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Public Health Weekly Report 2024; 17(49): 2177-2196
Published online November 18, 2024
https://doi.org/10.56786/PHWR.2024.17.49.1
© The Korea Disease Control and Prevention Agency
Analysis of Laboratory Diagnostic Tests for Scrub Typhus from 2019 to 2023
Kyeong-Ah Lee 
, Byoungchul Gill *, Hwa Su Kim , Jaeil Yoo
Division of Bacterial Diseases, Department of Laboratory Diagnosis and analysis, Korea Disease Control and Prevention Agency, Cheongju, Korea
*Corresponding author: Byoungchul Gill, Tel: +82-43-719-8113, E-mail: gilri@korea.kr
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Scrub typhus is a tick-borne infectious disease caused by Orientia tsutsugamushi. When bitten by an infected tick, an eschar forms and symptoms such as fever, chills, and rash appear. This study systematically analyzed the prevalence of scrub typhus reported from 2019 to 2023 by the Korea Disease Control and Prevention Agency and 19 regional institutes of public health and the environment, examining trends according to diagnostic methods, sex, age, and region. Of the 2,607 individuals with suspected infection, 12.4% (322 patients) tested positive for scrub typhus, with 17.9% (466 patients) undergoing both polymerase chain reaction (PCR) and indirect immunofluorescence assay (IFA) tests. Among the 466 patients who were tested using both methods, Group A (positive PCR and IFA test results) demonstrated the highest positivity rate of 68.9%, suggesting a combination of past infections and reinfections. The positivity rate of the gene detection test (20.0%) was higher than that of the antibody detection test (7.8%), suggesting that PCR-based gene detection has a relatively higher sensitivity for confirming current infections. Analysis by sex and age revealed the highest positivity rate in women in their 70s (18.2%), with women consistently exhibiting higher positivity rates than men across all age groups. A regional analysis indicated that Gwangju contributed to 26.8% of patients with a positive test result, while Gangwon Province had the highest testing rate per capita (343.2%). Performing both IFA antibody detection and PCR gene detection tests simultaneously to diagnose scrub typhus is valuable for more accurately distinguishing current infections, offering critical insights to enhance the diagnostic and management strategies for scrub typhus.
Key words Scrub typhus; Orientia tsutsugamushi; Tick-borne diseases; Laboratory diagnosis
Key messages
① What is known previously?
Scrub typhus is a tick-borne infectious disease caused by Orientia tsutsugamushi. When bitten by an infected tick, an eschar forms, and symptoms such as fever, chills, and rash appear.
② What new information is presented?
The positivity rate of the gene detection test (20.0%) was higher than that of the antibody detection test (7.8%), suggesting that polymerase chain reaction (PCR)-based gene detection has relatively higher sensitivity in confirming current infections.
③ What are implications?
Performing both immunofluorescence assay antibody detection and PCR gene detection tests simultaneously in the diagnosis of scrub typhus is considered useful for more accurately distinguishing current infections.
Scrub typhus is a vector-borne disease caused by Orientia tsutsugamushi and is primarily transmitted through chiggers or mites, such as Leptotrombidium scutellare or Leptotrombidium pallidum [1]. When an O. tsutsugamushi-infected chigger adheres to the skin and ingests bodily fluids, the pathogen enters the body, causing symptoms, such as high fever, headache, myalgia, and eschar formation. An eschar can be a main finding in the diagnosis of scrub typhus, making it possible to differentiate it from other febrile illnesses [2]. If no eschar is identified, diagnosis may be delayed because initial symptoms are nonspecific and are similar to those of the common cold. Without appropriate antibiotic treatment, the disease may progress to severe conditions, including interstitial pneumonia, myocarditis, delirium, and central nervous system complications [3].
Scrub typhus is found in a particular geographic region known as the tsutsugamushi triangle. This triangle includes Japan, Republic of Korea (ROK), northeastern China, Pakistan, Thailand, Vietnam, northern Indonesia, and northern Australia. However, there are recent reports of scrub typhus cases outside of the tsutsugamushi triangle, including Chile, the United States, and the Netherlands [4]. These cases suggest the potential for scrub typhus to occur globally, beyond previously known areas. In Japan and Taiwan, aggressive prevention and health promotion activities have led to a slight decrease in the incidence of scrub typhus over the past two decades [5,6]. On the other hand, the incidence in China has been increasing annually due to geographical characteristics, socioeconomic factors, and differences in education levels. Thus, effective prevention and control are needed during outbreaks [7].
In ROK, the first infection case was reported during the Korean War in 1951 [8]. Since 1994, the Korea Disease Control and Prevention Agency (KDCA) has conducted systematic patient surveillance and control. Scrub typhus is diagnosed by antibody detection tests using indirect immunofluorescence assays (IFA) and genetic testing based on polymerase chain reaction (PCR). Previously, genetic testing was performed only at KDCA. However, the technology was transferred to the Public Health and Environment Research Institute in 2024, and public health laboratories nationwide are now capable of performing antibody detection and genetic testing simultaneously.
In antibody detection tests, until 2019, a single antibody titer of 1:256 or more for IgG or 1:16 or more for IgM was considered positive. In 2020, the diagnostic criterion was changed to a 4-fold or greater increase in the antibody titer. This was designed to improve diagnostic accuracy by distinguishing between past and recent infections. No other country uses a single antibody level as a confirmatory criterion, even in international cases. In ROK, it was reported that IgG antibodies continue to be detectable at low titers (median 1:128) from 18 months to 2 years after infection, and IgM antibodies remained detectable in some patients (29%) for 1 year after infection [9].
However, few studies have evaluated the impact of this change in diagnostic criteria on actual diagnostic results. It is necessary to analyze disease incidence with respect to demographic factors and to compare the positive rate of each test method. Based on the results of antibody detection tests and genetic testing conducted by KDCA and Public Health and Environment Research Institutes in 19 cities and provinces in ROK from 2019 to 2023, this study aimed to analyze patterns of scrub typhus diagnosis with respect to sex, age, and region and to evaluate the impact of changes in diagnostic criteria on test results. In this way, we evaluated the effectiveness of each test method and provided basic data for improving the diagnosis and management system for scrub typhus.
1. Study Subjects and Sample Collection
In total, 2,607 patients with suspected scrub typhus referred to KDCA and the Public Health and Environment Research Institute in cities and provinces nationwide from 2019 to 2023 were enrolled. Patients had symptoms, such as fever, tick bites, and low platelets; however, data for antibiotics use were not available. The requested samples included serum, whole blood, eschar, etc. Patients were categorized by test method into those who underwent only antibody detection tests (1,458), only genetic testing (683), or both tests (466).
2. Diagnostic Criteria
Scrub typhus was diagnosed by IFA and PCR. For IFA antibody detection tests, acute-phase serum (first referral) was compared with convalescent-phase serum (second or more referrals). Acute-phase serum (first referral) samples were determined to be inconclusive regardless of antibody titer, while convalescent serum was identified as positive if the antibody titer increased by more than 4-fold over that in the acute phase. If the increase in antibody titer was <4-fold, it was considered negative. Genetic testing was performed using whole blood and eschar, targeting the 56-KDa outer membrane protein gene (TSA56). If this gene was detected, the case was judged positive.
3. Classification of Test Results
Of 2,607 patients with suspected scrub typhus, 1,458 patients were evaluated using the IFA antibody detection test only and 683 patients were evaluated through genetic testing only. Results of antibody detection tests were evaluated based on the IFA antibody titer criteria, and genetic tests were classified as positive or negative based on the presence or absence of the TSA56 gene. Additionally, 466 patients who underwent both genetic testing and an antibody detection test were divided into four groups on the basis of genetic testing results. Group A consisted of patients who tested positive in genetic testing and had an IgG or IgM antibody titer of 1:16 or higher in the antibody detection test. Group B was composed of patients who tested positive on genetic testing but had both IgG and IgM antibody titers less than 1:16 in antibody detection tests. Group C included patients who tested negative on genetic testing but had an IgG or IgM antibody titer of 1:16 or higher in antibody detection tests. Finally, Group D included patients who were negative on genetic testing and had both IgG and IgM antibody titers less than 1:16 in antibody detection tests. Patients who underwent only one test were classified as positive or negative based on the results of their respective tests, and these results were compared with those for patients evaluated using both tests.
4. Data Analysis
A total of 3,515 test results were analyzed, including results for 2,334 antibody detection tests and 1,181 genetic tests. The results for each sample were analyzed according to sex, age, and region. The number of tests per population by region was analyzed based on the ‘2022 Population and Housing Census’ from Statistics Korea. In addition, the median antibody titer, which refers to the mean antibody titer for samples showing an immune response of IgG 1:16 or higher or IgM 1:16 or higher, was calculated based on 5 years of information. Statistical analyses, including comparisons of the proportion of positive cases in each group and infection rates by age and sex, were performed using IBM SPSS Statistics 26 (IBM Corp.). The results were derived from the diagnosis and regional characteristics.
1. Analysis of Single Test Results
Of 1,458 patients evaluated using the IFA antibody detection test alone, 63 (4.3%) tested positive. When using the 2019 criterion for a single antibody titer (IgG 1:256 or greater or IgM 1:16 or greater), however, the number of positive tests increased to 90 (6.2%). Of the 683 patients evaluated using PCR-based genetic testing alone, 158 (23.1%) tested positive; this was higher than the positive rate (6.2%) obtained using antibody detection tests alone.
2. Analysis of Combined Test Results
The 466 patients who underwent both genetic testing and antibody detection test were classified into four groups based on test results. Groups A and B tested positive for a specific gene on genetic testing, among which 67 of 74 (90.5%) positive cases were confirmed. In Group A, 27 of 51 patients showed an IgG antibody titer of 1:2,048 or higher, suggesting the possibility of reinfection. Group B, which included 16 patients, had no immune response in the antibody detection test, while genetic testing results were positive, indicating a highly probable early infection. Group C consisted of patients who did not undergo genetic testing and showed a 4-fold or greater increase in antibody titers in convalescent serum compared with acute phase serum in the antibody detection test, identified as 37 inconclusive, 7 positive, and 2 negative. Group D was composed of patients who were negative on both genetic testing and antibody detection tests, with 348 patients classified as inconclusive (Table 1). When applying the 2019 single antibody titer criteria (IgG 1:256 or higher or IgM 1:16 or higher), the number of patients who tested positive in Group C increased from 7 to 12. In Group A, 2 patients tested positive on genetic testing, leaving the results of the analysis unchanged.
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Table 1. Results of concurrent genetic and antibody tests in patients (2019–2023) Group PCR IFA titer (IgG/IgM) Total
(Pos./rate [%])Diagnosis case +/–a) Under 1:16 1:16 1:32 1:64 1:128 1:256 1:512 1:1,024 Over 1:2,048 Group A + 9/11
(9/11)1/0
(1/0)2/0
(2/0)3/3
(3/3)2/9
(2/9)2/4
(2/4)1/7
(1/7)4/4
(4/4)27/13
(27/13)51
(51/68.9)Pos. Neg. Und. 51 0 0 Group B + 16/16
(16/16)0/0
(0/0)0/0
(0/0)0/0
(0/0)0/0
(0/0)0/0
(0/0)0/0
(0/0)0/0
(0/0)0/0
(0/0)16
(16/21.6)Pos. Neg. Und. 16 0 0 Group C – 8/20
(1/3)2/6
(1/0)4/6
(0/1)1/1
(0/0)6/4
(0/2)7/3
(1/0)7/1
(1/0)3/2
(1/0)8/3
(2/1)46
(7/9.5)Pos. Neg. Und. 7 2 37 Group D – 353/353
(0/0)0/0
(0/0)0/0
(0/0)0/0
(0/0)0/0
(0/0)0/0
(0/0)0/0
(0/0)0/0
(0/0)0/0
(0/0)353
(0/0.0)Pos. Neg. Und. 0 5 348 Total (positive case) 386/400
(26/30)3/6
(2/0)6/6
(2/1)4/4
(3/3)8/13
(2/11)9/7
(3/4)8/8
(2/7)7/6
(5/4)35/16
(29/14)466
(74/100.0)Pos. Neg. Und. 74 7 385 Values are presented as patients cases (positive cases). PCR=polymerase chain reaction; IFA=indirect immunofluorescence assay; Und.=Undetermined. a)‘+’: Positive or Pos., ‘–’: Negative or Neg..
3. Diagnostic Test Results by Test Method
A total of 3,515 diagnostic tests were performed on 2,607 patients with suspected scrub typhus from 2019 to 2023. The positivity rate for genetic testing was lowest in 2020 at 8.7% (11/126) and increased to 25.8% (71/275) in 2022 (Figure 1A). For antibody detection tests, the positivity rate was 17.4% (78/449) based on the single antibody titer criterion in 2019 and decreased to 9.8% (44/449) when applying the revised criterion after 2020. However, this was still the highest rate in the last 5 years. The 44 patients with a 4-fold increase in antibody titers were suspected to be recently infected, while 34 patients who tested positive based on a single antibody titer were likely antibody carriers from past infections (Figure 1B).
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Figure 1. Number of diagnosed and positive cases by year
(A) Annual trends in diagnostic results using genetic test, (B) yearly diagnostic trends in antibody test.
4. Diagnostic Test Results by Sex and Age
A total of 3,515 diagnostic tests were conducted on 2,607 patients with suspected scrub typhus between 2019 and 2023. The sex ratio was nearly 1:1, with 1,745 (49.6%) male patients and 1,770 (50.4%) female patients. Of the 418 positive patients, however, there were 160 (38.3%) males and 258 (61.7%) females, indicating a higher positive rate in females than in males (Table 2).
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Table 2. Number of diagnostic tests by age and sex and number of positive cases (2019–2023) Sex Year Age (yr) 0–19 20–29 30–39 40–49 50–59 60–69 70–79 Over 80 Total Male 2019 2/17
(0.6)2/24
(0.6)2/24
(0.6)4/40
(1.2)1/54
(0.3)13/72
(3.8)17/80
(4.9)8/34
(2.3)49/345
(14.2)2020 2/14
(0.8)1/27
(0.4)0/12
(0.0)0/22
(0.0)1/47
(0.4)5/65
(1.9)6/53
(2.3)0/17
(0.0)15/257
(5.8)2021 0/5
(0.0)0/28
(0.0)0/27
(0.0)1/30
(0.4)3/30
(1.1)6/53
(2.2)7/67
(2.5)0/35
(0.0)17/275
(6.2)2022 3/18
(0.8)4/39
(1.1)0/27
(0.0)0/24
(0.0)2/43
(0.6)10/69
(2.8)14/82
(3.9)4/55
(1.1)37/357
(10.4)2023 0/25
(0.0)2/56
(0.4)0/32
(0.0)0/46
(0.0)9/73
(1.8)14/120
(2.7)15/114
(2.9)2/45
(0.4)42/511
(8.2)Total 7/79
(8.9)9/174
(5.2)2/122
(1.6)5/162
(3.1)16/247
(6.5)48/379
(12.7)59/396
(14.9)14/186
(7.5)160/1,745
(9.2)Female 2019 1/7
(0.3)1/16
(0.3)2/14
(0.5)4/25
(1.1)17/72
(4.5)16/86
(4.2)29/101
(7.7)13/57
(3.4)83/378
(22.0)2020 0/11
(0.0)1/13
(0.4)1/25
(0.4)2/16
(0.8)2/17
(0.8)0/36
(0.0)9/92
(3.4)3/53
(1.1)18/263
(6.8)2021 0/6
(0.0)0/11
(0.0)2/16
(0.6)0/19
(0.0)2/43
(0.6)16/79
(5.2)13/76
(4.2)4/60
(1.3)37/310
(11.9)2022 2/5
(0.5)2/24
(0.5)0/21
(0.0)7/33
(1.8)7/50
(1.8)18/72
(4.7)19/93
(5.0)11/84
(2.9)66/382
(17.3)2023 1/22
(0.2)2/19
(0.5)1/23
(0.2)2/33
(0.5)10/54
(2.3)11/112
(2.5)14/99
(3.2)13/75
(3.0)54/437
(12.4)Total 4/51
(7.8)6/83
(7.2)6/99
(6.1)15/126
(11.9)38/236
(16.1)61/385
(15.8)84/461
(18.2)44/329
(13.4)258/1770
(14.6)Total 11/130
(0.3)15/257
(0.4)8/221
(0.2)20/288
(0.6)54/483
(1.5)109/764
(3.1)143/857
(4.1)58/515
(1.7)418/3,515
(11.9)Values are presented as number/total (%).
By age, a total of 3,515 scrub typhus cases were referred, with 857 patients (24.4%) aged 70–79 years, followed by 764 (21.7%) aged 60–69 years and 515 (14.7%) aged 80 years and older. The mean age of scrub typhus-positive patients was 64.7 years, with 143 (34.2%) aged 70–79 years, followed by 109 (26.1%) aged 60–69 years and 58 (13.9%) aged 80 years and older. The positive rate relative to the number of diagnoses was highest for individuals aged 70–79 years at 4.1% (143/857), followed by 3.1% (109/764) for 60–69 years, 1.7% (58/515) for 80 years and older, and 1.5% (54/483) for 50–59 years. In particular, the 5-year age breakdown of positive patients showed that females had a higher positive rate than that for males in all age groups, except 19 years and younger (7.8%) (Table 2).
5. Diagnostic Test Results by Region
Of the 3,515 diagnostic tests performed from 2019 to 2023, the highest number of tests was performed in Incheon (1,149, 32.7%), followed by Gwangju (500, 14.2%), Seoul (471, 13.4%), and Gangwon (302, 8.6%). The number of positive test results was highest in Gwangju (112, 26.8%), followed by Incheon (60, 14.4%) and Jeollanam-do (57, 13.6%). The positive rate relative to the number of tests was highest in Daejeon (62.5%, 5/8), followed by Jeju (27.6%, 24/87) and Jeollanam-do (26.1%, 57/218) (Table 3).
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Table 3. Number of diagnosed and positive cases by region (2019–2023) Region Year 2019 2020 2021 2022 2023 Total Gangwon 5/30 (16.7) 3/54 (5.6) 3/93 (3.2) 5/49 (10.2) 7/76 (9.2) 23/302 (7.6) Gyeonggi 0/20 (0.0) 2/35 (5.7) 1/46 (2.2) 4/46 (8.7) 7/114 (6.1) 14/261 (5.4) Gyeongsangbuk-do 1/9 (11.1) 1/2 (50.0) 0/2 (0.0) 0/4 (0.0) 0/0 (0.0) 2/17 (11.8) Gyeongsangnam-do 0/4 (0.0) 0/1 (0.0) 1/8 (12.5) 3/6 (50.0) 0/5 (0.0) 4/24 (16.7) Gwangju 35/111 (31.5) 16/120 (13.3) 23/113 (20.4) 28/107 (26.2) 10/49 (20.4) 112/500 (22.4) Daegu 0/3 (0.0) 0/0 (0.0) 0/1 (0.0) 0/1 (0.0) 1/6 (16.7) 1/11 (9.1) Busan 1/15 (6.7) 0/1 (0.0) 1/1 (100.0) 9/27 (33.3) 5/27 (18.5) 16/71 (22.5) Seoul 3/56 (5.4) 0/28 (0.0) 13/96 (13.5) 11/121 (9.1) 22/170 (12.9) 49/471 (10.4) Incheon 4/193 (2.1) 9/208 (4.3) 8/124 (6.5) 14/246 (5.7) 25/378 (6.6) 60/1,149 (5.2) Jeollanam-do 52/143 (36.4) 1/25 (4.0) 1/28 (3.6) 2/19 (10.5) 1/3 (33.3) 57/218 (26.1) Jeollanam-do 1/39 (2.6) 1/27 (3.7) 1/36 (2.8) 24/73 (32.9) 15/87 (17.2) 42/262 (16.0) Jeju 24/66 (36.4) 0/1 (0.0) 0/8 (0.0) 0/9 (0.0) 0/3 (0.0) 24/87 (27.6) Chungcheongnam-do 1/19 (5.3) 0/7 (0.0) 2/17 (11.8) 2/10 (20.0) 2/13 (15.4) 7/66 (10.6) Chungcheongbuk-do 0/9 (0.0) 0/9 (0.0) 0/8 (0.0) 1/21 (4.8) 1/17 (5.9) 2/64 (3.1) Daejeon 5/6 (83.3) 0/2 (0.0) 0/0 (0.0) 0/0 (0.0) 0/0 (0.0) 5/8 (62.5) Ulsan 0/0 (0.0) 0/0 (0.0) 0/4 (0.0) 0/0 (0.0) 0/0 (0.0) 0/4 (0.0) Sejong 0/0 (0.0) 0/0 (0.0) 0/0 (0.0) 0/0 (0.0) 0/0 (0.0) 0/0 (0.0) Total 132/723 (18.3) 33/520 (6.3) 54/585 (9.2) 103/739 (13.9) 96/948 (10.1) 418/3,515 (11.9) Values are presented as number/total (%).
In a re-analysis of the 2019 tests applying the 2020 revised criteria, the positive rates in Jeju and Jeollanam-do decreased to 19.7% (13/66) and 24.5% (35/143), respectively. This indicates that the single antibody titer criterion changed the positivity rate in each region. The number of diagnoses relative to the population in each region was highest in Gangwon (343.2%, 302/88), Jeollanam-do (159.1%, 218/137), and Jeollabuk-do (124.8%, 262/210), in order, which might be due to the large forested areas in these regions (Figure 2).
-
Figure 2. Status of diagnostic tests relative to population density by region
Diagnosis rate (%): number of diagnostic tests/population density by region (people/Km2).
In the IFA antibody detection test, IgG and IgM antibody titers below 1:16 accounted for the highest proportion of patients, 60.8% (1,419/2,334) and 76.4% (1,783/2,334), respectively (Table 4). The proportions of antibody titers were 11.4% (266/2,334) for IgG 1:2,048 or higher and 4.1% (96/2,334) for IgM 1:64. In Gwangju, antibody titers of IgG 1:2,048 or higher and IgM 1:64 were most frequent, while IgG 1:1,024 and IgM 1:512 and IgG 1:256 and IgM 1:32 were predominant in Jeju and Jeollanam-do, respectively. The antibody titers in Gangwon were mainly IgG 1:16 and IgM 1:16 (Table 4).
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Table 4. Number of IgG/IgM antibody titers by region (2019–2023) Region IFA titers (IgG/IgM) Total Under 1:16 1:16 1:32 1:64 1:128 1:256 1:512 1:1,024 Over 1:2,048 Gangwon 125/156 36/33 17/20 11/10 12/5 12/3 6/3 10/10 16/5 245 Gyeonggi 150/171 3/8 8/5 10/4 14/4 3/8 6/2 5/3 9/3 208 Gyeongsangbuk-do 6/7 2/2 1/4 3/2 0/0 2/0 1/1 0/0 2/1 17 Gyeongsangnam-do 9/12 0/0 1/1 1/0 0/0 1/0 0/0 0/0 1/0 13 Gwangju 204/373 1/12 7/24 12/47 32/15 28/10 40/2 34/8 138/5 496 Daegu 5/5 0/0 0/0 2/2 0/0 0/0 1/0 0/1 3/3 11 Busan 3/18 1/0 1/1 10/0 2/1 3/1 0/0 1/0 0/0 21 Seoul 261/269 4/1 3/6 4/7 6/5 3/6 7/4 3/4 19/8 310 Incheon 532/525 1/4 5/4 6/6 6/11 3/14 10/9 6/10 38/24 607 Jeollanam-do 36/67 1/5 6/9 3/8 12/1 21/3 8/0 4/1 7/4 98 Jeollabuk-do 24/101 1/6 18/8 21/5 26/6 21/8 14/6 9/5 16/5 150 Jeju 13/17 2/3 5/5 5/2 4/4 4/4 5/7 6/5 3/0 47 Chungcheongnam-do 10/17 2/9 2/3 3/1 8/6 3/1 3/2 3/1 12/6 46 Chungcheongbuk-do 41/45 3/4 4/2 2/2 0/3 4/1 3/2 3/1 0/0 60 Daejeon 0/0 1/1 0/0 0/0 0/0 0/0 0/0 0/0 0/0 1 Ulsan 0/0 0/0 0/0 0/0 0/1 1/0 0/0 1/3 2/0 4 Sejong 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0 Total (%) 1,419/1,783
(60.8/76.4)58/88
(2.5/3.8)78/92
(3.3/3.9)93/96
(4.0/4.1)122/62
(5.2/2.7)109/59
(4.7/2.5)104/38
(4.5/1.6)85/52
(3.6/2.2)266/64
(11.4/2.7)2,334
(100.0/100.0)Values are presented as number/total.
For 2,334 IFA antibody detection tests performed between 2019 and 2023, the median titers were 1:256 for IgG and 1:64 for IgM (Figure 3).
-
Figure 3. Distribution of diagnostic test counts and median IFA titer levels
IFA=indirect immunofluorescence assay.
The present study analyzed 466 cases of scrub typhus diagnosed in the last 5 years using a combination of PCR genetic testing and IFA antibody detection tests. We identified a high number of patients in Group A with high antibody titers (IgG≥1:2,048 and IgM≥1:2,048), indicating an association with pre-existing infection or delayed treatment. These findings suggest that the potential for reinfection should be considered in patients with high antibody titers at the time of diagnosis, and indicate that there is a need to establish criteria to distinguish long-term retention of antibody titers in the future. Group B represents cases in which a specific gene was detected in the absence of an antibody response, demonstrating that PCR genetic testing can be useful for early detection prior to antibody production. PCR-base gene detection is a useful tool for early diagnosis, before the appearance of antibodies; therefore, it may play an important role in the early diagnosis of infections that are not associated with an antibody response. Group C included cases with an antibody response but no detection of the specific gene. This is consistent with previous results showing a decrease in PCR sensitivity after antibiotic administration [10], suggesting that samples should be obtained prior to antibiotic administration for the diagnosis of scrub typhus. Thus, we recommend that healthcare institutions establish clear guidelines on the timing of sample collection.
In the comparison of positivity rates by test method, PCR genetic testing (20.0%) had a higher positivity rate than that of IFA antibody detection tests (7.8%) (Figure 1), suggesting that PCR could be more widely adopted for the diagnosis of scrub typhus in the future. We noticed that social distancing and reduced visits to green spaces during the coronavirus disease 2019 pandemic affected the number of tests, which may be an important factor in tracking the regional incidence of tick-borne diseases in the future [11].
Analyses by sex and age showed a particularly high positivity rate among middle-aged and elderly females, which could be related to various factors, such as the demographic composition in rural areas and the rise of the elderly population (Table 2). In this regard, tick prevention measures should be focused on gathering places, especially among females, to reduce the risk of infection.
With respect to region, Gangwon Province had the highest number of referrals for tests, suggesting that there is a link between large forest areas and the tick distribution (Figure 2). However, owing to the limited information about the occupation or activity of patients, further analyses of regional characteristics are needed [12].
Based on a previous study on long-term retention of scrub typhus antibody titers, a single antibody titer criterion was not used in this study, enabling us to more accurately distinguish between past infections and the possibility of reinfection. By combining genetic testing, we were able to more reliably identify patients who would be difficult to diagnose based on antibodies alone, which may have contributed to improving the diagnostic accuracy of current scrub typhus infections.
For the diagnosis of scrub typhus, thus, we recommend a comprehensive test that can simultaneously detect a 4-fold increase in antibody titers and a specific gene. Furthermore, healthcare providers should be educated on protocols to request these tests in combination. The combination of PCR genetic testing and IFA antibody detection tests is expected to be a useful approach to clearly differentiate the scrub typhus infection status.
Ethics Statement: Not applicable.
Funding Source: None.
Acknowledgments: None.
Conflict of Interest: The authors have no conflicts of interest to declare.
Author Contributions: Conceptualization: HSK, BCG. Data curation: HSK, BCG, KAL. Formal analysis: BCG, KAL. Investigation: KAL. Writing – original draft: KAL. Writing – review & editing: JIY, HSK.
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- Akaike T, Ishizuka K, Tominaga N, Motohashi I. Scrub typhus: the clinical significance of the eschar. BMJ Case Rep 2023;16:e255404.
- Chakraborty S, Sarma N. Scrub typhus: an emerging threat. Indian J Dermatol 2017;62:478-85.
- Jiang J, Richards AL. Scrub typhus: no longer restricted to the tsutsugamushi triangle. Trop Med Infect Dis 2018;3:11.
- National Institute of Infectious Diseases (NIID). Scrub typhus and Japanese spotted fever in Japan 2007-2016 [Internet]. NIID; 2017 [cited 2024 Aug 16].
Available from: https://www.niid.go.jp/niid/en/basic-science/865-iasr/7342-448te.html - Taiwan Centers for Disease Control. Scrub typhus (tsutsugamushi disease) [Internet]. Taiwan Centers for Disease Control; 2017 [cited 2024 Aug 16].
Available from: https://www.cdc.gov.tw/En/Category/ListContent/bg0g_VU_Ysrgkes_KRUDgQ?uaid=nx2oarjBljCn_78hBlI5fA - Qian J, Wu Y, Zhu C, et al. Spatiotemporal heterogeneity and long-term impact of meteorological, environmental, and socio-economic factors on scrub typhus in China from 2006 to 2018. BMC Public Health 2024;24:538.
- Chung MH, Kang JS. History of tsutsugamushi disease in Korea. Infect Chemother 2019;51:196-209.
- Kim DM, Lee YM, Back JH, et al. A serosurvey of Orientia tsutsugamushi from patients with scrub typhus. Clin Microbiol Infect 2010;16:447-51.
- Kim DM. Clinical features and diagnosis of scrub typhus. Infect Chemother 2009;41:315-22.
- Chang C, Park SH, Seol A. Factors affecting changes in forest recreational activities during the COVID-19 pandemic. J Korean Soc For Sci 2021;110:711-23.
- Korea Disease Control and Prevention Agency (KDCA). 2024 KDCA management guidelines for tick-borne and rodent-borne diseases. Cheongju: KDCA; 2024 Mar. Report No.: 11-1790387-000525-10.
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Public Health Weekly Report 2024; 17(49): 2177-2196
Published online December 19, 2024 https://doi.org/10.56786/PHWR.2024.17.49.1
Copyright © The Korea Disease Control and Prevention Agency.
Analysis of Laboratory Diagnostic Tests for Scrub Typhus from 2019 to 2023
Kyeong-Ah Lee , Byoungchul Gill *, Hwa Su Kim , Jaeil Yoo
Division of Bacterial Diseases, Department of Laboratory Diagnosis and analysis, Korea Disease Control and Prevention Agency, Cheongju, Korea
Correspondence to:*Corresponding author: Byoungchul Gill, Tel: +82-43-719-8113, E-mail: gilri@korea.kr
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Scrub typhus is a tick-borne infectious disease caused by Orientia tsutsugamushi. When bitten by an infected tick, an eschar forms and symptoms such as fever, chills, and rash appear. This study systematically analyzed the prevalence of scrub typhus reported from 2019 to 2023 by the Korea Disease Control and Prevention Agency and 19 regional institutes of public health and the environment, examining trends according to diagnostic methods, sex, age, and region. Of the 2,607 individuals with suspected infection, 12.4% (322 patients) tested positive for scrub typhus, with 17.9% (466 patients) undergoing both polymerase chain reaction (PCR) and indirect immunofluorescence assay (IFA) tests. Among the 466 patients who were tested using both methods, Group A (positive PCR and IFA test results) demonstrated the highest positivity rate of 68.9%, suggesting a combination of past infections and reinfections. The positivity rate of the gene detection test (20.0%) was higher than that of the antibody detection test (7.8%), suggesting that PCR-based gene detection has a relatively higher sensitivity for confirming current infections. Analysis by sex and age revealed the highest positivity rate in women in their 70s (18.2%), with women consistently exhibiting higher positivity rates than men across all age groups. A regional analysis indicated that Gwangju contributed to 26.8% of patients with a positive test result, while Gangwon Province had the highest testing rate per capita (343.2%). Performing both IFA antibody detection and PCR gene detection tests simultaneously to diagnose scrub typhus is valuable for more accurately distinguishing current infections, offering critical insights to enhance the diagnostic and management strategies for scrub typhus.
Keywords: Scrub typhus, Orientia tsutsugamushi, Tick-borne diseases, Laboratory diagnosis
Body
Key messages
① What is known previously?
Scrub typhus is a tick-borne infectious disease caused by Orientia tsutsugamushi. When bitten by an infected tick, an eschar forms, and symptoms such as fever, chills, and rash appear.
② What new information is presented?
The positivity rate of the gene detection test (20.0%) was higher than that of the antibody detection test (7.8%), suggesting that polymerase chain reaction (PCR)-based gene detection has relatively higher sensitivity in confirming current infections.
③ What are implications?
Performing both immunofluorescence assay antibody detection and PCR gene detection tests simultaneously in the diagnosis of scrub typhus is considered useful for more accurately distinguishing current infections.
Introduction
Scrub typhus is a vector-borne disease caused by Orientia tsutsugamushi and is primarily transmitted through chiggers or mites, such as Leptotrombidium scutellare or Leptotrombidium pallidum [1]. When an O. tsutsugamushi-infected chigger adheres to the skin and ingests bodily fluids, the pathogen enters the body, causing symptoms, such as high fever, headache, myalgia, and eschar formation. An eschar can be a main finding in the diagnosis of scrub typhus, making it possible to differentiate it from other febrile illnesses [2]. If no eschar is identified, diagnosis may be delayed because initial symptoms are nonspecific and are similar to those of the common cold. Without appropriate antibiotic treatment, the disease may progress to severe conditions, including interstitial pneumonia, myocarditis, delirium, and central nervous system complications [3].
Scrub typhus is found in a particular geographic region known as the tsutsugamushi triangle. This triangle includes Japan, Republic of Korea (ROK), northeastern China, Pakistan, Thailand, Vietnam, northern Indonesia, and northern Australia. However, there are recent reports of scrub typhus cases outside of the tsutsugamushi triangle, including Chile, the United States, and the Netherlands [4]. These cases suggest the potential for scrub typhus to occur globally, beyond previously known areas. In Japan and Taiwan, aggressive prevention and health promotion activities have led to a slight decrease in the incidence of scrub typhus over the past two decades [5,6]. On the other hand, the incidence in China has been increasing annually due to geographical characteristics, socioeconomic factors, and differences in education levels. Thus, effective prevention and control are needed during outbreaks [7].
In ROK, the first infection case was reported during the Korean War in 1951 [8]. Since 1994, the Korea Disease Control and Prevention Agency (KDCA) has conducted systematic patient surveillance and control. Scrub typhus is diagnosed by antibody detection tests using indirect immunofluorescence assays (IFA) and genetic testing based on polymerase chain reaction (PCR). Previously, genetic testing was performed only at KDCA. However, the technology was transferred to the Public Health and Environment Research Institute in 2024, and public health laboratories nationwide are now capable of performing antibody detection and genetic testing simultaneously.
In antibody detection tests, until 2019, a single antibody titer of 1:256 or more for IgG or 1:16 or more for IgM was considered positive. In 2020, the diagnostic criterion was changed to a 4-fold or greater increase in the antibody titer. This was designed to improve diagnostic accuracy by distinguishing between past and recent infections. No other country uses a single antibody level as a confirmatory criterion, even in international cases. In ROK, it was reported that IgG antibodies continue to be detectable at low titers (median 1:128) from 18 months to 2 years after infection, and IgM antibodies remained detectable in some patients (29%) for 1 year after infection [9].
However, few studies have evaluated the impact of this change in diagnostic criteria on actual diagnostic results. It is necessary to analyze disease incidence with respect to demographic factors and to compare the positive rate of each test method. Based on the results of antibody detection tests and genetic testing conducted by KDCA and Public Health and Environment Research Institutes in 19 cities and provinces in ROK from 2019 to 2023, this study aimed to analyze patterns of scrub typhus diagnosis with respect to sex, age, and region and to evaluate the impact of changes in diagnostic criteria on test results. In this way, we evaluated the effectiveness of each test method and provided basic data for improving the diagnosis and management system for scrub typhus.
Methods
1. Study Subjects and Sample Collection
In total, 2,607 patients with suspected scrub typhus referred to KDCA and the Public Health and Environment Research Institute in cities and provinces nationwide from 2019 to 2023 were enrolled. Patients had symptoms, such as fever, tick bites, and low platelets; however, data for antibiotics use were not available. The requested samples included serum, whole blood, eschar, etc. Patients were categorized by test method into those who underwent only antibody detection tests (1,458), only genetic testing (683), or both tests (466).
2. Diagnostic Criteria
Scrub typhus was diagnosed by IFA and PCR. For IFA antibody detection tests, acute-phase serum (first referral) was compared with convalescent-phase serum (second or more referrals). Acute-phase serum (first referral) samples were determined to be inconclusive regardless of antibody titer, while convalescent serum was identified as positive if the antibody titer increased by more than 4-fold over that in the acute phase. If the increase in antibody titer was <4-fold, it was considered negative. Genetic testing was performed using whole blood and eschar, targeting the 56-KDa outer membrane protein gene (TSA56). If this gene was detected, the case was judged positive.
3. Classification of Test Results
Of 2,607 patients with suspected scrub typhus, 1,458 patients were evaluated using the IFA antibody detection test only and 683 patients were evaluated through genetic testing only. Results of antibody detection tests were evaluated based on the IFA antibody titer criteria, and genetic tests were classified as positive or negative based on the presence or absence of the TSA56 gene. Additionally, 466 patients who underwent both genetic testing and an antibody detection test were divided into four groups on the basis of genetic testing results. Group A consisted of patients who tested positive in genetic testing and had an IgG or IgM antibody titer of 1:16 or higher in the antibody detection test. Group B was composed of patients who tested positive on genetic testing but had both IgG and IgM antibody titers less than 1:16 in antibody detection tests. Group C included patients who tested negative on genetic testing but had an IgG or IgM antibody titer of 1:16 or higher in antibody detection tests. Finally, Group D included patients who were negative on genetic testing and had both IgG and IgM antibody titers less than 1:16 in antibody detection tests. Patients who underwent only one test were classified as positive or negative based on the results of their respective tests, and these results were compared with those for patients evaluated using both tests.
4. Data Analysis
A total of 3,515 test results were analyzed, including results for 2,334 antibody detection tests and 1,181 genetic tests. The results for each sample were analyzed according to sex, age, and region. The number of tests per population by region was analyzed based on the ‘2022 Population and Housing Census’ from Statistics Korea. In addition, the median antibody titer, which refers to the mean antibody titer for samples showing an immune response of IgG 1:16 or higher or IgM 1:16 or higher, was calculated based on 5 years of information. Statistical analyses, including comparisons of the proportion of positive cases in each group and infection rates by age and sex, were performed using IBM SPSS Statistics 26 (IBM Corp.). The results were derived from the diagnosis and regional characteristics.
Results
1. Analysis of Single Test Results
Of 1,458 patients evaluated using the IFA antibody detection test alone, 63 (4.3%) tested positive. When using the 2019 criterion for a single antibody titer (IgG 1:256 or greater or IgM 1:16 or greater), however, the number of positive tests increased to 90 (6.2%). Of the 683 patients evaluated using PCR-based genetic testing alone, 158 (23.1%) tested positive; this was higher than the positive rate (6.2%) obtained using antibody detection tests alone.
2. Analysis of Combined Test Results
The 466 patients who underwent both genetic testing and antibody detection test were classified into four groups based on test results. Groups A and B tested positive for a specific gene on genetic testing, among which 67 of 74 (90.5%) positive cases were confirmed. In Group A, 27 of 51 patients showed an IgG antibody titer of 1:2,048 or higher, suggesting the possibility of reinfection. Group B, which included 16 patients, had no immune response in the antibody detection test, while genetic testing results were positive, indicating a highly probable early infection. Group C consisted of patients who did not undergo genetic testing and showed a 4-fold or greater increase in antibody titers in convalescent serum compared with acute phase serum in the antibody detection test, identified as 37 inconclusive, 7 positive, and 2 negative. Group D was composed of patients who were negative on both genetic testing and antibody detection tests, with 348 patients classified as inconclusive (Table 1). When applying the 2019 single antibody titer criteria (IgG 1:256 or higher or IgM 1:16 or higher), the number of patients who tested positive in Group C increased from 7 to 12. In Group A, 2 patients tested positive on genetic testing, leaving the results of the analysis unchanged.
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Results of concurrent genetic and antibody tests in patients (2019–2023) Group PCR IFA titer (IgG/IgM) Total
(Pos./rate [%])Diagnosis case +/–a) Under 1:16 1:16 1:32 1:64 1:128 1:256 1:512 1:1,024 Over 1:2,048 Group A + 9/11
(9/11)1/0
(1/0)2/0
(2/0)3/3
(3/3)2/9
(2/9)2/4
(2/4)1/7
(1/7)4/4
(4/4)27/13
(27/13)51
(51/68.9)Pos. Neg. Und. 51 0 0 Group B + 16/16
(16/16)0/0
(0/0)0/0
(0/0)0/0
(0/0)0/0
(0/0)0/0
(0/0)0/0
(0/0)0/0
(0/0)0/0
(0/0)16
(16/21.6)Pos. Neg. Und. 16 0 0 Group C – 8/20
(1/3)2/6
(1/0)4/6
(0/1)1/1
(0/0)6/4
(0/2)7/3
(1/0)7/1
(1/0)3/2
(1/0)8/3
(2/1)46
(7/9.5)Pos. Neg. Und. 7 2 37 Group D – 353/353
(0/0)0/0
(0/0)0/0
(0/0)0/0
(0/0)0/0
(0/0)0/0
(0/0)0/0
(0/0)0/0
(0/0)0/0
(0/0)353
(0/0.0)Pos. Neg. Und. 0 5 348 Total (positive case) 386/400
(26/30)3/6
(2/0)6/6
(2/1)4/4
(3/3)8/13
(2/11)9/7
(3/4)8/8
(2/7)7/6
(5/4)35/16
(29/14)466
(74/100.0)Pos. Neg. Und. 74 7 385 Values are presented as patients cases (positive cases). PCR=polymerase chain reaction; IFA=indirect immunofluorescence assay; Und.=Undetermined. a)‘+’: Positive or Pos., ‘–’: Negative or Neg...
3. Diagnostic Test Results by Test Method
A total of 3,515 diagnostic tests were performed on 2,607 patients with suspected scrub typhus from 2019 to 2023. The positivity rate for genetic testing was lowest in 2020 at 8.7% (11/126) and increased to 25.8% (71/275) in 2022 (Figure 1A). For antibody detection tests, the positivity rate was 17.4% (78/449) based on the single antibody titer criterion in 2019 and decreased to 9.8% (44/449) when applying the revised criterion after 2020. However, this was still the highest rate in the last 5 years. The 44 patients with a 4-fold increase in antibody titers were suspected to be recently infected, while 34 patients who tested positive based on a single antibody titer were likely antibody carriers from past infections (Figure 1B).
-
Figure 1. Number of diagnosed and positive cases by year
(A) Annual trends in diagnostic results using genetic test, (B) yearly diagnostic trends in antibody test.
4. Diagnostic Test Results by Sex and Age
A total of 3,515 diagnostic tests were conducted on 2,607 patients with suspected scrub typhus between 2019 and 2023. The sex ratio was nearly 1:1, with 1,745 (49.6%) male patients and 1,770 (50.4%) female patients. Of the 418 positive patients, however, there were 160 (38.3%) males and 258 (61.7%) females, indicating a higher positive rate in females than in males (Table 2).
-
Number of diagnostic tests by age and sex and number of positive cases (2019–2023) Sex Year Age (yr) 0–19 20–29 30–39 40–49 50–59 60–69 70–79 Over 80 Total Male 2019 2/17
(0.6)2/24
(0.6)2/24
(0.6)4/40
(1.2)1/54
(0.3)13/72
(3.8)17/80
(4.9)8/34
(2.3)49/345
(14.2)2020 2/14
(0.8)1/27
(0.4)0/12
(0.0)0/22
(0.0)1/47
(0.4)5/65
(1.9)6/53
(2.3)0/17
(0.0)15/257
(5.8)2021 0/5
(0.0)0/28
(0.0)0/27
(0.0)1/30
(0.4)3/30
(1.1)6/53
(2.2)7/67
(2.5)0/35
(0.0)17/275
(6.2)2022 3/18
(0.8)4/39
(1.1)0/27
(0.0)0/24
(0.0)2/43
(0.6)10/69
(2.8)14/82
(3.9)4/55
(1.1)37/357
(10.4)2023 0/25
(0.0)2/56
(0.4)0/32
(0.0)0/46
(0.0)9/73
(1.8)14/120
(2.7)15/114
(2.9)2/45
(0.4)42/511
(8.2)Total 7/79
(8.9)9/174
(5.2)2/122
(1.6)5/162
(3.1)16/247
(6.5)48/379
(12.7)59/396
(14.9)14/186
(7.5)160/1,745
(9.2)Female 2019 1/7
(0.3)1/16
(0.3)2/14
(0.5)4/25
(1.1)17/72
(4.5)16/86
(4.2)29/101
(7.7)13/57
(3.4)83/378
(22.0)2020 0/11
(0.0)1/13
(0.4)1/25
(0.4)2/16
(0.8)2/17
(0.8)0/36
(0.0)9/92
(3.4)3/53
(1.1)18/263
(6.8)2021 0/6
(0.0)0/11
(0.0)2/16
(0.6)0/19
(0.0)2/43
(0.6)16/79
(5.2)13/76
(4.2)4/60
(1.3)37/310
(11.9)2022 2/5
(0.5)2/24
(0.5)0/21
(0.0)7/33
(1.8)7/50
(1.8)18/72
(4.7)19/93
(5.0)11/84
(2.9)66/382
(17.3)2023 1/22
(0.2)2/19
(0.5)1/23
(0.2)2/33
(0.5)10/54
(2.3)11/112
(2.5)14/99
(3.2)13/75
(3.0)54/437
(12.4)Total 4/51
(7.8)6/83
(7.2)6/99
(6.1)15/126
(11.9)38/236
(16.1)61/385
(15.8)84/461
(18.2)44/329
(13.4)258/1770
(14.6)Total 11/130
(0.3)15/257
(0.4)8/221
(0.2)20/288
(0.6)54/483
(1.5)109/764
(3.1)143/857
(4.1)58/515
(1.7)418/3,515
(11.9)Values are presented as number/total (%)..
By age, a total of 3,515 scrub typhus cases were referred, with 857 patients (24.4%) aged 70–79 years, followed by 764 (21.7%) aged 60–69 years and 515 (14.7%) aged 80 years and older. The mean age of scrub typhus-positive patients was 64.7 years, with 143 (34.2%) aged 70–79 years, followed by 109 (26.1%) aged 60–69 years and 58 (13.9%) aged 80 years and older. The positive rate relative to the number of diagnoses was highest for individuals aged 70–79 years at 4.1% (143/857), followed by 3.1% (109/764) for 60–69 years, 1.7% (58/515) for 80 years and older, and 1.5% (54/483) for 50–59 years. In particular, the 5-year age breakdown of positive patients showed that females had a higher positive rate than that for males in all age groups, except 19 years and younger (7.8%) (Table 2).
5. Diagnostic Test Results by Region
Of the 3,515 diagnostic tests performed from 2019 to 2023, the highest number of tests was performed in Incheon (1,149, 32.7%), followed by Gwangju (500, 14.2%), Seoul (471, 13.4%), and Gangwon (302, 8.6%). The number of positive test results was highest in Gwangju (112, 26.8%), followed by Incheon (60, 14.4%) and Jeollanam-do (57, 13.6%). The positive rate relative to the number of tests was highest in Daejeon (62.5%, 5/8), followed by Jeju (27.6%, 24/87) and Jeollanam-do (26.1%, 57/218) (Table 3).
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Number of diagnosed and positive cases by region (2019–2023) Region Year 2019 2020 2021 2022 2023 Total Gangwon 5/30 (16.7) 3/54 (5.6) 3/93 (3.2) 5/49 (10.2) 7/76 (9.2) 23/302 (7.6) Gyeonggi 0/20 (0.0) 2/35 (5.7) 1/46 (2.2) 4/46 (8.7) 7/114 (6.1) 14/261 (5.4) Gyeongsangbuk-do 1/9 (11.1) 1/2 (50.0) 0/2 (0.0) 0/4 (0.0) 0/0 (0.0) 2/17 (11.8) Gyeongsangnam-do 0/4 (0.0) 0/1 (0.0) 1/8 (12.5) 3/6 (50.0) 0/5 (0.0) 4/24 (16.7) Gwangju 35/111 (31.5) 16/120 (13.3) 23/113 (20.4) 28/107 (26.2) 10/49 (20.4) 112/500 (22.4) Daegu 0/3 (0.0) 0/0 (0.0) 0/1 (0.0) 0/1 (0.0) 1/6 (16.7) 1/11 (9.1) Busan 1/15 (6.7) 0/1 (0.0) 1/1 (100.0) 9/27 (33.3) 5/27 (18.5) 16/71 (22.5) Seoul 3/56 (5.4) 0/28 (0.0) 13/96 (13.5) 11/121 (9.1) 22/170 (12.9) 49/471 (10.4) Incheon 4/193 (2.1) 9/208 (4.3) 8/124 (6.5) 14/246 (5.7) 25/378 (6.6) 60/1,149 (5.2) Jeollanam-do 52/143 (36.4) 1/25 (4.0) 1/28 (3.6) 2/19 (10.5) 1/3 (33.3) 57/218 (26.1) Jeollanam-do 1/39 (2.6) 1/27 (3.7) 1/36 (2.8) 24/73 (32.9) 15/87 (17.2) 42/262 (16.0) Jeju 24/66 (36.4) 0/1 (0.0) 0/8 (0.0) 0/9 (0.0) 0/3 (0.0) 24/87 (27.6) Chungcheongnam-do 1/19 (5.3) 0/7 (0.0) 2/17 (11.8) 2/10 (20.0) 2/13 (15.4) 7/66 (10.6) Chungcheongbuk-do 0/9 (0.0) 0/9 (0.0) 0/8 (0.0) 1/21 (4.8) 1/17 (5.9) 2/64 (3.1) Daejeon 5/6 (83.3) 0/2 (0.0) 0/0 (0.0) 0/0 (0.0) 0/0 (0.0) 5/8 (62.5) Ulsan 0/0 (0.0) 0/0 (0.0) 0/4 (0.0) 0/0 (0.0) 0/0 (0.0) 0/4 (0.0) Sejong 0/0 (0.0) 0/0 (0.0) 0/0 (0.0) 0/0 (0.0) 0/0 (0.0) 0/0 (0.0) Total 132/723 (18.3) 33/520 (6.3) 54/585 (9.2) 103/739 (13.9) 96/948 (10.1) 418/3,515 (11.9) Values are presented as number/total (%)..
In a re-analysis of the 2019 tests applying the 2020 revised criteria, the positive rates in Jeju and Jeollanam-do decreased to 19.7% (13/66) and 24.5% (35/143), respectively. This indicates that the single antibody titer criterion changed the positivity rate in each region. The number of diagnoses relative to the population in each region was highest in Gangwon (343.2%, 302/88), Jeollanam-do (159.1%, 218/137), and Jeollabuk-do (124.8%, 262/210), in order, which might be due to the large forested areas in these regions (Figure 2).
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Figure 2. Status of diagnostic tests relative to population density by region
Diagnosis rate (%): number of diagnostic tests/population density by region (people/Km2).
In the IFA antibody detection test, IgG and IgM antibody titers below 1:16 accounted for the highest proportion of patients, 60.8% (1,419/2,334) and 76.4% (1,783/2,334), respectively (Table 4). The proportions of antibody titers were 11.4% (266/2,334) for IgG 1:2,048 or higher and 4.1% (96/2,334) for IgM 1:64. In Gwangju, antibody titers of IgG 1:2,048 or higher and IgM 1:64 were most frequent, while IgG 1:1,024 and IgM 1:512 and IgG 1:256 and IgM 1:32 were predominant in Jeju and Jeollanam-do, respectively. The antibody titers in Gangwon were mainly IgG 1:16 and IgM 1:16 (Table 4).
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Number of IgG/IgM antibody titers by region (2019–2023) Region IFA titers (IgG/IgM) Total Under 1:16 1:16 1:32 1:64 1:128 1:256 1:512 1:1,024 Over 1:2,048 Gangwon 125/156 36/33 17/20 11/10 12/5 12/3 6/3 10/10 16/5 245 Gyeonggi 150/171 3/8 8/5 10/4 14/4 3/8 6/2 5/3 9/3 208 Gyeongsangbuk-do 6/7 2/2 1/4 3/2 0/0 2/0 1/1 0/0 2/1 17 Gyeongsangnam-do 9/12 0/0 1/1 1/0 0/0 1/0 0/0 0/0 1/0 13 Gwangju 204/373 1/12 7/24 12/47 32/15 28/10 40/2 34/8 138/5 496 Daegu 5/5 0/0 0/0 2/2 0/0 0/0 1/0 0/1 3/3 11 Busan 3/18 1/0 1/1 10/0 2/1 3/1 0/0 1/0 0/0 21 Seoul 261/269 4/1 3/6 4/7 6/5 3/6 7/4 3/4 19/8 310 Incheon 532/525 1/4 5/4 6/6 6/11 3/14 10/9 6/10 38/24 607 Jeollanam-do 36/67 1/5 6/9 3/8 12/1 21/3 8/0 4/1 7/4 98 Jeollabuk-do 24/101 1/6 18/8 21/5 26/6 21/8 14/6 9/5 16/5 150 Jeju 13/17 2/3 5/5 5/2 4/4 4/4 5/7 6/5 3/0 47 Chungcheongnam-do 10/17 2/9 2/3 3/1 8/6 3/1 3/2 3/1 12/6 46 Chungcheongbuk-do 41/45 3/4 4/2 2/2 0/3 4/1 3/2 3/1 0/0 60 Daejeon 0/0 1/1 0/0 0/0 0/0 0/0 0/0 0/0 0/0 1 Ulsan 0/0 0/0 0/0 0/0 0/1 1/0 0/0 1/3 2/0 4 Sejong 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0 Total (%) 1,419/1,783
(60.8/76.4)58/88
(2.5/3.8)78/92
(3.3/3.9)93/96
(4.0/4.1)122/62
(5.2/2.7)109/59
(4.7/2.5)104/38
(4.5/1.6)85/52
(3.6/2.2)266/64
(11.4/2.7)2,334
(100.0/100.0)Values are presented as number/total..
For 2,334 IFA antibody detection tests performed between 2019 and 2023, the median titers were 1:256 for IgG and 1:64 for IgM (Figure 3).
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Figure 3. Distribution of diagnostic test counts and median IFA titer levels
IFA=indirect immunofluorescence assay.
Discussion
The present study analyzed 466 cases of scrub typhus diagnosed in the last 5 years using a combination of PCR genetic testing and IFA antibody detection tests. We identified a high number of patients in Group A with high antibody titers (IgG≥1:2,048 and IgM≥1:2,048), indicating an association with pre-existing infection or delayed treatment. These findings suggest that the potential for reinfection should be considered in patients with high antibody titers at the time of diagnosis, and indicate that there is a need to establish criteria to distinguish long-term retention of antibody titers in the future. Group B represents cases in which a specific gene was detected in the absence of an antibody response, demonstrating that PCR genetic testing can be useful for early detection prior to antibody production. PCR-base gene detection is a useful tool for early diagnosis, before the appearance of antibodies; therefore, it may play an important role in the early diagnosis of infections that are not associated with an antibody response. Group C included cases with an antibody response but no detection of the specific gene. This is consistent with previous results showing a decrease in PCR sensitivity after antibiotic administration [10], suggesting that samples should be obtained prior to antibiotic administration for the diagnosis of scrub typhus. Thus, we recommend that healthcare institutions establish clear guidelines on the timing of sample collection.
In the comparison of positivity rates by test method, PCR genetic testing (20.0%) had a higher positivity rate than that of IFA antibody detection tests (7.8%) (Figure 1), suggesting that PCR could be more widely adopted for the diagnosis of scrub typhus in the future. We noticed that social distancing and reduced visits to green spaces during the coronavirus disease 2019 pandemic affected the number of tests, which may be an important factor in tracking the regional incidence of tick-borne diseases in the future [11].
Analyses by sex and age showed a particularly high positivity rate among middle-aged and elderly females, which could be related to various factors, such as the demographic composition in rural areas and the rise of the elderly population (Table 2). In this regard, tick prevention measures should be focused on gathering places, especially among females, to reduce the risk of infection.
With respect to region, Gangwon Province had the highest number of referrals for tests, suggesting that there is a link between large forest areas and the tick distribution (Figure 2). However, owing to the limited information about the occupation or activity of patients, further analyses of regional characteristics are needed [12].
Based on a previous study on long-term retention of scrub typhus antibody titers, a single antibody titer criterion was not used in this study, enabling us to more accurately distinguish between past infections and the possibility of reinfection. By combining genetic testing, we were able to more reliably identify patients who would be difficult to diagnose based on antibodies alone, which may have contributed to improving the diagnostic accuracy of current scrub typhus infections.
For the diagnosis of scrub typhus, thus, we recommend a comprehensive test that can simultaneously detect a 4-fold increase in antibody titers and a specific gene. Furthermore, healthcare providers should be educated on protocols to request these tests in combination. The combination of PCR genetic testing and IFA antibody detection tests is expected to be a useful approach to clearly differentiate the scrub typhus infection status.
Declarations
Ethics Statement: Not applicable.
Funding Source: None.
Acknowledgments: None.
Conflict of Interest: The authors have no conflicts of interest to declare.
Author Contributions: Conceptualization: HSK, BCG. Data curation: HSK, BCG, KAL. Formal analysis: BCG, KAL. Investigation: KAL. Writing – original draft: KAL. Writing – review & editing: JIY, HSK.
Fig 1.
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(A) Annual trends in diagnostic results using genetic test, (B) yearly diagnostic trends in antibody test.
Fig 2.
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Diagnosis rate (%): number of diagnostic tests/population density by region (people/Km2).
Fig 3.
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IFA=indirect immunofluorescence assay.
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Results of concurrent genetic and antibody tests in patients (2019–2023) Group PCR IFA titer (IgG/IgM) Total
(Pos./rate [%])Diagnosis case +/–a) Under 1:16 1:16 1:32 1:64 1:128 1:256 1:512 1:1,024 Over 1:2,048 Group A + 9/11
(9/11)1/0
(1/0)2/0
(2/0)3/3
(3/3)2/9
(2/9)2/4
(2/4)1/7
(1/7)4/4
(4/4)27/13
(27/13)51
(51/68.9)Pos. Neg. Und. 51 0 0 Group B + 16/16
(16/16)0/0
(0/0)0/0
(0/0)0/0
(0/0)0/0
(0/0)0/0
(0/0)0/0
(0/0)0/0
(0/0)0/0
(0/0)16
(16/21.6)Pos. Neg. Und. 16 0 0 Group C – 8/20
(1/3)2/6
(1/0)4/6
(0/1)1/1
(0/0)6/4
(0/2)7/3
(1/0)7/1
(1/0)3/2
(1/0)8/3
(2/1)46
(7/9.5)Pos. Neg. Und. 7 2 37 Group D – 353/353
(0/0)0/0
(0/0)0/0
(0/0)0/0
(0/0)0/0
(0/0)0/0
(0/0)0/0
(0/0)0/0
(0/0)0/0
(0/0)353
(0/0.0)Pos. Neg. Und. 0 5 348 Total (positive case) 386/400
(26/30)3/6
(2/0)6/6
(2/1)4/4
(3/3)8/13
(2/11)9/7
(3/4)8/8
(2/7)7/6
(5/4)35/16
(29/14)466
(74/100.0)Pos. Neg. Und. 74 7 385 Values are presented as patients cases (positive cases). PCR=polymerase chain reaction; IFA=indirect immunofluorescence assay; Und.=Undetermined. a)‘+’: Positive or Pos., ‘–’: Negative or Neg...
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Number of diagnostic tests by age and sex and number of positive cases (2019–2023) Sex Year Age (yr) 0–19 20–29 30–39 40–49 50–59 60–69 70–79 Over 80 Total Male 2019 2/17
(0.6)2/24
(0.6)2/24
(0.6)4/40
(1.2)1/54
(0.3)13/72
(3.8)17/80
(4.9)8/34
(2.3)49/345
(14.2)2020 2/14
(0.8)1/27
(0.4)0/12
(0.0)0/22
(0.0)1/47
(0.4)5/65
(1.9)6/53
(2.3)0/17
(0.0)15/257
(5.8)2021 0/5
(0.0)0/28
(0.0)0/27
(0.0)1/30
(0.4)3/30
(1.1)6/53
(2.2)7/67
(2.5)0/35
(0.0)17/275
(6.2)2022 3/18
(0.8)4/39
(1.1)0/27
(0.0)0/24
(0.0)2/43
(0.6)10/69
(2.8)14/82
(3.9)4/55
(1.1)37/357
(10.4)2023 0/25
(0.0)2/56
(0.4)0/32
(0.0)0/46
(0.0)9/73
(1.8)14/120
(2.7)15/114
(2.9)2/45
(0.4)42/511
(8.2)Total 7/79
(8.9)9/174
(5.2)2/122
(1.6)5/162
(3.1)16/247
(6.5)48/379
(12.7)59/396
(14.9)14/186
(7.5)160/1,745
(9.2)Female 2019 1/7
(0.3)1/16
(0.3)2/14
(0.5)4/25
(1.1)17/72
(4.5)16/86
(4.2)29/101
(7.7)13/57
(3.4)83/378
(22.0)2020 0/11
(0.0)1/13
(0.4)1/25
(0.4)2/16
(0.8)2/17
(0.8)0/36
(0.0)9/92
(3.4)3/53
(1.1)18/263
(6.8)2021 0/6
(0.0)0/11
(0.0)2/16
(0.6)0/19
(0.0)2/43
(0.6)16/79
(5.2)13/76
(4.2)4/60
(1.3)37/310
(11.9)2022 2/5
(0.5)2/24
(0.5)0/21
(0.0)7/33
(1.8)7/50
(1.8)18/72
(4.7)19/93
(5.0)11/84
(2.9)66/382
(17.3)2023 1/22
(0.2)2/19
(0.5)1/23
(0.2)2/33
(0.5)10/54
(2.3)11/112
(2.5)14/99
(3.2)13/75
(3.0)54/437
(12.4)Total 4/51
(7.8)6/83
(7.2)6/99
(6.1)15/126
(11.9)38/236
(16.1)61/385
(15.8)84/461
(18.2)44/329
(13.4)258/1770
(14.6)Total 11/130
(0.3)15/257
(0.4)8/221
(0.2)20/288
(0.6)54/483
(1.5)109/764
(3.1)143/857
(4.1)58/515
(1.7)418/3,515
(11.9)Values are presented as number/total (%)..
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Number of diagnosed and positive cases by region (2019–2023) Region Year 2019 2020 2021 2022 2023 Total Gangwon 5/30 (16.7) 3/54 (5.6) 3/93 (3.2) 5/49 (10.2) 7/76 (9.2) 23/302 (7.6) Gyeonggi 0/20 (0.0) 2/35 (5.7) 1/46 (2.2) 4/46 (8.7) 7/114 (6.1) 14/261 (5.4) Gyeongsangbuk-do 1/9 (11.1) 1/2 (50.0) 0/2 (0.0) 0/4 (0.0) 0/0 (0.0) 2/17 (11.8) Gyeongsangnam-do 0/4 (0.0) 0/1 (0.0) 1/8 (12.5) 3/6 (50.0) 0/5 (0.0) 4/24 (16.7) Gwangju 35/111 (31.5) 16/120 (13.3) 23/113 (20.4) 28/107 (26.2) 10/49 (20.4) 112/500 (22.4) Daegu 0/3 (0.0) 0/0 (0.0) 0/1 (0.0) 0/1 (0.0) 1/6 (16.7) 1/11 (9.1) Busan 1/15 (6.7) 0/1 (0.0) 1/1 (100.0) 9/27 (33.3) 5/27 (18.5) 16/71 (22.5) Seoul 3/56 (5.4) 0/28 (0.0) 13/96 (13.5) 11/121 (9.1) 22/170 (12.9) 49/471 (10.4) Incheon 4/193 (2.1) 9/208 (4.3) 8/124 (6.5) 14/246 (5.7) 25/378 (6.6) 60/1,149 (5.2) Jeollanam-do 52/143 (36.4) 1/25 (4.0) 1/28 (3.6) 2/19 (10.5) 1/3 (33.3) 57/218 (26.1) Jeollanam-do 1/39 (2.6) 1/27 (3.7) 1/36 (2.8) 24/73 (32.9) 15/87 (17.2) 42/262 (16.0) Jeju 24/66 (36.4) 0/1 (0.0) 0/8 (0.0) 0/9 (0.0) 0/3 (0.0) 24/87 (27.6) Chungcheongnam-do 1/19 (5.3) 0/7 (0.0) 2/17 (11.8) 2/10 (20.0) 2/13 (15.4) 7/66 (10.6) Chungcheongbuk-do 0/9 (0.0) 0/9 (0.0) 0/8 (0.0) 1/21 (4.8) 1/17 (5.9) 2/64 (3.1) Daejeon 5/6 (83.3) 0/2 (0.0) 0/0 (0.0) 0/0 (0.0) 0/0 (0.0) 5/8 (62.5) Ulsan 0/0 (0.0) 0/0 (0.0) 0/4 (0.0) 0/0 (0.0) 0/0 (0.0) 0/4 (0.0) Sejong 0/0 (0.0) 0/0 (0.0) 0/0 (0.0) 0/0 (0.0) 0/0 (0.0) 0/0 (0.0) Total 132/723 (18.3) 33/520 (6.3) 54/585 (9.2) 103/739 (13.9) 96/948 (10.1) 418/3,515 (11.9) Values are presented as number/total (%)..
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Number of IgG/IgM antibody titers by region (2019–2023) Region IFA titers (IgG/IgM) Total Under 1:16 1:16 1:32 1:64 1:128 1:256 1:512 1:1,024 Over 1:2,048 Gangwon 125/156 36/33 17/20 11/10 12/5 12/3 6/3 10/10 16/5 245 Gyeonggi 150/171 3/8 8/5 10/4 14/4 3/8 6/2 5/3 9/3 208 Gyeongsangbuk-do 6/7 2/2 1/4 3/2 0/0 2/0 1/1 0/0 2/1 17 Gyeongsangnam-do 9/12 0/0 1/1 1/0 0/0 1/0 0/0 0/0 1/0 13 Gwangju 204/373 1/12 7/24 12/47 32/15 28/10 40/2 34/8 138/5 496 Daegu 5/5 0/0 0/0 2/2 0/0 0/0 1/0 0/1 3/3 11 Busan 3/18 1/0 1/1 10/0 2/1 3/1 0/0 1/0 0/0 21 Seoul 261/269 4/1 3/6 4/7 6/5 3/6 7/4 3/4 19/8 310 Incheon 532/525 1/4 5/4 6/6 6/11 3/14 10/9 6/10 38/24 607 Jeollanam-do 36/67 1/5 6/9 3/8 12/1 21/3 8/0 4/1 7/4 98 Jeollabuk-do 24/101 1/6 18/8 21/5 26/6 21/8 14/6 9/5 16/5 150 Jeju 13/17 2/3 5/5 5/2 4/4 4/4 5/7 6/5 3/0 47 Chungcheongnam-do 10/17 2/9 2/3 3/1 8/6 3/1 3/2 3/1 12/6 46 Chungcheongbuk-do 41/45 3/4 4/2 2/2 0/3 4/1 3/2 3/1 0/0 60 Daejeon 0/0 1/1 0/0 0/0 0/0 0/0 0/0 0/0 0/0 1 Ulsan 0/0 0/0 0/0 0/0 0/1 1/0 0/0 1/3 2/0 4 Sejong 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0 Total (%) 1,419/1,783
(60.8/76.4)58/88
(2.5/3.8)78/92
(3.3/3.9)93/96
(4.0/4.1)122/62
(5.2/2.7)109/59
(4.7/2.5)104/38
(4.5/1.6)85/52
(3.6/2.2)266/64
(11.4/2.7)2,334
(100.0/100.0)Values are presented as number/total..
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