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  Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 36  |  Issue : 1  |  Page : 108-112
 

Diagnostic performance of serological tests to detect antibodies against acute scrub typhus infection in central India


1 Department of Microbiology, Mahatma Gandhi Institute of Medical Sciences, Sewagram, Wardha, India
2 Department of Community Medicine, Mahatma Gandhi Institute of Medical Sciences, Sewagram, Wardha, India

Date of Web Publication2-May-2018

Correspondence Address:
Dr. Rahul Narang
Department of Microbiology, Mahatma Gandhi Institute of Medical Sciences, Sewagram, Wardha, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_17_405

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 ~ Abstract 

Background: Differentiating scrub typhus from other acute febrile illness is difficult due to non specificity of clinical symptoms and relative absence of eschar in Indian population. The diagnosis thus relies mainly on laboratory tests. Antibody based serological tests are mainstay of scrub typhus diagnosis. Here, we evaluated the diagnostic performance of IgM ELISA, IgM IFA and ICT to detect antibodies against O. tsutsugamushi in acute serum of febrile patients. Methodology: The serum samples from 600 randomly selected patients suffering from acute undifferentiated fever were tested by all the three tests mentioned above. We used latent class analysis to generate unbiased results as all the tests for scrub typhus diagnosis are imperfect and none of them can be considered as reference standard. Results: We found that IgM ELISA with cutoff titer 0.5 OD has high diagnostic accuracy (sensitivity 99.9% and specificity 99.15) than IgM IFA (sensitivity 96.8% and specificity 99.7%) for scrub typhus diagnosis. ICT used in our study had very high specificity 100% but low sensitivity (38%) which would limit its use for acute serum samples. ICT being a screening or point of care test, has to be more sensitive while some compromise with specificity is affordable. Hence, optimal cutoff for ICT should be evaluated under different settings. Conclusion: IgM ELISA being simple and affordable could be an alternative diagnostic test to IgM IFA which is subjective and costly.


Keywords: Immunoglobulin M enzyme-linked immunosorbent assay, immunochromatographic test, indirect immunofluorescence assay, latent class analysis, scrub typhus, serology


How to cite this article:
Pote K, Narang R, Deshmukh P. Diagnostic performance of serological tests to detect antibodies against acute scrub typhus infection in central India. Indian J Med Microbiol 2018;36:108-12

How to cite this URL:
Pote K, Narang R, Deshmukh P. Diagnostic performance of serological tests to detect antibodies against acute scrub typhus infection in central India. Indian J Med Microbiol [serial online] 2018 [cited 2018 Nov 15];36:108-12. Available from: http://www.ijmm.org/text.asp?2018/36/1/108/231673



 ~ Introduction Top


In India, scrub typhus is being considered as re-emerging disease owing to several reports of its outbreak from the different parts of the country.[1],[2],[3],[4],[5] Diagnostic testing, in particular, early detection, is critical for scrub typhus, as most infected individuals have non-specific symptoms that are easily confused with other acute febrile illnesses such as malaria, dengue and leptospirosis. Rapid and accurate diagnosis enables specific and effective antibiotic treatment of scrub typhus. Several methods are currently available for diagnosing scrub typhus including isolation of Orientia tsutsugamushi, molecular methods as well as serological techniques such as Weil–Felix test, indirect immunofluorescence assay (IFA), indirect immunoperoxidase assay, enzyme-linked immunosorbent assay (ELISA), immunochromatographic test (ICT) etc.

O. tsutsugamushi can be isolated from blood of patient with scrub typhus by culturing in vitro on cell lines; however, it requires biosafety Level-3 facilities.[6],[7] In serology, though Weil–Felix test is most commonly used for scrub typhus diagnosis, it lacks both sensitivity and specificity.[8] IFA is the current reference standard for serological diagnosis of scrub typhus; however, it has limitations due to non-availability of standard slides, need of paired sera, the absence of consensus on cut-off titres, presence of cross-reactivity and subjectivity in end-point determination.[6],[9],[10] Thus, it is very difficult to consider it as reference standard.[9] ELISA techniques, on the other hand, are relatively easy to standardise, reading is done objectively and has high sample throughput. The Indian Council of Medical Research (ICMR) has recommended ELISA techniques, particularly, immunoglobulin M (IgM) capture ELISA with cut-off OD of 0.5 as the most sensitive test available for scrub typhus diagnosis for Indian population.[8] For point-of-care tests, rapid diagnostic test in the form of ICT have been developed and are available for testing. Usefulness and limitations of ICT-based rapid tests have been reported from many countries.[11],[12],[13],[14] Nucleic acid-based detection is the most accurate test among all in acute phase of disease, but has limitations, especially in areas with limited resources, sensitivity decreases with duration of fever and assay has lower limit of detection.[15],[16] However, combination of nucleic acid-based method and serology for diagnosis of disease was found to increase the sensitivity with minimal reduction in specificity.[15]

In the present study, we assessed the diagnostic performance of commercially available rapid ICT (SD Bioline, South Korea), IgM ELISA (InBios International Inc. USA) and IgM IFA (Fuller laboratories, USA) to detect antibodies against O. tsutsugamushi in acute serum of patients with acute febrile illness.


 ~ Materials and Methods Top


Study setting and patients

A hospital-based surveillance of zoonotic diseases was carried out in a tertiary care hospital situated in central India, during the period from July 2015 to June 2016. Patients of any age who presented with history of fever (>38°C) of ≥ 5 days and negative for other causes of fever, namely malaria, dengue and enteric fever were recruited after obtaining written informed consent. Total number of patients recruited was 1680. From each patient 5 ml of blood was drawn into a vacutainer and transferred to the microbiology laboratory immediately. Serum was separated and tests such as ICT, IgM ELISA and IgM IFA for scrub typhus were performed on the serum samples. For the present study, 600 patients were randomly selected to overcome the selection bias and also to reduce cost associated with testing all the patients' serum sample (1680) by IgM IFA.

The study protocol was approved by the Institutional Ethics Committee.

IgM enzyme-linked immunosorbent assay

We used commercial Scrub typhus Detect IgM ELISA Kit (InBios International Inc. USA) which employs a recombinant p56-kDa type-specific antigen for the detection of O. tsutsugamushi strains Karp, Kato, Gilliam and TA716 for the purpose of IgM antibodies detection. The procedure was performed as per manufacturer's recommendation. All serum samples were diluted at 1:100 dilution. Absorbance was read at 450 nm using Multiskan FC reader (Thermo Scientific, USA). Cut-off values were determined by performing IgM ELISA using same kit on the 100 serum samples of healthy volunteers from this region and calculated using following formula: cut-off value = average OD of normal human sera + 3SD of normal human sera = 0.5.

Samples having OD >0.5 were considered as positive, and those below 0.5 were reported as negative.

IgM indirect immunofluorescence assay

IgM IFA was performed using a kit from Fullers laboratory (USA). In this kit, IFA slide is coated with four different prototype antigens of O. tsutsugamushi, namely, Karp, Kato, Gilliam and Boryong. The assay was performed as per manufacturer's recommendations. The slides were read under fluorescent microscope Evos FL (Invitrogen, USA), and the results were recorded as positive or negative. End-point titres up to 1: 512 were calculated. The criteria for positivity as recommended by kit literature were kept at 1: 64.

Immunochromatographic test

This rapid test (SD Bioline Tsutsugamushi) detects total IgM, IgG or IgA antibodies in human serum against O. tsutsugamushi. The test was performed as per recommendation of manufacturer. Briefly, 10 μl serum was applied to sample well and three drops of assay diluent were added. Results were read after 10–15 min. Test had two-coloured lines, a control line “C” and test line “T”. Color band observed only at “C” was recorded as negative while if observed at both “C” and “T” recorded as positive. Absence of “C” line indicated invalid result.

Data analysis

All the tests available for the diagnosis of scrub typhus are imperfect, and none of them can be considered as reference standard. Imperfection of reference standard test can lead to serious bias into evaluation of other tests. Hence, to overcome this problem, we used latent class analysis to estimate the indicators of diagnostic accuracy (sensitivity and specificity) using randomLCA package in R along with their 95% confidence interval. We used dichotomised result of IgM IFA, IgM ELISA and ICT for modelling a latent variable, i.e., presence and absence of scrub typhus. It also gives an estimate of magnitude of disease in the study subjects. We also calculated probabilities of the presence of disease using different permutation and combination of results of the three tests.


 ~ Results Top


Out of 600participants, 55% were females. Nearly 18.5% of the study participants were <10 years and 9.2% were in the age group of 51–60 years. Distribution across the age groups was almost equal [Table 1].
Table 1: Age and sex distribution of study participants

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Proportion of scrub typhus among the study participants was 15.2% by IgM IFA. It was 14.3% by IgM ELISA and 5.2% by ICT. Proportion of disease by latent class model (LCM) was 13.6% [Table 2].
Table 2: Magnitude of scrub typhus

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Sensitivity and specificity of IgM IFA was 96.8% (95% CI: 80.4%–99.5%) and 99.7% (95% CI: 95.9%–98.7%), respectively. Performance of IgM ELISA was marginally better than IgM IFA, and it had sensitivity of 99.9% (95% CI: 90.4%–100%) and specificity of 99.1% (95% CI: 96.8%–99.8%). However, the sensitivity of ICT was very low 38% (95% CI: 28%-49%) while it had very high specificity 100% (95% CI: 98.5%–100%) [Table 3].
Table 3: Performance of the three tests for diagnosis of scrub typhus

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[Table 4] shows the probability of a person having the disease given different combinations of results of the three tests under evaluation. If all the three tests are positive, the disease probability is >0.99999. When the results of all the three tests are negative, probability of patient having the disease is <0.0001.
Table 4: Probability of disease for a given combination of results of three tests

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 ~ Discussion Top


Serological tests are the backbone of scrub typhus diagnosis and have their own advantages and limitations. Despite of lower specificity and sensitivity, Weil–Felix test is widely used test in India.[8] IFA is considered as reference standard for diagnosis, but its use is limited due to the cost of consumables and need of technical expertise. Comparatively, ELISA is easy to perform and cheaper; however, facilities are not available beyond secondary health-care centres such as district hospitals in India.[8] Scrub typhus being an occupational disease among rural population in Asia-Pacific region,[17] even tertiary care hospitals catering to rural areas may not have most of these tests. In such a condition, use of ICT which is a rapid diagnostic test provides a point-of-care test. Hence, we assessed the performance of commercially available ICT (SD Bioline Orientia tsutsugamushi), IgM ELISA (InBios Scrub typhus detect) and IgM IFA (Fuller laboratories, USA) to detect the presence of antibodies against O. tsutsugamushi in acute serum of patients with acute febrile illness.

Conventionally, performance of test of interest is determined by comparing its results with reference test, assuming that reference test has perfect sensitivity and specificity. If not so, there will be bias in the performance of test under evaluation. Even though IFA has been considered as reference serological test for diagnosis, it is suggested that validation of newer test should not be done only against IFA but must be validated against a panel of both serological and antigen detection tests.[5] Thus, in the absence of perfect gold standard, we used latent class analysis to estimate the indicators of diagnostic accuracy (sensitivity and specificity). The latent class analysis is used to estimate the performance and accuracy of diagnostic test under evaluation since it does not have to assume that accuracy of the reference test is perfect.[18],[19] In recent years a study conducted using Bayesian LCM found that the use of imperfect gold standard would underestimate the true diagnostic accuracy of certain scrub typhus infection criteria and overestimation of serological endpoints.[16]

We found performance of IgM ELISA with cut-off titre of 0.5 OD for the diagnosis of scrub typhus to be marginally better than IgM IFA. Other researchers have also validated IgM ELISA (InBios) to perform satisfactorily and it could be an alternative to IgM IFA.[20],[21],[22] IgM ELISA is easy to perform and does not require specific instrument like IFA. As mentioned earlier, in India, IgM ELISA with cutoff OD 0.5 has been recommended to be most sensitive test to indicate recent infection with O. tsutsugamushi.[8] The use of ELISA will result in reduced subjectivity and diagnosis can be provided with single acute serum unlike IFA which needs paired sera for confirmed diagnosis (≥4 fold rise in IgM titer). In our setting, we had determined the appropriate diagnostic cut-off titre (0.5 OD) using serum of healthy volunteers to eliminate the background levels of antibodies which may lead to false positivity. Blacksell et al. have recommended that geographically specific diagnostic cut-off should be determined and applied, so that IgM ELISA could be an alternative diagnostic serological test.[23]

The SD Bioline ICT did perform fairly in our study with 100% specificity but should be used carefully due to its lower sensitivity 38% (CI 28%–49%) in the present format. Watthanaworawit et al. have also reported the performance of SD Bioline ICT with lower sensitivity (20.9%) in acute serum and 76.7% for convalescent serum with equivalent specificity for both acute and convalescent serum 74.4% and 76.6%, respectively, in population of Thailand.[24] Earlier study conducted in Thailand using SD Bioline ICT had reported sensitivity and specificity to be 66.7% and 98.4%, respectively.[11] On the contrary, Lee et al. reported higher sensitivity (72.6%) of SD Bioline ICT in Korean population.[25] Another ICT, ImmuneMed RDT was found to be more sensitive (98.6%) than SD Bioline RDT (84.8%) in Korean population.[26]

Lower sensitivity of SD Bioline ICT observed in our study could be attributed to the antigens used in the kit. SD Bioline tsutsugamushi test utilised antigens from three serotypes of O. tsutsugamushi including Gilliam, Karp and Kato. There is only one study from India reporting identification of Kato, Karp, Gilliam, Ikeda and Neimeng-65 genotype strains circulating in South, Northern and Northeast India [27] while still there is no knowledge about the circulating serotypes/genotypes of O. tsutsugamushi in the central part of India. The incorporation of locally circulating serotype/genotype antigen into assay might increase the accuracy of the assay. Recently, a study conducted in South India has reported the performance of two ICTs with better sensitivities and specificity, namely, Scrub Typhus Detect IgM Rapid test, InBios International, USA (sensitivity 99.2% and specificity 93.0%) and ImmuneMed ST Rapid kit, ImmuneMed, Chuncheon, South Korea (sensitivity 94.8% and specificity 94.1%).[14] InBios scrub typhus detect IgM rapid test uses recombinant 56-kD type-specific antigen of O. tsutsugamushi strain Karp, Kato, Gilliam and TA716 while ImmuneMed scrub typhus RDT uses antigens from five serotypes of O. tsutsugamushi including Gilliam, Karp, Kato, Kangwon and Boryong. More information on the prevalence of serotypes of O. tsutsugamushi in the different geographical areas of the country is thus required.

Our study highlights the usefulness of scrub typhus InBios IgM ELISA and limitations of SD Bioline ICT. InBios IgM ELISA is a sensitive and specific test and could be a substitute for IgM IFA in resource-limited settings. Our findings corroborates with the recently published DHR-ICMR guidelines for the diagnosis and management of rickettsial diseases in India which discourages the use of ICTs for diagnosis of scrub typhus.[8] The present cut-off of ICT gives lower sensitivity and higher specificity. As the ICT is screening or POC test, it is required to be more sensitive and some compromise with the specificity may be affordable. Hence, it is recommended that optimal cut-off of ICT should be evaluated under different settings.

Acknowledgement

We are grateful to MGIMS and Kasturba Health Society, Sewagram. We are also thankful to the ICMR Zoonosis project staff for technical help.

Financial support and sponsorship

This study was funded by ICMR New Delhi vide Grant Letter No. ZON/15/11/2014-ECD-II.

Conflicts of interest

There are no conflicts of interest.

 
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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