Lack of the IFN-γ signal leads to lethal Orientia tsutsugamushi infection in mice with skin eschar lesions.

Scrub typhus is an acute febrile disease due to Orientia tsutsugamushi (Ot) infection and can be life-threatening with organ failure, hemorrhage, and fatality. Yet, little is known as to how the host reacts to Ot bacteria at early stages of infection; no reports have addressed the functional roles of type I versus type II interferon (IFN) responses in scrub typhus. In this study, we used comprehensive intradermal (i.d.) inoculation models and two clinically predominant Ot strains (Karp and Gilliam) to uncover early immune events. Karp infection induced sequential expression of Ifnb and Ifng in inflamed skin and draining lymph nodes at days 1 and 3 post-infection. Using double Ifnar1-/-Ifngr1-/- and Stat1-/- mice, we found that deficiency in IFN/STAT1 signaling resulted in lethal infection with profound pathology and skin eschar lesions, which resembled to human scrub typhus. Further analyses demonstrated that deficiency in IFN-γ, but not IFN-I, resulted in impaired NK cell and macrophage activation and uncontrolled bacterial growth and dissemination, leading to metabolic dysregulation, excessive inflammatory cell infiltration, and exacerbated tissue damage. NK cells were found to be the major cellular source of innate IFN-γ, contributing to the initial Ot control in the draining lymph nodes. In vitro studies with dendritic cell cultures revealed a superior antibacterial effect offered by IFN-γ than IFN-ß. Comparative in vivo studies with Karp- and Gilliam-infection revealed a crucial role of IFN-γ signaling in protection against progression of eschar lesions and Ot infection lethality. Additionally, our i.d. mouse models of lethal infection with eschar lesions are promising tools for immunological study and vaccine development for scrub typhus.

Global seroprevalence of scrub typhus: a systematic review and meta-analysis.

Scrub typhus, a neglected disease, is a significant health concern in the Tsutsugamushi triangle of the Asia-Pacific and has raised global concerns due to recent cases occurring outside this region. To estimate the global prevalence of scrub typhus, we conducted a systematic review and meta-analysis. We conducted a systematic search of PubMed, Scopus, and Embase databases for observational studies on scrub typhus. Using a random-effects model, we combined the prevalence estimates with inverse-variance weights while also evaluating heterogeneity and publication bias. Among 3551 reports screened, we identified 181 studies with 1,48,251 samples for inclusion in our synthesis. The overall pooled seroprevalence (95% confidence intervals) of scrub typhus infections was 24.93% (23.27-26.60). Gender-wise pooled prevalence was estimated to be 50.23% (47.05-53.40) for males and 48.84% (45.87-51.80) for females. Eschar prevalence was observed to be 30.34% (22.54-38.15) among the positive cases. One-fourth of all the samples tested positive for scrub typhus and eschar was present in one-third of these total positive cases, encompassing regions beyond the Tsutsugamushi triangle. This estimation underlines the importance of this neglected disease as a public health problem. Strengthening surveillance and implementing disease control measures are needed in the affected regions.

Dominant B cell-T cell epitopes instigated robust immune response in-silico against Scrub Typhus.

Scrub typhus, a potentially life-threatening infectious disease, is attributed to bacteria Orientia tsutsugamushi (O. tsutsugamushi). The transmission of this illness to humans occurs through the bite of infected chiggers, which are the larval forms of mites belonging to the genus Leptotrombidium. In this research, we developed a subunit vaccine specifically designed to target outer membrane proteins. Immunodominant cytotoxic T-lymphocytes (CTLs), B- lymphocytes (BCLs), and major histocompatibility complex (MHC)- II epitopes were identified using machine learning and bioinformatics approaches. These epitopes were arranged in different combinations with the help of suitable linkers like AAY, KK, GPGPG and adjuvant (cholera toxin B) that resulted in a vaccine construct. Physiochemical properties were assessed, where the predicted solubility (0.571) was higher than threshold value. Tertiary structure was predicted using I-TASSER web server and evaluated using Ramachandran plot (94 % residues in most favourable region) and z-score (-6.04), which had shown the structure to have good stability and residue arrangement. Molecular docking with immune receptors, Toll-like receptor (TLR)-2 and -4 showed good residue interaction with 13 and 5 hydrogen bonds respectively. Molecular dynamics simulations of receptor-ligand complex provided the idea about the strong interaction having 1.524751 × 10-5 eigenvalue. Amino acid sequence of vaccine was converted to nucleotide sequence and underwent codon optimization. The optimized codon sequence was used for in-silico cloning, which provided idea about the possibility of synthesis of vaccine using E. coli as host. Overall, this study provided a promising blueprint for a scrub typhus vaccine, although experimental validation is needed for confirmation. Furthermore, it is crucial to acknowledge that while bioinformatics provides valuable insights, in-vitro and in-vivo studies are imperative for a comprehensive evaluation of vaccine candidate. Thus, the integration of computational predictions with empirical research is essential to validate the efficacy, safety, and real-world applicability of the designed vaccine against Scrub Typhus. Nevertheless, the findings are good to carry forward for in-vitro and in-vivo investigations.

Scrub typhus in Indonesia: A cross-sectional analysis of archived fever studies samples.

BACKGROUND: Scrub typhus is an understudied vector-borne bacterial infection. METHODS: We tested archived fever samples for scrub typhus seropositivity to begin charting its geographic distribution in Indonesia. We analysed 1033 serum samples from three sites. IgM and IgG enzyme-linked immunosorbent assay (ELISA) against Orientia tsutsugamushi was performed using Karp, Kato, Gilliam, TA 716 antigens. To determine the cutoff in the absence of a presumed unexposed population and gold standard tests, we identified the visual inflection point, performed change point analysis, and used finite mixture models. RESULTS: The optical density cutoff values used for IgM and IgG were 0.49 and 0.13, respectively. Across all sites, IgM seropositivity was 4.6% (95% CI: 3.4 to 6.0%) while IgG seropositivity was 4.4% (95% CI: 3.3 to 5.8%). The overall seropositivity across sites was 8.8% (95% CI: 8.1 to 11.7%). The overall seropositivity for Jambi, Denpasar, Tabanan were 9.7% (95% CI: 7.0 to 13.3%), 8.0% (95% CI: 5.7 to 11.0%), 9.0% (95% CI: 6.1 to 13.0%), respectively. CONCLUSIONS: We conclude that O. tsutsugamushi exposure in humans occurred at all sites analysed and could be the cause of illness in some cases. Though it was not the main cause of acute fever in these locations, it is still important to consider scrub typhus in cases not responding to beta-lactam antibiotics. Future seroprevalence surveys and testing for scrub typhus in acute febrile illness studies will be essential to understand its distribution and burden in Indonesia.

Development of a Scrub Typhus Diagnostic Platform Incorporating Cell-Surface Display Technology.

Scrub typhus (ST), also known as tsutsugamushi disease and caused by rickettsia Orientia tsutsugamushi, is an underestimated fatal epidemic in the Asia-Pacific region, resulting in a million human infections each year. ST is easily misdiagnosed as clinical diagnosis is based on non-specific skin eschar and flu-like symptoms. Thus, the lack of accurate, convenient, and low-cost detection methods for ST poses a global health threat. To address this problem, we adopted baculovirus surface-display technology to express three variants of TSA56, the major membrane antigen of O. tsutsugamushi, as well as the passenger domain of ScaC (ScaC-PD), on insect Sf21 cell surfaces rather than biosafety level 3 bacteria in an enzyme-linked immunosorbent assay (ELISA). Recombinant TSA56 and ScaC-PD were all properly expressed and displayed on Sf21 cells. Our cell-based ELISA comprising the four antigen-displaying cell types interacted with monoclonal antibodies as well as serum samples from ST-positive field-caught rats. This cell-based ELISA presented high accuracy (96.3%), sensitivity (98.6%), and specificity (84.6%) when tested against the ST-positive rat sera. Results of a pilot study using human sera were also highly consistent with the results of immunofluorescence analyses. By adopting this approach, we circumvented complex purification and refolding processes required to generate recombinant O. tsutsugamushi antigens and reduced the need for expensive equipment and extensively trained operators. Thus, our system has the potential to become a widely used serological platform for diagnosing ST.

Orientia tsutsugamushi selectively stimulates the C-type lectin receptor Mincle and type 1-skewed proinflammatory immune responses.

Orientia tsutsugamushi is an obligately intracellular bacterium and the etiological agent of scrub typhus. The lung is a major target organ of infection, displaying type 1-skewed proinflammatory responses. Lung injury and acute respiratory distress syndrome are common complications of severe scrub typhus; yet, their underlying mechanisms remain unclear. In this study, we investigated whether the C-type lectin receptor (CLR) Mincle contributes to immune recognition and dysregulation. Following lethal infection in mice, we performed pulmonary differential expression analysis with NanoString. Of 671 genes examined, we found 312 significantly expressed genes at the terminal phase of disease. Mincle (Clec4e) was among the top 5 greatest up-regulated genes, accompanied with its signaling partners, type 1-skewing chemokines (Cxcr3, Ccr5, and their ligands), as well as Il27. To validate the role of Mincle in scrub typhus, we exposed murine bone marrow-derived macrophages (MΦ) to live or inactivated O. tsutsugamushi and analyzed a panel of CLRs and proinflammatory markers via qRT-PCR. We found that while heat-killed bacteria stimulated transitory Mincle expression, live bacteria generated a robust response in MΦ, which was validated by indirect immunofluorescence and western blot. Notably, infection had limited impact on other tested CLRs or TLRs. Sustained proinflammatory gene expression in MΦ (Cxcl9, Ccl2, Ccl5, Nos2, Il27) was induced by live, but not inactivated, bacteria; infected Mincle-/- MΦ significantly reduced proinflammatory responses compared with WT cells. Together, this study provides the first evidence for a selective expression of Mincle in sensing O. tsutsugamushi and suggests a potential role of Mincle- and IL-27-related pathways in host responses to severe infection. Additionally, it provides novel insight into innate immune recognition of this poorly studied bacterium.

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.

Misdiagnosis of scrub typhus as hemorrhagic fever with renal syndrome and potential co-infection of both diseases in patients in Shandong Province, China, 2013-2014.

BACKGROUND: Scrub typhus, caused by Orientia tsutsugamushi, an obligate intracellular gram-negative bacterium, along with hemorrhagic fever with renal syndrome (HFRS), caused by hantaviruses, are natural-focus infectious diseases prevalent in Shandong Province, China. Both diseases have similar clinical manifestations in certain disease stages and similar epidemic seasons, which has caused difficulties for physicians in distinguishing them. The aim of this study was to investigate whether misdiagnosis of scrub typhus as HFRS occurred in patients in Shandong Province. METHODS: Serum samples (N = 112) of clinically suspected HFRS patients from 2013 to 2014 in Shandong Province were analyzed with enzyme-linked immunosorbent assay (ELISA) for antibodies to both hantavirus and Orientia tsutsugamushi. RESULTS: ELISA showed that 56.3% (63/112) and 8.0% (9/112) of clinically suspected HFRS patients were IgM antibody positive to hantavirus and O. tsutsugamushi, respectively. Among the hantavirus IgM antibody positive patients, 7.9% (5/63) were also IgM antibody positive to O. tsutsugamushi. Among the hantavirus IgM antibody negative sera, 8.2% (4/49) of sera were positive to O. tsutsugamushi. CONCLUSIONS: We concluded that some scrub typhus patients were misdiagnosed as HFRS and co-infection of scrub typhus and HFRS might exist in China. Due to the different treatments for scrub typhus and HFRS, physicians should carefully differentiate between scrub typhus and HFRS and consider administering anti-rickettsia antibiotics if treatment for HFRS alone does not work.

High initial IgG antibody levels against Orientia tsutsugamushi are associated with an increased risk of severe scrub typhus infection.

BACKGROUND: Scrub typhus is a dominant cause of febrile illness in many parts of Asia. Immunity is limited by the great strain diversity of Orientia tsutsugamushi. It is unclear whether previous infection protects from severe infection or enhances the risk. METHODS/PRINCIPAL FINDINGS: We studied IgG antibody levels against O. tsutsugamushi at presentation in 636 scrub typhus patients using enzyme-linked immunosorbent assays (ELISA). The association between ELISA optical density (OD) and risk of severe infection was modelled using Poisson regression. OD was categorised as low (<1.0), intermediate (1.0 to 2.9), and high (≥3.0). OD was also modelled as a continuous variable (cubic spline). Median age of cases was 41 years (range 0-85), with 37% having severe infection. Compared to the low category, the age-adjusted risk of severe infection was 1.5 times higher in the intermediate category (95%CI 1.2, 1.9), and 1.3 times higher in the high category (95%CI 1.0, 1.7). The effect was stronger in cases <40 years, doubling the risk in the intermediate and high categories compared to the low category. The effect was more pronounced in cases tested within 7 days of fever onset when IgG ODs are more likely to reflect pre-infection levels. CONCLUSIONS/SIGNIFICANCE: Intermediate and high IgG antibody levels at the time of diagnosis are associated with a higher risk of severe scrub typhus infection. The findings may be explained by severe infection eliciting an accelerated IgG response or by previous scrub typhus infection enhancing the severity of subsequent episodes.

Follow-up investigation of antibody titers and diagnostic antibody cutoff values in patients with scrub typhus in South Korea.

BACKGROUND: Scrub typhus is a mite-borne infectious disease caused by Orientia tsutsugamushi. Few follow-up studies have assessed antibody titers using serologic tests from various commercial laboratories and the Korea Centers for Disease Control and Prevention (KCDC). METHODS: A prospective study to assess the antibody titers in patients with scrub typhus and seroprevalence in individuals undergoing health checkups was conducted using results of immunofluorescence antibody assays (IFAs) and serologic tests, used by the KCDC and commercial laboratories, respectively. The following tests were performed simultaneously: (i) indirect IFA used by the KCDC to detect immunoglobulin (Ig) M and IgG, (ii) IFA used by a commercial laboratory to detect total Ig, and (iii) antibody tests using two commercially available kits. RESULTS: When the IgM and IgG cutoff values (≥1:16 and ≥1:256, respectively) used in the IFA and the total IgG cutoff values (≥1:40) were used in prospective follow-up investigations, the antibody positivity rates of 102 patients with scrub typhus were 44.1, 35.3, and 57.6%, respectively, within 5 days of symptom onset. Among 91 individuals who recovered from scrub typhus, the follow-up IgM, IgG, and total Ig positivity rates for 13 years were 37.4% (34/91), 22.0% (20/91), and 76.9% (70/91), respectively. Among 216 individuals undergoing health checkups, the seroprevalence of IgM was 4.2% (9/216); no seroprevalence of IgG was observed. CONCLUSIONS: IFAs used by the KCDC and the commercial laboratory and rapid commercial kits could not distinguish between patients who had recovered from scrub typhus and those who are currently infected with O. tsutsugamushi. In South Korea and other countries, where low antibody cutoff values are used, upward adjustments of cutoff values may be necessary.

Epidemiological Characteristics of Rodents and Chiggers with Orientia Tsutsugamushi in the Republic of Korea.

A survey of rodents and chiggers associated with Orientia tsutsugamushi was conducted in a rural region of the Republic of Korea (Korea) between 2014 and 2018. Overall Apodemus agrarius 15.2% had the highest seropisitive for O. tsutsugamushi, followed by Myodes regulus 11.4%. Monthly risk factors using logistic regression analysis were not associated with O. tsutsugamushi infections in rodents. The overall prevalence rate of O. tsutsugamushi among chiggers was 0.3%. The chigger (Leptotrombidium scutellare) and monthly (October) risk factors were associated with O. tsutsugamushi human infections (P<0.05). Orientia tsutsugamushi infections are endemic in rodents in Korea and people, for example, soldiers who are active outdoors, must employ preventive measures, especially during October (P<0.05). When there are many reports of O. tsutsugamushi infections in Korea. The Boryong strain 85.7% (2/14) was the most common strain detected in chiggers, followed by the Shimokoshi 7.1% (1/14) and Karp 7.1% strains.

Performance of molecular and serologic tests for the diagnosis of scrub typhus.

Diagnosis of scrub typhus, caused by the bacterium Orientia tsutsugamushi, is challenging because of the overlap of its non-specific symptoms with other infections coupled with the lack of sufficient data on the performance of diagnostic tests. Early diagnosis of scrub typhus is crucial to improve outcomes and this study evaluates the diagnostic performance of various tests. The present study aims at assessing the accuracy of various rapid diagnostic tests, serologic tests, and nucleic acid amplification methods on well-characterized patient samples. Adult patients with acute febrile illness and manifestations suggestive of scrub typhus confirmed by positive PCR in the blood, eschar or tissue were characterized as cases. Patients with acute febrile illness and a confirmed alternate etiology such as culture-confirmed typhoid, smear/PCR positive for malaria, PCR/NS1 antigen positive for dengue, PCR positive for influenza, PCR/MAT positive for leptospirosis, PCR positive for spotted fever were characterized as controls with other infections. The healthy controls consisted of subjects from the same geographic region. We performed the following tests on blood samples for scrub typhus and calculated the sensitivity, specificity, positive predictive value, and negative predictive value: (1) Quantitative real time PCR using 47kDa gene (qPCR); (2) Conventional PCR using 56kDa gene (cPCR); (3) Loop-mediated isothermal amplification assay (LAMP assay); (4) Immunofluorescence assay (IFA); (5) Enzyme-linked immunosorbent assay (ELISA); (6) Weil-Felix test(WF test); and (7) Immunochromatographic Rapid Diagnostic Test (RDT).Among the 316 participants, 158 had confirmed scrub typhus (cases) and 158 were controls. ELISA and RDT detecting Orientia tsutsugamushi specific IgM antibodies had excellent discriminative potential with sensitivities and specificities of 92%, 94% and 92%, 92% respectively. The sensitivity and specificity of IFA were found to be 95% and 74% respectively. IgM serology had a false positivity rate of 8% with other acute febrile illnesses such as dengue, leptospirosis and spotted fever due to the nonspecific binding of the pentavalent IgM. LAMP assay had 91.7% sensitivity and 77.2% specificity while qPCR provided excellent sensitivity (97%) and perfect specificity. In conclusion, ELISA and RDT detecting Orientia tsutsugamushi specific IgM antibodies have excellent sensitivity and specificity while the accuracy of IFA is suboptimal for the diagnosis of scrub typhus. Given its perfect specificity and superior sensitivity, qPCR is preferred for diagnostic confirmation in reference laboratories particularly for diagnosis of early disease with less than 7 days duration. This study provides a comprehensive evaluation of all currently available diagnostic tests for scrub typhus.

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.

Serum C-reactive protein and procalcitonin values in acute Q fever, scrub typhus, and murine typhus.

BACKGROUND: Although C-reactive protein (CRP) and procalcitonin (PCT) are widely used inflammatory markers for infectious diseases, their role and potential application for rickettsioses were rarely studied. METHODS: A retrospective chart review and serological study were conducted in patients with rickettsioses. The clinical presentations, characteristics, laboratory data, and treatment responses were recorded and their associations with CRP and PCT values were analyzed. RESULTS: A total of 189 cases of rickettsioses, including 115 cases of acute Q fever (60.8%), 55 cases of scrub typhus (29.1%), and 19 cases of murine typhus (10.1%) were investigated. Both CRP and PCT values increased in the acute phase and declined in the convalescent phase. In the acute phase, mean CRP and PCT values were 78.2 ± 63.7 mg/L and 1.05 ± 1.40 ng/mL, respectively. Percentages of patients falling under different cut-off values of CRP and PCT were calculated systematically. Only 10.8% of CRP was > 150 mg/L and 14.2% of PCT was > 2.0 ng/mL. Patients with delayed responses to doxycycline treatment (> 3 days from treatment to defervescence) had significantly higher CRP values (102.7 ± 77.1 vs. 72.2 ± 58.2 mg/L, p = 0.041) and more PCT > 1.0 ng/ml (48.4% vs. 26.0%, p = 0.019) in the acute phase; higher CRP values (19.1 ± 37.4 vs. 3.6 ± 13.1 mg/L, p = 0.049) and more PCT > 0.5 ng/ml (19.2% vs. 1.4%, p = 0.005) in the convalescent phase. Correlation analysis was conducted for patients with acute Q fever. CRP and PCT values were positively correlated to each other, and both markers also had a positive correlation with serum aspartate transaminase values. Both CRP and PCT values and white blood cell counts were positively correlated to the days needed from doxycycline treatment to defervescence. CONCLUSION: CRP and PCT values might be useful in clinical investigations for patients with suspected rickettsioses and in predicting the response to doxycycline treatment for rickettsioses.

Evaluation of Recombinant Type-Specific Antigens of Orientia tsutsugamushi Expressed by a Baculovirus-Insect Cell System as Antigens for Indirect Immunofluorescence Assay in the Serological Diagnosis of Scrub Typhus.

Scrub typhus (ST) is a mite-borne rickettsiosis caused by the intracellular bacterium Orientia tsutsugamushi (OTS), which is classified as a biosafety level-3 (BSL-3) pathogen. For serological tests of ST, mouse fibroblast cells infected with the five prevalent serotypes of OTS in Japan are generally used as antigens for indirect immunofluorescence assay (IFA). In this study, Spodoptera frugiperda derived insect cell line (Sf9) cells infected with recombinant type-specific antigen (rTSA)-expressing baculovirus were used for IFA. The paired serum samples of 15 ST patients, 10 rickettsiosis patients, and 10 control individuals were used. IgM and IgG titers determined by the rTSA-based IFA were correlated with those determined by the OTS-infected cell-based IFA (R2 = 0.7319 to 0.7956). Based on the criteria for serological diagnosis, such as a suitable cutoff for single serum samples (IgM ≥ 1:160) and/or a significant increase in IgG titers between paired sera (≥ 4-fold), all 15 ST patients diagnosed as positive with the OTS-infected cell-based IFA were also diagnosed as positive by the rTSA-based IFA, whereas all 10 rickettsiosis patients and 10 control individuals were not. Thus, the rTSAs, which can be prepared in BSL-2 laboratories, are efficacious for the serological diagnosis of ST.