Primary Amebic Meningoencephalitis Associated With Rafting on an Artificial Whitewater River: Case Report and Environmental Investigation.

Background: Naegleria fowleri is a thermophilic ameba found in freshwater that causes primary amebic meningoencephalitis (PAM) when it enters the nose and migrates to the brain. Patient exposure to water containing the ameba typically occurs in warm freshwater lakes and ponds during recreational water activities. In June 2016, an 18-year-old woman died of PAM after traveling to North Carolina, where she participated in rafting on an artificial whitewater river. Methods: We conducted an epidemiologic and environmental investigation to determine the water exposure that led to the death of this patient. Results: The case patient's most probable water exposure occurred while rafting on an artificial whitewater river during which she was thrown out of the raft and submerged underwater. The approximately 11.5 million gallons of water in the whitewater facility were partially filtered, subjected to ultraviolet light treatment, and occasionally chlorinated. Heavy algal growth was noted. Eleven water-related samples were collected from the facility; all were positive for N. fowleri. Of 5 samples collected from the nearby natural river, 1 sediment sample was positive for N. fowleri. Conclusions: This investigation documents a novel exposure to an artificial whitewater river as the likely exposure causing PAM in this case. Conditions in the whitewater facility (warm, turbid water with little chlorine and heavy algal growth) rendered the water treatment ineffective and provided an ideal environment for N. fowleri to thrive. The combination of natural and engineered elements at the whitewater facility created a challenging environment to control the growth of N. fowleri.

Potentially pathogenic genera of free-living amoebae coexisting in a thermal spring.

Little is known about the prevalence of Balamuthia mandrillaris within the environment due to its difficult isolation, but once an axenic culture is established, it is relatively easy to maintain. As most of the time researchers are interested mainly in isolating B. mandrillaris from environmental samples, the flora that accompanies it becomes second in importance. Therefore, this study aimed to determine which potentially pathogenic free-living amoebae, in addition to B. mandrillaris, could be found co-inhabiting a source of natural thermal water called "Agua Caliente" (Mexico), where this amoeba has previously been detected twice by molecular methods. A third sampling from this same source was carried out to try to isolate B. mandrillaris and other free-living amoebae using 37 and 45 °C as isolation temperatures. For PCR techniques, specific primers were used for B. mandrillaris, Naegleria fowleri, and Acanthamoeba species, plus a universal primer set for the eukaryotic 18S SSU rRNA gene for other isolated amoebae. PCR products were sequenced for final identification. 42 strains of the primary isolate were obtained, but only 34 could be kept in culture. Of them, 23 strains were identified as Naegleria lovaniensis, eight strains as Acanthamoeba jacobsi, two strains as Stenamoeba sp. and only one was identified as Vermamoeba vermiformis. The isolation of B. mandrillaris was once again not successful, but the presence of potentially pathogenic and nonpathogenic free-living amoebae is reported for the first time in this type of water in Mexico thanks to molecular methodology.

Development of nanoparticle-assisted PCR assay in the rapid detection of brain-eating amoebae.

Brain-eating amoebae (Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri) have gained increasing attention owing to their capacity to produce severe human and animal infections involving the brain. Early detection is a pre-requisite in successful prognosis. Here, we developed a nanoPCR assay for the rapid detection of brain-eating amoebae using various nanoparticles. Graphene oxide, copper and alumina nanoparticles used in this study were characterized using Raman spectroscopy measurements through excitation with a He-Ne laser, while powder X-ray diffraction patterns were taken on a PANanalytical, X'Pert HighScore diffractometer and the morphology of the materials was confirmed using high-resolution transmission electron microscopy (HRTEM). Using nanoparticle-assisted PCR, the results revealed that graphene oxide, copper oxide and alumina nanoparticles significantly enhanced PCR efficiency in the detection of pathogenic free-living amoebae using genus-specific probes. The optimal concentration of graphene oxide, copper oxide and alumina nanoparticles for Acanthamoeba spp. was determined at 0.4, 0.04 and 0.4 µg per mL respectively. For B. mandrillaris, the optimal concentration was determined at 0.4 µg per mL for graphene oxide, copper oxide and alumina nanoparticles, and for Naegleria, the optimal concentration was 0.04, 4.0 and 0.04 µg per mL respectively. Moreover, combinations of these nanoparticles proved to further enhance PCR efficiency. The addition of metal oxide nanoparticles leads to excellent surface effect, while thermal conductivity property of the nanoparticles enhances PCR productivity. These findings suggest that nanoPCR assay has tremendous potential in the clinical diagnosis of parasitic infections as well as for studying epidemiology and pathology and environmental monitoring of other microbes.

First report of the isolation of Balamuthia mandrillaris in the northern region of Japan.

Balamuthia mandrillaris is a free-living amoeba that lives in soil and water near human settlements. B. mandrillaris was first isolated from a mandrill baboon that died at the San Diego Zoo Wildlife Park in California in 1986, and the first human infection was reported in 1990. Although reported B. mandrillaris infections are often not properly characterized, it appears that B. mandrillaris invades the living body from the soil and water, either via a wound or the nasal cavity. Most confirmed infections have originated in South and North America. B. mandrillaris inhabits warm climates and is recognized as a pathogen in warm areas such as desert climates and tropical climates. B. mandrillaris has been isolated from environmental samples since 2000, most of which originated from warm areas such as step climates, tropical climates, and desert climates. However, B. mandrillaris may survive in diverse environments, although fewer granulomatous amebic encephalitis patients have been reported in colder Japanese and Northern European regions. In the present study, we conducted a survey of 13 soil samples in Aomori Prefecture located at the northernmost tip of Japan Honshu and successfully isolated one strain of B. mandrillaris from soil for the first time in Japan. In addition, B. mandrillaris gene was detected from several soils. This confirms that B. mandrillaris is capable of spreading to a wider climatic region.

[Isolation of Balamuthia mandriallaris parasite from air conditioning of the houses in south and southeast of Turkey]. / Türkiye'nin güney ve güneydogusundaki evlerin iklimlendirmelerinde Balamuthia mandrillaris parazitinin izolasyonu.

The free living amoebae cause various infections such as Acanthamoeba keratitis, granulomatous amoebic encephalitis, primer amoebic meningoencephalitis in humans and animals. The free living amoebae Acanthamoeba, Balamuthia mandriallis, Naegleria fowleria and Sappinia species that cause disease in humans have been isolated from many environmental materials until today. However, no isolation has been reported from the filters of the air conditions from the houses used for ventilation. The aim of this study was toinvestigate the existence of free living amoebae using molecular methods in the filters of air-conditions used in the study living area of the people. A total of 30 dust samples were taken from the filtersof air-conditions in Adana and Gaziantep province of Turkey. DNA isolation of the dust samples was performed using the DNeasy PowerSoil kit (Qiagen, Germany) and polymerase chain reaction was done with specific primers of Acanthamoeba spp., B.mandriallis, N.fowleria and Sappinia species. As a result of this study, Acanthamoeba spp. was determined as 33.3% (5/15) and B.mandriallis was determined as6.6% (1/15) in Adana province. On the other hand, Acanthamoeba species was determined as 26.6% (4/15) and B.mandriallis was determined as 13.3% (2/15) in Gaziantep province. N.fowleria and Sappina species were not detectedin both of the cities. DNA sequence analysis was performed for the confirmation of the species and 99% of the results were similar to the other species in GenBank. The rates of Acanthamoeba castellanii and Acanthamoeba griffinii (T3) were determined as %66.6 (6/9) and 33.3% (3/9), respectively by DNA sequencing. Distribution of Acanthamoeba species according to the cities were 33.3% (3/9) for A.castellanii and 22.2% (2/9) for A.griffini in Adana. It was 33.3% (3/9) for A.castellanii and 11.1% (1/9) for A.griffini in Gaziantep. There was no significant difference in the distribution of the parasite species among cities (p> 0.1). It is important to raise awareness of the diseases caused by free living amoebae among people. Acanthamoeba species have been reported frequently from environmental materials in Turkey, but B.mandriallis has not been reported from any environmental sample since this study. The presence of B.mandriallis has been reported in the air-conditions of houses in this study. This result shows that people have risk in terms of illness of free living amoebae in living areas. Our study emphasized that firstly the health personnel and then the people should be informed about the deadly parasites and the cleaning of the air conditions should be done in certain periods.

Brain-Eating Amoebae: Predilection Sites in the Brain and Disease Outcome.

Acanthamoeba spp. and Balamuthia mandrillaris are causative agents of granulomatous amoebic encephalitis (GAE), while Naegleria fowleri causes primary amoebic meningoencephalitis (PAM). PAM is an acute infection that lasts a few days, while GAE is a chronic to subacute infection that can last up to several months. Here, we present a literature review of 86 case reports from 1968 to 2016, in order to explore the affinity of these amoebae for particular sites of the brain, diagnostic modalities, treatment options, and disease outcomes in a comparative manner.

Molecular identification of waterborne free living amoebae (Acanthamoeba, Naegleria and Vermamoeba) isolated from municipal drinking water and environmental sources, Semnan province, north half of Iran.

The present study tested 80 samples of municipal, geothermal and recreational water samples for the occurrence of waterborne free living amoebae (FLA) including Acanthamoeba, Balamuthia mandrillaris, Vahlkampfiids and Vermamoeba in Semnan province, North half of Iran. Four sets of primers including JDP1,2 primers, ITS1,2 primers (Vahlkampfiids), 16S rRNABal primers (Balamuthia mandrillaris) and NA1,2 primers (Vermamoeba) were used to confirm the morphological identification. From the 80 water samples tested in the present study, 16 (20%) were positive for the outgrowth of free living amoebae based on the morphological page key. Out of the 34 municipal water samples, 7 (20.6%) were positive for outgrowth of Free living amoeba, belonging to Vermamoeba, Naegleria and Acanthamoeba using molecular tools. Three out of the six investigated hot springs were also contaminated with Naegleria spp. Sequencing of the ITS1,2 region of the Vahlkampfiid isolates revealed the highest homology with N. gruberi (2 isolates), N. australiensis (1 isolate) and N. pagei (3 isolates). This is the first report of N. gruberi in the country. Using morphological and molecular analysis, Balamuthia mandrillaris was undetected in all the water samples. The present study further confirmed the occurrence of potentially pathogenic waterborne free living amoebae in habitats with high human activity. It is of utmost importance that more studies are conducted to evaluate the niches of B. mandrillaris and N. fowleri in Iran and worldwide. Such investigations regarding the relevance of FLA as a hazard to humans, should be brought to the notice of the health authorities.

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.

Isolation of Balamuthia mandrillaris-specific antibody fragments from a bacteriophage antibody display library.

Balamuthia mandrillaris is a protist pathogen that can cause encephalitis with a mortality rate of more than 95%. Early diagnosis followed by aggressive treatment is a pre-requisite for successful prognosis. Current methods for identifying this organism rely on culture and microscopy, antibody-based methods using animals, or involve the use of molecular tools that are expensive. Here, we describe the isolation of antibody fragments that can be used for the unequivocal identification of B. mandrillaris. B. mandrillaris-specific antibody fragments were isolated from a bacteriophage antibody display library. Individual clones were studied by enzyme-linked immunosorbent assay, and immunofluorescence. Four antibody clones showed specific binding to B. mandrillaris. The usefulness of phage antibody display technology as a diagnostic tool for isolating antibody fragments against B. mandrillaris antigens and studying their biological role(s) is discussed further.

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.

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-time polymerase 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.

Disseminated Balamuthia mandrillaris Infection.

Balamuthia mandrillaris is a rare cause of human infection, but when infections do occur, they result in high rates of morbidity and mortality. A case of disseminated Balamuthia infection is presented. Early diagnosis and initiation of recommended therapy are essential for increased chances of successful outcomes.

Acute encephalitis in the immunocompromised individual.

PURPOSE OF REVIEW: This article describes recent advances in the diagnosis and management of encephalitis in immunocompromised individuals. RECENT FINDINGS: Herpes simplex virus (HSV) and varicella zoster virus (VZV) are common causes of encephalitis in immunocompromised individuals, although clinical manifestations may be atypical, and thus challenging to recognize. Recently, an increased incidence of HSV and VZV central nervous system infections has been reported in association with novel immunosuppressive and immunomodulatory treatments. The free-living ameba Balamuthia mandrillaris causes granulomatous encephalitis predominantly in immunocompromised individuals and is associated with nearly uniform fatality. In the setting of organ transplantation, the recipient's immunocompromised state along with the potential for donor-transmitted infections can result in a unique epidemiology of encephalitis, including infection by human herpes virus-6 and BK virus. Recent studies utilizing next-generation sequencing techniques have identified several pathogens, including Leptospira santarosai and a neurotropic astrovirus, as causes of encephalitis in immunocompromised individuals. SUMMARY: Diagnosis and management of encephalitis is challenging in immunocompromised individuals, in part because of atypical clinical presentations and the presence of uncommon or novel infectious agents. Unbiased techniques for pathogen discovery are likely to play an increasing role in the diagnosis of central nervous system infections in immunocompromised individuals.

Balamuthia mandrillaris therapeutic mud bath in Jamaica.

Balamuthia mandrillaris is an emerging cause of encephalitis in humans. The transmission dynamics are poorly understood due to the high fatality rate and the sporadic nature of cases. Seventy-two soil samples were collected from beaches and the banks of lagoons, rivers, ponds, mineral springs and streams from across Jamaica and assayed for the presence of B. mandrillaris. Seventy-nine sites were sampled and the mitochondrial 16S rDNA gene of B. mandrillaris was amplified and sequenced to confirm the presence of the amoeba. One isolate of B. mandrillaris was recovered from soil from mineral spring which hosts an informal therapeutic mud bath business. Although B. mandrillaris is less frequently isolated from soil than other free-living amoebae, rubbing mud containing the organism onto the skin increases the likelihood of exposure and infection. This first report on the isolation of B. mandrillaris in the Caribbean and its presence in soil where human contact is likely warrants further investigation using serological methods to elucidate exposure patterns.

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.

Assessment of blood-brain barrier penetration of miltefosine used to treat a fatal case of granulomatous amebic encephalitis possibly caused by an unusual Balamuthia mandrillaris strain.

Balamuthia mandrillaris, a free-living ameba, causes rare but frequently fatal granulomatous amebic encephalitis (GAE). Few patients have survived after receiving experimental drug combinations, with or without brain lesion excisions. Some GAE survivors have been treated with a multi-drug regimen including miltefosine, an investigational anti-leishmanial agent with in vitro amebacidal activity. Miltefosine dosing for GAE has been based on leishmaniasis dosing because no data exist in humans concerning its pharmacologic distribution in the central nervous system. We describe results of limited cerebrospinal fluid (CSF) and serum drug level testing performed during clinical management of a child with fatal GAE who was treated with a multiple drug regimen including miltefosine. Brain biopsy specimens, CSF, and sera were tested for B. mandrillaris using multiple techniques, including culture, real-time polymerase chain reaction, immunohistochemical techniques, and serology. CSF and serum miltefosine levels were determined using a liquid chromatography method coupled to tandem mass spectrometry. The CSF miltefosine concentration on hospital admission day 12 was 0.4 µg/mL. The serum miltefosine concentration on day 37, about 80 h post-miltefosine treatment, was 15.3 µg/mL. These are the first results confirming some blood-brain barrier penetration by miltefosine in a human, although with low-level CSF accumulation. Further evaluation of brain parenchyma penetration is required to determine optimal miltefosine dosing for Balamuthia GAE, balanced with the drug's toxicity profile. Additionally, the Balamuthia isolate was evaluated by real-time polymerase chain reaction (PCR), demonstrating genetic variability in 18S ribosomal RNA (18S rRNA) sequences and possibly signaling the first identification of multiple Balamuthia strains with varying pathogenicities.

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.

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.