Asian Journal of Transfusion Science
Home About Journal Editorial Board Search Current Issue Ahead of print Back Issues Instructions Subscribe Login  Users: 213 Print this page  Email this page Small font sizeDefault font sizeIncrease font size 


 
CASE REPORT Table of Contents   
Year : 2018  |  Volume : 12  |  Issue : 2  |  Page : 165-168
Comparative evaluation of enzyme-linked immunosorbent assay with rapid plasma reagin for screening of syphilis in blood donors


1 Department of Transfusion Medicine, PGIMER, Chandigarh, India
2 Department of Gastroenterology, PGIMER, Chandigarh, India
3 Department of Medical Microbiology, PGIMER, Chandigarh, India
4 Department of Blood Bank (Pathology), Kalpana Chawla Government Medical College, Karnal, Haryana, India

Click here for correspondence address and email

Date of Submission25-Sep-2017
Date of Acceptance04-Mar-2018
Date of Web Publication19-Dec-2018
 

   Abstract 

World Health Organization (WHO) recommends screening of syphilis in low prevalence populations of blood donors by treponemal tests like enzyme-linked immunosorbent assay (ELISA), whereas in India screening is done by rapid plasma reagin (RPR). The present pilot study evaluated the performance of ELISA compared to RPR, keeping Treponema pallidum hemagglutination assay as a reference test. ELISA was equally sensitive (100%), more specific (56.3% vs. 0%), more accurate (83.7% vs. 62.7%), had better positive predictive value (79.4% vs. 62.8%) and negative predictive value (100% vs. 0%), and less biological false positivity (37.2% vs. 20.6%) when compared to RPR. The WHO recommendations of screening for syphilis in low prevalence population of blood donors using ELISA may be adopted for usage in transfusion services that have the facility of ELISA.

Keywords: Biological false positivity, enzyme-linked immunosorbent assay, negative predictive value, positive predictive value, rapid plasma reagin, syphilis, Treponema pallidum hemagglutination assay, Venereal Disease Research Laboratory

How to cite this article:
Sachdev S, Sharma AK, Sethi S, Garg S, Lamba DS, Sharma RR, Marwaha N. Comparative evaluation of enzyme-linked immunosorbent assay with rapid plasma reagin for screening of syphilis in blood donors. Asian J Transfus Sci 2018;12:165-8

How to cite this URL:
Sachdev S, Sharma AK, Sethi S, Garg S, Lamba DS, Sharma RR, Marwaha N. Comparative evaluation of enzyme-linked immunosorbent assay with rapid plasma reagin for screening of syphilis in blood donors. Asian J Transfus Sci [serial online] 2018 [cited 2019 Jul 17];12:165-8. Available from: http://www.ajts.org/text.asp?2018/12/2/165/247981





   Introduction Top


In India, Drugs and Cosmetics Act of 1940 and the Rules of 1945 as amended from time to time, mandate screening for syphilis in blood donors by Venereal Disease Research Laboratory (VDRL) test.[1] The World Health Organisation (WHO) however, recommends screening of syphilis in blood donors by treponemal tests like enzyme linked immunosorbent assay (ELISA).[2] VDRL and rapid plasma reagin (RPR) are nontreponemal tests and detect antiphospholipid antibodies against the lipids released from damaged host cells and the lipid in the cell wall of treponeme.[2] These tests are simple to perform, are rapid, inexpensive and indicate active infection. But these tests have certain limitations. These tests are less specific and may show false negative (FN) results due to prozone phenomenon.[3],[4] The print-outs of results are not available for documentation purpose and the ratio of VDRL antigen and test serum can vary especially while testing large number of samples in high throughput blood banks. Another problem with these tests is the phenomenon of biological false positivity (BFP).[3],[4] Treponemal tests like ELISA detects human antibodies against Treponema pallidum and are mainly used as confirmatory tests to verify the reactivity seen in nontreponemal tests. These tests are more specific, provide objective printout of test results and the testing platform is amenable to automation.[3],[4] ELISA test runs may be subjected to quality control by using in house and external controls. Calculating E ratios and plotting Levy-Jennings charts helps in tracking intra and inter assay variations.

Keeping in view the WHO recommendation for syphilis screening, this pilot study was conducted to evaluate the performance of ELISA as a screening tool for screening of syphilis in blood donors compared to RPR, keeping T. pallidum hemagglutination assay (TPHA) as reference test.


   Materials and Methods Top


This study was conducted after taking approval from Institutional Ethical Committee of the institute. In phase 1 (one), we conducted simultaneous screening of 600 consecutive donor serum samples by ELISA and RPR, in order to evaluate the technical performance of ELISA at our centre. In phase 2, repository of 43 repeat reactive samples (on two RPR kits) was tested on ELISA kits at Department of Transfusion Medicine and Gastroenterology Virology (GE Virology) independently. TPHA was done at Department of Medical Microbiology. All those samples which initially came negative by TPHA were again repeated and reconfirmed. The study methodology was designed after reviewing the literature.[3],[4],[5],[6],[7],[8],[9] RPR test was performed using Carbogen (Tulip Diagnostics (P) Ltd., India) and RPR test (Span, Arkray Healthcare Private Limited, India). ELISA was done using ERBALISA SYPHILIS kits (Transasia Bio-medicals Ltd., India) which detect total (IgG, IgM, IgA etc.) antibodies using a mixture of recombinant Treponemal proteins coated on microwells. TPHA was done using IMMUTREP TPHA (Omega Diagnostics Limited, U.K). All tests were performed strictly as per manufacturer's instructions.

The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), accuracy, and the percentage of false positive (FP) results obtained by RPR and ELISA were calculated keeping TPHA as a reference test.

True positive (TP) is when the test results are reactive; if the disease is present i.e., TPHA is positive.

True negative (TN) is when the test results are nonreactive; if the disease is not present i.e., TPHA is negative.

FN is when the test results are nonreactive; if the disease is present i.e., TPHA is positive.

FP is when the test results are reactive; if the disease is not present i.e., TPHA is negative.

Sensitivity is defined as the probability that a test result will be positive when the disease is present (TP rate).

Sensitivity = TP/TP + FN

Specificity is defined as the probability that a test result will be negative when the disease is not present (TN rate).

Specificity = TN/TN + FP

PPV is defined as the probability that the disease is present when the test is positive.

PPV = TP/TP + FP

NPV is defined as the probability that the disease is not present when the test is negative.

NPV = TN/TN + FN

Accuracy = TP + TN/total number of samples

BFP = Total number of FP results/total positive results given by the test under evaluation

Sensitivity, specificity, positive and NPVs were expressed as percentages for ease of interpretation. Their confidence intervals (CI) were “exact” Clopper-Pearson CI. All the statistical analysis in this study was done using MedCalc online diagnostic software (MEDCALC®, Acacialaan 22, Ostend, Belgium).


   Results Top


In phase 1, 600 consecutive donor serum samples which were nonreactive on RPR test were also nonreactive on ELISA.

In phase 2, 43 repeat reactive repository samples (reactive on two RPR kits) were screened by ELISA and confirmed by TPHA. On analysis TPHA gave the positive result in 27 serum samples while it was negative in 16 samples. ELISA gave reactive results on 34 samples and was nonreactive in 9 samples. The results are depicted in [Table 1].
Table 1: Comparison of results obtained on rapid plasma reagin and enzyme linked immunosorbant assay with Treponema pallidum haemagglutination assay

Click here to view


Based on the analysis of the above data; the sensitivity, specificity, PPV, NPV and accuracy of the RPR and ELISA were calculated keeping TPHA as a reference test, and the results are shown in [Table 2]. In the present study the sensitivity of the two methods was same, ELISA was more specific, had better PPV and NPV, and was more accurate when compared to RPR as a screening method. The results of BFP obtained with RPR and ELISA are shown in [Table 3]. It was observed that BFP was more with RPR as compared to ELISA.
Table 2: Sensitivity, specificity, positive predictive value, negative predictive value and accuracy of rapid plasma reagin and enzyme linked immunosorbant assay compared to Treponema pallidum haemagglutination assay

Click here to view
Table 3: Biological false positivity with rapid plasma reagin and enzyme linked immunosorbant assay

Click here to view


The comparison of the various parameters assessed to evaluate ELISA and RPR with TPHA with other studies is represented in [Table 4].
Table 4: Comparison of enzyme linked immunosorbant assay and rapid plasma reagin in different studies

Click here to view


Optical density of each of the 43 repeat RPR reactive serum samples obtained by ELISA was divided by cut-off value and the signal to cut-off obtained was evaluated against the TPHA. receiver operating curve curves was applied to find out sensitivity, specificity, NPV, PPV (95% CI) for different values of signal to cut-off ratio. It was observed that the signal to cut-off ratio of 7.72 on ELISA, had a sensitivity of 100% (95% CI: 87.2%–100%), 93.75% specificity (95% CI: 69.8%–99.8%), PPV of 96.4% (95% CI: 81.7%–99.9%) and NPV of 100% (95% CI: 78.2%–100%) with Area under the curve of 99.3% (95% CI: 90.5%–100%) at P = 0.0001.


   Discussion Top


No single test is ideal for diagnosing syphilis since each test has some limitations in terms of sensitivity, specificity and the results of a particular test varies depending upon the stage of syphilis. The sensitivity of RPR in the present study was found to be comparable with ELISA at 100% while specificity was found to be 0% for RPR and 56.2% for ELISA. Saral et al. have reported a lower sensitivity of 58%, and a comparable specificity of 0% of RPR compared to TPHA.[5] The 0% specificity of RPR obtained in the present study has to be interpreted keeping in view that this is obtained in a population of blood donors and thus may not be extrapolated to other clinical settings where in clinical suspicion is present and the test is ordered based on the clinical suspicion. Naidu et al. reported the sensitivity of RPR as 70% and specificity as 54.28%.[6] The PPV of ELISA in the present study was 79.4% is lower than 99.3% reported by Woznicová and Valisová.[9] Whereas the PPV of RPR in the present study was 62.8% which is lower than 92% reported by Saral et al.[5] The NPV of ELISA in the present study was 100% comparable with 97.2% reported by Woznicová and Valisová.[9] Further the 0% NPV of RPR obtained in the present study has earlier been also reported by Saral et al.[5] The accuracy of ELISA in the present study was 83.7% compared to 62.7% of RPR. Saral et al. have reported accuracy of 56% with RPR.[5] Previous studies by Young et al. using wild type of treponemal pallidum antigens reported the sensitivity of ELISA to be 98.4% and specificity of 99.3% compared to TPHA and fluorescent treponemal antibody absorption.[10] Young et al. while using recombinant antigen of treponema reported sensitivity of ELISA to be 99% and specificity of 91.4%.[11] A comparative evaluation of ten different enzyme immunoassay using wild or recombinant antigens by Cole et al. the sensitivity of ELISA was found to vary between 94.7%-99.1% while specificity was 100%.[12]

The BFP obtained in the present study is more with RPR (37.2%) compared to ELISA (20.5%) with odds of 2.28 (0.81–6.44) at P = 0.13, which is much less than 56.4 % for RPR and 50% for ELISA reported by Naidu et al. from Mumbai, India.[6] BFP (short-term or long-term persistence) has been attributed to strong immunological stimulus like viral infections, systemic lupus erythematosis, thyroiditis, rheumatoid arthritis, atopic dermatitis, vaccination, or may occur even in apparently healthy people with advancing age.[3],[13],[14]

Importantly the characteristic of treponemal tests like ELISA of test being reactive despite treatment could be utilized to significant advantage in blood donor screening as a surrogate marker of high risk behavior of the blood donor at any point of time during the lifetime. This is not the case when using nontreponemal tests like VDRL/RPR, which usually turn negative after successful treatment. Therefore a negative treponemal screening test could help blood bank to select a low risk donor. Further keeping in view that for blood donors screening a test of high sensitivity and high specificity is recommended, the preliminary results of the present study of very low specificity of RPR do not support the usage of RPR in blood donor setting. It may be reemphasized that quality control practices can be put in place by using ELISA and this will ensure compliance with accreditation and good manufacturing and laboratory practices.

In the present study, at a signal to cut-off ratio value of 7.72 or above on ELISA, it was found that the possibility of the test result was more likely to be TP, though this value may be limited by the main limitation of the present pilot study in terms of sample size. The other limitation of the present study was the inability to detect FNs on RPR testing because the study utilized a repository of stored repeat reactive RPR samples. Further the specificity obtained for RPR in the study could be biased by the inclusion of samples from apparently healthy blood donors without and clinical symptomatology. However the results support the need to evaluate the efficacy of ELISA as a screening tool in blood donors on a large scale preferably including different geographical regions.


   Conclusion Top


ELISA was equally sensitive (100%), more specific (56.3% vs. 0%), more accurate (83.7% vs. 62.7%), had better PPV (79.4% vs. 62.8%) and NPV (100% vs. 0%), and less BFP (37.2% vs. 20.6%) when compared to RPR in the present evaluation. The WHO recommendations of screening for syphilis in low prevalence population of blood donors using ELISA may be adopted for usage in transfusion services that have facility of ELISA.

Acknowledgment

The authors acknowledge the contribution of M/S Transasia Bio-medicals Ltd., for the ELISA kits.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Ministry of Health and Family Welfare, Government of India. The Drugs and Cosmetics Act, 1940 and the Drugs and Cosmetics Rules, 1945. Schedule F. Part XIIB; 2005. Available from: http://www.cdsco.nic.in/Drugs&CosmeticAct.pdf. [Last accessed on 2016 Dec 27].  Back to cited text no. 1
    
2.
World Health Organization. Screening Donated Blood for Transfusion Transmissible Infections: Recommendations; 2010. Available from: http://www.who.int/bloodsafety/ScreeningDonatedBloodforTransfusion.pdf. [Last accessed on 2016 Dec 27].  Back to cited text no. 2
    
3.
Ratnam S. The laboratory diagnosis of syphilis. Can J Infect Dis Med Microbiol 2005;16:45-51.  Back to cited text no. 3
    
4.
Seña AC, White BL, Sparling PF. Novel Treponema pallidum serologic tests: A paradigm shift in syphilis screening for the 21st century. Clin Infect Dis 2010;51:700-8.  Back to cited text no. 4
    
5.
Saral Y, Dilek AR, Dilek N, Bahçeci I, Ulusan DZ. Serologic diagnosis of syphilis: Comparison of different diagnostic methods. Acta Dermatovenerol Croat 2012;20:84-8.  Back to cited text no. 5
    
6.
Naidu NK, Bharucha ZS, Sonawane V, Ahmed I. Comparative study of treponemal and non-treponemal test for screening of blood donated at a blood center. Asian J Transfus Sci 2012;6:32-5.  Back to cited text no. 6
  [Full text]  
7.
Castro R, Prieto ES, Santo I, Azevedo J, Exposto Fda L. Evaluation of an enzyme immunoassay technique for detection of antibodies against Treponema pallidum. J Clin Microbiol 2003;41:250-3.  Back to cited text no. 7
    
8.
Schmidt BL, Edjlalipour M, Luger A. Comparative evaluation of nine different enzyme-linked immunosorbent assays for determination of antibodies against Treponema pallidum in patients with primary syphilis. J Clin Microbiol 2000;38:1279-82.  Back to cited text no. 8
    
9.
Woznicová V, Valisová Z. Performance of CAPTIA SelectSyph-G enzyme-linked immunosorbent assay in syphilis testing of a high-risk population: Analysis of discordant results. J Clin Microbiol 2007;45:1794-7.  Back to cited text no. 9
    
10.
Young H, Moyes A, McMillan A, Robertson DH. Screening for treponemal infection by a new enzyme immunoassay. Genitourin Med 1989;65:72-8.  Back to cited text no. 10
    
11.
Young H, Moyes A, Seagar L, McMillan A. Novel recombinant-antigen enzyme immunoassay for serological diagnosis of syphilis. J Clin Microbiol 1998;36:913-7.  Back to cited text no. 11
    
12.
Cole MJ, Perry KR, Parry JV. Comparative evaluation of 15 serological assays for the detection of syphilis infection. Eur J Clin Microbiol Infect Dis 2007;26:705-13.  Back to cited text no. 12
    
13.
Wicher K, Horowitz HW, Wicher V. Laboratory methods of diagnosis of syphilis for the beginning of the third millennium. Microbes Infect 1999;1:1035-49.  Back to cited text no. 13
    
14.
Larsen SA, Steiner BM, Rudolph AH. Laboratory diagnosis and interpretation of tests for syphilis. Clin Microbiol Rev 1995;8:1-21.  Back to cited text no. 14
    

Top
Correspondence Address:
Suchet Sachdev
Department of Transfusion Medicine, PGIMER, Chandigarh
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ajts.AJTS_126_17

Rights and Permissions



 
 
    Tables

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



 

Top
 
  Search

  
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Email Alert *
    Add to My List *
* Registration required (free)  


    Abstract
   Introduction
    Materials and Me...
   Results
   Discussion
   Conclusion
    References
    Article Tables

 Article Access Statistics
    Viewed812    
    Printed46    
    Emailed0    
    PDF Downloaded6    
    Comments [Add]    

Recommend this journal

Association Contact us | Sitemap | Advertise | What's New | Feedback | Copyright and Disclaimer

2006 - Asian Journal of Transfusion Science | Published by Wolters Kluwer - Medknow
Online since 10th November, 2006