Orientia tsutsugamushi dynamics in vectors and hosts: ecology and risk factors for foci of scrub typhus transmission in northern Thailand.

BACKGROUND: Scrub typhus is an important neglected vector-borne zoonotic disease across the Asia-Pacific region, with an expanding known distribution. The disease ecology is poorly understood, despite the large global burden of disease. The key determinants of high-risk areas of transmission to humans are unknown. METHODS: Small mammals and chiggers were collected over an 18-month period at three sites of differing ecological profiles with high scrub typhus transmission in Chiang Rai Province, northern Thailand. Field samples were identified and tested for Orientia tsutsugamushi by real-time PCR. The rates and dynamics of infection were recorded, and positive and negative individuals were mapped over time at the scale of single villages. Ecological analyses were performed to describe the species richness, community structure and interactions between infected and uninfected species and habitats. Generalised linear modelling (GLM) was applied to examine these interactions. RESULTS: The site with the highest rates of human infection was associated with the highest number of infected chigger pools (41%), individual chiggers (16%), proportion of the known vector species Leptotrombidium deliense (71%) and chigger index (151). Chigger species diversity was lowest (Shannon diversity index H': 1.77) and rodent density appeared to be high. There were no consistent discrete foci of infection identified at any of the study sites. The small mammals Rattus tanezumi and Bandicota indica and the chiggers L. deliense and Walchia kritochaeta emerged as central nodes in the network analysis. In the GLM, the end of the dry season, and to a lesser extent the end of the wet season, was associated with O. tsutsugamushi-infected small mammals and chiggers. A clear positive association was seen between O. tsutsugamushi-positive chigger pools and the combination of O. tsutsugamushi-positive chigger pools and O. tsutsugamushi-positive small mammals with lowland habitats. CONCLUSIONS: These findings begin to reveal some of the factors that may determine high-risk foci of scrub typhus at a fine local scale. Understanding these factors may allow practical public health interventions to reduce disease risk. Further studies are needed in areas with diverse ecology.

CCR2 Deficiency Impairs Ly6Clo and Ly6Chi Monocyte Responses in Orientia tsutsugamushi Infection.

Orientia (O.) tsutsugamushi, the causative agent of scrub typhus, is a neglected, obligate intracellular bacterium that has a prominent tropism for monocytes and macrophages. Complications often involve the lung, where interstitial pneumonia is a typical finding. The severity of scrub typhus in humans has been linked to altered plasma concentrations of chemokines which are known to act as chemoattractants for myeloid cells. The trafficking and function of monocyte responses is critically regulated by interaction of the CC chemokine ligand 2 (CCL2) and its CC chemokine receptor CCR2. In a self-healing mouse model of intradermal infection with the human-pathogenic Karp strain of O. tsutsugamushi, we investigated the role of CCR2 on bacterial dissemination, development of symptoms, lung histology and monocyte subsets in blood and lungs. CCR2-deficient mice showed a delayed onset of disease and resolution of symptoms, higher concentrations and impaired clearance of bacteria in the lung and the liver, accompanied by a slow infiltration of interstitial macrophages into the lungs. In the blood, we found an induction of circulating monocytes that depended on CCR2, while only a small increase in Ly6Chi monocytes was observed in CCR2-/- mice. In the lung, significantly higher numbers of Ly6Chi and Ly6Clo monocytes were found in the C57BL/6 mice compared to CCR2-/- mice. Both wildtype and CCR2-deficient mice developed an inflammatory milieu as shown by cytokine and inos/arg1 mRNA induction in the lung, but with delayed kinetics in CCR2-deficient mice. Histopathology revealed that infiltration of macrophages to the parenchyma, but not into the peribronchial tissue, depended on CCR2. In sum, our data suggest that in Orientia infection, CCR2 drives blood monocytosis and the influx and activation of Ly6Chi and Ly6Clo monocytes into the lung, thereby accelerating bacterial replication and development of interstitial pulmonary inflammation.

Orientia tsutsugamushi modulates cellular levels of NF-κB inhibitor p105.

BACKGROUND: Scrub typhus is a neglected tropical disease that threatens more than one billion people. If antibiotic therapy is delayed, often due to mis- or late diagnosis, the case fatality rate can increase considerably. Scrub typhus is caused by the obligate intracellular bacterium, Orientia tsutsugamushi, which invades phagocytes and endothelial cells in vivo and diverse tissue culture cell types in vitro. The ability of O. tsutsugamushi to replicate in the cytoplasm indicates that it has evolved to counter eukaryotic host cell immune defense mechanisms. The transcription factor, NF-κB, is a tightly regulated initiator of proinflammatory and antimicrobial responses. Typically, the inhibitory proteins p105 and IκBα sequester the NF-κB p50:p65 heterodimer in the cytoplasm. Canonical activation of NF-κB via TNFα involves IKKß-mediated serine phosphorylation of IκBα and p105, which leads to their degradation and enables NF-κB nuclear translocation. A portion of p105 is also processed into p50. O. tsutsugamushi impairs NF-κB translocation into the nucleus, but how it does so is incompletely defined. PRINCIPAL FINDINGS: Western blot, densitometry, and quantitative RT-PCR analyses of O. tsutsugamushi infected host cells were used to determine if the pathogen's ability to inhibit NF-κB is linked to modulation of p105. Results demonstrate that p105 levels are elevated several-fold in O. tsutsugamushi infected HeLa and RF/6A cells with only a nominal increase in p50. The O. tsutsugamushi-stimulated increase in p105 is bacterial dose- and protein synthesis-dependent, but does not occur at the level of host cell transcription. While TNFα-induced phosphorylation of p105 serine 932 proceeds unhindered in infected cells, p105 levels remain elevated and NF-κB p65 is retained in the cytoplasm. CONCLUSIONS: O. tsutsugamushi specifically stabilizes p105 to inhibit the canonical NF-κB pathway, which advances understanding of how it counters host immunity to establish infection.

Analysis of 262 Children with Scrub Typhus Infection: A Single-Center Experience.

Scrub typhus, a vector-borne rickettsiosis, is the leading treatable cause of non-malarial febrile illness in Asia. The myriad of typical and atypical features poses a clinical conundrum. We aimed to study the clinical and laboratory profile of children with scrub typhus infection diagnosed by IgM ELISA. Data of children < 12 years presenting with undifferentiated fever to the pediatric services of a tertiary teaching institute between January 2012 and December 2018 were retrieved. Children with seropositive IgM ELISA (InBios International Kit, Seattle, WA) for scrub typhus were enrolled in the study. Clinical features, laboratory investigations, treatment received, and the outcome recorded were obtained. Objective evidence of organ dysfunction was taken as severe scrub typhus. In total, 262 children were diagnosed with scrub typhus. The mean age was 5 years, with male preponderance (65%). And, 13 children presented during infancy. Fever was universal, and generalized lymphadenopathy (93.5%) and hepatomegaly (70%) were the common clinical signs. Eschar was identified in 31%, with greater predilection for groin and axilla. Thrombocytopenia was striking in one-third of children. Also, 25 children (9.5%) had severe scrub typhus and 18 required intensive care stay. Elevated aspartate aminotransferase enzyme levels was a predictor of severity ([OR 3.9], P value 0.005) by multivariate analysis. Lymphadenopathy was found significantly associated with eschar (P < 0.005). No mortality was recorded. This 6-year study underscores the varied spectrum of pediatric scrub typhus infection. Zero mortality in our cohort signifies the excellent outcome with judicious first-line antibiotics.

Ideal Criteria for Accurate Mouse Models of Vector-Borne Diseases with Emphasis on Scrub Typhus and Dengue.

Nine criteria regarding the infectious agent, mode of transmission, portal of entry, route of spread, target organs, target cells, pathologic lesions, incubation period, and modifiable spectrum of disease and outcomes appropriate to the intended experimental purpose are described. To provide context for each criterion, mouse models of two vector-borne zoonotic infectious diseases, scrub typhus and dengue, are summarized. Application of the criteria indicates that intravenous inoculation of Orientia tsutsugamushi into inbred mice is the best current model for life-threatening scrub typhus, and intradermal inoculation accurately models sublethal human scrub typhus, whereas the immunocompromised mouse models of dengue provide disease outcomes most closely associated with human dengue. In addition to addressing basic questions of immune and pathogenic mechanisms, mouse models are useful for preclinical testing of experimental vaccines and therapeutics. The nine criteria serve as guidelines to evaluate and compare models of vector-borne infectious diseases.

Dual RNA-seq of Orientia tsutsugamushi informs on host-pathogen interactions for this neglected intracellular human pathogen.

Studying emerging or neglected pathogens is often challenging due to insufficient information and absence of genetic tools. Dual RNA-seq provides insights into host-pathogen interactions, and is particularly informative for intracellular organisms. Here we apply dual RNA-seq to Orientia tsutsugamushi (Ot), an obligate intracellular bacterium that causes the vector-borne human disease scrub typhus. Half the Ot genome is composed of repetitive DNA, and there is minimal collinearity in gene order between strains. Integrating RNA-seq, comparative genomics, proteomics, and machine learning to study the transcriptional architecture of Ot, we find evidence for wide-spread post-transcriptional antisense regulation. Comparing the host response to two clinical isolates, we identify distinct immune response networks for each strain, leading to predictions of relative virulence that are validated in a mouse infection model. Thus, dual RNA-seq can provide insight into the biology and host-pathogen interactions of a poorly characterized and genetically intractable organism such as Ot.

Sequelae Following Acute Encephalitis Syndrome Caused by Orientia Tsutsugamushi.

We evaluated 146 patients with acute encephalitis syndrome caused by Orientia tsutsugamushi, after median interval of 5 months of hospitalization, to estimate the proportion and spectrum of disability. Fifty-six (38.4%) had mild, whereas 19 (13%) had moderate to severe degree of disability. Most patients had impairment in the domain of cognition and behavior.

Spatiotemporal and demographic characteristics of scrub typhus in Southwest China, 2006-2017: An analysis of population-based surveillance data.

Scrub typhus is a life-threatening vector-borne disease. During the past decade, the number of areas affected by this disease has expanded in many countries. In this study, we aim to identify the spatiotemporal and demographic characteristics of scrub typhus in Southwest China, an emerging endemic region for scrub typhus. Population-based surveillance data capturing scrub typhus cases in two provinces of Southwest China during 2006-2017 were retrieved. Descriptive temporal and spatial analyses were conducted and stratified by age group. The space-time scan statistic was used to identify spatiotemporal clusters of scrub typhus occurrence at the county level. During the study period, 30,001 scrub typhus cases were recorded in Southwest China, with a total of 61.0% (191/313) of counties being affected; most cases (94.3%) occurred in rural areas. The annual incidence rate increased substantially from 0.25/100,000 in 2006 to 5.38/100,000 in 2017 (>21-fold change). The 0- to 4-year-old and 45- to 64-year-old subgroups had the highest cumulative incidence rates (57.46 and 32.98/100,000, respectively). Furthermore, since 2006, the 0- to 4-year-old (slope = 0.83, p < .01) and 45-64-year-old (slope = 0.69, p < .01) age groups have had the highest increases in incidence of all age groups. The most likely spatial cluster of overall cases (relative risk = 4.13, p < .01) occurred in the southern region of Southwest China and included 41 high-risk counties. In conclusion, scrub typhus appears to be widely distributed and rapidly increasing in Southwest China. Young children and middle-aged adults were the most severely affected groups, and the disease appeared to predominantly cluster in the southern part of Southwest China. Further in-depth surveys to determine the epidemiological characteristics and driving factors of this emerging disease and to facilitate effective control programmes among high-risk groups in the affected areas should be promoted.

Scrub typhus with multi-organ dysfunction syndrome and immune thrombocytopenia: a case report and review of the literature.

BACKGROUND: Scrub typhus is an acute infectious zoonotic disease caused by Orientia tsutsugamushi. Multi-organ dysfunction secondary to scrub typhus is hard to diagnose and has a high mortality rate. Only one case of scrub typhus with multi-organ dysfunction syndrome and immune thrombocytopenia has been reported thus far. In this study, we report a second case of scrub typhus with multi-organ dysfunction syndrome and immune thrombocytopenia, and we summarize its diagnosis and treatment. CASE PRESENTATION: A 43-year-old Han Chinese woman, a sanitation worker, was admitted to our hospital after 7 days of a skin infection and 5 days of a sore throat with fever and dizziness. A physical examination revealed the presence of an eschar on the right side of her neck. She had a history of insect bites during her sanitation work. A diagnostic evaluation identified scrub typhus as the primary illness, which was associated with multi-organ dysfunction syndrome and immune thrombocytopenia. She recovered completely after 15 days of treatment and extensive symptomatic supportive care. CONCLUSION: We report a second case of tsutsugamushi disease with multi-organ dysfunction syndrome and immune thrombocytopenia, which resolved after treatment and extensive care.

Biosafety and biosecurity requirements for Orientia spp. diagnosis and research: recommendations for risk-based biocontainment, work practices and the case for reclassification to risk group 2.

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.

Mortality due to scrub typhus - report of five cases.

Scrub typhus is largely ignored in India particularly during outbreaks of viral fever. The disease course is often complicated leading to fatalities in the absence of treatment. However, if diagnosed early and a specific treatment is initiated, the cure rate is high. We report here five cases of scrub typhus to highlight the fact that high clinical suspicion for such a deadly disease is an absolute necessity.

Scrub typhus caused by Shimokoshi type Orientia tsutsugamushi showing variant 56-kDa type-specific antigen gene sequence in Tohoku region, Japan.

In 2018, a patient was diagnosed with Shimokoshi type scrub typhus in Yamagata Prefecture, Japan. The causative pathogen was likely a variant type because 43 (8.3%) of 521 deduced amino acid sequences of the 56-kDa type-specific antigen (TSA) were different from those of the Shimokoshi prototype strain. The patient's paired sera showed low antibody titers against the Shimokoshi prototype strain. Two cases of scrub typhus reported in the Tohoku region during 2011-2012 also involved the same 56-kDa TSA gene sequence. These findings suggest the presence of diversity in Shimokoshi type Orientia tsutsugamushi, which may impede the laboratory diagnosis of scrub typhus.

Severe fever with thrombocytopenia syndrome: comparison with scrub typhus and clinical diagnostic prediction.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is emerging in Asian 3 countries, China, Japan and Korea, which are scrub typhus endemic areas, and its incidence is increasing. As the two infections overlap epidemiologically and clinically and the accessibility or sensitivity of diagnostic tests is limited, early clinical prediction may be useful for diagnostic and therapeutic purposes. METHODS: Patients aged ≥16 years who were clinically suspected and laboratory-confirmed to be infected with Orientia tsutsugamushi or the SFTS virus in South Korea were enrolled. Clinical and laboratory parameters were compared. Scrub typhus was further subclassified according to the status of eschar and skin rash. An SFTS prediction scoring tool was generated based on a logistic regression analysis of SFTS compared with scrub typhus. RESULTS: The analysis was performed on 255 patients with scrub typhus and 107 patients with SFTS. At initial presentation, subjective symptoms except for gastrointestinal symptoms, were more prominent in scrub typhus patients. In addition to the characteristic eschar and skin rash, headache was significantly more prominent in scrub typhus, while laboratory abnormalities were more prominent in SFTS. Leukopenia (white blood cell count < 4000/mm3; odds ratio [OR] 30.13), thrombocytopenia (platelet count < 80,000 /mm3; OR 19.73) and low C-reactive protein (< 1 mg/dL; OR 67.46) were consistent risk factors for SFTS (all P < 0.001). A prediction score was generated using these 3 variables, and a score ≥ 2 had a sensitivity of 93.1% (95% confidence interval [CI], 87.9-96.4%) and a specificity of 96.1% (95% CI, 93.8-97.6%) for SFTS. CONCLUSION: This prediction scoring tool may be useful for differentiating SFTS from eschar- or skin rash-negative scrub typhus. It is a simple and readily applicable tool with potential for use in primary care settings.

Scrub Typhus and Abnormal Electrocardiography.

This study compared the frequency of abnormal electrocardiogram (ECG) types between scrub typhus patient group and age- and gender-matched health checkup group and their associations with disease severity in scrub typhus patient. Demographic characteristics and ECG and laboratory findings of patients with scrub typhus admitted to Chosun University Hospital, and normal subjects visiting the hospital for health checkup from January 2008 to December 2012 were retrospectively studied. Electrocardiogram abnormalities at admission were observed in 72 of 165 (43.6%) scrub typhus confirmed patients. The following ECG abnormalities were observed: arrhythmic group (31 cases, 18.8%), ischemic change group (25 cases, 15.1%), prolonged QT group (32 cases, 19.4%).Compared with the age and gender-matched health checkup group, ECG abnormalities were more commonly observed in scrub typhus patient group (13.9% versus 43.6%, P < 0.001). In addition, when compared with the normal ECG group, scrub typhus in the abnormal ECG group showed greater disease severity and this phenomenon was particularly prominent in the prolonged QT group. Based on our study prolonged QT observed in approximately 20% of patients with scrub typhus, clinicians should pay additional attention to drugs that affect QT interval.

Bacterial microbiome of the chigger mite Leptotrombidium imphalum varies by life stage and infection with the scrub typhus pathogen Orientia tsutsugamushi.

Scrub typhus is a mites-borne rickettsiosis caused by the obligate intracellular Gram-negative bacterium Orientia tsutsugamushi. The disease is potentially life threatening and is prevalent in tropical Asia, islands of the western Pacific Ocean and northern Australia where an estimated one million cases occur annually. Orientia tsutsugamushi is transmitted by the bite of larval mites in the genus Leptotrombidium. In the present study, the composition of the microbiome in larvae, deutonymphs and adult males and females from laboratory colonies of L. imphalum that were infected as well as uninfected with O. tsutsugamushi were investigated by high-throughput sequencing of the bacterial 16S rRNA gene. Notably, the bacterial microbiomes of infected adult females were dominated by sequences of O. tsutsugamushi and an unidentified species of Amoebophilaceae, which together comprised 98.2% of bacterial sequences. To improve the taxonomic resolution of the Amoebophilaceae OTU a nearly full length sequence of the 16S rRNA gene was amplified, cloned, and Sanger sequenced. Infected female mites had 89 to 92% nucleotide identity with the Amoebophilaceae family, indicating that the bacterium was likely to be a species of a novel genus. The species composition of bacterial communities varied between mite life stages regardless of their infection status. Uninfected adults exhibited greater species diversity than adults infected with O. tsutsugamushi. In the infected colony, the rate of filial infection with Orientia was less than 100%. Larval and male mites that were PCR-negative for Orientia contained low numbers of sequences of Amoebophilaceae (0.01 and 0.06%, respectively) in their taxonomic profiles, suggesting that a mutualistic relationship exists between the novel species of Amoebophilaceae and O. tsutsugamushi. Our study findings provide the basis for further research to determine the influence of the novel Amoebophilaceae species on the bacterial microbiome and on vector susceptibility to and transovarial transmission of O. tsutsugamushi.

Survey and Phylogenetic Analysis of Rodents and Important Rodent-Borne Zoonotic Pathogens in Gedu, Bhutan.

Rodents are well-known reservoirs and vectors of many emerging and re-emerging infectious diseases, but little is known about their role in zoonotic disease transmission in Bhutan. In this study, a cross-sectional investigation of zoonotic disease pathogens in rodents was performed in Chukha district, Bhutan, where a high incidence of scrub typhus and cases of acute undifferentiated febrile illness had been reported in people during the preceding 4-6 months. Twelve rodents were trapped alive using wire-mesh traps. Following euthanasia, liver and kidney tissues were removed and tested using PCR for Orientia tsutsugamushi and other bacterial and rickettsial pathogens causing bartonellosis, borreliosis, human monocytic ehrlichiosis, human granulocytic anaplasmosis, leptospirosis, and rickettsiosis. A phylogenetic analysis was performed on all rodent species captured and pathogens detected. Four out of the 12 rodents (33.3%) tested positive by PCR for zoonotic pathogens. Anaplasma phagocytophilum, Bartonella grahamii, and B. queenslandensis were identified for the first time in Bhutan. Leptospira interrogans was also detected for the first time from rodents in Bhutan. The findings demonstrate the presence of these zoonotic pathogens in rodents in Bhutan, which may pose a risk of disease transmission to humans.