Am. J. Trop. Med. Hyg., 76(3), 2007, pp. 553-558
Copyright © 2007 by The American Society of Tropical Medicine and Hygiene
PREPARATION OF RECOMBINANT ANTIGEN OF O. TSUTSUGAMUSHI PTAN STRAIN AND DEVELOPMENT OF RAPID DIAGNOSTIC REAGENT FOR SCRUB TYPHUS
MIN CAO,
HENGBIN GUO,
TANG TANG,
CHANGJUN WANG,
XIANFU LI,
XIUZHEN PAN,
ZHU JIN, AND
JIAQI TANG*
Department of Epidemiology and Microbiology, Research Institute for Medicine of Nanjing Command, Jiangsu, China; School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
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ABSTRACT
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Spring scrub typhus has frequently occurred in Pingtan Island, China, since 2000. In this study, we amplified a 1352-bp DNA fragment encoding a truncated 56-kDa outer membrane protein of the Ptan strain, which was isolated from a serum sample of a patient with spring scrub typhus, and cloned it into the pET28a vector for expression. The expression product was a recombinant polypeptide containing a His-tag to facilitate purification on a Ni2+ chromatography column. The recombinant protein was further identified by Western blotting and enzyme-linked immunosorbent assay (ELISA) and appeared to be a good diagnostic antigen candidate. A rapid colloidal gold immunochromatographic assay (CIA) for detecting serum total antibodies, IgG and IgM, which are anti-Orientia tsutsugamushi, was developed, using a mixture of the r56 of the Gilliam and Ptan strains as the diagnostic antigen. CIA performance was tested on a panel of 112 control sera from confirmed cases of scrub typhus. The detection sensitivities of CIA against anti-O. tsutsugamushi total antibodies, IgM, and IgG were 98.2%, 81.2%, and 94.6%, respectively, while that of IFA (using the lysate of the O. tsutsugamushi Gilliam-infected chicken yolk sac as the antigen) against IgG was 85.7%. One hundred five serum samples from healthy individuals and patients with other febrile diseases were tested with CIA as negative controls. Specificities of CIA against anti-O. tsutsugamushi total antibodies, IgM, and IgG were 98.1%, 100%, and 98.9%, respectively, while the specificity of IFA against IgG was 98.9%. These results indicated that CIA was a good assay and could substitute for conventional immunofluorescence assays for diagnosis of scrub typhus.
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INTRODUCTION
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Scrub typhus, an acute natural focal infectious disease, is caused by Orientia tsutsugamushi infection. The disease is often characterized by fever, eschar, lymphadenopathy, and rash. Before 1986, the epidemic region of this disease was restricted to regions on the south bank of the Yangtze River. Since 1986, however, the distribution of its natural infection has expanded beyond the Yangtze River to many northern areas of China. Very recently, several cases were reported in the spring (March to May) from Pingtan Island, Fujian, China, a traditionally endemic region of scrub typhus. Previous records showed that the epidemic season in the Pingtan region was typically only in the summer.1 Because of the similarities in symptoms, it is usually difficult to distinguish scrub typhus from other acute febrile diseases, such as murine typhus, dengue fever, and viral hemorrhagic fevers. Misdiagnosis of scrub typhus is common, and direct consequences include serious damage to patients’ health or even death due to delayed or inappropriate treatment. This change in the epidemic season may exacerbate this situation.
Current diagnostic assays for scrub typhus in China have several disadvantages that limit their applications. Serodiagnostic assays, such as Weil-Felix (WF) assay,2 immunofluorescence assay (IFA),3 dot-blot assay,4 and indirect immunoperoxidase assay,5 are technically challenging, because of the required propagation of rickettsiae in infected yolk sacs of embryonic chicken eggs or antibiotic-free cell cultures. The specificity of the WF method is also relatively poor. A few diagnostic assays, including IFA, ELISA,6 and conventional and real-time PCR, appeared to be suitable for diagnosis of scrub typhus with high sensitivity and specificity. Conventional PCR can even detect O. tsutsugamushi at the onset of illness, when antibody titers are not yet high enough for detection.7,8 Real-time PCR9 is very helpful in diagnosis of relapse, as it has the same sensitivity as standard PCR, and it provides more rapid, quantitative results. Down sides of these methods are that they require special instruments and reagents and they are generally not convenient for field operations, such as rural hospitals. Therefore, it is an urgent task for researchers to find a simple, rapid diagnostic assay for O. tsutsugamushi. In this study, we obtained the truncated 56-kDa recombinant protein from the Ptan strain, which had been isolated from a blood sample of a patient with spring scrub typhus. After identifying its antigenicity, we developed a rapid colloidal gold immunochromatograpic assay (CIA) to detect the total antibodies, IgM, and IgG against O. tsutsugamushi in serum samples from the patients, using a mixture of the truncated 56-kDa recombinant antigen from Gilliam and Ptan strains as diagnostic antigens.
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MATERIALS AND METHODS
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Bacteria and vector plasmid.
Escherichia coli TG1 was used for cloning, and E. coli BL21(DE3) was used for over-expression of proteins under the control of phage T7 lac promoter. The plasmid vector used was pET28a (Novagen, Darmstadt, Germany).
Extraction of Ptan strain
O. tsutsugamushi DNA. The procedure was performed with a method as introduced by Cao and others.10 The extracted DNA was dissolved in 1x TE buffer. Primers were designed based on the gene sequence of the 56-kDa protein from the reference strains Karp, Gilliam, and Kato11,12:
Forward primer: 5'-ggg gat mLg gat tta gag cag ag-3';
Backward primer: 5'-ggc gaa ttc aaa aac tag aag tta tag cg-3'.
The italic portions are inserted BamHI and EcoRI restriction sites. The primers were synthesized by Sangon Co. (Shanghai, China). The coding sequence of the truncated 56-kDa protein from the Ptan strain was amplified by PCR. The PCR reaction had final concentrations of 200 µmol/L of each of the deoxynucleoside triphosphate, 0.3 µmol/L of each of the primer, and 0.6 unit of Taq polymerase (in 10 mmol/L Tris-HCl working buffer containing 2.0 mmol/L MgCl2 and 50 mmol/L KCl, pH 8.3, provided by TaKaRa Co., Dalian, China). The PCR started with a step of 5 min at 95°C, followed by 35 cycles of 50 s at 94°C, 50 s at 56°C, and 60 s at 72°C. The reaction ended with a final step of 7 min at 72°C. The PCR products were analyzed on 1% agarose gel, and the size of the 56-kDa protein gene from Ptan strain was determined with the standard molecular weight marker (Fermentas MBI Co., Lithuania, and TaKaRa Co.; see Figure 1c
).
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FIGURE 1. Recombinant plasmid screened by restriction enzyme, PCR results: M1, DNA marker 1, GeneRuler DNA Ladder Mix; a, recombinant plasmid digested with EcoRI and BamHI; b, undigested recombinant plasmid; M2, DNA Marker DL2000; c, PCR product of r56 of Ptan strain.
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Construction and identification of recombinant plasmid.
All of restriction enzymes were purchased from TaKaRa Co. Both the purified PCR products and the plasmid pET28a were digested with BamHI and EcoRI and ligated at 16°C overnight to form the reconstructed plasmid carrying the inserted fragment. E. coli TGI was transformed with the plasmid. E. coli TGI colonies carrying the recombinant plasmid were selected with kanamycin. The recombinant plasmid was further confirmed by BamHI and EcoRI digestion analysis (Figure 1
).
Expression and identification of the truncated Ptan strain 56-kDa protein.
Plasmids carrying the inserts were transformed into the expression host E. coli Bl21 (DE3). Transformed cells from a single colony were propagated at 37°C with shaking in 3 mL of 2x YT medium (1 L medium contains 16 g bactotryptone, 10 g bacto-yeast extract, 5 g NaCl, pH 7.0) containing 100 mg/mL kanamycin. Fifty microliters of the overnight culture was transferred to 5 mL of 2x YT containing 100 mg/mL kanamycin and cultured at 37°C with agitation until A600 reached 0.6–0.8.
Isopropyl-ß-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mmol/L to induce expression of the recombinant protein. Post-induction incubation usually lasted 4 h, with agitation. The expression product was identified with SDS-PAGE as the truncated recombinant protein (Ptan r56). SDS-PAGE analysis was performed with the Basic Unit (8 x 9 cm, Amersham Biosciences Corp., Piscataway, NJ). The stacking gel and separation gel contained 5% and 12% ac-rylamide (acrylamide/bisacrylamide ratio was 29:1), respectively. Electrophoresis was carried out at a constant voltage (80 V) for 75 min. The proteins were either stained with Coomassie brilliant blue R250 or electroblotted onto nitro-cellulose membranes.
Purification of Ptan r56.
E. coli Bl21(DE3) carrying truncated r56-expressing recombinant plasmid was cultured at 37°C in 100 mL of 2x YT with 100 mg/mL kanamycin, until the optical density (OD) A600 reached 0.5–0.8. After expression of the recombinant protein in the E. coli host, the bacteria were harvested by centrifugation at 8000 rpm for 30 min. The cell pellets were resuspended in 1/20 of the total volume PB buffer (20 mmol/L phosphate buffer, pH 7.4) containing 0.05% Triton X-100. The suspended cells were sonicated for 30 cycles, with each cycle consisted of 20 s of sonication and a 1 min pause, after a 15 min incubation in ice. The lysate was centrifuged for 20 min at 12,000g, 4°C. The pellets were completely resuspended in PB buffer (containing 2 mol/L urea, 2% Triton X-100), placed under room temperature for an additional 10 min, and centrifuged for 10 min at 12,000g, 4°C. The pellets were finally dissolved in 8 mol/L urea solution containing 20 mmol/L PB buffer, 0.1 mmol/L dithiothreitol, and 2% Triton X-100, placed under room temperature for 1 h, and centrifuged for 30 min at 12,000g. The supernatant was loaded onto a Ni-His tagged affinity chromatography column (Amersham Biosciences Corp., Piscataway, NJ). The column was eluded with a series of gradient washing solutions (20 mmol/LPB, 8 mol/L urea, pH 7.4, 40–500 mmol/L imidazole). The purified proteins were identified with SDS-PAGE.
Refolding of Ptan r56.
Refolding of r56 was achieved by sequential dialysis with a series of urea buffers of decreasing concentration gradients. Collected peak fractions were combined and dialyzed in 8 volumes of PB buffer containing 6 mol/L urea for 2 h at room temperature. The r56 fractions were then dialyzed in PB buffer containing 4 mol/L urea for 30 min, followed by an additional 30 min of dialysis with a fresh solution of the same concentration. The same procedure was repeated in 20 mmol/L PB buffer containing 2 mol/L urea. The final dialysis was carried out in PB solutions containing no urea 3 times, 30 min each time. The product was placed under 4°C overnight.
Western blot.
SDS-PAGE analysis was performed as previously described. The gel was then electroblotted onto nitro-cellulose membranes. After transfer to the nitrocellulose membrane, the membrane were blocked with 1% bovine serum albumin (BSA) at room temperature for 1 h. The membrane was then incubated at room temperature for 2 h, with a positive serum from a patient with scrub typhus at 1:100 dilution. A secondary peroxidase-conjugated goat anti-human IgG (H+L) antibody (ZSGB-BIO Inc., Beijing, China) was used at 1:1000 dilution to detect the antibody–antigen complexes. Signals were developed with 3,3'-diaminobenzidine (DAB).
Serum samples for ELISA.
Thirty-six IFA-positive serum samples from 18 confirmed cases of spring scrub typhus1 in Pingtan Island were used for the ELISA test. Twenty serum samples from healthy individuals in Pingtan Island were used as negative controls.
ELISA.
Microtiter plates (96 well) were coated with anti-g/well) gens r56(Ptan) diluted in pH 9.6 Na2CO3 buffer (0.1 µ overnight at 4°C, blocked with 1% BSA for 1 h, and rinsed with PBS for 2 x 5 min. Patient sera were diluted 1:400 in PBS with control protein extracts (20 µg/mL) purified from E. coli BL21. The plates were incubated for 1 h at room temperature and washed four times with PBS containing 0.1% Triton X-100. Peroxidase-conjugated goat anti-human IgG (H+L; ZSGB-BIO Inc.) and peroxidase-conjugated goat anti-human IgM(µ) (Sigma, St. Louis, MO) were added at 1:5000 and 1:10,000 dilutions, respectively. After 1 h of incubation at room temperature, the plates were washed three times with PBS containing 0.1% Triton X-100 and once more with PBS before the o-phenylenediamine substrate (AMRESCO, Solon, OH) was added. OD415 was measured after 15 min of incubation at room temperature by a Bio-Rad Model 550 Microplate Reader (Bio-Rad, Hercules, CA). One positive and two negative control sera were run with each experiment.
Human serum samples for IFA and CIA.
We have performed the CIA and IFA using 112 sera from laboratory-confirmed cases of scrub typhus in Dongtai county, Jiangsu province, in 1987. Thirty-eight sera samples from 18 confirmed cases of spring scrub typhus were also tested by CIA and IFA. Negative control serum samples, including samples from 89 healthy individuals and 16 samples from non-scrub typhus patients (2 from hemorrhagic fever with renal syndrome, 6 from Toxoplasma gondii infections, 2 from cysticercosis cellulosae, 1 from sparganosis, and 5 from clonorchiasis).
Indirect IFA assay.
The lysate of the O. tsutsugamushi Gilliam-infected chicken yolk sac was pipetted onto slides coated with 1% BSA then air-dried and fixed with acetone. A series of 2-fold dilutions, beginning at 1:16, were made with PBS (with 1% BSA, pH 7.4). The diluted samples were loaded to slides, incubated at 37°C for 30 min, and then washed with PBS for 3 x 5 min. Goat anti-human IgG (G) tagged with FITC was then applied to the sample for 35 min at the optimal dilution of 1:150 at 37°C, followed by 3 x 5 min PBS washes. The sample was air-dried and read with an epifluorescence UV microscope. Samples with an antibody titer 
1:64 were considered positive.
Preparation of diagnostic antigen.
The recombinant truncated r56 proteins of Ptan and Gilliam strains described previously13 were mixed at a ratio of 1:1 to a total concentration of 1.0 mg/mL. The mixture was used as the diagnostic antigen for CIA.
CIA for detecting total sera anti-O. tsutsugamushi antibodies.
The assay was to use the double-antigen sandwich technique to facilitate detection of the anti-O. tsutsugamushi antibody in sera of patients with scrub typhus. The prepared diagnostic antigen–colloidal gold conjugate and mouse IgG–colloidal gold conjugate were coated onto a fiberglass pad. The diagnostic antigen was also deposited on a nitrocellulose membrane as the recognition probe to form a test line. The goat anti-mouse IgG (purchased from Bosheng Co., Xiamen, China) was deposited on the membrane to form a control line. The anti-O. tsutsugamushi antibody in the patient’s serum could react with the colloidal gold-conjugated diagnostic antigen and form a complex of Ab-Ag-Au. According to the principle of chromatography, when the complexes are wicked along the nitrocellulose membrane, they are captured by the diagnostic antigen immobilized on the test line, resulting in a red line (Ag-Ab-Ag-Au). A control line is also presented by mouse IgG-colloidal gold conjugate reacting with the goat anti-mouse IgG. Ten microliters of the test serum and a drop of reaction buffer (20 mmol/L PB, pH 7.4) were added on the strip. The result was read after 10 min. When both the test line and the control line show red, it can be determined that the corresponding sample was positive; if only the control line turned red, a negative result is indicated; if the control line fails to appear, the result is concluded to be invalid (Figure 4).
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FIGURE 4. CIA strip for detecting anti-O. tsutsugamushi total antibodies in serum: a, positive result (+); b, negative result (–). This figure appears in color at www.ajtmh.org.
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CIA for detection of anti-O. tsutsugamushi IgG and IgM.
In this test, the diagnostic antigen conjugated to colloidal gold and mouse IgG–colloidal gold conjugate were deposited on a pad, and the anti-human IgG and IgM monoclonal antibodies (purchased from Bosheng Co.) were bound on the nitrocellulose as the IgG and IgM capture reagents, respectively. The goat anti-mouse IgG was deposited on control line. The anti-O. tsutsugamushi IgG and IgM in the patient’s serum could react with the colloidal gold-conjugated r56 and later be captured together by the anti-human IgG and IgM monoclonal antibodies as they are wicked along the nitrocellulose membrane. The captured IgG-r56/IgM-r56 complex forms IgG and IgM test lines, respectively. Only 5 µL of the test serum and a drop of reaction buffer (20 mmol/L PB, pH 7.4) were added on the strip. The result was read after 10 min. When both the test line and the control line show red, it can be determined that the corresponding sample was positive; if only the control line turns red, a negative result is indicated; failure of the control line to appear indicates an invalid result (Figure 5).
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FIGURE 5. CIA strip for diagnosis of anti-O. tsutsugamushi IgM and IgG in serum: a, IgG(+) and IgM(+); b, IgG(+) and IgM(–); c, IgG(–) and IgM(–). This figure appears in color at www.ajtmh.org.
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RESULTS
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Identification and purification of the recombinant 56-kDa protein.
As described in Materials and Methods, the recombinant expression plasmid was transformed into E. coli BL21(DE3), protein expression was induced by the addition of IPTG, and the protein was analyzed with SDS-PAGE. Expression of a protein of 
52-kDa was detected, and no other similar protein was observed when the empty plasmid of pET28a was transformed into the same host. This is consistent with the predicted molecular weight of the truncated 56-kDa protein (Figure 2a and b). The majority of the recombinant protein was expressed as inclusion bodies dissolved in 8 M urea. The lysate was subjected to Ni-His tag affinity chromatograph, and the desired protein was eluted with 200 mol/L imidazole elution buffer (Figure 2c). After the protein was renatured with a series of dialyses in PB buffers, the majority of the protein was active. Protein yield was 10 mg/L.
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FIGURE 2. Expressed products of recombinant plasmid analyzed by SDS-PAGE: M, protein marker; a, pET28a induced by IPTG for 4 h; b, recombinant plasmid induced by IPTG for 4 h; c, recombinant protein purified by Ni2+ chromatography column. This figure appears in color at www.ajtmh.org.
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Results of Western blot.
The results of Western blot showed that the recombinant protein could be recognized by the positive serum from the patient (Figure 3). It suggested that this recombinant protein is a good antigen for diagnosis of scrub typhus.
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FIGURE 3. Expressed products of recombinant plasmid analyzed by Western blot: a, products of IPTG-induced Pet28a react with positive serum; b, products of IPTG-induced recombinant plasmid pETp-tan react with positive serum; c, recombinant protein purified by Ni2+ chromatography column and refolded react with positive serum. This figure appears in color at www.ajtmh.org.
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Ptan r56 was identified by ELISA.
Twenty sera from healthy Pingtan soldiers were used to establish an ELISA cut-off for positive reactions (mean plus 2 SD) with Ptan r56 0.16 (0.05 as the antigen. These cut-off values were OD415 = 0.12 (0.03 plus 0.09) for IgM plus 0.11) for IgG and OD415 = at 1:400 serum dilution. The OD415 measurement of r56 ELISA on 38 sera from 18 patients confirmed to have scrub typhus from Pingtan Island were obtained and compared with the result of CIA, with IgG titers determined by an IFA using the lysate of a chicken yolk sac infected by O. tsutsugamushi Gilliam as the diagnostic antigen.1 The results of ELISA correlate well with those of IFA and CIA (Table 1).
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TABLE 1 Comparison of ELISA with folded r56 (ptan) as antigen, CIA and IFA reactivity using Gilliam-infected chicken yolk sac as the antigen with 18 laboratory-confirmed sera from Pingtan Island (+, positive; –, negative)
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Sensitivity and specificity of CIA.
A total of 112 serum samples from confirmed scrub typhus cases (Dongtai, Jiangsu) were tested with CIA. The sensitivities of CIA react against anti-O. tsutsugamushi total antibodies, IgM, and IgG were 98.2%, 81.2%, and 94.6%, respectively, while that of IFA against IgG was 85.7%. A total of 105 serum samples from healthy individuals and confirmed patients of other febrile illness were used to evaluate the specificity of CIA in detecting anti-O. tsutsugamushi total antibodies; its specificity was 98.1%. Ninety-one serum samples from 89 health individuals and 2 patients with hemorrhagic fever with renal syndrome were used to evaluate the specificity of CIA detecting IgM and IgG and IFA detecting IgG. The specificities were 100%, 98.9%, and 98.9%, respectively (Table 2).
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DISCUSSION
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The 56-kDa protein is one of the major outer membrane proteins of O. tsutsugamushi, accounting for 10–15% of its total proteins. Sera from most patients with scrub typhus can recognize this protein, suggesting that it is a good candidate for a diagnostic antigen for scrub typhus. Analysis of the genes encoding the 56-kDa protein from various serotypes has revealed that the fragment contains four variable domains and four conserved domains,14,15 demonstrating that the conserved domains are important for the responses of isotype antibody and that the variable domains are important for the allotype antibody responses. At present, the 56-kDa protein has been widely used as a diagnostic antigen in many countries. Kim et al. used a recombinant 56-kDa protein from the Boryong strain fused with maltose binding protein as the antigen in ELISA and a passive hemagglutination test for scrub typhus.6,14 Ching and others have developed a rapid immunochromatographic flow test (RFA) to detect the anti-O. tsutsugamushi IgG and IgM in patients’ sera for diagnosis of scrub typhus, by employing a karp r56 protein that contained deletions of 79 and 77 amino acid residues at the N and C terminals, respectively, as the diagnostic antigen.15,16 In this work, a 1352-bp gene fragment was selected and cloned into expression vector pET28a, which covered the four variable domains and most of the conserved domains of the 56-kDa protein of the Ptan strain and the regions of epitopes for the recognition of isotype and allotype antibodies.
The expression of the fragment yielded a 52-kDa, His-tagged protein with a deletion of 80 amino acid residues at the N terminal. ELISA and Western blot results on the Ptan r56 protein indicated that the recombinant polypeptide was a potential diagnostic antigen. It was previously documented that most isolates of O. tsutsugamushi in China were serotyped as Gilliam or Karp strains.17 To broaden the reaction spectrum, we used a mixture of Ptan r56 and the previously expressed Gilliam r56 as diagnostic antigens in the rapid CIA to detect the anti-O. tsutsugamushi total antibodies, IgG, and IgM in serum. CIA demonstrated better sensitivity in detecting anti-O. tsutsugamushi total antibodies and IgG than did the IFA (P < 0.001) using 112 sera from confirmed cases of scrub typhus. We also found that all sera that were IgM positive also presented as IgG positive but that no serum presented as both IgM positive and IgG negative. This may be attributed to the fact that all sera were collected at least 10 days after the onset of the illness, when the titer of IgG had begun to increase. In 106 IgG-positive sera, 15 were IgM negative; these sera were mainly collected at least 30 days after the onset of illness, when the titer of IgM may have begun to decrease. However, not all sera collected 30 days after the onset of illness presented as IgM negative, which may due to the different immune status with different patients. According to the test for CIA specificity, only 1 healthy serum showed false-positive results in detecting anti-O. tsutsugamushi total antibodies and IgG, but was IgM negative. The provider of the serum sample that yielded the false-positive result had once been suspected of being infected with O. tsutsugamushi and had received scrub typhus treatment. Another serum from a patient infected with Toxoplasma gondii also showed positive with anti-O. tsutsugamushi total antibodies. No further IgG and IgM detection with the above serum was performed due to lack of sufficient sample. The false-positive result could be caused by the cross reactivity of anti-T. gondii antibody with 56-kDa antigen or a dual infection of T. gondii and O. tsutsugamushi in the patient. The actual specificity of CIA could be higher. CIA was more convenient than the RFA method developed by Ching and others16 because, with a CIA test, the procedure is as easy as applying the serum and the buffer directly to the test strip. We tried to examine the sera of patients confirmed with scrub typhus infection from 4 regions in China, including the cities of Dali, Guangzhou, Changchun, and Pingtan Island, Fujian. Detection rates were 86.6% (26/30), 87.5% (28/ 32), 94.7% (18/19), and 100% (28/28), respectively. These results suggest that CIA may be used for the diagnosis of scrub typhus in various regions in China.
We conclude that CIA is a simple, rapid, and reliable assay for diagnosis of scrub typhus, capable of providing accurate results in less than 10 minutes without needing special equipment or techniques. It is highly suitable for field deployment in remote areas with limited medical support. Therefore, CIA is an ideal substitute for IFA in diagnosis of scrub typhus.
Received December 31, 2005.
Accepted for publication October 27, 2006.
Acknowledgment: We thank Dr. Qiajia Huang, Fuzhou General Hospital of Nanjing Command stationed in Fuzhou, Fujian province, China for critically reading the manuscript and helpful corrections in grammar and writing.
Financial support: This study was supported by the Bureau of Hygiene, Nanjing Command, PLA.
* Address correspondence to Jiaqi Tang, 293 East Zhongshan Road, Nanjing, China 210002. E-mail: tjiaqi2006{at}yahoo.com.cn 
Authors’ addresses: Min Cao, Hengbin Guo, Changjun Wang, Xianfu Li, Xiuzhen Pan, Zhu Jin, and Jiaqi Tang, Department of Epidemiology and Microbiology, Research Institute for Medicine of Nanjing Command, Jiangsu, China, Telephone: +86-25-84526002, Fax: +86-25-84507094, E-mail: tjiaqi2006{at}yahoo.com.cn. Tang Tang, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, E-mail: olivertt{at}gmail.com.
Min Cao, Hengbin Guo, Tang Tang, and Jiaqi Tang contributed equally to the work.
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ABSTRACT
INTRODUCTION
