Epidemiological,clinical and genetic characterization of scrub typhus in patients presenting with acute febrile illness in New Delhi

PurposeScrub typhus (ST) is a zoonotic disease, caused by O. tsutsugamushi is a major cause of acute febrile illness (AFI) in India. There is a need to study the prevalence and risk factors in various regions of India.MethodsA study to estimate the prevalence and study the risk factors of ST in patients presenting with acute febrile illness (AFI) was performed. All patients underwent serology for IgM antibodies to Orientia tsutsugamushi (In Bios International Inc, Seattle, WA) as per the manufacturers’ protocol. Following this, Polymerase Chain reaction (PCR) (real time SYBR green based targeting groEL gene and conventional PCR targeting 56 ?kDa type specific antigen gene) was performed from stored serum samples.ResultsDuring the study period, 473 patients were admitted. Of these 56 (11.8%) patients were ST positive by IgM serology. The conventional PCR targeting 56 ?kDa type specific antigen gene of O. tsutsugamushi was positive in six patients while Ot groEL SYBR green based PCR) was positive in five. PCR was positive in patients who had demonstrated a higher OD value in ELISA. Conventional PCR positive amplicons were sent for Sanger sequencing and confirmed to be O. tsutsugamushi. The mean age of the patients was 49 ?± ?18.3 years and males constituted a higher number of patients (67.9%, n ?= ?38). The pathognomonic eschar was present in 7 (12.5%) patients. Phylogenetic analysis revealed that sequences clustered close to Kato-like Hualein-20 strain and Karp-like Linh DT strains. All patients were administered doxycycline in our study. Mortality was recorded in 8.9% of the patients.ConclusionsIn patients presenting with acute febrile illness, ST should be considered as a differential diagnosis, especially in post-monsoon season. Along with serology, serum can also be used as sample for PCR in an intracellular bacterium like O. tsutsugamushi.     

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.     

Evaluation of an Immunoglobulin M Capture Enzyme-Linked Immunosorbent Assay for Diagnosis of Orientia tsutsugamushi Infection

To differentiate scrub typhus from other acute febrile diseases, a rapid and reliable serological diagnosis is important. We developed an immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) for diagnosis of recent Orientia tsutsugamushi infections in humans. The 56-kDa major outer membrane protein of O. tsutsugamushi is well known as the most immunodominant antigen in scrub typhus. The test is based on the use of the biotinylated recombinant 56-kDa protein of O. tsutsugamushi Boryong, Bor56, which was expressed as a fusion protein with a maltose-binding protein in Escherichia coli. In the test, the serum IgM antibodies were captured by anti-human IgM antibodies coated onto a microtiter plate. The captured IgM antibodies were revealed through sequential addition of biotinylated Bor56 antigen and peroxidase-conjugated streptavidin to the plate. The IgM capture ELISA was compared with the immunofluorescence antibody assay (IFA) by testing 176 serum samples from patients with diagnosed cases of rickettsial disease and patients with other acute febrile diseases. Of the 81 IgG IFA-positive samples, 78 tested positive (sensitivity, 96.3%) and all 31 IgM IFA-positive samples tested positive (sensitivity, 100%) by the IgM capture ELISA. The specificity of the IgM capture ELISA was 99%, and 1 of the 95 IFA-negative samples was positive in the assay. These results strongly suggest that IgM capture ELISA using the recombinant Bor56 antigen is a reliable and detailed method for the detection of early O. tsutsugamushi infection.     

Comparison of Nested Polymerase Chain Reaction and Real-Time Polymerase Chain Reaction Targeting 47kDa Gene for the Diagnosis of Scrub Typhus

Introduction: Scrub typhus is a zoonotic infection caused by Orientia tsutsugamushi which is transmitted by Leptotrombidium mites. The disease manifests as a mild-to-severe illness with non-specific clinical symptoms. Rapid diagnosis and prompt treatment are essential for patient management. Both serological and molecular methods are used for the diagnosis of scrub typhus. The present study assessed the usefulness of detection of the gene encoding the 47kDa outer-membrane protein (OMP) for the laboratory diagnosis of scrub typhus. Materials and Methods: Nested polymerase chain reaction (nPCR) and real-time PCR targeting 47 kDa OMP antigen gene of O. tsutsugamushi were performed on ethylenediaminetetraacetic acid blood samples. Results: Six of the 103 (5.8%) patients showed the presence of 47kDa gene by nPCR. Seventy of 103 (67.9%) cases showed the presence of 47kDa gene by qPCR. Among the 70 positive cases, the majority of them were females (40/70, 57.1%). The highest number of positive cases was observed during October–February. Conclusion: Real-time PCR targeting O. tsutsugamushi-specific 47-kDa gene is more sensitive than nPCR and may be the assay of choice for the detection of the organism in patients with suspected scrub typhus.     

Evaluation of an in-house PCR for diagnosing scrub typhus along with a preliminary study of genotypic characterization of Orientia tsutshugamushi circulating in Chhattisgarh: A prospective clinico-microbiological study

PurposeScrub typhus, caused by Orientia tsutsugamushi (O. tsutsugamushi) present nonspecific clinical features during manifestation of acute undifferentiated febrile illness (AUFI) to render its early diagnosis difficult. Accordingly, this study was undertaken to assess an in-house groEL PCR versus IgM ELISA for the diagnosis of scrub typhus and to genotypically characterise the randomly selected scrub typhus positive cases.MethodsBlood samples, collected from two hundred twenty one (221) AUFI cases were subjected to groEL PCR and IgM ELISA for diagnosis of scrub typhus. Eleven randomly selected PCR positive cases were processed for DNA sequencing to determine the genetic diversity of O. tsutsugamushi in Chhattisgarh.ResultsScrub typhus prevalence of 35.2% were detected among AUFI cases using both in-house groEL PCR and IgM ELISA. PCR alone showed sensitivity, specificity, positive and negative predictive values of 66.6% (CI: 55.08–76.94), 100% (CI: 90 to 100),100% (CI: 93.15 to 100) and 57.37% (CI: 44.05 to 69.96) while for IgM ELISA, these parameters were 62.8% (CI: 51.13–73.50), 100% (CI: 90 to 100), 100% (CI: 92.75 to 100) and 54.68% (CI: 41.75 to 67.18) respectively. PCR and ELISA could detect scrub typhus in 37.2% and 33.3% cases, when tested alone. groEL PCR detected the O. tsutsugamushi throughout the course of infection. Phylogenetic analysis depicted 5 of 11 positive cases belonged to Kuroki, Japan strain of O. tsutsugamushi, followed by Gilliam and Karp strain in 4 and 2 cases respectively.ConclusionScrub typhus should be considered in differential diagnosis of AUFI. groEL PCR may aid on to IgM ELISA test for optimum laboratory diagnosis of scrub typhus by its implementation especially in seronegative cases. Predominance of Kuroki-like strain followed by Gillian and Karp strains of O. tsutsugamushi in Chhattisgarh confirm variable geographical distribution of O. tsutsugamushi and provide the baseline epidemiological data which will eventually be used to help the researchers for developing better diagnostic tests and vaccine covering the predominant genotypes.     

Phylogenetic Analysis of the 56-kDa Type-Specific Protein Genes of Orientia tsutsugamushi in Central Korea

There are several antigenic variants of Orientia tsutsugamushi. The 56-kDa type-specific antigen (TSA) is responsible for the antigenic variation. Nucleotide sequences of the 56-kDa TSA obtained from 44 eschar samples of Korean scrub typhus patients and from 40 representative strains retrieved from the GenBank database were analyzed phylogenetically. Clinical patient data were assessed based on the genotyping results. Of the 44 nucleotide sequences, 32 (72.7%) clustered with the Boryong genotype, which is the major genotype in Korea. Eleven nucleotide sequences (25%) clustered with the Kawasaki genotype, not identified in Korea until 2010. One nucleotide sequence was consistent with the Karp genotype. The clinical course of the patients infected with each genotype showed no differences. Diagnostic performance of the immunofluorescence assay (IFA) using the 56-kDa TSA from Gilliam, Karp and Boryong as test antigens were not different for the Boryong and Kawasaki genotypes. Although Boryong is still the predominant genotype, the results suggest that Kawasaki genotype is quite prevalent in Chungbuk province of Korea.     

Identification of Outer Membrane Vesicles Derived from Orientia tsutsugamushi

Orientia tsutsugamushi, a causative pathogen of Scrub typhus, is a gram-negative intracellular bacterium. Outer membrane vesicles (OMVs) are produced from the membrane of bacteria and play many roles related to the survival of the pathogen. However, there have been no reports confirming whether O. tsutsugamushi indeed produce OMVs. O. tsutsugamushi boryong was cultured in ECV-304 cells for the purification of OMVs. Western blot analysis and immunoenrichment using anti-O. tsutsugamushi monoclonal antibody and electron microscopy were employed for identification and characterization of OMVs. We confirm the presence of OMVs derived from O. tsutsugamushi, and also found that those OMVs contain a major surface antigen of 56-kDa protein and variant immunogenic antigens.     

Association of High Orientia tsutsugamushi DNA Loads with Disease of Greater Severity in Adults with Scrub Typhus

Orientia tsutsugamushi, the cause of scrub typhus, is a major pathogen in the Asia-Pacific region. The severity of infection ranges from mild features to multiorgan failure and death. The aim of this prospective study was to define the O. tsutsugamushi loads in the blood samples of patients with scrub typhus on the day of hospital admission and to determine whether this was associated with disease severity. Quantitation was performed using a real-time PCR assay targeting the 16S rRNA gene of O. tsutsugamushi. A total of 155 patients with a confirmed diagnosis of scrub typhus had a median (interquartile range [IQR], range) O. tsutsugamushi DNA load in blood of 13 (0 to 334, 0 to 310,253) copies/ml. This included 74 patients who had undetectable bacterial loads. An analysis of bacterial load versus clinical features for all 155 patents demonstrated that duration of illness (P < 0.001), presence of eschar (P = 0.004), and concentrations of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase (P < 0.001 for all three) were positively correlated with bacterial load. Patients who died had a significantly higher bacterial load than those who survived (mean [standard deviation] values: 17,154 [12.7] versus 281 [5.2] copies/ml; P < 0.001). This study has demonstrated a relationship between bacterial load and disease severity in adults with scrub typhus.     

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.     

Involvement of lipid rafts in the budding-like exit of Orientia tsutsugamushi

Orientia tsutsugamushi is an intracellular parasite that causes scrub typhus. After entering the cytoplasm by induced phagocytosis, O. tsutsugamushi escapes from the primary phagosome into the host cytosol, where it replicates slowly. Subsequently, it is released from the host cells by a process resembling viral budding with a remaining bacterial aggregate near the nucleus. Lipid rafts have been implicated in the life cycle of a wide variety of pathogenic microorganisms. We have observed that proteins of O. tsutsugamushi were co-fractionated with the lipid rafts over a sucrose density gradient, suggesting the possible involvement of lipid rafts during the intracellular life cycle of O. tsutsugamushi. The entry of O. tsutsugamushi into the host cells was shown to be independent on lipid rafts as judged by the inability of lipid raft-disrupting agents to inhibit bacterial entry and no co-localization of bacterial proteins with caveolin. To our interest, a 47-kDa protein (HtrA) was observed to be co-localized with caveolin at the cell membrane at 72 h after infection, when bacterial particles move to the cell membrane and initiate the exit into the extracellular environment. Our results suggest that O. tsutsugamushi involves lipid rafts of the host cells in the budding-like process to exit from host cells.     

First case series of emerging Rickettsial neonatal sepsis identified by polymerase chain reaction-based deoxyribonucleic acid sequencing

Purpose: To detect and identify the aetiological agent in the peripheral blood from the cases of neonatal sepsis. Materials and Methods: Four neonates from geographically different regions of South India presented with signs of neonatal sepsis and all the routine clinical and laboratory investigations were performed. Blood culture by Bac T Alert 3D was negative. To establish the aetiology, polymerase chain reaction (PCR) for eubacterial genome and subsequent amplification with Gram positive and Gram negative primers were performed followed by deoxyribonucleic acid (DNA) sequencing. Results: PCR for the detection of eubacterial genome was positive in all the four neonates and further amplification with designed Gram positive and Gram negative primers revealed the presence of Gram negative bacteria. The amplicons were identified as Orientia tsutsugamushi in three neonates and Coxiella burnetti in the other neonate. Multalin analysis was done to further characterise the strain variation among the three strains. Conclusion: PCR-based DNA sequencing is a rapid and reliable diagnostic tool to identify the aetiological agents of neonatal sepsis. This is the first case series of emerging Rickettsial neonatal sepsis in India.     

In Vitro Bacteriostatic Effects of Rifampin on Orientia tsutsugamushi

We performed an in vitro cell culture experiment to ascertain whether rifampin exhibits bactericidal effects against Orientia tsutsugamushi, the causative agent of scrub typhus. ECV304 cells were infected with the Boryong or AFSC-4 strain of O. tsutsugamushi and then, the cultures were maintained in media with increasing concentrations of rifampin, azithromycin, doxycycline, or chloramphenicol for 4 days. On day 5, the media were replaced with fresh antibiotic-free medium and the cultures were maintained until day 28. On days 5, 13, and 28, immunofluorescence (IF) staining of O. tsutsugamushi was performed. IF staining on days 13 and 28 revealed increasing numbers of IF-positive foci in all cultures, even in cultures initially exposed to the highest concentration of rifampin (80 µg/mL), azithromycin (80 µg/mL), doxycycline (20 µg/mL), or chloramphenicol (100 µg/mL). The present study reveals that rifampin has no bactericidal effect against O. tsutsugamushi as observed for azithromycin, doxycycline, and chloramphenicol. A subpopulation of the bacteria that are not killed by high concentrations of the antibiotics may explain the persistence of O. tsutsugamushi in humans even after complete recovery from scrub typhus with antibiotic therapy.

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Development of a Recombinant Protein-Based Enzyme-Linked Immunosorbent Assay and Its Applications in Field Surveillance of Rodent Mice for Presence of Immunoglobulin G against Orientia tsutsugamushi

A recombinant protein containing the immunodominant conserved epitope region of the 56-kDa outer membrane protein of the Karp strain of Orientia tsutsugamushi was purified to near homogeneity using recombinant DNA techniques. The purified protein was used to immunize rabbits and produced an antibody that could recognize different strains of O. tsutsugamushi, as demonstrated both by Western blotting and immunofluorescence assay. An enzyme-linked immunosorbent assay (ELISA) based on this recombinant protein was developed to detect antibody (immunoglobulin G [IgG]) against O. tsutsugamushi in mice captured in different districts of Taiwan during 2000 to 2001. A significant difference was found in the antibody seroprevalence rates of Suncus murinus mice captured in different districts of Taiwan (χ²_{4, 0.95} = 26.64; P < 0.05). Furthermore, a significant difference of IgG seropositivity rates was observed among different kinds of mice (χ²_{5, 0.95} = 93.85; P < 0.05). Antibody seropositivity rates were higher in Bandicota indica (100%), Rattus flavipectus (96.17%), and Rattus losea (95.83%) than in Rattus norvegicus (86.05%) and Rattus mindanensis (83.67%) (χ²_{diff, 5, 0.95} = 12.59, P < 0.05). The lowest antibody seropositivity rate (54.4%) was observed in Suncus murinus. Antibody seropositivity rates of mice from different districts differed significantly because of the significant difference in antibody seroprevalence rates for S. murinus. The results of this study indicated that the recombinant protein ELISA developed in this study could be used to conduct large-scale surveillance of rodent mice for the presence of antibody against O. tsutsugamushi. The high seroprevalence rates in rodent mice (except S. murinus) suggest that people residing in these districts are at increased risk of developing O. tsutsugamushi infection.     

In Vitro Activity of Tigecycline Against Orientia tsutsugamushi

Scrub typhus is a zoonosis caused by Orientia tsutsugamushi (O. tsutsugamushi) occurring mainly in autumn in Korea. The need of new antibiotics has arisen with a report on strains resistant to antibiotics and chronic infection. This study aims to identify susceptibility of tigecycline in-vitro as a new therapeutic option for O. tsutsugamushi. Antibacterial activity of tigecycline against the O. tsutsugamushi was compared with doxycycline using flow cytometry assay. The inhibitory concentration 50 (IC₅₀) was 3.59×10^-3 µg/mL in doxycycline-treated group. Whereas in 0.71×10^-3 µg/mL tigecycline-treated group. These findings indicate that tigecycline may be a therapeutic option for the treatment of scrub typhus.     

Phylogenetic analysis of canine parvovirus partial VP2 gene in India

A total of 85 samples (58.0 %) were found to be positive for Canine parvovirus (CPV) by PCR assay (Hfor/Hrev primers) out of 158 suspected faecal samples of dogs collected from various states/union territories of India. Nine CPV isolates could be obtained in A-72 cell line. The sequencing of the partial VP2 gene of CPV identified the predominant CPV strain as CPV-2a (Ser297Ala) with one CPV-2b (Ser297Ala) and another CPV-2a variant strain (Ser297Gly). Several non-synonymous and synonymous mutations were also recorded in this study. The phylogenetic tree revealed that most of the CPV sequences from Tamil Nadu (Southern India) and Maharashtra (Western India) obtained during 2011 and few sequences from Northern India obtained during 2012 were grouped together along with CPV-2a (Ser297Ala) strains from China and India and followed the same evolution; although there was definitive indication of separate lineages too by few other sequences.     

Shiga toxin-producing Escherichia coli strains from cattle as a source of the Stx2a bacteriophages present in enteroaggregative Escherichia coli O104:H4 strains

Enteroaggregative, Shiga toxin-producing E. coli (EAEC-STEC) O104:H4 strains are emerging pathogens causing life threatening diseases in humans. EAEC-STEC O104:H4 strains isolated between 2001 and 2011 were found to harbor a distinct type of Shiga toxin 2a- (Stx2a) encoding prophage. This phage type shows only <65% genetic similarity to so far described viable Stx phages due to differences in the modules for DNA replication, metabolism, regulation and host specificity. Stx production in EAEC is rarely observed and the source of the Stx2a phage in the EAEC-STEC O104:H4 strains is not known. We identified two DNA segments derived from orf15 and the cI gene of the O104:H4 Stx2a phage P13374 that are characteristic for Stx2a prophages present in EAEC-STEC O104:H4 strains. By PCR, these sequences were detected in 14 (5.8%) of 241 Stx2-positive STEC from animals and food. Infectious Stx2a phages could be isolated from four bovine STEC strains. These were found highly similar to P13374 for orf15, cI and stx2a sequences, the chromosomal integration site (wrbA), for phage DNA restriction profiles, virion morphology and superinfection immunity. Stx2a phages of the four bovine STEC strains formed lysogens on the E. coli K-12 strain C600. Phage P13374 from an EAEC-STEC O104:H4 outbreak strain and one of the bovine STEC phages (P13803) lysogenized the Stx-negative EAEC O104:H4 strain CB14647 by integrating in the wrbA gene of CB14647 and converted it into a Stx2a producer. Our findings provide experimental evidence that EAEC-STEC O104:H4 strains have evolved by uptake of Stx2a phages from the bovine reservoir.     

Scrub Typhus in Previously Unrecognized Areas of Endemicity in China

Scrub typhus, caused by Orientia tsutsugamushi, has emerged recently in areas of northern China where the disease had not been known to exist. We analyzed epidemiological, clinical, and laboratory data for 104 patients who were admitted to a hospital in Fuyang City between 26 September and 1 November 2008. We showed that the major clinical manifestations of the patients were fever (100%), headache (82%), myalgias (77%), eschar (67%), rash (52%), and unusual facial flushing (62%). Among the 104 patients, the sera of 98% contained IgM antibodies to O. tsutsugamushi detected by indirect immunofluorescence assays (IFA), and DNA of the O. tsutsugamushi 56-kDa gene was amplified by PCR from the blood of 36 patients. We conclude that 104 patients were infected with scrub typhus in Fuyang City, Anhui Province. Our study indicates that physicians need to consider the diagnosis of scrub typhus for febrile patients living in northern China, where scrub typhus had not been considered to exist in the past.Scrub typhus, also known as tsutsugamushi disease, is an acute, febrile infectious illness. It is a zoonosis that is widespread in southern Asia, in a triangle from northern Japan and far-eastern Russia in the north to northern Australia in the south and to Pakistan and Afghanistan in the west, as well as in the islands of the western Pacific and Indian Oceans. More than half (55%) of the world''s population lives in areas where scrub typhus is endemic. The causative agent, Orientia tsutsugamushi (formerly Rickettsia tsutsugamushi), is an obligately intracellular bacterium and is transmitted to humans by the bite of a larval trombiculid mite, popularly known as a chigger. Scrub typhus has been known in southern China for thousands of years (5). However, the disease has emerged in northern China only in the last 2 decades (3). Here we report an outbreak of scrub typhus cases in the fall of 2008 in northwestern Anhui Province in central China, where the disease had not been known to occur previously.     

Do Escherichia coli strains causing acute cystitis have a distinct virulence repertoire?

Bacterial virulence factors influence the site and severity of urinary tract infections. While pyelonephritis-associated molecular traits have been defined, virulence factors specific for acute cystitis strains have not been identified. This study examined the virulence factor repertoire of 247 Escherichia coli strains, prospectively isolated from women with community-acquired acute cystitis. Fim sequences were present in 96% of the isolates, which also expressed Type 1 fimbriae. Curli were detected in 75%, 13% of which formed cellulose. Pap sequences were present in 47%, 27% were papG+, 23% were prsG+ and 42% expressed P fimbriae. TcpC was expressed by 33% of the strains, 32% in a subgroup of patients who only had symptoms of cystitis and 42% in patients with signs of upper urinary tract involvement; most frequently by the papG+/prsG+ subgroup. Strains with the full fim, pap and TcpC and curli virulence profile were more common in cystitis patients with than in patients without upper tract involvement (p < 0.05). The varied virulence profile of E. coli strains causing acute cystitis suggests that diverse bacterial strains, expressing Type 1 fimbriae trigger a convergent host response, involving pathways that give rise to the characteristic symptoms of acute cystitis.     

Coagulation and inflammation in scrub typhus and murine typhus—a prospective comparative study from Laos

Scrub typhus (caused by Orientia tsutsugamushi) and murine typhus (caused by Rickettsia typhi) cause up to 28% of febrile episodes in Thailand and Laos. The current understanding of coagulation and inflammation in the pathogenesis of these clinically very similar vasculotropic diseases is limited. This study compared human in vivo changes in 15 coagulation, inflammation and endothelial activation markers in prospectively collected admission and follow-up samples of 121 patients (55 scrub typhus, 55 murine typhus, and 11 typhus-like illness) and 51 healthy controls from Laos. As compared with controls, all but one of the markers assessed were significantly affected in typhus patients; however, the activation patterns differed significantly between scrub and murine typhus patients. The levels of markers of coagulation activation and all inflammatory cytokines, except for interleukin-12, were significantly higher in patients with scrub typhus than in those with murine typhus. In patients with murine typhus, however, the levels of endothelium-derived markers were significantly higher. Anticoagulant factors were inhibited in both typhus patient groups. This is the first study demonstrating that, in scrub typhus, in vivo coagulation activation is prominent and is related to a strong proinflammatory response, whereas in murine typhus, changes in coagulant and fibrinolytic pathways are suggestive of endothelial cell perturbation. These data suggest that, although late-stage endothelial infection is common in both diseases, the in vivo pathogenic mechanisms of R. typhi and O. tsutsugamushi could differ in the early phase of infection and may contribute to disease differentiation.