Am. J. Trop. Med. Hyg., 101(2), 2019, pp. 332–335 doi:10.4269/ajtmh.18-0631 Copyright © 2019 by The American Society of Tropical Medicine and Hygiene

Case Report: Coinfection with monacensis and

Seok Won Kim,1† Choon-Mee Kim,2† Dong-Min Kim,3* and Na Ra Yun3 1Department of Neurosurgery, College of Medicine, Chosun University, Gwangju, Republic of Korea; 2Premedical Science, College of Medicine, Chosun University, Gwangju, Republic of Korea; 3Department of Internal Medicine, College of Medicine, Chosun University, Gwangju, Republic of Korea

Abstract. Rickettsia monacensis and Orientia tsutsugamushi are of the family , which causes fever, rash, and eschar formation; outdoor activities are a risk factor for Rickettsiaceae infection. A 75-year-old woman presented with fever, rash, and eschar and was confirmed as being scrub based on a nested-polymerase chain reaction (N-PCR) test for a 56-kDa gene of O. tsutsugamushi; the genome was identified as the Boryong genotype. In addition, a pan-Rickettsia real-time PCR test was positive and a N-PCR test using a Rickettsia-specific partial outer membrane protein A (rOmpA) confirmed R. monacensis. This is the first case wherein a patient suspected of having owing to the presence of rash and eschar was also found to be coinfected with O. tsutsugamushi and R. monacensis based on molecular testing.

INTRODUCTION leukocyte count, 7,200/mm3; hemoglobin, 11.6 g/dL; platelet count, 232,000/mm3; and erythrocyte sedimentation rate, 31 Rickettsia monacensis is a pathogen that causes spotted mm/hours. C-reactive protein and procalcitonin levels were fever group rickettsial infection; the main symptoms of in- elevated at 9.26 mg/dL and 0.836 ng/mL (0–0.5 ng/mL), re- fection include fever, headache, and myalgia, as well as es- 1 spectively. Levels of blood electrolytes (Na/K/Cl) were 129/ char or rash. Orientia tsutsugamushi, the causative bacterium 4.1/95 mEq/L. Biochemical examination of the serum revealed of scrub typhus, is a rickettsial infectious agent; clinical 2 a ratio of 59.1:62.9 (mg/dL) for aspartate transaminase:alanine manifestations of infection include fever, eschar, and rash. aminotransferase; total bilirubin, 0.78 mg/dL; blood urea Although R. monacensis was previously isolated from a pa- 3 nitrogen/creatinine, 14.1/1.0 mg/dL); serum lactate dehydro- tient in Korea, there has been no report of R. monacensis and genase, 584 U/L; and adenosine deaminase, 54.4 U/L; there O. tsutsugamushi coinfection to date. Herein, we report the were no abnormal urinalysis findings. Arterial blood test re- first case of a patient admitted owing to fever who was found sults at the time of visit revealed pH, 7.533; pCO2/pO2, 27.4/ to have rash and eschar, thus leading to suspicion of scrub 89.3 mmHg; HCO , 26.3 mmol/L; and O saturation 98.5%, fi 3 2 typhus; however, subsequent molecular testing con rmed indicating metabolic alkalosis. Cardiac enzyme levels were R. monacensis and O. tsutsugamushi coinfection. within normal ranges with creatine kinase MB of 4.9 ng/mL and high-sensitivity troponin T of 0.011 ng/mL. Brain computed CASE REPORT tomography was conducted because of her drowsiness, but showed no abnormal results. Cerebrospinal fluid (CSF) ex- A 75-year-old woman presented with chills as her main amination revealed two white blood cells (WBCs), zero red complaint. Her chills continuously persisted for 4 days before blood cells (RBCs), protein 47.3 mg/dL, and glucose 91.6 mg/ her hospital visit on October 16. Moreover, she had engaged in dL (serum 208 mg/dL). frequent outdoor activity of collecting persimmons or chest- The patient was involved in outdoor activities and displayed nuts in the forest. Her medical history indicated that the patient characteristic eschar findings. Using the patient’s buffy coat from had been taking medications for 10 years after being di- the blood sample, polymerase chain reaction (PCR) test was agnosed with hypertension and diabetes, but she had no positive for a 56-kDa target gene of O. tsutsugamushi4;sub- history of hepatitis or tuberculosis. Her symptoms started on sequent sequencing analysis confirmed the Boryong genotype October 13. (accession no. MK613928). Immunofluorescence antibody as- Vital signs at the time of visit were as follows: blood pres- say (IFA) examination for O. tsutsugamushi5 revealed an IgM ratio sure, 110/70 mmHg; pulse rate, 98 beats/minutes; breathing of 1:16 and an IgG ratio of 1:128, whereas total Ig antibody titer ° rate, 22 times/minutes; and body temperature, 38.4 C. De- examined by Green Cross Laboratories (Youngin, Korea) was fi spite her drowsy state, there were no abnormal ndings on elevated at 1:640; IFA testing for R. conorii by the Korea Centers neurologic examination, although cervical lymphadenopathy for Disease Control and Prevention (KCDC) revealed values was observed. Results of heart and lung auscultation were of < 1:32 for both IgM and IgG antibodies on admission (October normal, and abdominal examination revealed no hepato- 21) and at follow-up (December 18). IFA testing for R. japonica megaly, splenomegaly, or abdominal pain. There was an er- revealed an IgM ratio of < 1:16 and an IgG ratio of 1:32 on ad- ythematous macular rash, and the scalp had eschar (Figure 1). mission(October21)andanIgMratioof1:16andanIgGratioof Test results of peripheral blood collected on October 21 and 1:128 at follow-up (December 18). The patient was diagnosed those of blood collected on admission were as follows: with scrub typhus infection and was administered 200 mg doxycycline daily. From the second day of hospitalization, her fever subsided, and her symptoms improved; although the * Address correspondence to Dong-Min Kim, Department of Internal weakness remained, she was discharged. Medicine, School of Medicine, Chosun University, 588 Seosuk-dong, Dong-gu, Gwangju 501-717, Republic of Korea. E-mail: drongkim@ To detect Rickettsia species, pan-rickettsial real-time PCR chosun.ac.kr that targeted the 23S rRNA was performed using blood col- † These authors contributed equally to this work. lected at the time of hospitalization; as the results were positive,6 332 COINFECTION WITH R. MONACENSIS AND O. TSUTSUGAMUSHI 333

FIGURE 1. A 75-year-old woman was admitted with chills and rash as the primary complaints. (A) Eschar lesion identified on the head. (B) Erythematous rash on the face. (C) Conjunctival suffusion in both the eyes. (D) Maculopapular rash on the abdomen. (E) Phylogenetic tree for Rickettsia spp. based on the partial outer membrane protein A (ompA) gene (521 nucleotides). The number at the node represents the bootstrap confidence levels for 1,000 replicates. The black triangles represent the isolates obtained in our study. (F) Phylogenetic tree for Orientia tsutsugamushi based on the partial 56-kDa protein gene (435 nucleotides). The number at the node represents bootstrap confidence levels for 1,000 replicates. The black triangles represent the isolates obtained in our study. This figure appears in color at www.ajtmh.org. 334 KIM, KIM, AND OTHERS the real-time PCR amplicon was subjected to sequencing no Rickettsia-related symptoms; thus, the pathogenic role of analysis, which confirmedthepresenceofR. monacensis.To R. monacensis was unclear. Interestingly, CSF from the patient in reconfirm the result using an additional target gene, nested-PCR the present case was negative for O. tsutsugamushi using PCR, testing using Rickettsia-specific rOmpA was performed and was whereas it was positive for R. monacensis. Thus, further studies positive; subsequent sequencing analysis reconfirmed the on the pathogenicity of R. monacensis are required. presence of R. monacensis (accession no. MK613926). Using In a study conducted in southwestern Slovakia using rodents CSF, a N-PCR test for the Rickettsia-specific partial outer and ectoparasitic , 112 of 345 pool samples after mite membrane protein A (rOmpA) also confirmed R. monacensis pooling (32.46%) and 46 of 487 rodent blood samples (9.44%) (Figure 1) (accession no. MK613927). Homology was tested were positive for Rickettsia DNA, and sequencing analysis based on sequence alignment, in which R. monacensis identified confirmed the presence of R. helvetica and R. monacensis.13 in the buffy coat showed 99.6% homology with R. monacensis Another study from China revealed a unique Rickettsia species identified in CSF. The buffy coat isolate showed 96.9% homology that was detected in Leptotrombidium scutellare;thisstudy with the Korea R. monacensis isolate CN45kr (accession no. expanded the knowledge on the vector distribution of Rickettsia KC993862), 98.3% homology with the Chinese R. monacensis spp.14 These findings suggested that mites per se act as a isolate (accession no. EU665232), and 98.7% homology with vector and a reservoir of R. monacensis.InKorea,scrubtyphus Rickettsia sp. ZJ42/2007 (accession no. EU258734). Finally, caused by O. tsutsugamushi is the most common rickettsial coinfection with O. tsutsugamushi and R. monacensis was infection, for which the vector and reservoir are larval trombi- confirmed. culid mites, a Leptotrombidium spp. Because there are various mites in Korea, coinfection is theoretically possible, and the DISCUSSION roles of mites in Korea as a cause of coinfection and a vector of R. monacensis should be further studied. Korea is an endemic region of scrub typhus, with about In conclusion, the present study reports the first confirma- 10,000 cases reported to the KCDC every year. Most inci- tion of coinfection with O. tsutsugamushi and R. monacensis dences occur from late October to early November.7 Rick- based on molecular testing in a patient suspected of having ettsia monacensis distribution is widely prevalent in European scrub typhus due to the presence of fever, rash, and eschar. regions such as Spain, Slovakia, Germany, and Italy8–10; Thus, clinicians should note that patients diagnosed with R. monacensis was first characterized and isolated from Ixo- scrub typhus in an endemic region may be coinfected with des ricinus collected in Munich, Germany, in 2002.8 In R. monacensis. 2005, R. monacensis was reported as the causative human pathogen in Mediterranean spotted fever-like illness (MSF-like Received August 1, 2018. Accepted for publication May 6, 2019. illness) in Spain, where the patient had maculopapular rash but Published online June 17, 2019. no eschar. Moreover, R. monacensis was isolated from the same species of ticks in Portugal and Hungary.9 In addition, Financial support: This research was supported by Basic Science Research Program through the National Research Foundation there was a case report in Italy of a 28-year-old man who had of Korea (NRF) funded by the Ministry of Education (NRF- eschar without rash; the organism detected was similar to 2016R1D1A1B03930956). R. monacensis isolate N72 (accession no. FJ919650.1), the 10 Disclosure: The authors do not have any commercial interests or other causative agent of MSF-like illness. associations that might pose a conflict of interest. In Korea, R. monacensis was isolated from I. nippiness ticks 11 Authors’ addresses: Seok Won Kim, Chosun University College of collected from rats. In addition, it was reported in Korea that Medicine, Departments of Neurosurgery Chosun University College a patient presented with a main complaint of fever, along with of Medicine, Gwangju, Republic of Korea, E-mail: chosunns@ eschar and rash; R. monacensis was isolated from the pa- chosun.ac.kr. Choon-Mee Kim, College of Medicine, Premedical tient.3 The patient also had eschar and rash similar to that of Science, Gwangju, Republic of Korea, E-mail: choonmee@ the scrub typhus patient, and testing for antibodies against chosun.ac.kr. Dong-Min Kim and Na Ra Yun, Chosun University College of Medicine, Departments of Internal Medicine Chosun Uni- O. tsutsugamushi was positive. 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