• View in gallery

    Screening for serological diagnostic antigens with immunoinformatics. From the total of 1,408 proteins in the Orientia tsutsugamushi Boryong strain, putative antigenic proteins were predicted by a bioinformatic analysis of protein localization, antigenicity, adhesion probability, number of transmembrane domains, and similarity to host proteins.

  • View in gallery

    Experimental verification of five serological candidate antigens. Candidate antigens were expressed in Escherichia coli, and the presence of antibodies (immunoglobulin G and M) to each candidate antigen in the sera from scrub typhus patients (A) or negative control patients (B) was examined by immunoblotting. This figure appears in color at www.ajtmh.org.

  • View in gallery

    Amino acid sequence analysis of Orientia tsutsugamushi 27-kDa protein. (A) Multiple sequence alignment of the 27-kDa antigen in different O. tsutsugamushi strains. Identical amino acid residues are shown highlighted in black, and similar residues are shown highlighted in gray. Dashes indicate gaps inserted for maximal alignment score. (B) Amino acid sequence identity and similarity for the 27-kDa antigen in different O. tsutsugamushi strains.

  • View in gallery

    Evaluation of the diagnostic potential of the Orientia tsutsugamushi 27-kDa antigen. O. tsutsugamushi 27- and 56-kDa proteins were purified, and their diagnostic performance with human samples was compared by Western blot. One microgram of purified antigens were loaded and the presence of immunoglobulin M or G for each antigen was examined separately from serum samples of scrub typhus negative patients (A) and positive patients (B).

  • View in gallery

    Lateral flow immunoassay (LFIA) test using the 27-kDa antigen. LFIA strips after testing with sera from negative control patients (A) or scrub typhus patients (B). The control lane is upper and test lane is lower. This figure appears in color at www.ajtmh.org.

  • 1.

    Luce-Fedrow A, Lehman ML, Kelly DJ, Mullins K, Maina AN, Stewart RL, Ge H, John HS, Jiang J, Richards AL, 2018. A review of scrub typhus (Orientia tsutsugamushi and related organisms): then, now, and tomorrow. Trop Med Infect Dis 3: 8.

    • Search Google Scholar
    • Export Citation
  • 2.

    Peter JV, Sudarsan TI, Prakash JA, Varghese GM, 2015. Severe scrub typhus infection: clinical features, diagnostic challenges and management. World J Crit Care Med 4: 244250.

    • Search Google Scholar
    • Export Citation
  • 3.

    Taylor AJ, Paris DH, Newton PN, 2015. A systematic review of mortality from untreated scrub typhus (Orientia tsutsugamushi). PLoS Negl Trop Dis 9: e0003971.

    • Search Google Scholar
    • Export Citation
  • 4.

    WHO , 1999. WHO Recommended Surveillance Standards, 2nd ed. Geneva, Switzerland: World Health Organization.

  • 5.

    Sonthayanon P et al., 2006. Rapid diagnosis of scrub typhus in rural Thailand using polymerase chain reaction. Am J Trop Med Hyg 75: 10991102.

    • Search Google Scholar
    • Export Citation
  • 6.

    Paris DH, Blacksell SD, Newton PN, Day NP, 2008. Simple, rapid and sensitive detection of Orientia tsutsugamushi by loop-isothermal DNA amplification. Trans R Soc Trop Med Hyg 102: 12391246.

    • Search Google Scholar
    • Export Citation
  • 7.

    Koh GC, Maude RJ, Paris DH, Newton PN, Blacksell SD, 2010. Diagnosis of scrub typhus. Am J Trop Med Hyg 82: 368370.

  • 8.

    Ro HJ, Lee H, Park EC, Lee CS, Il Kim S, Jun S, 2018. Ultrastructural visualization of Orientia tsutsugamushi in biopsied eschars and monocytes from scrub typhus patients in South Korea. Sci Rep 8: 17373.

    • Search Google Scholar
    • Export Citation
  • 9.

    Blacksell SD, Jenjaroen K, Phetsouvanh R, Tanganuchitcharnchai A, Phouminh P, Phongmany S, Day NP, Newton PN, 2010. Accuracy of rapid IgM-based immunochromatographic and immunoblot assays for diagnosis of acute scrub typhus and murine typhus infections in Laos. Am J Trop Med Hyg 83: 365369.

    • Search Google Scholar
    • Export Citation
  • 10.

    Park SW, Lee CK, Kwak YG, Moon C, Kim BN, Kim ES, Kang JM, Lee CS, 2010. Antigenic drift of Orientia tsutsugamushi in South Korea as identified by the sequence analysis of a 56-kDa protein-encoding gene. Am J Trop Med Hyg 83: 930935.

    • Search Google Scholar
    • Export Citation
  • 11.

    Krogh A, Larsson B, von Heijne G, Sonnhammer ELL, 2001. Predicting transmembrane protein topology with a hidden markov model: application to complete genomes. J Mol Biol 305: 567580.

    • Search Google Scholar
    • Export Citation
  • 12.

    Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ, 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 33893402.

    • Search Google Scholar
    • Export Citation
  • 13.

    Sachdeva G, Kumar K, Jain P, Ramachandran S, 2004. SPAAN: a software program for prediction of adhesins and adhesin-like proteins using neural networks. Bioinformatics 21: 483491.

    • Search Google Scholar
    • Export Citation
  • 14.

    He Y, Xiang Z, Mobley HLT, 2010. Vaxign: the first web-based vaccine design program for reverse vaccinology and applications for vaccine development. J Biomed Biotechnol 2010: 297505.

    • Search Google Scholar
    • Export Citation
  • 15.

    Doytchinova IA, Flower DR, 2007. VaxiJen: a server for prediction of protective antigens, tumour antigens and subunit vaccines. BMC Bioinformatics 8: 4.

    • Search Google Scholar
    • Export Citation
  • 16.

    Koczula KM, Gallotta A, 2016. Lateral flow assays. Essays Biochem 60: 111120.

  • 17.

    Kim YJ et al., 2016. Clinical evaluation of rapid diagnostic test kit for scrub typhus with improved performance. J Korean Med Sci 31: 11901196.

    • Search Google Scholar
    • Export Citation
  • 18.

    Ha NY, Kim Y, Min CK, Kim HI, Yen NTH, Choi MS, Kang JS, Kim YS, Cho NH, 2017. Longevity of antibody and T-cell responses against outer membrane antigens of Orientia tsutsugamushi in scrub typhus patients. Emerg Microbes Infect 6: e116.

    • Search Google Scholar
    • Export Citation
  • 19.

    Kim DM, Lee YM, Back JH, Yang TY, Lee JH, Song HJ, Shim SK, Hwang KJ, Park MY, 2010. A serosurvey of Orientia tsutsugamushi from patients with scrub typhus. Clin Microbiol Infect 16: 447451.

    • Search Google Scholar
    • Export Citation
  • 20.

    Sengupta M, Anandan S, Daniel D, Prakash JA, 2015. Scrub typhus seroprevalence in healthy Indian population. J Clin Diagn Res 9: DM01DM02.

 
 
 

 

 

 

 

 

 

Identification of a Novel Antigen for Serological Diagnosis of Scrub Typhus

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  • 1 Research Center for Bioconvergence Analysis, Korea Basic Science Institute, Cheongju, Republic of Korea;
  • | 2 Center for Convergent Research of Emerging Virus Infection, Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea;
  • | 3 Department of Bio-Analysis Science, University of Science & Technology, Daejeon, Republic of Korea,
  • | 4 Manufacture Business Division Curebio Ltd, Seoul, Republic of Korea,
  • | 5 Center for Research Equipment, Korea Basic Science Institute, Cheongju, Republic of Korea;
  • | 6 Department of Internal Medicine, Jeonbuk National University Medical School, Jeonju, Republic of Korea;
  • | 7 Biomedical Research Institute of Jeonbuk National University Hospital, Jeonju, Republic of Korea

ABSTRACT.

Scrub typhus is an acute infectious disease caused by the bacterium Orientia tsutsugamushi, which is widely distributed in northern, southern, and eastern Asia. Early diagnosis is essential because the average case fatality rate is usually >10% but can be as high as 45% if antimicrobial treatment is delayed. Although an O. tsutsugamushi 56-kDa type-specific antigen (TSA) is commonly used for serological diagnosis of scrub typhus, the 56-kDa TSA shows variations among O. tsutsugamushi strains, which may lead to poor diagnostic results. Therefore, the discovery of new antigenic proteins may improve diagnostic accuracy. In this study, we identified an O. tsutsugamushi 27 kDa antigen through an immunoinformatic approach and verified its diagnostic potential using patient samples. Compared with the O. tsutsugamushi 56-kDa antigen, the new 27-kDa antigen showed better diagnostic specificity with similar diagnostic sensitivity. Therefore, the O. tsutsugamushi 27-kDa antigen shows potential as a novel serological diagnostic antigen for scrub typhus, providing higher diagnostic accuracy for O. tsutsugamushi than the 56-kDa antigen.

INTRODUCTION

Scrub typhus (also known as tsutsugamushi disease) is an acute and febrile infectious disease caused by the Gram‐negative α‐proteobacterium Orientia tsutsugamushi, which is transmitted through the bites of infected chiggers of the Trombiculidae mite. O. tsutsugamushi is distributed in the “Tsutsugamushi triangle” that extends northward to Japan, Russia, and the Primorske Karal region in the Russian far East, Southward to Northern Australia and the Western Pacific islands, and Westward to Afghanistan, Pakistan, and areas bordering the central Asian republics.1 Once bitten by an infected chigger, the pathogen spreads through the lymphatic fluid and blood, and an eschar develops at the site of inoculation.2 Systemic manifestations of infection by O. tsutsugamushi include fever, headache, myalgia, lymphadenopathy, and a skin rash. If untreated, patients develop complications with systemic involvement and disseminated vasculitis, including organ dysfunction.2 Patients with mild symptoms can be easily treated with common antibiotics, such as doxycycline. However, the mortality can be as high as 45% if antimicrobial treatment is delayed in patients over 50 years old.3 The WHO considers scrub typhus to be one of the most underdiagnosed and underreported febrile illnesses requiring hospitalization because the symptoms of scrub typhus are similar to those of many other diseases.4 Thus, early diagnosis of scrub typhus is essential to prevent complications and reduce the mortality rate.

The current methods used to diagnose scrub typhus are isolation of the pathogen in cell culture, detection of pathogen-specific nucleic acids by polymerase chain reaction (PCR), or identification of the antigen or antibodies through immunoassays. Identification of O. tsutsugamushi in cell culture is definitive; however, it takes days to confirm the presence of the pathogen. For PCR assays, diagnostic sensitivity is relatively low. The sensitivity of nested PCR ranges from 22.5% to 36.1%, and the sensitivity of real-time PCR ranges from 45% to 82%.2,57 Detection of antigens by immunoassays also has low sensitivity and specificity because the O. tsutsugamushi bacteria exist at low concentrations in monocytes.8

In the clinical field, the standard diagnostic methods for scrub typhus are serological immunoassays, such as an indirect immunofluorescence assay (IFA), an enzyme-linked immunosorbent assay, or a lateral flow immunoassay (LFIA).1 The O. tsutsugamushi 56-kDa type-specific antigen (TSA) is commonly used to detect antibodies. The 56-kDa TSA is an immunodominant major outer membrane protein that is unique to O. tsutsugamushi. However, a LFIA test using recombinant 56-kDa antigen showed low specificity (71.4%) during the detection of antibodies.9 Furthermore, frequent sequence variations of 56-kDa TSA, which can cause antigenic drift, were reported.10 Therefore, the discovery of novel antigenic proteins is necessary to improve the diagnostic sensitivity and specificity of tests for scrub typhus.

In this study, we used an immunoinformatic approach to uncover novel serological diagnostic antigens for scrub typhus. We identified the O. tsutsugamushi 27-kDa protein and demonstrated its diagnostic potential.

MATERIALS AND METHODS

Ethics statement and patient sera.

The serum samples used in this study were collected from 40 subjects (20 scrub typhus–negative patients and 20 scrub typhus–positive patients, Supplemental Table 1) as part of registered protocols approved by Chonbuk National University Hospital (institutional review board registration no. 2016-04-007). The diagnosis of scrub typhus was confirmed by an increase in the indirect IFA immunoglobulin (Ig)M titer ≥1:160 against O. tsutsugamushi, a ≥4-fold increase in the IFA titer for O. tsutsugamushi, or when a positive reaction was observed in a nested PCR targeting the 56-kDa gene of O. tsutsugamushi.

Immunoinformatic analysis.

We used the immunoinformatics methods to screen for putative antigenic proteins, based on the assessment on the predicted number of transmembrane domains, the similarity to host proteins, the adhesion probability, and the antigenicity score. The number of transmembrane helices was predicted using TMHMM,11 with a threshold of one. Host similarity was calculated by BlastP,12 with an e-value less than 1e-05. SPAAN13 was used to predict the adhesion probability. These three programs were run internally in Vaxign,14 a web-based vaccine design program for reverse vaccinology. The antigenicity score was calculated by VaxiJen antigen prediction server,15 with a cutoff value of 0.5.

DNA cloning and recombinant protein production.

The genes encoding the five O. tsutsugamushi proteins, WP_011944239.1, WP_011945097.1, WP_011944458.1, WP_011944821.1, and WP_011944571.1, were codon-optimized for protein expression in E. coli, and then synthesized and cloned into pET21a with the 6 × His affinity tag at the C-terminus using the Nde I and Xho I restriction enzyme sites (Bionics, South Korea). The primer sequence used for subcloning is listed in Supplemental Table 2. The cloned plasmids were transformed into the Escherichia coli strain BL21 (DE3), and the cells were grown in LB medium containing 100 μg/mL ampicillin at 37°C until the OD600 reached 0.6. Recombinant protein expression was induced by 0.5 mM isopropyl β-D-1-thiogalactopyronoside (IPTG) at 18°C for 20 h. The recombinant proteins were expressed as inclusion bodies.

The cells expressing the WP_011944571.1 (27 kDa) protein were harvested by centrifugation at 5,000 rpm for 30 min, resuspended in lysis buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM β-mercaptoethanol, and 2 mM phenylmethylsulfonyl fluoride), and disrupted by sonication. After centrifugation at 18,000 rpm for 40 min, the pellet of cell lysate was solubilized by homogenizer in the denaturation buffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, and 8 M urea), and the denatured sample was incubated at room temperature for 2 h. The solubilized fraction was loaded onto a HisTrap FF column (GE Healthcare, Chicago, IL), equilibrated with 50 mM Tris-HCl, pH 8.0, 300 mM NaCl, and 8 M urea. The recombinant proteins were eluted with elution buffer (20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 200 mM imidazole, and 8 M urea). The molecular weight and purity of WP_011944571.1 protein was verified with sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS-PAGE). The eluted proteins were diluted in phosphate-buffered saline before further experimentation.

Immunoblotting.

Samples were separated by 10% SDS-PAGE and transferred to a PVDF membrane (Millipore, Burlington, MA). The membrane was washed with 30 mM Tris-HCl(pH 7.5), 100 mM NaCl and 0.1% Tween-20(TBST) containing 5% skim milk for 1 h. After incubation with patient serum (1:1000 in 5% skim milk in TBST) overnight at 4°C, the membrane was washed with TBST. The specific IgG or IgM binding was visualized by incubation with anti-human-IgG or -IgM peroxidase conjugate (1:5,000 in 5% skim milk in TBST) and development with SuperSignal™ West Pico PLUS Chemiluminescent Substrate (ThermoFisher Scientific, Waltham, MA). The chemiluminescent signal was detected using an ChemiDoc MP(Bio-rad, Hercules, CA).

LFIA test prototype.

The LIFA tests were prepared as previously described.16 Briefly, the LIFA test strips were made of a plastic adhesive backing pad, a sample pad, a conjugation pad, a detection pad, and an absorbent pad.

For the detection pad, O. tsutsugamushi 27-kDa protein (1 mg/mL) and anti-goat IgG/M antibodies (1 mg/mL) were dispensed onto the test line and control line on a nitrocellulose membrane, respectively, using a line dispenser (BTM Inc., Seoul, South Korea) at a dispensing speed of 50 mm/sec and a dispensing rate of 1 μL/cm. The membrane was dried at 37°C for 1 h, blocked for 1 h at 37°C with blocking solution, and then dried again at 37°C for 1 h in a vacuum drying oven. For the conjugation pad, the conjugate pad was soaked in 0.1% triton X-100 and dried for 1 h at 37°C. the goat anti-human IgG/M antibodies conjugated with gold colloid were sprayed on a glass fiber. After all pads were assembled, the test strips were cut to a width of 4 mm and placed in plastic cases.

RESULTS

Immunoinformatic screening for novel antigens.

To screen for potential serological diagnostic antigen proteins of the O. tsutsugamushi Boryong strain, we first predicted the subcellular localization of proteins to select for extracellular, outer membrane, and periplasmic proteins. Of the total 1408 O. tsutsugamushi proteins, 31 were predicted to be localized in these three regions. Next, we performed an immunoinformatic analysis using the Vaxign and VaxiJen in silico tools. The screening process for antigenicity and adhesion probability identified five proteins as potential serological diagnostic antigen proteins for O. tsutsugamushi (Figure 1). These five proteins consisted of one extracellular, one periplasmic, and three outer membrane proteins with one or fewer transmembrane helices (Table 1). In addition, the five potential antigenic proteins showed no similarity to human proteins.

Figure 1.
Figure 1.

Screening for serological diagnostic antigens with immunoinformatics. From the total of 1,408 proteins in the Orientia tsutsugamushi Boryong strain, putative antigenic proteins were predicted by a bioinformatic analysis of protein localization, antigenicity, adhesion probability, number of transmembrane domains, and similarity to host proteins.

Citation: The American Journal of Tropical Medicine and Hygiene 105, 5; 10.4269/ajtmh.20-0129

Table 1

List of candidate antigens for serological diagnosis of Orientia tsutsugamushi

Protein accession no.Locus_tagLocalizationAntigenicy scoreAdhesin probabilityNo. of trans-membrane helicesCalculated molecular weight
WP_011944239.1OTBS_RS00095Periplasmic0.59230.587150,246
WP_011945097.1OTBS_RS08730Outer membrane0.56880.5551110,963
WP_011944458.1OTBS_RS01880Outer membrane0.71810.54169,372
WP_011944821.1OTBS_RS05725Extracellular0.64210.578144,843
WP_011944571.1OTBS_RS02790Outer membrane0.69770.516026,812

Recombinant protein production and preliminary assessment by immunoblotting.

Constructs encoding the five selected proteins were cloned into a pET21a plasmid and transfected into E. coli BL21. To test for the presence of antibodies for each protein in patients, the total lysate of the transformed E. coli was used for immunoblotting with the anti-sera from scrub typhus patients. The immunoblotting results show that antibodies for O. tsutsugamushi WP_011944571.1 protein were detected in all patients (Figure 2A). Antibodies for the other four proteins, however, were detected in only a few patients (Figure 2A); no antibodies for the five selected O. tsutsugamushi proteins were found in negative control patients (Figure 2B). To ensure the result, we performed additional immunoblot assay using purified antigen proteins. The results showed that antibodies for WP_011944571.1 and WP_011944821.1 protein were detected in all patients (Supplemental Figure 1A). In the case of WP_011944821.1 protein, however, its antibodies were also detected in negative control patients (Supplemental Figure 1B). This suggests that the O. tsutsugamushi WP_011944571.1 protein could be used as a serological antigen for the diagnosis of scrub typhus. The WP_011944571.1 protein consists of 243 amino acids and has a molecular weight of 27 kDa. This protein is a type of β-barrel protein localized in the outer membrane; however, its biological function has not yet been determined. We hereafter refer to this protein as O. tsutsugamushi 27 kDa. Amino acid sequence alignment of this protein revealed that O. tsutsugamushi 27-kDa is well conserved among O. tsutsugamushi substrains (Figure 3).

Figure 2.
Figure 2.

Experimental verification of five serological candidate antigens. Candidate antigens were expressed in Escherichia coli, and the presence of antibodies (immunoglobulin G and M) to each candidate antigen in the sera from scrub typhus patients (A) or negative control patients (B) was examined by immunoblotting. This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene 105, 5; 10.4269/ajtmh.20-0129

Figure 3.
Figure 3.

Amino acid sequence analysis of Orientia tsutsugamushi 27-kDa protein. (A) Multiple sequence alignment of the 27-kDa antigen in different O. tsutsugamushi strains. Identical amino acid residues are shown highlighted in black, and similar residues are shown highlighted in gray. Dashes indicate gaps inserted for maximal alignment score. (B) Amino acid sequence identity and similarity for the 27-kDa antigen in different O. tsutsugamushi strains.

Citation: The American Journal of Tropical Medicine and Hygiene 105, 5; 10.4269/ajtmh.20-0129

Evaluation of the diagnostic potential of 27 kDa.

To evaluate the potential of O. tsutsugamushi 27-kDa protein as a serological antigen for diagnosis of scrub typhus, we purified the 27 kDa protein (Supplemental Figure 2), determined the presence of antibodies in clinical samples, and compared the results with those for the 56 kDa previously known serological antigen. Anti-27 kDa IgG antibodies were detected in all patients with scrub typhus, and anti-27 kDa IgM antibodies were detected in 16 of 20 patients (Figure B). Anti-56-kDa IgM and IgG antibodies were observed in all scrub typhus patients (Figure 4B). Anti-27-kDa IgM or IgG antibodies were not detected in any of the negative control patients (Figure 4A). However, anti-56-kDa antibodies were detected in six of 20 negative control patients (Figure 4A). This suggests that the O. tsutsugamushi 27-kDa protein may have greater diagnostic specificity than the 56-kDa protein and similar diagnostic sensitivity.

Figure 4.
Figure 4.

Evaluation of the diagnostic potential of the Orientia tsutsugamushi 27-kDa antigen. O. tsutsugamushi 27- and 56-kDa proteins were purified, and their diagnostic performance with human samples was compared by Western blot. One microgram of purified antigens were loaded and the presence of immunoglobulin M or G for each antigen was examined separately from serum samples of scrub typhus negative patients (A) and positive patients (B).

Citation: The American Journal of Tropical Medicine and Hygiene 105, 5; 10.4269/ajtmh.20-0129

Preliminary evaluation of a LFIA test prototype.

To test whether O. tsutsugamushi 27-kDa protein could be used for rapid diagnosis of scrub typhus, we made prototype LFIA test strips using the 27 kDa protein. When we tested these strips with clinical samples (Supplemental Table 3), a positive line was observed only in scrub typhus patients (Figure 5). This indicates that an LFIA test with the 27-kDa protein can successfully diagnose scrub typhus.

Figure 5.
Figure 5.

Lateral flow immunoassay (LFIA) test using the 27-kDa antigen. LFIA strips after testing with sera from negative control patients (A) or scrub typhus patients (B). The control lane is upper and test lane is lower. This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene 105, 5; 10.4269/ajtmh.20-0129

DISCUSSION

We performed an immunoinformatic analysis to identify potential serological diagnostic markers for scrub typhus. We included only extracellular, outer membrane, and periplasmic proteins of O. tsutsugamushi in this process because these proteins are more easily recognized by the host immune system and thus generate antibodies more effectively than cytoplasmic or inner membrane proteins. We discovered five potential antigenic proteins through the bioinformatics analysis and verified that one of the five candidates was suitable for use as a serological diagnostic antigen. This suggests that immunoinformatics is a useful method for discovering novel serological diagnostic markers for bacterial pathogens with larger genomes than viruses. Immunoinformatics may be no better than immunoproteomics as a screening tool for finding the best serological diagnostic markers; however, immunoinformatics can be easily used even for high-risk pathogens because it is not necessary to culture the target pathogens with this method.

In this study, we identified an O. tsutsugamushi 27-kDa antigen using immunoinformatics and verified its diagnostic potential using patient samples. The O. tsutsugamushi 27-kDa antigen is a type of β-barrel protein that is expressed in the outer membrane, and the amino acid sequence of the protein is well conserved among O. tsutsugamushi strains. The 27-kDa antigen protein may not be applicable to distinguish O. tsutsugamushi substrains; however, this antigen would be useful to diagnose scrub typhus because using a 27-kDa protein from a single O. tsutsugamushi strain could detect all the O. tsutsugamushi substrains. In fact, amino acid sequence identity of 27-kDa protein is much higher than that of 56-kDa protein among O. tsutsugamushi strains. Compared with the previously identified and well-known O. tsutsugamushi 56-kDa antigen, the new 27-kDa antigen showed better diagnostic specificity and similar diagnostic sensitivity. Therefore, the O. tsutsugamushi 27-kDa protein has potential as a novel serological diagnostic antigen for scrub typhus, providing higher diagnostic accuracy for O. tsutsugamushi than the 56-kDa antigen.

We observed anti-56-kDa antibodies in several negative control patients (Figure 4A). The O. tsutsugamushi 56 kDa protein is a well-known diagnostic marker and is currently used in commercial LFIA tests.17 Recent reports showed that anti-56-kDa antibodies can remain in the human body for up to 2 years18,19 and are detected in approximately 15% of healthy subjects.20 This means that diagnostic tests that use the O. tsutsugamushi 56-kDa protein may have trouble distinguishing newly infected scrub typhus patients from patients previously infected by O. tsutsugamushi. However, antibodies to the O. tsutsugamushi 27-kDa protein were not detected in any of the negative control patients (Figure 4A). This suggests that the O. tsutsugamushi 27-kDa protein may be a better antigen in terms of diagnostic specificity than the 56-kDa protein and may be especially useful for diagnosing patients newly infected with scrub typhus.

ACKNOWLEDGMENTS

This work was supported by Korea Basic Science Institute (KBSI) Research Programs (grants C39931 and C39123), National Research Council of Science and Technology (NST) grant by the Korea government (no. CRC‐16‐01‐KRICT), and Korea Health Technology R&D Project through the Korea Health Industry Development Institute, funded by the Ministry of Health & Welfare, Republic of Korea (grants HI20C0033 and HI20C363).

REFERENCES

  • 1.

    Luce-Fedrow A, Lehman ML, Kelly DJ, Mullins K, Maina AN, Stewart RL, Ge H, John HS, Jiang J, Richards AL, 2018. A review of scrub typhus (Orientia tsutsugamushi and related organisms): then, now, and tomorrow. Trop Med Infect Dis 3: 8.

    • Search Google Scholar
    • Export Citation
  • 2.

    Peter JV, Sudarsan TI, Prakash JA, Varghese GM, 2015. Severe scrub typhus infection: clinical features, diagnostic challenges and management. World J Crit Care Med 4: 244250.

    • Search Google Scholar
    • Export Citation
  • 3.

    Taylor AJ, Paris DH, Newton PN, 2015. A systematic review of mortality from untreated scrub typhus (Orientia tsutsugamushi). PLoS Negl Trop Dis 9: e0003971.

    • Search Google Scholar
    • Export Citation
  • 4.

    WHO , 1999. WHO Recommended Surveillance Standards, 2nd ed. Geneva, Switzerland: World Health Organization.

  • 5.

    Sonthayanon P et al., 2006. Rapid diagnosis of scrub typhus in rural Thailand using polymerase chain reaction. Am J Trop Med Hyg 75: 10991102.

    • Search Google Scholar
    • Export Citation
  • 6.

    Paris DH, Blacksell SD, Newton PN, Day NP, 2008. Simple, rapid and sensitive detection of Orientia tsutsugamushi by loop-isothermal DNA amplification. Trans R Soc Trop Med Hyg 102: 12391246.

    • Search Google Scholar
    • Export Citation
  • 7.

    Koh GC, Maude RJ, Paris DH, Newton PN, Blacksell SD, 2010. Diagnosis of scrub typhus. Am J Trop Med Hyg 82: 368370.

  • 8.

    Ro HJ, Lee H, Park EC, Lee CS, Il Kim S, Jun S, 2018. Ultrastructural visualization of Orientia tsutsugamushi in biopsied eschars and monocytes from scrub typhus patients in South Korea. Sci Rep 8: 17373.

    • Search Google Scholar
    • Export Citation
  • 9.

    Blacksell SD, Jenjaroen K, Phetsouvanh R, Tanganuchitcharnchai A, Phouminh P, Phongmany S, Day NP, Newton PN, 2010. Accuracy of rapid IgM-based immunochromatographic and immunoblot assays for diagnosis of acute scrub typhus and murine typhus infections in Laos. Am J Trop Med Hyg 83: 365369.

    • Search Google Scholar
    • Export Citation
  • 10.

    Park SW, Lee CK, Kwak YG, Moon C, Kim BN, Kim ES, Kang JM, Lee CS, 2010. Antigenic drift of Orientia tsutsugamushi in South Korea as identified by the sequence analysis of a 56-kDa protein-encoding gene. Am J Trop Med Hyg 83: 930935.

    • Search Google Scholar
    • Export Citation
  • 11.

    Krogh A, Larsson B, von Heijne G, Sonnhammer ELL, 2001. Predicting transmembrane protein topology with a hidden markov model: application to complete genomes. J Mol Biol 305: 567580.

    • Search Google Scholar
    • Export Citation
  • 12.

    Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ, 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 33893402.

    • Search Google Scholar
    • Export Citation
  • 13.

    Sachdeva G, Kumar K, Jain P, Ramachandran S, 2004. SPAAN: a software program for prediction of adhesins and adhesin-like proteins using neural networks. Bioinformatics 21: 483491.

    • Search Google Scholar
    • Export Citation
  • 14.

    He Y, Xiang Z, Mobley HLT, 2010. Vaxign: the first web-based vaccine design program for reverse vaccinology and applications for vaccine development. J Biomed Biotechnol 2010: 297505.

    • Search Google Scholar
    • Export Citation
  • 15.

    Doytchinova IA, Flower DR, 2007. VaxiJen: a server for prediction of protective antigens, tumour antigens and subunit vaccines. BMC Bioinformatics 8: 4.

    • Search Google Scholar
    • Export Citation
  • 16.

    Koczula KM, Gallotta A, 2016. Lateral flow assays. Essays Biochem 60: 111120.

  • 17.

    Kim YJ et al., 2016. Clinical evaluation of rapid diagnostic test kit for scrub typhus with improved performance. J Korean Med Sci 31: 11901196.

    • Search Google Scholar
    • Export Citation
  • 18.

    Ha NY, Kim Y, Min CK, Kim HI, Yen NTH, Choi MS, Kang JS, Kim YS, Cho NH, 2017. Longevity of antibody and T-cell responses against outer membrane antigens of Orientia tsutsugamushi in scrub typhus patients. Emerg Microbes Infect 6: e116.

    • Search Google Scholar
    • Export Citation
  • 19.

    Kim DM, Lee YM, Back JH, Yang TY, Lee JH, Song HJ, Shim SK, Hwang KJ, Park MY, 2010. A serosurvey of Orientia tsutsugamushi from patients with scrub typhus. Clin Microbiol Infect 16: 447451.

    • Search Google Scholar
    • Export Citation
  • 20.

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Author Notes

Address correspondence to Edmond Changkyun Park or Hye-Yeon Kim, Research Center for Bioconvergence Analysis, Korea Basic Science Institute, Cheongju 28119, Republic of Korea. E-mails: edpark@kbsi.re.kr or hyeyeon@kbsi.re.kr

These authors contributed equally to this work.

Authors’ addresses: Wooyoung Kim, Sang-Yeop Lee, Seung Il Kim, Sun Cheol Park, Hye-Yeon Kim, and Edmond Changkyun Park, Research Center for Bioconvergence Analysis, Korea Basic Science Institute, Cheongju, Republic of Korea, E-mails: kwy91@kbsi.re.kr, yop0214@gmail.com, ksi@kbsi.re.kr, psc1234@kbsi.re.kr, hyeyeon@kbsi.re.kr, and edpark@kbsi.re.kr. In-Kook Sohng, Manufacture Business Division Curebio Ltd, Seoul, Republic of Korea, E-mail: iksohng@gmail.com. Sangmi Jun, Center for Research Equipment, Korea Basic Science Institute, Cheongju, Republic of Korea, E-mail: smjun@kbsi.re.kr. Chang-Seop Lee, Department of Internal Medicine, Chonbuk National University Medical School, Jeonju 54986, Republic of Korea, E-mail: lcsmd@jbnu.ac.kr.

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