The Status of Molecular Analyses of Isolates of Acanthamoeba Maintained by International Culture Collections.

Acanthamoeba is among the most ubiquitous protistan groups in nature. Knowledge of the biological diversity of Acanthamoeba comes in part from the use of strains maintained by the major microbial culture collections, ATCC and CCAP. Standard strains are vital to ensure the comparability of research. The diversity of standard strains of Acanthamoeba in the culture collections is reviewed, emphasizing the extent of genotypic studies based on DNA sequencing of the small subunit ribosomal RNA from the nucleus (18S rRNA gene; Rns) or the mitochondria (16S-like rRNA gene; rns). Over 170 different strains have been maintained at some time by culture centers. DNA sequence information is available for more than 70% of these strains. Determination of the genotypic classification of standard strains within the genus indicates that frequencies of types within culture collections only roughly mirror that from clinical or environmental studies, with significant differences in the frequency of some genotypes. Culture collections include the type of isolate from almost all named species of Acanthamoeba, allowing an evaluation of the validity of species designations. Multiple species are found to share the same Sequence Type, while multiple Sequence Types have been identified for different strains that share the same species name. Issues of sequence reliability and the possibility that a small number of standard strains have been mislabeled when studied are also examined, leading to potential problems for comparative analyses. It is important that all species have reliable genotype designations. The culture collections should be encouraged to assist in completing the molecular inventory of standard strains, while workers in the Acanthamoeba research community should endeavor to ensure that strains representative of genotypes that are missing from the culture collection are provided to the culture centers for preservation.

Molecular identification and phylogenetic analysis of free-living amoeba in the water resources of Arak, Iran.

The aim of the present study was to detect free-living amoeba (FLA) in the water resources of Arak, Iran using molecular tools. A total of 154 samples were collected from different water supplies. Molecular analyses, sequencing, and phylogenetic study were conducted to confirm the species and genotypes of FLA. Fisher exact test was used to determine the significance. Of 154 water samples, 19 (12.3%) samples were tested positive for FLA. Three genotypes of Acanthamoeba including T4, subtype D, and T5 were identified among the isolates. The pathogenicity assay showed that the isolate of Acanthamoeba in drinking water was highly pathogenic. Three species of Naegleria, including N. australiensis, N. pagei, and N. gruberi were found among the samples. Six isolates of Vermamoeba were identified as V. vermiformis. Meanwhile, three other species including Vannella sp., Vahlkampfia avara, and Stenamoeba polymorpha were also recovered from the water samples. Statistical analysis showed a significant difference between the various water resources contaminated with FLA. This is the first study to reveal the presence of S. polymorpha in water sources in Iran. According to the findings of the present study, health officials should be beware of potential public health impacts of FLA in water resources.

A Brief History of the Major Rickettsioses in the Asia-Australia-Pacific Region: A Capstone Review for the Special Issue of TMID.

The rickettsioses of the "Far East" or Asia-Australia-Pacific region include but are not limited to endemic typhus, scrub typhus, and more recently, tick typhus or spotted fever. These diseases embody the diversity of rickettsial disease worldwide and allow us to interconnect the various contributions to this special issue of Tropical Medicine and Infectious Disease. The impact of rickettsial diseases-particularly of scrub typhus-was substantial during the wars and "police actions" of the last 80 years. However, the post-World War II arrival of effective antibiotics reduced their impact, when recognized and adequately treated (chloramphenicol and tetracyclines). Presently, however, scrub typhus appears to be emerging and spreading into regions not previously reported. Better diagnostics, or higher population mobility, change in antimicrobial policies, even global warming, have been proposed as possible culprits of this phenomenon. Further, sporadic reports of possible antibiotic resistance have received the attention of clinicians and epidemiologists, raising interest in developing and testing novel diagnostics to facilitate medical diagnosis. We present a brief history of rickettsial diseases, their relative importance within the region, focusing on the so-called "tsutsugamushi triangle", the past and present impact of these diseases within the region, and indicate how historically, these often-confused diseases were ingeniously distinguished from each another. Moreover, we will discuss the importance of DNA-sequencing efforts for Orientia tsutsugamushi, obtained from patient blood, vector chiggers, and rodent reservoirs, particularly for the dominant 56-kD type-specific antigen gene (tsa56), and whole-genome sequences, which are increasing our knowledge of the diversity of this unique agent. We explore and discuss the potential of sequencing and other effective tools to geographically trace rickettsial disease agents, and develop control strategies to better mitigate the rickettsioses.

Stenamoeba dejonckheerei sp. nov., a Free-Living Amoeba Isolated from a Thermal Spring.

Two amoeboid organisms were obtained from water samples taken from a thermal spring, "Agua Caliente", in Northwestern Mexico. The isolates were obtained when samples were cultivated at 37 °C on non-nutrient agar coated with Escherichia coli. The initial identification of the isolates was performed morphologically using light microscopy. The samples were found to have trophozoite morphology consistent with members of the genus Stenamoeba, a genus derived in 2007 from within the abolished polyphyletic genus Platyamoeba. Further analysis was performed by sequencing PCR products obtained using universal eukaryotic primers for the small subunit ribosomal ribonucleic acid (SSU rRNA) gene. Sequencing primers were designed to allow the comparison of the 18S rRNA gene sequences of the new isolates with previous sequences reported for Stenamoeba. Phylogenetic relationships among sequences from Stenamoeba were determined using Maximum Likelihood analysis. The results showed the two "Agua Caliente" sequences to be closely related, while clearly separating them from those of other Stenamoeba taxa. The degrees of sequence differentiation from other taxa were considered sufficient to allow us to propose that the Mexican isolates represent a new species.

Species, Sequence Types and Alleles: Dissecting Genetic Variation in Acanthamoeba.

Species designations within Acanthamoeba are problematic because of pleomorphic morphology. Molecular approaches, including DNA sequencing, hinted at a resolution that has yet to be fully achieved. Alternative approaches were required. In 1996, the Byers/Fuerst lab introduced the concept of sequence types. Differences between isolates of Acanthamoeba could be quantitatively assessed by comparing sequences of the nuclear 18S rRNA gene, ultimately producing 22 sequence types, designated T1 through T22. The concept of sequence types helps our understanding of Acanthamoeba evolution. Nevertheless, substantial variation in the 18S rRNA gene differentiates many isolates within each sequence type. Because the majority of isolates with sequences in the international DNA databases have been studied for only a small segment of the gene, designated ASA.S1, genetic variation within this hypervariable region of the 18S rRNA gene has been scrutinized. In 2002, we first categorized variation in this region in a sample of T3 and T4 isolates from Hong Kong, observing ten "alleles" within type T4 and five "alleles" within T3. Subsequently, confusion occurred when different labs applied redundant numerical labels to identify different alleles. A more unified approach was required. We have tabulated alleles occurring in the sequences submitted to the international DNA databases, and determined their frequencies. Over 150 alleles have occurred more than once within 3500+ isolates of sequence type T4. Results from smaller samples of other sequence types (T3, T5, T11 and T15, and supergroup T2/6) have also been obtained. Our results provide new insights into the evolutionary history of Acanthamoeba, further illuminating the degree of genetic separation between significant taxonomic units within the genus, perhaps eventually elucidating what constitutes a species of Acanthamoeba.

Origins, Importance and Genetic Stability of the Prototype Strains Gilliam, Karp and Kato of Orientia tsutsugamushi.

Scrub typhus, a chigger-borne febrile illness, occurs primarily in countries of the Asia-Pacific rim and islands of the Western Pacific. The etiologic agent is the obligate intracellular rickettsial bacterium Orientia tsutsugamushi. Research on O. tsutsugamushi has relied on the availability of several prototype strains, which were isolated from human cases of scrub typhus in the 1940s and 1950s. We review the history of the three original, and most important, prototype strains, Gilliam, Karp and Kato, including information on their isolation, their culture history, their clinical characteristics, their importance within the research literature on scrub typhus, and recent advances in elucidating their molecular genomics. The importance of these strains to the research and development of clinical tools related to scrub typhus is also considered. Finally, we examine whether the strains have been genetically stable since their isolation, and whether prototype strains maintained in separate laboratories are identical, based on pairwise comparisons of several sequences from four genes. By using genetic information archived in international DNA databases, we show that the prototype strains used by different laboratories are essentially identical, and that the strains have retained their genetic integrity at least since the 1950s. The three original prototype strains should remain a standard by which new diagnostic procedures are measured. Given their fundamental position in any comparative studies, they are likely to endure as a critical part of present and future research on scrub typhus and Orientia.

The Historical Case for and the Future Study of Antibiotic-Resistant Scrub Typhus.

Scrub typhus is an acute, and sometimes fatal, human febrile illness, typically successfully treated using chloramphenicol or one of the tetracyclines. Over the past several years, descriptions of strains of Orientia tsutsugamushi with reduced susceptibility to antibiotics have appeared. Because case-fatality ratios approached 50% during the pre-antibiotic era, antibiotic-resistant scrub typhus is concerning. Herein, we review the data on resistant scrub typhus, describe how the theoretical existence of such resistance is affected by interpretation of treatment outcomes, and propose a plan to further identify whether true drug resistance is present and how to deal with drug resistance if it has evolved. Limited resistance is not unambiguous, if present, and antibiotic resistance in scrub typhus is not a dichotomous trait. Rather, evidence of resistance shows a continuous gradation of increasing resistance. The availability of genomes from isolates of O. tsutsugamushi allows the search for loci that might contribute to antibiotic resistance. At least eighteen such loci occur in all genomes of O. tsutsugamushi examined. One gene (gyrA) occurs as a quinolone-resistant form in the genome of all isolates of O. tsutsugamushi. At least 13 other genes that are present in some members of the genus Rickettsia do not occur within O. tsutsugamushi. Even though reports of scrub typhus not responding appropriately to chloramphenicol or a tetracycline treatment have been in the literature for approximately 23 years, the existence and importance of antibiotic-resistant scrub typhus remains uncertain.

Phylogenetic analysis and the evolution of the 18S rRNA gene typing system of Acanthamoeba.

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.

The prevalence of rickettsial and ehrlichial organisms in Amblyomma americanum ticks collected from Ohio and surrounding areas between 2000 and 2010.

The lone star tick, Amblyomma americanum, feeds upon a variety of hosts and is a known vector of several human pathogens. In Ohio, populations of A. americanum have been expanding their range and increasing in abundance and distribution, thereby elevating the public health concerns regarding bites from this species. We used a set of PCR assays to detect the presence of ehrlichial and rickettsial species in A. americanum ticks submitted to the Ohio Department of Health Zoonotic Disease Program over an 11-year period (2000-2010). We did not detect the presence of known pathogens Rickettsia rickettsii or Ehrlichia chaffeensis, but we did identify the presence of two other bacterial species: 'Candidatus Rickettsia amblyommii', and Ehrlichia sp. Panola Mountain. 'Candidatus R. amblyommii' was the most common species identified (30.2%), whereas the ehrlichiae was quite rare (0.6%). With growing evidence implicating both 'Candidatus Rickettsia amblyommii' and Ehrlichia sp. Panola Mountain in mild to moderate human disease, our results support the importance of continued monitoring of A. americanum ticks for the presence of potential pathogens.

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.

Insights from the DNA databases: approaches to the phylogenetic structure of Acanthamoeba.

Species of Acanthamoeba have been traditionally described using morphology (primarily cyst structure), or cytology of nuclear division (used by Pussard and Pons, 1977). Twenty-plus putative species were proposed based on such criteria. Morphology, however, is often plastic, dependent upon culture conditions. DNA sequences of the nuclear small subunit (18S) rRNA that can be used for the study of the phylogeny of Acanthamoeba have increased from a single sequence in 1986 to more than 1800 in 2013. Some of the patterns of the sequence data for Acanthamoeba are reviewed, and some of the insights that this data illuminates are illustrated. In particular, the data suggest the existence of 20 or more genotypic types, a number not dissimilar to the number of named species of Acanthamoeba. However, molecular studies make clear that the relationship between phylogenetic relatedness and species names as we know them for Acanthamoeba is tenuous at best.

The effects of locus number, genetic divergence, and genotyping error on the utility of dominant markers for hybrid identification.

In surveys of hybrid zones, dominant genetic markers are often used to identify individuals of hybrid origin and assign these individuals to one of several potential hybrid classes. Quantitative analyses that address the statistical power of dominant markers in such inference are scarce. In this study, dominant genotype data were simulated to evaluate the effects of, first, the number of loci analyzed, second, the magnitude of differentiation between the markers scored in the groups that are hybridizing, and third, the level of genotyping error associated with the data when assigning individuals to various parental and hybrid categories. The overall performance of the assignment methods was relatively modest at the lowest level of divergence examined (F st ˜ 0.4), but improved substantially at higher levels of differentiation (F st ˜ 0.67 or 0.8). The effect of genotyping error was dependent on the level of divergence between parental taxa, with larger divergences tempering the effects of genotyping error. These results highlight the importance of considering the effects of each of the variables when assigning individuals to various parental and hybrid categories, and can help guide decisions regarding the number of loci employed in future hybridization studies to achieve the power and level of resolution desired.

Performance evaluation and optimization of multiplex PCRs for the highly discriminating OSU 10-locus set Y-STRs.

In a previous study, a new set of Y-chromosome short tandem repeats, the OSU 10-locus set (MPM1 and MPM2), was shown to have a higher discrimination power when evaluated against the 10 SWGDAM loci on a common population panel. Here, we describe the optimization of the multiplex reactions using dye-labeled primers followed by performance evaluations. The loci exhibited high precision, human male specificity, reliability in different body fluids, high sensitivity, stability, and the ability to amplify nonprobative casework and mixture samples. Stutter for the all of the loci, with the exception of the highly polymorphic locus DYS688, was similar to that observed for autosomal loci. The results of the performance evaluations reinforced the utility of these loci.

Endemic scrub typhus-like illness, Chile.

We report a case of scrub typhus in a 54-year-old man who was bitten by several terrestrial leeches during a trip to Chiloé Island in southern Chile in 2006. A molecular sample, identified as related to Orientia tsutsugamushi based on the sequence of the16S rRNA gene, was obtained from a biopsy specimen of the eschar on the patient's leg. Serologic analysis showed immunoglobulin G conversion against O. tsutsugamushi whole cell antigen. This case and its associated molecular analyses suggest that an Orientia-like agent is present in the Western Hemisphere that can produce scrub typhus-like illness. The molecular analysis suggests that the infectious agent is closely related, although not identical, to members of the Orientia sp. from Asia.

Multisystemic infection with an Acanthamoeba sp in a dog.

UNLABELLED: 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.

Isolation, morphologic, serologic and molecular identification of Acanthamoeba T4 genotype from the liver of a Temminck's tragopan (Tragopan temminckii).

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.

Molecular detection of Rickettsia bellii, Rickettsia montanensis, and Rickettsia rickettsii in a Dermacentor variabilis tick from nature.

Rickettsial diseases, such as Rocky Mountain spotted fever, pose a public health threat because of humans' interrelationship with common arthropod species, such as ticks, mites, fleas, and lice. Individuals may come in contact with these vectors of disease on a fairly regular basis either directly or indirectly through pets or wildlife species, at home or in recreational areas. Therefore, it is of vital importance to know and understand the geographical distribution and prevalence of disease and rickettsial-infected arthropods. We analyzed Dermacentor variabilis ticks from nature found positive for Rickettsia sp. to determine the specific species present. Rickettsiae were detected through a 17-kDa surface antigen seminested PCR. Seminested PCR represents a sensitive and specific molecular technique in which to identify the presence of bacteria within arthropod hosts. Through sequence analysis of this gene, three Rickettsia species, Rickettsia bellii, Rickettsia montanensis, and Rickettsia rickettsii, were detected in a single tick specimen. Further molecular analyses of the 17-kDa surface antigen and the citrate synthase gene were also performed to support this finding. This is the first report of the detection of multiple Rickettsia species from a single D. variabilis tick in nature.

A comparative analysis of two different sets of Y-chromosome short tandem repeats (Y-STRs) on a common population panel.

A comparative analysis of two Y-STR loci sets was conducted on a population sample of 224 individuals, 114 Caucasians and 110 African Americans. One set of loci, designated the OSU 10-locus set, comprises variable, single copy, male-specific loci that are dispersed across the Y-chromosome. Parallel evaluations were performed using the 10 Y-chromosome loci most frequently used for forensic analysis, the loci chosen as the SWGDAM Y-STR loci. The OSU 10-locus set had a greater average number of alleles per locus and higher average gene diversity than the SWGDAM loci. The OSU 10-locus set found 220 unique haplotypes in 224 individuals. In approximately 6000 pairwise haplotype comparisons for each population with each set of loci, the OSU 10-locus set also yielded a greater average number of allelic differences per pair than the SWGDAM loci. Finally, the overall linkage disequilibrium levels were lower for the OSU 10-locus set in the Caucasian population. In general, the OSU 10-locus set revealed a higher power of discrimination than the SWGDAM set.

Genotypic identification of Acanthamoeba sp. isolates associated with an outbreak of acanthamoeba keratitis.

PURPOSE: To determine whether increased rates of Acanthamoeba keratitis (AK) are due to changes in municipal water treatment or to emergence of a more pathogenic strain of Acanthamoeba. METHODS: Previous sequence analysis of the 18S ribosomal DNA of Acanthamoeba isolates resulted in the identification of 15 different genotypic classes. These analyses indicate that AK cases are associated predominantly ( approximately 97%) with a single genotype (designated T4) of Acanthamoeba and rarely with other genotypes (eg, T3 and T11). In this study, we test the hypothesis that a new or more pathogenic genotype of Acanthamoeba is the cause of the recent surge in AK. RESULTS: We determined the genotype of 15 Acanthamoeba sp. isolates from AK cases associated with this outbreak using sequence analysis of a region of the 18S ribosomal DNA. Our results indicate that these isolates are predominantly genotype T4 (87%), with the remaining isolates being genotype T3 (13%). Both genotypes have previously been observed in AK cases. CONCLUSIONS: There is no support for the hypothesis that the current AK outbreak is associated with infection by a new more pathogenic Acanthamoeba genotype. In addition, these results offer support for the hypothesis that the increased AK incidence may be because of changes in water treatment protocols leading to increased bacterial colonization of the water supply and subsequent increases of already present Acanthamoeba sp, ultimately culminating in an increase of AK cases.

Scrub typhus: the geographic distribution of phenotypic and genotypic variants of Orientia tsutsugamushi.

Orientia tsutsugamushi is the etiological agent of scrub typhus, an acute, mite-borne, febrile illness that occurs in the Asia-Pacific region. Historically, strain characterization used serological analysis and revealed dramatic antigenic diversity. Eyeing a recommendation of potential vaccine candidates for broad protection, we review geographic diversity and serological and DNA prevalences. DNA analysis together with immunological analysis suggest that the prototype Karp strain and closely related strains are the most common throughout the region of endemicity. According to serological analysis, approximately 50% of isolates are seroreactive to Karp antisera, and approximately one-quarter of isolates are seroreactive to antisera against the prototype Gilliam strain. Molecular methods reveal greater diversity. By molecular methods, strains phylogenetically similar to Karp make up approximately 40% of all genotyped isolates, followed by the JG genotype group (Japan strains serotypically similar to the Gilliam strain but genetically non-Gilliam; 18% of all genotyped isolates). Three other genotype groups (Kato-related, Kawasaki-like, and TA763-like) each represent approximately 10% of genotyped isolates. Strains genetically similar to the Gilliam strain make up only 5% of isolates. Strains from these groups should be included in any potential vaccine.