Balamuthia Research Articles

Novel culture medium for the axenic growth of Balamuthia mandrillaris

Until now, for axenic cultivation of Balamuthia mandrillaris, the BM-3 culture medium and the Modified Chang's special medium have been the only ones recommended, but they have some disadvantages, as both require many components and their preparations are laborious. Therefore, we developed a novel culture medium for B. mandrillaris axenic cultivation. Each one of the 11 components of BM-3 was combined with Cerva's medium as basal culture medium. Ten strains of B. mandrillaris including the reference strain CDC:V039 and 9 environmental isolates were used during trials. After testing all combinations, the basal medium complemented with 10× Hank's balanced salt solution was the only one that supported confluent growth of B. mandrillaris. Cell shape and motility of trophozoites were normal. This developed medium is as useful as BM-3 for axenization. The development of a cheaper and easy-to-prepare medium for B. mandrillaris opens the possibility of increasing its study.

Balamuthia mandrillaris: Morphology, biology, and virulence.

Balamuthia mandrillaris is a protist pathogen that can cause encephalitis with a fatality rate of >95%. This is due to our incomplete understanding of the pathogenesis and pathophysiology of B. mandrillaris encephalitis. B. mandrillaris has two stages in its life cycle, an active trophozoite stage during which it divides mitotically. However, under unfavorable conditions, the trophozoite transforms into a dormant cyst stage. A major concern during the course of therapy is that B. mandrillaris can transform into cysts. Cysts are highly resistant to physical and chemical conditions and present a problem in successful antimicrobial chemotherapy. Several lines of evidence suggest that B. mandrillaris encephalitis develops as a result of hematogenous spread, but it is unclear how circulating amoebae enter the central nervous system and cause inflammation, blood-brain barrier disruption, and neuronal injury. Recent studies have identified several parasite-host determinants for B. mandrillaris translocation of the blood-brain barrier, and host inflammatory markers that may be associated with neuronal injury. These determinants may provide important targets for the prevention and treatment of this devastating infection. Here, we present a brief overview of the current understanding of the morphology, biology, pathogenesis, and pathophysiology of B. mandrillaris encephalitis.

Management of granulomatous amebic encephalitis: Laboratory diagnosis and treatment.

Granulomatous amebic encephalitis is a life-threatening central nervous system (CNS) infection caused by the free-living amoebae Acanthamoeba spp., Balamuthia mandrillaris and Sappinia pedata. The disease has a subacute to chronic onset affecting commonly the immunocompromised population with high mortality rate. The diagnosis of this disease entity requires high suspicion with appropriate sample collection and testing by the laboratory experts. Radiological investigations are nonspecific and commonly confused with CNS tuberculosis, neurocysticercosis, disseminated encephalomyelitis, viral encephalitis etc., delaying the accurate diagnosis of these cases. Early diagnosis plays a crucial role in the survival of these cases since appropriate management can be initiated. No single drug is effective; hence multiple antibiotics targeting various proteins or receptors are required for successful treatment. A combination of surgical and medical interventions involving multiple specialty experts is required to prevent death and morbidity in survivors.

Vannellid Species Isolated from Freshwater Source in a Park in Jamaica, West Indies.

Free-living amoebae (FLA) occupy a wide range of freshwater, marine, and soil habitats, and are opportunistic pathogens in human beings. While Acanthamoeba spp., Naegleria fowleri, and Balamuthia mandrillaris are well-known opportunistic organisms, Vannella epipetala is nonpathogenic. Sediments were collected from a freshwater source from a park in Jamaica to investigate the presence of FLA. Acanthamoeba and Naegleria spp. were not recovered; however, a Vannellid species identified by microscopy and PCR analysis as V. epipetala was isolated. These nonpathogens pose a threat to human beings as they may act as Trojan horses for microsporidian parasites and other pathogens, thereby facilitating their transmission to human beings.

Resistance to intranasal infection with Balamuthia mandrillaris amebae is T-cell dependent.

T and B cell-deficient BALB/c SCID mice become severely ill and die of amebic encephalitis after intranasal infection with Balamuthia mandrillaris, while adult immunocompetent BALB/c wild-type (WT) mice are resistant. To further investigate the role of lymphocytes in protection from Balamuthia amebic encephalitis (BAE), SCID mice were reconstituted with and WT mice selectively depleted of lymphocytes before infection. Reconstitution of SCID mice with whole spleen cells from WT mice rendered the recipients as resistant to BAE as WT mice. SCID mice that had received spleen cells depleted of CD4(+) T cells remained susceptible. When adult WT mice were depleted of both CD4(+) and CD8(+) T cells or of CD4(+) T cells alone, these mice also became susceptible to BAE. Depletion of CD8(+) T cells alone increased susceptibility only marginally. All morbidity and mortality data were underpinned by histological analysis of the brain.

Molecular Identification of Free-living Amoebae Isolated from Artificial Water Bodies Located in Poland

Free living amoebae (FLA) are amphizoic protozoa that are widely found in various environmental sources. They are known to cause serious human infections, including a fatal encephalitis, a blinding keratitis, and pneumonia. The main aim of the study was detection and molecular identification of Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris, Sappinia pedata, and Vermoamoeba vermiformis (formerly Hartmannella vermiformis) in artificial water bodies in North-Western Poland. We examined 86 water samples collected during 2-year period from 43 water bodies, including outdoor and indoor swimming pools, firefighting reservoirs, fountains, as well as water network. The samples were filtrated using Filta-Max® membrane filters (IDEXX Laboratories, USA) and, in order to select potentially pathogenic, thermophilic strains and to limit the number of PCR examined samples, the thermal tolerance test was carried out. Obtained filtrates were transferred to non-nutrient agar plates with E. coli. The agar plates were incubated at 37°C and then proliferated amoebae were passaged at 42°C. DNA was extracted from the thermophilic trophozoites and then polymerase chain reactions and sequence analysis were performed for molecular identification of FLA. From the 86 collected water samples 57 strains of FLA were able to proliferate at 37°C and 7 of them showed ability to proliferate at 42°C. For molecular identification of Acanthamoeba spp. and V. vermiformis, regions of 18S rDNA were amplified. In order to detect B. mandrillaris DNA, we used mitochondrial 16S rDNA as a marker, and for detection of N. fowleri and S. pedata – ITS regions. Based on molecular analysis, isolates were classified to the genus Acanthamoeba (T4 and T11 genotypes, as well as the new genotypes detected earlier in clinical samples and named T16) and V. vermiformis species. Detected strains were highly similar or identical to pathogenic strains detected earlier in patients. Our results show a wide distribution of potential pathogenic FLA, as Acanthamoeba T4, T11, T16 genotypes, and V. vermiformis species in various artificial water bodies located in North-Western Poland and suggest a potential threat to health of humans in this part of the country.

Solid Organ Transplant Donors With Central Nervous System Infection

While donor-derived infections (DDI) remain uncommon, multiple reports describe DDI with pathogens that cause central nervous system (CNS) infection resulting in significant recipient disease. The Ad Hoc Disease Transmission Advisory Committee (DTAC) reviewed the records of potential donor-derived disease transmission events (PDDTE) to describe donor characteristics and outcomes associated with DDI from CNS pathogens. All PDDTE reported from January 2008 to September 2010 were reviewed for characteristics suggesting CNS infection in the donor or the recipient. Identified cases were further examined to determine if donor CNS infection resulted in recipient infection. Ninety-one PDDTE cases in which there was concern for CNS infection in the donor or recipient were identified. Further review confirmed CNS infection in 12 donors, six of whom transmitted infection to 10 of 15 exposed recipients with five recipient deaths. Pathogens included Balamuthia mandrillaris, Cryptococcus neoformans, lymphocytic choriomeningitis virus, and West Nile virus. Listed cause of death at procurement for these donors included stroke, anoxia, acute disseminated encephalomyelitis, and meningoencephalitis. Confounding diagnoses were present in 6 of 12 donors that would have allowed them to be considered at low risk of transmitting a CNS pathogen. Of the six donors with no confounding conditions, three exhibited at least two suspicious "DTAC warning criteria" for CNS infection. Careful clinical assessment of donors combined with a high index of suspicion for ambiguous or misleading findings associated with CNS infection can reduce, but not eliminate, DDI with CNS pathogens.

Atomic force microscopic imaging of Acanthamoeba castellanii and Balamuthia mandrillaris trophozoites and cysts.

Light microscopy and electron microscopy have been successfully used in the study of microbes, as well as free-living protists. Unlike light microscopy, which enables us to observe living organisms or the electron microscope which provides a two-dimensional image, atomic force microscopy provides a three-dimensional surface profile. Here, we observed two free-living amoebae, Acanthamoeba castellanii and Balamuthia mandrillaris under the phase contrast inverted microscope, transmission electron microscope and atomic force microscope. Although light microscopy was of lower magnification, it revealed functional biology of live amoebae such as motility and osmoregulation using contractile vacuoles of the trophozoite stage, but it is of limited value in defining the cyst stage. In contrast, transmission electron microscopy showed significantly greater magnification and resolution to reveal the ultra-structural features of trophozoites and cysts including intracellular organelles and cyst wall characteristics but it only produced a snapshot in time of a dead amoeba cell. Atomic force microscopy produced three-dimensional images providing detailed topographic description of shape and surface, phase imaging measuring boundary stiffness, and amplitude measurements including width, height and length of A. castellanii and B. mandrillaris trophozoites and cysts. These results demonstrate the importance of the application of various microscopic methods in the biological and structural characterization of the whole cell, ultra-structural features, as well as surface components and cytoskeleton of protist pathogens.

Encephalitis caused by pathogens transmitted through organ transplants, United States, 2002-2013.

The cause of encephalitis among solid organ transplant recipients may be multifactorial; the disease can result from infectious or noninfectious etiologies. During 2002-2013, the US Centers for Disease Control and Prevention investigated several encephalitis clusters among transplant recipients. Cases were caused by infections from transplant-transmitted pathogens: West Nile virus, rabies virus, lymphocytic choriomeningitis virus, and Balamuthia mandrillaris amebae. In many of the clusters, identification of the cause was complicated by delayed diagnosis due to the rarity of the disease, geographic distance separating transplant recipients, and lack of prompt recognition and reporting systems. Establishment of surveillance systems to detect illness among organ recipients, including communication among transplant center physicians, organ procurement organizations, and public health authorities, may enable the rapid discovery and investigation of infectious encephalitis clusters. These transplant-transmitted pathogen clusters highlight the need for greater awareness among clinicians, pathologists, and public health workers, of emerging infectious agents causing encephalitis among organ recipients.

A multisystemic Acanthamoeba infection in a dog in Tenerife, Canary Islands, Spain

A 22-month-old male Spanish water dog was hospitalized after its physical examination revealed fever and movement difficulty. After 24h, the dog was found to have a high fever (39.5°C) and was treated empirically with doxycycline/ciprofloxacin. At 48h, after submission the fever rose to 41°C and the animal presented with a stiff neck and dehydration. Peripheral blood and cerebrospinal fluid (CSF) were sampled and trophozoites with an Acanthamoeba-like morphology were observed in the CSF. PCR specific for Acanthamoeba, Naegleria fowleri and Balamuthia mandrillaris were performed and the CSF sample found positive for Acanthamoeba. Lungs, kidney, liver and spleen samples were collected post mortem. All collected organ samples were positive for Acanthamoeba by PCR, thus confirming a multisystemic infection. Water samples taken at a suspected site of infection yielded an almost identical PCR fragment to those of the clinical samples, indicating that this was probably where the infection originated. This is the first report of a fatal case of Acanthamoeba disseminated infection in a dog in Spain.

Isolation and molecular characterization of Acanthamoeba and Balamuthia mandrillaris from combination shower units in Costa Rica.

Free living amoebae (FLA) are ubiquitous protozoa, which may behave as parasites under certain conditions. Four genera are recognized as causal agents of infections in humans and animals: Naegleria, Sappinia, Acanthamoeba and Balamuthia. This work determines the presence of FLA in combination shower units and employs molecular biology for the characterization of isolates. The morphological analysis and partial sequencing of the 18S rDNA gene revealed the presence of Acanthamoeba genotype T4 in 30% of the units sampled. In addition to Acanthamoeba cysts, trophozoites with morphological characteristics similar to Balamuthia were identified. PCR assay using the mitochondrial 16S rRNA gene as a target confirmed the identification of the amoeba as Balamuthia mandrillaris. Up to date, this is the first report of the isolation of B. mandrillaris in Central America and the fifth report worldwide.

Genetic analysis among environmental strains of Balamuthia mandrillaris recovered from an artificial lagoon and from soil in Sonora, Mexico

Since the first report of Balamuthia mandrillaris as a causative agent of granulomatous amoebic encephalitis in humans, the environmental niche of this amoeba was assumed to be restricted to soil and dust. A single isolation from water was recently made independently by us from Northern Mexico. Now we report the isolation of 8 new strains of B. mandrillaris from Mexico. This continues the pattern of an excess of isolates from North America, compared to other parts of the world. All of the new isolates are environmental isolates, 7 from water samples and one from soil. The identity of each isolate was confirmed by PCR and by examining the sequences of the mitochondrial 16S-like rRNA gene. Success in amplification was determined using comparisons of amplifications of DNA from the strain CDC: V039 and the water strain (ITSON-BM1) as positive controls. The DNA sequences of the new isolates were compared to older strains from clinical cases using phylogenetic analysis, showing very high sequence similarity. The similarity among the new isolates and with previous clinical and environmental isolates of B. mandrillaris was also examined using biochemical and immunological studies. High homogeneity of total protein products, and similarity in antigenic moiety among the eight new isolates and two controls was found. Taken together, the molecular and biochemical studies indicate very low levels of genetic variation within B. mandrillaris.

Longest Known Survival of a Kidney Transplant Recipient After Donor Transmission of Balamuthia Mandrillaris.

Longest Known Survival of a Kidney Transplant Recipient After Donor Transmission of Balamuthia Mandrillaris.

Presence of potentially pathogenic free-living amoebae strains from well water samples in Guinea-Bissau

Free-living amoebae (FLA) include opportunistic pathogens such as Naegleria fowleri, Balamuthia mandrillaris, and the genera Sappinia and Acanthamoeba. In this study, a survey was conducted in order to evaluate the presence of potentially pathogenic amoebic strains in water samples collected from wells located in the western part of Guinea-Bissau. The samples were left to precipitate for 48 hours and then the sediments were seeded on non-nutrient agar plates containing Escherichia coli spread and cultures were checked daily for the presence of FLA. Identification of FLA strains was based on the morphological and polymerase chain reaction (PCR) using the 18S rDNA or 16S mitochondrial rDNA genes in the case of Naegleria and Balamuthia genera, respectively. In the case of positive samples of Acanthamoeba, strains were further classified at the genotype level by sequencing the diagnostic fragment 3 (DF3) region located in the 18S rDNA gene as previously described. Sappinia sp. was not isolated during the study and thus, no molecular analysis was performed for this genus. The obtained results revealed the presence of Acanthamoeba (genotypes T3 and T4), Naegleria fowleri, and Balamuthia mandrillaris. To the best of our knowledge, this is the first report demonstrating the presence of FLA in water bodies from Guinea-Bissau and the first report on the isolation of Balamuthia mandrillaris from environmental sources in Africa.

Transmission of Balamuthia mandrillaris Through Solid Organ Transplantation: Utility of Organ Recipient Serology to Guide Clinical Management

A liver, heart, iliac vessel and two kidneys were recovered from a 39-year-old man who died of traumatic head injury and were transplanted into five recipients. The liver recipient 18 days posttransplantation presented with headache, ataxia and fever, followed by rapid neurologic decline and death. Diagnosis of granulomatous amebic encephalitis was made on autopsy. Balamuthia mandrillaris infection was confirmed with immunohistochemical and polymerase chain reaction (PCR) assays. Donor and recipients' sera were tested for B. mandrillaris antibodies. Donor brain was negative for Balamuthia by immunohistochemistry and PCR; donor serum Balamuthia antibody titer was positive (1:64). Antibody titers in all recipients were positive (range, 1:64-1:512). Recipients received a four- to five-drug combination of miltefosine or pentamidine, azithromycin, albendazole, sulfadiazine and fluconazole. Nausea, vomiting, elevated liver transaminases and renal insufficiency were common. All other recipients survived and have remained asymptomatic 24 months posttransplant. This is the third donor-derived Balamuthia infection cluster described in solid organ transplant recipients in the United States. As Balamuthia serologic testing is only available through a national reference laboratory, it is not feasible for donor screening, but may be useful to determine exposure status in recipients and to help guide chemotherapy.

Laboratory testing of clinically approved drugs against Balamuthia mandrillaris.

Balamuthia mandrillaris is a free-living protist pathogen that can cause life-threatening granulomatous amoebic encephalitis. Given the lack of effective available drugs against B. mandrillaris encephalitis with a mortality rate of more than 90%, here we screened drugs, targeting vital cellular receptors and biochemical pathways, that are already in approved clinical use for their potential clinical usefulness. Amoebicidal assays were performed by incubating B. mandrillaris with drugs (3 × 10(5) cells/0.5 mL/well) in phosphate buffered saline for 24 h and viability was determined using Trypan blue exclusion staining. For controls, amoebae were incubated with the solvent alone. To determine whether effects are reversible, B. mandrillaris were pre-exposed to drugs for 24 h, washed twice, and incubated with human brain microvascular endothelial cells, which constitute the blood-brain barrier as food source, for up to 48 h. Of the ten drugs tested, amlodipine, apomorphine, demethoxycurcumin, haloperidol, loperamide, prochlorperazine, procyclidine, and resveratrol showed potent amoebicidal effects, while amiodarone and digoxin exhibited minimal effectiveness. When pre-treated with these drugs, no viable trophozoites re-emerged, suggesting that drugs destroyed parasite irreversibly. Based on the in vitro assay, amlodipine, apomorphine, demethoxycurcumin, haloperidol, loperamide, prochlorperazine, procyclidine, and resveratrol are potential antimicrobials for further testing against B. mandrillaris encephalitis. These findings may provide novel strategies for therapy but further research is needed to determine clinical usefulness of aforementioned drugs against granulomatous amoebic encephalitis caused by B. mandrillaris, and other free-living amoebae, such as Acanthamoeba spp., and Naegleria fowleri.

Balamuthia mandrillaris in South America: An emerging potential hidden pathogen in Perú

Balamuthia mandrillaris is a free living amoeba that can be isolated from soil. It is an emerging pathogen causing skin lesions as well as CNS involvement with a fatal outcome if untreated. Further, infections can sometimes can also appear in peripheral areas such as extremities (usually knee), or trunk.Moreover, it often progresses to an infiltrative lesion that occasionally becomes ulcerated. In countries like Peru, a skin lesion will precede other symptoms. This primary cutaneous lesion can be present for weeks or even months. However, the appearance of neurological disease predicts a poor prognosis. Diagnosis requires a high level of suspicion.

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.

Combined drug therapy in the management of granulomatous amoebic encephalitis due to Acanthamoeba spp., and Balamuthia mandrillaris

Granulomatous amoebic encephalitis (GAE) is caused by two protist pathogens, Acanthamoeba spp., and Balamuthia mandrillaris. Although rare, it almost always results in death. In the present study, amoebae were treated with various combinations of clinically-approved drugs, targeting vital cellular receptors and biochemical pathways. The results revealed that among the seven different combinations tested, three proved highly effective against both Acanthamoeba castellanii as well as B. mandrillaris at a concentration of 100μM. These combinations included (i) prochlorperazine plus loperamide; (ii) prochlorperazine plus apomorphine; and (iii) procyclidine plus loperamide. In viability assays, none of the drug-treated amoebae emerged as viable trophozoites, suggesting irreversible amoebicidal effects. Four combinations of drugs tested showed varied potency against A. castellanii and B. mandrillaris at 100μM. The combination of haloperidol and loperamide was highly effective against A. castellanii at 100μM, but potent effects against B. mandrillaris were observed only at 250μM. Digoxin and amlodipine were effective against A. castellanii and B. mandrillaris at 100μM and 250μM, respectively. In contrast, the combination of apomorphine and haloperidol was effective against B. mandrillaris and A. castellanii at 100μM and 250μM, respectively. At 100μM, the combination of procyclidine and amiodarone was effective against neither A. castellanii nor B. mandrillaris. In this case, amoebicidal properties were observed at 750μM for A. castellanii, and 950μM for B. mandrillaris. As these drugs are used clinically against non-communicable diseases, the findings reported here have the potential to be tested in a clinical setting against amoebic encephalitis caused by A. castellanii and B. mandrillaris.

Serologic survey for exposure following fatal Balamuthia mandrillaris infection

Granulomatous amebic encephalitis (GAE) from Balamuthia mandrillaris, a free-living ameba, has a case fatality rate exceeding 90% among recognized cases in the USA. In August 2010, a GAE cluster occurred following transplantation of infected organs from a previously healthy landscaper in Tucson, AZ, USA, who died from a suspected stroke. As B. mandrillaris is thought to be transmitted through soil, a serologic survey of landscapers and a comparison group of blood donors in southern Arizona was performed. Three (3.6%) of 83 serum samples from landscapers and 11 (2.5%) of 441 serum samples from blood donors were seropositive (p = 0.47). On multivariable analysis, county of residence was associated with seropositivity, whereas age, sex, and ethnicity were not. Exposure to B. mandrillaris, previously unexamined in North America, appears to be far more common than GAE in Southern Arizona. Risk factors for disease progression and the ameba's geographic range should be examined.