Orientia tsutsugamushi Ank5 promotes NLRC5 cytoplasmic retention and degradation to inhibit MHC class I expression.

How intracellular bacteria subvert the major histocompatibility complex (MHC) class I pathway is poorly understood. Here, we show that the obligate intracellular bacterium Orientia tsutsugamushi uses its effector protein, Ank5, to inhibit nuclear translocation of the MHC class I gene transactivator, NLRC5, and orchestrate its proteasomal degradation. Ank5 uses a tyrosine in its fourth ankyrin repeat to bind the NLRC5 N-terminus while its F-box directs host SCF complex ubiquitination of NLRC5 in the leucine-rich repeat region that dictates susceptibility to Orientia- and Ank5-mediated degradation. The ability of O. tsutsugamushi strains to degrade NLRC5 correlates with ank5 genomic carriage. Ectopically expressed Ank5 that can bind but not degrade NLRC5 protects the transactivator during Orientia infection. Thus, Ank5 is an immunoevasin that uses its bipartite architecture to rid host cells of NLRC5 and reduce surface MHC class I molecules. This study offers insight into how intracellular pathogens can impair MHC class I expression.

Tick-borne pathogens isolated from ticks, rodents, and a shrew in Gangwon and Gyeonggi provinces in the Republic of Korea.

The prevalence of tick-borne pathogens (TBP), Orientia tsutsugamushi, Rickettsia and Borrelia spp. in wild small animals, namely wild rodents, is now widely investigated. This study is to present the prevalence and distribution of O. tsutsugamushi, Rickettsia and Borrelia spp. in wild small animals and ticks collected from Gyeonggi and Gangwon provinces, Republic of Korea (ROK) in 2014. A total of 131 wild small animals, rodents and shrews, and 2,954 ticks were collected from Gyeonggi and Gangwon provinces from May to November 2014. The wild small animals (KR1-9) and ticks (K1-17) were grouped in accordance with capture dates and locations. Among the wild small animals, a total of 393 tissues and blood samples were extracted from six selected small animal series (KR1-3, KR6-8). Also, each date and location-grouped ticks were identified for its species and pooled according to the stage of development. Molecular identification for Rickettsia, Orientia, and Borrelia species was performed using polymerase chain reaction (PCR). To detect TBPs among wild small animals and ticks, primer sets targeting the 56 kDa protein encoding gene of Orientia spp., outer membrane protein B gene (OmpB) of Rickettsia spp., and 5S-23S intergenic spacer region (IGS) gene of Borrelia spp. were used. Of the 393 wild small animals' blood and tissue samples, 199 (50.6%) were positive for Orientia spp., 158 (40.2%) were positive for Borrelia spp., and 55 (14.0%) were positive for Rickettsia spp. Moreover, a total of 14 tick pools (n = 377) was positive for Rickettsia spp. (n=128, 34.0%) and Borrelia spp. (n=33, 8.8%). High prevalence of Orientia spp. and Rickettsia spp. in rodents and shrews were observed. This study presents significant insights by presenting data collected in 2014 that the prevalence of TBP was already high in mid 2010s. This study highlights the sustainable routine surveillance model for TBP.

Discovery of a novel spotted fever group Rickettsia, "Candidatus Rickettsia kedanie," in unfed larval chigger mites, Leptotrombidium scutellare.

Spotted fever group (SFG) rickettsia, the causative agent of SFG rickettsiosis, is predominantly carried by ticks, whereas Orientia tsutusgamushi, the causative agent of scrub typhus, is primarily transmitted by chigger mites in Japan. In this study, we attempted to isolate intracellular eubacteria from Leptotrombidium scutellare, a major vector of O. tsutsugamushi; moreover, we isolated an SFG rickettsia using a mosquito-derived cell line. Draft genome sequences of this unique isolate, by applying criteria for species delimitation, classified this isolate as a novel strain, proposed as "Rickettsia kedanie." Further genetic analysis identified conserved virulence factors, and the isolate successfully propagated in mammalian cells, suggesting its ability to cause diseases in humans. The presence of SFG rickettsia in unfed larvae implies potential dual-pathogen carriage and reflects a symbiotic relationship similar to that between the mites and O. tsutsugamushi, indicating possibility of its transovarial transmission from female adults. Furthermore, conserved genomic similarity of the novel isolate to known SFG rickettsia suggests potential multiple hosts, including chiggers and ticks. In the natural environment, ticks, chigger mites, and wild animals may carry new isolates, complicating the infection cycle and increasing the transmission risks to humans. This discovery challenges the conventional association of SFG rickettsia with ticks, emphasizing its implications for research and disease control. However, this study was confined to a particular species of chigger mites and geographic area, underscoring the necessity for additional studies to comprehend the ecological dynamics, host interactions, and health implications linked to this newly identified SFG rickettsia.

A cross sectional study on molecular prevalence of Orientia tsutsugamushi in household rat population of South India.

This study aimed to assess the molecular prevalence of mite-borne zoonotic pathogen O. tsutsugamushi in household rats of South India through nested polymerase chain reaction amplification of O. tsutsugamushi 47-kDa htrA gene and to determine the most suitable sample type for screening of O. tsutsugamushi in rats. Out of 85 rats trapped in Tamil Nadu, Karnataka, and Puducherry regions, 47 rats were found positive for the O. tsutsugamushi genome with prevalence of 55.29 %. Among different sample types screened, faecal samples exhibited the highest positivity rate, followed by liver, spleen, kidney, and blood samples. Agreement between faecal and spleen samples of rats for the presence of O. tsutsugamushi was the highest. Principal component analysis revealed a positive correlation between the spleen, liver, and faeces and a negative correlation between blood and faeces for the presence of O. tsutsugamushi genome. These findings underscore the varied distribution of O. tsutsugamushi among different samples and indicate that the faecal and liver samples of rats are an ideal choice of samples for epidemiological studies. This is the first study to report a high level of presence of O. tsutsugamushi in faecal samples of rats.

Orientia tsutsugamushi: An Unusual Intracellular Bacteria-Adaptation Strategies, Available Antibiotics, and Alternatives for Treatment.

During evolution Orientia tsutsugamushi became a smarter obligate bacterium to establish as intracellular pathogens. O. tsutsugamushi is a human pathogenic bacterium responsible for 1 billion infections of scrub typhus. Several novel mechanisms make this bacterium unique (cell wall, genetic constitutions, secretion system, etc.). In 2007, O. tsutsugamushi Boryong was pioneer strain for whole-genome sequencing. But the fundamental biology of this bacterial cell is a mystery till date. The unusual biology makes this organism as model for host cell interaction. Only a few antibiotics are effective against this intracellular pathogen but emergence of less susceptibility toward antibiotics make the situation alarming. The review was captivated to highlight the unusual aspects of adaptation, antibiotics, and drugs beyond antibiotics.

mNGS helped diagnose scrub typhus-associated HLH in children: a report of two cases.

Background: Scrub typhus, caused by the Orientia tsutsugamushi (Ot), is a widespread vector-borne disease transmitted by chigger mites. Hemophagocytic lymphohistiocytosis (HLH) is considered to be one of the potentially severe complications. The diagnosis of scrub typhus-associated HLH may be overlooked due to the non-specific clinical characteristics and the absence of pathognomonic eschar. Case presentation: We obtained clinical data from two patients in the South of Sichuan, China. The first case involved a 6-year-old girl who exhibited an unexplained fever and was initially diagnosed with sepsis, HLH, and pulmonary infection. The other patient presented a more severe condition characterized by multiple organ dysfunction and was initially diagnosed with septic shock, sepsis, HLH, acute kidney injury (AKI), and pulmonary infection. At first, a specific examination for scrub typhus was not performed due to the absence of a characteristic eschar. Conventional peripheral blood cultures yielded negative results in both patients, and neither of them responded to routine antibiotics. Fortunately, the causative pathogen Orientia tsutsugamushi (Ot) was detected in the plasma samples of both patients using metagenomics next-generation sequencing (mNGS) and further confirmed by polymerase chain reaction. Subsequently, they both were treated with doxycycline and recovered quickly. Conclusion: The unbiased mNGS provided a clinically actionable diagnosis for an uncommon pathogen-associated infectious disease that had previously evaded conventional diagnostic approaches.

A molecular survey of zoonotic pathogens of public health importance in rodents/shrews and their ectoparasites trapped in Puducherry, India.

BACKGROUND: Globally, India has a high zoonotic disease burden and lacks surveillance data in humans and animals. Rodents are known reservoirs for many zoonotic diseases and their synanthropic behavior poses a great public health threat. METHODS: In this study, trapped rodents/shrews from randomly selected villages within Puducherry, India, and their ectoparasites were screened for zoonotic pathogens, namely, Orientia tsutsugamushi, other pathogenic rickettsiae, Leptospira spp., Cryptosporidium spp., Coxiella burnetii and methicillin-resistant Staphylococcus aureus (MRSA) using conventional PCR. A total of 58 rodents/shrews were trapped from 11 villages. The species trapped were Suncus murinus (49/58, 84.48%), Rattus rattus (8/58, 13.79%) and Rattus norvegicus (1/58, 1.72%). All ectoparasites collected were identified as mites and its infestation rate was 46.55% (27/58). RESULTS: Real-time PCR targeting the 47 kDa gene of O. tsutsugamushi revealed positivity in one rodent and one shrew (3.45%) and two mite pools (7.41%). Conventional PCR targeting the 56 kDa gene revealed positivity in one shrew and two mite pools and the phylogenetic analysis of all three amplicons indicated the circulation of the Gilliam-related serotype. MRSA was detected in the alimentary tract of a shrew (1/32, 3.13%). Leptospira spp., Rickettsia, Cryptosporidium spp. and Co. burnetii tested negative. CONCLUSIONS: The detection of zoonotic pathogens within reservoir hosts and vectors poses a risk of transmission to humans. This study signifies the need for zoonotic pathogen surveillance in synanthropic rodents/shrews.

Microbiome and mitogenomics of the chigger mite Pentidionis agamae: potential role as an Orientia vector and associations with divergent clades of Wolbachia and Borrelia.

BACKGROUND: Trombiculid mites are globally distributed, highly diverse arachnids that largely lack molecular resources such as whole mitogenomes for the elucidation of taxonomic relationships. Trombiculid larvae (chiggers) parasitise vertebrates and can transmit bacteria (Orientia spp.) responsible for scrub typhus, a zoonotic febrile illness. Orientia tsutsugamushi causes most cases of scrub typhus and is endemic to the Asia-Pacific Region, where it is transmitted by Leptotrombidium spp. chiggers. However, in Dubai, Candidatus Orientia chuto was isolated from a case of scrub typhus and is also known to circulate among rodents in Saudi Arabia and Kenya, although its vectors remain poorly defined. In addition to Orientia, chiggers are often infected with other potential pathogens or arthropod-specific endosymbionts, but their significance for trombiculid biology and public health is unclear. RESULTS: Ten chigger species were collected from rodents in southwestern Saudi Arabia. Chiggers were pooled according to species and screened for Orientia DNA by PCR. Two species (Microtrombicula muhaylensis and Pentidionis agamae) produced positive results for the htrA gene, although Ca. Orientia chuto DNA was confirmed by Sanger sequencing only in P. agamae. Metagenomic sequencing of three pools of P. agamae provided evidence for two other bacterial associates: a spirochaete and a Wolbachia symbiont. Phylogenetic analysis of 16S rRNA and multi-locus sequence typing genes placed the spirochaete in a clade of micromammal-associated Borrelia spp. that are widely-distributed globally with no known vector. For the Wolbachia symbiont, a genome assembly was obtained that allowed phylogenetic localisation in a novel, divergent clade. Cytochrome c oxidase I (COI) barcodes for Saudi Arabian chiggers enabled comparisons with global chigger diversity, revealing several cases of discordance with classical taxonomy. Complete mitogenome assemblies were obtained for the three P. agamae pools and almost 50 SNPs were identified, despite a common geographic origin. CONCLUSIONS: P. agamae was identified as a potential vector of Ca. Orientia chuto on the Arabian Peninsula. The detection of an unusual Borrelia sp. and a divergent Wolbachia symbiont in P. agamae indicated links with chigger microbiomes in other parts of the world, while COI barcoding and mitogenomic analyses greatly extended our understanding of inter- and intraspecific relationships in trombiculid mites.

Molecular epidemiological study of Scrub Typhus in residence, farm and forest habitats from Yunnan Province, China.

The number of people suffering from scrub typhus, which is not of concern, is increasing year by year, especially in Yunnan Province, China. From June 1, 2021 to August 15, 2022, a total of 505 mammalian samples were collected from farm, forest, and residential habitats with high incidence of scrub typhus in Yunnan, China, for nPCR (nested PCR) and qPCR (quantitative real-time PCR) detection of Orientia tsutsugamushi. A total of 4 orders of murine-like animals, Rodentia (87.52%, n = 442), Insectivora (10.29%, n = 52), Lagomorpha (1.79%, n = 9) and Scandentia (0.40%, n = 2) were trapped. Comparing the qPCR infection rates in the three habitats, it was no significant difference that the infection rate of residential habitat (44.44%) and that of the farm habitat (45.05%, P>0.05), which is much larger than that of the forest habitat (3.08%) (P<0.001). Three genotypes (Karp-like, Kato-like and TA763-like) of O. tsutsugamushi were found from Yunnan, China in this study.

Molecular detection of Orientia tsutsugamushi in ectoparasites & their small mammal hosts captured from scrub typhus endemic areas in Madurai district, India.

BACKGROUND OBJECTIVES: Scrub typhus, caused by Orientia tsutsugamushi present in small mammals harbouring the ectoparasites. A study was undertaken to detect the pathogen present in small mammals and its ectoparasites in the scrub typhus-reported areas. METHODS: The small mammals (rodents/shrews) and its ectoparasites were screened for O. tsutsugamushi using nested PCR amplification of the groEL gene. Small mammals were collected by trapping and screened for ectoparasites (mites, ticks and fleas) by combing method. RESULTS: All the chigger mites collected were tested negative for O. tsutsugamushi . Interestingly, adult non-trombiculid mites ( Oribatida sp., Dermanyssus gallinae ), fleas ( Xenopsylla astia, X. cheopis, Ctenophalides felis and Ctenophalides sp.) and ticks ( Rhipicephalus sanguineus , R. haemaphysaloides ) screened were found to be positive for O. tsutsugamushi , which the authors believe is the first report on these species globally. Bandicota bengalensis with O. tsutsugamushi infection is reported for the first time in India. The O. tsutsugamushi groEL sequences from the positive samples were similar to the reference strains, Karp and Ikeda and phylogenetically clustered in clade IV with less evolutionary divergence. The blood samples of Rattus rattus , Suncus murinus and B. bengalensis collected from this area were tested positive for O. tsutsugamushi ; interestingly, the sequence similarity was much pronounced with their ectoparasites indicating the transmission of the pathogen to host or vice versa . INTERPRETATION CONCLUSIONS: The outcome of the present investigations widened our scope on the pathogens present in ectoparasites and rodents/shrews from this area. This will help to formulate the required vector control methods to combat zoonotic diseases.

Comparative Evaluation of Different Tissues and Molecular Techniques for the Zoonotic Surveillance of Scrub Typhus.

Background and Objectives: Scrub typhus (ST) is detected in one-fourth of patients with acute febrile illnesses, confirming its nationwide re-emergence. The disease, if not diagnosed, can lead to multiple organ dysfunction and mortality. Being a vector-borne zoonotic disease, the molecular survey for pathogens in animal hosts is essential to predict the risk of its transmission to humans. Hence, this study aimed at identifying the effective animal tissue and molecular technique for zoonotic surveillance of ST infection in small animal hosts. Methods: Rodents/shrews were trapped from seventeen randomly selected villages in Puducherry between July and September, 2022. The presence of Orientia tsutsugamushi in ectoparasites and tissues including blood, lung, liver, spleen, kidney, heart, brain, and intestine retrieved from the animals was screened by nested PCR targeting 56 kDa, real-time PCR (qPCR) targeting 47 kDa and traD, and conventional PCR targeting groEL. The Weil-Felix test was carried out to detect antibodies against O. tsutsugamushi in rodent/shrew serum samples. Diagnostic accuracy measures of the molecular tests were calculated for each of the tissues by latent class modeling. Results: O. tsutsugamushi detected in the rodents/shrews were identified to be Karp-like and Kawasaki-like strains. Upon statistical analysis, qPCR targeting 47 kDa exhibited the highest accuracy measures in most of the tissues analyzed, with perfect sensitivity and specificity of 100% and 97% for intestine and lung samples for the epidemiological surveillance, respectively. Interpretation and Conclusion: The study recommends qPCR targeting 47 kDa gene and analysis of intestine and lung along with blood for the zoonotic surveillance of ST infection.

Transmission Cycle of Shimokoshi-Type Orientia tsutsugamushi in Shimane Prefecture, Japan.

To demonstrate the transmission cycle of Shimokoshi-type Orientia tsutsugamushi in Shimane Prefecture, field rodents were captured from areas where four human infections caused by the pathogen have been reported. The rodents were investigated for the transmission cycle of the pathogen based on the pathogen's genome, antibodies against the pathogen, and the vector of the pathogen (Leptotrombidium palpale). In addition, the vector was captured from the soil in the study area. A total of 44 rodents were captured. No O. tsutsugamushi DNA was detected in the blood or spleen samples by real-time polymerase chain reaction. However, a specific antibody against the pathogen was detected in 2 out of 44 (4.5%) rodents using the indirect immunoperoxidase method, indicating the presence of the pathogen in the study area. Although 29 L. palpale were identified, DNA detection was not performed because of the insufficient number of vectors, based on the DNA detection rate in previous studies. However, the identification of the vector, as well as the specific antibody in rodents, suggests the presence of the transmission cycle of Shimokoshi-type O. tsutsugamushi in Shimane Prefecture.

Genotyping of Orientia tsutsugamushi circulating in and around Vellore (South India) using TSA 56 gene.

PURPOSE: The immunodominant TSA 56 gene of Orientia tsutsugamushi, (scrub typhus agent) has four variable regions (VD-I to VD-IV) making it useful for genotyping. This study was undertaken to determine Orientia tsutsugamushi genotypes circulating in and around Vellore using complete and partial TSA 56 gene. METHODS: Of the 162 patients positive by 47 â€‹kDa qPCR, on 21 samples PCR to amplify the complete TSA 56 gene (≈1605 bp: Long protocol) and the partial gene sequence using the Horinouchi (≈650bp) and the Furuya (≈480 bp) protocol was performed. Sanger and Nanopore sequencing was performed to obtain sequence data for assigning genotype. For 13 amplicons partial and complete gene data was obtained. RESULTS: Phylogenetic analysis of the complete gene (Long protocol) which includes VD-I to VD-IV region and partial gene (Horinouchi) which amplifies the VD-I to VD-III regions showed identical genotypes. Twelve belonged to TA763 genotype and one belongs to Karp genotype. The Furuya sequence (in silico) correctly identified the Karp genotype and 10 of the TA763 genotypes. Two TA763 genotypes (identified by complete and 650 bp partial gene analysis) were misidentified by Furuya sequence analysis as Karp genotype. CONCLUSIONS: Analysis of the 13 complete 56 â€‹kDa gene sequences suggests that TA763 is the commonest genotype in Vellore. Sanger sequencing of the 650 bp fragment gives similar results. However, these results need to be validated by larger prospective multi-centric studies.

Clinical, laboratory profile and molecular characterization of Orientia tsutsugamushi among fatal scrub typhus patients from Karnataka, India.

BACKGROUND: Scrub typhus is a vector-borne infection caused by the obligate intracellular organism Orientia tsutsugamushi. In some cases, scrub typhus can result in severe complications, multiorgan failure and death. OBJECTIVE: To study the clinical and laboratory profiles of patients who succumbed to scrub typhus. METHODS: A prospective cohort study was conducted from August 2019 through April 2023 on scrub typhus patients admitted to our hospital. Clinical and laboratory parameters of all the patients were recorded, and blood samples were drawn. To confirm scrub typhus, a nested polymerase chain reaction (nPCR) was performed in collected samples. Viable amplicons were sequenced, and phylogenetic analyses were performed to identify infecting genotypes. RESULTS: A total of 261 patients were enrolled. Of these, nine (3.45%) patients succumbed at a median (Interquartile Range) duration of 5 (1.5, 10.5) days after admission. Sepsis with septic shock (9, 100%) and acute kidney injury (AKI) (6, 66%) were noted among the succumbed patients. All the succumbed patients (100%) required intensive care admission, inotropic and ventilatory support. While 5 (55%) patients required dialysis, two (22%) required blood transfusion. Three (33%) patient samples were co-positive for Leptospira IgM, and four (44%) patients had superinfection with Candida tropicalis, multi-drug-resistant (MDR) E. Coli sepsis, pan drug-resistant (PDR) Acinetobacter Baumanii, and Klebsiella pneumoniae. Phylogenetic analysis revealed Orientia tsutsugamushi Japanese Gilliam-variant (JG-v) like (50%), Karp-like (37.5%), and Japanese Gilliam (JG) like (12.5%) strains among succumbed patients. CONCLUSION: Delay in scrub typhus diagnosis can result in severe complications, septic shock, and multisystem organ failure, culminating in death.

Differential cellular immune responses against Orientia tsutsugamushi Karp and Gilliam strains following acute infection in mice.

Scrub typhus is a leading cause of febrile illness in endemic countries due to infection with Orientia tsutsugamushi (Ot), a seriously understudied intracellular bacterium. Pulmonary involvement associated with vascular parasitism in patients is common and can develop into life threatening interstitial pneumonia. The diverse antigenicity of Ot genotypes and inter-strain differences in genome content are connected to varied virulence and clinical outcomes; however, detailed studies of strain-related pulmonary immune responses in human patients or small animal models of infection are lacking. In this study, we have used two clinically prevalent bacterial strains (Karp and Gilliam) to reveal cellular immune responses in inflamed lungs and potential biomarkers of disease severity. The results demonstrate that outbred CD-1 mice are highly susceptible to both Karp and Gilliam strains; however, C57BL/6 (B6) mice were susceptible to Karp, but resistant to Gilliam (with self-limiting infection), corresponding to their tissue bacterial burdens and lung pathological changes. Multicolor flow cytometric analyses of perfused B6 mouse lungs revealed robust and sustained influx and activation of innate immune cells (macrophages, neutrophils, and NK cells), followed by CD4+ and CD8+ T cells, during Karp infection, but such responses were greatly attenuated during Gilliam infection. The robust cellular responses in Karp-infected B6 mice positively correlated with significantly early and high levels of serum cytokine/chemokine protein levels (CXCL1, CCL2/3/5, and G-CSF), as well as pulmonary gene expression (Cxcl1/2, Ccl2/3/4, and Ifng). In vitro infection of B6 mouse-derived primary macrophages also revealed bacterial strain-dependent immune gene expression profiles. This study provided the lines of evidence that highlighted differential tissue cellular responses against Karp vs. Gilliam infection, offering a framework for future investigation of Ot strain-related mechanisms of disease pathogenesis vs. infection control.

CRISPR/Cas12a-Based Detection Platform for Early and Rapid Diagnosis of Scrub Typhus.

Orientia tsutsugamushi is responsible for causing scrub typhus (ST) and is the leading cause of acute encephalitis syndrome (AES) in AES patients. A rapid and sensitive method to detect scrub typhus on-site is essential for the timely deployment of control measures. In the current study, we developed a rapid, sensitive, and instrument-free lateral flow assay (LFA) detection method based on CRISPR/Cas12a technology for diagnosing ST (named LoCIST). The method is completed in three steps: first, harnessing the ability of recombinase polymerase for isothermal amplification of the target gene; second, CRISPR/Cas12a-based recognition of the target; and third, end-point detection by LFA. The detection limit of LoCIST was found to be one gene copy of ST genomic DNA per reaction, and the process was complete within an hour. In 81 clinical samples, the assay showed no cross-reactivity with other rickettsial DNA and was 100% consistent with PCR detection of ST. LoCIST demonstrated 97.6% sensitivity and 100% specificity. Overall, the LoCIST offers a novel alternative for the portable, simple, sensitive, and specific detection of ST, and it may help prevent and control AES outbreaks due to ST. In conclusion, LoCIST does not require specialized equipment and poses a potential for future applications as a point-of-care diagnostic.

Molecular characterization of Orientia tsutsugamushi causing fatal scrub typhus in a young male affected by so-called "mystery fever" in North India.

Scrub typhus is a zoonotic disease caused by Orientia tsutsugamushi. Although the presence of eschar is considered pathognomic, diagnosis of scrub typhus is challenging due to overlapping presentation. The diagnosis is based on the serological and molecular assay. Here, we describe a case of a young male patient who was diagnosed with scrub typhus and developed complications in the course of the disease. We also performed molecular characterization of the strain which revealed a close relatedness to Karp-like Linh.DT strains were previously reported from Vietnam.

Orientia tsutsugamushi detected by microbiome diagnostics.

Scrub typhus is caused by the mite-borne bacterium Orientia tsutsugamushi. Imported cases have been suspected in Denmark but no diagnostic method has yet been available to confirm the diagnosis. This is a case report of a 38-year-old male admitted to hospital with high fever, severe malaise and headache after returning from Malaysia. Scrub typhus was suspected and the patient recovered after one week of doxycycline treatment. The pathogen was identified by use of microbiome 16S/18S rRNA next-generation sequencing on ethylenediamine tetraacetic acid (EDTA) blood, which in the future may serve an important role in the investigation of travel-associated infections.

Orientia tsutsugamushi: comprehensive analysis of the mobilome of a highly fragmented and repetitive genome reveals the capacity for ongoing lateral gene transfer in an obligate intracellular bacterium.

IMPORTANCE: Obligate intracellular bacteria, or those only capable of growth inside other living cells, have limited opportunities for horizontal gene transfer with other microbes due to their isolated replicative niche. The human pathogen Ot, an obligate intracellular bacterium causing scrub typhus, encodes an unusually high copy number of a ~40 gene mobile genetic element that typically facilitates genetic transfer across microbes. This proliferated element is heavily degraded in Ot and previously assumed to be inactive. Here, we conducted a detailed analysis of this element in eight Ot strains and discovered two strains with at least one intact copy. This implies that the element is still capable of moving across Ot populations and suggests that the genome of this bacterium may be even more dynamic than previously appreciated. Our work raises questions about intracellular microbial evolution and sounds an alarm for gene-based efforts focused on diagnosing and combatting scrub typhus.