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Infection with either Rickettsia prowazekii or Orientia tsutsugamushi is common, yet diagnostic capabilities are limited due to the short window for positive identification. Until now, although targeted enrichment had been applied to increase sensitivity of sequencing-based detection for various microorganisms, it had not been applied to sequencing of R. prowazekii in clinical samples. Additionally, hybridization-based targeted enrichment strategies had only scarcely been applied to qPCR of any pathogens in clinical samples. Therefore, we tested a targeted enrichment technique as a proof of concept and found that it dramatically reduced the limits of detection of these organisms by both qPCR and high throughput sequencing. The enrichment methodology was first tested in contrived clinical samples with known spiked-in concentrations of R. prowazekii and O. tsutsugamushi DNA. This method was also evaluated using clinical samples, resulting in the simultaneous identification and characterization of O. tsutsugamushi directly from clinical specimens taken from sepsis patients. We demonstrated that the targeted enrichment technique is helpful by lowering the limit of detection, not only when applied to sequencing, but also when applied to qPCR, suggesting the technique could be applied more broadly to include other assays and/or microbes for which there are limited diagnostic or detection modalities.

Rickettsioses, often underreported, pose public health challenges. Rickettsia asembonensis is a potential emerging pathogen that was previously detected in humans, animals, and a variety of arthropods. While its pathogenicity in humans remains unclear, it poses a potential public health threat. Here, we present an extended epidemiological, diagnostic, and genetic analysis of the information provided in a preliminary report on the investigation of rickettsiae in Peru. In particular, we report the detection of R. asembonensis in blood specimens collected from four human patients with an acute undifferentiated fever of a seven- to nine-day duration, all of whom tested negative for other vector-borne pathogens. Additionally, we describe the replicative capacity of the R. asembonensis isolates in cell cultures.

Background Scrub typhus is a vector-borne febrile illness caused by Orientia tsutsugamushi transmitted by the bite of Trombiculid mites. O. tsutsugamushi has a high genetic diversity and is increasingly recognized to have a wider global distribution than previously assumed. Methodology/principle findings We evaluated the clinical outcomes and host immune responses of the two most relevant human pathogenic strains of O. tsutsugamushi; Karp (n = 4) and Gilliam (n = 4) in a time-course study over 80 days post infection (dpi) in a standardized scrub typhus non-human primate rhesus macaque model. We observed distinct features in clinical progression and immune response between the two strains; Gilliam-infected macaques developed more pronounced systemic infection characterized by an earlier onset of bacteremia, lymph node enlargement, eschar lesions and higher inflammatory markers during the acute phase of infection, when compared to the Karp strain. C-reactive protein (CRP) plasma levels, interferon gamma (IFN-γ, interleukin-1 receptor antagonist (IL-1ra), IL-15 serum concentrations, CRP/IL10- and IFN-γ/IL-10 ratios correlated positively with bacterial load in blood, implying activation of the innate immune response and preferential development of a T helper-type 1 immune response. The O. tsutsugamushi-specific immune memory responses in cells isolated from skin and lymph nodes at 80 dpi were more markedly elevated in the Gilliam-infected macaques than in the Karp-infected group. The comparative cytokine response dynamics of both strains revealed significant up-regulation of IFN-γ, tumor necrosis factor (TNF), IL-15, IL-6, IL-18, regulatory IL-1ra, IL-10, IL-8 and granulocyte-colony-stimulating factor (G-CSF). These data suggest that the clinical outcomes and host immune responses to scrub typhus could be associated with counter balancing effects of pro- and anti-inflammatory cytokine-mediated responses. Currently, no data on characterized time-course comparisons of O. tsutsugamushi strains regarding measures of disease severity and immune response is available. Our study provides evidence for the strain-specificity of host responses in scrub typhus, which supports our understanding of processes at the initial inoculation site (eschar), systemic disease progression, protective and/or pathogenic host immune mechanisms and cellular immune memory function. Conclusions/significance This study characterised an improved intradermal rhesus macaque challenge model for scrub typhus, whereby the Gilliam strain infection associated with higher disease severity in the rhesus macaque model than the previous Karp strain infection. Difficulties associated with inoculum quantitation for obligate-intracellular bacteria were overcome by using functional inoculum titrations in outbred mice. The Gilliam-based rhesus macaque model provides improved endpoint measurements and contributes towards the identification of correlates of protection for future vaccine development. Author summary Scrub typhus is a mite-borne rickettsial illness, which is potentially severe if untreated or diagnosis is delayed or missed. This easily treatable disease is endemic in southeastern and eastern parts of Asia, but is increasingly recognised to have a global distribution. In endemic areas, where infected vectors transmit different strains of Orientia tsutsugamushi—the causative pathogen of scrub typhus—the diversity of these bacterial strains poses a serious obstacle for developing effective diagnostics and a universal scrub typhus vaccine. Comparison studies on the host immune responses against various strains are rare and there is limited evidence on strain-specific immune responses over the disease course. In this study, we characterized the clinical disease progression from the start of infection to full convalescence in a well-characterised non-human primate rhesus macaque model, infected with the two most prevalent O. tsutsugamushi strains; Karp and Gillam, and compared the time course dynamics of bacteremia with correlations of various immune response mechanisms. We found that Gilliam strain infection associated with higher disease severity, earlier onset of bacteremia, lymph node enlargement, eschar lesions and higher inflammatory markers during the acute phase of infection, when compared to the Karp strain. The findings suggest that a Gilliam strain infection in rhesus macaques provides improved endpoint measurements when compared to Karp strain infections, which is useful for future vaccine development.

Epidemic typhus, caused by Rickettsia prowazekii bacteria and transmitted through body lice (Pediculus humanus corporis), was a major public health threat in Eastern Europe as a consequence of World War II. In 2022, war and the resulting population displacement in Ukraine risks the return of this serious disease.

Scrub typhus is a potentially severe rickettsiosis, caused by Orientia tsutsugamushi in the Asia-Pacific region. Recently, however, two distinct pathogens, “Candidatus Orientia chuto” and “Candidatus Orientia chiloensis”, have been discovered in the Middle East and South America, respectively. Since the novel pathogens differ significantly from O. tsutsugamushi, many established diagnostic methods are unreliable. This work describes the development and validation of a new quantitative real-time PCR (qPCR) assay (Orien16S) for the detection of all known Orientia species. Based on a 94 bp sequence of the 16S rRNA gene (rrs), Orien16S recognized DNA samples from O. tsutsugamushi (n = 41), Ca. O. chiloensis (n = 5), and Ca. O. chuto (n = 1), but was negative for DNA preparations from closely related rickettsiae and other members of the order Rickettsiales (n = 22) as well as unrelated bacterial species (n = 11). After its implementation in Chile, the assay was verified, correctly identifying all tested eschar and buffy coat samples (n = 28) of clinical suspected cases. Furthermore, Orien16S detected Orientia DNA in trombiculid mites collected in endemic regions in southern Chile. The presented novel qPCR assay provides a useful tool for detecting Orientia and diagnosing scrub typhus from all geographical regions.

Scrub typhus is a potentially fatal rickettsiosis caused by Orientia species intracellular bacteria of the genus Orientia. Although considered to be restricted to the Asia Pacific region, scrub typhus has recently been discovered in southern Chile. We analyzed Orientia gene sequences of 16S rRNA (rrs) and 47-kDa (htrA) from 18 scrub typhus patients from Chile. Sequences were ≥99.7% identical among the samples for both amplified genes. Their diversity was 3.1%–3.5% for rrs and 11.2%–11.8% for htrA compared with O. tsusugamushi and 3.0% for rrs and 14.8% for htrA compared with Candidatus Orientia chuto. Phylogenetic analyses of both genes grouped the specimens from Chile in a different clade from other Orientia species. Our results indicate that Orientia isolates from Chile constitute a novel species, which, until they are cultivated and fully characterized, we propose to designate as Candidatus Orientia chiloensis, after the Chiloé Archipelago where the pathogen was identified.

Abstract Scrub typhus is an important arthropod-borne disease causing significant acute febrile illness by infection with Orientia spp. Using a risk-based approach, this review examines current practice, the evidence base and regulatory requirements regarding matters of biosafety and biosecurity, and presents the case for reclassification from Risk Group 3 to Risk Group 2 along with recommendations for safe working practices of risk-based activities during the manipulation of Orientia spp. in the laboratory. We recommend to reclassify Orientia spp. to Risk Group 2 based on the classification for RG2 pathogens as being moderate individual risk, low community risk. We recommend that low risk activities, can be performed within a biological safety cabinet located in a Biosafety Level (BSL) 2 core laboratory using standard personal protective equipment. But when the risk assessment indicates, such as high concentration and volume, or aerosol generation, then a higher biocontainment level is warranted. For, the majority of animal activities involving Orientia spp., Animal BSL 2 (ABSL2) is recommended however where high risk activities are performed including necropsies, Animal BSL (ABSL3) is recommended.

Cooperative research programs aimed at reducing biological threats have increased scientific capabilities and capacities in Kazakhstan. The German Federal Foreign Office's German Biosecurity Programme, the United Kingdom's International Biological Security Programme and the United States Defense Threat Reduction Agency's Biological Threat Reduction Program provide funding for partner countries, like Kazakhstan. The mutual goals of the programs are to reduce biological threats and enhance global health security. Our investigation examined these cooperative research programs, summarizing major impacts they have made, as well as common successes and challenges. By mapping various projects across the three programs, research networks are highlighted which demonstrate best communication practices to share results and reinforce conclusions. Our team performed a survey to collect results from Kazakhstani partner scientists on their experiences that help gain insights into enhancing day-to-day approaches to conducting cooperative scientific research. This analysis will serve as a basis for a capability maturity model as used in industry, and in addition builds synergy for future collaborations that will be essential for quality and sustainment.

Endemic scrub typhus was recently detected on Chiloé Island in southern Chile. We report a series of cases, acquired over a wide geographical range in continental Chile during 2016–2018, demonstrating that this emerging rickettsial infection is also found on the mainland of South America.

Rickettsioses are endemic to Vietnam; however, only a limited number of clinical studies have been performed on these vectorborne bacteria. We conducted a prospective hospital-based study at 2 national referral hospitals in Hanoi to describe the clinical characteristics of scrub typhus and murine typhus in northern Vietnam and to assess the diagnostic applicability of quantitative real-time PCR assays to diagnose rickettsial diseases. We enrolled 302 patients with acute undifferentiated fever and clinically suspected rickettsiosis during March 2015–March 2017. We used a standardized case report form to collect clinical information and laboratory results at the time of admission and during treatment. We confirmed scrub typhus in 103 (34.1%) patients and murine typhus in 12 (3.3%) patients. These results highlight the need for increased emphasis on training for healthcare providers for earlier recognition, prevention, and treatment of rickettsial diseases in Vietnam.

Cooperative research that addresses infectious disease surveillance and outbreak investigations relies heavily on availability and effective use of appropriate diagnostic tools, including serological and molecular assays, as exemplified by the current COVID-19 pandemic. In this paper, we stress the importance of using these assays to support collaborative epidemiological studies to assess risk of rickettsial disease outbreaks among international partner countries. Workforce development, mentorship, and training are important components in building laboratory capability and capacity to assess risk of and mitigate emerging disease outbreaks. International partnerships that fund cooperative research through mentoring and on-the-job training are successful examples for enhancing infectious disease surveillance. Cooperative research studies between the Naval Medical Research Center's Rickettsial Diseases Research Program (RDRP) and 17 institutes from nine countries among five continents were conducted to address the presence of and the risk for endemic rickettsial diseases. To establish serological and molecular assays in the collaborative institutes, initial training and continued material, and technical support were provided by RDRP. The laboratory methods used in the research studies to detect and identify the rickettsial infections included (1) group-specific IgM and IgG serological assays and (2) molecular assays. Twenty-six cooperative research projects performed between 2008 and 2020 enhanced the capability and capacity of 17 research institutes to estimate risk of rickettsial diseases. These international collaborative studies have led to the recognition and/or confirmation of rickettsial diseases within each of the partner countries. In addition, with the identification of specific pathogen and non-pathogen Rickettsia species, a more accurate risk assessment could be made in surveillance studies using environmental samples. The discoveries from these projects reinforced international cooperation benefiting not only the partner countries but also the scientific community at large through presentations (n = 40) at international scientific meetings and peer-reviewed publications (n = 18). The cooperative research studies conducted in multiple international institutes led to the incorporation of new SOPs and trainings for laboratory procedures; biosafety, biosurety, and biosecurity methods; performance of rickettsia-specific assays; and the identification of known and unknown rickettsial agents through the introduction of new serologic and molecular assays that complemented traditional microbiology methods.

In Vietnam, the public health burden of rickettsial infections continues to be underestimated due to knowledge gaps in the epidemiology of these diseases. We conducted a systematic study among 27 hospitals from 26 provinces in eight ecological regions throughout Vietnam to investigate the prevalence, distribution, and clinical characteristics of rickettsial diseases. We recruited 1834 patients in the study from April 2018 to October 2019. The findings showed that rickettsial diseases were common among undifferentiated febrile patients, with 564 (30.8%) patients positive by qPCR for scrub typhus, murine typhus or spotted fever. Scrub typhus (484, 85.8%) was the most common rickettsial disease, followed by murine typhus (67, 11.9%) and spotted fever (10, 1.8%). Rickettsial diseases were widely distributed in all regions of Vietnam and presented with nonspecific clinical manifestations.

Abstract Background Scrub typhus is a neglected vector-borne zoonosis causing life-threatening illnesses, endemic in the Asian-Pacific region and, as recently discovered, in southern Chile. Scrub typhus is rarely reported in travelers, most probably due to the lack of clinical experience and diagnostic tests in non-endemic countries. We report the first case of imported scrub typhus in South America. Case presentation A 62-year-old tourist from South Korea presented severely ill with fever, rash, and eschar in Santiago, Chile. Laboratory exams showed thrombocytopenia and elevated inflammation parameters, hepatic enzymes, and LDH. With the clinical suspicion of scrub typhus, empirical treatment with doxycycline was initiated and the patient recovered rapidly and without complications. The diagnosis was confirmed by IgM serology and by real-time PCR, which demonstrated infection with Orientia tsutsugamushi (Kawasaki clade). Conclusions Only due to the emerging clinical experience with endemic South American scrub typhus and the recent implementation of appropriate diagnostic techniques in Chile, were we able to firstly identify and adequately manage a severe case of imported scrub typhus in South America. Physicians attending febrile travelers need to be aware of this rickettsiosis, since it requires prompt treatment with doxycycline to avoid complications.

Investigation of a scrub typhus outbreak in Thailand during September 2013 found that 9.1% of Thai soldiers and 11.1% of residents living in areas surrounding training sites had antibodies against the causative agent, Orientia tsutsugamushi. Sequence analysis of O. tsutsugamushi from rodents and chiggers identified 7 genogroups and 3 genotypes.

Recently, an intradermal inoculation of the rhesus macaque model of scrub typhus has been characterized at our institution. The current project was to establish a rhesus macaque model of scrub typhus using the naturally infected chigger challenge method that faithfully mimics the natural route of pathogen transmission to fully understand the host-pathogen-vector interactions influencing pathogen transmission. Unlike the needle-based inoculation route, Orientia tsutsugamushi-infected chiggers introduce both pathogen and chigger saliva into the host epidermis at the bite site. However, information on the interaction or influence of chigger saliva on pathogenesis and immunity of host has been limited, consequently hindering vaccine development and transmission-blocking studies. To characterize chigger inoculated O. tsutsugamushi in rhesus macaques, we determined the minimum chigger attachment time required to efficiently transmit O. tsutsugamushi to the immunocompetent hosts and preliminary assessed clinical parameters, course of bacterial infection, and host’s immunological response to identifying potential factors influencing pathogen infection. Chigger infestation on hosts resulted in: (i) Rapid transmission of O. tsutsugamushi within 1 h and (ii) antigen-specific type I and II T-cell responses were markedly increased during the acute phase of infection, suggesting that both systems play critical roles in response to the pathogen control during the primary infection. In summary, we demonstrate that O. tsutsugamushi infection in rhesus macaques via chigger challenge recapitulates the time of disease onset and bacteremia observed in scrub typhus patients. Levels of proinflammatory cytokines and chemokines were positively correlated with bacteremia.

The antigenic diversity of Orientia tsutsugamushi as well as the interstrain difference(s) associated with virulence in mice impose the necessity to dissect the host immune response. In this study we compared the host response in lethal and non-lethal murine models of O. tsutsugamushi infection using the two strains, Karp (New Guinea) and Woods (Australia). The models included the lethal model: Karp intraperitoneal (IP) challenge; and the nonlethal models: Karp intradermal (ID), Woods IP, and Woods ID challenges. We monitored bacterial trafficking to the liver, lung, spleen, kidney, heart, and blood, and seroconversion during the 21-day challenge. Bacterial trafficking to all organs was observed in both the lethal and nonlethal models of infection, with significant increases in average bacterial loads observed in the livers and hearts of the lethal model. Multicolor flow cytometry was utilized to analyze the CD4+ and CD8+ T cell populations and their intracellular production of the cytokines IFNγ, TNF, and IL2 (single, double, and triple combinations) associated with both the lethal and nonlethal murine models of infection. The lethal model was defined by a cytokine signature of double- (IFNγ-IL2) and triple-producing (IL2-TNF-IFNγ) CD4+ T-cell populations; no multifunctional signature was identified in the CD8+ T-cell populations associated with the lethal model. In the nonlethal model, the cytokine signature was predominated by CD4+ and CD8+ T-cell populations associated with single (IL2) and/or double (IL2-TNF) populations of producers. The cytokine signatures associated with our lethal model will become depletion targets in future experiments; those signatures associated with our nonlethal model are hypothesized to be related to the protective nature of the nonlethal challenges.

The genus Bartonella contains >40 species, and an increasing number of these Bartonella species are being implicated in human disease. One such pathogen is Bartonella ancashensis, which was isolated in blood samples from 2 patients living in Caraz, Peru, during a clinical trial of treatment for bartonellosis. Three B. ancashensis strains were analyzed by using whole-genome restriction mapping and high-throughput pyrosequencing. Genome-wide comparative analysis of Bartonella species showed that B. ancashensis has features seen in modern and ancient lineages of Bartonella species and is more related to B. bacilliformis. The divergence between B. ancashensis and B. bacilliformis is much greater than what is seen between known Bartonella genetic lineages. In addition, B. ancashensis contains type IV secretion system proteins, which are not present in B. bacilliformis. Whole-genome analysis indicates that B. ancashensis might represent a distinct Bartonella lineage phylogenetically related to B. bacilliformis.

While studying rickettsial infections in Peru, we detected Rickettsia asembonensis in fleas from domestic animals. We characterized 5 complete genomic regions (17kDa, gltA, ompA, ompB, and sca4) and conducted multilocus sequence typing and phylogenetic analyses. The molecular isolate from Peru is distinct from the original R. asembonensis strain from Kenya.

In this study, we used a metagenomic approach to analyze bacterial communities from diverse populations (humans, animals, and vectors) to investigate the role of these microorganisms as causative agents of disease in human and animal populations. Wild rodents and ectoparasites were collected from 2014 to 2018 in Nan province, Thailand where scrub typhus is highly endemic. Samples from undifferentiated febrile illness (UFI) patients were obtained from a local hospital. A total of 200 UFI patient samples were obtained and 309 rodents and 420 pools of ectoparasites were collected from rodents (n = 285) and domestic animals (n = 135). The bacterial 16S rRNA gene was amplified and sequenced with the Illumina. Real-time PCR and Sanger sequencing were used to confirm the next-generation sequencing (NGS) results and to characterize pathogen species. Several pathogens were detected by NGS in all populations studied and the most common pathogens identified included Bartonella spp., Rickettsia spp., Leptospira spp., and Orientia tsutsugamushi. Interestingly, Anaplasma spp. was detected in patient, rodent and tick populations, although they were not previously known to cause human disease from this region. Candidatus Neoehrlichia, Neorickettsia spp., Borrelia spp., and Ehrlichia spp. were detected in rodents and their associated ectoparasites. The same O. tsutsugamushi genotypes were shared among UFI patients, rodents, and chiggers in a single district indicating that the chiggers found on rodents were also likely responsible for transmitting to people. Serological testing using immunofluorescence assays in UFI samples showed high prevalence (IgM/IgG) of Rickettsia and Orientia pathogens, most notably among samples collected during September–November. Additionally, a higher number of seropositive samples belonged to patients in the working age population (20–60 years old). The results presented in this study demonstrate that the increased risk of human infection or exposure to chiggers and their associated pathogen (O. tsutsugamushi) resulted in part from two important factors; working age group and seasons for rice cultivation and harvesting. Evidence of pathogen exposure was shown to occur as there was seropositivity (IgG) in UFI patients for bartonellosis as well as for anaplasmosis. Using a metagenomic approach, this study demonstrated the circulation and transmission of several pathogens in the environment, some of which are known causative agents of illness in human populations.

Rickettsia asembonensis, the most well-characterized rickettsia of the Rickettsia felis-like organisms (RFLO), is relatively unknown within the vector-borne diseases research community. The agent was initially identified in peri-domestic fleas from Asembo, Kenya in an area in which R. felis was associated with fever patients. Local fleas collected from domestic animals and within homes were predominately infected with R. asembonensis with < 10% infected with R. felis. Since the identification of R. asembonensis in Kenya, it has been reported in other locations within Africa, Asia, the Middle East, Europe, North America, and South America. With the description of R. asembonensis-like genotypes across the globe, a need exists to isolate these R. asembonensis genotypes in cell culture, conduct microscopic, and biological analysis, as well as whole genome sequencing to ascertain whether they are the same species. Additionally, interest has been building on the potential of R. asembonensis in infecting vertebrate hosts including humans, non-human primates, dogs, and other animals. The current knowledge of the presence, prevalence, and distribution of R. asembonensis worldwide, as well as its arthropod hosts and potential as a pathogen are discussed in this manuscript.

To increase knowledge of undifferentiated fevers in Kenya, we tested paired serum samples from febrile children in western Kenya for antibodies against pathogens increasingly recognized to cause febrile illness in Africa. Of patients assessed, 8.9%, 22.4%, 1.1%, and 3.6% had enhanced seroreactivity to Coxiella burnetii, spotted fever group rickettsiae, typhus group rickettsiae, and scrub typhus group orientiae, respectively.

Using a large, passive, febrile surveillance program in Iquitos, Peru, we retrospectively tested human blood specimens for scrub typhus group orientiae by ELISA, immunofluorescence assay, and PCR. Of 1,124 participants, 60 (5.3%) were seropositive, and 1 showed evidence of recent active infection. Our serologic data indicate that scrub typhus is present in the Peruvian Amazon.

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.

Scrub typhus and its etiological agents, Orientia species, have been around for a very long time. Historical reference to the rickettsial disease scrub typhus was first described in China (313 AD) by Hong Ge in a clinical manual (Zhouhofang) and in Japan (1810 AD) when Hakuju Hashimoto described tsutsuga, a noxious harmful disease in the Niigata prefecture. Other clinicians and scientists in Indonesia, Philippines, Taiwan, Australia, Vietnam, Malaysia, and India reported on diseases most likely to have been scrub typhus in the early 1900s. All of these initial reports about scrub typhus were from an area later designated as the Tsutsugamushi Triangle—an area encompassing Pakistan to the northwest, Japan to the northeast and northern Australia to the south. It was not until the 21st century that endemic scrub typhus occurring outside of the Tsutsugamushi Triangle was considered acceptable. This report describes the early history of scrub typhus, its distribution in and outside the Tsutsugamushi Triangle, and current knowledge of the causative agents, Orientia species.

To evaluate the containment of antimicrobial-resistant (AMR) Salmonella contaminations of a HACCP slaughterhouse (HACCP SH) and a non-HACCP slaughterhouse (non-HACCPSH), 360 paired pig rectal (representing the farm pig status) and carcass samples (representing the contamination) were collected equally from the two slaughterhouses that serviced 6 and 12 farms, respectively, in Northeast Thailand (n = 720). The purified Salmonella isolates were serotype identified, antimicrobial susceptibility tested, and pulsed-field gel electrophoresis (PFGE) assessed. Four evaluations of two slaughterhouses were examined: (1) the means of slaughtering contamination rates (SCR) (to evaluate the contamination level by averaged farm SCRs): the HACCP SH decreased contamination (SCR: −48.89% ± 8.80%, n = 6), whereas the non-HACCP SH increased (SCR: 14.31% ± 9.35%, n = 12). (2) The serotype diversity: the HACCP SH decreased the diversity from the rectal group (110 isolates, 9 serotypes) to carcass group (23 isolates, 3 serotypes), whereas there was no decrease in the non-HACCP SH (rectal group (66 isolates, 14 serotypes) and carcass group (31 isolates, 10 serotypes)). (3) The AMR patterns: the HACCP SH decreased from rectal group (96 isolates, 7 patterns) to carcass group (22 isolates, 1 pattern), whereas there was no decrease from the non-HACCP SH rectal group (22 isolates, 7 patterns) to carcass group (48 isolates, 8 patterns). (4) The estimated indirect contamination rate (by serotype screening and PFGE confirmation): the HACCP SH was 60.87% (14/23), whereas the non-HACCP SH was 98.48% (65/66). This study indicates that both the slaughterhouses keep a high level of indirect contamination; the HACCP SH decreases Salmonella contaminations and reduces the AMR patterns, the non-HACCP SH increases contaminations.

Serum samples from patients in Kenya with febrile illnesses were screened for antibodies against bacteria that cause spotted fever, typhus, and scrub typhus. Seroprevalence was 10% for spotted fever group,

We assessed serum samples from 1,000 US Marines deployed to Afghanistan during 2001–2010 to find evidence of 4 rickettsial pathogens. Analysis of predeployment and postdeployment samples showed that 3.4% and 0.5% of the Marines seroconverted for the causative agents of Q fever and spotted fever group rickettsiosis, respectively.

Kazakhstan and the United States have partnered since 2003 to counter the proliferation of weapons of mass destruction. The US Department of Defense (US DoD) has funded threat reduction programs to eliminate biological weapons, secure material in repositories that could be targeted for theft, and enhance surveillance systems to monitor infectious disease outbreaks that would affect national security. The cooperative biological research (CBR) program of the US DoD’s Biological Threat Reduction Program has provided financing, mentorship, infrastructure, and biologic research support to Kazakhstani scientists and research institutes since 2005. The objective of this paper is to provide a historical perspective for the CBR involvement in Kazakhstan, including project chronology, successes and challenges to allow lessons learned to be applied to future CBR endeavors. A project compendium from open source data and interviews with partner country Kazakhstani participants, project collaborators, and stakeholders was developed utilizing studies from 2004 to the present. An earlier project map was used as a basis to determine project linkages and continuations during the evolution of the CBR program. It was determined that consistent and effective networking increases the chances to collaborate especially for competitive funding opportunities. Overall, the CBR program has increased scientific capabilities in Kazakhstan while reducing their risk of biological threats. However, there is still need for increased scientific transparency and an overall strategy to develop a capability-based model to better enhance and sustain future research. Finally, we offer a living perspective that can be applied to further link related studies especially those related to One Health and zoonoses and the assessment of similar capability-building programs.

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.

While studying chronic verruga peruana infections in Peru from 2003, we isolated a novel Bartonella agent, which we propose be named Candidatus Bartonella ancashi. This case reveals the inherent weakness of relying solely on clinical syndromes for diagnosis and underscores the need for a new diagnostic paradigm in developing settings.

During a scrub typhus outbreak investigation in Thailand, 4 isolates of O. tsutsugamushi were obtained and established in culture. Phylogenetic analysis based on the 56-kDa type-specific antigen gene demonstrated that the isolates fell into 4 genetic clusters, 3 of which had been previously reported and 1 that represents a new genotype.

To determine previous exposure and incidence of rickettsial infections in western Kenya during 2007–2010, we conducted hospital-based surveillance. Antibodies against rickettsiae were detected in 57.4% of previously collected serum samples. In a 2008–2010 prospective study, Rickettsia felis DNA was 2.2× more likely to be detected in febrile than in afebrile persons.

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.

We found that 14.3% (15/105) of Amblyomma maculatum and 3.3% (10/299) of Dermacentor variabilis ticks collected at 3 high-use military training sites in west-central Kentucky and northern Tennessee, USA, were infected with Rickettsia parkeri and Rickettsia montanensis, respectively. These findings warrant regional increased public health awareness for rickettsial pathogens and disease.

To determine the cause of acute febrile illnesses other than malaria in the North Eastern Province, Kenya, we investigated rickettsial infection among patients from Garissa Provincial Hospital for 23 months during 2006–2008. Nucleic acid preparations of serum from 6 (3.7%) of 163 patients were positive for rickettsial DNA as determined by a genus-specific quantitative real-time PCR and were subsequently confirmed by molecular sequencing to be positive for Rickettsia felis. The 6 febrile patients’ symptoms included headache; nausea; and muscle, back, and joint pain. None of the patients had a skin rash.

Scrub typhus threatens one billion people in the Asia-Pacific area and cases have emerged outside this region. It is caused by infection with any of the multitude of strains of the bacterium Orientia tsutsugamushi. A vaccine that affords heterologous protection and a commercially-available molecular diagnostic assay are lacking. Herein, we determined that the nucleotide and translated amino acid sequences of outer membrane protein A (OmpA) are highly conserved among 51 O. tsutsugamushi isolates. Molecular modeling revealed the predicted tertiary structure of O. tsutsugamushi OmpA to be very similar to that of the phylogenetically-related pathogen, Anaplasma phagocytophilum, including the location of a helix that contains residues functionally essential for A. phagocytophilum infection. PCR primers were developed that amplified ompA DNA from all O. tsutsugamushi strains, but not from negative control bacteria. Using these primers in quantitative PCR enabled sensitive detection and quantitation of O. tsutsugamushi ompA DNA from organs and blood of mice that had been experimentally infected with the Karp or Gilliam strains. The high degree of OmpA conservation among O. tsutsugamushi strains evidences its potential to serve as a molecular diagnostic target and justifies its consideration as a candidate for developing a broadly-protective scrub typhus vaccine.

Scrub typhus is the most important rickettsial disease in the world. Its previous endemic region was considered to be in Asia, Australia and islands in the Indian and Pacific Oceans; this area was referred to as the Tsutsugamushi Triangle. Accumulation of serological, molecular, genetic, and culture data have shown that not only is scrub typhus not limited to the Tsutsugamushi Triangle, but can be caused by orientiae other than Orientia tsutsugamushi. This review describes evidence currently available that will be instrumental to researchers, healthcare providers and medical leaders in developing new research projects, performing diagnosis, and preventing scrub typhus in locations not previously thought to be endemic.

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.

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.

Murine typhus was diagnosed by PCR in 50 (7%) of 756 adults with febrile illness seeking treatment at Patan Hospital in Kathmandu, Nepal. Of patients with murine typhus, 64% were women, 86% were residents of Kathmandu, and 90% were unwell during the winter. No characteristics clearly distinguished typhus patients from those with blood culture–positive enteric fever.

The typical clinical presentation of several spotted fever group Rickettsia infections includes eschars. Clinical diagnosis of the condition is usually made by analysis of blood samples. We describe a more sensitive, noninvasive means of obtaining a sample for diagnosis by using an eschar swab specimen from patients infected with Rickettsia parkeri.

We report evidence that Amblyomma maculatum tick populations are well established in southeastern Virginia. We found that 43.1% of the adult Gulf Coast ticks collected in the summer of 2010 carried Rickettsia parkeri, suggesting that persons living in or visiting southeastern Virginia are at risk for infection with this pathogen.

Rickettsia typhi and R. felis, etiologic agents of murine typhus and fleaborne spotted fever, respectively, were detected in Oriental rat fleas (Xenopsylla cheopis) collected from rodents and shrews in Java, Indonesia. We describe the first evidence of R. felis in Indonesia and naturally occurring R. felis in Oriental rat fleas.

Human spotted fever rickettsiosis was detected molecularly by 2 real-time polymerase chain reaction (PCR) assays performed on DNA extracted from a Thai patient's serum sample. Sequences of PCR amplicons from 5 rickettsial genes used for multilocus sequence typing were 100% identical with those deposited with GenBank for Rickettsia honei TT-118.

Serum specimens from patients at 4 sites in Peru were tested for evidence of spotted fever group rickettsial infection. Results showed that 30 (18%) of 170 patients had spotted fever group rickettsial infections, which likely caused their illnesses. These findings document laboratory-confirmed spotted fever from diverse areas of Peru.

We describe a man with a febrile illness and an eschar that developed at the site of a tick bite. Rickettsia parkeri was detected and isolated from the eschar. This report represents the second documented case of R. parkeri rickettsiosis in a US serviceman in eastern Virginia.