Antigenic Relationships among Human Pathogenic Orientia tsutsugamushi Isolates from Thailand.

BACKGROUND: Scrub typhus is a common cause of undiagnosed febrile illness in certain tropical regions, but can be easily treated with antibiotics. The causative agent, Orientia tsutsugamushi, is antigenically variable which complicates diagnosis and efforts towards vaccine development. METHODOLOGY/PRINCIPAL FINDINGS: This study aimed to dissect the antigenic and genetic relatedness of O. tsutsugamushi strains and investigate sero-diagnostic reactivities by titrating individual patient sera against their O. tsutsugamushi isolates (whole-cell antigen preparation), in homologous and heterologous serum-isolate pairs from the same endemic region in NE Thailand. The indirect immunofluorescence assay was used to titrate Orientia tsutsugamushi isolates and human sera, and a mathematical technique, antigenic cartography, was applied to these data to visualise the antigenic differences and cross-reactivity between strains and sera. No functional or antigen-specific analyses were performed. The antigenic variation found in clinical isolates was much less pronounced than the genetic differences found in the 56kDa type-specific antigen genes. The Karp-like sera were more broadly reactive than the Gilliam-like sera. CONCLUSIONS/SIGNIFICANCE: Antigenic cartography worked well with scrub typhus indirect immunofluorescence titres. The data from humoral responses suggest that a Karp-like strain would provide broader antibody cross-reactivity than a Gilliam-like strain. Although previous exposure to O. tsutsugamushi could not be ruled out, scrub typhus patient serum antibody responses were characterised by strong homologous, but weak heterologous antibody titres, with little evidence for cross-reactivity by Gilliam-like sera, but a broader response from some Karp-like sera. This work highlights the importance of antigenic variation in O. tsutsugamushi diagnosis and determination of new serotypes.Wellcome TrustThis is the final version of the article. It first appeared from PLOS via http://dx.doi.org/10.1371/journal.pntd.000472

Accuracy of Rapid IgM-Based Immunochromatographic and Immunoblot Assays for Diagnosis of Acute Scrub Typhus and Murine Typhus Infections in Laos

We studied the diagnostic accuracy of a rapid immunochromatographic test (ICT) for detection of IgM against scrub typhus (ST ICT) and an immunoblot test for the detection of IgM against murine typhus (MT IBT) by using admission serum samples from 1,030 febrile patients in Laos. Sensitivity and specificity for the ST ICT determined by using the diagnostic criteria of a four-fold increase in IgM against Orientia tsutsugamushi between paired samples were 23.8% (95% confidence interval [CI] = 15.9–33.3%) and 86.2% (95% CI = 84.1–88.6%), respectively. Sensitivity and specificity for the ST ICT determined by using an admission IgM titer ≥ 1:400 were 39.1% (95% CI = 34.1–44.2%) and 99.5% (95% CI = 98.7–99.9%), respectively. Sensitivity and specificity for the MT IBT determined by using the criteria of a four-fold increase in IgM against Rickettsia typhi between paired serum samples were 61.2% (95% CI = 53.7–68.3%) and 86.5% (95% CI = 84.1–88.8%), respectively. Sensitivity and specificity for the MT IBT determined by using an admission IgM titer ≥ 1:400 were 54.6% (95% CI = 49.1–60.0%) and 94.1% (95% CI = 92.0–95.7%), respectively. Both assays had relatively good specificity but low sensitivity and thus have limited utility for admission diagnosis

Antigenic Relationships among Human Pathogenic Orientia tsutsugamushi Isolates from Thailand

Scrub typhus is a common cause of undiagnosed febrile illness in certain tropical regions, but can be easily treated with antibiotics. The causative agent, Orientia tsutsugamushi, is antigenically variable which complicates diagnosis and efforts towards vaccine development. Methodology/Principal Findings: This study aimed to dissect the antigenic and genetic relatedness of O. tsutsugamushi strains and investigate sero-diagnostic reactivities by titrating individual patient sera against their O. tsutsugamushi isolates (whole-cell antigen preparation), in homologous and heterologous serum-isolate pairs from the same endemic region in NE Thailand. The indirect immunofluorescence assay was used to titrate Orientia tsutsugamushi isolates and human sera, and a mathematical technique, antigenic cartography, was applied to these data to visualise the antigenic differences and cross-reactivity between strains and sera. No functional or antigen-specific analyses were performed. The antigenic variation found in clinical isolates was much less pronounced than the genetic differences found in the 56kDa type-specific antigen genes. The Karp-like sera were more broadly reactive than the Gilliam-like sera. Conclusions/Significance: Antigenic cartography worked well with scrub typhus indirect immunofluorescence titres. The data from humoral responses suggest that a Karp-like strain would provide broader antibody cross-reactivity than a Gilliam-like strain. Although previous exposure to O. tsutsugamushi could not be ruled out, scrub typhus patient serum antibody responses were characterised by strong homologous, but weak heterologous antibody titres, with little evidence for cross-reactivity by Gilliam-like sera, but a broader response from some Karp-like sera. This work highlights the importance of antigenic variation in O. tsutsugamushi diagnosis and determination of new serotypes

Antigenic Relationships among Human Pathogenic Orientia tsutsugamushi Isolates from Thailand

Scrub typhus is a common cause of undiagnosed febrile illness in certain tropical regions, but can be easily treated with antibiotics. The causative agent, Orientia tsutsugamushi, is antigenically variable which complicates diagnosis and efforts towards vaccine development. Methodology/Principal Findings: This study aimed to dissect the antigenic and genetic relatedness of O. tsutsugamushi strains and investigate sero-diagnostic reactivities by titrating individual patient sera against their O. tsutsugamushi isolates (whole-cell antigen preparation), in homologous and heterologous serum-isolate pairs from the same endemic region in NE Thailand. The indirect immunofluorescence assay was used to titrate Orientia tsutsugamushi isolates and human sera, and a mathematical technique, antigenic cartography, was applied to these data to visualise the antigenic differences and cross-reactivity between strains and sera. No functional or antigen-specific analyses were performed. The antigenic variation found in clinical isolates was much less pronounced than the genetic differences found in the 56kDa type-specific antigen genes. The Karp-like sera were more broadly reactive than the Gilliam-like sera. Conclusions/Significance: Antigenic cartography worked well with scrub typhus indirect immunofluorescence titres. The data from humoral responses suggest that a Karp-like strain would provide broader antibody cross-reactivity than a Gilliam-like strain. Although previous exposure to O. tsutsugamushi could not be ruled out, scrub typhus patient serum antibody responses were characterised by strong homologous, but weak heterologous antibody titres, with little evidence for cross-reactivity by Gilliam-like sera, but a broader response from some Karp-like sera. This work highlights the importance of antigenic variation in O. tsutsugamushi diagnosis and determination of new serotypes

Orientia tsutsugamushi in Human Scrub Typhus Eschars Shows Tropism for Dendritic Cells and Monocytes Rather than Endothelium

Scrub typhus is a common and underdiagnosed cause of febrile illness in Southeast Asia, caused by infection with Orientia tsutsugamushi. Inoculation of the organism at a cutaneous mite bite site commonly results in formation of a localized pathological skin reaction termed an eschar. The site of development of the obligate intracellular bacteria within the eschar and the mechanisms of dissemination to cause systemic infection are unclear. Previous postmortem and in vitro reports demonstrated infection of endothelial cells, but recent pathophysiological investigations of typhus patients using surrogate markers of endothelial cell and leucocyte activation indicated a more prevalent host leucocyte than endothelial cell response in vivo. We therefore examined eschar skin biopsies from patients with scrub typhus to determine and characterize the phenotypes of host cells in vivo with intracellular infection by O. tsutsugamushi, using histology, immunohistochemistry, double immunofluorescence confocal laser scanning microscopy and electron microscopy. Immunophenotyping of host leucocytes infected with O. tsutsugamushi showed a tropism for host monocytes and dendritic cells, which were spatially related to different histological zones of the eschar. Infected leucocyte subsets were characterized by expression of HLADR+, with an “inflammatory” monocyte phenotype of CD14/LSP-1/CD68 positive or dendritic cell phenotype of CD1a/DCSIGN/S100/FXIIIa and CD163 positive staining, or occasional CD3 positive T-cells. Endothelial cell infection was rare, and histology did not indicate a widespread inflammatory vasculitis as the cause of the eschar. Infection of dendritic cells and activated inflammatory monocytes offers a potential route for dissemination of O. tsutsugamushi from the initial eschar site. This newly described cellular tropism for O. tsutsugamushi may influence its interaction with local host immune responses

Rickettsial illnesses as important causes of febrile illness in Chittagong, Bangladesh

We conducted a yearlong prospective study of febrile patients admitted to a tertiary referral hospital in Chittagong, Bangladesh, to assess the proportion of patients with rickettsial illnesses and identify the causative pathogens, strain genotypes, and associated seasonality patterns. We diagnosed scrub typhus in 16.8% (70/416) and murine typhus in 5.8% (24/416) of patients; 2 patients had infections attributable to undifferentiated Rickettsia spp. and 2 had DNA sequence-confirmed R. felis infection. Orientia tsutsugamushi genotypes included Karp, Gilliam, Kato, and TA763-like strains, with a prominence of Karp-like strains. Scrub typhus admissions peaked in a biphasic pattern before and after the rainy season, whereas murine typhus more frequently occurred before the rainy season. Death occurred in 4% (18/416) of cases; case-fatality rates were 4% each for scrub typhus (3/70) and murine typhus (1/28). Overall, 23.1% (96/416) of patients had evidence of treatable rickettsial illnesses, providing important evidence toward optimizing empirical treatment strategies

Risk factors analysis for neglected human rickettsioses in rural communities in Nan province, Thailand: A community-based observational study along a landscape gradient

International audienceIn this study, we estimated exposure for Scrub typhus (STG), Typhus (TG) and Spotted fever groups (SFG) rickettsia using serology at a fine scale (a whole sub-district administration level) of local communities in Nan Province, Thailand. Geographical characteristics of the sub-district were divided into two landscape types: lowland agricultural area in an urbanized setting (lowland-urbanized area) and upland agricultural area located close to a protected area of National Park (upland-forested area). This provided an ideal contrast between the two landscapes with low and high levels of human-altered habitats to study in differences in disease ecology. In total, 824 serum samples of participants residing in the eight villages were tested by screening IgG ELISA, and subsequently confirmed by the gold standard IgG Immunofluorescent Assay (IFA). STG and TG IgG positivity were highest with seroprevalence of 9.8% and 9.0%, respectively; whereas SFG positivity was lower at 6.9%. Inhabitants from the villages located in upland-forested area demonstrated significantly higher STG exposure, compared to those villages in the lowland-urbanized area (chi-square = 51.97, p < 0.0001). In contrast, TG exposure was significantly higher in those villagers living in lowland-urbanized area (chi-square = 28.26, p < 0.0001). In addition to the effect of landscape types, generalized linear model (GLM) analysis identified socio-demographic parameters, i.e., gender, occupation, age, educational level, domestic animal ownership (dog, cattle and poultry) as influential factors to explain the level of rickettsial exposure (antibody titers) in the communities. Our findings raise the public health awareness of rickettsiosis as a cause of undiagnosed febrile illness in the communities

A Use of 56-kDa Recombinant Protein of <i>Orientia tsutsugamushi</i> Karp Serotype in Serodiagnosis of Scrub Typhus by Enzyme-Linked Immunosorbent Assay in Thais

Scrub typhus is a mite-borne disease caused by a Gram-negative obligately intracellular bacillus, Orientia tsutsugamushi. The disease is endemic in the Asia–Australia–Pacific region, including Thailand. Scrub typhus generally manifests as acute undifferentiated febrile fever along with myalgia, rash, and lymphadenopathy. An eschar can be a valuable diagnostic clue, but this skin lesion may be missed in some patients. The disease symptoms resemble those of other febrile illnesses such as leptospirosis, typhoid, murine typhus, malaria, and dengue fever, making a laboratory diagnosis necessary for the definitive diagnosis. In this study, we expressed a recombinant protein derived from 56-kDa type-specific antigen of O. tsutsugamushi Karp serotype and tested its ability to detect and differentiate scrub typhus infection. IgM and IgG antibodies were determined in sera from scrub typhus (n = 92) and other febrile illness patients (murine typhus (n = 25), melioidosis (n = 36), leptospirosis (n = 42), and dengue (n = 35)) from Thailand. Sensitivities of 87.0% and 59.8% with a specified assay cut-off were obtained for IgM and IgG indirect ELISAs, respectively, with a specificity of 100% in both tests. The sensitivity was increased to 95.7% when a combination of IgM and IgG ELISAs results was considered. Our study suggested a potential of the 56-kDa recombinant protein for further development and evaluation for use in scrub typhus serodiagnosis

Diagnosis of Murine Typhus by Serology in Peninsular Malaysia: A Case Report Where Rickettsial Illnesses, Leptospirosis and Dengue Co-Circulate

Murine typhus is a rarely diagnosed cause of acute febrile illness in Malaysia, and its true disease burden is unknown. We report a case of an acute murine typhus infection in a patient living in a small city in Peninsular Malaysia, presenting with fever, rash, and headache. Unresponsive to the initial empirical treatment for leptospirosis, he showed a rapid response to doxycycline when murine typhus was diagnosed later. This case highlights the importance of considering murine typhus as a diagnostic in cases of acute febrile illness in urban and sub-urban areas, such as that of in Peninsular Malaysia

Serological detection of Rickettsia spp. and evaluation of blood parameters in pet dogs and cats from Bangkok and neighboring provinces.

Rickettsiosis is caused by Orientia spp. and Rickettsia spp., arthropod-borne zoonotic intracellular bacteria. The close relationships between pet dogs, cats and owners increase the risk of rickettsial transmission, with limited studies on the seroprevalence in pets. This study investigated the prevalence of rickettsia exposure among dogs and cats in Bangkok and neighboring provinces. The samples from 367 dogs and 187 cats used in this study were leftover serum samples from routine laboratory testing stored at the Veterinary Teaching Hospital. In-house Enzyme-linked immunosorbent assay (ELISA) tests included IgG against the scrub typhus group (STG), typhus group (TG), and spotted fever group (SFG). The seroprevalence in pet dogs was 30.25% (111/367), including 21.53% for STG, 4.36% for TG, and 1.09% for SFG. Co-seroprevalence consisted of 2.72% for STG and TG, 0.27% for STG and SFG, and 0.27% for pangroup infection. The prevalence in cats was 62.56% (117/187), including 28.34% for STG, 4.28% for TG, and 6.42% for STG. Co-seroprevalence in cats consisted of STG and TG (4.28%), STG and SFG (5.35%), TG and SFG (3.21%), and three-group infection (10.69%). No significant difference in seroprevalence for the three serogroups was observed in any of the 64 districts sampled. The mean hematocrit level significantly decreased in seropositive dogs (P<0.05). Seropositive dogs and cats were detected in significantly greater numbers of anemia cases than nonanemia cases (P<0.05) (odds ratio: 7.93, 0.44, p = 0.00, p = 0.01). A significantly higher number of seropositive cats had decreased hemoglobin levels (P<0.05) (odds ratio: 3.63, p = 0.00). The seropositive samples significantly differed among older cats (P<0.05). These high exposures in pet dogs and cats could constitute important relationship dynamics between companion animals and rickettsial vectors. Significantly decreased hematocrit and hemoglobin levels indicated anemia in the exposed dogs and cats. The study findings will raise awareness of this neglected disease among pet owners and veterinary hospital personnel and aid in future public health preventative planning