Case Report: Encephalitis Caused by Balamuthia mandrillaris in a 3-Year-Old Iranian Girl.

It is about half a century since free-living amoebae were recognized as pathogenic organisms, but there is still much we should learn about these rare fatal human infectious agents. A recently introduced causative agent of granulomatous amoebic encephalitis, Balamuthia mandrillaris, has been reported in a limited number of countries around the world. A 3-year-old girl was referred to our tertiary hospital because of inability to establish a proper diagnosis. She had been experiencing neurologic complaints including ataxia, altered level of consciousness, dizziness, seizure, and left-sided hemiparesis. The patient's history, physical examination results, and laboratory investigations had led to a wide differential diagnosis. Computed tomography (CT) scan and magnetic resonance imaging analyses revealed multiple mass lesions. As a result, the patient underwent an intraoperative frozen section biopsy of the brain lesion. The frozen section study showed numerous cells with amoeba-like appearances in the background of mixed inflammatory cells. Medications for free-living amoebic meningoencephalitis were administered. PCR assay demonstrated B. mandrillaris as the pathogenic amoeba. Unfortunately, the patient died 14 days after her admission. To our knowledge, this is the first report of B. mandrillaris meningoencephalitis in the Middle East and the first time we have captured the organism during a frozen-section study.

Balamuthia mandrillaris-Related Primary Amoebic Encephalitis in China Diagnosed by Next Generation Sequencing and a Review of the Literature.

BACKGROUND: Encephalitis is caused by infection, immune mediated diseases, or primary inflammatory diseases. Of all the causative infectious pathogens, 90% are viruses or bacteria. Granulomatous amoebic encephalitis (GAE), caused by Balamuthia mandrillaris, is a rare but life-threatening disease. Diagnosis and therapy are frequently delayed due to the lack of specific clinical manifestations. METHOD: A healthy 2 year old Chinese male patient initially presented with a nearly 2 month history of irregular fever. We present this case of granulomatous amoebic encephalitis caused by B. mandrillaris. Next generation sequencing of the patient's cerebrospinal fluid (CSF) was performed to identify an infectious agent. RESULT: The results of next generation sequencing of the CSF showed that most of the mapped reads belonged to Balamuthia mandrillaris. CONCLUSION: Next generation sequencing (NGS) is an unbiased and rapid diagnostic tool. The NGS method can be used for the rapid identification of causative pathogens. The NGS method should be widely applied in clinical practice and help clinicians provide direction for the diagnosis of diseases, especially for rare and difficult cases.

Saksenaea infection masquerading as a brain tumor in an immunocompetent child.

Saksenaea species are a rare cause of mucormycosis, the majority associated with cutaneous and subcutaneous infections resulting from trauma in both immunocompromised and immunocompetent individuals. Unlike other causative agents of mucormycosis, cerebral infections are exceptionally rare. We describe the first case of isolated cerebral infection by Saksenaea in a 4-year-old previously healthy male child who presented with headaches. He had no past medical history other than an episode of febrile seizures. In addition to raising the awareness of an unusual presentation of infection by Saksenaea, this case highlights the importance of pathologic examination for the prompt diagnosis of mucormycosis as well as the specific fungal identification for treatment as Saksenaea spp. may be more susceptible to posaconazole and less susceptible to amphotericin B compared to more common causes of mucormycosis.

Report: Effect of macrophage alone or primed with cytokines on Balamuthia mandrillaris interactions with human brain microvascular endothelial cells in vitro.

Balamuthia mandrillaris is well known to cause fatal Balamuthia amoebic encephalitis (BAE). Amoebic transmission into the central nervous system (CNS), haematogenous spread is thought to be the prime step, followed by blood-brain barrier (BBB) dissemination. Macrophages are considered to be the foremost line of defense and present in excessive numbers during amoebic infections. The aim of the present investigation was to evaluate the effects of macrophages alone or primed with cytokines on the biological characteristics of Balamuthia in vitro. Using human brain microvascular endothelial cells (HBMEC), which constitutes the BBB, we have shown that Balamuthia demonstrated <90% binding and <70% cytotoxicity to host cells. However, macrophages further increased amoebic binding and Balamuthia-mediated cell cytotoxicity. Furthermore macrophages exhibited no amoebicidal effect against Balamuthia. Zymography assay demonstrated that macrophages exhibited no inhibitory effect on proteolytic activity of Balamuthia. Overall we have shown for the first time macrophages has no inhibitory effects on the biological properties of Balamuthia in vitro. This also strengthened the concept that how and why Balamuthia can cause infections in both immuno-competent and immuno-compromised individuals.

An In Vitro Model of the Blood-Brain Barrier: Naegleria fowleri Affects the Tight Junction Proteins and Activates the Microvascular Endothelial Cells.

Naegleria fowleri causes a fatal disease known as primary amoebic meningoencephalitis. This condition is characterized by an acute inflammation that originates from the free passage of peripheral blood cells to the central nervous system through the alteration of the blood-brain barrier. In this work, we established models of the infection in rats and in a primary culture of endothelial cells from rat brains with the aim of evaluating the activation and the alterations of these cells by N. fowleri. We proved that the rat develops the infection similar to the mouse model. We also found that amoebic cysteine proteases produced by the trophozoites and the conditioned medium induced cytopathic effect in the endothelial cells. In addition, N. fowleri can decrease the transendothelial electrical resistance by triggering the destabilization of the tight junction proteins claudin-5, occludin, and ZO-1 in a time-dependent manner. Furthermore, N. fowleri induced the expression of VCAM-1 and ICAM-1 and the production of IL-8, IL-1ß, TNF-α, and IL-6 as well as nitric oxide. We conclude that N. fowleri damaged the blood-brain barrier model by disrupting the intercellular junctions and induced the presence of inflammatory mediators by allowing the access of inflammatory cells to the olfactory bulbs.

An Acute Case of Granulomatous Amoebic Encephalitis-Balamuthia mandrillaris Infection.

A 74-year-old woman who exhibited drowsiness was referred to our hospital. Enhanced head magnetic resonance imaging (MRI) revealed multiple ring-enhancing lesions and lesions showing partial mild hemorrhaging. The patient gradually progressed to a comatose condition with notable brain deterioration of unknown cause on follow-up MRI. On day nine, the patient inexplicably died, although brain herniation was suspected. Autopsy and histopathology revealed numerous amoebic trophozoites in the perivascular spaces and within the necrotic tissue. Brain immunostaining tested positive for Balamuthia mandrillaris. Infection due to free-living amoeba is rare in Japan; however, it may increase in the near future due to unknown reasons.

Relation between acetylcholinesterase and Na+, K+-ATPase activities with impaired memory of mice experimentally infected by Trypanosoma cruzi.

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and causes severe cardiac and brain damage, leading to behavioral alterations in humans and animals. However, the mechanisms involved in memory impairment during T. cruzi infection remain unknown. It has long been recognized that the enzymatic activities of acetylcholinesterase (AChE) and Na+, K+-ATPase are linked with memory dysfunction during other trypanosomiasis. Thus, the aim of this study was to evaluate the involvement of cerebral AChE and Na+, K+-ATPase activities in the memory impairment during T. cruzi (Colombian strain) infection. A significant decrease on latency time during the inhibitory avoidance task was observed in animals infected by T. cruzi compared to uninfected animals, findings compatible to memory dysfunction. Moreover, the cerebral AChE activity increased, while the Na+, K+-ATPase decreased in T. cruzi infected compared to uninfected animals. Histopathology revealed mild to moderate multifocal gliosis in the cerebral cortex and light focal meningeal lymphoplasmacytic infiltrate, which may have contributed to memory loss. Based on these evidences, we can conclude that T. cruzi (Colombian strain) causes memory impairment in mice experimentally infected. Moreover, the changes in AChE and Na+, K+-ATPase activities may be considered a mechanism involved in disease pathogenesis.

Identification of Naegleria fowleri proteins linked to primary amoebic meningoencephalitis.

Naegleria fowleri (N. fowleri) causes primary amoebic meningoencephalitis, a rapidly fatal disease of the central nervous system. N. fowleri can exist in cyst, flagellate or amoebic forms, depending on environmental conditions. The amoebic form can invade the brain following introduction into the nasal passages. When applied intranasally to a mouse model, cultured N. fowleri amoebae exhibit low virulence. However, upon serial passage in mouse brain, the amoebae acquire a highly virulent state. In the present study, a proteomics approach was applied to the identification of N. fowleri amoeba proteins whose expression was associated with the highly virulent state in mice. Mice were inoculated intranasally with axenically cultured amoebae or with mouse-passaged amoebae. Examination by light and electron microscopy revealed no morphological differences. However, mouse-passaged amoebae were more virulent in mice as indicated by exhibiting a two log10 titre decrease in median infective dose 50 (ID50). Scatter plot analysis of amoebic lysates revealed a subset of proteins, the expression of which was associated with highly virulent amoebae. MS-MS indicated that this subset contained proteins that shared homology with those linked to cytoskeletal rearrangement and the invasion process. Invasion assays were performed in the presence of a select inhibitor to expand on the findings. The collective results suggest that N. fowleri gene products linked to cytoskeletal rearrangement and invasion may be candidate targets in the management of primary amoebic meningoencephalitis.

Meningoencephalitis caused by pathogenic Sarcocystis species in a naturally infected sheep in Turkey.

A 3-year-old sheep was examined after an acute onset of hind limb paralysis and ataxia. At necropsy, central nervous system, pulmonary and intestinal hyperaemia and ecchymoses in the aortic arch were observed. Main microscopic lesions were confined to the heart, cerebrum and cerebellum. There were a multifocal mild myocarditis and nonsuppurative meningoencephalitis together with protozoal cysts in the heart and the brain. Protozoal cystic structures were observed within many of the myocardial fibers as well as in the cerebrum and cerebellum. Using light microscopy it could not be morphologically determined whether these organisms were Toxoplasma (T.) gondii or Neospora (N.) caninum. Additional diagnostic methods like immunohistochemistry and polymerase chain reaction provided differentiation of Sarcocystis from T. gondii and N. caninum. Transmission electron microscopy demonstrated characteristic features of Sarcocystis sp. as previously described. This is the first confirmed diagnosis of Sarcocystis sp. in the central nervous system of a sheep from Turkey.

Cutaneous Balamuthia mandrillaris infection as a precursor to Balamuthia amoebic encephalitis (BAE) in a healthy 84-year-old Californian.

Soil and freshwater-dwelling amoebae may opportunistically infect the skin and evoke a granulomatous dermatitis that camouflages their underlying morphology. Amoebic infestations are incredibly rare in the U.S., predominantly occurring in the young, elderly, and immunocompromised. Sadly, because diagnosis is difficult and unsuspected, most cases are diagnosed at autopsy. The following case is of a healthy 84-year-old man with a non-healing nodulo-ulcerative cutaneous lesion on his left forearm that appeared following a gardening injury. Lesional punch biopsies repeatedly showed non-specific granulomatous inflammation with no pathogens evident histologically or by culture. Histopathologic diagnosis was made five months after initial presentation via identification of amoebic trophozoite forms in tissue from a large excisional specimen. Anti-amoebic therapy was initiated immediately. The patient experienced mental status changes three days following lesion excision, with evidence of a cystic mass in the left medial parieto-occipital lobe by CT. Both intraoperative brain biopsies and cutaneous tissue samples tested positive for Balamuthia mandrillaris by indirect immunofluorescent antibody assay performed at the Centers for Disease Control. The patient achieved a full recovery on a triple antibiotic regimen. Clinical suspicion and thorough histopathologic analysis may determine the difference between survival and death for a patient presenting with a treatment-refractory localized granulomatous lesion.

Iron-Binding Protein Degradation by Cysteine Proteases of Naegleria fowleri.

Naegleria fowleri causes acute and fulminant primary amoebic meningoencephalitis. This microorganism invades its host by penetrating the olfactory mucosa and then traveling up the mesaxonal spaces and crossing the cribriform plate; finally, the trophozoites invade the olfactory bulbs. During its invasion, the protozoan obtains nutrients such as proteins, lipids, carbohydrates, and cationic ions (e.g., iron, calcium, and sodium) from the host. However, the mechanism by which these ions are obtained, particularly iron, is poorly understood. In the present study, we evaluated the ability of N. fowleri to degrade iron-binding proteins, including hololactoferrin, transferrin, ferritin, and hemoglobin. Zymography assays were performed for each substrate under physiological conditions (pH 7 at 37°C) employing conditioned medium (CM) and total crude extracts (TCEs) of N. fowleri. Different degradation patterns with CM were observed for hololactoferrin, transferrin, and hemoglobin; however, CM did not cause ferritin degradation. In contrast, the TCEs degraded only hololactoferrin and transferrin. Inhibition assays revealed that cysteine proteases were involved in this process. Based on these results, we suggest that CM and TCEs of N. fowleri degrade iron-binding proteins by employing cysteine proteases, which enables the parasite to obtain iron to survive while invading the central nervous system.

Pathogenesis of amoebic encephalitis: Are the amoebae being credited to an 'inside job' done by the host immune response?

Pathogenic free living amoeba like Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris are known to cause fatal "amoebic meningoencephalitis" by acquiring different route of entries to the brain. The host immune response to these protist pathogens differs from each another, as evidenced by the postmortem gross and microscopic findings from the brains of the affected patients. Cited with the expression of 'brain eating amoeba' when the infection is caused by N. fowleri, this expression is making its way into parasitology journals and books. The impression that it imparts is, as if the brain damage is substantially due to the enzymes and toxins produced by this amoeba. A detailed review of the literature, analysis of archived specimens and with our experimental assays, here we establish that with N. fowleri, Acanthamoeba and Balamuthia spp., the infections result in an extensive brain damage that in fact is substantially caused by the host immune response rather than the amoeba. Due to the comparatively larger sizes of these pathogens and the prior exposure of the amoebal antigen to the human body, the host immune system launches an amplified response that not only breaches the blood brain barrier (BBB), but also becomes the major cause of brain damage in Amoebic meningoencephalitis. It is our understanding that for N. fowleri the host immune response is dominated by acute inflammatory cytokines and that, in cases of Acanthamoeba and Balamuthia spp., it is the type IV hypersensitivity reaction that fundamentally not only contributes to disruption and leakiness of the blood brain barrier (BBB) but also causes the neuronal damage. The further intensification of brain damage is done by toxins and enzymes secreted by the amoeba, which causes the irreversible brain damage.

An autopsy case of Balamuthia mandrillaris amoebic encephalitis, a rare emerging infectious disease, with a brief review of the cases reported in Japan.

Balamuthia mandrillaris is an amoeba found in fresh water and soil that causes granulomatous amoebic encephalitis. We report herein an autopsy case of B. mandrillaris amoebic encephalitis, which was definitely diagnosed by PCR. An 81-year-old man, who had Sjögren's syndrome, manifested drowsiness 2 months before his death with progressive deterioration. Neuroimaging demonstrated foci of T2- and fluid-attenuated inversion recovery high and T1 low-intensity with irregular post-contrast ring enhancement in the cerebral hemisphere, thalamus and midbrain. Pathologically, multiple hemorrhagic and necrotic lesions were found in the cerebrum, thalamus, midbrain, pons, medulla and cerebellum, which were characterized by liquefactive necrosis, marked edema, hemorrhage and necrotizing vasculitis associated with the perivascular accumulation of amoebic trophozoites, a few cysts, and the infiltration of numerous neutrophils and microglia/macrophages. The trophozoites were ovoid or round, 10-60 µm in diameter, and they showed foamy cytoplasm and a round nucleus with small karyosome in the center. The PCR and immunohistochemistry from paraffin-embedded brain specimens revealed angioinvasive encephalitis due to B. mandrillaris. Human cases of B. mandrillaris brain infection are rare in Japan, with only a few brief reports in the literature.

Autopsy case of amebic granulomatous meningoencephalitis caused by Balamuthia mandrillaris in Japan.

Balamuthia mandrillaris is a free-living ameba that causes amebic encephalitis. Herein, we report an autopsy case of Balamuthia encephalitis proven with polymerase chain reaction (PCR) and immunohistochemistry from paraffin-embedded brain biopsy specimens. A 68-year-old Japanese male presented at a hospital with progressive right hemiparesis approximately 3 months before his death. An open-brain biopsy specimen showed diffuse meningitis with massive coagulative necrosis. The perivascular spaces contained numerous lymphocytes, histiocytes and giant cells, although the etiology was not determined. The patient deteriorated into coma and died from cerebral herniation. Autopsy revealed abundant trophozoites and cysts in the subarachnoid and Virchow-Robin's spaces. Electron-micrographs of the amebic cysts showed a characteristic triple-walled envelope. The amebas were identified as Balamuthia mandrillaris based on immunohistochemical analysis from the autopsy and biopsy specimens. Primer sets designed to amplify approximately 200 bp bands of mitochondrial 16S rRNA gene of Balamuthia by PCR produced positive results from the biopsy specimens but negative results from the autopsy specimens. In summary, PCR to amplify shorter segments of DNA may be of diagnostic value in detecting suspected cases of balamuthiasis in formalin-fixed, paraffin-embedded specimens. Increased awareness and timely diagnosis of Balamuthia encephalitis might lead to earlier initiation of therapy and improved outcome.

Granulomatous amebic encephalitis: an under-recognized cause of infectious mortality after hematopoietic stem cell transplantation.

Granulomatous amebic encephalitis (GAE) is a rare, nearly always fatal form of encephalitis that occurs mostly in the setting of immune compromise or chronic disease. The prevalence and clinical characteristics of this Acanthamoeba infection in hematopoietic stem cell transplant (HSCT) recipients are not well described. We present an HSCT patient in whom the diagnosis of GAE was made at autopsy. A systematic review of previously reported cases is provided to highlight the clinical presentation and early diagnostic features of GAE in HSCT recipients. Amebic infection usually initially involves the skin or lungs over a period of months, and becomes rapidly fatal once it crosses the blood-brain barrier. GAE is usually discovered postmortem owing to lack of awareness of this deadly infection and delay in diagnosis. Subacute presentation of multiple recurrent panniculitis-like subcutaneous nodules associated with eosinophilia and a history of chronic rhinitis or sinusitis warrant investigation for a possible amebic infection. Prolonged corticosteroid use and a recent exposure to unhygienic water are potential risk factors for GAE. Successful outcomes may be achieved with early intensive treatment using a combination of effective drugs.

Acanthamoeba meningoencephalitis in an immunocompetent patient: an autopsy case report.

Chronic granulomatous CNS infections may be caused by tuberculosis, fungi and rarely by free-living amoeba, especially in immunocompromised individuals. We report a rare, fatal case of granulomatous amoebic encephalitis in an immunocompetent patient mimicking CNS tuberculosis, and review the imageological features and diagnostic tests.

Diagnostic exercise. Cerebral mass in a puppy with respiratory distress and progressive neurologic signs.

A 5-month-old mongrel puppy with a history of respiratory disease presented with progressive neurologic dysfunction. Hematologic results included leukocytosis (neutrophilia with a left shift) and lymphopenia. A mass in the right forebrain, identified by magnetic resonance imaging, was biopsied during decompressive craniectomy. The histologic diagnosis was granulomatous meningoencephalitis with intralesional amoebae. The dog died within 24 hours of surgery. At necropsy, a well-demarcated granuloma was confined to the cerebrum, but granulomatous pneumonia was disseminated through all lobes of the lung. Concurrent infections included canine distemper, canine adenoviral bronchiolitis, and oral candidiasis. Canine distemper virus probably caused immunosuppression and increased susceptibility to secondary infections.

Astrocytic and microglial response and histopathological changes in the brain of horses with experimental chronic Trypanosoma evansi infection / Resposta astrocítica e microglial e alterações histopatológicas no sistema nervoso central de eqüinos infectados cronicamente com Trypanosoma evansi

This study aimed to characterize astrocytic and microglial response in the central nervous system (CNS) of equines experimentally infected with T. evansi. The experimental group comprised males and females with various degrees of crossbreeding, ages between four and seven years. The animals were inoculated intravenously with 10(6) trypomastigotes of T. evansi originally isolated from a naturally infected dog. All equines inoculated with T. evansi were observed until they presented symptoms of CNS disturbance, characterized by motor incoordination of the pelvic limbs, which occurred 67 days after inoculation (DAI) and 124 DAI. The animals in the control group did not present any clinical symptom and were observed up to the 125th DAI. For this purpose the HE histochemical stain and the avidin biotin peroxidase method was used. Lesions in the CNS of experimentally infected horses were those of a wide spread non suppurative meningoencephalomyelitis.The severity of lesions varied in different parts of the nervous system, reflecting an irregular distribution of inflammatory vascular changes. The infiltration of mononuclear cells was associated with anisomorphic gliosis and reactive microglia was identified. The intensity of the astrocytic response in the CNS of the equines infected by T. evansi characterizes the importance of the performance of these cells in this trypanosomiasis. The characteristic gliosis observed in the animals in this experiment suggests the ability of these cells as mediators of immune response. The parasite, T. evansi, was not identified in the nervous tissues.

Este estudo objetivou caracterizar a participação astrocítica e microglial no sistema nervoso central (SNC) de eqüinos experimentalmente infectados com T. evansi. O grupo experimental foi formado por machos e fêmeas com vários graus de cruzamentos e idade variando entre quatro e sete anos. Os animais foram inoculados com 10(6) tripomastigotas de T. evansi, originalmente isolada de um cão infectado naturalmente. Todos os eqüinos inoculados foram observados até o aparecimento dos sintomas neurológicos, caracterizados por incoordenação motora dos membros pélvicos, o qual ocorreu entre 67 e 124 dias após a inoculação (DPI). Os animais do grupo controle não apresentaram sinais clínicos e foram observados até o 125º DPI. Para este propósito, foram utilizados os métodos histoquímicos (HE) e imunoistoquímicos do complexo avidina-biotina peroxidase (ABC). A lesão no sistema nervoso central (SNC) dos eqüinos infectados com T. evansi foi caracterizada como meningoencefalomielite não supurativa. A gravidade das lesões variou em diferentes segmentos do SNC, refletindo distribuição irregular das alterações vasculares. Infiltrado perivascular e meníngeo foi associado a gliose anisomórfica e microgliose reativa. A intensidade da resposta astrocítica no SNC dos equinos infectados com T. evansi caracteriza a importância da performance destas células nas tripanossomíases. A gliose observada nos animais deste experimento sugerem a habilidade destas células como mediadoras da resposta imune. T. evansi não foi identificado no parênquima do SNC.