Tsutsugamushi Strains Research Articles

Antigenic Relationships among Human Pathogenic Orientia tsutsugamushi Isolates from Thailand

BackgroundScrub 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 FindingsThis 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/SignificanceAntigenic 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.

Seroepidemiological Survey of Zoonotic Diseases in Small Mammals with PCR Detection of Orientia tsutsugamushi in Chiggers, Gwangju, Korea.

Serosurveillance for zoonotic diseases in small mammals and detection of chiggers, the vector of Orientia tsutsugamushi, were conducted from September 2014 to August 2015 in Gwangju Metropolitan Area. Apodemus agrarius was the most commonly collected small mammals (158; 91.8%), followed by Myodes regulus (8; 4.6%), and Crocidura lasiura (6; 3.5%). The highest seroprevalence of small mammals for O. tsutsugamushi (41; 26.3%) was followed by hantaviruses (24; 15.4%), Rickettsia spp. (22; 14.1%), and Leptospira (2; 1.3%). A total of 3,194 chiggers were collected from small mammals, and 1,236 of 3,194 chiggers were identified with 7 species of 3 genera: Leptotrombidium scutellare was the most commonly collected species (585; 47.3%), followed by L. orientale (422; 34.1%), Euchoengastia koreaensis (99; 8.0%), L. palpale (58; 4.7%), L. pallidum (36; 2.9%), Neotrombicula gardellai (28; 2.3%), and L. zetum (8; 0.6%). L. scutellare was the predominant species. Three of 1,236 chigger mites were positive for O. tsutsugamushi by PCR. As a result of phylogenetic analysis, the O. tsutsugamushi strain of chigger mites had sequence homology of 90.1-98.2% with Boryong. This study provides baseline data on the distribution of zoonotic diseases and potential vectors for the development of prevention strategies of vector borne diseases in Gwangju metropolitan area.

Determination of Scrub typhus Suggests a New Epidemic Focus in the Anhui Province of China.

In 2007, 19 cases of a scrub typhus epidemic occurred within a week at a sports school in Mingguang County, Anhui Province, where no previous incidence of this mite borne disease had been reported. Sero-surveillance in 2009 indicated that 10 of the 100 school students possessed anti-Orientia tsutsugamushi antibodies. From 2009 to 2013, 60 small animals and 2250 mites were collected in the vicinity of the school. 5 of the Apodemus agrarius samples and 1 group of Leptotrombidium linhuaikongense tested positive via PCR for O. tsutsugamushi. Two strains of O. tsutsugamushi were identified by injecting Kun Ming (KM) mice peritoneally with the organs of either Apodemus agrarius or Leptotrombidium linhuaikongense. Apart from sharing 98% homology with the O. tsutsugamushi Yongworl strain, genes encoding the membrane protein from the two O. tsutsugamushi isolates shared >99% sequence homology with each other, reflecting the consistency of the pathogen in both the vector and the host. In addition, we also characterized a chronic scrub typhus infection in a local patient. The membrane protein gene fragment from the patient’s blood shared 99% homology with O. tsutsugamushi Gilliam strain, suggesting that more than one O. tsutsugamushi strain is present at this location.

Mixed Scrub Typhus Genotype, Shandong, China, 2011

Mixed Scrub Typhus Genotype, Shandong, China, 2011

Decontamination of mycoplasma-contaminated Orientia tsutsugamushi strains by repeating passages through cell cultures with antibiotics

BackgroundMycoplasmas-contamination of Orientia tsutsugamushi, one of the obligated intracellular bacteria, is a very serious problem in in vitro studies using cell cultures because mycoplasmas have significant influence on the results of scientific studies. Only a recommended decontamination method is to passage the contaminated O. tsutsugamushi strains through mice to eliminate only mycoplasmas under influence of their immunity. However, this method sometimes does not work especially for low virulent strains of O. tsutsugamushi which are difficult to propagate in mice. In this study, we tried to eliminate mycoplasmas contaminants from both high virulent and low virulent strains of the contaminated O. tsutsugamushi by repeating passage through cell cultures with antibiotics in vitro.ResultsWe cultured a contaminated, high virulent strain of O. tsutsugamushi using a mouse lung fibroblasts cell line, L-929 cell in the culture medium containing lincomycin at various concentrations and repeated passages about every seven days. At the passage 5 only with 10 μg/ml of lincomycin, we did not detect mycoplasmas by two PCR based methods whereas O. tsutsugamushi continued good growth. During following four passages without lincomycin, mycoplasmas did not recover. These results suggested that mycoplasmas were completely eliminated from the high virulent strain of O. tsutsugamushi. Furthermore, by the same procedures with 10 μg/ml of lincomycin, we also eliminated mycoplasmas from a contaminated, low virulent strain of O. tsutsugamushi. Our additional assay showed that 50 μg/ml of lyncomycin did not inhibit the growth of O. tsutsugamushi, although MICs of many mycoplasmas contaminants were less than 6 μg/ml as shown previously.ConclusionOur results showed an alternative method to eliminate mycoplasmas from the contaminated O. tsutsugamushi strains in place of in vivo passage through mice. Especially this notable method works for the decontamination not only from the high virulent strain also from the low virulent strain of O. tsutsugamushi. For further elimination, lincomycin at the limit concentration, which does not inhibit the growth of O. tsutsugamushi, can possibly eliminate most mycoplasmas from contaminated O. tsutsugamushi strains.

High In Vitro Infectivity of a Doxycycline-Insensitive Strain of Orientia tsutsugamushi.

We compared the infectivity and growth rates of 12 strains of Orientia tsutsugamushi in a cell culture system. ECV304 cells were infected with O. tsutsugamushi strains for 4 hr, and the culture was maintained for 72 hr. Immunofluorescence (IF) staining was performed at 4, 24, 48, and 72 hr after inoculation (hpi), and IF-positive foci were enumerated. The AFSC-4 strain, which is known to be insensitive to doxycycline, showed higher numbers of IF-positive foci than the other 11 strains at 4, 24, 48, and 72 hpi, which may explain its apparent insensitivity to antibiotics.

Prokaryotic expression and immunogenicity of 56‐kDa protein of Orientia tsutsugamushi strain Karp

To express the 56-kDa protein of O. tsutsugamushi strain Karp, this protein gene was cloned into pET30a(+) before transforming into host bacteria, E. coli Rossetta. Specificity of the recombinant protein was assessed by ELISA using rabbit sera against common members of the order Rickettsiae and 10 other pathogenic bacteria. After IPTG induction, SDS-PAGE analysis of isolated protein demonstrated a band at approximately 46-kDa. Western blot and mass spectrometry analysis proved that the recombinant protein was expressed successfully. Specificity analysis demonstrated that all sera were negative, except sera against O. tsutsugamushi strains TA763, TH1817 and Kato, B. quintana, A. phagocytophilum, E. chaffeensis and B. bacilliformis. The purified protein was used to immunize BALB/c mice and polyclonal antisera were harvested. By examination of IFA and ELISA, the highest titer of the polyclonal antibodies reaches 1:1600. The recombinant 56-kDa protein in the study is valuable for developing a simple and rapid diagnostic test and vaccine for O. tsutsugamushi.

Molecular epidemiology of Orientia tsutsugamushi in Cambodia and Central Vietnam reveals a broad region-wide genetic diversity

Scrub typhus is an acute infectious disease caused by an obligate intracellular bacterium Orientia tsutsugamushi following the bite of infected trombiculid mites of the genus Leptotrombidium. This zoonotic disease is a major cause of febrile illness in the Asia-Pacific region, with a large spectrum of clinical manifestations from unapparent or mild disease to fatal disease. O. tsutsugamushi is characterized by a very high genomic plasticity and a large number of antigenic variants amongst strains. The 56-kDa type specific antigen (TSA) gene, encoding the major antigenic protein, was used as reference to investigate the genetic relationships between the strains and to genotype O. tsutsugamushi isolates. The open reading frame of the 56-kDa TSA gene of 41 sequences (28 Cambodian and 13 Vietnamese strains) from patient samples were sequenced and used for genotyping. The 28 Cambodian isolates clustered into 5 major groups, including Karp (43.5%), JG-v (25%), Kato/TA716 (21.5%), TA763 (3.5%) and Gilliam (3.5%). Karp (77%), TA763 (15.5%) and JG-v (7.5%) strains were identified amongst the 13 Vietnamese isolates. This is the first countrywide genotyping description in Cambodia and in Central Vietnam. These results demonstrate the considerable diversity of genotypes in co-circulation in both countries. The genotyping result might raise awareness amongst Cambodian and Vietnamese clinicians of the high genetic diversity of circulating O. tsutsugamushi strains and provides unique and beneficial data for serological and molecular diagnosis of scrub typhus infections as well as raw materials for future studies and vaccine development.

First Report of anOrientia tsutsugamushiType TA716–Related Scrub Typhus Infection in Thailand

Orientia tsutsugamushi causes scrub typhus and is a rural zoonosis endemic in the Asia Pacific region. This is the first report of O. tsutsugamushi TA716-like strain in a human in Thailand. The patient was in the 1st trimester of pregnancy when she developed scrub typhus. The O. tsutsugamushi strain TA716 was detected from her admission blood sample, and the pregnancy ended in spontaneous abortion. The effects of scrub typhus in pregnant women and the pregnancy outcome are sparsely documented in the published medical literature. Improved clinical recognition and laboratory diagnosis will be essential to better define the morbidity caused by this zoonosis especially in pregnancy.

Intrinsic fluoroquinolone resistance in Orientia tsutsugamushi

Scrub typhus is a public health concern for a population of over a billion humans, with an estimated incidence of one million cases/year in endemic areas. Although doxycycline remains the standard therapy, fluoroquinolones have been used successfully in a few patients. However, there is also clinical evidence that fluoroquinolones are ineffective in the treatment of scrub typhus. To clarify this matter, we determined the in vitro susceptibility of Orientia tsutsugamushi strain Kato to ciprofloxacin and ofloxacin and sequenced the quinolone resistance-determining region (QRDR) of the gyrA gene, the target of fluoroquinolones, of 18 fresh isolates from the Lao PDR. Orientia tsutsugamushi strain Kato was resistant to ciprofloxacin and ofloxacin in vitro (minimum inhibitory concentration = 8 μg/mL). All sequences obtained, including those from the two available genomes of O. tsutsugamushi (strains Boryong and Ikeda), had a Ser83Leu mutation in their QRDR domain that is known to be associated with fluoroquinolone resistance. These findings re-emphasise the usefulness of in silico analysis for the prediction of antibiotic resistance and suggest that fluoroquinolones should not be used in the treatment of scrub typhus.

The Whole-genome Sequencing of the Obligate Intracellular Bacterium Orientia tsutsugamushi Revealed Massive Gene Amplification During Reductive Genome Evolution

Scrub typhus (‘Tsutsugamushi’ disease in Japanese) is a mite-borne infectious disease. The causative agent is Orientia tsutsugamushi, an obligate intracellular bacterium belonging to the family Rickettsiaceae of the subdivision alpha-Proteobacteria. In this study, we determined the complete genome sequence of O. tsutsugamushi strain Ikeda, which comprises a single chromosome of 2 008 987 bp and contains 1967 protein coding sequences (CDSs). The chromosome is much larger than those of other members of Rickettsiaceae, and 46.7% of the sequence was occupied by repetitive sequences derived from an integrative and conjugative element, 10 types of transposable elements, and seven types of short repeats of unknown origins. The massive amplification and degradation of these elements have generated a huge number of repeated genes (1196 CDSs, categorized into 85 families), many of which are pseudogenes (766 CDSs), and also induced intensive genome shuffling. By comparing the gene content with those of other family members of Rickettsiacea, we identified the core gene set of the family Rickettsiaceae and found that, while much more extensive gene loss has taken place among the housekeeping genes of Orientia than those of Rickettsia, O. tsutsugamushi has acquired a large number of foreign genes. The O. tsutsugamushi genome sequence is thus a prominent example of the high plasticity of bacterial genomes, and provides the genetic basis for a better understanding of the biology of O. tsutsugamushi and the pathogenesis of ‘Tsutsugamushi’ disease.

In vitro Study on the Dose and Duration of Doxycycline Treatment against Orientia tsutsugamushi

Background :Doxycycline, azithromycin, and chloramphenicol have been used for the treatment of scrub typhus; however, the duration of treatment for this disease is still controversial. Most clinical studies on the treatment of scrub typhus focus on the effectiveness of antibiotics; few studies have focused on the duration of the treatment. There has been no in vitro evaluation of the optimal duration of treatment of scrub typhus. We performed an experiment to evaluate the regrowth of Orientia tsutsugamushi after exposure to doxycycline of various concentration and exposure time. Materials and Methods :O. tsutsugamushi strain Boryong was inoculated into the ECV304 cell line. The infected cells were stained with FS15, a monoclonal antibody reacting against a linear epitope on the 56-kDa major outer membrane protein of O. tsutsugamushi; thereafter, the antimicrobial susceptibilities were measured by flow cytometry and expressed as a growth index (total mass of Orientia). Incubation in media containing 0.1 (the minimal inhibitory concentration of doxycycline against O. tsutsugamushi strain Boryong), 0.2, and 2 μg/mL doxycycline for 1, 3, 5, and 7 day was followed by incubation in antibiotic-free media for 5 day. Two inocula sizes were employed; low inoculum (45.18% of cells were infected) and high inoculum (87.21% of cells). Results : The incubation with 0.1 μg/mL doxycycline resulted in O. tsutsugamushi regrowth irrespective of doxycycline treatment duration. When the concentration of doxycycline was increased to 0.2 or 2 μg/mL, regrowth was observed in short duration treatments (1-5 day at 0.2 μg/mL; 1 day at 2 μg/mL). Higher inoculum size of O. tsutsugamushi increased regrowth (1 day at the low inoculum; 1-5 day at the high inoculum). Conclusions : The regrowth of O. tsutsugamushi after doxycycline treatment depends on the doxycycline dose; the higher the dose, the lower the relapse rate. If this result is extrapolated to humans, higher dose of doxycycline might be preferred to lower the relapse rate. The inoculum effect in O. tsutsugamushi infection needs further investigations.

In Vitro Antibiotic Susceptibility of Orientia tsutsugamushi strain Boryong Measured by Flow Cytometry

Background : Scrub typhus an infectious disease caused by Orientia tsutsugamushi is endemic in Korea. With the introduction of tetracycline and chloramphenicol in clinical practice the mortality due to scrub typhus has markedly decreased. In 1995 scrub typhus poorly responsive to doxycycline was reported in Thailand; the need for safe antibiotics for the treatment of scrub typhus acquired during pregnancy or for children is emerging; also broader spectrum antibiotics having anti-Orientia activity may be preferred for empirical therapy of enteric fever syndrome and for complicated scrub typhus. The anti-Orientia activities of various antibiotics including recently licensed antibiotics were investigated by flow cytometry. Materials and Methods : O. tsutsugamushi strain Boryong was inoculated into the ECV304 cell line. The infected cells were stained with FS15 a monoclonal antibody reacting against a linear epitope on 56-kDa major outer membrane protein of O. tsutsugamushi. Then the antimicrobial susceptibilities were measured by flow cytometry and expressed as a growth index (total mass of Orientia. A concentration at which no further decrease in growth index occurred wa sd efined as the minimal inhibitory concentration (MIC. Microbial susceptibilities to the following antibiotics were measured: quinupristin-dalfopristin (Synercid levofloxacin ciprofloxacin moxifloxacin metronidazole linezolid clindamycin chloramphenicol doxycycline azithromycin and rifampin. Results : Considering the usual serum concentrations of rifampin (MIC=0.025-0.05 g/mL azithromycin (MIC=0.05-0.5 g/mL and doxycycline (MIC=0.05-0.1 g/mL these antibiotics exhibited very low MICs. Synercid (MIC=0.2-1.0 g/mL clindamycin (MIC=1.0 g/mL and chloramphenicol (MIC=1-2 g/mL exhibited moderately low MICs; moxifloxacin (MIC=8 g/mL ciprofloxacin (MIC=25.6 g/mL or more and levofloxacin (MIC=30 g/mL exhibited relatively high MICs; and cefotaxime (MIC＞50 g/mL metronidazole (MIC＞30 g/mL and linezolid (＞30 g/mL exhibited high MICs. Conclusions : Among the new antibiotics none was superior to doxycycline azithromyci no r rifampin with respect to anti-Orientia activity. Synercid clindamycin and moxifloxacin may show moderate therapeutic efficacies in human.

Spring Scrub Typhus, People's Republic of China

Spring Scrub Typhus, People's Republic of China

Cloning and Sequence Analysis of the 22‐kDa Antigen Genes of Orientia tsutsugamushi Strains Kato, TA763, AFSC 7, 18–032460, TH1814, and MAK 119

The 22-kDa protein antigen is one of several antigens recognized by sera from scrub typhus patients infected with Orientia tsutsugamushi. The 22-kDa protein genes from six O. tsutsugamushi strains (Kato, TA763, AFSC 7, 18-032460, TH1814, MAK119) were cloned and their sequences were determined and compared to each other and to the Karp strain sequence listed in GenBank. The sequence alignment revealed that the promoter regions of these seven strains were highly conserved. However, the ORFs exhibited some variation. The phylogenetic analysis of the DNA sequences indicated that among the seven strains assessed, Kato and TA763 were the most closely related, while Karp and TH1814 were the most distantly related. The information gained from this analysis will facilitate our selection of O. tsutsugamushi strains from which antigens should be derived to be included in a multivalent vaccine candidate for scrub typhus.

Do pyrogenic cytokines mediate low-grade fever that developed in stressful situation?

Scrub typhus, caused by Orientia tsutsugamushi, has recently emerged in northern China where the disease had not been known to exist. Although doxycycline and azithromycin are the recommended agents for the treatment of scrub typhus, clinical responses depend both on the susceptibilities of various O. tsutsugamushi strains and the severity of the disease. A retrospective analysis was conducted on patients diagnosed with mild scrub typhus from August 2013 to January 2016 in the Affiliated Hospital of Nantong University, northern China. A total of 40 patients who received minocycline treatment and 34 patients who received azithromycin treatment were included in the analysis. All patients except one defervesced within 120 h after initiating antimicrobial therapy. Kaplan–Meier curves in association with log-rank test showed that the median time to defervescence was significantly shorter for the minocycline-treated group than the azithromycin-treated group (P = 0.003). There were no serious adverse events during treatment. No relapse occurred in either group during the 1-month follow-up period. In conclusion, both minocycline and azithromycin are effective and safe for the treatment of mild scrub typhus, but minocycline is more active than azithromycin against O. tsutsugamushi infection acquired in northern China.

Epidemiological study of autumn-winter type scrub typhus in a new endemic focus of Fei County, Shandong Province, China

To characterize the epidemiology of autumn-winter type scrub typhus in a new endemic focus of Fei County, Shandong Province, China, field and laboratory studies were conducted to determine the species composition and seasonal fluctuations of chigger mites and their animal hosts. Orientia tsutsugamushi was isolated, and serotypes of antibodies to O. tsutsugamushi were detected and identified. A total of 1044 rodents and insectivores were field collected: 751 Apodemus agrarius (71.94%), 266 Tscherskia triton (25.48%), 1 Cricetulus barabensis (0.09%), 11 Rattus norvegicus (1.05%), 3 Mus musculus (0.29%), and 12 Crocidura suaveolens (1.15%). Apodemus agrarius, T. triton and R. norvegicus were shown to be naturally infected with O. tsutsugamushi. The serum antibody positive rate of rodents was 14.78%. A total of 11762 chigger mites were collected: 2547 Leptotrombidium scutellare (21.65%), 1596 L. palpale (13.57%), 466 L. taishanicum (3.96%), 2833 L. linhuaikongense (24.09%), and 4320 Walchia pacifica (36.73%). Leptotrombidium scutellare was most numerous from September to November and was the predominant species during the local scrub typhus epidemic season. Natural infection with O. tsutsugamushi was detected in L. scutellare, and transovarial transmission of the pathogen by this chigger was demonstrated. 38 strains of O. tsutsugamushi were isolated from 33 patients (14 strains), 3 species of rodents (9 strains), and 4 species of mites (15 strains). All O. tsutsugamushi strains isolated in this endemic focus were of low virulence. Serotype identification showed that 33 strains were of the Gilliam type, and 3 strains were of the Karp type. Another 2 strains remain to be identified. In the Fei County focus of scrub typhus, the main reservoir host is A. agrarius, the predominant mite vector is L. scutellare, the principal serotype of O. tsutsugamushi is Gilliam, and all strains of O. tsutsugamushi areof low virulence. Most cases of scrub typhus occurred from September to November; therefore, scrub typhus in this focus is autumn-winter type. This focus is located in the premontane flatlands of Fei County.

Isolation of Orientia tsutsugamushi from patients in Shikoku and finding of a strain which grows preferentially at low temperatures.

Three strains of Orientia tsutsugamushi were isolated from patients in Anan City, Tokushima Prefecture. The strains were identified as Karp type by analyses of reactivities with type-specific monoclonal antibodies. One strain, Okazaki, was isolated in L cells cultivated at 31 C, but not in cells at 36 C or in mice. This strain showed better growth at 31 C than 36 C. This is the first report of an O. tsutsugamushi strain which grows preferentially at low temperatures.

Transmission of Rickettsia tsutsugamushi strains among humans, wild rodents, and trombiculid mites in an area of Japan in which tsutsugamushi disease is newly endemic.

Thirty-two newly isolated strains of Rickettsia tsutsugamushi, 14 from patients with tsutsugamushi disease, 12 from wild rodents, and 6 from trombiculid mites parasitizing rodents in Gifu Prefecture, Japan, were examined for reactivities to 12 monoclonal antibodies by an indirect fluorescent-antibody test to classify their antigenicities. All of the isolates could be classified into one of six groups (KN-1, KN-2, KN-3, GJ-1, R158, and R161) according to their reactivities to the monoclonal antibodies. The KN-1 and GJ-1 strains that are prevalent among patients from Gifu Prefecture had the same reactivities as the Kawasaki and Kuroki strains, respectively, which have been isolated and are prevalent in the Miyazaki and Kagoshima prefectures in southwest Japan. The isolates from patients were different in serotype from those from rodents and mites (Leptotrombidium pallidum). The KN-2 and KN-3 strains were most prevalent among patients and among rodents and mites, respectively. No close similarity between KN-2 and other strains tested was observed. KN-3 is only a minor contributor to diseases in patients in Gifu Prefecture; however, it was proven that the same strain was prevalent in Niigata Prefecture in northern Japan. Thus, Gifu Prefecture is an area where southern, northern, and local strains are found. We hypothesize that humans are prone to infection with KN-2, GJ-1 (very similar to Kuroki), and KN-1 (very similar to Kawasaki), probably by infestation with Leptotrombidium scutellare. While both L. scutellare and L. pallidum parasitize wild rodents and may carry any rickettsial strain, the most virulent strain, KN-3, is predominant among wild rodents. Antigenic analysis using monoclonal antibodies to R. tsutsugamushi should be useful for epidemiological studies of infection with this organism.

Diagnosis of typhus infection with Rickettsia tsutsugamushi by polymerase chain reaction

Two sets of oligonucleotide primers were used to amplify the genomic DNA of Rickettsia tsutsugamushi, the causative agent of scrub typhus (tsutsugamushi disease), by the polymerase chain reaction. Each set of primers amplified 538-bp and 109-bp products, representing part of a gene encoding a possible major 58-kDa immunogenic protein, from whole genomic DNA extracted from R. tsutsugamushi strains Karp, Kato, Gilliam, Kuroki and Kawasaki. No amplification was observed from R. sibirica, R. rickettsii, mouse and human genomic DNA. DNA amplification was observed from crude lysates of peripheral whole blood, tissue homogenates and paraffin-embedded skin biopsy sections obtained from patients with scrub typhus disease. Southern blot analysis demonstrated the specificity of the amplified DNA fragments following hybridisation with a DNA probe generated from R. tsutsugamushi strain Karp. By means of this procedure, a rapid and sensitive diagnosis of scrub typhus disease can be made during the acute stage of this infection.