Orientia tsutsugamushi in eschars from scrub typhus patients.

To verify the value of eschars for the diagnosis of scrub typhus and to characterize genotypes of Orientia tsutsugamushi in patients, we examined eschars and blood specimens of 7 patients from Shandong Province, People's Republic of China, for O. tsutsugamushi by polymerase chain reaction targeting the Sta56 gene. All 7 eschars and acute-phase blood samples were positive, while no specific DNA amplicons were obtained from the 7 convalescent-phase blood samples collected after antimicrobial drug therapy. The findings indicate that patients' eschars can be used for detection and genetic characterization of O. tsutsugamushi during the convalescent phase.

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Scrub typhus, a widely endemic disease in Asian Pacific regions, is caused by Orientia tsutsugamushi, a gram-negative obligate intracellular bacterium in the family Rickettsiaceae. When the rickettsia is transmitted to a human through the bite of an infected mite, it begins to multiply at the bite site, and a characteristic skin lesion known as an eschar is formed. The pathogen then spreads systemically by the hematogenous and lymphogenous routes. Various clinical manifestations develop, including fever, rash, and lymphadenopathy (1).

Before 1986, scrub typhus was only found in southern China (south of the Yangtze River) primarily in the summer. O. tsutsugamushi, which causes "summer-type scrub typhus," is highly virulent and usually transmitted by the Leptotrombidium deliense mite. In 1986, scrub typhus was first reported in Mengyin County, Shandong Province, north of the Yangtze River. This newly recognized "autumn-winter type scrub typhus" is caused by a less virulent strain of O. tsutsugamushi and transmitted by the L. scutellare mite (1,2). Since then, cases of autumn-winter scrub typhus have been increasingly reported in many northern areas of China; eschars developed in 82%-91% of those infected (1,2).

Traditionally, the diagnosis of scrub typhus mainly relied on serologic tests. The disease could be retrospectively diagnosed in cases of seroconversion or a [greater than or equal to] 4-fold rise in antibody titers between acute-phase and convalescent-phase serum specimens. The requirement of double serum specimens has limited its usage for diagnosis. Recently a polymerase chain reaction (PCR) assay was developed for detecting O. tsutsugamushi Sta56 gene in blood samples or isolates from patients (3-8). However, the test often gave a false-negative result because hemoglobin and other components in blood may inhibit PCR amplification (3,4,9). The commonly seen eschars in scrub typhus patients were suggested as alternative specimens for diagnosis (9). The objectives of this study were to verify the value of eschars for the diagnosis of scrub typhus by PCR assay and to characterize the genotype of O. tsutsugamushi during the convalescent phase.

Materials and Methods

Sample Collection

Seven scrub typhus patients reported at Feixian County (116[degrees] 11'-118[degrees] 18'E, 35[degrees]01'-35[degrees] 33'N), Shandong Province, China, in September, October, and November of 2003-2004 were included in the study. The identification (ID) codes, age, sex, the locations of eschars, and other clinical characteristics on admission were documented (Table). The typical eschars of 2 patients (03PE1 and 04PE5) are shown in the Figure. After informed consent was obtained, 5 mL acute-phase blood was collected from each patient before treatment. Chloramphenicol was then administered orally at a dosage of 1.5-2.5 g 4x/day for 4-5 days. Fever resolved for all 7 patients within 2 days of treatment. Eschar specimens and 5-mL convalescent-phase blood sample from each patient were collected at the time that the eschar spontaneously desquamated (6-15 days after treatment). Serum specimens were separated by centrifugation at 2,500x g for 10 min. All specimens from eschars, serum, and residual blood clots were kept at -70[degrees]C until use.

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Detection of IgG Antibodies against O. tsutsugamushi

An indirect immunofluorescent antibody assay (IFA) was performed as described previously (10), by using mixed Gilliam, Karp, and Kato strains of O. tsutsugamushi as diagnostic antigen. Scrub typhus was diagnosed in the case of seroconversion or a [greater than or equal to] 4-fold rise in IgG antibody titers between acute-phase and convalescent-phase sera.

DNA Extraction

Complete eschars (30-60 mg in weight) or 0.3 mL blood clot was homogenized with TE (10 mmol/L Tris Cl [pH 8.0] and 1 mmol/L EDTA) buffer and centrifuged at 3,000x g for 5 min; the supernatant was discarded. For the blood clot, the precipitate was resuspended and washed with TE buffer 3 times to eliminate the residual inhibitors in blood. Then 400 [micro]L lysis buffer (10 mmol/L Tris [pH 8.0], 0.1 mol/L EDTA, 0.5% sodium dodecyl sulfate), 10 [micro]L proteinase K (20 mg/mL; Promega Corp., Madison, WI, USA), and 2 [micro]L lysozyme (4 mg/mL; DingGuo Biotech. Co. Ltd, Beijing, People's Republic of China) were added, and incubated at 50[degrees]C for 6 h. DNA was extracted with phenol/chloroform/isoamylalcohol (25: 24:1) and precipitated in ethanol. Finally, the DNA was washed with 75% ethanol and dissolved in 20 [micro]L distilled water.

PCR Amplification

PCR amplification of the Sta56 gene was performed by using species-specific primers, Pr1 (5'-tac att agc tgc agg tat gac-3') and Pr2 (5'-AAT TCT TCA ACC AAG CGA TCC-3') (3,4,10). The amplifications were performed in a volume of 50 [micro]L with a Perkin-Elmer model 2400 thermal cycler (Perkin-Elmer, Norwalk, CT, USA). The amplification program consisted of 1 cycle for 5 min at 94[degrees]C, 35 cycles of denaturation at 94[degrees]C for 30 s, annealing at 56[degrees]C for 1 min, and extension at 72[degrees]C for 1 min. This process was followed by a final extension at 72[degrees]C for 5 min. The amplification products then underwent electrophoresis in a 2% agarose gel containing ethidium bromide and visualized trader UV light.

The acute- and convalescent-phase blood samples were processed and run through the PCR instrument at the same time as the eschar specimens. DNA from reference strains of Gilliam, Karp, and Kato was used as positive controls, and distilled water was used as a negative control in each amplification. To avoid contamination, DNA extraction, reagent setup, PCR, and electrophoresis were performed in separate rooms.

Sequence Analysis

The purified PCR amplicons of all the positive samples were sequenced by Shanghai Invitrogen Biotechnology Co. Ltd (Shanghai, People's Republic of China). The sequences were compared with all available reported O. tsutsugamushi Sta56 gene sequences in GenBank by using BLAST (Basic Local Alignment Search Tool) program (available from www.ncbi.nlm.nih.gov/BLAST). The GenBank accession numbers of sequences obtained from the 7 patients in this study are DQ188085, DQ188086, DQ188087, DQ188088, DQ188089, DQ188090, and DQ188091, respectively.

Results

Seroconversion or a >4-fold rise in titers of IgG antibody to O. tsutsugamushi was observed in all 7 patients (Table), thus confirming the diagnosis of scrub typhus. Seven eschars and 7 acute-phase blood samples from the patients were positive by PCR targeting the Sta56 gene, while 7 convalescent-phase blood samples collected after antimicrobial drug treatment were all PCR-negative.

The 317-bp partial sequence of the O. tsutsugamushi Sta56 gene amplified from each eschar was identical to that of its corresponding acute-phase blood sample. The sequences from the 7 patients differed from each other by 1 or 2 bp; two sequences were identical. The nucleotide sequences were 95.6%-97.8% homologous with the corresponding parts of Kawasaki strain Sta56 gene deposited in GenBank (accession no. M63383), while the sequence homologies with other strains such as Karp, Kato, Kuroki, Shimokoshi, and Je-cheon were all <75.87%.

Discussion

Previous studies used spleen tissues of infected mammals or acute-phase blood from patients to detect O. tsutsugamushi by PCR (3,4). However, PCR amplification of O. tsutsugamushi DNA from blood often lacks sensitivity because some hemoglobin, iron porphyrin, and other factors may inhibit the PCR, although obtaining and processing the blood that avoids the inhibitors is possible (3,4,9).

Ono et al. previously found O. tsutsugamushi DNA (identified as Kawasaki type) in only 1 patient's eschar before antimicrobial drug treatment but not in the acute-phase blood sample (9). In the present study, 7 scrub typhus patients were examined, and O. tsutsugamushi DNA was successfully detected in their spontaneously desquamated eschars and acute-phase blood samples. These findings further proved that eschars could be used as an alternative, easily acquired, and sensitive sample for the diagnosis of O. tsutsugamushi infection, particularly when persons are reluctant to provide a blood sample because of cultural or other reasons.

We described a new simple confirmatory diagnostic assay in which eschars are used as an alternative to serologic tests such as IFA, which usually requires double blood samples from acute and convalescent phases. In addition, from the successful and efficient detection of the O. tsutsugamushi DNA in naturally desquamated eschars, we can infer the presence of the agent in eschars before beginning antimicrobial drug therapy. If eschars had been sampled during the acute phase by punch biopsy, this method could be used for the early diagnosis of scrub typhus.

A previous study carried out in Thailand detected O. tsutsugamushi DNA in convalescent-phase blood of patients after a single dose of doxycycline (10). However, in the present study, O. tsutsugamushi DNA was not persistent in the convalescent-phase blood of patients after 4-5 days of chloramphenicol treatment. Whether a lack of PCR sensitivity or difference in the treatment regimens explains the apparent lack of O. tsutsugamushi DNA in the convalescent-phase blood samples is not known. A possible reason is that the patients in the present study were infected with the less virulent strain of O. tsutsugamushi (11), which may only persist in blood for a short period after antimicrobial drug treatment. Our previous study indicated that to isolate O. tsutsugamushi from patients with autumn-winter scrub typhus, cyclophosphamide (0.25 mg/g of body weight) had to be injected into the experimental mice after injection of patients' blood to suppress immunity (11).

Sequence analysis of partial Sta56 gene clarified that the genotypes of O. tsutsugamushi in the scrub typhus patients from Shandong Province, China, were more closely related to Kawasaki type, which is less virulent than other genotypes and only caused a mild syndrome (1). The finding has applications for physicians to treat patients and prescribe medicine.

This research was supported by the National Nature and Science Foundation of the People's Republic of China (no. 30371237) and EU Project (no. SP22--CT--2004--003824).

References

(1.) Chen XR. Scrub typhus and Orientia tsutsugamushi. Beijing: Military Medical Science Press, 2001. p. 176-9.

(2.) Wu GH. The epidemiological characteristics and prevention and cure of scrub typhus in China. Chin J Public Health. 2000;16:777-9.

(3.) Guo HB, Pan SZ, Li XF, Tang JQ, Zhang Y, Wu GH. Studies on detecting and typing of Rickettsia tsutsugamushi by polymerase chain reaction. Chin J Zoon. 1997;13:8-11.

(4.) Guo HB, Tang JQ, Li XF, Pan SZ, Zhang Y, Yu MM, et al. Study on target gene of new type Rickettsia tsutsugamushi in China by PCR/RFLP and sequence analysis. Chin J Public Health. 1997;6:193-6.

(5.) Ohashi N, Koyama Y, Urakami H, Fukuhara M, Tamura A, Kawamori F, et al. Demonstration of antigenic and genotypic variation of Orientia tsutsugamushi which were isolated in Japan, and their classification into type and subtype. Microbiol Immunol. 1996;40: 627-38.

(6.) Furuya Y, Yoshida Y, Katayama T, Yamamoto S, Kawamura JR. Serotype-specific amplification of Rickettsia tsutsugamushi DNA by nested polymerase chain reaction. J Clin Microbiol. 1993;31: 1637-40.

(7.) Ohashi N, Nashimoto H, Ikeda H, Tamura A. Cloning and sequencing of the gene (tsg56) encoding a type-specific antigen from Rickettsia tsutsugamushi. Gene. 1990;91:119-22.

(8.) Stover CK, Marana DP, Carter JM, Roe BA, Mardis E, Oaks EV. The 56-kilodation major protein antigen gene of Rickettsia tsutsugamushi: molecular cloning and sequencing analysis of the Sta56 gene and precise identification of a strain-specific epitope. Infect Immun. 1990;58:2076-84.

(9.) Ono A, Nakamura K, Hihuchi S, Miwa Y, Nakamura K, Tsunoda T, et al. Successful diagnosis using eschar for PCR specimen in tsutsugamushi disease. Intern Med. 2002;41:408-11.

(10.) Saisongkorh W, Chenchittikul M, Silpapojakul K. Evaluation of nested PCR for the diagnosis of scrub typhus among patients with acute pyrexia of unknown origin. Trans R Soc Trop Med Hyg. 2004;98:360-6.

(11.) Liu YX, Zhao ZT, Gao Y, Jia CQ, Zhang JL, Yang ZQ, et al. Characterization of Orientia tsutsugamushi strains isolated in Shandong Province, China by immunofluorescence and restriction fragment length polymorphism (RFLP) analyses. Southeast Asian J Trop Med Public Health. 2004;35:353-7.

Yun-Xi Liu, * Wu-Chun Cao, * Yuan Gao, ([dagger]) Jing-Lan Zhang, ([dagger]) Zhan-Qing Yang, ([dagger]) Zhong-Tang Zhao, ([double dagger]) and Janet E. Foley ([section])

* Beijing Institute of Microbiology and Epidemiology, Beijing, People's Republic of China; ([dagger]) Center for Disease Control and Prevention of Jinan, Jinan, People's Republic of China; ([double dagger]) Shandong University, Jinan, People's Republic of China; and ([section]) University of California, Davis, Davis, California, USA

Address for correspondence: Wu-Chun Cao, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, 20 Dong-Da St, Fengtai District, Beijing 100071, People's Republic of China; email: [e-mail]

Dr Liu is a professor of epidemiology at the Beijing Institute of Microbiology and Epidemiology. His research interests focus on vectorborne infectious diseases and highly pathogenic avian influenza.

Table. Clinical characteristics on admission and serologic
results of 7 patients with scrub typhus *

Patient           Age, y             Fever,
ID                (sex)            [degrees]C     Rash

03PE1             35 (F)               38          +
03PE2             42 (M)               40          +
03PE3             48 (M)               40          +
03PE4             28 (F)               38          +
04PE5             53 (M)               38          +
04PE6             36 (M)               40          +
04PE7             31 (F)               39          +

Patient                              Location of
ID            Lymphadenitis            eschars

03PE1               +                    Neck
03PE2               +                 Umbilicus
03PE3               +                Right groin
03PE4               +                Left papilla
04PE5               +                   Waist
04PE6               +              Behind right ear
04PE7               +                Left axilla

                       Acute phase

              Blood samples
Patient         collected,         IgG titers
ID            d after onset         of sera

03PE1                3                  N
03PE2                7                 80
03PE3                5                  N
03PE4                8                160
04PE5                2                  N
04PE6               10                 80
04PE7                7                  N

                   Convalescent phase

            Eschars and blood
            samples collected,
Patient         ([dagger])         IgG titers
ID            d after onset         of sera

03PE1                9                 320
03PE2                8                 320
03PE3               10                 320
03PE4               11                 640
04PE5                6                 320
04PE6               15               1,280
04PE7                9                 640

* F, female; M, male; Ig, immunoglobulin, N, negative.

([dagger]) Chloramphenicol was given to a patient immediately
after the acute-phase blood was collected.