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.

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.

A Review of Scrub Typhus (Orientia tsutsugamushi and Related Organisms): Then, Now, and Tomorrow.

Scrub typhus and the rickettsial diseases represent some of the oldest recognized vector-transmitted diseases, fraught with a rich historical aspect, particularly as applied to military/wartime situations. The vectors of Orientia tsutsugamushi were once thought to be confined to an area designated as the Tsutsugamushi Triangle. However, recent reports of scrub typhus caused by Orientia species other than O. tsutsugamushi well beyond the limits of the Tsutsugamushi Triangle have triggered concerns about the worldwide presence of scrub typhus. It is not known whether the vectors of O. tsutsugamushi will be the same for the new Orientia species, and this should be a consideration during outbreak/surveillance investigations. Additionally, concerns surrounding the antibiotic resistance of O. tsutsugamushi have led to considerations for the amendment of treatment protocols, and the need for enhanced public health awareness in both the civilian and medical professional communities. In this review, we discuss the history, outbreaks, antibiotic resistance, and burgeoning genomic advances associated with one of the world's oldest recognized vector-borne pathogens, O. tsutsugamushi.

Comparative pan-genomic analyses of Orientia tsutsugamushi reveal an exceptional model of bacterial evolution driving genomic diversity.

Orientia tsutsugamushi, formerly Rickettsia tsutsugamushi, is an obligate intracellular pathogen that causes scrub typhus, an underdiagnosed acute febrile disease with high morbidity. Scrub typhus is transmitted by the larval stage (chigger) of Leptotrombidium mites and is irregularly distributed across endemic regions of Asia, Australia and islands of the western Pacific Ocean. Previous work to understand population genetics in O. tsutsugamushi has been based on sub-genomic sampling methods and whole-genome characterization of two genomes. In this study, we compared 40 genomes from geographically dispersed areas and confirmed patterns of extensive homologous recombination likely driven by transposons, conjugative elements and repetitive sequences. High rates of lateral gene transfer (LGT) among O. tsutsugamushi genomes appear to have effectively eliminated a detectable clonal frame, but not our ability to infer evolutionary relationships and phylogeographical clustering. Pan-genomic comparisons using 31 082 high-quality bacterial genomes from 253 species suggests that genomic duplication in O. tsutsugamushi is almost unparalleled. Unlike other highly recombinant species where the uptake of exogenous DNA largely drives genomic diversity, the pan-genome of O. tsutsugamushi is driven by duplication and divergence. Extensive gene innovation by duplication is most commonly attributed to plants and animals and, in contrast with LGT, is thought to be only a minor evolutionary mechanism for bacteria. The near unprecedented evolutionary characteristics of O. tsutsugamushi, coupled with extensive intra-specific LGT, expand our present understanding of rapid bacterial evolutionary adaptive mechanisms.

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 Spatiotemporal Database to Track Human Scrub Typhus Using the VectorMap Application.

Scrub typhus is a potentially fatal mite-borne febrile illness, primarily of the Asia-Pacific Rim. With an endemic area greater than 13 million km2 and millions of people at risk, scrub typhus remains an underreported, often misdiagnosed febrile illness. A comprehensive, updatable map of the true distribution of cases has been lacking, and therefore the true risk of disease within the very large endemic area remains unknown. The purpose of this study was to establish a database and map to track human scrub typhus. An online search using PubMed and the United States Armed Forces Pest Management Board Literature Retrieval System was performed to identify articles describing human scrub typhus cases both within and outside the traditionally accepted endemic regions. Using World Health Organization guidelines, stringent criteria were used to establish diagnoses for inclusion in the database. The preliminary screening of 181 scrub typhus publications yielded 145 publications that met the case criterion, 267 case records, and 13 serosurvey records that could be georeferenced, describing 13,739 probable or confirmed human cases in 28 countries. A map service has been established within VectorMap (www.vectormap.org) to explore the role that relative location of vectors, hosts, and the pathogen play in the transmission of mite-borne scrub typhus. The online display of scrub typhus cases in VectorMap illustrates their presence and provides an up-to-date geographic distribution of proven scrub typhus cases.

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.

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.

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.

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.

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.

Fatal granulomatous Acanthamoeba encephalitis mimicking a stroke, diagnosed by correlation of results of sequential magnetic resonance imaging, biopsy, in vitro culture, immunofluorescence analysis, and molecular analysis.

Amebic infections involving the central nervous system are rare and difficult to diagnose. Magnetic resonance imaging (MRI) at timed intervals may be helpful, where scans reveal enhancing lesions and increased signal. We report a unique case of granulomatous amebic encephalitis that was proven pathologically with progressive radiological findings on MRI.

History of U.S. military contributions to the study of rickettsial diseases.

Rickettsial diseases have affected the military throughout history. Efforts such as those of the Joint U.S. Typhus Commission near the beginning of World War II and of military researchers since have reduced the impact of these diseases on U.S. and Allied forces. Despite the postwar development of effective antibiotic therapies, the newly emerging antibiotic-resistant scrub typhus rickettsial strains of the Asian Pacific region mandate continued research and surveillance. Similarly, tick-infested training areas in the United States and similar exposure abroad render the spotted fevers and the ehrlichioses problematic to deployed troops. The military continues to work on countermeasures to control the arthropod vectors, as well as actively participating in the development of rapid accurate diagnostic tests, vaccines, and improved surveillance methods. Several rickettsial diseases, including epidemic typhus, scrub typhus, the ehrlichioses, and the spotted fevers, are reviewed, with emphasis on the military historical significance and contributions.

Identification and distribution of Acanthamoeba species genotypes associated with nonkeratitis infections.

Acanthamoeba is a free-living protozoan genus found in a wide variety of natural habitats, including water, soil, and air. Pathogenic isolates of Acanthamoeba are medically relevant as the causative agent of sight- threatening Acanthamoeba keratitis (AK), serious infections of other organs, and fatal granulomatous amebic encephalitis. Previous work employing DNA sequences of nuclear and mitochondrial small-subunit rRNA genes (SSU rRNA genes) determined the genotypic diversity of Acanthamoeba and found that many named species of Acanthamoeba are associated with particular genotypes. These studies also concluded that nearly all AK infections result from a single molecular genotype: T4. Here, we asked whether Acanthamoeba clinical isolates from non-AK infections are also associated with particular genotypes. DNA sequence determination of nuclear SSU rRNA genes was employed for genotypic identification of 29 isolates of Acanthamoeba from non-AK infections. Sequence analysis demonstrates that T4 is the predominant genotype in non-AK infections, including those in brain, cerebrospinal fluid, nasal passages, skin, and lung. Rare genotypes (T1, T10, and T12) have been isolated from brain infections. We conclude that genotype T4 is the primary genotype in non-AK Acanthamoeba infections, as was the case in AK infections. However, the genotypes that were isolated from brains have not been observed in environmental isolates of Acanthamoeba, and their natural ecological niche is unknown.

Molecular and physiological evaluation of subtropical environmental isolates of Acanthamoeba spp., causal agent of Acanthamoeba keratitis.

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 = 20), soil (n = 3), and tap water (n = 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 tap-water acanthamoebae reproduced at 3.2%. All isolates grew at 37 degrees C and two (T5) at 42 degrees C. 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.

Serologic evidence of infection with ehrlichiae and spotted fever group rickettsiae among residents of Gag Island, Indonesia.

The causative agents of scrub and murine typhus are considered endemic to Indonesia. However, the presence of spotted fever group rickettsiae and ehrlichiae have not been previously described in this country. During an investigation of arthropod-borne diseases on Gag Island, located northwest of the island of New Guinea in eastern Indonesia, the prevalence of antibody to the etiologic agents of monocytic ehrlichiosis, spotted fever rickettsiosis, and scrub and murine typhus were determined. Analysis of 55 blood samples from residents of Gag Island showed seroreactivity to antigen preparations of Ehrlichia chaffeensis (7 of 48, 14.6%), two spotted fever group rickettsiae: Rickettsia rickettsii (5 of 48, 10.4%) and R. conorii (10 of 49, 20.4%), Orientia tsutsugamushi (5 of 53, 9.4%), and R. typhi (1 of 48, 2.1% [by an indirect immunofluorescence assay] and 1 of 50, 2.0% [by an enzyme-linked immunosorbent assay]). These results show serologic evidence of infection with ehrlichiae and spotted fever group rickettsiae for the first time in Indonesia in a location where the prevalence of antibody to O. tsutsugamushi and R. typhi was lower.

Evidence of Rickettsia typhi and the potential for murine typhus in Jayapura, Irian Jaya, Indonesia.

Murine typhus (etiologic agent: Rickettsia typhi) is endemic to Indonesia, especially on the highly populated island of Java. A survey of rodents from Irian Jaya, the eastern-most province of Indonesia, indicated striking geographic variation in risk factors associated with murine typhus. Murid rodents (n = 112) collected from two villages in the Arso district of northeastern Irian Jaya, were found to be free of ectoparasites normally associated with transmission of R. typhi (i.e., Xenopsylla cheopis). All rodents (n = 72) tested by enzyme-linked immunosorbent assay were negative for antibodies to R. typhi, whereas 12.5% (9/72) were positive for antibodies to Orienta tsutsugamushi. In contrast, both Rattus norvegicus and R. rattles (combined n = 87) from the harbor area of the provincial capital, Jayapura, were infested with flea ectoparasites. X. cheopis was found on 31 (35.6%) of the live-captured rodents. Serum samples from nine of 82 rodents contained antibodies reactive to R. typhi (11.0%). These data show for the first time that rodents exposed to R. typhi are well established in Jayapura, and that some of these rodents harbor fleas potentially capable of transmitting murine typhus and plague.

The past and present threat of rickettsial diseases to military medicine and international public health.

Morbidity and mortality caused by rickettsioses have had a major influence on military activities and public health for >2000 years. The threat posed by the rickettsioses is reviewed, focusing on the impact and epidemiology of those that have adversely influenced wartime operations and the current challenges posed by these diseases. With their uneven worldwide distribution, the discovery of drug-refractory strains of Orientia tsutsugamushi, the increased threat of their use in acts of bioterrorism, frequent deployment of troops to regions of endemicity, and exposures due to increased humanitarian missions, these diseases continue to be a threat to military personnel in the field. Effective strategies to reduce the impact of these diseases include development of effective vaccines, enhanced surveillance, and development of new safe, effective, and odorless repellants. The continuation of a proven, highly productive military infectious disease research program is essential for providing solutions to these daunting tasks.