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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The indirect immunofluorescent antibody assay (IFA) and the indirect immunoperoxidase test (IIP) are generally accepted as the gold standard serologic assays. However, IFA and IIP assays are time consuming and they require specialized equipment and trained personnel. There is also a delay of several days between the onset of the illness and the increased antibody titer, and a positive reaction is often detected only after the acute illness has resolved. Diagnosis can be difficult in the early stage of illness when the antibody titers are not yet high enough to be detected. Therefore, a simple and more rapid laboratory diagnostic method for scrub typhus is needed. Performing a polymerase chain reaction (PCR) assay on the blood sample has proven useful for the early diagnosis of scrub typhus.4 PCR for detecting the DNA from O. tsutsugamushi is both sensitive and specific, and this method has been studied in the laboratory; however, there is a lack of data to evaluate this assay in the clinical setting.5
In this study, we conducted a prospective study on 135 patients with possible scrub typhus who were admitted to a tertiary care hospital or one of three community hospitals that are located in southwestern Korea to assess the clinical usefulness of performing nested PCR as a diagnostic technique. We also compared the PCR results with the passive hemagglutination assay (PHA) results and the gold standard IFA results.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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18 years of age) who had the history of fever together with eschar or a maculopapular skin rash and who also had at least two symptoms such as headache, malaise, myalgia, coughing, nausea, and abdominal discomfort. Informed consent was obtained from the patients or their guardians. All the patients were admitted from September 1, 2004 to December 31, 2004 to Chosun University Hospital or one of its three community branch hospitals (Haenam General Hospital, Jangheung Hospital, and Muan Hospital); these three branch hospitals are all located in the southwestern part of Korea. Blood samples were also collected from 22 medical school students who showed no evidence of recent illness. Scrub typhus was confirmed on the basis of either a single indirect immunofluorescent-specific IgM titer of 1:10 against O. tsutsugamushi or a 4-fold or greater rise in the IFA IgG titer.6 Whole blood samples were collected for PCR testing, and sera were obtained for performing serology evaluation with using the indirect IFA and PHA. Additional blood samples were collected at 7-day intervals for 1 month after treatment, if possible. IFA and PCR were performed at the Korea Center for Disease Control and Prevention (Seoul, Korea). PHA was performed at the Green Cross Corporation in Korea using Genedia Tsutsu PHA II test kits (GreenCross SangA, Yongin-shi Kyunggi-do, Korea). The laboratory personnel who carried out these assays did not know any of the clinical information and definitive diagnoses, and the physicians who treated the patients did not know the PCR results and the IFA results. The study was approved by the Ethics in Human Research Committee of Chosun University Hospital. The enrolled patients were evaluated for diseases other than scrub typhus by using clinical or laboratory evidence of such diseases as murine typhus, leptospirosis, hemorrhagic fever with renal syndrome, and systemic lupus erythematosus. Nested PCR assay. Because Orientia is an intracellular organism, the buffy coat of the whole blood samples was used for the nested PCR tests. DNA was prepared from the buffy coat (0.2 mL) of the patients with using the QIA amp DNA mini kit (Qiagen. Hilden, Germany). The final volume of the extracted DNA was 50 µL. We used the 50-µL volume of DNA instead of manufacturer’s recommended volume of 200 or 400 µL to concentrate the DNA.
The nested PCR assay was performed as described previously by Furuya and others4 with some slight modification. The nucleotide primers were based on the nucleotide sequences of a gene encoding for the 56-kDa antigen of the Gilliam strain of O. tsutsugamushi.4 Primers 34 (5'-TCAAGCTTATTGCTAGTGCAATGTCTGC-3') and 55 (5'-AGGGATCCCTGCTGCTGTGCTTGCTGCG-3') were used for the first PCR, and nested PCR primers 10 (5'-GATCAAGCTTCCTCAGCCTACTATAATGCC-3') and 11 (5'-CTAGGGATCCCGACAGATGCACTATTAGGC-3') were used to amplify a 483-bp fragment.
The first PCR step was carried out with 5 µL of DNA that was extracted from the buffy coat. The PCR amplification mixture in a final volume of 50 µL contained 2 mmol/L MgCl2, 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 8.3), 200 µmol/L of deoxynucleoside triphosphates (dNTPs), and 200 µmol/L of 1.25 U of Taq DNA polymerase (TAKARA, Shiga, Japan). The amount of primer added to the reaction mixtures was 0.1 µmol/L for the first step and 0.2 µmol/L for the second step. The reactions were carried out for 30 cycles in both the first and second amplifications. The amplification conditions consisted of initial polymerase activation at 94°C for 5 minutes, 35 cycles at 94°C for 30 seconds, 55°C for 2 minutes, and 72°C for 2 minutes, and a final elongation step at 72°C was done for 7 minutes using a DNA pelter thermal cycler (MJ Research, Boston, MA). The PCR products were analyzed by electrophoresis on a 1.5% agarose gel; the products were stained with ethidium bromide and observed under an ultraviolet transilluminator. The sample was designated as positive when the 483 bp–specific band was detectable. To avoid cross-contamination, we adhered to the strict regulations recommended for PCR diagnostic laboratories.7
Immunofluorescent antibody assay. Four dots of the reference strains of O. tsutsugamushi such as the Gilliam, Karp, Kato, and Boryong strains were spotted on each well of the slides. The slides were dried at room temperature and fixed with acetone for 10 minutes; they were kept in a deep freezer until IFA was carried out. Before testing, the slides were warmed to room temperature. The patients’ sera were assayed for the IgM and IgG antibody titers against the reference strains of O. tsutsugamushi using a modified IFA test as described by Robinson and others.8 Briefly, the sera were diluted 1/32 with phosphate-buffered saline (PBS), and 2-fold serial dilutions were dropped on each well of the slides. Known positive and negative sera were used as internal quality controls for each test. The test slides were incubated in a humidified chamber at 37°C for 30 minutes, and next they were washed with PBS and distilled water. The fluorescein-conjugated anti-human IgG (whole molecule) and fluorescein-conjugated anti-human IgM (5FC µ; Cappel/ICN Biomedicals, Irvine, CA) were diluted 1/300 in PBS and applied on the slides; the slides were allowed to incubate, and after this, they were washed as described above. The slides were air dried and mounted with FA mounting solution (Bacto, Sparks, MD), and they were examined by using a fluorescence microscope (Axioskop 2; Carl Zeiss, Jena, Germany) at x400 magnification. The highest titers of the four strains (i.e., the Gilliam, Karp, Kato, and Boryong) obtained by the IFA were used. The titers of antibodies were expressed as the reciprocal of the highest dilution.
Passive hemagglutination assay.
PHA was performed at the Green Cross Corporation in Korea using Genedia Tsutsu PHA II test kits. Genedia Tsutsu PHA II is a test kit for the quantitative and qualitative detection of antibodies against O. tsutsugamushi in human serum based on the PHA. For the kit, we used sheep erythrocytes that were sensitized with recombinant 56-kDa proteins from the O. tsutsugamushi Karp and Gilliam strains, including the Boryong strain found in Korea.9 A positive result was defined as a 4-fold or greater increased titer in the paired sera from patients or a titer of 1:80 in a single serum sample.
Data analysis. We computed the sensitivities and specificities, the positive and negative predictive values, and the 95% CIs for nested PCR and PHA. For all statistical analyses, P values < 0.05 were considered statistically significant. The data were analyzed using SPSS 10.0 software (SPSS, Chicago, IL).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1:10 or by a 4-fold or greater rise in the IFA IgG titer. Ten patients had negative IFA results during both the acute and recovery phases, and they were confirmed as suffering from other diseases. Seven patients were undetermined because their IgM tests were negative or there was a failure to show a 4-fold or greater rise in titer because of follow-up loss. The demographics of the patient population are shown in Table 1
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. Among the 47 patients who were tested within 6 days of the initiation of symptoms, an IgM-positive finding was detected in 34 patients (70.4%), and an IgG titer > 32 was detected in 44 patients (93.6%; Table 2).
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summarizes the sensitivities, specificities, the positive and negative predictive values, and the corresponding 95% CIs for each test. Ninety-six patients of the 118 patients confirmed with IFA were positive for IgM on their admission day. Of the 23 patients who were negative for IgM antibody at admission, 19 patients had positive results on the nested PCR of the buffy coat. All 23 patients with negative results for IgM antibody at admission were followed up, and the IgM of 22 of these 23 patients seroconverted to positive. Although the IgM of the one other patient was negative at 7 days after hospital admission, the IgG of this patient also seroconverted to > 4-fold. Of the seven undetermined patients, six patients had eschar, and all their buffy coat PCRs showed positive results.
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Duration of the PCR positive results. Of the 118 patients confirmed with IFA, 97 were positive on the nested PCR at the time of admission. Sixty-nine patients were followed to the end of 7-day therapy, and 28 patients were lost to follow-up. Eight of these 69 patients were positive on nested PCR; the sampling time of these 8 patients were as follows (2 patients at 5 days after the administration of antibiotics, 3 patients at 6 days after the administration of antibiotics, 2 patients at 7 days after the administration of antibiotics, and 1 patient at 11 days after the administration of antibiotics). Of the eight patients who had positive results on follow-up nested PCR, five patients were followed for another 1 week, and three patients were lost to follow-up. All five patients who were followed had negative PCR results at another 7 days after the end of treatment.
PHA for the diagnosis of scrub typhus.
Forty-nine of the 118 patients who were confirmed by IFA were positive with a titer of 1:80 on their admission serum sample. After 7 days of therapy, 77 of the 118 patients were followed up and tested. Among the 77 patients, 34 patients remained negative on the PHA results. Only 30 patients (47.14%) among the 77 followed-up patients displayed seroconversion of a 4-fold or greater rise in the PHA titer. A total of 64 of the confirmed 118 patients were positive with a titer of 1:80 on the admission and follow-up serum samples. When comparing the PHA results to an admission serum sample tested with IFA, which is the gold standard for the diagnosis of scrub typhus, a sensitivity of 42% and a specificity of 100% were seen. The positive and negative predictive values were 1 and 0.13, respectively (Table 2
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because O. tsutsugamushi is an intracellular organism, the whole blood or the buffy coat is considered to be the preferred samples for PCR tests. In this study, we used the buffy coat for PCR assay, and we used nested PCR to enhance the detection rate. The nested PCR test was based on specific primers derived from the 56-kDa major outer membrane protein antigen of O. tsutsugamushi. This nested PCR method is known to be 100 times more sensitive than performing single PCR for detecting Orientia DNA.2 It has been reported that nested PCR enabled the detection of five copies of O. tsutsugamushi DNA in a specimen. This study was conducted around the season of a scrub typhus epidemic in Korea; hence, our cohort might represent a relatively high prevalence group.
PHA has been widely used for the diagnosis of scrub typhus in Korea. This method is convenient to perform12,13; however, in our group of patients, the PHA assay showed a positive seroconversion of a 4-fold or great rise in the PHA titer for only 30 of the 77 followed-up patients. The PHA assay showed a low sensitivity of 42%, although it showed a high specificity of 100% when the PHA titer–positive result was defined as a titer of 1:80 in a single serum sample at the time of admission compared with the IFA for the patients with suspected scrub typhus. On the other hand, the results of nested PCR assay showed a sensitivity of 82.2% (95% CI: 0.74–0.88), a specificity of 100% (95% CI: 0.66–1), a positive predictive value of 1 (95% CI: 0.95–1), and a negative predictive value of 0.32 (95% CI: 0.17–0.51). In addition, of the 23 patients who were negative for IFA-tested IgM antibody at the time of admission, all 23 patients seroconverted to positive and 19 had positive results for the buffy coat nested PCR. Of the seven undetermined patients who were lost to follow-up, six patients had eschar, and their buffy coat PCRs all showed positive results. Therefore, we found that nested PCR showed positive results during the acute rickettsemia phase, which occurs before the antibody titer increases during scrub typhus infection.
It’s been shown in a study from Thailand that O. tsutsugamushi DNA can to be detected by the nested PCR technique as early as day 3 of the fever phase; this is before the appearance of specific antibodies in the blood.5 However, in our study, O. tsutsugamushi DNA could be detected in the blood by nested PCR as early as the first day of fever onset. We also assessed how long the PCR remains positive for the patients after the clinical presentation. Around 7 days (range: 5–11 days) after antibiotic administration, 11.6% of the patients (8 of 69 patients who were followed) remained positive on nested PCR. Of the eight patients who showed positive PCR results, five patients were followed, and all five patients had negative PCR results after another 7 days. We found that the blood PCR results can remain positive for up to 11 day after the administration of antibiotics.
Twenty-one of the 118 patients who were confirmed with IFA showed false-negative PCR results. Twelve of the 118 patients had received antibiotics before admission. We observed the trend of a false-negative result in those patients who had high immunoglobulin titers on IFA. To clarify the relevance between the high titers of immunoglobulin and the negative PCR results, we subgrouped the patients into two groups according to their IgM antibody titer (i.e., IgM < 1:640 and IgM 1:640), except for the 12 patients who had received prior antibiotic therapy before admission, therefore minimizing the effects caused by antibiotics on the PCR results. Among the 106 confirmed patients who had not received antibiotics, 7 of the 65 patients whose IgM titer was < 1:640 showed negative results on PCR (10.8%). In contrast, 12 of 41 patients whose IgM titers were > 1:640 showed negative PCR results (29.3%, P = 0.031). This high rate of false-negative PCR results for the patients whose IgM antibody titers were high might have been caused by the clearance of Orientia by the immune system.5,14
The failure to detect O. tsutsugamushi DNA in the healthy volunteers suggests that the PCR test has high specificity. Therefore, judicious use of nested PCR for the evaluation of patients with suspected scrub typhus, and especially for the serologically unconfirmed fatal cases, could be a specific diagnostic tool for the early detection of scrub typhus.
In conclusion, the results of our study suggest that nested PCR of the buffy coat, when used in conjunction with IFA, is a rapid and reliable method for diagnosing scrub typhus in patients who are undergoing medical evaluation for fever and rash during the season of epidemic scrub typhus.
Received January 26, 2006. Accepted for publication May 16, 2006.
Disclosure: The authors do not have any commercial interest or other associations that might pose a conflict of interest. The research described in this manuscript did not benefit from any external financial support.
* Address correspondence to Mi-Yeoun Park, Division of Zoonoses, Center for Immunology and Pathology, National Institute of Health, #5 Nokbun-Dong, Eunpyung-Gu, Seoul 122-701, Korea. E-mail: miyeoun{at}nih.go.kr 
Authors’ addresses: Dong-Min Kim and Na Ra Yun, Department of Internal Medicine, Chosun University College of Medicine, 588 Seosuk-dong, Dong-gu, Gwangju 501-717, Korea, Telephone: 82-62-220-3108, Fax: 82-62-234-9653, E-mail: drongkim{at}chosun.ac.kr. Tae Young Yang, Departments of Internal Medicine, Haenam General Hospital, 182-1 Haenam, Jellanamdo 536-809, Korea. Ji Hyun Lee, Departments of Internal Medicine, Jangheung General Hospital, 383-5 Jangheung, Jellanamdo 529-800, Korea. Jong Tae Yang, Departments of Internal Medicine, Muan General Hospital, 165 Muan, Jellanamdo 534-804, Korea. Soo-Kyoung Shim, Eun-Na Choi, and Mi-Yeoun Park, Division of Zoonoses, Center for Immunology and Pathology, National Institute of Health, #5 Nokbun-Dong, Eunpyung-Gu, Seoul 122-701, Korea. Seung-Hyun Lee, Department of Pediatrics, Seonam University College of Medicine, 720 Gwangchi-dong, Nam-won, Jeollabukdo 590-711, Korea, Telephone: 82-62-370-7660, Fax: 82-62-371-3092, E-mail: 31gmlakd{at}hanmil.net.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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