Laboratory Diagnosis of Primary Amoebic Meningoencephalitis.

Primary amebic meningoencephalitis (PAM) is a fulminant fatal human disease caused by the free-living amoeba Naegleria fowleri. Infection occurs after inhalation of water containing the amoeba, typically after swimming in bodies of warm freshwater. N. fowleri migrates to the brain where it incites meningoencephalitis and cerebral edema leading to death of the patient 7 to 10 days postinfection. Although the disease is rare, it is almost always fatal and believed to be underreported. The incidence of PAM in countries other than the United States is unclear and possibly on track to being an emerging disease. Poor prognosis is caused by rapid progression, suboptimal treatment, and underdiagnosis. As diagnosis is often performed postmortem and testing is only performed by a few laboratories, more accessible testing is necessary. This article reviews the current methods used in the screening and confirmation of PAM and makes recommendations for improved diagnostic practices and awareness.

Aislamiento de Acanthamoeba spp. en salas cerradas de un hospital de la provincia de Buenos Aires, Argentina / Isolation of Acanthamoeba spp. in closed rooms of a hospital in Buenos Aires province, Argentina

Resumen Las amebas de vida libre (AVL) son protozoos ubicuos con cuatro géneros patógenos para el ser humano: Acanthamoeba, Naegleria, Balamuthia, y Sappinia. Acanthamoeba puede actuar como reservorio de microorganismos (endosimbiontes), por lo cual, en medio hospitalario, implicaría un riesgo para la transmisión de bacterias, virus y hongos intranosocomiales. Se investigó la presencia de AVL, con énfasis en Acanthamoeba spp., en un hospital pediátrico de la provincia de Buenos Aires, Argentina. Se colectaron 22 muestras de lavamanos e incubadoras en salas de Neonatología y Terapia Intensiva, las que fueron cultivadas a 37 y 42 °C. Los aislados fueron identificados molecularmente. El 63,64% de las muestras presentaron Acanthamoeba spp. Esta investigación representa el primer estudio realizado en la Argentina sobre la detección de Acanthamoeba spp. en salas cerradas de un hospital. Su presencia es una señal de alarma y resulta un blanco útil para investigar posibles reservorios de microorganismos patógenos en ambientes hospitalarios.

Abstract Free-living amoebae (FLA) are ubiquitous protozoa with four pathogenic genera for humans: Acanthamoeba, Naegleria, Balamuthia, and Sappinia. Acanthamoeba can act as a reservoir of microorganisms (endosymbionts), for which reason, in a hospital environment, it would imply a risk for transmission of nosocomial bacteria, viruses and fungi. The presence of AVL, with emphasis on Acanthamoeba spp., was investigated in a pediatric hospital. Twenty-two samples were collected from sinks and incubators in Neonatology and Intensive Care rooms, which were cultured at 37 and 42 °C. The isolates found were molecularly identified. A total of 63.64% of the samples presented Acanthamoeba spp. This research represents the first study in Argentina on the detection of Acanthamoeba spp. in closed rooms of a hospital. Its presence is an alarm signal, and it is a useful target to investigate possible reservoirs of pathogenic microorganisms in hospital environments.

Resumo As amebas de vida livre (AVL) são protozoários ubíquos com quatro gêneros patogênicos para o ser humano: Acanthamoeba, Naegleria, Balamuthia, e Sappinia. Acanthamoeba pode atuar como um reservatório de microrganismos (endossimbiontes), e portanto, em um ambiente hospitalar, representaria um risco de transmissão de bactérias, vírus e fungos intra-nosocomiais. A presença de AVL, com em Acanthamoeba spp. em um hospital pediátrico da província de Buenos Aires, Argentina, foi investigada. Vinte e duas amostras foram coletadas em lavatórios e incubadoras em Salas de Neonatologia e Cuidados Intensivos, cultivadas a 37 e 42 °C. Os isolados foram identificadas molecularmente. Foram encontradas Acanthamoeba spp. em 63,64% das amostras. Esta investigação representa o primeiro estudo realizado na Argentina sobre a detecção de Acanthamoeba spp. em salas fechadas de um hospital. A sua presença é um sinal de alarme e um alvo para investigar possíveis reservatórios de microrganismos patogênicos em ambientes hospitalares.

Distribution and Current State of Molecular Genetic Characterization in Pathogenic Free-Living Amoebae.

Free-living amoebae (FLA) are protozoa widely distributed in the environment, found in a great diversity of terrestrial biomes. Some genera of FLA are linked to human infections. The genus Acanthamoeba is currently classified into 23 genotypes (T1-T23), and of these some (T1, T2, T4, T5, T10, T12, and T18) are known to be capable of causing granulomatous amoebic encephalitis (GAE) mainly in immunocompromised patients while other genotypes (T2, T3, T4, T5, T6, T10, T11, T12, and T15) cause Acanthamoeba keratitis mainly in otherwise healthy patients. Meanwhile, Naegleria fowleri is the causative agent of an acute infection called primary amoebic meningoencephalitis (PAM), while Balamuthia mandrillaris, like some Acanthamoeba genotypes, causes GAE, differing from the latter in the description of numerous cases in patients immunocompetent. Finally, other FLA related to the pathologies mentioned above have been reported; Sappinia sp. is responsible for one case of amoebic encephalitis; Vermamoeba vermiformis has been found in cases of ocular damage, and its extraordinary capacity as endocytobiont for microorganisms of public health importance such as Legionella pneumophila, Bacillus anthracis, and Pseudomonas aeruginosa, among others. This review addressed issues related to epidemiology, updating their geographic distribution and cases reported in recent years for pathogenic FLA.

Cutaneous balamuthiasis: A clinicopathological study.

INTRODUCTION: Balamuthia mandrillaris, a free-living amoeba, causes an uncommon infection that is characterized by cutaneous and neurological involvement, which carries a poor prognosis. METHODS: This is a retrospective observational study including patients with clinical suspicion of cutaneous balamuthiasis, their skin biopsies, and/or a positive direct immunofluorescence test. The data were collected from the Dermatology and Pathology service of the Hospital Cayetano Heredia and the Instituto Tropical Alexander von Humboldt, Lima, Peru, from January 1985 to June 2007. We identified 60 biopsies from 35 patients, from which clinical data were available in 30. RESULTS: Twenty-two (73%) patients had centrofacial lesions, mostly located on the nose. The classical lesion was an asymptomatic, erythematous, or violaceous infiltrated plaque. Twenty-two (73%) patients had neurologic involvement. Fifty (83%) biopsies showed granulomatous dermatitis and 75% showed ill-defined tuberculoid granulomas without caseous necrosis. Multinucleated giant cells were observed in 52 (87%) biopsies. Trophozoite forms were identified in the biopsies of 25 (71%) patients. Direct immunofluorescence was positive in 25 (71%) patients. CONCLUSION: B. mandrillaris is a pathogen that is capable of inducing a characteristic skin lesion with a reaction pattern of ill-defined tuberculoid granulomas and many giant cells.

Application of the omics sciences to the study of Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris: current status and future projections.

In this review, we focus on the sequenced genomes of the pathogens Naegleria fowleri, Acanthamoeba spp. and Balamuthia mandrillaris, and the remarkable discoveries regarding the pathogenicity and genetic information of these organisms, using techniques related to the various omics branches like genomics, transcriptomics, and proteomics. Currently, novel data produced through comparative genomics analyses and both differential gene and protein expression in these free-living amoebas have allowed for breakthroughs to identify genes unique to N. fowleri, genes with active transcriptional activity, and their differential expression in conditions of modified virulence. Furthermore, orthologous genes of the various nuclear genomes within the Naegleria and Acanthamoeba genera have been clustered. The proteome of B. mandrillaris has been reconstructed through transcriptome data, and its mitochondrial genome structure has been thoroughly described with a unique characteristic that has come to light: a type I intron with the capacity of interrupting genes through its self-splicing ribozymes activity. With the integration of data derived from the diverse omic sciences, there is a potential approximation that reflects the molecular complexity required for the identification of virulence factors, as well as crucial information regarding the comprehension of the molecular mechanisms with which these interact. Altogether, these breakthroughs could contribute to radical advances in both the fields of therapy design and medical diagnosis in the foreseeable future.

TITLE: Application des sciences de l'omique à l'étude de Naegleria fowleri, Acanthamoeba spp. et Balamuthia mandrillaris : état actuel et projections futures. ABSTRACT: Dans cette revue, l'accent est mis sur les génomes séquencés des agents pathogènes Naegleria fowleri, Acanthamoeba spp. et Balamuthia mandrillaris, et les découvertes remarquables concernant la pathogénicité et l'information génétique de ces organismes, en utilisant des techniques liées aux diverses branches de l'omique comme la génomique, la transcriptomique et la protéomique. Actuellement, de nouvelles données produites par des analyses génomiques comparatives et l'expression différentielle des gènes et des protéines dans ces amibes libres ont permis des percées pour identifier des gènes uniques à N. fowleri, des gènes avec une activité transcriptionnelle active et leur expression différentielle dans des conditions de virulence modifiée. En outre, les gènes orthologues des divers génomes nucléaires des genres Naegleria et Acanthamoeba ont été regroupés. Le protéome de B. mandrillaris a été reconstruit grâce aux données du transcriptome, et la structure de son génome mitochondrial décrite de manière détaillée, mettant ainsi une caractéristique unique à jour : un intron de type I avec la capacité d'interrompre les gènes par son activité d'auto-épissage des ribozymes. Avec l'intégration des données issues des diverses sciences omiques, il existe une approximation potentielle qui reflète la complexité moléculaire requise pour l'identification des facteurs de virulence, ainsi que des informations cruciales concernant la compréhension des mécanismes moléculaires avec lesquels ceux-ci interagissent. Dans l'ensemble, ces percées pourraient contribuer à des progrès notables à la fois dans les domaines de la conception de la thérapie et du diagnostic médical dans un avenir proche.

Miltefosine: A Miracle Drug for Meningoencephalitis Caused by Free-Living Amoebas.

Meningoencephalitis caused by free-living amoebas (FLA) has a high mortality rate, and most treatments are ineffective. FLA includes Naegleria, Fowleri, Acanthamoeba, and Balamuthia mandrillaris (M). We explore the use of miltefosine in the treatment of one of these infections. The concerning mortality of the infection obligates us to look for more effective treatments for meningoencephalitis caused by FLA. During this review, we will consolidate the knowledge of using miltefosine in these three infections. We will investigate the mechanism by which the drug is effective in these infections as well. After this comprehensive review, we should assess if miltefosine improves the mortality and prognosis of the infection with the information collected. We used a Medical Subject Headings (MeSH) search on PubMed. Inclusion criteria included papers written in the English language and human subjects research for the past 25 years. Until today, there are no definitive guidelines to be followed when treating such patients. However, miltefosine has demonstrated promising results. Miltefosine decreases the usual mortality rate in the three infections; however, there are few reports due to the low frequency of these infections. Almost all cases we documented have survived. More information needs to be gathered for the use of miltefosine for these infections.

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.

Non-granulomatous cerebellar infection by Acanthamoeba spp. in an immunocompetent host.

Acanthamoeba spp. is a free-living amoeba, frequently involved in keratitis by contact lens in immunocompetent hosts. Anecdotal reports associate Acanthamoeba spp. as a cause of severe granulomatous encephalitis in immunocompromised and, less frequently, in immunocompetent subjects. Data regarding clinical and therapeutic management are scanty and no defined therapeutic guidelines are available. We describe an unusual case of non-granulomatous Acanthamoeba cerebellitis in an immunocompetent adult male, with abrupt onset of neurological impairment, subtle hemorrhagic infarction at magnetic resonance imaging, and initial suspicion of cerebellar neoplasm. Histopathological findings of excised cerebellar mass revealed the presence of necrosis and inflammation with structure resembling amoebic trophozoites, but without granulomas. Polymerase chain reaction from cerebellar tissue was positive for Acanthamoeba T4 genotype. Due to gastrointestinal intolerance to miltefosine, the patient was treated with long-term course of fluconazole and trimethoprim/sulphamethoxazole, obtaining complete clinical and neuroradiological resolution.

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...

Hallazgo de acanthamoeba spp en biopsia cerebral de un paciente con adenocarcinoma metastásico / Acanthamoeba spp found in a cerebral biopsy from a patient with metastasic adenocarcinoma

Las amibas de vida libre (AVL) del género Acanthamoeba son parásitos facultativos ampliamente distribuidos en el ambiente, por lo cual el contacto del humano con ellos es frecuente, sin embargo, los reportes de enfermedades producidas por AVL son escasos. La gravedad de éstas justifica su búsqueda, sobre todo cuando la infección es del sistema nervioso central (SNC) y cuando hay compromiso in munológico. En este trabajo se reporta el hallazgo de Acanthamoeba spp en una biopsia cerebral, tomada a un paciente de 44 años de edad con adenocarcinoma. Se exa minó la muestra bajo el protocolo del Laboratorio de Amibiasis, de la Cátedra de Parasitología, Escuela de Bioanálisis, Facultad de Medicina, Universidad Central de Venezuela, para la investigación de AVL y Entamoeba histolytica. Se realizó examen directo y coloraciones especiales, inducción de la flagelación y cultivos a diferentes temperaturas. Se observó crecimiento de Acanthamoeba spp en el medio de Page modificado por Chinchilla y col. (1979), incubado a 37 °C a las 96 horas. La descripción morfológica coincide con la morfología del grupo 3 de Acanthamoeba, según Visvesvara (1991). Se destaca la importancia de la realización del cultivo para el crecimiento e identificación de estos organismos en aquellos laboratorios donde los métodos basados en biología molecular aun no se hayan implementado como parte del diagnóstico y así aplicar el tratamiento oportuno y específico al paciente.

Free-living amoebas (FLA) of genus Acanthamoeba are parasitic facultative organisms whose are widely distributed in the environment, thus the human contact with them is frequent; nevertheless, there are few reports of diseases produced by FLA. The severity of these diseases justifies its re search, mainly when the infection is at central nervous system (CNS) and when there is an immunocompromised host. This work reports the finding of Acanthamoeba spp in a cerebral biopsy from a 44 year-old patient with adenocarcinoma. We followed the protocol established by the Amibiasis Laboratory, Parasitology Chair, School of Bioanalisis, Medicine Faculty, Central University of Venezuela, for the investigation of FLA and Entamoeba histolytica. It wasmade by direct examination of the sample and special stains, flagellation induction and cultures incubated at different temperatures. We observed Acanthamoeba spp growth in biphasic Page media modified by Chinchilla and col. (1979), incubated for 96 hours at 37 °C. The morphologic des cription agrees with the morphology of group 3 of Acanthamoeba, according to Visvesvara (1991). We emphasize the importance of using growth culture for isolation and identification of these organisms in those laboratories where molecular methods has not been implemented yet as part of the diagnosis scheme and thus for applying the opportune and specific treatment to the patient.

Amibas de vida libre en seres humanos / Free-living amoebae in humans

Entre las enfermedades graves, peligrosas y de gran importancia epidemiológica se encuentran la meningitis y la encefalitis, que pueden llevar a invalidez o muerte. Los agentes etiológicos que las producen generalmente son bacterias, virus y hongos. En cuanto a los protozoos causantes de meningitis y encefalitis, se encuentran las amibas de vida libre. Los individuos infectados con estas amibas tienen como antecedente el haber tenido contacto con aguas contaminadas en días recientes, especialmente de piscinas o aguas termales. Naegleria fowleri parasita a individuos aparentemente saludables, niños o jóvenes y es la causante de meningoencefalitis amebiana primaria, que se caracteriza por su rápida evolución y luego la muerte. Algunas especies del género Acanthamoeba, Balamuthia mandrillaris, y recientemente Sappinia pedata, producen encefalitis amebiana granulomatosa, que se presenta de forma subaguda o crónica; generalmente parasitan a individuos inmunosuprimidos. Además, ciertas especies de Acanthamoeba producen queratitis, que se confunden con las producidas por Herpes simplex u hongos. Estas amibas también causan infecciones severas en pulmones, oídos, piel y nariz. El diagnóstico de estas amibas en la mayoría de los casos se hace después de la muerte de los individuos infectados. En cuanto a su tratamiento, se han empleado combinaciones de varios antimicrobianos con resultados poco alentadores. Estas amibas no son lo suficientemente conocidas por el personal de salud, por lo tanto, en esta revisión se pretende evidenciar los aspectos más relevantes de éstas, ya que puede haber subdiagnóstico o confusión con otros agentes etiológicos y no sospechar de la presencia de amibas de vida libre...

Meningitis and Encephalitis are amongst the most dangerous disabling or death causing diseases. The etiological causes of these diseases include bacterium, virus and fungi. Protozoan which causes Meningitis and Encephalitis tend to be free-living amoebae. Patients identified with these amoebae infections had recently been exposed to contaminated water in swimming pools or steam baths. Healthy children and youth are vulnerable to Naegleria fowleri which causes Primary Amoebic Meningoencephalitis which evolution rapidly, causing death. Some species of Acanthamoeba, Balamuthia mandrillaris and recently Sappinia cause Granulomatous Amoebic Encephalitis in both acute and sub-acute forms, primarily affecting case with immunosuppression. Furthermore, some species produce Keratitis, which is confounded with diseases caused by Herpes Simplex or fungi. These amoebae are also infection causing agents in the lungs, ears, skin and nose. In most cases, diagnosis of these amoebae is posthumous. Combinations of antimicrobial treatment have had little effect, combating infections. This study seeks to enhance approaches towards diagnosing diseases caused by these agents and overcoming confounding them with other etiological agents without doubting the presence of free living amoebae...

Frequência de amebas de vida livre em fezes de indivíduos de uma creche da cidade de São Paulo / Frequency free-living amebas in the feces of persons from a nursery in Säo Paulo city

A ocorrência de amebas de vida livre foi pesquisada em 90 amostras de fezes de indivíduos pertencentes a uma creche da cidade de São Paulo. As amostras foram semeadas em placas de Petri contendo ágar não nutriente, ou ágar não nutriente - sal, com um tapete de Ent.erobacter aerogenes, morto pelo calor. As placas foram incubadas a temperaturas de 28, 37 e 400 C, e observadas diariamente durante sete dias. A positividade foi de 22,71% em 66 crianças, e de 8,33% em 24 adultos. Foram isoladas 21 amostras do gênero Acanthamoeba, 4 de Naegleria, 1 de Echinamoeba, e 12 de amebídeos não identificados. Foram examinadas mais 3 amostras fecais de cada indivíduo com exame positivo, com intervalo de um mês entre as coletas. Amostras de Acanthamoeba sp. foram reisoladas em dois indivíduos, o que nos leva a supor que amebídeos estavam sendo por eles albergados, e não em simples trânsito intestinal (AU).