Am. J. Trop. Med. Hyg., 77(4), 2007, pp. 719-722
Copyright © 2007 by The American Society of Tropical Medicine and Hygiene
Severe Scrub Typhus Confirmed Early via Immunohistochemical Staining
Dong-Min Kim,
Sung-Chul Lim,
Kyung Jun Won,
Yeon-Joo Choi,
Kyung-Hee Park, AND
Won-Jong Jang*
Division of Infectious Diseases, Department of Internal Medicine, Chosun University College of Medicine, Gwangju, Korea; Department of Pathology and Research Center for Resistant Cells, Chosun University College of Medicine, Gwangju, Korea; Department of Microbiology, Konkuk University College of Medicine, Choongju, Republic of Korea
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ABSTRACT
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We describe a case of interstitial pneumonia that was confirmed as scrub typhus by immunohistochemical (IHC) staining of an eschar. When a patient presents with interstitial pneumonia accompanied by generalized lymphadenopathy on the thoracic CT scan, clinicians should suspect scrub typhus, especially when the patient has a history of travel to a scrub typhus-endemic area. IHC staining on an eschar revealed invasion by Orientia tsutsugamushi coccobacilli of the patient’s sweat ducts and glands as well as vascular endothelium. IHC staining of an eschar could be used as an early-confirmation diagnostic method to establish scrub typhus.
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INTRODUCTION
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Scrub typhus is an acute febrile illness characterized by the presence of a vasculitis caused by Orientia tsutsugamushi. It occurs frequently in Asia and the Pacific islands, but because of increasing worldwide travel, cases have also been identified recently in the West.1,2 Typical physical findings are eschars and maculopapular skin rashes, which develop over the trunk and limbs. It has a poor prognosis when complications such as hemorrhagic gastritis, acute respiratory distress syndrome (ARDS), and meningitis are present.3 Here we describe a patient who presented with pneumonia and was confirmed to have scrub typhus by immunohistochemical (IHC) staining of a suspected eschar.
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CASE REPORT
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A 79-year-old female farmer was admitted to the Department of Surgery at the researchers’ hospital for claudication that had been worsening over the past 6 days. When the patient was diagnosed with arteriosclerosis obliterans, a stent was inserted into her left iliac artery, with patient heparinization. Three days before the patient was transferred to the hospital’s Department of Internal Medicine, she developed a fever of > 38°C, and 1 day before the transfer, a rash was detected over her trunk and limbs. The patient complained of dyspnea and was thus transferred to the Department of Internal Medicine’s Infectious Diseases Division.
The patient’s laboratory tests gave the following results: leukocyte count, 12,120/mm3 (> 90% neutrophils by differential count); hemoglobin count, 7.3 g/dL; platelet count, 142,000/mm3; AST/ALT, 151/54 IU; and LDH/CPK, 620/314 U/L (180–460/26–200). ABGA showed that the patient’s blood pH was 7.43, PaCO2 was 38 mm of Hg, PaO2 was 71 mm of Hg, O2Sat was 94.8%, and HCO3 was 24.9 mmol/L. A chest x-ray revealed that the patient had interstitial pneumonia and that both lung fields had pleural effusions (Figure 1
). Thoracic computed tomography (CT) showed multiple ground-glass–like lesions, bronchial wall thickening, and bilateral pleural fluid, with diffuse lymphadenopathy observed in the parahilum, the mediastinum, the posterior neck, and the axilla (Figure 1). Examination of the pleural fluid gave a leukocyte (WBC) count of 713/mm3 (poly 30%, mono 70%) and a pH of 7.455; albumin was 1.69 g/dL, protein was 2.53 g/dL, glucose 130 mg/dL, LDH 282 U/L, and ADA was 36 IU/L. To rule out pulmonary congestion due to heart problems, echocardiography was performed. No functional or structural abnormalities of the heart were detected. Physical examination of the patient at the time of transfer showed that not only were there rashes all over the patient’s body but there were also lesions like cigarette burns on her right shoulder, resembling eschar lesions (Figure 2a). With the patient’s consent, a 3-mm punch biopsy of an eschar-like lesion and subsequent IHC staining thereof were performed. The primary antibody used in the study was the ICR mouse hyper-immune serum against O. tsutsugamushi strain Boryong at a dilution of 1:200. This antibody also detects various strains of O. tsutsugamushi in immunofluorescence antibody (IFA) assays (data now shown). IHC staining was performed using a streptavidin–biotin immunoperoxidase method, according to the supplier’s protocol (LSAB kit, DAKO, Carpinteria, CA). At the same time, serological tests for leptospirosis, hemorrhagic fever with renal syndrome (HFRS), scrub typhus, and murine typhus were performed. The IHC staining of the eschar-like lesion revealed brown-colored coccobacilli, confirming the researchers’ suspicions (Figure 3). After this early confirmation of scrub typhus by IHC staining, doxycycline was administered. IFA assays using paired sera and L929 cells infected with O. tsutsugamushi strain Boryong revealed a positive conversion against the Orientia antigen (IgM < 1:10, IgG 1:2,048 at the time of the transfer, and IgM < 1:10, IgG 1:10,240, 10 days later). The antibody tests against HFRS, leptospirosis, and murine typhus were all negative.
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FIGURE 1. Thin-section CT image of a 79-year-old woman confirmed to have scrub typhus. (a) Both lung fields show thickening of the interlobular septum (black arrow) and pleural effusion. (b) Peribronchial wall thickening (black arrow), a large amount of bilateral pleural effusion, and a nodular ground-glass appearance in the periphery of both lower lung fields. (c) Lymphadenopathy in the posterior neck and axilla (white arrow). (d) Lymphadenopathy in the parahilum and mediastinum on the CT image (white arrow).
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FIGURE 2. (a) Atypical eschar on the right shoulder of the subject, who was confirmed to have scrub typhus. (b) Typical eschar on another patient with scrub typhus, confirmed by immunohistochemical staining. This figure appears in color at www.ajtmh.org.
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FIGURE 3. Histopathologic findings and immunohistochemical staining of the eschar on the right shoulder of the patient with scrub typhus. (a) Note the confluent epidermal necrosis with dermal vascular dilatation and perivascular inflammation. Mononuclear cell infiltration with lymphocytes and macrophages around the sweat ducts and glands is also visible (H & E; original magnification, 40x). (b) Strong intracytoplasmic immunohistochemical staining of the sweat duct’s epithelial lining (LSAB method, counterstained with Mayer’s hematoxylin; original magnification, 400x). (c) Positive immunohistochemical staining of the vascular endothelial cells and a few mononuclear cells scattered around the small blood vessels (LSAB method, counterstained with Mayer’s hematoxylin; original magnification, 1,000x). (d) Orientia coccobacilli within the lumina or cytoplasm of the sweat ducts and glands’ epithelial lining as seen by indirect immunofluorescence staining (FITC; original magnification, 400x). This figure appears in color at www.ajtmh.org.
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After administration of antibiotics, the patient’s pneumonia improved, the ground-glass–like lesions and pneumonic infiltration detected at the time of her transfer disappeared, and her effusion and congestion improved. The patient was discharged without complications.
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DISCUSSION
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Scrub typhus is confirmed by isolating O. tsutsugamushi from the blood of patients during the acute febrile period or by an increase of antibody titer against O. tsutsugamushi in the serum during the acute or recovery phase.4 However, isolation of O. tsutsugamushi from cultured cells or infected rats is only practical in research institutes, where biologic safety is assured, and Orientia culture takes a minimum of several weeks.4,5 As reported previously, the passive hemagglutination test, frequently used for diagnosis of scrub typhus, has low sensitivity and often cross-reacts with other diseases.6 IFA or immunoperoxidase assays are the gold standards for the diagnosis of scrub typhus. However, because the increase of the antibody titer after disease onset generally takes from several days to several weeks, it would be difficult to diagnose the disease during its early stages by these methods, and a delay in administration of effective antibiotics can cause fatal complications.7 Therefore a rapid and accurate diagnosis is required.8 It is well known that the isolation of O. tsutsugamushi DNA from the blood or eschar, using the polymerase chain reaction, can be used as a rapid early diagnostic method.6,9,10 However, with the PCR assay, there is a potential problem of false positives caused by DNA contamination; this can be a potential problem with nested PCR assays, but it rarely happens with state-of-the-art real-time PCR assays. Indeed, the latter could be a useful and promising study for diagnosing scrub typhus and eschar producing spotted fever-group rickettsioses. Real-time PCR assays are useful as they give fast results, can potentially be automated for high throughput, and provide quantitative information useful for assessing prognosis and response to treatment.11,12 The benefit of IHC staining is that it provides a diagnosis faster than waiting for a 4-fold rise in titer or seroconversion to occur in an acutely ill patient, especially in such settings where PCR assays are not available. A method using IHC staining on a skin biopsy has been reported to be a sensitive and specific diagnostic method for diagnosis of Rocky Mountain spotted fever or Rickettsialpox.13–15 However, to date, no such test has been used with an eschar to confirm a diagnosis of scrub typhus. The typical eschar is a lesion with a black crust at its center and slightly elevated erythema on its periphery (Figure 2b
). The eschar-like lesions of the patient in this study, however, differed from typical eschar lesions. Hence to confirm that they were due to scrub typhus, we performed IHC staining.
Evaluation of the eschar skin biopsy confirmed the presence of O. tsutsugamushi coccobacilli in the cytoplasm of the patient’s infected vascular endothelial cells and also within the macrophages in the vicinity of the blood vessels. The presence of fragmented, granular, and fused Orientia was also confirmed. Of note was the finding of numerous coccobacilli in the cytoplasm of the epithelial lining covering the surface of the sweat ducts and glands in the mid-dermis and deep dermis. This result was confirmed through IFA assay using mouse hyperimmune serum against O. tsutsugamushi strain Boryong at a dilution of 1:200 and the fluorescein isothiocyanate (FITC)-conjugated goat anti-human immunoglobulin (Ig) G (heavy plus light chains; cat. no. 109-095-003, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) antibody diluted in PBS at 1:100 (Figure 3d).
According to a report by Song and others, when lung lesions are present, interstitial pneumonia is a frequent complication in patients with scrub typhus. It was also proposed that interstitial pneumonia is a clinical index of the severity of the patient’s condition.16 For patients with scrub typhus, the basic pathology of the lungs is vasculitis as well as interstitial pneumonia. Common radiologic findings in scrub typhus are reported to be diffuse bilateral reticulonodular or ground-glass opacities with lower lung predominance.17 In the present case, we observed bronchial wall thickening, interlobular septal thickening, and ground-glass opacities consistent with interstitial pneumonia; we also noted generalized lymphadenopathy including the mediastinum, posterior neck, and the axilla on the thoracic CT. Thus, when a clinician encounters the symptoms of interstitial pneumonia, together with generalized lymphadenopathy as in this case, scrub typhus or other rickettsiosis should be considered in the differential diagnosis, especially in patients with a history of travel to scrub typhus-endemic areas. Further systemic studies, including thin-section CT, must be conducted to confirm the relevance of the radiologic findings.
In conclusion, the case of scrub typhus presented herein has clinical significance in 2 respects. First, IHC staining of an eschar was used in the human diagnosis of scrub typhus, and the clinical efficacy of this procedure for early diagnosis was demonstrated. Our findings suggest that systemic studies of the sensitivity and specificity of papule or eschar biopsy-specific IHC staining, which have not yet been evaluated, should be carried out. Our findings also show that the sweat ducts and glands as well as the vascular endothelium are major sites of involvement of O. tsutsugamushi. Second, this paper is regarded as the first report to suggest that clinicians should suspect scrub typhus when patients with a history of travel to scrub typhus-endemic areas present with interstitial pneumonia accompanying generalized lymphadenopathy on their CT scans.
Received January 23, 2007.
Accepted for publication June 12, 2007.
Acknowledgment: This study was supported by research funds from Chosun University, 2006.
* Address correspondence to Won-Jong Jang, Department of Microbiology, Konkuk University College of Medicine, 322 Danwol-dong, Choongju-si, Choongbuk 380-701, Republic of Korea. E-mail: wjjang{at}kku.ac.kr 
Authors’ addresses: Dong-Min Kim and Kyung Jun Won, Department of Internal Medicine, Chosun University College of Medicine, 588 Seosuk-dong, Dong-Gu, Gwangju 501-717, Republic of Korea, Telephone: +82-62-220-3108, Fax: +82-62-234-9653, E-mail: drongkim{at}chosun.ac.kr. Sung-Chul Lim, Department of Pathology and Research Center for Resistant Cells, 588 Seosuk-dong, Dong-gu, Gwangju, 501-717, Republic of Korea, Telephone: +82-62-230-6343, Fax: +82-62-234-4584, E-mail: sclim{at}chosun.ac.kr. Yeon-Joo Choi, Kyung-Hee Park, and Won-Jong Jang, Department of Microbiology, Konkuk University College of Medicine, 322 Danwol-dong, Choongju-si, Choongbuk 380-701, Republic of Korea, Telephone: +82-43-840-3724, Fax: +82-43-851-9329, E-mail: wjjang{at}kku.ac.kr.
D. M. Kim and S. C. Lim contributed equally to this work.
Reprint requests: Dong-Min Kim, Division of Infectious Diseases, Department of Internal Medicine, Chosun University College of Medicine, 588 Seosuk-dong, Dong-gu, Gwangju, 501-717, Republic of Korea. E-mail: drongkim{at}chosun.ac.kr.
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ABSTRACT
INTRODUCTION
