Balamuthia Amoebic Encephalitis Research Articles

Metagenomic next-generation sequencing for diagnosis of fatal Balamuthia amoebic encephalitis

IntroductionBalamuthia amoebic encephalitis (BAE), caused by Balamuthia mandrillaris, is a rare and life-threatening infectious disease with no specific and effective treatments available. The diagnosis of BAE at an early stage is difficult because of the non-specific clinical manifestations and neuroimaging. Case descriptionA 52-year-old male patient, who had no previous history of skin lesions, presented to the emergency department with an acute headache, walking difficulties, and disturbance of consciousness. The patient underwent a series of examinations, including regular cerebrospinal fluid (CSF) studies and magnetic resonance imaging, and tuberculous meningoencephalitis was suspected. Despite being treated with anti-TB drugs, no clinical improvement was observed in the patient. Following corticosteroid therapy, the patient developed a rapid deterioration in consciousness with dilated pupils. Metagenomic next-generation sequencing (mNGS) revealed an unexpected central nervous system (CNS) amoebic infection, and the patient died soon after the confirmed diagnosis. ConclusionThis study highlights the application of mNGS for the diagnosis of patients with suspected encephalitis or meningitis, especially those caused by rare opportunistic infections.

Pathogenic free-living amoebic encephalitis from 48 cases in China: A systematic review.

Free-living amoebae (FLA) including Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris can become pathogenic and cause severe cerebral infections, named primary amoebic meningoencephalitis (PAM), granulomatous amoebic encephalitis (GAE), and balamuthia amoebic encephalitis (BAE), respectively. FLA encephalitis has been reported across China, but the clinical data descriptions and analytical results of these different reports vary widely. Currently, no consensus treatment has been established. We conduct a systematic review to evaluate the exposure location, clinical symptoms, diagnosis, treatment, and prognosis of three FLA encephalitis and aim to reveal the differences between three FLA encephalitis in China. We used MEDLINE (PubMed interface), EMBASE, China National Knowledge Infrastructure (CNKI), Wanfang database, and China Biology Medicine disc (CBMdisc) databases for literatures published and manually retrieve the hospital records of our hospital. The search time was up to August 30, 2022, with no language restrictions. After excluding possible duplicate cases, a total of 48 patients of three FLA encephalitis were collected. One from the medical records of our hospital and 47 patients from 31 different studies. There were 11 patients of PAM, 10 patients of GAE, and 27 patients of BAE. The onset of PAM is mostly acute or subacute, and the clinical symptoms are acute and fulminant hemorrhagic meningoencephalitis. Most patients with GAE and BAE have an insidious onset and a chronic course. A total of 21 BAE patients (77.8%) had skin lesions before onset of symptoms. Additionally, 37 cases (77.1%) were diagnosed with FLA encephalitis before death. And there were 4 of PAM, 2 of GAE, and 10 of BAE diagnosed using next generation sequencing. No single agent can be proposed as the ideal therapy by itself. Only 6 cases were successfully treated. This review provides an overview of the available data and studies of FLA encephalitis in China and identify some potential differences. FLA encephalitis is a rare but pathogenic infection, and physicians should early identify this encephalitis to improve survival.

Drugs used for the treatment of cerebral and disseminated infections caused by free-living amoebae.

Free‐living amoebae (FLAs) are protozoa developing autonomously in diverse natural or artificial environments. The FLAs Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria fowleri represent a risk for human health as they can become pathogenic and cause severe cerebral infections, named granulomatous amoebic encephalitis (GAE), Balamuthia amoebic encephalitis (BAE), and primary amoebic meningoencephalitis (PAM), respectively. Additionally, Acanthamoeba sp. can also rarely disseminate to diverse organs, such as the skin, sinuses, or bones, and cause extracerebral disseminated acanthamebiasis (EDA). No consensus treatment has been established for cerebral FLA infections or EDA. The therapy of cerebral and disseminated FLA infections often empirically associates a large diversity of drugs, all exhibiting a high toxicity. Nevertheless, these pathologies lead to a high mortality, above 90% of the cases, even in the presence of a treatment. In the present work, a total of 474 clinical cases of FLA infections gathered from the literature allowed to determine the frequency of usage, as well as the efficacy of the main drugs and drug combinations used in the treatment of these pathologies. The efficacy of drug usage was determined based on the survival rate after drug administration. The most efficient drugs, drug combinations, and their mechanism of action were discussed in regard to the present recommendations for the treatment of GAE, EDA, BAE, and PAM. At the end, this review aims to provide a useful tool for physicians in their choice to optimize the treatment of FLA infections.

Discovery of repurposing drug candidates for the treatment of diseases caused by pathogenic free-living amoebae.

Diseases caused by pathogenic free-living amoebae include primary amoebic meningoencephalitis (Naegleria fowleri), granulomatous amoebic encephalitis (Acanthamoeba spp.), Acanthamoeba keratitis, and Balamuthia amoebic encephalitis (Balamuthia mandrillaris). Each of these are difficult to treat and have high morbidity and mortality rates due to lack of effective therapeutics. Since repurposing drugs is an ideal strategy for orphan diseases, we conducted a high throughput phenotypic screen of 12,000 compounds from the Calibr ReFRAME library. We discovered a total of 58 potent inhibitors (IC50 <1 μM) against N. fowleri (n = 19), A. castellanii (n = 12), and B. mandrillaris (n = 27) plus an additional 90 micromolar inhibitors. Of these, 113 inhibitors have never been reported to have activity against Naegleria, Acanthamoeba or Balamuthia. Rapid onset of action is important for new anti-amoeba drugs and we identified 19 compounds that inhibit N. fowleri in vitro within 24 hours (halofuginone, NVP-HSP990, fumagillin, bardoxolone, belaronib, and BPH-942, solithromycin, nitracrine, quisinostat, pabinostat, pracinostat, dacinostat, fimepinostat, sanguinarium, radicicol, acriflavine, REP3132, BC-3205 and PF-4287881). These compounds inhibit N. fowleri in vitro faster than any of the drugs currently used for chemotherapy. The results of these studies demonstrate the utility of phenotypic screens for discovery of new drugs for pathogenic free-living amoebae, including Acanthamoeba for the first time. Given that many of the repurposed drugs have known mechanisms of action, these compounds can be used to validate new targets for structure-based drug design.

Diagnosing Balamuthia mandrillaris encephalitis via next-generation sequencing in a 13-year-old girl

ABSTRACT Balamuthia amoebic encephalitis has a subacute-to-chronic course and is almost invariably fatal owing to delayed diagnosis and a lack of effective therapy. Here, we report a 13-year-old girl with cutaneous lesions and multifocal granulomatous encephalitis. The patient underwent a series of tests and was suspected as having tuberculosis. She was treated with various empiric therapies without improvement. She was finally correctly diagnosed via next-generation sequencing of the cerebrospinal fluid. The patient deteriorated rapidly and died 2 months after being diagnosed with Balamuthia mandrillaris encephalitis. This study highlights the important clinical significance of next-generation sequencing, which provides better diagnostic testing for unexplained paediatric encephalitis, especially that caused by rare or emerging pathogens.

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.

Presence of Balamuthia mandrillaris in hot springs from Mazandaran province, northern Iran.

Balamuthia mandrillaris is an opportunistic free-living amoeba that has been reported to cause cutaneous lesions and Balamuthia amoebic encephalitis. The biology and environmental distribution of B. mandrillaris is still poorly understood and isolation of this pathogen from the environment is a rare event. Previous studies have reported that the presence of B. mandrillaris in the environment in Iran may be common. However, no clinical cases have been reported so far in this country. In the present study, a survey was conducted in order to evaluate the presence of B. mandrillaris in hot-spring samples of northern Iran. A total of 66 water samples were analysed using morphological and molecular tools. Positive samples by microscopy were confirmed by performing PCR amplification of the 16S rRNA gene of B. mandrillaris. Sequencing of the positive amplicons was also performed to confirm morphological data. Two of the 66 collected water samples were positive for B. mandrillaris after morphological and molecular identification. Interestingly, both positive hot springs had low pH values and temperatures ranging from 32°C to 42°C. Many locals and tourists use both hot springs due to their medicinal properties and thus contact with water bodies containing the organism increases the likelihood of infection. To the best of our knowledge, this is the first report on the isolation of B. mandrillaris from hot-spring sources related to human activity. Therefore, B. mandrillaris should be considered as a possible causative agent if cases of encephalitis are suspected following immersion in hot springs in addition to Acanthamoeba and Naegleria.

Detection of Balamuthia mandrillaris DNA in the storage case of contact lenses in Germany.

Acanthamoeba spp. are frequently the etiological agents of a severe form of sight-threatening keratitis, called Acanthamoeba keratitis. The contact lens storage solution of a patient with keratitis of unknown genesis was screened using our diagnostic tools to detect potentially pathogenic free-living amoebae (FLA). Culture methods and a triplex quantitative real-timepolymerase chain reaction (qPCR) targeting Acanthamoeba spp., Naegleria fowleri, and Balamuthia mandrillaris were used in context of this routine screening. While no amoebae were detected by culture, qPCR specifically detected DNA of B. mandrillaris. This FLA is known as the etiological agent of a fatal form of encephalitis in humans and other mammals, Balamuthia amoebic encephalitis (BAE). A fragment of the 18S rDNA gene was amplified from the sample and showed 99% sequence identity to B. mandrillaris sequences from GenBank. To the best of our knowledge, this is the first report of B. mandrillaris found in association with contact lenses. Although no viable amoeba was obtained by culturing efforts, the verification of B. mandrillaris DNA in the contact lens storage solution demonstrates how easily this pathogen might come into close contact with humans.

Isolation of Balamuthia mandrillaris from soil samples in North-Western Iran

Balamuthia mandrillaris is an opportunistic free-living amoebae (FLA) which has been reported as the causative agent of the fatal Balamuthia amoebic encephalitis (BAE). Currently, the transmission dynamics of this pathogen remain poorly understood although the organism has been associated with soils, and thus, soil exposure has been identified as a risk factor for this pathogenic amoeba. Nevertheless, environmental isolation of B. mandrillaris is a rare event and strains of this amoebic species have been isolated from soil and dust sources only in seven previous reports (Iran, Mexico, USA, Peru, Costa Rica, Guinea Bissau, and Jamaica). In Iran, a previous study reported the isolation of B. mandrillaris from dust collected in the city of Tehran and free of known infectious involvement. Therefore, in this work, 55 soil samples collected from public and recreational areas of East Azerbaijan, North-Western Iran, were investigated for the presence of this pathogen. Samples were cultured in 2% non-nutrient agar plates and were monitored for the presence of B. mandrillaris-like trophozoites and/or cysts. Those samples that were positive for these amoebae by microscopic criteria were then confirmed by PCR amplification and DNA sequencing of the mitochondrial 16S ribosomal DNA (rDNA) of B. mandrillaris. The obtained results revealed the presence of this emerging pathogen in 5 of 55 samples included in the study. Homology analysis of the obtained DNA sequences revealed high similarity with previously isolated strains from clinical and environmental sources. To the best of our knowledge, this is the first report on the isolation of B. mandrillaris from soil sources in Iran related to human activity and the second time that this pathogen is isolated from the environment in this country.

The isolation of Balamuthia mandrillaris from environmental sources from Peru

Balamuthia mandrillaris is an opportunistic free-living amoeba that has been reported to cause skin lesions and the fatal Balamuthia amoebic encephalitis (BAE) in humans and other animals. Currently, around 200 human BAE cases have been reported worldwide, although this number is considered to be underestimated. The highest number of BAE cases has been reported in the American continent, mainly in the southwest of the USA. Peru seems to be another hotspot for BAE with around 55 human cases having been identified, usually involving cutaneous infection, especially lesions in the central face area. The isolation of Balamuthia from environmental sources has been reported on only three prior occasions, twice from Californian soils and once from dust in Iran and so it seems that this amoeba is relatively rarely encountered in samples from the environment. We investigated that possibility of finding the amoebae in soil samples from different regions where clinical cases have been reported in Peru. Twenty-one samples were cultured in non-nutrient agar plates and were checked for the presence of B. mandrillaris-like trophozoites and/or cysts. Those samples that were positive for these amoebae by microscopic criteria were then confirmed by PCR amplification and DNA sequencing of the mitochondrial 16S rDNA gene of B. mandrillaris. We have detected the presence of B. mandrillaris in four samples collected in the regions of Piura (3) and Lima (1) where infection cases have been previously reported. We hypothesize that B. mandrillaris is present in Peru in soil and dust which therefore constitutes a source of the infection for the BAE cases previously reported in this country. Further studies should be carried out in the area to confirm the generality of this finding.

Essentials of Veterinary ParasitologyEssentials of Veterinary Parasitology. Hany M. Elsheikha and Naveed Ahmed Khan . Editors, Caister Academic Press, Norfolk, UK. 2011. 221 pp. ISBN 978-1-908230-07-2. US $169 (hardback), $99 (paperback).

Essentials of Veterinary Parasitology is an attempt by the authors to provide a concise, readily accessible source of clinically oriented information on the most important veterinary parasites of domesticated animals. Only those parasites of wildlife that also infect livestock and companion animals are included for discussion, limiting the value of this book for those seeking information specifically on wildlife. The book is intended for use by veterinary students as a study aid and by veterinarians working in a traditional food production or companion animal practice as a reference source. For anyone involved in teaching in a veterinary school curriculum, the exercise of contemplating which specific parasites and what amount of information should be considered ‘‘essential’’ in the training of veterinarians is both useful and necessary, and in this respect I found the book thought provoking. In my experience in teaching, students tend to be overwhelmed by the sheer weight of numbers and the volume of information associated with the study of veterinary parasitology and would welcome a source offering just the ‘‘essentials’’ on this subject. The book is divided into chapters on basic concepts, diseases associated with specific parasites, diagnostic methodology, and principles of control. I think there would be a broad consensus of agreement among veterinary parasitologists with the majority of the parasites chosen for inclusion in this book. Perhaps a personal preference on my part, I would rather have seen the inclusion of some of the trematodes infecting companion animals (of which none are mentioned) instead of the sections on Balamuthia amoebic encephalitis and Rhinosporidiosis. For the most part, the text is clearly written, and the life cycle diagrams and many of the parasite images are well done, although in general, I would have liked to see more included. In some sections, brevity comes at the expense of necessary detail (e.g., the treatment for caval syndrome due to Dirofilaria immitis infection is given, but there is no mention of pathogenesis or diagnosis of the syndrome; the discussion on Isospora spp. is likely to mislead readers into thinking they are zoonotic parasites). Additionally, the information presented in the chapters on mange mites and lice is almost entirely from a food production animal practice perspective and would not serve the needs of a clinician in companion animal practice. The authors have done an admirable job of maintaining a worldwide perspective in most of the chapters; however, the North American reader will find the chapter on tick-borne diseases to be focused almost entirely on the situation in Europe and the United Kingdom. Particularly well done are the chapters on control. Provided the veterinary student or clinician is mindful that this book is not intended to be a complete encyclopedic treatise on the subject, he or she may find this book a helpful reference source or study aid, although on occasion further information will need to be sought elsewhere.

Impact of macrophages on Balamuthia mandrillaris virulence properties using human brain microvascular endothelial cells in vitro

Balamuthia amoebic encephalitis (BAE) is a serious human disease almost always leading to death. An important step in BAE is amoebae invasion of the bloodstream, followed by their haematogenous spread. Balamuthia mandrillaris entry into the central nervous system (CNS) most likely occurs at the blood–brain barrier (BBB) sites. Macrophages are thought to be the first line of defense in many infectious diseases and are present in high numbers during infections. The objective of the present study was to determine the impact of cytokines and macrophages on the virulence characteristics of B. mandrillaris in vitro.

Association of ABO blood groups and complicated Plasmodium falciparum malaria in Accra, Ghana

Background: Balamuthia amoebic encephalitis (BAE) is a serius human disease (caused by Balamuthia mandrillaris) almost lways leading to death. An important step in BAE is amoebae nvasion of the bloodstream, followed by their haematogenous pread. Balamuthia mandrillaris entry into the central nervous sysem (CNS)most likely occurs at the blood–brain barrier (BBB) sites. acrophages are thought to be the first line of defense in many nfectious diseases and are present in high numbers during infecions. The objective of the present study was to determine the mpact of cytokines and macrophages on the virulence characterstics of B. mandrillaris in vitro. Methods: In vitro, B. mandrillaris were used to demonstrate he effects of cytokines and macrophages on the physiological nd morphological characteristics of amoeba. Using human brain icrovascular endothelial cells (HBMEC), which constitutes the lood–brain barrier, adhesion and cytotoxicity assays were perormed. To investigate the engulfing property of the amoeba, hagocytosis assays were performed using fluorescein isothioyanate (FITC) labeledE. coliK12.Moreover zymographyassaywere lso used to observe the proteolytic activity of amoeba. Results: It was observed B. mandrillaris exhibited >90% binding nd>70% cytotoxicity toHBMECwhichwas further enhanced in the resence of cytokines and macrophages. It has also been observed hat cytokines TNFand TGFsignificantly increased the B. manrillaris numbers in the presence ofmacrophages. It is important to ote that amoebic numbers were more than doubled in the presnce of cytokines and macrophages within 24h. We have shown in he past the bacteria uptake by B. mandrillaris is limited which is urther significantly inhibited in the presence of cytokines during hagocytosis assays. Zymography assays revealed that cytokines nd macrophages have no inhibitory effect on proteolytic activty of B. mandrillaris. In addition the activated macrophages also ould not show any vital inhibitory effects on amoebic virulence roperties. Conclusion: Overall we described for the first time that ytokines and macrophages has no inhibitory effects on the virlence properties of B. mandrillaris in vitro.

Impact of macrophages on Balamuthia mandrillaris virulence properties using human brain microvascular endothelial cells in vitro

Balamuthia amoebic encephalitis (BAE) is a serious human disease almost always leading to death. An important step in BAE is amoebae invasion of the bloodstream, followed by their haematogenous spread. Balamuthia mandrillaris entry into the central nervous system (CNS) most likely occurs at the blood–brain barrier (BBB) sites. Macrophages are thought to be the first line of defense in many infectious diseases and are present in high numbers during infections. The objective of the present study was to determine the impact of cytokines and macrophages on the virulence characteristics of B. mandrillaris in vitro.

Balamuthia and Acanthamoeba-binding antibodies in West African human sera

Little is known about the prevalence of Balamuthia mandrillaris amoebae and Balamuthia amoebic encephalitis in Africa. As an approach, relative concentrations of amoebae-binding serum antibodies (Ab) were assessed by flow cytometry using formaldehyde-fixed B. mandrillaris, Acanthamoeba lenticulata 72-2 and Acanthamoeba castellanii 1BU amoebae for specific Ab capture (B.m.-Ab, A.l.-Ab, A.c.-Ab). One hundred and ninety-two sera from West African (Côte d’Ivoire) donors aged 11–95 years (mean 38 a; 51% males), and living in villages surrounded by rainforest near the Liberian border, were tested and related to reference sera from Berlin. While B.m.-Ab tended to increase with donor age, A.l.-Ab and A.c.-Ab did not. Accordingly, B.m.-Ab correlated only weakly with A.l.-Ab or A.c.-Ab. Of the nine individuals with the highest B.m.-Ab concentrations, most were elderly (mean 58 a), male (78%), and professed intensive outdoor activity (hunting, farming). Only three of these sera also showed elevated A.l.-Ab, and none elevated A.c.-Ab.

Isolation of Balamuthia mandrillaris from urban dust, free of known infectious involvement

The free-living amoeba Balamuthia mandrillaris can cause fatal encephalitis in humans and other mammals. The organism is associated with soils, and soil exposure has been identified as a risk factor for this pathogen. However, B. mandrillaris has been isolated only once from soils believed to be the source of the infection in child from California, USA who died of Balamuthia amoebic encephalitis and once from another unrelated soil source. We report for a third time the isolation of B. mandrillaris from the environment and for the second time its isolation from a sample not known to be involved with pathogenicity. We have established the new clonal B. mandrillaris strain (ID-19) in axenic media. The identity of our isolate was originally by morphology using a light microscope and this has been confirmed by 16S rRNA gene PCR. The new strain ID-19 groups with others of the species. The fact that our isolate came from dust particles deposited on surfaces from the air in an urban environment may suggest that it is not just soil exposure that constitutes a risk factor for Balamuthia infection. This is the first report of this organism from Iran.

The cyst wall carbohydrate composition of Balamuthia mandrillaris

Balamuthia mandrillaris is an opportunistic cyst-producing amoeba that can cause rare, but fatal, Balamuthia amoebic encephalitis (BAE). Cysts are resistant to harsh environmental conditions and many antimicrobial compounds and thus can contribute to BAE recurrence. However, little is known of cyst wall synthesis, cyst wall composition, or how encystment is induced. In this study, we examined the carbohydrate composition of the cyst wall. The major components were mannose (20.9 mol%) and glucose (79.1 mol%), with trace amounts of galactose present in the cyst wall samples analysed. The linkage analysis showed cyst wall carbohydrates with apparently linear and branching saccharides and suggested the presence of cellulose. These components may play an important protective role by creating a permeability barrier around the cyst.

Balamuthia mandrillaris interactions with human brain microvascular endothelial cells in vitro

Balamuthia amoebic encephalitis (BAE) is a serious human disease almost always leading to death. An important step in BAE is amoebae invasion of the bloodstream, followed by their haematogenous spread. Balamuthia mandrillaris entry into the central nervous system most likely occurs at the blood-brain barrier sites. Using human brain microvascular endothelial cells (HBMECs), which constitute the blood-brain barrier, this study determined (i) the ability of B. mandrillaris to bind to HBMECs and (ii) the associated molecular mechanisms. Adhesion assays revealed that B. mandrillaris exhibited greater than 90 % binding to HBMECs in vitro. To determine whether recognition of carbohydrate moieties on the surface of the HBMECs plays a role in B. mandrillaris adherence to the target cells, adhesion assays were performed in the presence of the saccharides mannose, galactose, xylose, glucose and fucose. It was observed that adherence of B. mandrillaris was significantly reduced by galactose, whilst the other saccharides had no effect. Acetone fixation of amoebae, but not of HBMECs, abolished adhesion, suggesting that B. mandrillaris adhesin(s) bind to galactose-containing glycoproteins of HBMECs. B. mandrillaris also bound to microtitre wells coated with galactose-BSA. By affinity chromatography using a galactose-Sepharose column, a galactose-binding protein (GBP) was isolated from detergent extracts of unlabelled amoebae. The isolation of a GBP from cell-surface-biotin-labelled amoebae suggested its membrane association. One-dimensional SDS-PAGE confirmed the proteinaceous nature of the GBP and determined its molecular mass as approximately 100 kDa. This is the first report suggesting the role of a GBP in B. mandrillaris interactions with HBMECs.

Successful Treatment of Balamuthia Amoebic Encephalitis: Presentation of 2 Cases

Successful Treatment of Balamuthia Amoebic Encephalitis: Presentation of 2 Cases

Balamuthia mandrillaris from soil samples.

Balamuthia mandrillaris amoebas are recognized as a causative agent of granulomatous amoebic encephalitis, a disease that is usually fatal. They were first recognized when isolated from the brain of a mandrill baboon that died in the San Diego Zoo Wild Life Animal Park. Subsequently, the amoebas have been found in a variety of animals, including humans (young and old, immunocompromised and immunocompetent persons), in countries around the world. Until recently, the amoebas had not been recovered from the environment and their free-living status was in question. The recovery of a Balamuthia amoeba from a soil sample taken from a plant at the home of a child from California, USA, who died of Balamuthia amoebic encephalitis, was reported previously. In a continued investigation, a second amoeba was isolated from soil that was obtained from an outdoor potted plant in a spatially unrelated location. A comparison of these two environmental amoebas that were isolated from different soils with the amoeba that was obtained from the child's clinical specimen is reported here. Included are the isolation procedure for the amoebas, their growth requirements, their immunological response to anti-Balamuthia serum, their sensitivity to a selection of antimicrobials and sequence analysis of their 16S rRNA gene. The evidence is consistent that the amoebas isolated from both soil samples and the clinical isolate obtained from the Californian child are B. mandrillaris.