Determination of a cut-off value for the serological diagnosis of scrub typhus by detecting anti-Orientia tsutsugamushi immunoglobulin M

Background & objectives: The diagnosis of scrub typhus (ST) is usually done using enzyme-linked immunosorbent assay (ELISA) due to its ease of performance and reading objectivity. The cut-off value for ELISA needs to be calculated for each geographical location as it depends on zonal endemicity of the disease. This study was, therefore, undertaken to calculate the pan-India cut-off for anti-Orientia tsutsugamushi (OT) immunoglobulin M (IgM) by ELISA. Methods: Samples from cases (cases of ST) and controls (voluntary, consenting, healthy adults) were collected by a network of 29 laboratories across India and tested for anti-OT IgM by immunofluorescence assay (IFA), the considered gold standard test. These samples were retested by ELISA for anti-OT IgM and their optical densities (ODs) were used for cut-off estimation by receiver operating characteristic (ROC) curve. Results: Anti-OT IgM ELISA ODs from 273 controls and 136 cases were used for the cut-off estimation. The ODs of the anti-OT IgM ELISA on healthy individuals and those of confirmed ST cases ranged from 0.1 to 0.75 and 0.5 to 4.718, respectively. ROC curve-based cut-off for ELISA was calculated as 0.554 at a sensitivity of 95.2 per cent and specificity of 95.1 per cent. A value of >1 was noted to have a specificity of 100 per cent in diagnosing ST. Interpretation & conclusions: The cut-off calculated for India was similar to the previous cut-off that was used until now.

Early diagnostic value of mNGS for 15 cases with critical rickettsial disease in Hainan / 中华检验医学杂志

Objective:To explore the clinical utility of metagenomic next-generation sequencing (mNGS) for patients with critically ill atypical rickettsial infections in the early diagnosis and therapy.Methods:From Jan 2020 to Aug 2022, clinical features, blood biochemical results, imaging data and mNGS results in patients with unexplained critical illnesses were collected and analyzed retrospectively. Fisher's exact test was used to compare the positive rate of mNGS and weil felix reaction.Results:All 15 patients with rickettsial disease had fever, 12 cases had headache, but only 3 had a typical rash or scab of diagnostic significance, 6 had septic shock and all had multi-organ dysfunction; blood mNGS tests were positive in 15 cases, of which 10 had Orientia tsutsugamushi detected in their blood and the remaining five had Rickettsia moschata detected in their blood. The positive rate of mNGS was significantly higher than that of the weil felix reaction (15/15 vs 0, P<0.001). All patients were given doxycycline and other treatments after diagnosis, of which 14 improved and were discharged, and one died 1 week after discharge due to critical condition and abandonment of treatment. Conclusion:mNGS can improve the detection rate of atypical rickettsiae in patients with negative routine test results, which can provide valuable reference basis for early diagnosis and early anti-infection treatment of patients with critical rickettsial disease.

Emerging trends of scrub typhus disease in southern Rajasthan, India: A neglected public health problem

Scrub typhus is the oldest known vector-borne zoonotic infectious disease in the world which is life-threatening for all age groups as it presents acute febrile illness along with multi-organ involvements and spread with the biting of infectious ‘Trombiculid mite’ (chigger mite). The pathogen of this disease is an obligatory coccobacillus gram-negative rickettsial bacteria Orientia tsutsugamushi. Scrub typhus disease was previously confined geographically only to the Asia Pacific region (tsutsugamushi triangle), but in recent years it has crossed its limit and has spread in other countries beyond the tsutsugamushi triangle and has become more hazardous for the community. The objective of this study is to explore the scrub typhus disease outbreak trends with existing information in southern Rajasthan state, India. This study concluded that scrub typhus disease is being re-emerged again and again in various Indian geographical regions with new species of vectors. The disease has been raised in tremendous amounts in Rajasthan within the last five years especially in the hilly zone and has led to major public health problems with other zoonotic diseases

Scrub Typhus and Myocarditis: A Rare Complication

Scrub typhus, also called bush typhus, is a zoonotic disease a Gram-negative bacterium. Its presentation may range from nonspecific febrile illness to severe disease, with cardiovascular, renal, hepatic and neurological involvement. Myocarditis is one of the rare complications of scrub typhus. Hence, we are reporting a case of a 50-year-old male, farmer by occupation, presenting to us in multiorgan dysfunction syndrome who developed myocarditis during second week of his illness.

Scrub Typhus with Multiple Cranial Nerve Palsy: A Rare Case Presentation

Scrub typhus, a rickettsial disease endemic in several parts of India, usually presents with acute symptoms. It is caused by small intracellular Gram-negative bacteria belonging to the Rickettsiaceae family. Optic neuritis and lateral rectus palsy may be associated with a range of autoimmune disorders, infectious diseases and raised intracranial tension. In this case, we report optic neuritis and lateral rectus palsy induced by Orientia tsutsugamushi. We report a case of a 23-year-old woman presenting with complaints of high-grade fever, vomiting and generalized swelling since 5 days. During this febrile period, on 4/10/2021, she complained of difficulty in vision and double vision. She was found to be positive for scrub typhus on 5/10/2021. Optic neuritis was diagnosed on the basis of ophthalmologic examination and magnetic resonance imaging (MRI) brain. Investigation was done to rule out autoimmune disorders (vasculitis and connective tissue diseases). Rickettsial optic neuritis was confirmed by detection of specific antibodies in serum and the negativity of other serologic tests. Fever, eschar, history of tick exposure and supportive diagnostic tests usually lead to the diagnosis. This case aims to raise awareness among the healthcare providers for this type of association. Scrub typhus should be included in the differential diagnosis when a patient presents with fever with or without eschar and isolated or multiple cranial nerve palsy

Epidemiology and clinical characteristics of scrub typhus based on 680 unexplained fever patients in Hainan / 中国热带医学

@#Abstract: Objective　To analyze the epidemiological characteristics of scrub typhus in 680 patients with unexplained fever in Hainan Island and provide guidance for local scrub typhus prevention and control. Methods　Blood samples from patients with clinically diagnosed unexplained fever in the First Affiliated Hospital of Hainan Medical University, Second Affiliated Hospital of Hainan Medical University, Haikou Municipal People's Hospital and Central South University Xiangya Medical College Affiliated Hospital, People's Hospital of Qiongzhong Li Miao Autonomous County were collected between 2018 and 2021, then samples were tested for Orientia tsutsugamushi specific IgM, IgG antibodies and Orientia tsutsugamushi 56kD-specific antigen genes using colloidal gold immunochromatography and PCR methods. The clinical and epidemiological information of the samples were also collected. Epidemiological analysis was carried out on the obtained sample information and test results. Results　A total of 680 samples from patients with clinically unknown fever were collected. The positive rates of scrub typhus IgM antibody, IgG antibody, PCR were 23.97% (163/680), 36.62% (249/680), 20.88% (142/680), respectively, and eschar or rash was observed in 12.06% (82/680) of all patients. According to the diagnostic criteria, 223 patients were finally diagnosed with scrub typhus. Among them, there were 111 males (49.78%) and 109 females (48.88%); the average age was (53.14±15.12) years old, and the 40-<60 years old was the main incidence group (98 cases, accounting for 43.95%). The ethnicity of the patient with scrub typhus was mainly Han ethnicity (136 cases, accounting for 60.99%). Farmers were the main morbidity group (93 cases, 41.70%). The patient with scrub typhus distributed throughout the island except for Sanya and Wuzhishan, with Haikou and Qiongzhong show the highest number of cases. The number of cases in autumn was the highest (114 cases, 51.12%). The clinical manifestations of infected patients were mainly non-specific symptoms and signs, 36.77% of patients developed skin-specific ulcers or eschars, which were mainly distributed in the shoulder and neck, armpits, elbow fossa, chest and abdomen, back, buttocks, groin, scrotum and other joints and skin folds of the upper and lower limbs. Conclusions　Hainan Island is the foci of scrub typhus and the epidemic time has obvious seasonality. Its prevention should focus on rural areas in autumn, and its clinical features are mainly high fever and eschar or ulceration.

Circulatory and hepatic failure at admission predicts mortality of severe scrub typhus patients: A prospective cohort study

Objective: To study the spectrum and outcome of severe scrub typhus in adult patients and to predict the hospital mortality by organ failure on admission. Methods: This was a prospective observational cohort study conducted between July 2017 and October 2020 at the medical emergency centre of PGIMER, Chandigarh, India. One hundred and twenty-six patients aged =13 years were diagnosed with scrub typhus. Severe disease was defined as the presence of organ failure based on the Sequential Organ Failure Assessment (SOFA) score on admission. Results: About two-thirds of the patients were from geographic regions outside the endemic sub-Himalayan belt. Fever (99.21%) and dyspnea (79.36%) were the most frequent complaints. Respiratory failure (81.75%) was the most common organ failure, followed by hepatic (52.38%), coagulative (47.62%), circulatory (33.33%), renal (21.43%), and cerebral dysfunction (13.49%). The median (Q1, Q3) SOFA score was 8 (6, 9), and 48.41% of the patients had a quick-SOFA score =2. Organ supports with invasive ventilation (40.48%), vasopressors (36.51%), and renal replacement therapy (7.14%) were frequently required. The in-hospital mortality was 11.90% and was independently predicted by circulatory and hepatic failures on multivariate logistic regression (OR 11.12, 95% CI 1.73-71.31 and OR 8.49, 95% CI 1.18-61.41, respectively). Conclusions: Most patients had pulmonary dysfunction; circulatory or hepatic failure on admission strongly predicts death.

Scrub typhus mimicking the clinical course of infectious mononucleosis: a case report / Journal of Rural Medicine

Objective: Scrub typhus is a relatively common life-threating disease; its symptoms are non-specific and similar to those of other viral infections. Therefore, scrub typhus might be underdiagnosed.Patient: Herein, we report a patient with scrub typhus whose clinical course mimicked that of infectious mononucleosis. A 63-year-old male patient with hypertension presented to our hospital complaining of symptoms including prolonged fever, pharyngeal discomfort, and a mild headache. He showed the appearance of a rash after amoxicillin administration. At the same time, he did not show a crusted rash on his body surface.Results: After a comprehensive examination, Epstein-Barr virus and cytomegalovirus infections were ruled out. We suspected that this patient suffered from scrub typhus on the basis of his usual lifestyle. Finally, polymerase chain reaction analysis showed a positive result for deoxyribonucleic acid of Orientia tsutsugamushi in his blood sample. Fortunately, he recovered naturally with only supportive treatment during his hospitalization.Conclusion: We should observe and monitor patients with infectious mononucleosis-like symptoms and emphasize the importance of a clinical interview.

Comparison of diagnostic performance between serological tests (Scrub IgM-ELISA) and Scrub-PCR for scrub typhus fever in an Indian tertiary care teaching hospital

Scrub typhus is a mite borne transmittable disease caused by the obligate intracellular bacterium known as Orientiatsutsugamushi. Although it presents with nonspecific clinical manifestation, early diagnosis and treatment oftenprevents it’s complication. Here in this study, we used two different diagnostic tools Scrub-polymerase chainreaction (PCR) and serologic Scrub IgM antibody based enzyme-linked immunosorbent assay (IgM ELISA) test.Out of 154 cases, total of 101 cases were found Scrub IgM-ELISA positive. Both serology and Scrub-PCR wasdone in total 96 cases. Out of 96 cases, 41.6% (n−40) were Scrub-PCR positive and 58.3% (n−56) cases werePCR negative. Eighty percent cases become serology positive (IgM ELISA) those who presented after 7 days butbefore 14 days of illness and least (54%) when presented before 1 week of illness. Twenty cases were found to beIgM positive but PCR negative. The mean duration of illness among those 20 patients who were IgM positive butPCR negative was found to 12.55 days. Only four cases were PCR positive but IgM negative. The mean durationof illness was 6.2 days among those four patients so that the antibody conversion was not detectable at that phase.Therefore, the combination of molecular and serological tests needs to be used, so that it can cover the entireduration of illness for early and accurate diagnosis so as to prevent the complications.

Evidence of natural infection of Orientia tsutsugamushi in vectors and animal hosts � Risk of scrub typhus transmission to humans in Puducherry, South India

Background: Scrub typhus infection is endemic in India and reported to be the major cause for acute encephalitis syndrome (AES) in humans. Periodic occurrence of scrub typhus cases and presence of pathogen in rodents were also reported in areas with human cases of scrub typhus in Puducherry. Objectives: This study was carried out to screen Orientia tsutsugamushi in rodent/shrew reservoirs and vectors in villages of Puducherry with no reports of human scrub typhus cases. Methods: This study was conducted during October 2017 to January 2018 in ten randomly selected villages in Puducherry. Rodents/shrews in the peridomestic area were trapped using Sherman traps. Screening of O. tsutsugamushi in rodents/shrews and mite vectors was done by polymerase chain reaction (PCR). Weil-Felix test was done to screen antibodies against O. tsutsugamushi in rodent serum samples. Results: Among the 54 rodents trapped, Suncus murinus was the major small animal and Leptotrombidium deliense was the major mite species retrieved. PCR screening revealed pathogen positivity in 8 rodent blood and 3 pooled mite samples. Phylogenetic analysis has shown that Kato was the circulating serotype of O. tsutsugamushi. None of the rodent serum samples was tested positive for antibodies against O. tsutsugamushi by Weil-Felix test. Conclusions: The presence of pathogen in both vectors and reservoir animal hosts imposes a risk for scrub typhus transmission to the inhabitants; hence, initiation of vector control measures before the start of winter is recommended in the study area. It is also recommended to screen scrub typhus in patients with undifferentiated acute febrile illness and AES.

A single-centre experience of scrub typhus in a tertiary care centre - a re-emerging infection

Background: Scrub typhus is a documented disease in Himachal Pradesh, but there have been few clinico-epidemiological studies in this area. The present study is done with IgM ELISA as a diagnostic test which has higher sensitivity and specificity as most of the previous studies had used Weil Felix test as a diagnostic test. Methodology: This was a prospective observational study. All the patients more than 18 years of age with positive IgM ELISA for scrub typhus with/without eschar were included. The clinical profile was observed. IgM scrub typhus was done by ELISA. Results: Total of 39 patients were observed between July 2016 to Dec 2016. Maximum patients were observed in August, September, and October. Fever with Headache was the most common presenting complaint. Eschar was present in 10 % patients. Complications were seen in 76.92 %. The mortality rate was 0 %. Conclusion: The varied presentations and high rate of complications require a high index of suspicion for Scrub Typhus. The general physicians should be sensitized for the early diagnosis and treatment to reduce morbidity and mortality.

Assessment of Predictors for Severity in Scrub Typhus-A Case-Control Study

Objective: Scrub typhus is a rickettsial febrile condition caused by bacteria called Orientia tsusugamushi. It spreads to people through bites of infected larval mites. The objective of this study was to predict the severity of scrub typhus. Methods: A case-control study was done at Kasturba Medical College Hospital (2012-2015) retrospectively on patients admitted with scrub typhus. Patient demographics, lab parameters, investigations and treatment courses were noted. Subjects were divided into two groups, non-severe and severe group. Patient clinical details and laboratory parameters were compared in both the groups. The lab parameters associated with the severity of disease and mortality were also analyzed. A total of 210 patients out of which 140 controls (non-severe group) and 70 were cases (severe group). Results: The overall mortality due to scrub typhus infection was 14.3%. Eschar was present in 21.4% of the patients. Among the cases, the common symptoms were cough, chest pain, abdominal pain and distension, pedal edema and facial puffiness. Lymphadenopathy was observed with non-severe cases of scrub typhus and was statistically significant whereas, icterus, maculopapular rash, and hepatomegaly were associated with severe scrub typhus infection. Hemoglobin, platelet count and serum albumin were considerably lower in patients who died, whereas AST, aPTT, serum urea and creatinine were higher in them. Conclusion: Lower levels of hemoglobin, platelet count, serum albumin and higher levels of total leucocyte count, hepatic transaminases and serum creatinine correlated with severity. Doctors need to be watchful for such lab parameters and act quickly.

Phylogenetic diversity of Orientia tsutsugamushi isolates in patients with scrub typhus in Bengaluru, India

Scrub typhus has re-emerged as an important cause of acute febrile illness in India. There is a dearth of information on strain diversity of Orientia tsutsugamushi from Karnataka, India, hence the present study sought to address this issue. One hundred clinically suspected cases of scrub typhus/rickettsiosis (as per the DHR-ICMR guidelines) were included. Nested-polymerase chain reaction (PCR) for 56-kDa gene and phylogenetic analysis was performed. PCR was positive in 22 cases and phylogenetic analysis showed the presence of different strains, with predominance of clustering (57%) with Gilliam-type for the first time in Karnataka. Knowledge of genetic diversity has implications in development of diagnostics and vaccine.

Prediction of T cell and B cell epitopes of the 22-, 47-, 56-, and 58-kDa proteins of Orientia tsutsugamushi

Objective: To predict B cell and T cell epitopes of 22-kDa, 47-kDa, 56-kDa and 58-kDa proteins. Methods: The sequences of 22-kDa, 47-kDa, 56-kDa and 58-kDa proteins which were derived from Orientia tsutsugamushi were analyzed by SOPMA, DNAstar, Bcepred, ABCpred, NetMHC, NetMHC II and IEDB. The 58-kDa tertiary structure model was built by MODELLER9.17. Results: The 22-kDa B-cell epitopes were located at positions 194-200, 20-26 and 143-154, whereas the T-cell epitopes were located at positions 154-174, 95-107, 17-25 and 57-65. The 47-kDa protein B-cell epitopes were at positions 413-434, 150-161 and 283-322, whereas the T-cell epitopes were located at positions 129-147, 259-267, 412-420 and 80-88. The 56-kDa protein B-cell epitopes were at positions 167-173, 410-419 and 101-108, whereas the T-cell epitopes were located at positions 88-104, 429-439, 232-240 and 194-202. The 58-kDa protein B-cell epitopes were at positions 312-317, 540-548 and 35-55, whereas the T-cell epitopes were located at positions 415-434, 66-84 and 214-230. Conclusions: We identified candidate epitopes of 22-kDa, 47-kDa, 56-kDa and 58-kDa proteins from Orientia tsutsugamushi. In the case of 58-kDa, the dominant antigen is displayed on tertiary structure by homology modeling. Our findings will help target additional recombinant antigens with strong specificity, high sensitivity, and stable expression and will aid in their isolation and purification.

Epidemiology and clinical features of scrub typhus in Hainan, China

Objective: To identify the epidemiology and clinical features of scrub typhus in Hainan Province, China. Methods: A total of 258 patients with acute febrile illness were recruited from four hospitals in Hainan between 1st June 2018 and 31th August 2019. Orientia tsutsugamushi-specific IgM/IgG and DNA were detected by goldimmuno- chromatographic assay and PCR on the patients' serum samples. The clinical and epidemiological characteristics of the laboratory-confirmed cases were recorded using a predesigned database. Results: The positive rates of IgM and IgG were 22.5% (58/258) and 39.9% (103/258), respectively, and the positive rate of PCR was 19.37% (50/258). There were 55 males and 48 females among the 103 IgG-positive patients. Patients of all age groups were affected (10 to 86 years); 65.0% of the adults aged 41 to 70 years (67/103); 64.1% of the patients were farmers (66/103). The patients resided in 12 counties and cities of Hainan, and the highest number of cases being reported among residents in Qiongzhong County and Haikou City (54/103, 52.4%). Seasonal variations in the incidence rate of scrub typhus was observed, with the peak incidence (53/103, 51.5%) occurring in the summer (June to August). The common presenting symptoms were fever (103/103, 100.0%), eschar or skin ulcer (38/103, 36.9%), and chills and rigors (30/103, 29.1%). The major complications were liver dysfunction (9/103, 8.7%). Four genotypes of Orientia tsutsugamushi were identified among these patients, including Karp, Kato, Gilliam and Ta763. The predominant genotype was Karp (33/51, 64.7%). Conclusions: Scrub typhus is endemic in Hainan, which predominantly affects residents with prolonged outdoor activities such as farming.

Prediction of T cell and B cell epitopes of the 22-, 47-, 56-, and 58-kDa proteins of Orientia tsutsugamushi

Objective: To predict B cell and T cell epitopes of 22-kDa, 47-kDa, 56-kDa and 58-kDa proteins. Methods: The sequences of 22-kDa, 47-kDa, 56-kDa and 58-kDa proteins which were derived from Orientia tsutsugamushi were analyzed by SOPMA, DNAstar, Bcepred, ABCpred, NetMHC, NetMHCⅡ and IEDB. The 58-kDa tertiary structure model was built by MODELLER9.17. Results: The 22-kDa B-cell epitopes were located at positions 194-200, 20-26 and 143-154, whereas the T-cell epitopes were located at positions 154-174, 95-107, 17-25 and 57-65. The 47-kDa protein B-cell epitopes were at positions 413-434, 150-161 and 283-322, whereas the T-cell epitopes were located at positions 129-147, 259-267, 412-420 and 80-88. The 56-kDa protein B-cell epitopes were at positions 167-173, 410-419 and 101-108, whereas the T-cell epitopes were located at positions 88-104, 429-439, 232-240 and 194-202. The 58-kDa protein B-cell epitopes were at positions 312-317, 540-548 and 35-55, whereas the T-cell epitopes were located at positions 415-434, 66-84 and 214-230. Conclusions: We identified candidate epitopes of 22-kDa, 47-kDa, 56-kDa and 58-kDa proteins from Orientia tsutsugamushi. In the case of 58-kDa, the dominant antigen is displayed on tertiary structure by homology modeling. Our findings will help target additional recombinant antigens with strong specificity, high sensitivity, and stable expression and will aid in their isolation and purification.

Annual Fluctuation in Chigger Mite Populations and Orientia Tsutsugamushi Infections in Scrub Typhus Endemic Regions of South Korea

OBJECTIVES: Chigger mites are vectors for scrub typhus. This study evaluated the annual fluctuations in chigger mite populations and Orientia tsutsugamushi infections in South Korea.METHODS: During 2006 and 2007, chigger mites were collected monthly from wild rodents in 4 scrub typhus endemic regions of South Korea. The chigger mites were classified based on morphological characteristics, and analyzed using nested PCR for the detection of Orientia tsutsugamushi.RESULTS: During the surveillance period, the overall trapping rate for wild rodents was 10.8%. In total, 17,457 chigger mites (representing 5 genera and 15 species) were collected, and the average chigger index (representing the number of chigger mites per rodent), was 31.7. The monthly chigger index was consistently high (> 30) in Spring (March to April) and Autumn (October to November). The mite species included Leptotrombidium pallidum (43.5%), L. orientale (18.9%), L. scutellare (18.1%), L. palpale (10.6%), and L. zetum (3.6%). L. scutellare and L. palpale populations, were relatively higher in Autumn. Monthly O. tsutsugamushi infection rates in wild rodents (average: 4.8%) and chigger mites (average: 0.7%) peaked in Spring and Autumn.CONCLUSION: The findings demonstrated a bimodal pattern of the incidence of O. tsutsugamushi infections. Higher infection rates were observed in both wild rodents and chigger mites, in Spring and Autumn. However, this did not reflect the unimodal incidence of scrub typhus in Autumn. Further studies are needed to identify factors, such as human behavior and harvesting in Autumn that may explain this discordance.

Survey of Rickettsia spp. and Orientia tsutsugamushi Pathogens Found in Animal Vectors (Ticks, Fleas, Chiggers) in Bangkaew District, Phatthalung Province, Thailand

Rickettsial infections (Rickettsioses) are the causes of acute fever found in Thailand. It is classified as acute febrile illnesses transmitted by bloodsucking arthropod vectors (tick, flea, and chigger). This research investigated pathogens of scrub typhus in vectors from Bangkaew District, Phatthalung Province. A total of 303 pools of vector samples were ticks (Rhipicephalus sanguineus, R. microplus, and Haemaphysalis sp.), fleas (Ctenocephalides felis orientis, C. f. felis, and C. canis), and chiggers (Leptotrombidium deliense, Aschoschoengastia indica, Blankaartia acuscutellaris and Walchia disparunguis pingue) collected from reservoir hosts (dogs and rodents). The 17 and 56 kDa gene of Rickettsia causing scrub typhus were found in 29% of ticks and 98% of flea. DNA sequence analysis reveeled the detected strains were R. asembonensis and Rickettsia sp. cf1 and 5.The chiggers, 1%, were infected with Rickettsia strain TA763, a pathogen of scrub typhus.

Use of eschar for the molecular diagnosis and genotypic characterisation of Orientia tsutsugamushi causing scrub typhus

Scrub typhus caused by Orientia tsutsugamushi presents as an acute febrile illness with a varied presentation from mild illness to fatal disease in the absence of appropriate antibiotic treatment. Performing polymerase chain reaction (PCR) on eschar sample acts a rapid diagnostic tool in the early stage of scrub typhus when blood is negative. A total of eight patients from whom both whole blood and eschar samples were collected and tested by nested PCR targeting 56 kDa trichostatin A (TSA) gene to detect O. tsutsugamushi DNA. All (100%) eschar samples and three whole blood samples tested positive. Genetic analysis of the 56 kDa TSA gene sequences showed that the majority were related to Karp reference strains, while one clustered with Kawasaki strain. When present, eschar should be favoured as a diagnostic sample over whole blood in the early phase of infection.

A surface antigen of Orientia tsutsugamushi activates human monocyte-derived dendritic cells via nuclear factor-kB & p38 mitogen-activated protein kinase pathways

Background & objectives: Scrub typhus is a chigger-borne disease caused by Orientia tsutsugamushi. The immunological reactions to O. tsutsugamushi infection are not completely understood. In this study, we investigated the response of dendritic cells (DCs) to a major 56-kDa scrub typhus antigen Sta56. Methods: Monocyte-derived human DCs were incubated with different concentrations of recombinant Sta56 and analyzed for maturation based on phagocytic capacity, the ability to induce T-cell proliferation, expression of surface markers, cytokine secretion and activation of toll-like receptor (TLR)-dependent signalling pathways. Results: Treatment of DCs with Sta56 induced cell surface expression of CD80, CD83, CD86 and MHC Class II increased the production of interleukin-12 (IL-12) p40, IL-12 p70 and IL-10 and decreased DC phagocytic capacity. Furthermore, Sta56 increased the ability of DCs to activate T-cell proliferation and interferon-? secretion. TLR4-specific antibodies neutralized Sta56-elicited effects on DC maturation, suggesting direct interaction between Sta56 and TLR4. Moreover, Sta56 activated nuclear factor (NF)-?B and p38 mitogen-activated protein kinase (MAPK) signalling as evidenced by decrease in Sta56-induced cytokine production and surface marker expression by specific inhibitors helenalin and SB203580, respectively, and increase in I?B? and p38 phosphorylation and NF-?B-DNA binding. Interpretation & conclusions: Our results showed that the surface antigen of O. tsutsugamushi activated DCs through interaction with TLR4 and activation of MAPK and NF-?B signalling, suggesting Sta56 as a potential candidate molecule for the development of vaccine against scrub typhus.