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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Survival of Acanthamoeba cysts after desiccation for more than 20 years.

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.

Geographic distribution and genetic diversity of the Ehrlichia sp. from Panola Mountain in Amblyomma americanum.

BACKGROUND: A novel Ehrlichia, closely related to Ehrlichia ruminantium, was recently discovered from Panola Mountain State Park, GA, USA. We conducted a study to determine if this agent was recently introduced into the United States. METHODS: We developed a sensitive PCR assay based on the conserved gltA (citrate synthase) gene and tested DNA samples extracted from 1964 field-collected and 1835 human-biting Amblyomma americanum from 23 eastern states of the USA. RESULTS: The novel agent was detected in 36 ticks collected from 10 states between 1998 and 2006. Infected ticks were collected both from vegetation (n = 14, 0.7%) and from humans (n = 22, 1.2%). Fragments of the conserved gltA gene and the variable map1 gene were sequenced from positive samples. Two distinct clades, with 10.5% nucleic acid divergence over the 730 bp map1 sequence, were identified. CONCLUSION: These data suggest that the Panola Mountain Ehrlichia was not recently introduced to the United States; this agent has an extensive distribution throughout the range of its tick vector, has been present in some locations for several years, and displays genetic variability. Furthermore, people in several states were exposed to this agent through the bite of infected ticks, underscoring the potential public health risk of this emerging ehrlichiosis.

The association of contact lens solution use and Acanthamoeba keratitis.

PURPOSE: To investigate Acanthamoeba keratitis (AK) risk factors. Diagnosis of AK, a rare but serious corneal infection, has recently increased significantly at the University of Illinois at Chicago (UIC) Cornea Service. DESIGN: Retrospective case-control study. METHODS: settings: University, tertiary care hospital. patients: Fifty-five AK cases with contact lens use were diagnosed between May 1, 2003 and September 15, 2006. Clinic-matched controls with contact lens use were recruited. Subjects completed surveys targeting lens hygiene, contact lens solution use, and water exposure. main outcome measure: Acanthamoeba keratitis. RESULTS: Thirty-nine (73.6%) cases and 113 (65.3%) controls participated; 38 cases had complete contact lens data. Thirty-five of 38 cases (92.1%) and 47 of 100 controls (47.0%) used soft lenses. Analysis was performed on 30 cases and 39 controls with matched pairs with soft lens use. Exclusive use of Advance Medical Optics (AMO) Complete MoisturePlus Multi-Purpose Solution was independently associated with AK in multivariable analysis (55.2% vs 10.5%; odds ratio [OR], 16.67; 95% confidence interval [CI] 2.11 to 162.63; P = .008). However, 38.8% of cases reported no use of AMO Complete MoisturePlus Multi-Purpose Solution either alone or in combination with other solutions. Although not statistically significant, additional hygiene-related variables (solution "reuse," lack of "rubbing," and showering with lenses) suggest a pattern of risk. CONCLUSIONS: AMO Complete MoisturePlus Multi-Purpose Solution use is independently associated with AK among soft contact lens users. However, it does not explain all cases, suggesting additional factors. Further research into environmental risk factors and hygiene practices is warranted, especially considering this is the second outbreak of an atypical, contact lens-related infection.

Infections caused by pathogenic free-living amebas (Balamuthia mandrillaris and Acanthamoeba sp.) in horses.

This article describes amebic infections in 4 horses: granulomatous amebic encephalitis caused by Balamuthia mandrillaris and Acanthamoeba culbertsoni and systemic infections caused by Acanthamoeba sp. The former infection occurred in 1 of 4 horses spontaneously without any underlying conditions; the latter amebic infection was perhaps "opportunistic" considering the visceral involvement by this protozoan in association with Aspergillus sp. and/or Escherichia coli and Pseudomonas sp. The clinicopathologic findings and demonstration of the amebic organisms using immunohistochemical techniques, culture, polymerase chain reactions, and electron microscopy are presented.

In vitro culture, serologic and molecular analysis of Acanthamoeba isolated from the liver of a keel-billed toucan (Ramphastos sulfuratus).

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.

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.

A rickettsial mixed infection in a Dermacentor variabilis tick from Ohio.

We present the first report of superinfection in a Dermacentor variabilis tick from nature. The single tick, collected in Ohio, was found infected with Rickettsia belli, R. nontanensis, and R. rickettsii.

Advantages of using mitochondrial 16S rDNA sequences to classify clinical isolates of Acanthamoeba.

PURPOSE: This work was intended to test the classification of Acanthamoeba into genotypes based on nuclear ribosomal RNA gene (18S rDNA, Rns) sequences. Nearly all Acanthamoeba keratitis (AK) isolates are genotype RnsT4. This marked phylogenetic localization is presumably either due to an innate potential for pathogenicity or to a peculiarity of the gene sequences used. To differentiate between these possibilities, relationships among isolates have been reexamined, using a second gene. METHODS: Phylogenetic relationships among isolates of Acanthamoeba were studied, using sequences of the mitochondrial small subunit ribosomal RNA gene (16S rDNA; rns). Genotypes based on complete sequences of approximately 1540 bp were determined for 68 strains, by using multiple phylogenetic analyses. RESULTS: Each strain's mitochondria contained a single intron-free rns sequence (allele). The 68 strains had 35 different sequences. Twenty-eight strains had unique sequences, and 40 strains each shared one of the seven remaining sequences. Eleven mitochondrial rns genotypes corresponding to 11 of 12 previously described nuclear Rns genotypes were identified. Genotype rnsT4 was subdivided into eight distinct clades, with seven including Acanthamoeba keratitis (AK) isolates. CONCLUSIONS: The phylogenetic clustering of AK isolates was confirmed and thus is not specific to the nuclear gene. Rns and rns sequences are both suitable for genotyping of ACANTHAMOEBA: However, the mitochondrial sequences are shorter and more consistent in length, have a higher percentage of alignable bases for sequence comparisons, and have none of the complications caused by multiple alleles or introns, which are occasionally found in Rns. In addition, the more common occurrence of strains with identical rns sequences simplifies identification and clustering of isolates.

Genotyping of Balamuthia mandrillaris based on nuclear 18S and mitochondrial 16S rRNA genes.

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.