The Canadian journal of infectious diseases & medical microbiology = Journal canadien des maladies infectieuses et de la microbiologie médicale / AMMI Canada, 2006
Acanthamoeba species keratitis has been associated with soft contact lens wear. In the present re... more Acanthamoeba species keratitis has been associated with soft contact lens wear. In the present report, an epidemiological link was established between the patient's isolate and well water from the home using molecular methods. To the authors' knowledge, this is the first case in Canada where such a link has been established. Primary care practitioners and specialists, including ophthalmologists and infectious diseases specialists, must maintain a high degree of clinical suspicion in soft contact lens wearers with keratitis unresponsive to conventional topical and systemic treatment.
Journal of the American Veterinary Medical Association, Jun 1, 2011
A 10-month-old castrated male Boxer (26 kg [57.2 lb]) was evaluated at the University of Georgia ... more A 10-month-old castrated male Boxer (26 kg [57.2 lb]) was evaluated at the University of Georgia Veterinary Teaching Hospital for signs of cervical pain and fever. The dog had been evaluated 2 days previously by the referring veterinarian for an acute onset of lethargy, difficulty rising from a lying position, and a stiff gait in all 4 limbs. On physical examination performed by the referring veterinarian, the dog was clinically normal except for fever (39.3°C [102.74°F]) and signs of cervical pain. Complete blood count revealed neutrophilic leukocytosis (WBC count, 25.08 X 10 3 cells/µL [reference range, 5.50 X 10 3 cells/µL to 16.90 X 10 3 cells/µL]; neutrophil count, 19.92 X 10 3 cells/µL [reference range, 2.0 X 10 3 cells/µL to 12.0 X 10 3 cells/µL]) and monocytosis (2.68 X 10 3 cells/ µL; reference range, 0.30 X 10 3 cells/µL to 2.00 X 10 3 Case Description-A 10-month-old Boxer was evaluated for fever and signs of cervical pain. Clinical Findings-Physical examination revealed lethargy, fever, and mucopurulent ocular and preputial discharge. On neurologic examination, the gait was characterized by a short stride. The dog kept its head flexed and resisted movement of the neck, consistent with cervical pain. Clinicopathologic findings included neutrophilic leukocytosis, a left shift, and monocytosis. Cervical radiographs were unremarkable. Cerebrospinal fluid analysis revealed neutrophilic pleocytosis and high total protein content. On the basis of signalment, history, and clinicopathologic data, a diagnosis of steroid-responsive meningitis-arteritis was made. Treatment and Outcome-The dog was treated with prednisone (3.2 mg/kg [1.45 mg/lb], PO, q 24 h), for 3 weeks with limited response. Consequently, azathioprine (2 mg/kg [0.9 mg/lb], PO, q 24 h) was administered. Three weeks later, the dog was evaluated for tachypnea and lethargy. Complete blood count revealed leukopenia, neutropenia, and a left shift. Thoracic radiography revealed a diffuse bronchointerstitial pattern. The dog subsequently went into respiratory arrest and died. On histologic evaluation, amoebic organisms were observed in the lungs, kidneys, and meninges of the brain and spinal cord. A unique Acanthamoeba sp was identified by use of PCR assay. Clinical Relevance-This dog developed systemic amoebic infection presumed to be secondary to immunosuppression. The development of secondary infection should be considered in animals undergoing immunosuppression for immune-mediated disease that develop clinical signs unrelated to the primary disease. Although uncommon, amoebic infection may develop in immunosuppressed animals. Use of a PCR assay for identification of Acanthamoeba spp may provide an antemortem diagnosis. (J Am Vet Med
Contrary to data on Acanthamoeba infections in humans, little is known about infections in fishes... more Contrary to data on Acanthamoeba infections in humans, little is known about infections in fishes. The present study combines the description of strains isolated from fishes with presentation of an improved method for subgeneric classification. Acanthamoeba spp. were isolated aseptically from tissues of 14 (1.7%) of 833 asymptomatic fishes collected in rivers and streams in the Czech Republic. Acanthamoebae successfully cloned from 10 of the 14 isolated strains were examined here. Morphology of these isolates was evaluated using light optics plus scanning and transmission electron microscopy. Cyst morphology, which varied extensively within and among clones, was most like morphological group II, but species-level classification was considered impossible. A distance analysis based on 442 bases in an 18S rDNA polymerase chain reaction fragment of about 460 bp placed the isolates in a clade composed of sequence types T3, T4, and T11, the 3 subdivisions of morphological group II. Fluorescent in situ hybridization (FISH) using oligonucleotide probes indicated that all isolates belong to a single subdivision of group II, the T4 sequence type. It has been concluded that the fish isolates are most closely related to strains commonly isolated from human infections, especially Acanthamoeba keratitis. The shorter diagnostic fragment sequences have proved nearly as useful as complete 18S rDNA sequences for identification of Acanthamoeba isolates.
ABSTRACT Purpose: An outbreak of Acanthamoeba keratitis (AK), a rare infection with significant m... more ABSTRACT Purpose: An outbreak of Acanthamoeba keratitis (AK), a rare infection with significant morbidity caused by a ubiquitous waterborne organism, has persisted despite the recall of a contact lens solution strongly associated with disease. Environmental Protection Agency regulations reducing allowable disinfection byproducts in potable water were implemented concomitantly; a microbial shift may be an unintended consequence. The relationship between AK and potable water was evaluated in Chicago to understand the contribution. Methods: 65 AK cases diagnosed 6/03 6/08 and 175 controls matched on contact lens use, age, and service date were recruited from the UIC Cornea Service. Water samples collected 7/06 7/08 from subjects' homes were analyzed for Acanthamoeba microbiologic presence. Geographic information systems (GIS) and SaTScan software were used in spatial analysis. Among subjects with Lake Michigan water, conditional logistic regression was used to estimate the association between AK and the distance between water treatment and end usage, and also positive water samples and the distance from water treatment. Results: Acanthamoeba were identified in 17 of 64 (26.6%) case and 30 of 165 (18.2%) control homes with Lake Michigan water. Spatial analysis identified an area of elevated AK risk (OR: 3.62; p = 0.02) that was relatively distant from the water treatment facility. AK and Acanthamoeba-positive water sample risk increased significantly (OR: 2.38; 95%CI: 1.46 3.88 and OR: 2.63; 95%CI: 1.61 4.29, respectively) with each 10-mile increase between water treatment and end usage. Conclusion: Results support hypotheses that the domestic water supply is contributing to the persisting AK outbreak.
Species of Acanthamoeba were first described using morphological characters including cyst struct... more Species of Acanthamoeba were first described using morphological characters including cyst structure and cytology of nuclear division. More than 20 nominal species were proposed using these methods. Morphology, especially cyst shape and size, has proven to be plastic and dependent upon culture conditions. The DNA sequence of the nuclear small-subunit (18S) rRNA, the Rns gene, has become the most widely accepted method for rapid diagnosis and classification of Acanthamoeba. The Byers-Fuerst lab first proposed an Rns typing system in 1996. Subsequent refinements, with an increasing DNA database and analysis of diagnostic fragments within the gene, have become widely accepted by the Acanthamoeba research community. The development of the typing system, including its current state of implementation is illustrated by three cases: (i) the division between sequence types T13 and T16; (ii) the diversity within sequence supertype T2/T6, and (iii) verification of a new sequence type, designated T20. Molecular studies make clear the disconnection between phylogenetic relatedness and species names, as applied for the genus Acanthamoeba. Future reconciliation of genetic types with species names must become a priority, but the possible shortcomings of the use of a single gene when reconstructing the evolutionary history of the acanthamoebidae must also be resolved.
Contrary to data on Acanthamoeba infections in humans, little is known about infections in fishes... more Contrary to data on Acanthamoeba infections in humans, little is known about infections in fishes. The present study combines the description of strains isolated from fishes with presentation of an improved method for subgeneric classification. Acanthamoeba spp. were isolated aseptically from tissues of 14 (1.7%) of 833 asymptomatic fishes collected in rivers and streams in the Czech Republic. Acanthamoebae successfully cloned from 10 of the 14 isolated strains were examined here. Morphology of these isolates was evaluated using light optics plus scanning and transmission electron microscopy. Cyst morphology, which varied extensively within and among clones, was most like morphological group II, but species-level classification was considered impossible. A distance analysis based on 442 bases in an 18S rDNA polymerase chain reaction fragment of about 460 bp placed the isolates in a clade composed of sequence types T3, T4, and T11, the 3 subdivisions of morphological group II. Fluorescent in situ hybridization (FISH) using oligonucleotide probes indicated that all isolates belong to a single subdivision of group II, the T4 sequence type. It has been concluded that the fish isolates are most closely related to strains commonly isolated from human infections, especially Acanthamoeba keratitis. The shorter diagnostic fragment sequences have proved nearly as useful as complete 18S rDNA sequences for identification of Acanthamoeba isolates.
Balamuthia mandrillaris is a free-living ameba that causes granulomatous amebic encephalitis in b... more Balamuthia mandrillaris is a free-living ameba that causes granulomatous amebic encephalitis in both immunocompromised and immunocompetent individuals. Because of a lack of pathognomonic symptoms and the difficulty in recognizing amebas in biopsied tissues, most cases are not diagnosed or effectively treated, leading to a >95% mortality. We report here on five cases of balamuthiasis that were diagnosed by indirect immunofluorescence
The American journal of tropical medicine and hygiene
Balamuthia mandrillaris is an opportunistically pathogenic ameba that causes fatal granulomatous ... more Balamuthia mandrillaris is an opportunistically pathogenic ameba that causes fatal granulomatous amebic encephalitis (GAE) in vertebrates. Previous phylogenetic analyses that included the sequence of a single nuclear small subunit ribosomal RNA gene (18S or ssu rDNA) from this ameba suggested that Balamuthia is closely related to Acanthamoeba, another opportunistically pathogenic amebic genus, which includes multiple ssu rDNA genotypes. We tested whether this also is true for Balamuthia. The nuclear ssu rDNA from 4 isolates and the mitochondrial ssu rDNA from 7 isolates of B. mandrillaris have been sequenced. No variation in the nuclear rDNA sequences and low levels of variation in the mitochondrial rDNA were found. Both gene sequences were consistent with a single genotype for B. mandrillaris. The mitochondrial sequences of B. mandrillaris are unique and should be useful for development of genus-specific diagnostic probes for use with clinical, environmental, and archived specimens.
Members of the genus Acanthamoeba are usually free-living amebae and are found in a variety of ec... more Members of the genus Acanthamoeba are usually free-living amebae and are found in a variety of ecological niches including soil, fresh and brackish water, dust in air, filters of heating, ventilating, and air conditioning units, swimming pools and hot tubs, etc. Occasionally, they are also known to cause central nervous system infections in humans and other animals. We isolated into culture an amoeba from the liver tissue of a keel-billed toucan and identified it as Acanthamoeba sp. based on culture characteristics and immunofluorescent analysis. Further, we characterized the cultured amoeba and also the amoeba in the liver tissue as Acanthamoeba, genotype T4, by sequencing a diagnostic region of the nuclear small subunit ribosomal RNA gene.
A phylogenetic investigation of the autosporic chlorophycean alga species Selenastrum capricornut... more A phylogenetic investigation of the autosporic chlorophycean alga species Selenastrum capricornutum using the small subunit (SSU) rRNA gene revealed the unusual presence of six group IC1 introns. Previous studies showed that numerous green algal taxa contain group IC1 introns. However, whereas some algal species harbor multiple introns in a single ribosomal gene, none have contained as many as S. capricornutum.
Members of the genus Acanthamoeba are usually free-living amoebae that are found in a variety of ... more Members of the genus Acanthamoeba are usually free-living amoebae that are found in a variety of ecological niches including soil, fresh and brackish water, dust in the air, heating, ventilating, and air conditioning filters, swimming pools and hot tubs. Occasionally they are also known to cause central nervous system infections in humans and animals. We isolated into culture an amoeba from the liver of a Temminck's tragopan (horned pheasant) (Tragopan temminckii) that died of amoebic infection. We identified the infecting amoeba as Acanthamoeba sp. based on culture characteristics, cyst morphology and immunofluorescence assays. Additionally, we identified the amoeba as Acanthamoeba, genotype T4, by sequencing a diagnostic region of the nuclear small subunit ribosomal RNA gene.
The genus Acunlhurnoeha is an assemblage of approximately 20 named species of free-living amoebae... more The genus Acunlhurnoeha is an assemblage of approximately 20 named species of free-living amoebae inhabiting a wide range of ecological niches, and has a worldwide distribution. Individual Acan-thurnorh~i havc been isolated from soil, fresh and saltwater, humans, domestic and feral animals. In addition to natural habitats, Acanthaniorhtr has heen identified as the cause of scveral diseases. It acts as an opportunistic pathogen in the painful and sight-thrcatening condition Ar~cinthunzoehu keratitis (AK) in otherwise healthy individuals. Acan-thui~zoc~ha is also responsible for life threatening infections in patients with immune defense deficiencies (IDD). Acantharnoeha strains have also heen associated with other infections, including those of the skin, eye, and brain (granulornatous amoebic encephalitis, GAE). These are generally observed in imiiiunocompromised individuals. Variations in the pathogenicity of Acanrhamoeha strains havc been recognized in various laboralory studies, but the relevance of these results to human disease is unclear.
Previous molecular examination of Acanthamoeba spp. has resulted in the determination of distinct... more Previous molecular examination of Acanthamoeba spp. has resulted in the determination of distinct genotypes in this genus (designated T1-T12, T14). Genotype T4 has been responsible for the majority of cases of Acanthamoeba keratitis. Here we examine the relative abundance of environmental T4 isolates on beaches and ask whether they have temperature and salinity tolerances that could enhance pathogenicity. Twenty-four Acanthamoeba strains were isolated from beach sand (n ϭ 20), soil (n ϭ 3), and tap water (n ϭ 1) in south Florida. Phylogenetic analysis identified 19 of 24 isolates as T4, the Acanthamoeba keratitis-associated genotype. The remaining isolates were genotype T5 (4) and T11 (1). Nearly all beach isolates were genotype T4, whereas the tap water and soil isolates were mostly T5. All amoebae grew at 0, 1.0, and 2.0% salt and 19 of 20 beach isolates also grew at 3.2%. No soil or tapwater acanthamoebae reproduced at 3.2%. All isolates grew at 37 ЊC and two (T5) at 42 ЊC. Little correlation existed between beach location, salt-tolerance, and genetic relatedness. Overall, the large majority of environmental isolates obtained were genotype T4, suggesting it may be the most common genotype in this environment and could be a potential source of Acanthamoeba keratitis infections.
Ultrastructural studies of tetrasporalean green algae have suggested the order is polyphyletic. T... more Ultrastructural studies of tetrasporalean green algae have suggested the order is polyphyletic. These features, including the absolute orientation of the flagellar apparatus and the bi-versus quadriflagellated motile cell morphology, suggest that Chaetopeltis as well as a number of others, may be ancestral to a group that includes Tetraspora. In this study, we examine the phylogenetic relationships of selected tetrasporalean taxa based on analysis of 18S ribosomal RNA gene sequences. Results show that the tetrasporalean taxa are polyphyletic. Biflagellated genera group with biflagellated volvocalean taxa, whereas the quadriflagellated species compose a distinct monophyletic clade not closely related to the biflagellated taxa. In addition, tetrasporalean taxa group with other chlorophycean algal species with similar flagellar apparatus absolute orientation, but the quadriflagellated Tetrasporales do not appear to be ancestral to the entire Chlorophyceae. These results are concordant with previous conclusions drawn from ultrastructural data and further confirm the utility of (small-subunit) ribosomal RNA gene sequences to discern green algal evolutionary relationships.
The order Chaetophorales includes filamentous green algae whose taxonomic relationships to other ... more The order Chaetophorales includes filamentous green algae whose taxonomic relationships to other chlorophycean orders is uncertain. Chaetophoralean taxa include filamentous species which are both branched and unbranched. Ultrastructural studies of zoospores have revealed similar flagellar apparatuses in a number of genera, including Uronema, Stigeoclonium, and Fritschiella, suggesting a close phylogenetic relationship among these taxa. The order Oedogoniales represents a second group of branched and unbranched filamentous green algae whose relationships to other chlorophycean orders also has been unclear. A possible close relationship between the Chaetophorales and Oedogoniales has been suggested. Using DNA sequences from the small-subunit ribosomal RNA gene (SSU rRNA) of several members of each order, we have examined the monophyly of the Chaetophorales and Oedogoniales, as well as the nature of their relationship to other chlorophycean orders. Our results show that chaetophoralean and oedogonialean taxa form separate monophyletic groups. Results also suggest that the two orders are not closely related to each other.
This report describes the first isolation of the ameba Balamuthia mandrillaris from an environmen... more This report describes the first isolation of the ameba Balamuthia mandrillaris from an environmental soil sample associated with a fatal case of amebic encephalitis in a northern California child. Isolation of the ameba into culture from autopsied brain tissue confirmed the presence of Balamuthia. In trying to locate a possible source of infection, soil and water samples from the child's home and play areas were examined for the presence of Balamuthia. The environmental samples (plated onto nonnutrient agar with Escherichia coli as a food source) contained, in addition to the ameba, a variety of soil organisms, including other amebas, ciliates, fungi, and nematodes, as contaminants. Presumptive Balamuthia amebas were recognized only after cultures had been kept for several weeks, after they had burrowed into the agar. These were transferred through a succession of nonnutrient agar plates to eliminate fungal and other contaminants. In subsequent transfers, axenic Naegleria amebas and, later, tissue cultures (monkey kidney cells) served as the food source. Finally, the amebas were transferred to cell-free axenic medium. In vitro, the Balamuthia isolate is a slow-growing organism with a generation time of ϳ30 h and produces populations of ϳ2 ؋ 10 5 amebas per ml. It was confirmed as Balamuthia by indirect immunofluorescence staining with rabbit anti-Balamuthia serum and human anti-Balamuthia antibody-containing serum from the amebic encephalitis patient. The environmental isolate is similar in its antimicrobial sensitivities and identical in its 16S ribosomal DNA sequences to the Balamuthia isolate from the deceased patient.
Acanthamoeba is a free-living ameba that is found throughout the world and that causes encephalit... more Acanthamoeba is a free-living ameba that is found throughout the world and that causes encephalitis, keratitis, and cutaneous infections in humans. It has two stages in its life cycle: a trophic stage and a resistant cyst stage. We describe here the ability of Acanthamoeba cysts to survive desiccation for more than 20 years.
The Canadian journal of infectious diseases & medical microbiology = Journal canadien des maladies infectieuses et de la microbiologie médicale / AMMI Canada, 2006
Acanthamoeba species keratitis has been associated with soft contact lens wear. In the present re... more Acanthamoeba species keratitis has been associated with soft contact lens wear. In the present report, an epidemiological link was established between the patient's isolate and well water from the home using molecular methods. To the authors' knowledge, this is the first case in Canada where such a link has been established. Primary care practitioners and specialists, including ophthalmologists and infectious diseases specialists, must maintain a high degree of clinical suspicion in soft contact lens wearers with keratitis unresponsive to conventional topical and systemic treatment.
Journal of the American Veterinary Medical Association, Jun 1, 2011
A 10-month-old castrated male Boxer (26 kg [57.2 lb]) was evaluated at the University of Georgia ... more A 10-month-old castrated male Boxer (26 kg [57.2 lb]) was evaluated at the University of Georgia Veterinary Teaching Hospital for signs of cervical pain and fever. The dog had been evaluated 2 days previously by the referring veterinarian for an acute onset of lethargy, difficulty rising from a lying position, and a stiff gait in all 4 limbs. On physical examination performed by the referring veterinarian, the dog was clinically normal except for fever (39.3°C [102.74°F]) and signs of cervical pain. Complete blood count revealed neutrophilic leukocytosis (WBC count, 25.08 X 10 3 cells/µL [reference range, 5.50 X 10 3 cells/µL to 16.90 X 10 3 cells/µL]; neutrophil count, 19.92 X 10 3 cells/µL [reference range, 2.0 X 10 3 cells/µL to 12.0 X 10 3 cells/µL]) and monocytosis (2.68 X 10 3 cells/ µL; reference range, 0.30 X 10 3 cells/µL to 2.00 X 10 3 Case Description-A 10-month-old Boxer was evaluated for fever and signs of cervical pain. Clinical Findings-Physical examination revealed lethargy, fever, and mucopurulent ocular and preputial discharge. On neurologic examination, the gait was characterized by a short stride. The dog kept its head flexed and resisted movement of the neck, consistent with cervical pain. Clinicopathologic findings included neutrophilic leukocytosis, a left shift, and monocytosis. Cervical radiographs were unremarkable. Cerebrospinal fluid analysis revealed neutrophilic pleocytosis and high total protein content. On the basis of signalment, history, and clinicopathologic data, a diagnosis of steroid-responsive meningitis-arteritis was made. Treatment and Outcome-The dog was treated with prednisone (3.2 mg/kg [1.45 mg/lb], PO, q 24 h), for 3 weeks with limited response. Consequently, azathioprine (2 mg/kg [0.9 mg/lb], PO, q 24 h) was administered. Three weeks later, the dog was evaluated for tachypnea and lethargy. Complete blood count revealed leukopenia, neutropenia, and a left shift. Thoracic radiography revealed a diffuse bronchointerstitial pattern. The dog subsequently went into respiratory arrest and died. On histologic evaluation, amoebic organisms were observed in the lungs, kidneys, and meninges of the brain and spinal cord. A unique Acanthamoeba sp was identified by use of PCR assay. Clinical Relevance-This dog developed systemic amoebic infection presumed to be secondary to immunosuppression. The development of secondary infection should be considered in animals undergoing immunosuppression for immune-mediated disease that develop clinical signs unrelated to the primary disease. Although uncommon, amoebic infection may develop in immunosuppressed animals. Use of a PCR assay for identification of Acanthamoeba spp may provide an antemortem diagnosis. (J Am Vet Med
Contrary to data on Acanthamoeba infections in humans, little is known about infections in fishes... more Contrary to data on Acanthamoeba infections in humans, little is known about infections in fishes. The present study combines the description of strains isolated from fishes with presentation of an improved method for subgeneric classification. Acanthamoeba spp. were isolated aseptically from tissues of 14 (1.7%) of 833 asymptomatic fishes collected in rivers and streams in the Czech Republic. Acanthamoebae successfully cloned from 10 of the 14 isolated strains were examined here. Morphology of these isolates was evaluated using light optics plus scanning and transmission electron microscopy. Cyst morphology, which varied extensively within and among clones, was most like morphological group II, but species-level classification was considered impossible. A distance analysis based on 442 bases in an 18S rDNA polymerase chain reaction fragment of about 460 bp placed the isolates in a clade composed of sequence types T3, T4, and T11, the 3 subdivisions of morphological group II. Fluorescent in situ hybridization (FISH) using oligonucleotide probes indicated that all isolates belong to a single subdivision of group II, the T4 sequence type. It has been concluded that the fish isolates are most closely related to strains commonly isolated from human infections, especially Acanthamoeba keratitis. The shorter diagnostic fragment sequences have proved nearly as useful as complete 18S rDNA sequences for identification of Acanthamoeba isolates.
ABSTRACT Purpose: An outbreak of Acanthamoeba keratitis (AK), a rare infection with significant m... more ABSTRACT Purpose: An outbreak of Acanthamoeba keratitis (AK), a rare infection with significant morbidity caused by a ubiquitous waterborne organism, has persisted despite the recall of a contact lens solution strongly associated with disease. Environmental Protection Agency regulations reducing allowable disinfection byproducts in potable water were implemented concomitantly; a microbial shift may be an unintended consequence. The relationship between AK and potable water was evaluated in Chicago to understand the contribution. Methods: 65 AK cases diagnosed 6/03 6/08 and 175 controls matched on contact lens use, age, and service date were recruited from the UIC Cornea Service. Water samples collected 7/06 7/08 from subjects' homes were analyzed for Acanthamoeba microbiologic presence. Geographic information systems (GIS) and SaTScan software were used in spatial analysis. Among subjects with Lake Michigan water, conditional logistic regression was used to estimate the association between AK and the distance between water treatment and end usage, and also positive water samples and the distance from water treatment. Results: Acanthamoeba were identified in 17 of 64 (26.6%) case and 30 of 165 (18.2%) control homes with Lake Michigan water. Spatial analysis identified an area of elevated AK risk (OR: 3.62; p = 0.02) that was relatively distant from the water treatment facility. AK and Acanthamoeba-positive water sample risk increased significantly (OR: 2.38; 95%CI: 1.46 3.88 and OR: 2.63; 95%CI: 1.61 4.29, respectively) with each 10-mile increase between water treatment and end usage. Conclusion: Results support hypotheses that the domestic water supply is contributing to the persisting AK outbreak.
Species of Acanthamoeba were first described using morphological characters including cyst struct... more Species of Acanthamoeba were first described using morphological characters including cyst structure and cytology of nuclear division. More than 20 nominal species were proposed using these methods. Morphology, especially cyst shape and size, has proven to be plastic and dependent upon culture conditions. The DNA sequence of the nuclear small-subunit (18S) rRNA, the Rns gene, has become the most widely accepted method for rapid diagnosis and classification of Acanthamoeba. The Byers-Fuerst lab first proposed an Rns typing system in 1996. Subsequent refinements, with an increasing DNA database and analysis of diagnostic fragments within the gene, have become widely accepted by the Acanthamoeba research community. The development of the typing system, including its current state of implementation is illustrated by three cases: (i) the division between sequence types T13 and T16; (ii) the diversity within sequence supertype T2/T6, and (iii) verification of a new sequence type, designated T20. Molecular studies make clear the disconnection between phylogenetic relatedness and species names, as applied for the genus Acanthamoeba. Future reconciliation of genetic types with species names must become a priority, but the possible shortcomings of the use of a single gene when reconstructing the evolutionary history of the acanthamoebidae must also be resolved.
Contrary to data on Acanthamoeba infections in humans, little is known about infections in fishes... more Contrary to data on Acanthamoeba infections in humans, little is known about infections in fishes. The present study combines the description of strains isolated from fishes with presentation of an improved method for subgeneric classification. Acanthamoeba spp. were isolated aseptically from tissues of 14 (1.7%) of 833 asymptomatic fishes collected in rivers and streams in the Czech Republic. Acanthamoebae successfully cloned from 10 of the 14 isolated strains were examined here. Morphology of these isolates was evaluated using light optics plus scanning and transmission electron microscopy. Cyst morphology, which varied extensively within and among clones, was most like morphological group II, but species-level classification was considered impossible. A distance analysis based on 442 bases in an 18S rDNA polymerase chain reaction fragment of about 460 bp placed the isolates in a clade composed of sequence types T3, T4, and T11, the 3 subdivisions of morphological group II. Fluorescent in situ hybridization (FISH) using oligonucleotide probes indicated that all isolates belong to a single subdivision of group II, the T4 sequence type. It has been concluded that the fish isolates are most closely related to strains commonly isolated from human infections, especially Acanthamoeba keratitis. The shorter diagnostic fragment sequences have proved nearly as useful as complete 18S rDNA sequences for identification of Acanthamoeba isolates.
Balamuthia mandrillaris is a free-living ameba that causes granulomatous amebic encephalitis in b... more Balamuthia mandrillaris is a free-living ameba that causes granulomatous amebic encephalitis in both immunocompromised and immunocompetent individuals. Because of a lack of pathognomonic symptoms and the difficulty in recognizing amebas in biopsied tissues, most cases are not diagnosed or effectively treated, leading to a >95% mortality. We report here on five cases of balamuthiasis that were diagnosed by indirect immunofluorescence
The American journal of tropical medicine and hygiene
Balamuthia mandrillaris is an opportunistically pathogenic ameba that causes fatal granulomatous ... more Balamuthia mandrillaris is an opportunistically pathogenic ameba that causes fatal granulomatous amebic encephalitis (GAE) in vertebrates. Previous phylogenetic analyses that included the sequence of a single nuclear small subunit ribosomal RNA gene (18S or ssu rDNA) from this ameba suggested that Balamuthia is closely related to Acanthamoeba, another opportunistically pathogenic amebic genus, which includes multiple ssu rDNA genotypes. We tested whether this also is true for Balamuthia. The nuclear ssu rDNA from 4 isolates and the mitochondrial ssu rDNA from 7 isolates of B. mandrillaris have been sequenced. No variation in the nuclear rDNA sequences and low levels of variation in the mitochondrial rDNA were found. Both gene sequences were consistent with a single genotype for B. mandrillaris. The mitochondrial sequences of B. mandrillaris are unique and should be useful for development of genus-specific diagnostic probes for use with clinical, environmental, and archived specimens.
Members of the genus Acanthamoeba are usually free-living amebae and are found in a variety of ec... more Members of the genus Acanthamoeba are usually free-living amebae and are found in a variety of ecological niches including soil, fresh and brackish water, dust in air, filters of heating, ventilating, and air conditioning units, swimming pools and hot tubs, etc. Occasionally, they are also known to cause central nervous system infections in humans and other animals. We isolated into culture an amoeba from the liver tissue of a keel-billed toucan and identified it as Acanthamoeba sp. based on culture characteristics and immunofluorescent analysis. Further, we characterized the cultured amoeba and also the amoeba in the liver tissue as Acanthamoeba, genotype T4, by sequencing a diagnostic region of the nuclear small subunit ribosomal RNA gene.
A phylogenetic investigation of the autosporic chlorophycean alga species Selenastrum capricornut... more A phylogenetic investigation of the autosporic chlorophycean alga species Selenastrum capricornutum using the small subunit (SSU) rRNA gene revealed the unusual presence of six group IC1 introns. Previous studies showed that numerous green algal taxa contain group IC1 introns. However, whereas some algal species harbor multiple introns in a single ribosomal gene, none have contained as many as S. capricornutum.
Members of the genus Acanthamoeba are usually free-living amoebae that are found in a variety of ... more Members of the genus Acanthamoeba are usually free-living amoebae that are found in a variety of ecological niches including soil, fresh and brackish water, dust in the air, heating, ventilating, and air conditioning filters, swimming pools and hot tubs. Occasionally they are also known to cause central nervous system infections in humans and animals. We isolated into culture an amoeba from the liver of a Temminck's tragopan (horned pheasant) (Tragopan temminckii) that died of amoebic infection. We identified the infecting amoeba as Acanthamoeba sp. based on culture characteristics, cyst morphology and immunofluorescence assays. Additionally, we identified the amoeba as Acanthamoeba, genotype T4, by sequencing a diagnostic region of the nuclear small subunit ribosomal RNA gene.
The genus Acunlhurnoeha is an assemblage of approximately 20 named species of free-living amoebae... more The genus Acunlhurnoeha is an assemblage of approximately 20 named species of free-living amoebae inhabiting a wide range of ecological niches, and has a worldwide distribution. Individual Acan-thurnorh~i havc been isolated from soil, fresh and saltwater, humans, domestic and feral animals. In addition to natural habitats, Acanthaniorhtr has heen identified as the cause of scveral diseases. It acts as an opportunistic pathogen in the painful and sight-thrcatening condition Ar~cinthunzoehu keratitis (AK) in otherwise healthy individuals. Acan-thui~zoc~ha is also responsible for life threatening infections in patients with immune defense deficiencies (IDD). Acantharnoeha strains have also heen associated with other infections, including those of the skin, eye, and brain (granulornatous amoebic encephalitis, GAE). These are generally observed in imiiiunocompromised individuals. Variations in the pathogenicity of Acanrhamoeha strains havc been recognized in various laboralory studies, but the relevance of these results to human disease is unclear.
Previous molecular examination of Acanthamoeba spp. has resulted in the determination of distinct... more Previous molecular examination of Acanthamoeba spp. has resulted in the determination of distinct genotypes in this genus (designated T1-T12, T14). Genotype T4 has been responsible for the majority of cases of Acanthamoeba keratitis. Here we examine the relative abundance of environmental T4 isolates on beaches and ask whether they have temperature and salinity tolerances that could enhance pathogenicity. Twenty-four Acanthamoeba strains were isolated from beach sand (n ϭ 20), soil (n ϭ 3), and tap water (n ϭ 1) in south Florida. Phylogenetic analysis identified 19 of 24 isolates as T4, the Acanthamoeba keratitis-associated genotype. The remaining isolates were genotype T5 (4) and T11 (1). Nearly all beach isolates were genotype T4, whereas the tap water and soil isolates were mostly T5. All amoebae grew at 0, 1.0, and 2.0% salt and 19 of 20 beach isolates also grew at 3.2%. No soil or tapwater acanthamoebae reproduced at 3.2%. All isolates grew at 37 ЊC and two (T5) at 42 ЊC. Little correlation existed between beach location, salt-tolerance, and genetic relatedness. Overall, the large majority of environmental isolates obtained were genotype T4, suggesting it may be the most common genotype in this environment and could be a potential source of Acanthamoeba keratitis infections.
Ultrastructural studies of tetrasporalean green algae have suggested the order is polyphyletic. T... more Ultrastructural studies of tetrasporalean green algae have suggested the order is polyphyletic. These features, including the absolute orientation of the flagellar apparatus and the bi-versus quadriflagellated motile cell morphology, suggest that Chaetopeltis as well as a number of others, may be ancestral to a group that includes Tetraspora. In this study, we examine the phylogenetic relationships of selected tetrasporalean taxa based on analysis of 18S ribosomal RNA gene sequences. Results show that the tetrasporalean taxa are polyphyletic. Biflagellated genera group with biflagellated volvocalean taxa, whereas the quadriflagellated species compose a distinct monophyletic clade not closely related to the biflagellated taxa. In addition, tetrasporalean taxa group with other chlorophycean algal species with similar flagellar apparatus absolute orientation, but the quadriflagellated Tetrasporales do not appear to be ancestral to the entire Chlorophyceae. These results are concordant with previous conclusions drawn from ultrastructural data and further confirm the utility of (small-subunit) ribosomal RNA gene sequences to discern green algal evolutionary relationships.
The order Chaetophorales includes filamentous green algae whose taxonomic relationships to other ... more The order Chaetophorales includes filamentous green algae whose taxonomic relationships to other chlorophycean orders is uncertain. Chaetophoralean taxa include filamentous species which are both branched and unbranched. Ultrastructural studies of zoospores have revealed similar flagellar apparatuses in a number of genera, including Uronema, Stigeoclonium, and Fritschiella, suggesting a close phylogenetic relationship among these taxa. The order Oedogoniales represents a second group of branched and unbranched filamentous green algae whose relationships to other chlorophycean orders also has been unclear. A possible close relationship between the Chaetophorales and Oedogoniales has been suggested. Using DNA sequences from the small-subunit ribosomal RNA gene (SSU rRNA) of several members of each order, we have examined the monophyly of the Chaetophorales and Oedogoniales, as well as the nature of their relationship to other chlorophycean orders. Our results show that chaetophoralean and oedogonialean taxa form separate monophyletic groups. Results also suggest that the two orders are not closely related to each other.
This report describes the first isolation of the ameba Balamuthia mandrillaris from an environmen... more This report describes the first isolation of the ameba Balamuthia mandrillaris from an environmental soil sample associated with a fatal case of amebic encephalitis in a northern California child. Isolation of the ameba into culture from autopsied brain tissue confirmed the presence of Balamuthia. In trying to locate a possible source of infection, soil and water samples from the child's home and play areas were examined for the presence of Balamuthia. The environmental samples (plated onto nonnutrient agar with Escherichia coli as a food source) contained, in addition to the ameba, a variety of soil organisms, including other amebas, ciliates, fungi, and nematodes, as contaminants. Presumptive Balamuthia amebas were recognized only after cultures had been kept for several weeks, after they had burrowed into the agar. These were transferred through a succession of nonnutrient agar plates to eliminate fungal and other contaminants. In subsequent transfers, axenic Naegleria amebas and, later, tissue cultures (monkey kidney cells) served as the food source. Finally, the amebas were transferred to cell-free axenic medium. In vitro, the Balamuthia isolate is a slow-growing organism with a generation time of ϳ30 h and produces populations of ϳ2 ؋ 10 5 amebas per ml. It was confirmed as Balamuthia by indirect immunofluorescence staining with rabbit anti-Balamuthia serum and human anti-Balamuthia antibody-containing serum from the amebic encephalitis patient. The environmental isolate is similar in its antimicrobial sensitivities and identical in its 16S ribosomal DNA sequences to the Balamuthia isolate from the deceased patient.
Acanthamoeba is a free-living ameba that is found throughout the world and that causes encephalit... more Acanthamoeba is a free-living ameba that is found throughout the world and that causes encephalitis, keratitis, and cutaneous infections in humans. It has two stages in its life cycle: a trophic stage and a resistant cyst stage. We describe here the ability of Acanthamoeba cysts to survive desiccation for more than 20 years.
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Papers by Gregory Booton