Parasitol Res (2012) 110:1291–1296 DOI 10.1007/s00436-011-2617-8
SHORT COMMUNICATION
Clinical case of cerebral amebiasis caused by E. histolytica Cinthya A. Maldonado-Barrera & Maria del Rosario Campos-Esparza & Luis Muñoz-Fernández & Joaquin A. Victoria-Hernández & Rafael Campos-Rodríguez & Patricia Talamás-Rohana & Javier Ventura-Juárez
Received: 17 June 2011 / Accepted: 5 August 2011 / Published online: 26 August 2011 # Springer-Verlag 2011
Abstract Although amebic brain abscess is a rare form of invasive amebiasis, when present, it is frequently lethal. This disorder always begins with the infection of the colon by Entamoeba histolytica trophozoites, which then travel to extra-intestinal tissues through the bloodstream. Amebic brain abscesses are produced when trophozoites invade the central nervous system. Computerized axial tomography scans can be used to diagnose the presence or absence of a brain abscess with a certainty of 100%. However, this diagnostic tool does not reveal the etiological agent of disease. By analyzing the clinical case of a patient that died due to untimely treatment of this malady, the present study aims to identify a diagnostic tool that can give a precise determination of the etiological agent and therefore permit adequate and opportune treatment. Currently, diagnosis of amebic brain abscess is often done by identification of the ameba in a biopsy or autopsy. By immunohistochemistry C. A. Maldonado-Barrera : M. R. Campos-Esparza : J. Ventura-Juárez (*) Departamento de Morfología, Centro de Ciencias Básicas, Universidad Autónoma de Aguascalientes, Av Universidad 940, Col. Cd. Universitaria, Aguascalientes CP. 20131, Mexico e-mail:
[email protected] L. Muñoz-Fernández : J. A. Victoria-Hernández Departamento de Patología, Centenario Hospital Miguel Hidalgo, Aguascalientes, Mexico R. Campos-Rodríguez Departamento de Bioquímica, ESM-IPN, México City, Mexico P. Talamás-Rohana Departamento de Infectómica y Patogénesis Molecular, CINVESTAV-IPN, México City, Mexico
and immunofluorescence with specific antibodies, we identified the existence of E. histolytica, which presents proteins similar to Naegleria fowleri in its membrane.
Introduction Although the incidence of protozoan infections in the central nervous system is rare, particular attention to this matter is justified by the over 95% mortality rate among those who suffer this malady. The most frequent protozoan infections are by free-living amebas (Naegleria fowleri, Acanthamoeba spp., Balamuthia mandrilaris, and Sappinia diploidea) and, to a lesser extent, by Entamoeba histolytica. Worldwide, the frequency of cerebral infections caused by E. histolytica is from 0.6% to 8.1% (Castillo de la Cruz et al. 2004; Parija et al. 2005). In a review of 3,577 autopsies carried out in Mexico, 210 cases of amebiasis were found, among which 8.1% (17 cases) corresponded to cerebral amebiasis, which was associated in all cases with hepatic amebiasis and in some cases with lesions in the lungs (Lombardo et al. 1964). Whereas N. fowleri causes primary amebic meningoencephalitis, with a period of incubation from 4 to 7 days, Acanthamoeba spp., B. mandrilaris, and S. diploidea produce amebic granulomatose encephalitis, with a period of incubation from 10 days to various months. On the other hand, E. histolytica produces brain abscess, with a period of incubation from a few days to several months (PeraltaRodriguez and Ayala-Oviedo 2009). Because cerebral infections by bacteria have a medical profile similar to that of protozoans (including headaches, partial muscular weakness, lethargy, and dizziness), diagnosis is often difficult (Sayhan Emil et al. 2008; Peralta-Rodriguez and Ayala-Oviedo 2009).
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Nevertheless, computerized axial tomography (CAT scans) and nuclear magnetic resonance (MRI) both provide data that can lead to the detection of this malady in 100% of the cases. These amebic brain abscesses always have a brownish black liquid (also called “anchovy” pus) due to the lytic effect of the amebas and vary greatly in size (Shah et al. 1994; Castillo de la Cruz et al. 2004; DíazMárquez et al. 2009; Viriyavejakul and Riganti 2009). In spite of the reliability of detection of the malady with these techniques, the etiological agent remains unidentified (Benito Leon et al. 2001; Castillo de la Cruz et al. 2004). In the current contribution, we present a case of a male patient with a brain abscess that is not associated with hepatic amebiasis. Ten months after surgery, the patient died without having received treatment for amebic invasion.
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specific treatment was not given. Two months later, the patient was admitted to another hospital (Hospital de Alta Especialidad de León, Guanajuato, Mexico), where the diagnosis was an astrocyte brain tumor. Radiotherapy and chemotherapy were carried out in that hospital, after which time the patient was transferred to a third hospital (Salubridad de Encarnación de Díaz Jalisco, Mexico) where he died in April of 2009. The aim of the current contribution was to determine the etiology of the brain abscess, taking the following aspects into account: (a) the patient’s symptoms, (b) the absence of an accurate etiologic diagnosis, and (c) the epidemiologically known fact that brain abscesses can be caused by parasites, most frequently by free-living amoebae as N. fowleri and less frequently by E. histolytica.
Presentation of the case There was a case of a patient with a brain abscess of unknown etiology. The patient had no previous history of intestinal, hepatic, or lung infection. Upon draining the abscess, a sample of injured brain tissue was obtained for microscopic study. A 55-year-old man had a history of previous neurocisticercosis. In June of 2008, he began to show a progressive muscular weakness on the left side of his body, which with time gave him trouble in walking. Moreover, he presented migraine headaches and limited alertness. In July of the same year, he was hospitalized. During the physical exploration, a neurological deterioration was discovered, as well as an evident weakness hemiplegia in the left extremities. The lab tests showed leukocytes with a predominance of polymorphonuclear cells (11,300 leukocytes/ μl, with 73% neutrophils). A CAT scan of the skull revealed a round, hypodense lesion measuring 6×5× 4 cm in the right temporal–parietal zone, a lesion compatible with the presence of an abscess. A diagnosis was made of intracranial hypertension with a focal lesion in the right cerebral hemisphere. A craniotomy was performed to evacuate the content of the lesion, obtaining 30 ml of sallow liquid and various fragments of brain tissue. A histopathological study revealed extensive zones of necrosis in liquefaction, in which round cellular structures were found with clear microvacuoles in the cytoplasm. Some of these structures had fragments of erythrocytes in their interior. After surgery, the patient evolved satisfactorily and he left the hospital (Hospital Centenario Miguel Hidalgo, Aguascalientes, Mexico). Therefore, the etiopathologic diagnosis of an undetermined brain abscess was not made, and
Materials and methods Biological material Material of brain abscess with an abscessed lesion, provided to the authors from a biopsy of the right hemisphere of the brain performed during the patient’s stay in the first hospital (Centenario Hospital Miguel Hidalgo de Aguascalientes, Mexico), was fixed in 10%formaldehyde and embedded in paraffin. A morphological study was performed with the hematoxylin/eosin (H&E) stain method (Castillo de la Cruz et al. 2004). Immunohistochemistry Each cut (5 μm thick) was incubated with its corresponding primary antibody for 24 h in a humidified chamber at 4°C. The primary antibodies (Ab) employed were the following: (a) a mouse antibody against the 220-kDa protein of E. histolytica, at the dilution recommended by Meza et al. (1987) to identify this pathogen, and (b) a mouse antibody against the gliofibroid acidic protein (Millipore, MAB360, dilution 1:200). All antibodies were diluted in PBS–0.2% triton X100–3% BSA (seric bovine albumin). After the aforementioned procedure and various washings, incubation with the secondary antibody was carried out in a humidified chamber for 2 h at room temperature with a cocktail of polymers conjugated with peroxidase (DAKO Envision, K1490), which are capable of binding to immunoglobulins of mouse and rabbit. After another washing, the samples were incubated with diaminobenzidine (Sigma fast 3,3′diaminobenzidine, D-4168) and chromogene until color appeared in order to identify the peroxidase reactions.
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Finally, samples were stained with hematoxylin, dehydrated, and mounted on Entellan®. Indirect immunofluorescence A double immunofluorescence was carried out to detect antigens of E. histolytica and N. fowleri in the brain tissue. The primary antibodies were: (a) a mouse anti-E. histolytica L220 kDa protein, aforementioned, and (b) a rabbit antibody against N. fowleri (diluted 1:200), previously absorbed by E. histolytica antigens to detect free-living N. fowleri amebas, as recommended by Shibayama et al. (2003). The secondary antibodies were a goat anti-mouse Alexa Fluor 594 (Invitrogen, 2 μg/ml) and a goat antirabbit-Alexa Fluor 488 (Invitrogen, 2 μg/ml). After washing with PBS, samples were incubated for 15 min with Hoechst 33258 (5 μg/ml) diluted in PBS, then washed again with PBS. For observation, the tissue was covered with glycergel (Glycergel Mounting Medium Dako C0563) and mounted on a slide. Samples were observed and analyzed with a visible light microscope and an epifluorescent Axioscop 40 microscope (Carl Zeiss). Photos were taken with Image Pro Plus software.
Results Staining was initially carried out with H&E, with the aim of observing the type and degree of lesion in the tissue under study. Extensive necrosis of tissue was found, but without inflammatory infiltrate (marked by a cross), and some areas of tissue with liquefaction abscess (marked by an asterisk) Fig. 1 Panoramic view of an amebic brain abscess with extensive necrotic (cross) and abscessed areas (asterisk) in the tissue. a Magnification of the necrotic area is shown with ameboid cellular structures that have clear microvacuoles in their cytoplasm (arrow) and with normal glial cells in the near vicinity (double arrow). b Trophozoite with ingested fragmented erythrocytes (arrow) is shown in the necrotic area
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were observed (Fig. 1). In the necrotic zone (Fig. 1a), there were spherical or oblong elements that had clear microvacuoles in the cytoplasm, and some of these had fragments of erythrocytes in their interior (Fig. 1b). Thus, these elements coincided with the morphology of E. histolytica in its trophozoital phase. Confirming this symptomology, the brain tissue showed no evidence of the presence of an astrocyte brain tumor. Glial cells were found to have normal morphology, without any atypical characteristics (double arrow in Fig. 1a). The immunoassay for GFAP did not show any signs of cellular proliferation for glial cells (data not shown). A peroxidase immunohistochemical method was performed to confirm the identity of the etiological agent. Stained trophozoites, dark brown with pseudopods and vacuoles inside (Fig. 2a, c), were found to be distributed throughout the necrotic (shown by a cross) areas, while being absent in the abscessed zones (shown by an asterisk) of the cerebral tissue sample (Fig 2). Some of these trophozoites were grouped together (Fig. 2b). Because the patient had no history of intestinal or hepatic amebiasis, it was possible that amebas other than E histolytica were the causal agent of this disease. Therefore, we developed a double immunofluorescence method using antibodies against the 220-kDa protein of E. histolytica and against N. fowleri. The results showed that all trophozoites were positive for E. histolytica (Fig. 3a) and some of them were also positive for N. fowleri (Fig. 3b). Also, a coexistence of antigens of N. fowleri and E. histolytica was observed in cerebral tissue, suggesting that the membranes of E. histolytica and N. fowleri contain similar antigens (Fig. 3c). As a positive control, we
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Fig. 2 Identification of the etiological agent of a brain abscess. Panoramic view of the necrotic (cross) and abscessed zone (asterisk) of the cerebral tissue, in which gray and white substances have lost their configuration. In (a) there are abundant trophozoites with vacuoles (arrows) immersed in the necrotic tissue (cross). a Localization of E. histolytica trophozoites in cerebral tissue through the use of a monoclonal anti-L220 kDa lectin antibody. b Necrotic zone with multiple accumulated (two asterisks) trophozoites (arrows). c A magnification of a dark brown E. histolytica trophozoite (immunoperoxidase staining), adopting the classical amoeboid form with pseudopods (yellow arrowhead) and several intracytoplasmic vacuoles (arrows)
analyzed samples of amebic liver abscess and fulminant amebic colitis by immunohistochemistry using the same primary antibodies (data not shown).
Discussion Studies on cerebral amebiasis in Mexico indicate a greater prevalence of primary amebic meningoencephalitis in the northeast of the country (Cervantes-Sandoval et al. 2007), where there is a lesser extent of cases of amebic granulomatose encephalitis (Riestra-Castaneda et al. 1997). There are no epidemiological data on amebic brain abscess. In this study, the symptomology of the patient was insidious, and no history of intestinal, hepatic, or pulmonary infection by E histolytica existed. The result of the first histopathological exam carried out in Hospital Hidalgo was not conclusive, finding only extensive zones of necrosis in liquefaction, containing round cellular structures with clear microvacuoles in the cytoplasm. Some of these structures had fragments of erythrocytes in their interior, located on the wall of the abscess. No etiopathological diagnosis was determined, nor was a specific treatment given. The lesions observed are similar to those described by Lombardo et al. (1964) who discovered the same histopathology in 17 patients with amebic brain abscess; however, these were associated with hepatic and/or pulmonary lesions (Yamasaki et al. 2007; Viriyavejakul and Riganti 2009), or with disenteria (Schmutzhard et al. 1986; Morishita et al. 2007). Similarly, the amebic abscess on
Fig. 3 N. fowleri and E. histolytica trophozoites found in the same cerebral tissue. Trophozoites in nerve tissue labeled with the antiL220 kDa protein of E. histolytica (Alexa Fluor 594) in red (arrows) (a) and labeled with antibodies against N. fowleri trophozoites (Alexa Fluor 488) in green (arrow) (b). c Combination of previous images shows a double positive trophozoite that was recognized by both antibodies (yellow arrow), showing a yellowish area, which indicates that this particular cell contains epitopes that are being recognized by anti-N. fowleri and anti-E. histolytica antibodies. Next to it, there is another trophozoite showing a positive signal only for E. histolytica
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the right lobe and the lack of association with hepatic and/ or pulmonary amebiasis coincide with a case of a male patient in the State of Mexico who was also diagnosed with amebic brain abscess. In a second study conducted on the patient 2 months later in Hospital de Alta Especialidad of Leon, Guanajuato, Mexico, an astrocyte brain tumor was diagnosed. Since we were not able to obtain this second sample, we used a tumor marker (GFAP) on the tissue of the first histopathological sample and did not find any indication of a malignant tumor. In accordance with what Lombardo et al. (1964) described, we suggest that the histopathological diagnosis corresponds to old lesions that present thickening of the abscess wall with irregular layers of neuroglia and a small amount of connective tissue surrounded by an intense astrocytic reaction. Determining the etiological agent is the decisive factor for the administration of an adequate treatment when the symptomology indicates the possible presence of parasitic antigens. Such antigens can present problems of interpretation due to cross-reactions and low test sensitivity. Hence, it is necessary to use immunohistology in order to discover the etiological agent, as well as its life cycle and pathogenicity. To determine the etiopathology of the brain abscess, we employed: (1) a monoclonal antibody that recognizes the lectin of E. histolytica at 220 kDa, which has been widely used with in vitro studies (Meza et al. 1987; HernandezRamirez et al. 2000), and (2) a polyclonal antibody that recognizes N. fowleri proteins (Shibayama et al. 2003; Rojas-Hernandez et al. 2004). These tests assured us that E. histolytica was responsible for the amebic abscess and also demonstrated the antigenic complexity of this protozoon. Recent studies show that E. histolytica induces not very well-defined brain damage. A clinical case of a 31-year-old man in Thailand describes an amebic abscess located in the right occipital lobe, concomitantly with acute supurative meningitis (Viriyavejakul and Riganti 2009). In the USA, a case was reported of a 2-year-old girl who had multiple brain abscesses in the right cerebral peduncle and in both hemispheres, with abundant inflammatory infiltrate, which came to be confused with bacterial meningitis (Sayhan Emil et al. 2008). Based on these data and our results, we suggest that E. histolytica possibly shares membrane antigens and therefore some pathogenic mechanisms with N. fowleri. Supporting this idea, Rivera-Aguilar et al. (2000) have demonstrated that IgA from the saliva and serum of some patients in endemic areas recognizes similar proteins for E. histolytica and N. fowleri. It is noteworthy that Entamoeba was recently reclassified to recognize that this genus comprises six species (E. histolytica, Entamoeba dispar, Entamoeba moshkovskii, Entamoeba poleki, Entamoeba coli, and Entamoeba hart-
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manni) that live in the human intestinal lumen. E. histolytica, E. dispar, and E. moshkovskii are morphologically identical, but are biochemically and genetically different, which makes it difficult to identify them in a histopathological study. Although E. histolytica is recognized as a pathogen, the ability of the other two species to cause disease is unclear. E. moshkovskii, for example, is considered primarily a free-living ubiquitous ameba found in anoxic sediments, and E. dispar is considered primarily a commensal of the human gut (Fotedar et al. 2008). Acceptance of this redefinition has dramatically changed both our understanding of the true epidemiology of E. histolytica and the optimal methods for diagnosing amebiasis. In order to distinguish infection with E. histolytica from infection with E. dispar, molecular-based diagnostic tests using polymerase chain reaction to amplify amebic DNA or enzyme-linked immunosorbent assay to identify amebic antigens in stool samples have been developed (Huston and Petri 1999). However, in most cases of cerebral amebiasis, this distinction is never made due to the fact that: (1) there is sometimes a lack of association between amebiasis in different organs, (2) the majority of patients die before a specific diagnosis is made, and (3) brain damage from protozoa is frequently confused with that from bacterial etiology. In order to correctly diagnose cases of cerebral amebiasis, when the physician detects symptomatology that could indicate this disorder, imaging (MRI and CAT) analyses should immediately be made. If these studies prove positive for a brain abscess, then it must be determined by immunohistochemistry and immunofluorescence with specific antibodies whether the etiological agent of disease is E. histolytica or a free-living ameba such as N. fowleri.
Conclusion Although amebic brain abscess is a rare form of invasive amebiasis, it must be diagnosed in a timely manner in order to preserve the life of the patient. With this aim, the only way to determine the adequate treatment is to identify the etiological agent of the disease. Acknowledgments We thank the biologist Fabiola del Rocío Villalobos Gómez for her technical assistance and Bruce Allan Larsen for reviewing the use of English in this manuscript. We are grateful for the support provided by Consejo Nacional de Ciencia y Tecnología (CONACYT) (project no. 49749), by the Universidad Autónoma de Aguascalientes (Project PIBB07-2), and by the Comité de Operaciones y Fomento a las Actividades Académicas (COFAA) and EDIIPN in Mexico City. María del Rosario Campos-Esparza was supported with a fellowship from the Consejo Nacional de Ciencia y Tecnología (CONACYT-Mexico) for a post-doctoral training in the program “Estancias Posdoctorales y Sabáticas Vinculadas al Fortalecimiento de la Calidad del Posgrado Nacional 2009–2010,” Mexico.
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References Benito Leon J, Alvarez Linera J, Escribano J, Ruiz Galiana J (2001) Differentiation between cerebral abscesses and necrotic or cystic tumours by means of diffusion sequences. Rev Neurol 32:137– 140 Castillo de la Cruz M, Gallegos-Barredo J, Mendizábal-Guerra R, Félix I, Rivas A (2004) Reporte de caso, absceso cerebral multicéntrico causado por Entamoeba histolytica. Arch Neurocien 9:59–62 Cervantes-Sandoval I et al (2007) Characterization of Naegleria fowleri strains isolated from human cases of primary amoebic meningoencephalitis in Mexico. Rev Invest Clin 59:342–347 Díaz-Márquez S, Bastardo N, Bello A, Dib J Jr, Chacón N (2009) Abscesos cerebelosos como complicación infrecuente de absceso hepático amebiano. Reporte de un caso. GEN Revista de la Sociedad Venezolana de Gastroenterología 63:121–122 Fotedar R, Stark D, Marriott D, Ellis J, Harkness J (2008) Entamoeba moshkovskii infections in Sydney, Australia. Eur J Clin Microbiol Infect Dis 27:133–137 Hernandez-Ramirez VI, Anaya-Ruiz M, Rios A, Talamas-Rohana P (2000) Entamoeba histolytica: tyrosine kinase activity induced by fibronectin through the beta1-integrin-like molecule. Exp Parasitol 95:85–95 Huston CD, Petri WA (1999) Amebiasis: clinical implications of the recognition of Entamoeba dispar. Curr Infect Dis Rep 1:441–447 Lombardo L, Alonso P, Saenzarroyo L, Brandt H, Humbertomateos J (1964) Cerebral amebiasis: report of 17 cases. J Neurosurg 21:704–709 Meza I, Cazares F, Rosales-Encina JL, Talamas-Rohana P, Rojkind M (1987) Use of antibodies to characterize a 220-kilodalton surface protein from Entamoeba histolytica. J Infect Dis 156:798–805 Morishita A, Yamamoto H, Aihara H (2007) A case of amebic brain abscess. No Shinkei Geka 35:919–925
Parasitol Res (2012) 110:1291–1296 Parija S, Lalmuanpuii J, Bhattacharya S, Chandrasekhar S (2005) Parasitic infections of the central nervous system. J Parasitic Dis 29:85–96 Peralta-Rodriguez M, Ayala-Oviedo J (2009) Amibas de vida libre en seres humanos. Salud Uninorte 25(2):280–292 Riestra-Castaneda JM et al (1997) Granulomatous amebic encephalitis due to Balamuthia mandrillaris (Leptomyxiidae): report of four cases from Mexico. Am J Trop Med Hyg 56:603–607 Rivera-Aguilar V et al (2000) Immunoblot analysis of IgA antibodies to Naegleria fowleri in human saliva and serum. Parasitol Res 86:775–780 Rojas-Hernandez S, Jarillo-Luna A, Rodriguez-Monroy M, MorenoFierros L, Campos-Rodriguez R (2004) Immunohistochemical characterization of the initial stages of Naegleria fowleri meningoencephalitis in mice. Parasitol Res 94:31–36 Sayhan Emil S, Altinel D, Bayol U, Ozcolpan OO, Tan A, Ganiusmen O (2008) Amebic cerebral abscess mimicking bacterial meningitis. Indian J Pediatr 75:1078–1080 Schmutzhard E, Mayr U, Rumpl E, Prugger M, Pohl P (1986) Secondary cerebral amebiasis due to infection with Entamoeba histolytica. A case report with computer tomographic findings. Eur Neurol 25:161–165 Shah AA, Shaikh H, Karim M (1994) Amoebic brain abscess: a rare but serious complication of Entamoeba histolytica infection. J Neurol Neurosurg Psychiatry 57:240–241 Shibayama M, Serrano-Luna Jde J, Rojas-Hernandez S, CamposRodriguez R, Tsutsumi V (2003) Interaction of secretory immunoglobulin A antibodies with Naegleria fowleri trophozoites and collagen type I. Can J Microbiol 49:164–170 Viriyavejakul P, Riganti M (2009) Undiagnosed amebic brain abscess. Southeast Asian J Trop Med Public Health 40:1183–1187 Yamasaki M, Taniguchi A, Nagai M, Sasaki R, Naito Y, Kuzuhara S (2007) Probable amebic brain abscess in a homosexual man with an Entamoeba histolytica liver abscess. Rinsho Shinkeigaku 47:672–675
