoencephalitis in an immunocompetent patient. Not fully understood the factors that it depends on diseases caused by free-living amoebae are present, influencing the presence of some diseases, contact with water where are these amoebae, and the presence of enzymes capable of the cytopathic effect. Furthermore, the use of contact lenses, some customs and human needs break the ecological balance, thereby increasing the likelihood of human contact with these amoebae. Have been evidenced in these microorganisms in the process of endosymbiosis that so the free-living amoebae in the transmission of certain pathogenic microorganisms and amoebas and become a potential incubator and transmission of infectious agents. Biological aspects should be investigated and the relationship with the human.

Detection of Legionella pneumophila in water and biofilm samples by culture and molecular methods from man-made systems in São Paulo - Brazil

Legionella pneumophila is a pathogenic bacteria associated to aquatic habitat of natural and artificial environments. Clinical cases of legionellosis have been reported in Brazil but there is a lack of information about the incidence and concentration of this bacterium in environmental sources. Thus, the present study was designed to evaluate the occurrence of legionellae in São Paulo city, Brazil, using different methods of detection and identification. Sixty-seven water and biofilm samples from natural reservoirs and man-made systems were collected and analyzed for the presence of Legionella spp by culturing onto a selective medium, coculture in axenic free-living amoebae and direct fluorescent antibody (DFA) assay. Results showed that freshwater of reservoirs did not contain legionellae, Legionella pneumophila was isolated from man-made systems, with predominance of Legionella pneumophila serogroup 1 strains. Although there was no statistical difference among the proposed detection methods, the plate culture method yielded a higher number of L. pneumophila positive samples, followed by amoebic coculture procedure and direct fluorescent antibody assay. Results of PCR and sequencing reactions revealed that application of macrophage infectivity potentiator gene as a molecular marker was an important tool for the identification of environmental isolates of L. pneumophila. The agreement among the three detection methods-when all methods yielded similar results- and the prevalence of a single Legionella species in the sampled man-made systems could suggest that the occurrence of this bacterium had been influenced by the higher concentration of metallic ions dissociated in water of those systems than in natural reservoirs. Thus, the results of this study revealed that the water of man-made systems in Sao Paulo may serve as a reservoir for L. pneumophila and other microorganism, including free-living protozoans.

Legionella pneumophila é uma bactéria patogênica associada à habitats aquáticos de ambientes naturais e artificiais. Casos clínicos de legionelose têm sido descritos no Brasil, mas a incidência e concentração desta bactéria em fontes ambientais ainda são pouco conhecidas. Assim, o presente estudo foi desenvolvido para avaliar a ocorrência de bactérias do gênero Legionella na cidade de São Paulo, Brasil, utilizando diferentes métodos de isolamento e identificação. Sessenta e sete amostras de água e biofilme de reservatórios naturais e sistemas artificiais de climatização de ambientes interiores foram coletadas e analisadas quanto à presença de Legionella spp por métodos de cultivo em meio-de-cultura seletivo, cocultivo com amebas de vida livre axênicas e ensaios de imunofluorescência direta (IFD). Os resultados demonstraram que bactérias do gênero Legionella não foram detectadas em reservatórios naturais de água, Legionella pneumophila foi isolada de sistemas artificiais de climatização, com predominância de cepas de Legionella pneumophila sorogrupo 1. Apesar de não ter havido diferença estatística significante entre os métodos de detecção propostos, o método de cultivo em placa produziu o melhor resultado quanto ao número de amostras positivas para L. pneumophila, seguido do procedimento de cocultivo com amebas e ensaio de imunofluorescência direta. Os resultados das reações de PCR e sequenciamento revelaram que a aplicação do gene potencializador de infecção em macrófagos como marcador molecular foi um importante implemento na identificação de isolados ambientais de L. pneumophila. A concordância existente entre os três métodos de detecção - quando todos os métodos produziram resultados similares - e a prevalência de uma espécie de Legionella nos sistemas artificiais amostrados poderia sugerir que a ocorrência desta bactéria tenha sido influenciada pela maior concentração de íons metálicos dissociados na água daqueles sistemas do que...

Isolamento e identificação de amebas de vida livre potencialmente patogênicas em amostras de ambientes de hospital público da cidade de Porto Alegre, RS / Isolation and identification of potentially pathogenic free-living amoebae in samples from environments in a public hospital in the city of Porto Alegre, Rio Grande do Sul

Um estudo sobre a presença de amebas de vida livre em um hospital público foi desenvolvido na Cidade de Porto Alegre, RS. Poeira e biofilmes de 15 ambientes hospitalares, incluindo CTI, UTI pediátrica, cozinha, emergência, centro cirúrgico ambulatorial e centro cirúrgico, reservatórios de água, torneira e 6 bebedouros coletivos foram coletados mensalmente, de julho de 2004 a março de 2005, usando-se suabes estéreis, preparados para a pesquisa. As AVL foram isoladas em cultivo, utilizando-se meio de ágar não nutriente adicionado de Escherichia coli, mortas pelo calor. A identificação dos protozoários foi feita pela observação morfológica de cistos e trofozoítos, segundo critérios morfológicos de Page (1988). Das 135 amostras coletadas dos 15 ambientes estudados, 47 (35 por cento) foram positivas para AVL. Destas, 34 por cento apresentaram características morfológicas próprias do gênero Acanthamoeba.

A study on the presence of free-living amoebae in a public hospital was developed in the city of Porto Alegre, State of Rio Grande do Sul. Dust and biofilms were collected using sterile swabs that had been prepared for this study, from 15 hospital environments, including the intensive care center, pediatric intensive care unit, kitchen, emergency room, outpatient surgical center, clinical surgical center, water storage tanks, taps and six drinking fountains for general use, every month from July 2004 to March 2005. The FLAs were isolated by culturing, using non-nutrient agar medium with the addition of heat-killed Escherichia coli. The protozoa were identified by morphological observation of cysts and trophozoites, in accordance with Page's morphological criteria (1988). Among the 135 samples collected from the 15 environments, 47 (35 percent) were positive for FLAs. Of these, thirty-four percent presented morphological characteristics particular to the genus Acanthamoeba.

Amibas de vida libre potencialmente patógenas en aguas del parque “Las Cocuizas”, Maracay, Venezuela / Potentially pathogenic free-living amoebae present in “Las Cocuizas” park waters, Maracay, Venezuela

Se determinó la presencia de amibas de vida libre, y la calidad microbiológica de las aguas para la recreación del parque “Las Cocuizas” en Maracay, Venezuela, en 30 muestras de agua captadas en diferentes lugares dentro del parque. Se investigó la presencia de amibas de vida libre y otros parásitos mediante exámenes directo y por coloración. También se determinaron los recuentos de aerobios mesófilos, coliformes totales y fecales. Se observó la presencia de formas parasitarias en 15 del total de las 30 muestras; en 8 se visualizaron formas compatibles con Naegleria fowleri y en las 7 restantes otros parásitos. Del total de las muestras 37,0 % reveló un número más probable de coliformes fecales superior a 200/100 mL-1 y 33,3 % mostró contaminación fecal activa con Escherichia coli. Se evidenció que existe riesgo de contaminación por diversos microorganismos patógenos transmitidos por el agua y esto puede ser peligroso para los usuarios del parque.

The objective of this study was to determine the presence of free-living amoebae and the microbiological quality of the recreational waters of Cocuizas Park in Maracay, Venezuela. Thirty samples taken from different park sites were analyzed. The presence of free-living amoebae and other parasitic was detected by fresh mounts and specific parasitic stain slides. Mesophilic aerobic bacteria and total faecal Coliforms were also determined. Parasitic forms were observed in 15 of the 30 samples that were analyzed. Forms compatible with Naegleria fowleri were seen in eight of the samples; other parasites were observed in the remaining seven samples. 37% of the samples revealed faecal coliforms above 200/100 mL-1 and 33,3% showed an active Escherichia coli faecal contamination. These results show risk of human infection by several water-borne pathogenic microorganisms, which could be dangerous for users of the park.

Cultivation of Pathogenic Free-living Amoebae / 中国寄生虫学与寄生虫病杂志

The isolation and culture of pathogenic free-living amoebae are useful in the diagnosis and research. This review focuses on the methods of isolation and cultivation of pathogenic free-living amoebae, including sample treatment, culture conditions, passage culture, pathogen detection, and maintenance.