Under-diagnosis of vector-borne diseases among individuals suspected of having Scrub Typhus in South Korea

Abstract

Due to environmental and ecological changes and suitable habitats, the occurrence of vector-borne diseases is increasing. We investigated the seroprevalence of four major vector-borne pathogens in human patients with febrile illness who were clinically suspected of having Scrub Typhus (ST) caused by Orientia tsutsugamushi. A total of 187 samples (182 patient whole blood and sera samples, including 5 follow-up) were collected. Antibodies to Anaplasma phagocytophilum, Ehrlichia chaffeensis, Borrelia burgdorferi, and Bartonella henselae were tested by using indirect immunofluorescence assays. Molecular diagnoses were performed using real-time PCR. Of the 182 cases, 37 (20.3%) cases were designated as confirmed cases of ST, and the remaining 145 (79.7%) cases as other febrile diseases (OFDs). The seroprevalence of A. phagocytophilum, E. chaffeensis, B. burgdorferi, and B. henselae was 51.4% (19/37), 10.8% (4/37), 86.5% (32/37), and 10.8% (4/37) among the ST group, and 42.8% (62/145), 10.4% (19/145), 57.7% (105/145), and 15.9% (29/145) among the OFD group, respectively. There were no significant differences in the seroprevalence between the ST and the OFD groups. Considering the co-occurrence, 89.0% (162/182) had at least one antibody to tick-borne pathogens, 37.0% (60/162) were positive for two pathogens, 17.3% (28/162) for three pathogens, and 6.2% (10/162) for four pathogens. In real-time PCR, O. tsutsugamushi was positive in 16 cases [15 (40.5%) in ST group and 1 (2.2%) in OFD group], and the four other pathogens were negative in all cases except one confirmed as anaplasmosis. In evaluating the five follow-up samples, the appearance of new antibodies or an increase in the pre-existing antibody titers was detected. Our data highlighted that acute febrile illness and manifestations suggestive of a vector-borne infection must be recognized and further considered for coinfections in clinical practice and the laboratory.

Introduction

Global warming, environmental and ecological changes, and suitable habitats have increased the impact that ticks and mites have on humans, and are associated with the frequent emergence or re-emergence of tick- or arthropod-borne diseases with zoonotic characteristics [1, 2]. The growing number of such vector-mediated infection cases, and in particular, fatal viral epidemics in humans, has recently increased the extent of public awareness [2]. The “One Health” initiative of the World Health Organization (WHO) also encourages the development of strategies inhibiting and controlling vector-borne infections in humans and animals.

Vectors such as ticks, fleas, mites, or mosquitos can transmit bacterial, parasitic, and viral pathogens, and such vectors often host more than one agent simultaneously. Rickettsiales (genera Anaplasma, Ehrlichia, and Rickettsia), Bartonella, and Borrelia are the most common vector-borne pathogens [3]. Anaplasma phagocytophilum is an emerging, Gram-negative, obligate intracellular bacteria transmitted by Ixodes ticks [4]. In humans, infection ranges from asymptomatic to severe disease that can present with pancytopenia, multi-organ failure, or death. In addition, Ehrlichia chaffeensis also causes the life-threatening disease called Human Monocytic Ehrlichiosis, with acute sepsis and toxic shock-like symptoms that can evolve into multi-organ failure or death [5, 6]. Early clinical and laboratory diagnoses are problematic due to non-specific flu-like symptoms and limitations in the current diagnostic testing [5, 7]. Lyme disease caused by pathogenic members of the Borrelia burgdorferi s.l. complex, typically begins with erythema migrans (˜80%), but ∼18% of patients have non-specific symptoms such as malaise, fatigue, headache, arthralgias, myalgias, fever, and regional lymphadenopathy without recognition of erythema migrans, for which differential diagnoses are required [8]. Likewise, infection of Bartonella henselae, which is a gram-negative, coccobacillus, facultative intracellular bacterium, also manifests diverse and nonspecific symptoms above mentioned, which could initially lead to misdiagnosis as other diseases [9]. People usually contract the disease from cats infected with B. henselae, but flea or tick bite cases have been reported [10]. B. henselae is known to be transmitted by cat’s scratch or bite, due to the contamination of saliva and nails with the bacteria, but the other Bartonella spp. can be transmitted by ticks or fleas bites, such as Bartonella birtlesii [10–12].

Scrub Typhus (ST) is an acute febrile disease caused by Orientia tsutsugamushi, which is transmitted by larval-stage trombiculid mites [13]. In South Korea, ST is endemic and is one of the leading public health concerns, with infections most frequently occurring between October and November [14]. The clinical presentations of ST and other vector-borne diseases are similar at the early stage of infection: signs and symptoms typically develop within 1 and 2 weeks of infection and include fever, headache, malaise, and gastrointestinal symptoms [15]. Therefore, it is difficult to identify the causative pathogen based on the clinical presentation. Moreover, the clinical vector-borne disease spectrum ranges from asymptomatic to fatal and is disproportionately high in children and older adults who may not show distinct features [16]. Thus, a broad view and an extensive examination are essential for diagnosing and treating vector-borne diseases.

Despite the clinical relevance of vector-borne disease, in-depth epidemiological studies and research investigations are still lacking in Korea. Here, we investigate the seropositivity and DNA detection to the four major vector-borne pathogens (Anaplasma phagocytophilum, Ehrlichia, Borrelia, and Bartonella henselae) in a cohort of patients with febrile illness who were clinically suspected of having ST.

Materials and methods

Study cohort and sample collection

From September 2019 to June 2020, residual samples of 187 whole blood in EDTA tubes and 187 sera in serum separator tubes from 182 patients were collected after the ordered routine laboratory testing at Chonnam National University Hospital (CNUH), South Korea. Of the 187 sets of whole blood and serum, 177 were single-drawn sample sets from 177 patients, and 10 were paired sample sets consisting of 5 first-drawn and 5 randomly collected follow-up samples from 5 patients. Most presented febrile illness and were clinically suspected of having ST. Medical data of the patients, including age, gender, clinical history, symptoms, laboratory findings, and the final clinical diagnosis, were obtained through retrospective electronic medical record reviews, with personally identifiable information removed. Collected laboratory parameters were as follows: white blood cell with differential count (neutrophils, lymphocytes, monocytes, eosinophils, and basophils), hemoglobin, platelet (Sysmex XN-1000; Sysmex Corporation, Kobe, Japan); erythrocyte sedimentation rate (TEST 1 BCL; Alifax, Polverara, Italy); fibrinogen, activated partial thromboplastin time (STA-R; Diagnostica Stago, Asnieres, France); C-reactive protein, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase, blood urea nitrogen, creatinine, glucose, total bilirubin, direct bilirubin (AU5800; Olympus, Tokyo, Japan) and antibody against O. tsutsugamushi. The anti-O.tsutsugamushi antibody test was performed using commercially available lateral-flow-format immunochromatographic assay kits (SD Bio-line, Youngin, Korea). The final diagnoses of ST were defined by physicians according to clinical and laboratory findings: history of outdoor activities, typical eschar or maculopapular rash, fever, therapeutic response to treatment, and anti-O.tsutsugamushi antibody test results [17].

Indirect immunofluorescent assays for antibodies to other vector-borne pathogens

Commercially available IFA test kits containing the positive and negative control reagents were used to analyze the immunoglobulin G (IgG) of anti-Borrelia burgdorferi, anti-Anaplasma phagocytophilum, anti-Bartonella henselae, and anti-Ehrlichia chaffeensis (Fuller Laboratories, Fullerton, CA, USA). All 187 sera were screened at a 1:64 dilution, according to the manufacturer’s instructions. We serially diluted the positive controls at ratios of 1:64, 1:128, 1:256, 1:512, and 1:1,024. The negative control and the serial dilutions of the positive control were assayed with the samples in each run. First, the samples were placed on a slide in contact with the substrate and incubated. The slide was then washed in phosphate-buffered saline to remove unbound antibodies. In the second stage, each well was overlaid with a solution of a fluorescein-labeled antibody to human IgG. The antigen–antibody complexes reacted with the anti-human IgG. Each slide was washed, dried, mounted, and interpreted under a fluorescence microscope (Fig 1). The manufacturer recommended the cutoff titer as 1:512; therefore, the fluorescence intensity of the 1:512 diluted positive control was set to the cutoff level to determine a positive test result. Samples with less fluorescence intensity than the 1:512 positive control were interpreted as negative. The fluorescence intensity of the positive samples was compared to the positive controls, and the titers were graded as follows: 1+, the intensity of the 1:512 diluted positive control; 2+, the intensity of the 1:256 positive control; 3+, the intensity of the 1:128 positive control; and 4+, the intensity of the 1:64 positive control. Titers graded as 1+ and 2+ were defined as low titers, and 3+ and 4+ were defined as high titers.

[Figure omitted. See PDF.]

(A) Positive anti-Anaplasma phagocytophilum IgG appears as one or more distinct apple-green phagosomes (morulae) within the cytoplasm of the infected cells (IFA, × 200). (B) Positive anti-Ehrlichia chaffeensis IgG appears as peripheral clusters of distinct apple-green inclusion bodies within the infected erythrocytes (IFA, × 200). (C) Positive anti-Borrelia burgdorferi IgG appears as bright staining of characteristic spirochetes (IFA, × 200). (D) Positive anti-Bartonella henselae IgG appears as brightly fluorescent sharp, regularly stained coccobacilli within the cytoplasm of the fixed Vero cells (IFA, × 200).

Real-time PCR for vector-borne pathogens

DNA was extracted from all 187 whole blood samples using the QIAamp DNA Mini Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s instructions. The real-time PCR was performed using commercial kits for vector-borne pathogens: the Power Chek O. tsutsugamushi Real-time PCR Kit, PowerChek Ehrlichia/Anaplasma Real-time PCR Kit, and PowerChek Rickettsia/Borrelia/Bartonella Real-time PCR Kit (Kogene Bio-tech, Seoul, South Korea). Any positive reaction was re-confirmed by additional PCR and sequencing as previously described (S1 Table) [14, 18–21].

Statistical analysis

The results of the 182 single and first-drawn samples were included in the statistics, and the 5 follow-up samples from 5 patients were not included. Fisher’s exact test or a chi-square test was used to compare categorical variables. Student’s t-test was used to compare the continuous variables. A P-value of < 0.05 was considered statistically significant. All statistical analyses were performed using either SPSS 27.0 (IBM Corp., Armonk, NY, USA) or the diagnostic test evaluation calculator (MedCalc, Ostend, Belgium).

Ethics statement

The collection of samples for this study was conducted in accordance with the guidelines and approval of the Institutional Review Board of Chonnam National University Hospital (approval no. CNUH-2020-117). A waiver of consent was granted by given the nature of the project dealing with the remaining samples, and no information was used that could lead to patient identification.

Results

Demographics and clinical characteristics

Overall, 37 (20.3%) cases were designated as confirmed cases of ST, and the remaining 145 (79.7%) cases were designated as OFD group (Table 1). The OFD group comprised gastrointestinal diseases (n = 33), respiratory infections (n = 24), central nervous system diseases (n = 17), allergic diseases (n = 17), heart disease (n = 9), malignancy (n = 8), leptospirosis (n = 2), hemorrhagic fever with renal syndrome (n = 2), anaplasmosis (n = 2), severe fever with thrombocytopenia syndrome virus (SFTS) (n = 1) and undifferentiated fever (n = 30). The median age of the ST group was significantly higher than the OFD group (73.1 vs. 64.0 years, P = 0.018), and female predominance was found in the ST group (67.6% vs. 44.8%, P = 0.017). In addition, fever, tick-bite history or eschar, and a history of outdoor activity were more frequent in the ST group. Differences in the laboratory parameters were not significant between the ST and OFD groups, except for in the % of lymphocytes and % of basophils.

[Figure omitted. See PDF.]

Seroprevalence and titers of antibodies to vector-borne pathogens

The overall seroprevalence of antibodies to B. burgdorferi was 75.3%; for A. phagocytophilum, 44.5%; for B. henselae, 18.1%; and for E. chaffeensis, 12.6% (Table 2). Among the ST group, the seroprevalence to A. phagocytophilum, E. chaffeensis, B. burgdorferi, and B. henselae was 51.4%, 10.8%, 86.5%, and 10.8%, respectively. Among the OFD group, the seroprevalence to A. phagocytophilum, E. chaffeensis, B. burgdorferi, and B. henselae was 42.8%, 10.4%, 57.7%, and 15.9%, respectively. There was no significant difference in the seropositivity to A. phagocytophilum, E. chaffeensis, B. burgdorferi, and B. henselae between the ST and the OFD groups. As for the antibody titers, the ST group more frequently exhibited high titers of A. phagocytophilum and B. burgdorferi, but a low titer of E. chaffeensis. Low titers of A. phagocytophilum, E. chaffeensis, and B. henselae were more frequently observed among the OFD group. The proportion of cases with B. burgdorferi having the highest titer (4+) was significantly higher in the ST group than in the OFD group (43.2% vs. 19.2%, P = 0.02).

[Figure omitted. See PDF.]

Co-occurrence rate of antibodies to vector-borne pathogens

Fig 2 displays the overall co-occurrence rate and the co-occurring pathogens of the 182 cases with febrile illness screened for vector-borne infection in this study, and 89.0% (162/182) of them harbored at least one antibody to vector-borne pathogens. Among them, 39.5% (64/162) were antibody positive for one pathogen, 37.0% (60/162) were positive for two pathogens, 17.3% (28/162) for three pathogens, 6.2% (10/162) for four pathogens, and none for five pathogens. B. burgdorferi accounted for 67.2% (43/64) of the single antibody-positive group, followed by A. phagocytophilum at 14.1% (9/64). Those two pathogens were also the most common co-occurring pathogens in the double-antibody-positive group at 56.7% (34/60). The simultaneous incidence of A. phagocytophilum and B. burgdorferi were the highest among the triple antibody-positive group for 89.3% (25/28), frequently accompanied by O. tsutsugamushi or B. henselae. The co-occurrence of O. tsutsugamushi, A. phagocytophilum, and B. burgdorferi was observed in 70.0% of the quadruple antibody-positive groups. Additionally, the co-occurrence rate of each antibody among the 60.5% (98/162) cases seropositive for multiple pathogens was accessed and is shown in Table 3.

[Figure omitted. See PDF.]

The antibodies to Anaplasma phagocytophilum, Ehrlichia chaffeensis, Borrelia burgdorferi, and Bartonella henselae were tested with commercial IFA assay kits (Fuller Laboratories, Fullerton, CA, USA). Abbreviations: n—number.

[Figure omitted. See PDF.]

Real-time PCR for vector-borne pathogens

The real-time PCR for O. tsutsugamushi was positive in 16 cases; 12 were antibody-positive cases, and 4 were antibody-negative cases. Real-time PCR assays for E. chaffeensis, Borrelia burgdorferi, B. henselae were all negative, but only one case was positive. This positive reaction for A. phagocytophilum was confirmed by additional sequencing, and the sequence was deposited to GenBank (Accession no. OQ750553) (S1 Fig). This positive sample was derived from the patient who was clinically diagnosed with anaplasmosis by morulae found within granulocytes in the peripheral blood smear (PBS).

Paired follow-up sample data

In addition, we further investigated the paired follow-up samples from five patients (Table 4). They were all clinically diagnosed with ST. In case 1, antibodies for A. phagocytophilum and B. burgdorferi appeared on day 18, which were negative on day 0. In case 2, antibodies for O. tsutsugamushi and A. phagocytophilum appeared. The increase in titer was also observed in case 3. In case 4, the antibodies for O. tsutsugamushi, A. phagocytophilum, and B. henselae turned positive on day 10. In case 5, the antibody for E. chaffeensis appeared on day 2. The follow-up samples demonstrated new antibody appearance or some titer changes, but there were no cases of negative changes in existing antibodies.

[Figure omitted. See PDF.]

Discussion

Tick- and arthropod-borne diseases in humans often share similar clinical features but are epidemiologically and etiologically distinct. In South Korea, it is estimated that more than 10,000 patients are treated every year for tick- or arthropod-borne diseases, such as ST or spotted fever. Despite the clinical relevance of vector-borne infections, in-depth epidemiological studies and research investigations are lacking in Korea. The antibodies to several vector-borne pathogens may overlap because trombiculid mites and ticks share many of the same habitats, and even a single elevated antibody titer can be evidence of exposure to a pathogen, although not sufficient to confirm acute infection. In this study, we demonstrated that many ST patients have the possibility of co-exposure to multiple vector-borne pathogens.

Ticks can transmit both the Borrelia Lyme group and Borrelia relapsing fever group, including the Borrelia hard tick-borne relapsing fever group (Borrelia miyamotoi) [22]. The B. burgdorferi sensu lato (s.l.) complex is a diverse group of worldwide-distributed bacteria, comprised of 21 different species [8]. Eleven species from the B. burgdorferi s.l. complex were identified in and strictly associated with Eurasia and, until now, only B. afzelii, B. garinii and B. valaisiana have been reported in Korea [23–25]. Lyme disease, caused by pathogenic members of the B. burgdorferi s.l. complex, typically begins with erythema migrans (˜80%), but ∼18% of patients have non-specific symptoms such as malaise, fatigue, headache, arthralgias, myalgias, fever, and regional lymphadenopathy without recognition of erythema migrans, for which differential diagnoses are required [26]. Recent Korean data reported 8.1% seroprevalence to B. burgdorferi antibodies among forestry workers in National Park Offices, and they used an in-house IFA method [27]. At the present study, commercially available IFA kits were used, and the high seropositive rate of B. burgdorferi was noticed in both ST and OFD groups, in addition to high titers. Interpreting a single positive serologic result as an infection is challenging, especially in endemic areas [28]. However, in non-endemic areas with low seropositivity, like South Korea, a single elevated antibody titer can support the possibility of infection with vector-borne pathogens, especially for those with suspicious clinical manifestations and history. Therefore, an additional serologic evaluation for B. burgdorferi antibody might help identify the infection in Korea.

It should also be noted that the co-occurrence of B. burgdorferi with A. phagocytophilum or O. tsutsugamushi antibodies was the most frequently found in this study. Several ecological factors could be considered for these serological results. O. tsutsugamushi is transmitted through bites of trombiculid mites, but not through ticks’ bites. However, the co-occurrence of antibodies to several vector-borne bacteria could be predicted since trombiculid mites and ticks transmit B. burgdorferi or Anaplasma spp. and share many of the same habitats [29]. In a previous report, A. phagocytophilum was detected both in Ixodes persulcatus ticks and the blood of humans after tick bites [30]. In addition, several researchers previously announced that Rickettsiae could be transmitted by Ixodes sp. (Rickettsia helvetica and R. monacensis), Haemaphysalis longicornis and Dermacentor marginatus ticks (Rickettsia raoultii), or other ticks [31, 32]. Regarding climate change, it is known that warming impacts the activity and aggressiveness of ticks, causing human attacks and the possibility of transmission of severe tick-borne pathogens to increase [33]; thus, further caution to infection of various VBDs should be taken as concerns grow in Korea.

In addition to Borrelia, both A. phagocytophilum and E. chaffeensis were found in H. longicornis and I. persulcatus ticks throughout Korea [34]. Although several seroepidemiological and molecular studies have shown that these agents are present in Asia [35, 36], suspecting and diagnosing those infections is not easy. Previous seroprevalence studies showed that 1.8% of serum samples from patients with acute fever were positive for A. phagocytophilum through IFA testing [35]. In 2003, the first Korean case of A. phagocytophilum was detected using molecular and serological methods in Chuncheon, Gangwon [36]. Afterward, Yi et al. found 5 (7.1%) human anaplasmosis cases among 70 Koreans who underwent bone marrow examination due to fever and hemocytopenia. The five anaplasmosis cases were confirmed by PCR, and one of them revealed morulae in the PBS [37]. The detection rate of morulae is known to be 25–75% in the first week of the disease [5, 38]. In our study, one of the anaplasmosis cases was also diagnosed by morulae found within granulocytes in the PBS. The patient presented fever, dizziness, and myalgia, and laboratory results showed pancytopenia and increased CRP, AST, ALT, LDH, and BUN. The leptangamushi antibody test was positive for O. tsutsugamushi. The IFA assays were all negative, but the PCR was positive for A. phagocytophilum. The patient samples were taken on the fifth day after symptom onset. In a previous case report, the A. phagocytophilum IFA result on day 5 was negative and the PCR was positive, and the IFA titer began to increase on day 10, whereas the PCR turned negative [39]. The results on day 5 were consistent with our case, but the follow-up sample of this case was not included in this study. Considering the negative O. tsutsugamushi PCR result, it can be inferred that the positive O. tsutsugamushi antibody was caused by the possibility of infection in the past.

In this study, 85.2% of the A. phagocytophilum seropositive group harbored antibodies to Borrelia, indicating presumptive evidence of sharing the same vector. However, only 17.3% of the A. phagocytophilum seropositive group harbored antibodies to E. chaffeensis, and the overall seropositivity to E. chaffeensis and titers were relatively low to other pathogens. Only five patients exhibited high titers of E. chaffeensis. A previous Korean study showed that 1.0% of 1,618 ticks (H. longicornis, I. persulcatus, and I. nipponensis) were E. chaffeensis positive via PCR [40–44], and they suggested the distribution of E. chaffeensis throughout South Korea [25]. Although the seroprevalence of E. chaffeensis may be low in Korea, it is necessary to be cautious in cases with a high titer of E. chaffeensis, which may indicate a high burden of tick-borne disease. The presence of the causative agents and potential tick vectors with the capacity to bite humans suggests that the serological data reflect a previously unrecognized but emerging problem in South Korea.

In this study, 87.9% of the B. henselae antibody-positive cases harbored multiple antibodies to vector-borne pathogens. A relatively low titer of B. henselae was observed in the OFD group, whereas a high titer was noticed in the ST group. In Korea, serologic and molecular evidence for B. henselae and B. quintana was observed in ticks and small animals [41, 44, 45]. According to a previous study, Bartonella DNA was isolated from H. longicornis, H. flava, I. persulcatus, and I. nipponensis [46]. Human infections of B. henselae and B. quintana were also described [47–49]. The precise incidence of bartonellosis in Korea has not yet been investigated; however, those reports, including this study, suggest that the burden of bartonellosis in Korea could be higher than expected.

We found the possibility of the coinfection of multiple vector-borne pathogens in febrile illness patients, demonstrating the seropositivity of those pathogens. Primarily, the high titers of antibodies to multiple pathogens support the possibility of co-existence. Follow-up cases also strengthened the possibility of coinfection of ST and other VBDs. Four of the five follow-up ST patients were already seropositive to other vector-borne pathogens, suggesting previous exposure. A previous study of 91 individuals who recovered from ST, the follow-up IgM, IgG, and total Ig positivity rates for 13 years were 37.4% (34/91), 22.0% (20/91), and 76.9% (70/91), respectively [50]. Almost all patients with ST had a frequent outdoor activities history, suggesting that they might be persistently exposed to the risk of tick or mite bites. In follow-up evaluation, the appearance of new antibodies or an increase in the pre-existing antibody titers was detected. Such changes support the possibility of a coinfection of O. tsutsugamushi and other vector-borne pathogens.

Meanwhile, the positive reaction in IFA assay may be due to the cross-reactive immune responses to vector-borne pathogen-related antigens. They are typically group-specific, although perhaps not species-specific. Previous reports announced that antibodies reactive against E. chaffeensis or A. phagocytophilum could react with other species, impeding epidemiologic distinction between the infections [51]. In our data, a cross-reactive effect might exist in E. chaffeensis, considering that its average titer was very low (1+). The possibility of a cross-reaction between the antibodies of those pathogens needs to be further evaluated.

The diagnosis of vector-borne infection generally relies on serologic tests using indirect immunofluorescence assays (IFAs) showing at least a 4-fold increase in the antibody titers between paired sera [52, 53]. However, the need for the paired serum samples to be taken over a specific period is the most crucial factor that explains the low effectiveness of IFA tests during the acute phase of the disease. Additionally, to perform the IFA test, conditions such as fluorescent microscopes, dark rooms, and trained laboratory personnel are required. Furthermore, serologic evaluations for vector-borne pathogens other than ST are not usually performed in general laboratories because it is not yet permitted by the Korean Ministry of Food and Drug Safety for clinical diagnosis. Real-time PCR has also been proposed for the early diagnosis of vector-borne infection [54], but buffy coat samples are needed, which require technical expertise for their preparation. The clinical sensitivity of a real-time PCR using serum samples is insufficient and is not commercially available [55].

In the clinical field in South Korea, there is little choice in choosing laboratory tests to diagnose VBDs. IFA assays and PCRs for most vector-borne pathogens are unavailable for routine tests and can only be used for research. Therefore, most clinical diagnoses are restricted to the pathogens only available for routine antibody testing, such as O. tsutsugamushi. Therefore, diagnosing VBDs usually depends on the physician’s experience and clinical evidence. With this study, we want to show the possibility of the co-infection of other VBD with O. tsutsugamushi and provoke the recognition of the need for further laboratory evaluation. We suggest that when a patient is suspected of VBD, the IFA tests should be performed for the major pathogens. As shown in our data, the positive rate of PCR is relatively low. Previous reports announced that PCR-negative results do not exclude infection, as the presence in the blood of some vector-borne pathogens can be temporal and transient [56]. In this study, the patient clinically diagnosed with anaplasmosis showed a positive PCR and a negative IFA for Anaplasma. Likewise, molecular diagnosis can be helpful in the acute infection stage when antibodies are at low titers or negative.

Our work had several limitations. First, the titers by the directly diluted test samples were not taken. We indirectly compared the sample fluorescence with the diluted positive control. However, by only counting the fluorescence stronger than that of the 1:256 diluted positive control as positive, the positive result has enough value to suggest the seropositivity. Second, the baseline seroprevalence in healthy controls was not evaluated. A Korean study about ST reported no seroprevalence of IgG with a cutoff value of ≥1:256 among 216 health checkup personnel [50]. The baseline seroprevalence of the other four vector-borne pathogens is unknown in this geographical region; therefore, a further study of antibody and titer analysis for the four vector-borne pathogens is required. Third, along with the positive IgG antibody result, we did not test the IgM antibody, which could provide further information to distinguish between present and past infections. Fourth, cross-reactions among the pathogens should be excluded using assays such as Western blot. Last, we did not evaluate any samples other than blood samples. Molecular studies using tissue samples, such as eschar, may aid in the determination of the causative agents [57].

Here, we found that the co-existence of vector-borne pathogens in ST and other febrile illnesses may be underestimated. Coinfections should be considered in actual clinical practice and also in the laboratory. Acute febrile illness and manifestations suggestive of vector-borne infection must be recognized and further explored in order to determine the appropriate treatment. Further evaluation methods, such as IFA antibody testing and PCR, are needed to be introduced for routine laboratory work.

Supporting information

S1 Fig. Representative curves of the real-time PCR commercial kits for vector-borne pathogens used in this study.

(A) Positive controls included in the kits. Real-time PCR assays for four other tick-borne pathogens were all negative (B), but only one case showed a positive amplification curve to the Anaplasma target (C). This positive reaction for A. phagocytophilum was confirmed by additional sequencing, and the sequence was deposited to GenBank (Accession no. OQ750553).

https://doi.org/10.1371/journal.pone.0286631.s001

S1 Table. Primer sequences used for confirmation of positive reaction in real-time PCR assay.

https://doi.org/10.1371/journal.pone.0286631.s002

(DOCX)

Citation: Won EJ, Kim SH, Byeon KH, Jeon C-H, Kang S-J, Park J-H, et al. (2023) Under-diagnosis of vector-borne diseases among individuals suspected of having Scrub Typhus in South Korea. PLoS ONE 18(6): e0286631. https://doi.org/10.1371/journal.pone.0286631

About the Authors:

Eun Jeong Won

Roles: Conceptualization, Data curation, Funding acquisition, Project administration, Writing – original draft, Writing – review & editing

Affiliations: Departments of Parasitology and Tropical Medicine, Chonnam National University Medical School, Hwasun, South Korea, Department of Laboratory Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea

ORICD: https://orcid.org/0000-0002-8750-4257

Seong Hoon Kim

Roles: Investigation, Methodology, Writing – review & editing

Affiliation: Departments of Parasitology and Tropical Medicine, Chonnam National University Medical School, Hwasun, South Korea

Kyeong Hwan Byeon

Roles: Investigation, Methodology, Writing – review & editing

Affiliation: Departments of Parasitology and Tropical Medicine, Chonnam National University Medical School, Hwasun, South Korea

Chae-Hyeon Jeon

Roles: Formal analysis, Investigation, Methodology, Writing – review & editing

Affiliation: Department of Laboratory Medicine, Chonnam National University Hospital, Gwangju, South Korea

Seung-Ji Kang

Roles: Formal analysis, Investigation, Methodology, Resources, Supervision, Writing – review & editing

Affiliation: Department of Infectious Disease, Chonnam National University Bitgoeul Hospital, Gwangju, South Korea

Joo-Heon Park

Roles: Data curation, Investigation, Writing – review & editing

Affiliation: Department of Laboratory Medicine, Chonnam National University Hospital, Gwangju, South Korea

Seung-Jung Kee

Roles: Project administration, Supervision, Writing – review & editing

Affiliation: Department of Laboratory Medicine, Chonnam National University Hospital, Gwangju, South Korea

Hyun-Woo Choi

Roles: Conceptualization, Funding acquisition, Project administration, Writing – original draft, Writing – review & editing

E-mail: [email protected]

Affiliation: Department of Laboratory Medicine, Chonnam National University Bitgoeul Hospital, Gwangju, South Korea

ORICD: https://orcid.org/0000-0002-9438-1603

References

1. Gilbert L. The impacts of climate change on ticks and tick-borne disease risk. Annu Rev Entomol. 2021;66:373–88. pmid:33417823

2. Piotrowski M, Rymaszewska A. Expansion of tick-borne rickettsioses in the world. Microorganisms. 2020;8(12):1906. pmid:33266186

3. Choi E, Pyzocha NJ, Maurer DM. Tick-borne illnesses. Curr Sports Med Rep. 2016;15(2):98–104. pmid:26963018

4. Rikihisa Y. Mechanisms of obligatory intracellular infection with Anaplasma phagocytophilum. Clin Microbiol Rev. 2011;24(3):469–89.

5. Dumler JS, Madigan JE, Pusterla N, Bakken JS. Ehrlichioses in humans: epidemiology, clinical presentation, diagnosis, and treatment. Clin Infect Dis. 2007;45 Suppl 1:S45–51. pmid:17582569

6. Tominello TR, Oliveira ERA, Hussain SS, Elfert A, Wells J, Golden B, et al. Emerging roles of autophagy and inflammasome in Ehrlichiosis. Front Immunol. 2019;10:1011. pmid:31134081

7. Seo JW, Kim D, Yun N, Kim DM. Clinical update of severe fever with thrombocytopenia syndrome. Viruses. 2021;13(7):1213. pmid:34201811

8. Kingry LC, Anacker M, Pritt B, Bjork J, Respicio-Kingry L, Liu G, et al. Surveillance for and discovery of Borrelia species in US patients suspected of tickborne illness. Clin Infect Dis. 2018;66(12):1864–71.

9. Kwon HY, Park YK, Lee SM, Baek JH, Kang JS, Chung MH, et al. Characterization of clinical isolates of Bartonella henselae strains, South Korea. Emerg Infect Dis. 2018;24(5):912–5.

10. Chomel BB, Boulouis HJ, Breitschwerdt EB. Cat scratch disease and other zoonotic Bartonella infections. J Am Vet Med Assoc. 2004;224(8):1270–9. pmid:15112775

11. Cheslock MA, Embers ME. Human bartonellosis: an underappreciated public health problem? Trop Med Infect Dis. 2019;4(2).69. pmid:31010191

12. Reis C, Cote M, Le Rhun D, Lecuelle B, Levin ML, Vayssier-Taussat M, et al. Vector competence of the tick Ixodes ricinus for transmission of Bartonella birtlesii. PLoS Negl Trop Dis. 2011;5(5):e1186.

13. Tantibhedhyangkul W, Ben Amara A, Textoris J, Gorvel L, Ghigo E, Capo C, et al. Orientia tsutsugamushi, the causative agent of scrub typhus, induces an inflammatory program in human macrophages. Microb Pathog. 2013;55:55–63. pmid:23088884

14. Bang MS, Kim CM, Park JW, Chung JK, Kim DM, Yun NR. Prevalence of Orientia tsutsugamushi, Anaplasma phagocytophilum and Leptospira interrogans in striped field mice in Gwangju, Republic of Korea. PLoS One. 2019;14(8):e0215526.

15. Yun Y, Heo ST, Kim G, Hewson R, Kim H, Park D, et al. Phylogenetic analysis of Severe Fever with Thrombocytopenia Syndrome Virus in South Korea and migratory bird routes between China, South Korea, and Japan. Am J Trop Med Hyg. 2015;93(3):468–74. pmid:26033016

16. Sanchez-Vicente S, Tagliafierro T, Coleman JL, Benach JL, Tokarz R. Polymicrobial nature of tick-borne diseases. Mbio. 2019;10(5):e02055–19. pmid:31506314

17. Ra SH, Kim JY, Cha HH, Kwon JS, Lee HJ, Jeon NY, et al. Coinfection of Severe Fever with Thrombocytopenia Syndrome and Scrub Typhus in patients with tick-borne illness. Am J Trop Med Hyg. 2019;101(6):1259–62. pmid:31549609

18. Massung RF, Levin ML, Munderloh UG, Silverman DJ, Lynch MJ, Gaywee JK, et al. Isolation and propagation of the Ap-Variant 1 strain of Anaplasma phagocytophilum in a tick cell line. J Clin Microbiol. 2007;45(7):2138–43.

19. Bereswill S, Hinkelmann S, Kist M, Sander A. Molecular analysis of riboflavin synthesis genes in Bartonella henselae and use of the ribC gene for differentiation of Bartonella species by PCR. J Clin Microbiol. 1999;37(10):3159–66.

20. Loh SM, Gofton AW, Lo N, Gillett A, Ryan UM, Irwin PJ, et al. Novel Borrelia species detected in echidna ticks, Bothriocroton concolor, in Australia. Parasit Vectors. 2016;9(1):339.

21. Wagner ER, Bremer WG, Rikihisa Y, Ewing SA, Needham GR, Unver A, et al. Development of a p28-based PCR assay for Ehrlichia chaffeensis. Mol Cell Probes. 2004;18(2):111–6.

22. Trevisan G, Cinco M, Trevisini S, di Meo N, Ruscio M, Forgione P, et al. Borreliae Part 2: Borrelia Relapsing Fever Group and Unclassified Borrelia. Biology (Basel). 2021;10(11): 1117. pmid:34827110

23. Choi YJ, Han SH, Park JM, Lee KM, Lee EM, Lee SH, et al. First molecular detection of Borrelia afzelii in clinical samples in Korea. Microbiol Immunol. 2007;51(12):1201–7.

24. Park KH, Chang WH, Schwan TG. Identification and characterization of Lyme disease spirochetes, Borrelia burgdorferi sensu lato, isolated in Korea. J Clin Microbiol. 1993;31(7):1831–7.

25. Im JH, Baek J, Durey A, Kwon HY, Chung MH, Lee JS. Current status of tick-borne diseases in South Korea. Vector Borne Zoonotic Dis. 2019;19(4):225–33. pmid:30328790

26. Steere AC, Sikand VK. The presenting manifestations of Lyme disease and the outcomes of treatment. N Engl J Med. 2003;348(24):2472–4. pmid:12802042

27. Acharya D, Park JH. Seroepidemiologic survey of Lyme disease among forestry workers in national park offices in South Korea. Int J Environ Res Public Health. 2021;18(6):2933. pmid:33809327

28. Brown Marusiak A, Hollingsworth BD, Abernathy H, Alejo A, Arahirwa V, Mansour O, et al. Patterns testing for tick-borne diseases and implications for surveillance in the Southeastern US. JAMA Netw Open. 2022;5(5):e2212334. pmid:35576005

29. Park JH, Heo EJ, Choi KS, Dumler JS, Chae JS. Detection of antibodies to Anaplasma phagocytophilum and Ehrlichia chaffeensis antigens in sera of Korean patients by western immunoblotting and indirect immunofluorescence assays. Clin Diagn Lab Immunol. 2003;10(6):1059–64.

30. Cao WC, Zhao QM, Zhang PH, Yang H, Wu XM, Wen BH, et al. Prevalence of Anaplasma phagocytophila and Borrelia burgdorferi in Ixodes persulcatus ticks from northeastern China. Am J Trop Med Hyg. 2003;68(5):547–50.

31. Tariq M, Kim DM. Hemorrhagic Fever with Renal Syndrome: Literature review, epidemiology, clinical picture and pathogenesis. Infect Chemother. 2022;54(1):1–19. pmid:35384417

32. Choi YJ, Jang WJ, Kim JH, Ryu JS, Lee SH, Park KH, et al. Spotted fever group and typhus group rickettsioses in humans, South Korea. Emerg Infect Dis. 2005;11(2):237–44. pmid:15752441

33. Parola P, Socolovschi C, Jeanjean L, Bitam I, Fournier PE, Sotto A, et al. Warmer weather linked to tick attack and emergence of severe rickettsioses. PLoS Negl Trop Dis. 2008;2(11):e338. pmid:19015724

34. Kim CM, Kim MS, Park MS, Park JH, Chae JS. Identification of Ehrlichia chaffeensis, Anaplasma phagocytophilum, and A. bovis in Haemaphysalis longicornis and Ixodes persulcatus ticks from Korea. Vector Borne Zoonotic Dis. 2003;3(1):17–26.

35. Heo EJ, Park JH, Koo JR, Park MS, Park MY, Dumler JS, et al. Serologic and molecular detection of Ehrlichia chaffeensis and Anaplasma phagocytophila (human granulocytic ehrlichiosis agent) in Korean patients. J Clin Microbiol. 2002;40(8):3082–5.

36. Kim KH, Yi J, Oh WS, Kim NH, Choi SJ, Choe PG, et al. Human granulocytic anaplasmosis, South Korea, 2013. Emerg Infect Dis. 2014;20(10):1708–11. pmid:25271737

37. Yi J, Kim KH, Ko MK, Lee EY, Choi SJ, Oh MD. Human granulocytic anaplasmosis as a cause of febrile illness in Korea since at least 2006. Am J Trop Med Hyg. 2017;96(4):777–82. pmid:28093540

38. Lee SH, Park SY, Jang MJ, Choi KJ, Lee HK, Cho YU, et al. Clinical isolation of Anaplasma phagocytophilum in South Korea. Am J Trop Med Hyg. 2017;97(6):1686–90.

39. Lee SH, Shin NR, Kim CM, Park S, Yun NR, Kim DM, et al. First identification of Anaplasma phagocytophilum in both a biting tick Ixodes nipponensis and a patient in Korea: a case report. BMC Infect Dis. 2020;20(1):826.

40. Kim CM, Seo JW, Kim DM, Yun NR, Park JW, Chung JK, et al. Detection of Borrelia miyamotoi in Ixodes nipponensis in Korea. PLoS One. 2019;14(7):e0220465.

41. Chae JS, Yu DH, Shringi S, Klein TA, Kim HC, Chong ST, et al. Microbial pathogens in ticks, rodents and a shrew in northern Gyeonggi-do near the DMZ, Korea. J Vet Sci. 2008;9(3):285–93. pmid:18716449

42. Yu DH, Li YH, Yoon JS, Lee JH, Lee MJ, Yu IJ, et al. Ehrlichia chaffeensis infection in dogs in South Korea. Vector Borne Zoonotic Dis. 2008;8(3):355–8.

43. Lee M, Yu D, Yoon J, Li Y, Lee J, Park J. Natural co-infection of Ehrlichia chaffeensis and Anaplasma bovis in a deer in South Korea. J Vet Med Sci. 2009;71(1):101–3.

44. Kang SW, Doan HT, Choe SE, Noh JH, Yoo MS, Reddy KE, et al. Molecular investigation of tick-borne pathogens in ticks from grazing cattle in Korea. Parasitol Int. 2013;62(3):276–82. pmid:23501057

45. Kim CM, Yi YH, Yu DH, Lee MJ, Cho MR, Desai AR, et al. Tick-borne rickettsial pathogens in ticks and small mammals in Korea. Appl Environ Microbiol. 2006;72(9):5766–76. pmid:16957192

46. Kim CM, Kim JY, Yi YH, Lee MJ, Cho MR, Shah DH, et al. Detection of Bartonella species from ticks, mites and small mammals in Korea. J Vet Sci. 2005;6(4):327–34.

47. Lim MH, Chung DR, Kim WS, Park KS, Ki CS, Lee NY, et al. First case of Bartonella quintana endocarditis in Korea. J Korean Med Sci. 2012;27(11):1433–5. pmid:23166430

48. Im JH, Baek JH, Lee HJ, Lee JS, Chung MH, Kim M, et al. First Case of Bartonella henselae Bacteremia in Korea. Infect Chemother. 2013;45(4):446–50.

49. Durey A, Kwon HY, Im JH, Lee SM, Baek J, Han SB, et al. Bartonella henselae infection presenting with a picture of adult-onset Still’s disease. Int J Infect Dis. 2016;46:61–3.

50. Kim CM, Kim DM, Yun NR. Follow-up investigation of antibody titers and diagnostic antibody cutoff values in patients with scrub typhus in South Korea. BMC Infect Dis. 2021;21(1):69. pmid:33441087

51. Comer JA, Nicholson WL, Olson JG, Childs JE. Serologic testing for human granulocytic ehrlichiosis at a national referral center. J Clin Microbiol. 1999;37(3):558–64. pmid:9986812

52. Blacksell SD, Bryant NJ, Paris DH, Doust JA, Sakoda Y, Day NP. Scrub typhus serologic testing with the indirect immunofluorescence method as a diagnostic gold standard: a lack of consensus leads to a lot of confusion. Clin Infect Dis. 2007;44(3):391–401. pmid:17205447

53. Noh JY, Song JY, Bae JY, Park MS, Yoon JG, Cheong HJ, et al. Seroepidemiologic survey of emerging vector-borne infections in South Korean forest/field workers. PLoS Negl Trop Dis. 2021;15(8):e0009687. pmid:34407077

54. Modarelli JJ, Ferro PJ, de Leon AAP, Esteve-Gasent MD. TickPath Layerplex: adaptation of a real-time PCR methodology for the simultaneous detection and molecular surveillance of tick-borne pathogens. Sci Rep. 2019;9(1):6950. pmid:31061487

55. Elelu N, Ferrolho J, Couto J, Domingos A, Eisler MC. Molecular diagnosis of the tick-borne pathogen Anaplasma marginale in cattle blood samples from Nigeria using qPCR. Exp Appl Acarol. 2016;70(4):501–10.

56. Swanson SJ, Neitzel D, Reed KD, Belongia EA. Coinfections acquired from Ixodes ticks. Clin Microbiol Rev. 2006;19(4):708–27.

57. Chung IH, Robinson LK, Stewart-Juba JJ, Dasch GA, Kato CY. Analytically sensitive Rickettsia species detection for laboratory diagnosis. Am J Trop Med Hyg. 2022;106(5):1352–7.