Advertisement

  • Loading metrics

Open Access

Peer-reviewed

Research Article

The validity of diagnostic cut-offs for commercial and in-house scrub typhus IgM and IgG ELISAs: A review of the evidence

  • Kartika Saraswati ,

    Roles Data curation, Formal analysis, Writing – original draft

    ‡ These authors are joint first authors on this work.

    Affiliations Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand, Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom, Eijkman-Oxford Clinical Research Unit, Eijkman Institute for Molecular Biology, Jakarta, Indonesia

    ⨯
  • Meghna Phanichkrivalkosil ,

    Roles Data curation, Formal analysis, Investigation, Writing – original draft

    ‡ These authors are joint first authors on this work.

    Affiliation Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand

    ⨯
  • Nicholas P. J. Day,

    Roles Funding acquisition, Supervision, Writing – review & editing

    Affiliations Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand, Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom

    ⨯
  • Stuart D. Blacksell

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

    stuart@tropmedres.ac

    Affiliations Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand, Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom

    ⨯

Abstract

Background

Scrub typhus is a neglected tropical disease that causes acute febrile illness. Diagnosis is made based upon serology, or detection of the causative agent–Orientia tsutsugamushi–using PCR or in vitro isolation. The enzyme-linked immunosorbent assay (ELISA) is an objective and reproducible means of detecting IgM or IgG antibodies. However, lack of standardization in ELISA methodology, as well as in the choice of reference test with which the ELISA is compared, calls into question the validity of cut-offs used in diagnostic accuracy studies and observational studies.

Methodology/Principal findings

A PubMed search and manual screening of reference lists identified 46 studies that used ELISA antibody cut-offs to diagnose scrub typhus patients, 22 of which were diagnostic accuracy studies. Overall, 22 studies (47.8%) provided little to no explanation as to how the ELISA cut-off was derived, and 7 studies (15.2%) did not even state the cut-off used. Variation was seen locally in reference standards used, in terms of both the diagnostic test and cut-off titer. Furthermore, with the exception of studies using ELISAs manufactured by InBios, there was no standardization of the selection of antigenic strains. As a result, no consensus was found for determining a cut-off, ELISA methodology, or for a single value diagnostic cut-off.

Conclusions/Significance

We have concluded that there is a lack of consensus in the determination of a cut-off. We recommend interpreting the results from these studies with caution. Further studies will need to be performed at each geographic location to determine region-specific cut-offs, taking into consideration background antibody levels to discriminate true disease from healthy individuals.

Author summary

Scrub typhus is a neglected tropical disease that causes acute fever and can cause serious complications without appropriate antibiotic treatment. Diagnosis is usually made by the detection of specific antibodies or the causative agent–Orientia tsutsugamushi. Specific antibodies can be detected using ELISA technology however there is an apparent lack of standardization in the development of cut-offs used in diagnostic accuracy studies and observational studies. This study assessed 46 studies that used ELISA antibody cut-offs to diagnose scrub typhus patients. Overall, 22 studies (47.8%) provided little to no explanation as to how the ELISA cut-off was derived, and 7 studies (15.2%) did not even state the cut-off used. Furthermore, with the exception of studies using ELISAs manufactured by InBios company, there was no standard approach to the selection of antigenic strains, and therefore they may not be representative of the local antigenic strains causing disease. As a result, we have concluded that there is a lack of consensus in the determination of a cut-off. We recommend interpreting the results from these studies with caution and further studies will need to be conducted at each geographic location to determine region-specific cut-offs take into consideration background antibody levels to discriminate true disease from healthy individuals.

Citation: Saraswati K, Phanichkrivalkosil M, Day NPJ, Blacksell SD (2019) The validity of diagnostic cut-offs for commercial and in-house scrub typhus IgM and IgG ELISAs: A review of the evidence. PLoS Negl Trop Dis 13(2): e0007158. https://doi.org/10.1371/journal.pntd.0007158

Editor: Pamela L. C. Small, University of Tennessee, UNITED STATES

Received: November 18, 2018; Accepted: January 15, 2019; Published: February 4, 2019

Copyright: © 2019 Saraswati et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: KS, NPJD, and SDB are funded by the Wellcome Trust of the United Kingdom. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Scrub typhus is a neglected tropical disease caused by the obligate intracellular bacterium Orientia tsutsugamushi [1]. Transmission of the bacteria to humans occurs via the bite of larval trombiculid mites, known commonly as chiggers [2]. It was formerly thought to be confined to the ‘tsutsugamushi triangle’, encompassing Pakistan, Northern Australia and parts of Russia. However, cases acquired in Chile [3, 4], possibly Africa [5, 6], as well as the Middle East [7] (by a proposed novel species O. chuto), have been reported, suggesting that its endemicity may be more widespread than previously thought.

Patients typically present with acute febrile illness, but if left untreated, this may progress to systemic infection and multi-organ failure, contributing to an estimated median mortality rate of 6.0% for untreated and 1.4% for treated scrub typhus [8] highlighting the importance of early and accurate diagnosis. A characteristic necrotic lesion, or eschar, at the inoculation site may serve as a diagnostic clue, however its presence varies, ranging from 9%-97% depending on the population [9, 10]. Given that other febrile illnesses such as typhoid, dengue and leptospirosis have similar clinical manifestations as scrub typhus, laboratory test is essential to differentiate scrub typhus from other undifferentiated fever [11].

Serological methods are more often used to diagnose scrub typhus due to their simplicity and cost-effectiveness [12]. The indirect immunofluorescence assay (IFA) is considered the “gold standard,” but the requirement of a fluorescent microscope and the subjective nature of reading slides limits its application in rural areas where this disease is most prevalent [8, 11–14]. The Weil-Felix test is convenient to perform but suffers from poor sensitivity and specificity [13, 14]. Given the limitations of other serological methods, the enzyme-linked immunosorbent assay (ELISA) is acknowledged as a reasonably simple to perform alternative, providing an objective optical density (OD) result using an automated plate reader, that is reproducible in most clinical laboratory settings [13].

Despite the apparently standardized and objective ELISA platform, the diagnostic accuracy is influenced by methodological and patient factors. Methodological factors may include the composition of antigenic strains and their origin, and the choice of diagnostic cut-off. Patient factors are mainly centered on elevated levels of background immunity in endemic areas that may give rise to false positive results. Therefore, to ensure accuracy of diagnosis, standardized methodologies and locally validated OD cut-off levels for ELISA are urgently needed [8].

This review therefore aims to summarize (1) the differences in ELISA methodologies, (2) the OD cut-offs used for diagnosing scrub typhus in research, and (3) the rationale behind the selection of certain OD cut-offs for scrub typhus diagnosis in previously published diagnostic accuracy studies and observational studies.

Materials and methods

Search strategy and eligibility criteria

A scoping review was performed. Searches were performed by one author (MP) on the PubMed electronic database using the following search terms: “scrub typhus,” “tsutsugamushi”, “immunoassay”, and “ELISA”. The search was restricted to papers published in English, up to 16th October 2017. The titles and abstracts were screened for relevance. The full-text of relevant articles were assessed to determine eligibility. Diagnostic accuracy and observational studies using ELISA to diagnose scrub typhus in human were included. We excluded co-infection studies, case studies and studies investigating variations of the conventional ELISA methods (e.g., dot-ELISA). Reference lists of the relevant articles were also screened in order to identify additional studies. The protocol of this review was registered in the International Prospective Register for Systematic Review (PROSPERO) with registration number CRD42017078596.

Data extraction and analysis

Data was extracted by one author (MP), and where the information was unclear a second researcher was consulted (SDB). Details of the sample size, location, study date, reference test, cut-off, method used to calculate the cut-off, and ELISA methodology (antigenic strain and antibody isotype) were compiled into summary tables. The studies were grouped according to study design (diagnostic accuracy study or observational study), type of ELISA (in-house or commercial), and study location. The data was summarized using narrative synthesis. We did not evaluate minutiae of individual ELISA protocols, but instead focusing on the wider issues such as the methodologies used to determined diagnostic cut-offs.

Results

Summary of studies

Study types.

Of the total of 46 studies included in this review (Fig 1), 24 (52.2%, 24/46) were observational studies and the remaining 22 (47.8%, 22/46) were diagnostic accuracy studies (Tables 1–3). Eighteen (81.8%, 18/22) of the diagnostic accuracy study tested the accuracy of ELISA against reference assays, while the remaining four (18.2%, 4/22) used ELISA as the reference assay.

thumbnail
Download:
Fig 1. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow diagram.

https://doi.org/10.1371/journal.pntd.0007158.g001

thumbnail
Download:
Table 1. Summary of ELISA diagnostic accuracy studies.

https://doi.org/10.1371/journal.pntd.0007158.t001

thumbnail
Download:
Table 2. Summary of observational studies with a diagnostic accuracy component using InBios ELISAs.

https://doi.org/10.1371/journal.pntd.0007158.t002

thumbnail
Download:
Table 3. Summary of observational studies using NMRC in-house ELISAs.

https://doi.org/10.1371/journal.pntd.0007158.t003

Patient and geographic details.

Study year of included articles varying from 1986 to 2016 (Tables 1–3). The total samples analyzed was 23,498, however one study did not provide the number of samples [15]. Geographically, the majority of the ELISA studies were conducted on serum samples from India (52.2%, 24/46), followed by Thailand (15.2%, 7/46), China (8.7%, 4/46) and Korea (8.7%, 4/46). The remaining study populations were recruited from Japan, the United States (US), Australia, Malaysia, Bangladesh, Sri Lanka, Peru and Nepal (Tables 1–3). Two studies investigated deployed US soldiers (one in Korea, and one in Japan) [16, 17]. Two studies investigated serum samples from two different countries [18, 19], and two studies did not specify a location [20].

ELISA methodology

Source.

The commercial ELISA kits manufactured by InBios (InBios International Inc., Seattle WA, USA) (referred to as InBios ELISAs) were the most numerous–being used in 30 studies (65.2%, 30/46) with the remainder being in-house assays. Ten studies (21.7%, 10/46) used ELISA methods developed by the US Naval Medical Research Centre (NMRC) (Tables 1 and 3).

Antigenic composition.

There was a wide variety of native and recombinant antigens used in the ELISAs examined. The 30 InBios ELISAs identified in the study used a pool of recombinant 56-kDa proteins from Karp, Kato, Gilliam, and TA716 strains (r56-kDa) (Tables 1 and 2). Seven studies (15.2%, 7/46) used in-house ELISAs developed by NMRC, that employed whole-cell native antigens from Karp, Kato, Gilliam strains (Tables 1 and 3). While one study (2.2%, 1/46) used only Karp and Gilliam strain for its NMRC-developed in-house ELISAs (Table 3). There were two NMRC studies (4.3%, 2/46) that used recombinant Karp, Kato, Gilliam and TA763 strain or Karp alone antigens and two studies (4.3%, 2/46) with a combination of both whole cell and recombinant antigens. One study (2.2%, 1/46) used whole-cell Karp, Kato, Gilliam, and adding Kuroki and Kawasaki to the pool [21]. One study (2.2%, 1/46) employed combination of chimeric r56-kDa of Karp, Kato, and Gilliam strain, 21-kDa Boryong, and 56-kDa Kangwon 87–61 strain proteins as antigens. Remaining in-house ELISAs used variations of Karp, Kato, Gilliam, Boryong, and Ptan strains antigens (Tables 1 and 3).

Diagnostic accuracy studies: Cut-offs used and methodology for selecting cut-offs

Diagnostic cut-offs.

Eighteen studies (81.8%, 18/22) stated diagnostic cut-offs with considerable variation noted between the cut-offs (Table 1). Diagnostic cut-offs for IgM ranged from 0.064 [22] to 1.474 [23] OD (both 1:400 sample dilution) and IgG cut-offs ranged from 0.11 [22] to 1.305 [24] OD (both 1:100 sample dilution) which were all from Thai studies (Table 1). Variation was also apparent in cut offs selected for Indian studies, despite the exclusive use of the InBios ELISA where the IgM cut-offs ranged from 0.41 [25] to 1.0 [14] OD (Table 1).

Ten (43.5%, 10/22) studies investigated both IgG and IgM. Four of them (40.0%, 4/10) determined higher cut-off values for IgG than IgM. In two cases, the same cut-off was applied to both isotypes. For example, Kim et al calculated a cut-off of 0.2 OD for both isotypes despite using different reference standard cut-offs (Table 1) [15].

Methodology for selecting cut-offs.

Using the reference comparator result to derive a diagnostic cut-off, six out of 22 diagnostic studies (27.3%, 6/22) performed Receiver Operator Characteristic (ROC) analysis, and one (4.5%, 1/22) used Bayesian latent class modelling (LCM).

Fifteen (83.3%, 15/18) diagnostic studies that determined ELISAs accuracy used IFA or IIP as the reference test. IFA/micro-IFA were used in 11 studies (61.1%, 1/18), with diagnostic cut-off titers ranging from 1:10 to 1:12,800 for IgM (Table 1). Five of these studies (45.5%, 5/11) also used a 4-fold rise between paired samples as a seropositivity criteria in addition to a defined cut-off titer. There were four (22.2%, 4/18) studies that used the indirect immunoperoxidase (IIP) assay as reference test. Two studies that assessed ELISA accuracy did not clearly mention the reference assay. Other reference modalities used include PCR, presence of eschar, in vitro isolation, and response to antibiotic treatment [12, 23, 24, 26]. In one study (4.5%, 1/22), a combination of the above was used in the form of scrub typhus infection criteria (STIC) composite [12].

The most commonly used method to determine a diagnostic cut-off was the addition of standard deviations (SD) to the mean OD of negative controls (72.7%, 16/22). There were four studies adding 2 SD, nine diagnostic studies adding 3 SD to the mean OD and one study calculated mean OD + 2, 3, and 4 SD (Table 1).

Variation in derived IgG cut-offs were noted within two studies that had each used both ROC curves and the mean OD to determine a cut-off for the same population [19, 24]. IgM cut-offs remained roughly the same when applying different methods (ROC curves and mean OD) in three studies [12, 24, 26]. The remaining 16 studies either gave unclear information on the method used, unjustified methods to determine seropositivity (four studies), arbitrarily selected cut-offs (one study), or cut-offs from unpublished data (one study).

Observational studies: Cut-offs used and methodology for selecting cut-offs

Out of a total of 24 observational studies, seven (29.2%, 7/24) stated the method to determine the diagnostic cut-off and four (16.7%, 4/24) studies were unclear about how they derived the cut-offs stating “as used in other studies” or similar wording (Tables 2 and 3). Of the remaining studies, 13 (54.2%, 13/24) provided no clear explanation as to how the cut-off was selected, however 0.5 OD was used for IgM and/or IgG diagnosis for 11 (45.8%, 11/24) of these studies (Tables 2 and 3). Of the 19 observational studies using the InBios ELISA, seven (36.8%, 7/19) (Table 2) obtained local controls to determine a region-specific cut-off using the mean + 2 or 3 SD method. In the case of the NMRC in-house ELISA studies, the majority of studies (80.0%, 4/5) (Table 3), instead of calculating a single cut-off, patients were diagnosed with scrub typhus if they passed two criteria: 1) IgM OD ≥0.5 at a 1:100 dilution, and 2) a summed total OD of ≥1.0 of 4 sequential 4-fold dilutions. i.e., 1:100, 1:400, 1:1,600, 1:6,400) (Table 3).

Discussion

The application of appropriate diagnostic cut-offs is important for timely scrub typhus patient management using appropriate antibiotic therapy and to prevent complications leading to significant detrimental effect. This review has determined that there was a significant lack of consensus regarding methodologies, application and diagnostic cut-offs for ELISAs used for the diagnosis of scrub typhus infections. However, the reasons are complex and require further investigation.

Approximately half of the observational studies provided no or insufficient justification for the OD cut-offs, and two studies did not specify the cut-off they used. Although the 0.5 OD cut-off was used commonly in InBios ELISAs studies and used by the Indian Council of Medical Research (ICMR), this is probably an appropriate estimation for certain parts of India with limited application in other geographic locations. This cut-off should be applied only in regions where it has been validated by testing samples from healthy controls to determine the level of background immunity in the normal population. In some cases, it is difficult to select a cut-off as demonstrated by Blacksell et al, where optimal OD cut-offs ranged from 0.2–0.6 OD depending on the reference standards used [12]. Several studies used the same cut-off for IgG and IgM, despite the differences in immunity dynamics of the different antibody isotypes–this should be taken into consideration when interpreting results of such tests [15, 24, 27, 28], as generally, upon infection a spike in IgM is seen, followed by increased levels of IgG, which also confers long-term protection.

There was a lack of uniformity of approach regarding the diagnostic accuracy studies to determine appropriate ELISA cut-offs for various geographic locations. The reference methodologies varied from Bayesian LCM using composite scrub typhus infection criteria (STIC), IFA, through to mean + SD in healthy controls. In most cases, there was no clear justification for the reference test cut-offs employed, and it is likely that some of these cut-offs were not appropriate for the location in which they were being used. For example, while an IFA cut-off of 1:400 is often set in Thailand, it has been suggested to have a high false-positivity rate [29]. Subsequently, the diagnostic accuracy of composites such STIC have also been suggested to overestimate scrub typhus positivity compared with index [30]. Bayesian LCM is being increasingly used to determine true diagnostic accuracies, as they do not assume any reference diagnostic test is perfect [30]. A recent study calculated–using this method–an admission IgM IFA cut-off of ≥1:3,200 or at least a 4-fold rise to ≥1: 3,200 in the convalescent-phase sample to provide the highest accuracy [29]. Only one study in this review used Bayesian LCM; combining IFA, PCR, eschar and culture results as reference standards to interpret ELISA results [23]. Given that the reference standards all have different accuracies, using a composite in a Bayesian approach helps to eliminate bias. Other studies used different approaches that may compromise accuracy. For example, in one study, a response to an unnamed antibiotic, along with positivity by either PCR or presence of an eschar, was used as the diagnostic criteria [26]. Generally, doxycycline is prescribed to treat scrub typhus, however since it is a broad-spectrum antibiotic and also used to treat leptospirosis and murine typhus, a response to treatment may not point specifically to scrub typhus as the cause of illness.

A number of factors may have an influence on the diagnostic accuracy of ELISAs including antigenic composition and sample population. Differences in ELISA methodologies were observed where studies used local antigenic strains or incorporated these into pooled Karp, Kato and Gilliam antigens to supposedly increase the accuracy of the test. In general, higher ODs were obtained when using homologous antigens, therefore variation in cut-offs were likely to be seen depending on the antigen being used and the locally circulating strains. In India, the use of the InBios ELISAs (which used Karp, Kato, Gilliam and TA716 strain antigens) was widely implemented, providing a more standardized means of diagnosing scrub typhus. Jiang et al demonstrated a trivalent r56-kDa protein to be superior to both monovalent r56-kDa Karp and whole-cell Karp, Kato and Gillam ELISAs [16]. The antigens used for deployed soldiers or travelers need to be carefully considered, and results need to be interpreted with caution, given their background immunity is likely to differ significantly from those living in endemic areas. Nevertheless, standardized, region-specific antigen preparations should be used in ELISAs, taking into consideration the circulating strains.

Regarding study populations, the use of samples from diseased or normal subjects as well as the geographic origin of the subjects can affect the derived diagnostic cut-off. In one study, serum samples were collected from Australia and Thailand, but it was unclear to which population the cut-off was applied, or whether the cut-off was calculated using results from both the populations despite differences in endemicity [18].

In addition to a lack of ELISA methodology standardization there was also lack of consensus in what is considered as the gold standard reference assay to determine diagnostic cut-offs. The absence of standardized methods and appropriate cut-offs has implications for seroepidemiology and clinical studies, as well as clinical decision making. On one hand, lower cut-off would result in false positives results risking unnecessary treatment and increasing probability of antimicrobial resistance. On the other hand, higher cut-off would result in false negative results risking cases to be missed.

This review has several limitations. First, it only investigated studies published in English, which may limit literature retrieval. Second, only one author performed the article selection and data extraction, however, any unclear data was discussed amongst the authors in order to limit bias. Lastly, the ELISA protocol was not examined as a factor. This needs to be considered when interpreting results, as differences in protocol (e.g. the amount of antigen used in plate coating) can influence the sensitivity and specificity of ELISA tests, that in turn influence the selection of optimal cut-offs. To limit the heterogeneity caused by different ELISA protocol, variations of the conventional ELISA were excluded from the review, and the InBios ELISA studies were grouped together in the analysis.

Further research will need to be conducted to determine local levels of background immunity, as well as to identify circulating strains, in order to make informed decisions for a region-specific, standardized ELISA methodology and cut-off.

References

  1. 1. Wangrangsimakul T, Althaus T, Mukaka M, Kantipong P, Wuthiekanun V, Chierakul W, et al. Causes of acute undifferentiated fever and the utility of biomarkers in Chiangrai, northern Thailand. PLoS Negl Trop Dis. 2018;12(5):e0006477. Epub 2018/06/01. pmid:29852003.
  2. 2. Lerdthusnee K, Khuntirat B, Leepitakrat W, Tanskul P, Monkanna T, Khlaimanee N, et al. Scrub Typhus. Annals of the New York Academy of Sciences. 2003;990:25–35. pmid:12860596
  3. 3. Weitzel T, Dittrich S, Lopez J, Phuklia W, Martinez-Valdebenito C, Velasquez K, et al. Endemic Scrub Typhus in South America. N Engl J Med. 2016;375(10):954–61. Epub 2016/09/08. pmid:27602667.
  4. 4. Balcells ME, Rabagliati R, Garcia P, Poggi H, Oddo D, Concha M, et al. Endemic scrub typhus-like illness, Chile. Emerg Infect Dis. 2011;17(9):1659–63. Epub 2011/09/06. pmid:21888791; PubMed Central PMCID: PMCPMC3322051.
  5. 5. Ghorbani RP, Ghorbani AJ, Jain MK, Walker DH. A case of scrub typhus probably acquired in Africa. Clin Infect Dis. 1997;25(6):1473–4. Epub 1998/02/07. pmid:9431401.
  6. 6. Osuga K, Kimura M, Goto H, Shimada K, Suto T. A case of Tsutsugamushi disease probably contracted in Africa. Eur J Clin Microbiol Infect Dis. 1991;10(2):95–6. Epub 1991/02/01. pmid:1907545.
  7. 7. Izzard L, Fuller A, Blacksell SD, Paris DH, Richards AL, Aukkanit N, et al. Isolation of a novel Orientia species (O. chuto sp. nov.) from a patient infected in Dubai. J Clin Microbiol. 2010;48(12):4404–9. Epub 2010/10/12. pmid:20926708; PubMed Central PMCID: PMCPMC3008486.
  8. 8. Bonell A, Lubell Y, Newton PN, Crump JA, Paris DH. Estimating the burden of scrub typhus: A systematic review. PLoS Negl Trop Dis. 2017;11(9):e0005838. Epub 2017/09/26. pmid:28945755; PubMed Central PMCID: PMCPMC5634655.
  9. 9. Silpapojakul K, Varachit B, Silpapojakul K. Paediatric scrub typhus in Thailand: a study of 73 confirmed cases. Transactions of the Royal Society of Tropical Medicine and Hygiene. 2004;98(6):354–9. Epub 2004/04/22. pmid:15099991.
  10. 10. Ogawa M, Hagiwara T, Kishimoto T, Shiga S, Yoshida Y, Furuya Y, et al. Scrub typhus in Japan: epidemiology and clinical features of cases reported in 1998. The American journal of tropical medicine and hygiene. 2002;67(2):162–5. Epub 2002/10/23. pmid:12389941.
  11. 11. Koh GC, Maude RJ, Paris DH, Newton PN, Blacksell SD. Diagnosis of scrub typhus. Am J Trop Med Hyg. 2010;82(3):368–70. Epub 2010/03/09. pmid:20207857; PubMed Central PMCID: PMCPMC2829893.
  12. 12. Blacksell SD, Tanganuchitcharnchai A, Nawtaisong P, Kantipong P, Laongnualpanich A, Day NP, et al. Diagnostic Accuracy of the InBios Scrub Typhus Detect Enzyme-Linked Immunoassay for the Detection of IgM Antibodies in Northern Thailand. Clin Vaccine Immunol. 2015;23(2):148–54. Epub 2015/12/15. pmid:26656118; PubMed Central PMCID: PMCPMC4744921.
  13. 13. Luce-Fedrow A, Mullins K, Kostik AP, St John HK, Jiang J, Richards AL. Strategies for detecting rickettsiae and diagnosing rickettsial diseases. Future Microbiol. 2015;10(4):537–64. Epub 2015/04/14. pmid:25865193.
  14. 14. Koraluru M, Bairy I, Varma M, Vidyasagar S. Diagnostic validation of selected serological tests for detecting scrub typhus. Microbiol Immunol. 2015;59(7):371–4. Epub 2015/05/27. pmid:26011315.
  15. 15. Kim YJ, Yeo SJ, Park SJ, Woo YJ, Kim MW, Kim SH, et al. Improvement of the diagnostic sensitivity of scrub typhus using a mixture of recombinant antigens derived from Orientia tsutsugamushi serotypes. J Korean Med Sci. 2013;28(5):672–9. Epub 2013/05/17. pmid:23678257; PubMed Central PMCID: PMCPMC3653078.
  16. 16. Jiang J, Marienau KJ, May LA, Beecham HJ III, Wilkinson R, Ching WM, et al. Laboratory diagnosis of two scrub typhus outbreaks at Camp Fuji, Japan in 2000 and 2001 by enzyme-linked immunosorbent assay, rapid flow assay, and Western blot assay using outer membrane 56-kD recombinant proteins Am J Trop Med Hyg. 2003;69(1):60–6. pmid:12932099
  17. 17. Jiang J, Myers TE, Rozmajzl PJ, Graf PC, Chretien JP, Gaydos JC, et al. Seroconversions to Rickettsiae in US Military Personnel in South Korea. Emerg Infect Dis. 2015;21(6):1073–4. Epub 2015/05/20. pmid:25989279; PubMed Central PMCID: PMCPMC4451913.
  18. 18. Land MV, Ching WM, Dasch GA, Zhang Z, Kelly DJ, Graves SR, et al. Evaluation of a Commercially Available Recombinant-Protein Enzyme-Linked Immunosorbent Assay for Detection of Antibodies Produced in Scrub Typhus Rickettsial Infections. Journal of clinical microbiology. 2000;38(7):2701–5. pmid:10878067
  19. 19. Suwanabun N, Chouriyagune C, Eamsila C, Watcharapichat P, Dasch GA, Howard RS, et al. Evaluation of an Enzyme-Linked Immunosorbent Assay in Thai Scrub Typhus patients. Am J Trop Med Hyg. 1997;56(1):38–43. pmid:9063359
  20. 20. Janardhanan J, Prakash JA, Abraham OC, Varghese GM. Comparison of a conventional and nested PCR for diagnostic confirmation and genotyping of Orientia tsutsugamushi. Diagn Microbiol Infect Dis. 2014;79(1):7–9. Epub 2014/02/26. pmid:24565850.
  21. 21. Ogawa M, Satoh M, Saijo M, Ando S. Evaluation of a broad-ranging and convenient enzyme-linked immunosorbent assay using the lysate of infected cells with five serotypes of Orientia tsutsugamushi, a causative agent of scrub typhus. BMC Microbiol. 2017;17(1):7. Epub 2017/01/07. pmid:28056811; PubMed Central PMCID: PMCPMC5217197.
  22. 22. Ching WM, Wang H, Eamsila C, Kelly DJ, Dasch GA. Expression and refolding of truncated recombinant major outer membrane protein antigen (r56) of Orientia tsutsugamushi and its use in enzyme-linked immunosorbent assays. Clin Diagn Lab Immunol. 1998;5(4):519–26. pmid:9665960
  23. 23. Blacksell SD, Lim C, Tanganuchitcharnchai A, Jintaworn S, Kantipong P, Richards AL, et al. Optimal Cutoff and Accuracy of an IgM Enzyme-Linked Immunosorbent Assay for Diagnosis of Acute Scrub Typhus in Northern Thailand: an Alternative Reference Method to the IgM Immunofluorescence Assay. Journal of clinical microbiology. 2016;54(6):1472–8. Epub 2016/03/25. pmid:27008880; PubMed Central PMCID: PMCPMC4879268.
  24. 24. Chao CC, Zhang Z, Belinskaya T, Thipmontree W, Tantibhedyangkul W, Silpasakorn S, et al. An ELISA assay using a combination of recombinant proteins from multiple strains of Orientia tsutsugamushi offers an accurate diagnosis for scrub typhus. BMC Infect Dis. 2017;17(1):413. Epub 2017/06/12. pmid:28601091; PubMed Central PMCID: PMCPMC5466769.
  25. 25. Patricia KA, Hoti SL, Kanungo R, Jambulingam P, Shashikala N, Naik AC. Improving the Diagnosis of Scrub Typhus by Combining groEL Based Polymerase Chain Reaction and IgM ELISA. J Clin Diagn Res. 2017;11(8):DC27–DC31. Epub 2017/10/04. pmid:28969124; PubMed Central PMCID: PMCPMC5620764.
  26. 26. Gupta N, Chaudhry R, Thakur CK. Determination of Cutoff of ELISA and Immunofluorescence Assay for Scrub Typhus. J Glob Infect Dis. 2016;8(3):97–9. Epub 2016/09/14. pmid:27621559; PubMed Central PMCID: PMCPMC4997800.
  27. 27. Sengupta M, Anandan S, Daniel D, Prakash JA. Scrub Typhus Seroprevalence in Healthy Indian Population. J Clin Diagn Res. 2015;9(10):DM01–2. Epub 2015/11/12. pmid:26557523; PubMed Central PMCID: PMCPMC4625242.
  28. 28. Hu J, Tan Z, Ren D, Zhang X, He Y, Bao C, et al. Clinical characteristics and risk factors of an outbreak with scrub typhus in previously unrecognized areas, Jiangsu province, China 2013. PLoS One. 2015;10(5):e0125999. Epub 2015/05/09. pmid:25954814; PubMed Central PMCID: PMCPMC4425678.
  29. 29. Lim C, Blacksell SD, Laongnualpanich A, Kantipong P, Day NP, Paris DH, et al. Optimal Cutoff Titers for Indirect Immunofluorescence Assay for Diagnosis of Scrub Typhus. J Clin Microbiol. 2015;53(11):3663–6. Epub 2015/09/12. pmid:26354819; PubMed Central PMCID: PMCPMC4609688.
  30. 30. Lim C, Paris DH, Blacksell SD, Laongnualpanich A, Kantipong P, Chierakul W, et al. How to Determine the Accuracy of an Alternative Diagnostic Test when It Is Actually Better than the Reference Tests: A Re-Evaluation of Diagnostic Tests for Scrub Typhus Using Bayesian LCMs. PLoS One. 2015;10(5):e0114930. pmid:26024375; PubMed Central PMCID: PMCPMC4449177.
  31. 31. Coleman RE, Sangkasuwan V, Suwanabun N, Eamsila C, Mungviriya S, Devine PL, et al. Comparative evaluation of selected diagnostic assays for the detection of IgG and IgM antibody to Orientia tsutsugamushi in Thailand. The American journal of tropical medicine and hygiene. 2002;67(5):497–503. pmid:12479551
  32. 32. Morch K, Manoharan A, Chandy S, Chacko N, Alvarez-Uria G, Patil S, et al. Acute undifferentiated fever in India: a multicentre study of aetiology and diagnostic accuracy. BMC Infect Dis. 2017;17(1):665. Epub 2017/10/06. pmid:28978319; PubMed Central PMCID: PMCPMC5628453.
  33. 33. Gupta N, Chaudhry R, Kabra SK, Lodha R, Mirdha BR, Das BK, et al. Comparative Evaluation of Serological and Molecular Methods for the Diagnosis of Scrub Typhus in Indian Settings. Jpn J Infect Dis. 2017;70(2):221–2. Epub 2016/09/02. pmid:27580576.
  34. 34. Stephen S, Sangeetha B, Ambroise S, Sarangapani K, Gunasekaran D, Hanifah M, et al. Outbreak of scrub typhus in Puducherry & Tamil Nadu during cooler months. Indian J Med Res. 2015;142(5):591–7. Epub 2015/12/15. pmid:26658595; PubMed Central PMCID: PMCPMC4743347.
  35. 35. Kim YJ, Park S, Premaratna R, Selvaraj S, Park SJ, Kim S, et al. Clinical Evaluation of Rapid Diagnostic Test Kit for Scrub Typhus with Improved Performance. J Korean Med Sci. 2016;31(8):1190–6. Epub 2016/08/02. pmid:27478327; PubMed Central PMCID: PMCPMC4951546.
  36. 36. Stephen S, Kim SH, Pradeep J, Kim YJ, Kim EY, Park S, et al. Evaluation of ImmuneMed scrub typhus rapid test kit, for diagnosis of scrub typhus. J Vector Borne Dis. 2016;53(3):283–7. Epub 2016/09/30. pmid:27681553.
  37. 37. Anitharaj V, Stephen S, Pradeep J, Park S, Kim SH, Kim YJ, et al. Serological Diagnosis of Acute Scrub Typhus in Southern India: Evaluation of InBios Scrub Typhus Detect IgM Rapid Test and Comparison with other Serological Tests. J Clin Diagn Res. 2016;10(11):DC07–DC10. Epub 2017/01/05. pmid:28050364; PubMed Central PMCID: PMCPMC5198317.
  38. 38. Kim IS, Seong SY, Woo SG, Choi MS, Chang WH. High-level expression of a 56-kilodalton protein gene (bor56) of Rickettsia tsutsugamushi Boryong and its application to enzyme-linked immunosorbent assays. J Clin Microbiol. 1993;31(3):598–605. Epub 1993/03/01. pmid:8458953; PubMed Central PMCID: PMCPMC262827.
  39. 39. Jang WJ, Huh MS, Park KH, Choi MS, Kim IS. Evaluation of an Immunoglobulin M Capture Enzyme-Linked Immunosorbent Assay for Diagnosis of Orientia tsutsugamushi Infection. Clinical and Vaccine Immunology. 2003;10(3):394–8.
  40. 40. Cao M, Guo H, Tang T, Wang C, Li X, Pan X, et al. Preparation of recombinant antigen of O. tsutsugamushi Ptan strain and development of rapid diagnostic reagent for scrub typhus. Am J Trop Med Hyg. 2007;76(3):553–8. Epub 2007/03/16. pmid:17360883.
  41. 41. Varghese GM, Trowbridge P, Janardhanan J, Thomas K, Peter JV, Mathews P, et al. Clinical profile and improving mortality trend of scrub typhus in South India. Int J Infect Dis. 2014;23:39–43. Epub 2014/03/26. pmid:24661931.
  42. 42. Attur RP, Kuppasamy S, Bairy M, Nagaraju SP, Pammidi NR, Kamath V, et al. Acute kidney injury in scrub typhus. Clin Exp Nephrol. 2013;17(5):725–9. Epub 2013/01/08. pmid:23292176.
  43. 43. Varghese GM, Janardhanan J, Trowbridge P, Peter JV, Prakash JA, Sathyendra S, et al. Scrub typhus in South India: clinical and laboratory manifestations, genetic variability, and outcome. Int J Infect Dis. 2013;17(11):e981–7. Epub 2013/07/31. pmid:23891643.
  44. 44. Astrup E, Janardhanan J, Otterdal K, Ueland T, Prakash JA, Lekva T, et al. Cytokine network in scrub typhus: high levels of interleukin-8 are associated with disease severity and mortality. PLoS Negl Trop Dis. 2014;8(2):e2648. Epub 2014/02/12. pmid:24516677; PubMed Central PMCID: PMCPMC3916254.
  45. 45. Kalal BS, Puranik P, Nagaraj S, Rego S, Shet A. Scrub typhus and spotted fever among hospitalised children in South India: Clinical profile and serological epidemiology. Indian J Med Microbiol. 2016;34(3):293–8. Epub 2016/08/16. pmid:27514949.
  46. 46. Varghese GM, Janardhanan J, Mahajan SK, Tariang D, Trowbridge P, Prakash JA, et al. Molecular Epidemiology and Genetic Diversity of Orientia tsutsugamushi from Patients with Scrub Typhus in 3 Regions of India. Emerging Infectious Diseases. 2015;21(1).
  47. 47. Sengupta M, Benjamin S, Prakash JA. Scrub typhus continues to be a threat in pregnancy. Int J Gynaecol Obstet 2014;127(2):212. pmid:25109770
  48. 48. Bhargava A, Kaushik R, Kaushik RM, Sharma A, Ahmad S, Dhar M, et al. Scrub typhus in Uttarakhand & adjoining Uttar Pradesh: Seasonality, clinical presentations & predictors of mortality. Indian J Med Res. 2016;144(6):901–9. Epub 2017/05/06. pmid:28474627; PubMed Central PMCID: PMCPMC5433283.
  49. 49. Sood AK, Chauhan L, Gupta H. CNS Manifestations in Orientia tsutsugamushi Disease (Scrub Typhus) in North India. Indian J Pediatr. 2016;83(7):634–9. Epub 2016/01/29. pmid:26817467.
  50. 50. Usha K, Kumar E, Kalawat U, Kumar BS, Chaudhury A, Gopal DV. Molecular detection of scrub typhus in Tirupati, Andhra Pradesh, India. J Vector Borne Dis. 2015;52(2):171–4. Epub 2015/06/30. pmid:26119551.
  51. 51. Roopa KS, Karthika K, Sugumar M, Bammigatti C, Shamanna SB, Harish BN. Serodiagnosis of Scrub Typhus at a Tertiary Care Hospital from Southern India. Journal of Clinical and Diagnostic Research. 2015;9(11).
  52. 52. Trowbridge P, Divya P, Premkumar PS, Varghese GM. Prevalence and risk factors for scrub typhus in South India. Tropical Medicine and International Health. 2017;22(5):576–82. pmid:28173608
  53. 53. De W, Jing K, Huan Z, Qiong ZH, Monagin C, Min ZJ, et al. Scrub typhus, a disease with increasing threat in Guangdong, China. PLoS One. 2015;10(2):e0113968. Epub 2015/02/18. pmid:25689778; PubMed Central PMCID: PMCPMC4331496.
  54. 54. Chen H, Ning Z, Qiu Y, Liao Y, Chang H, Ai Y, et al. Elevated levels of von Willebrand factor and high mobility group box 1 (HMGB1) are associated with disease severity and clinical outcome of scrub typhus. Int J Infect Dis. 2017;61:114–20. Epub 2017/06/28. pmid:28652214.
  55. 55. Tay ST, Mohamed Zan HA, Lim YA, Ngui R. Antibody prevalence and factors associated with exposure to Orientia tsutsugamushi in different aboriginal subgroups in West Malaysia. PLoS Negl Trop Dis. 2013;7(8):e2341. Epub 2013/08/13. pmid:23936576; PubMed Central PMCID: PMCPMC3731234.
  56. 56. Pradeepan JA, Ketheesan N, Murugananthan K. Emerging scrub typhus infection in the northern region of Sri Lanka. BMC Res Notes. 2014;7:719. Epub 2014/10/16. pmid:25316171; PubMed Central PMCID: PMCPMC4216347.
  57. 57. Upadhyaya BP, Shakya G, Adhikari S, Rijal N, Acharya J, Maharjan L, et al. Scrub Typhus: An Emerging Neglected Tropical Disease in Nepal. J Nepal Health Res Counc. 2016;14(33):122–7. Epub 2016/11/26. pmid:27885295.
  58. 58. Maude RR, Maude RJ, Ghose A, Amin MR, Islam MB, Ali M, et al. Serosurveillance of Orientia tsutsugamushi and Rickettsia typhi in Bangladesh. Am J Trop Med Hyg. 2014;91(3):580–3. Epub 2014/08/06. pmid:25092819; PubMed Central PMCID: PMCPMC4155564.
  59. 59. Kocher C, Jiang J, Morrison AC, Castillo R, Leguia M, Loyola S, et al. Serologic Evidence of Scrub Typhus in the Peruvian Amazon. Emerg Infect Dis. 2017;23(8):1389–91. Epub 2017/07/21. pmid:28726619; PubMed Central PMCID: PMCPMC5547797.
  60. 60. Khan SA, Bora T, Chattopadhyay S, Jiang J, Richards AL, Dutta P. Seroepidemiology of rickettsial infections in Northeast India. Trans R Soc Trop Med Hyg. 2016;110(8):487–94. Epub 2016/09/14. pmid:27618917.