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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 definitive diagnosis of scrub typhus is made by the isolation of O. tsutsugamushi from blood. This is not practical for routine diagnostic laboratories because culture has poor sensitivity and requires growth in a cell monolayer and containment level 3 facilities, and the organism may take more than 1 month to isolate. The indirect immunofluorescent antibody (IFA) test is used widely to confirm the diagnosis using acute and convalescent sera, but by its nature provides a retrospective diagnosis. Rapid diagnosis would contribute toward acute patient management; the two diagnostic options are detection of antibodies using a bedside test and detection of organisms or their nucleic acid or antigens in blood or other clinical samples. Rapid serology tests have been described,7 but their use in early diagnosis is not clear. Detection of O. tsutsugamushi in blood during human infection using a nested polymerase chain reaction (PCR) was reported for 12 patients presenting with a febrile illness to a hospital in southern Thailand; this targeted the gene encoding the major 56-kd surface antigen.8 A second study reported the use of real-time PCR to detect the 47-kd gene in 10 culture-positive patients and in 7 of 17 (41%) culture-negative individuals with serologically proven scrub typhus infection.9 Such techniques have not been evaluated for the rapid diagnosis of a large patient population. The purpose of this study was to compare PCR amplification of a region of the 16S rRNA gene of O. tsutsugamushi for the early diagnosis of 183 cases of scrub typhus in an endemic area of Thailand.
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MATERIALS AND METHODS |
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15 years of age, had fever (> 37.8°C) of unknown cause, had given written informed consent to participate, and agreed to out-patient follow-up and a further blood test at 2 weeks. Patients with a blood smear positive for malaria parasites or other definable infections such as pneumonia or urinary tract infection were excluded. Blood was drawn on admission for aerobic blood culture, serologic testing, and molecular diagnostics. A second (convalescent) serum sample was taken at ~2 weeks after presentation. Duration of symptoms before admission, antibiotic treatment at presentation, clinical features, and inhospital outcome (survival or death) were recorded using a standardized data collection form. Blood samples were maintained at –20°C before use. The study protocol was approved by the Ethical Review Sub-committee of the Ministry of Public Health, Thailand.
Serological testing.
The IFA assay was used as the reference standard for the diagnosis of scrub typhus. This was performed on paired (acute and convalescent) sera at Siriraj Hospital using established methodology.10 In brief, pooled antigens of O. tsutsugamushi strain Karp, Kato and Gilliam were spotted on a glass slide kindly provided by The National Research Institute of Health (NIH), Ministry of Public Health, Thailand. Initial screening was performed using a dilution of 1:50, after which positives were assayed using 2-fold serial dilutions from 1:100 to 1:6,400. Antibody binding was determined using a fluorescent microscope (Olympus BX50, Olympus Corporation, Tokyo, Japan). Known positive and negative control sera were run with each experiment. A positive result for scrub typhus infection was defined as a single IFA IgM titer against O. tsutsugamushi of 1:400 or a 4-fold or greater rise in IFA IgM titer,11 and/or a single IgG titer 1:800 or a 4-fold or greater rise in IgG titer.
Molecular testing. Total genomic DNA was extracted from a 5-mL EDTA admission blood sample using the Nucleon BACC 3 kit (Amersham, Biosciences UK Ltd, Buckinghamshire, UK). Bacterial genomic DNA was extracted from laboratory culture using the Wizard SV Genomic DNA purification kit (Promega, Madison, WI). Primers were designed to be specific for O. tsutsugamushi by aligning the 16S rRNA gene sequence for O. tsutsugamushi strain Kato (accession no. D38624), strain Karp (accession no. D38623), strain Gilliam (accession no. L36222), Rickettsia typhi (accession no. L36221), R. prowazekii (accession no. M21789), R. canadensis (accession no. L36104), and R. sibirica (accession no. D38628). Scrub typhus and leptospirosis can present with very similar clinical features and may cause a dual infection. In view of this and the fact that rapid serologic tests may show cross-reactivity between rickettsia and leptospires, the alignment further included Leptospira interrogans (accession no. Z12817), L. weilii (accession no. AY034037), and L. borgpetersenii (accession no. AY149231). Primers were designed using Primer 3 software and were predicted to amplify O. tsutsugamushi alone. The primers OT1-F (5'-CGAATTAAT-GCTGAGTTTGCTTAG-3') and OT1-R (5'-CTCTCAGA-CCAGCTACAGATCACA-3') gave a predicted amplification product of 220 bp (position 48–71 and 245–268 of sequence accession number L36222).
Verification of primer specificity was performed using genomic DNA from O. tsutsugamushi strain Kato, R. typhi, R. prowazekii, R. conorii, R. australis, R. honei, Burkholderia pseudomallei, B. mallei, Pseudomonas aeruginosa, Escherichia coli, Enterococcus sp., Aeromonas hydrophila, Klebsiella pneumoniae, Salmonella typhi, Staphylococcus aureus, and L. interrogans serovar autumnalis. The reaction mix contained 1x PCR buffer, 1.5 mmol/L MgCl2, 200 µmol/L of dNTP, 1 µmol/L of each OT1-F and OT1-R primers, 1.25 U Taq Polymerase I enzyme, and 50–100 ng genomic DNA as template in a total volume of 50 µL. Amplifications were performed using a DNA engine PTC-200 (MJ Research, Watertown, MA) and the following cycling conditions: 95°C for 3 minutes (1 cycle), followed by 40 cycles of 90°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds. Amplification products were run on a 3% agarose gel, stained with ethidium bromide, and photographed under UV light using the Gel Doc 1000 system (Bio-Rad Laboratories, Hercules, CA).
Testing of clinical samples from patients presenting with febrile illness was performed using total DNA extracted from admission blood samples. Amplification was performed using the Rotor-Gene 3000 real time thermal cycler (Corbett Research Ltd., Sydney, Australia). The reaction mixture consisted of 1x iQ SYBR Green Supermix (Bio-Rad), 0.5 µmol/L of each OT1-F and OT1-R, 5 µL DNA sample, and sterile distilled water to a total volume of 25 µL. Cycling conditions were 95°C for 3 minutes (1 cycle), followed by 40 cycles of 90°C for 30 seconds, 60°C for 10 seconds, and 72°C for 20 seconds. After standardization, a positive result on melt curve analysis was set at 84°C. DNA extracted from O. tsutsugamushi strain Kato culture and L. interrogans serovar Autumnalis was used as positive and negative controls, respectively.
Rapid diagnosis using conventional PCR to amplify a 483-bp region of the gene encoding the 56-kd outer membrane protein has been described previously.12 This methodology was applied to all clinical samples in this study. The reaction was performed using a DNA engine PTC-200 (MJ Research). Amplification products were run on a 2% agarose gel electrophoresis, stained with ethidium bromide, and photographed under UV light as before.
Testing strategy. All patients identified by IFA as having scrub typhus were selected as cases. A control group of two controls for each case was formed by selecting two patients who did not have scrub typhus who were admitted immediately after a scrub typhus case. In the event that sequential patients were cases, an appropriate number of consecutive controls were selected immediately after the cases. A database was created of cases and controls, after which these were randomized and blinded before testing.
Statistical analysis. Sensitivity and specificity were calculated with exact 95% confidence intervals (CIs). Proportions were compared using the Fisher exact test, and the association between PCR positivity and days of symptoms before hospital admission was analyzed by logistic regression using Stata/SE 8.0 (Stata Corp., College Station, TX).
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RESULTS |
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The number of days between onset of symptoms and hospital presentation for patients with positive scrub typhus serology ranged from 1 to 60 days (median, 4.5 days; interquartile range [IQR], 3–7 days). An eschar was noted in 11 of 183 patients (6%). Two patients were taking antibiotics on admission that were predicted to be effective treatment of scrub typhus in this geographic area (chloramphenicol, one patient; doxycycline, one patient). Seven patients (3.8%) died as a result of scrub typhus infection during hospital admission.
Verification of 16S rRNA PCR primer specificity was performed using genomic DNA from a range of bacteria chosen to represent those most likely to be encountered by our patient group. Amplification products were only seen for O. tsutsugamushi (Figure 1
). PCR amplification of clinical samples was positive in 82 of 183 (44.8%) patients with positive IFA serology indicative of scrub typhus and 1 of 366 controls (0.27%) who were negative on IFA. The single patient with a negative IFA in whom O. tsutsugamushi DNA was detected had an eschar on presentation and is likely to represent a false-negative IFA (IgM and IgG titers were negative for paired sera in this case). None of the 27 patients who were diagnosed as having scrub typhus on the basis of IFA IgG titer alone were positive by PCR. Concordance between IFA and PCR is detailed in Table 1. The number of days of illness before admission for PCR-positive and PCR-negative cases was 7.1 ± 3.0 and 5.3 ± 4.7 (SD) days, respectively. The rate of PCR positivity was positively related to the number of symptomatic days before hospital presentation (P < 0.001) and the presence of an eschar (P < 0.001). PCR positivity was not related to death (P = 1).
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. The sensitivity of the 56-kd gene PCR was lower than that for the 16S rRNA gene PCR (P < 0.0001, McNemar test). To further explore the basis for the low sensitivity of PCR amplification of the 16S rRNA gene, we tested 16 blood samples taken on the day of admission from Thai patients presenting with unexplained fever during 2003–2004 who were proven by us to be culture positive for O. tsutsugamushi. The 16S rRNA PCR was positive for 13 of 16 (81.3%) samples (data not shown). To detect the presence of PCR inhibitors in the DNA sample, the three samples that were PCR negative were spiked with known concentrations of O. tsutsugamushi genomic DNA. The 16S rRNA PCR detected spiked DNA samples at the same sensitivity level as genomic O. tsutsugamushi DNA, suggesting that PCR inhibitors in the extracted sample DNA did not contribute to reduce sensitivity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A further explanation for low PCR sensitivity is that the IFA titers used to diagnose scrub typhus in this study may lack specificity in this highly endemic setting. None of the patients diagnosed as having scrub typhus on the basis of IgG alone were positive by PCR, suggesting that this assay may be a poor predictor of disease or that reinfections are associated with more rapid clearance of the bacteria. IgM seems to be more specific, in that 30/79 patients (38%) with a fold rising IgM titer and 52/77 patients (68%) with a single high IgM titer were PCR positive.
The lower sensitivity of PCR amplification of a fragment of the 56-kd gene compared with PCR targeting the 16S rRNA gene may relate to the technology used (conventional PCR versus real-time PCR, respectively). The 56-kd gene also contains regions of genetic polymorphism, and it is possible that failure of amplification is related to variability in sequence in the region of primer annealing.
The results of this study are unlikely to alter routine clinical practice, because the PCR described here does not offer sufficient accuracy on which to base a definitive diagnosis. The high specificity of the 16S rRNA PCR could be used by research studies in which entry criteria depended on a definitive diagnosis. The single case with positive PCR but negative IFA is likely to represent a false-negative IFA and is consistent with previous studies in which PCR detection identifies cases of bacterial disease in patients who fail to raise a detectable antibody response. Further work is required to define the extent and patterns of shedding of rickettsia in the bloodstream and optimal timing for culture, together with techniques that increase the sensitivity of molecular techniques for the detection of bacterial DNA.
Received April 25, 2006. Accepted for publication August 15, 2006.
Acknowledgments: The authors thank our colleagues at Udon Thani Hospital and the Mahidol-Oxford Tropical Medicine Research Unit and Mongkol Chenchittikul from NIH, Ministry of Public Health of Thailand, who kindly provide antigen slides for IFA testing. The authors thank Daniel Paris for critical review of the manuscript.
Financial support: This study was funded by a grant awarded to P.S. by the Federation of Infection Society, UK, and by the Wellcome Trust of Great Britain. S.P. holds a Wellcome Trust Career Development Award in Clinical Tropical Medicine.
* Address correspondence to Sharon J. Peacock, Wellcome Unit, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok 10400, Thailand. E-mail: sharon{at}tropmedres.ac 
Authors’ addresses: Piengchan Sonthayanon, Department of Clinical Tropical Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok Thailand 10400, Telephone: 662-354-9100 ext. 2025, Fax: 662-354-9169, E-mail: piengchan{at}tropmedres.ac. Wirongrong Chierakul, Department of Clinical Tropical Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok Thailand 10400, Telephone: 662-354-9100 ext. 1427, Fax: 662-354-9169, E-mail: kae{at}tropmedres.ac. Vanaporn Wuthiekanun, Wellcome Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok Thailand 10400, Telephone: 662-354-1395, Fax: 662-354-9169, E-mail: lek{at}tropmedres.ac. Stuart D. Blacksell, Wellcome Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok Thailand 10400, Telephone: 662-354-1395, Fax: 662-354-9169, E-mail: stuart{at}tropmedres.ac. Kriangsak Pimda, Medical Department, Udon Thani Hospital, Udon Thani, 41000 Thailand. Telephone: 664-234-8888. Yupin Suputtamongkol, Department of Medicine, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, 10700 Thailand. Telephone; 662-419-7203, E-mail: siysp{at}mahidol.ac.th. Sasithon Pukrittayakamee, Department of Clinical Tropical Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok Thailand 10400, Telephone: 662-354-9100 ext., Fax: 662-354-9169, E-mail: yon{at}tropmedres.ac. Nicholas J. White, Wellcome Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok Thailand 10400, Telephone: 662-354-1395, Fax: 662-354-9169, E-mail: nickwdt{at}tropmedres.ac. Nicholas P. Day, Wellcome Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok Thailand 10400, Telephone: 662-354-1395, Fax: 662-354-9169, E-mail: Nickd{at}tropmedres.ac. Sharon J. Peacock, Wellcome Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok Thailand 10400, Telephone: 662-354-1395, Fax: 662-354-9169, E-mail: sharon{at}tropmedres.ac.
Reprint requests: Sharon Peacock, Wellcome Unit, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok 10400, Thailand. E-mail: sharon{at}tropmedres.ac.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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