Enzyme-linked immunosorbent assay as a substitute for immunofluorescence assay: An analysis of different serological tests in diagnosis of scrub typhus

Asfia Sultan; Afaf Shuaib; Meher Rizvi; Fatima Khan; Haris M Khan

doi:10.4103/jacm.jacm_41_21

ORIGINAL ARTICLE

Year : 2021 | Volume : 23 | Issue : 1 | Page : 9-13

Enzyme-linked immunosorbent assay as a substitute for immunofluorescence assay: An analysis of different serological tests in diagnosis of scrub typhus

Asfia Sultan¹, Afaf Shuaib¹, Meher Rizvi², Fatima Khan¹, Haris M Khan¹
¹ Department of Microbiology, Jawaharlal Nehru Medical College and Hospital, Aligarh Muslim University, Aligarh, Uttar Pradesh, India
² Department of Microbiology and Immunology, College of Medicine and Health Sciences, Sultan Qaboos University Hospital, Muscat, Oman

Date of Submission	12-Apr-2021
Date of Decision	15-May-2021
Date of Acceptance	01-Jun-2021
Date of Web Publication	16-Sep-2021

Correspondence Address:
Dr. Asfia Sultan
Department of Microbiology, J.N Medical College, Aligarh Muslim University, Aligarh, Uttar Pradesh
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/jacm.jacm_41_21

Abstract

BACKGROUND: Among the causes of acute febrile illness, scrub typhus is an important entity. Serology is the mainstay of diagnosis of scrub typhus, with immunoglobulin M (IgM) immunofluorescence assay (IFA) test being the gold standard. Because of cost constraint, IFA cannot be employed for screening or routine diagnosis. This study was planned to evaluate the diagnostic efficacy of different serological test available for Scrub typhus.
MATERIALS AND METHODS: Total of 414 cases of undiagnosed acute febrile illness of >5 days were evaluated for scrub typhus. The tests employed were Weil-Felix, rapid immunochromatographic test, IgM enzyme-linked immunosorbent assay (ELISA) and IgM IFA. Performance of all the tests was evaluated against gold standard IFA test by IBM SPSS version 20.0 (SPSS Inc. Chicago, USA) and MedCalcsoftware.
RESULTS: Total of 112 (27%) were positive by ELISA while 32 (7.7%) and 44 (10.6%) cases were positive by Weil-Felix and RDT, respectively. Of these, only 98 (23.6%) samples showed titre of ≥1:64 by IFA. Statistical analysis showed sensitivity and specificity of IgM ELISA, 100 (95% confidence intervals [CI]: 96.31–97.88) and 96.05 (95% CI: 93.34–97.88) respectively. The sensitivity and specificity of RDT and Weil Felix was 64.47 (95% CI: 56.31–72.06) and 57.65 (95% CI: 49.85–65.18) and 100% (95% CI: 98.84–100) and 98.14 (95.99–99.31), respectively. On analysis of association between OD values and IFA titres, Spearman correlation coefficient was 0.783 (P < 0.01) while Pearson correlation coefficient r²⁼ 0.879(P < 0.01). Youden's index of Weil-Felix, RDT and ELISA was 0.56, 0.64 and 0.96, respectively.
CONCLUSION: IgM ELISA has turned out to be a significant tool in diagnosis of scrub typhus.

Keywords: Diagnosis, enzyme-linked immunosorbent assay, Immunochromatographic test, immunofluorescence, Scrub typhus

How to cite this article:
Sultan A, Shuaib A, Rizvi M, Khan F, Khan HM. Enzyme-linked immunosorbent assay as a substitute for immunofluorescence assay: An analysis of different serological tests in diagnosis of scrub typhus. J Acad Clin Microbiol 2021;23:9-13

How to cite this URL:
Sultan A, Shuaib A, Rizvi M, Khan F, Khan HM. Enzyme-linked immunosorbent assay as a substitute for immunofluorescence assay: An analysis of different serological tests in diagnosis of scrub typhus. J Acad Clin Microbiol [serial online] 2021 [cited 2023 Jun 3];23:9-13. Available from: https://www.jacmjournal.org/text.asp?2021/23/1/9/326044

Introduction

Scrub typhus has a considerable incidence among acute febrile illness in India.^[1] With advancements in estimating the burden of acute febrile episodes, it is essential to assess the burden of scrub typhus all over India.^[2] Despite the whole inventory of diagnostic tests available for diagnosis of scrub typhus, there is lack of access to specific and sensitive diagnostic tests in most places and low index of suspicion among the clinicians has led to underdiagnosis of scrub typhus in India.^[3] Each diagnostic assay possesses advantages and limitations. Isolation of Orientia tsutsugamushi from blood by culture is definitive but is long and cumbersome requiring biosafety level 3 facilities, which is not practical for routine diagnosis.^[4] Molecular methods like nested and real-time polymerase chain reaction have proved their utility in diagnosis but sensitivity varies with type of sample, method and antigen used and also the duration of fever.^[5]

Serological test remains the main tool for timely and accurate diagnosis of scrub typhus. Among various serological tests available for diagnosis of scrub typhus are, i.e. Weil-Felix test, immunofluorescence assay (IFA), latex agglutination, rapid immunochromatographic test (RDT), immunoperoxidase assay, enzyme-linked immunosorbent assay (ELISA), western immunoblot. Weil-Felix agglutination test is inexpensive and commercially available serological test that detects immunoglobulin M (IgM) antibody.^[6] It lacks both specificity and sensitivity. RDT are available for diagnosis but sensitivity is variable and its use in diagnosis is discouraged.^[7] The ELISA is simple and cost-effective. It is more sensitive and specific in detecting IgM and immunoglobulin G antibodies but requires pooling of samples which may hold up the diagnosis.^[1] IFA is 'gold standard' technique but it has its own drawbacks which include requirement of well-trained personnel and equipment, controversial cut-off antibody titre, subjective determination of results.^[1],[8]

Although various serological tests are available their diagnostic sensitivity and specificity is still a matter of debate. In this study, comparative evaluation of the diagnostic capabilities of three serological assays, i.e. Weil-Felix test, RIT, ELISA was done against gold standard IFA test which in turn help in providing definitive and timely results to clinicians.

Materials and Methods

This is an observational cross-sectional study conducted at a 1024 bedded tertiary care hospital in Western part of Uttar Pradesh, India, over a period of 3 years from April 2017 to March 2020. Ethical approval was taken by institutional ethical committee.

Inclusion criteria

Patients aged >15 years with undiagnosed acute febrile illnesses with clinical suspicion of scrub typhus (with or without Acute renal failure and Acute respiratory failure) were included in the study.

Exclusion criteria

Patients diagnosed with other common febrile illness i.e. malaria, dengue, leptospirosis, chikungunya and enteric fever were excluded from the study.

Blood samples were collected from all the patients and serum was separated by centrifugation at 3000 rpm for 5 min. Serum samples were stored at −20°C if not used immediately, till further use. All the samples were subjected to the following serological tests.

Weil–Felix test

The Weil-Felix Proteus agglutination assay (OX-19, OX-2, OX-K strain agglutination), (Progen^TM Tulip diagnostics, Pvt. Ltd.,) was performed on all sample according to the manufacturer's instructions by diluting each serum 1/40–1/640. A single Weil-Felix titre 1:160 was considered as a positive test.

Rapid diagnostic test

Rapid diagnostic test (RDT) was applied on all the serum samples. RDTs assays were manufactured by InBios International, Inc., Seattle, USA, using a mixture of recombinant 56-kDa proteins from different O. tsutsugamushi serotypes being coated on Immunochromatographic strips.

Immunoglobulin M enzyme-linked immunosorbent assay for scrub typhus

IgM ELISA for Scrub Typhus was performed on all the serum samples to detect IgM antibodies against recombinant p56 KDa antigen of O. tsutsugamushi. The kit was procured from InBios International (Seattle, WA, USA) and test was performed according to the manufacturer's instruction. Serum samples from thirty healthy controls were also subjected to IgM ELISA. The optical density (OD) was measured at 450 nm and the results recorded as positive or negative. The cut-off value was calculated as per the kit protocol, by adding three times of standard deviation (SD) (0.047) to the average of OD (0.111) of healthy individuals, i.e. (0.111 + 3SD [0.047]). The OD OD.35 was considered as positive.

Immunoglobulin M immunofluorescence assay

An indirect fluorescence immunoassay (Fuller labs, California, U. S. A.) was performed to detect IgM against O. tsutsugamushion all samples positive by any of the above diagnostic test. In order to check the sensitivity of ELISA, samples with OD values between 0.25 and 0.35 were also cross-checked by IgM IFA.

The IFA slides in kit had 4 strains: Boryong, Gilliam, Karp and Kato. Serum samples of all the patients were put at dilutions of 1:32, 1:64, 1:128, 1:256, 1:512 and 1:1024. Slides were examined at × 400 magnification. Samples were regarded as positive when fluorescent, short; pleomorphic rod forms appeared in any of the 4 antigen dots.

Quality control

Positive results are indicated when fluorescence intensity was equal to or greater than the positive control. The titre of o1:64 was taken as positive. Negative results were reported when the sera did not fluoresce at a dilution of 1: 64.

Statistical analysis

Statistical analysis was done by IBM SPSS version 20.0 (SPSS Inc. Chicago, USA). The sensitivities, specificities, positive predictive values (PPV), negative predictive values (NPV) of the serological tests were calculated using MedCalc for Windows, version 18.11.3 (MedCalc, Software, Ostend, Belgium). Diagnostic accuracy was calculated using Youden's index.

Results

Total of 414 serum samples of febrile patients along with 30 healthy controls were tested by Weil-Felix, RDT, ELISA for scrub typhus. Among these samples tested 112 (27%) were positive by ELISA (OD (0.35) while 32 (7.7%) and 44 (10.6%) cases were positive by Weil-Felix and RDT, respectively.

All the samples which were positive by any of the above test were tested by IgM IFA. Of these positive samples, only 98 (23.6%) patient samples showed a significant titre of o1:64 while 30 healthy control serum showed titres below 1:32 (insignificant). Among the 112 ELISA-positive samples, 14 (12.5%) showed titre positivity at titre of 1:32 while the remaining 98 (87.5%) showed significant titres ranging from 1:64 to 1:1024. The frequency of IFA titres among ELISA positive samples is shown in [Table 1]. For better analysis of ELISA ODs (equivocal), samples showing OD value above 0.25 were tested by IgM IFA. None of the samples between OD 0.25 and 0.35 in ELISA showed positivity in the tested titres in IgM IFA.

All the 44 RDT-positive samples were positive by ELISA as well as IgM IFA. Whereas, among the 32 samples positive by Weil-Felix, only 26 (81.2%) showed significant titres in IgM IFA. All 26 cases were also positive by RDT and ELISA. Remaining 6 (18.7%) were negative by all the other tests.

Table 1: Frequency of immunofluorescence assay titres of enzyme-linked immunosorbent assay positive cases

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Considering IgM IFA as the gold standard diagnostic test, sensitivity and specificity of IgM ELISA was100 (95% confidence intervals [CI]: 96.31–97.88) and 96.05 (95% CI: 93.34–97.88), respectively. The specificity of RDT was very high 100% (95% CI: 98.84–100) but it had a low sensitivity of 64.47 (95% CI: 56.31–72.06) However, the sensitivity and specificity of Weil-Felix was low as compared to other tests, i.e. 57.65 (95% CI: 49.85–65.18) and 98.14 (95.99–99.31) respectively, as 6 samples turned out false positive by Weil-Felix [Table 2]. Among the different tests included in the study, RDT, ELISA were specific. The diagnostic accuracy of Weil-Felix, RDT and ELISA test using Youden's index was 0.56, 0.64 and 0.96, respectively.

Table 2: Performance of different serological tests in diagnosis of scrub typhus

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Comparison of immunoglobulin M enzyme-linked immunosorbent assay with gold standard immunofluorescence assay

[Figure 1], shows the distribution of OD values of positive ELISA cases against MIF titres. Receiver operating characteristic (ROC) curve of OD values plotted against MIF titre showed a significant correlation (AUC: 0.993). Increasing IFA titres correspond significantly to the increasing OD values of ELISA test (P < 0.001). Considering OD value of 0.35 as cut off, ROC curve revealed 99% sensitivity and 97% specificity of ELISA whereas at OD value of 0.4 as cut off sensitivity decreases to 94% with specificity of 98%. On further increasing the cut off OD, sensitivity of ELISA decreases further. On analysis of association between OD values and IFA titres, Spearman correlation coefficient was 0.783 (P < 0.0001) while Pearson correlation coefficient r²⁼ 0.879(P < 0.0001).

Figure 1: Distribution of optical density values of enzyme-linked immunosorbent assay positive cases plotted against immunofluorescence assay titres

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Discussion

Scrub typhus is an established cause of acute febrile illness in India. It is essential to promptly diagnose scrub typhus, to reduce associated morbidity and mortality. Among the various diagnostic tests, Weil-Felix is cheap and affordable tool in the diagnosis of scrub typhus. It is widely used in India.^[7] However, its effectiveness is always questionable because of the non-rickettsial origin of antigens used in this test. National Centre for Disease Control (NCDC) report on a comparative evaluation of Weil-Felix test and IgM ELISA showed Weil-Felix test to be equally sensitive with 89% specificity.^[9] In our study, sensitivity (57.65%) was very poor with specificity of 98.4% at titre of 1:160. It is the only test in our study which gave false-positive results. Venkategowda 2015 reported 100% specificity of Weil-Felix with 30% sensitivity at titres even <1:80.^[10] Issac 2004 reported variable sensitivities and specificities with decreasing cut-off of Weil-Felix titre.^[11] In our study also, diagnostic sensitivity of Weil-Felix test would have been better if we had taken lower cut-off titre. But specificity would have compromised further on lesser cut-off titres. Others have also reported considerable specificity at this titre.^[12],[13] Reported sensitivity in our study at titre of 1:160 is quite high as compared to other authors, who reported 30% sensitivity.^[11],[14] However, Koraluru et al reported even higher sensitivities than our study.[13]

Other tests such as a rapid immunochromatographic flow assay have become trendy because of their prompt result, less requirement of expertise and ease of performance even in rural settings. They can be used as a point of care (POC) test, especially in hospitals catering rural population as scrub typhus is mainly a rural disease.^[15] We have evaluated the performance of commercially available Scrub Typhus Detect IgM Rapid test, InBios International, assay with incorporated recombinant 56 KDa protein antigens. A recent study has reported the satisfactory performance of Inbios RDT with sensitivity 99.2%, specificity 93.0% and NPV of 98.77%.^[16] However, sensitivity and specificity in our study was 64.47% and 100% respectively with NPV of 85.41%. We observed the positivity of Inbios RDT only in cases with higher OD values in ELISA (OD >1.5) while RDT did not detect the samples with lower ODs. Despite the better specificity, we cannot recommend its use as POC test because of more false-negative reports in our study. There are reports illustrating the variable sensitivities of RDT from different manufacturer.^{[15],[16],[17]} Variation in sensitivities of RDT and Weil-Felix test could be attributable to disparity in incorporated antigens and strains and circulating strains. Knowledge of circulating serotypes in the different geographical areas of the country is thus important for better utilisation of RDTs.^[15]

ELISA detected 112 positives among 414 tested sera at OD >0.35. When compared against gold standard IFA (98 positive sera at >1:64 titre), sensitivity and specificity of IgM ELISA was 100% (95% CI: 96.31–97.88) and 96.05% (95% CI: 93.34–97.88) with PPV and NPV of 88.29% (81.57–92.78) and 100%, respectively. It is a general rule that the performance of any test is to be compared against gold standard assuming that it has perfect sensitivity and specificity. Despite, IFA being considered gold standard, there are studies highlighting its limitation in diagnosis of scrub typhus.^{[5],[15],[18]}

In our study, the performance of IgM ELISA with cut-off titre of 0.35 OD is marginally comparable to IgM IFA. As ELISA detected 14 more cases (112 ELISA cases vs. 98 IFA cases) among febrile patients and none among healthy sera, we considered these to be true positives because of their clinical profile and improvement on recommended antibiotic therapy. In such a case, ELISA performed better than IFA in the detection of scrub typhus. A recent study using latent class analysis also highlighted the better performance of ELISA than IFA.^[15],[19] ROC curves and correlation of OD values with IFA titres(Spearman correlation coefficient was 0.783 [P < 0.0001] while Pearson correlation coefficient r²=0.879 [P < 0.0001]) also showed comparable results. Satisfactory and legitimate performance of IgM ELISA has also been highlighted by other authors.^{[20],[21],[22]} Also recent guidelines by DHR-ICMR has recommended IgM ELISA to be the most sensitive test at cutoff OD 0.5.^[7] However, we had taken the diagnostic cut-off titre (0.35 OD) which was derived using serum of healthy volunteers to eliminate the background levels of antibodies. Researchers have recommended that geographically specific diagnostic cut-off should be determined for utilisation of IgM ELISA as an alternative diagnostic serological test.^[21],[22]

IFA being a reference standard for diagnosis cannot be used routinely due to the cost of kits, equipment, subjectivity and requirement of technical expertise. Weil-Felix has low and variable sensitivity and specificities whereas RDT despite of higher specific, its sensitivity is questionable as it detected only strongly positive cases with higher OD values and higher IFA titres. Recent DHR-ICMR guidelines also disconsolated on the use of ICTs in scrub typhus diagnosis.[7]

Conclusion

ELISA turned out to be a better diagnostic tool than any other serological tests available for diagnosis of scrub typhus, be it cost, expertise and equipment. The use of RDT as POC test is still questionable and need to be explored.

Acknowledgements

Authors are thankful to SERB, DST for providing the funds. Authors are also thankful to Mr, Sanjay Sharma, Department of Microbiology for helping with the technical Work.

Financial support and sponsorship

This work was funded by grants from SERB, Department of Science and Technology (DST), INDIA under the Early Career Research Advancement Scheme.

Conflicts of interest

There are no conflicts of interest.

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