Effects of Antibiotic Treatment on the Results of Nested PCRs for Scrub Typhus

In order to determine the effects of antibiotic treatment onthe results of nested PCRs for scrub typhus, we investigatedthe frequency of positive outcomes at various times after antibioticadministration in patients with scrub typhus. The rate of detectionby nested PCR with blood buffy coat before antibiotic administrationwas 90.5%. However, this decreased to 60.5% within 3 days afteradministration of antibiotics such as doxycycline and rifampinand to 10% by the fourth day following antibiotic administration.Since the sensitivity of nested PCR can be affected by antibioticadministration, clinicians should perform this PCR before antibioticadministration and at the latest within 3 days after antibioticadministration.

INTRODUCTION

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INTRODUCTION

Scrub typhus is transmitted to humans by chiggers infected withOrientia tsutsugamushi that attach to the skin and suck tissuefluid, and it is characterized by eschar, fever, headache, andskin rash (1, 3). It can be diagnosed by isolating O. tsutsugamushifrom patients' blood during the febrile stage or by measuringantibody titers against O. tsutsugamushi during the acute andconvalescent stages (5). However, it takes several weeks toisolate O. tsutsugamushi from cultured cells or infected rats(7, 9), and the sensitivity of isolation is reported to be only46.7% (6). Scrub typhus can be diagnosed on the basis of a fourfoldor greater increase in antibody titer using the passive hemagglutinationassay, indirect immunofluorescent-antibody assay (IFA), indirectimmunoperoxidase assay, or enzyme-linked immunosorbent assay(2, 5). However, it is difficult to make an early diagnosisusing these assays for patients who visit a clinic, becauseantibodies may not have appeared and a follow-up visit may beneeded.

Scrub typhus has also been diagnosed by PCR for O. tsutsugamushi-specificgenes, and this has been shown to be of considerable use inthe early diagnosis of scrub typhus (4, 5, 7). However, itspotential disadvantages include false positivity due to DNAcontamination and a decline in sensitivity following antibioticadministration.

This study was designed to determine the effects of antibioticadministration on the results of nested PCR in patients withscrub typhus; to this end, we measured the positivity rate ofthe nested PCR at various times after antibiotic administration.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Patients with acute febrile diseases who visited Chosun UniversityHospital between September 2006 and December 2007 were includedin the study. The patients were $≥$ 18 years old and had a historyof fever together with eschar or a maculopapular skin rash,as well as more than two of the following symptoms: headache,general weakness, myalgia, cough, nausea, and abdominal discomfort.Informed consent was obtained from the patients or their parentsafter they were given a complete description of the study protocol.The definitive diagnosis of scrub typhus was defined as an immunoglobulinM (IgM) antibody titer of $≥$ 1:160 or a fourfold or greater increasein the IgM or IgG titer measured by IFA. Patients who were diagnosedwith diseases with clinical features similar to those of scrubtyphus, such as murine typhus, leptospirosis, epidemic hemorrhagicfever, and systemic lupus erythematosis, were excluded fromthe study, which was approved by the Institutional Review Boardof our hospital. Nested PCR was performed with blood buffy coat,and IFA was conducted with serum by a method described previously(5).

RESULTS

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RESULTS

Of 141 patients with confirmed scrub typhus between September2006 and December 2007, 129 were investigated and 12 were excludedbecause they had received an antibiotic(s) with antimicrobialeffects against O. tsutsugamushi, such as tetracyclines, macrolides,quinolones, and rifamycins, before admission. We had blood samplesprior to antibiotic administration from 116 of the 129 patients,and 105 of these (90.5%) proved positive in nested PCR for scrubtyphus (Table 1). The positivity rate had declined to 60.5%in blood drawn within 3 days after administration of antibioticssuch as doxycycline and rifampin and was only 10% at approximately4 to 9 days after antibiotic administration. O. tsutsugamushiDNA was detected by nested PCR for a maximum of 13 days afterantibiotic administration (Table 1).

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TABLE 1. Positivity rates for nested PCR at various times after antibiotic treatment in 129 patients with confirmed scrub typhus

DISCUSSION

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DISCUSSION

Scrub typhus may fail to be diagnosed on the basis of serumantibody titers during the acute and convalescent stages becauseantibody formation and a fourfold or greater increase in specificantibody titer require several weeks to occur. In contrast,PCR can detect O. tsutsugamushi DNA in blood from the onsetof symptoms, thus allowing physicians to make an early diagnosis(5). Furthermore, since life-threatening complications or deathcan occur as a result of delays in antibiotic therapy, PCR couldbe very useful for early diagnosis of scrub typhus in the clinicalsetting (5).

It has been reported that the sensitivity and specificity ofconventional PCR targeting the 16S rRNA gene of O. tsutsugamushiwere 44.8% and 99.7%, respectively, in Thai patients (8). Wepreviously reported that the sensitivity and specificity ofnested PCR targeting the gene that encodes the 56-kDa majorouter membrane protein were 82.2% and 100%, respectively. Inour study, we used the buffy coat for PCR assay since O. tsutsugamushiis an intracellular organism, and we used nested PCR to enhancethe sensitivity. This nested PCR method is known to be 100 timesmore sensitive than use of a single PCR for detecting OrientiaDNA (7).

The sensitivity of PCR for the diagnosis of scrub typhus, unlikethat of antibody detection methods, can be affected by antibiotictherapy. In this study, when patients who received antibiotictherapy prior to presentation were excluded, nested PCR showeda positivity rate of 90.5% at diagnosis. However, the positivityrate decreased to 10% by the fourth day following antibioticadministration in the patients who received antibiotic therapy.

In scrub typhus patients, eschars develop at the mite bitingsites, where O. tsutsugamushi multiplies and spreads throughoutthe whole body. Since such eschars contain a large number ofmicroorganisms, PCR performed using eschar specimens can detectOrientia DNA even 1 to 2 weeks after antibiotic administration.Hence, PCR using eschars is less affected by antibiotic therapy.In our previous study (4), we reported that PCR using escharswas positive in 92% (46/52) of patients who had been treatedwith doxycycline for 7 days. In contrast, the present data indicatethat nested PCR with blood buffy coat should be conducted withinat most 3 days after the onset of antibiotic administration,because Orientia DNA is more easily eradicated by antibioticsin blood than in eschars.

In conclusion, the sensitivity of PCR with blood buffy coatcan be affected by antibiotic treatment. The rate of detectionby nested PCR with blood buffy coat before antibiotic administrationwas 90.5%. It had fallen to 60.5% within 3 days of antibioticadministration and was only 10% by the fourth day of antibioticadministration. Therefore, clinicians should perform PCR withblood drawn before antibiotic administration or at the latest3 days after antibiotic administration.

ACKNOWLEDGMENTS

This study was supported by research funds from Chosun University,2007.

The authors do not have any commercial interest or other associationthat might pose a conflict of interest.

FOOTNOTES

* Corresponding author. Mailing address: Department of Radiology, Chosun University College of Medicine, 588 Seosuk-dong, Dong-gu, Gwangju 501-717, Republic of Korea. Phone: 82-62-220-3731. Fax: 82-62-234-9653. E-mail: jnbyun{at}chosun.ac.kr

Published ahead of print on 20 August 2008.

REFERENCES

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