|Year : 2019 | Volume
| Issue : 1 | Page : 69-73
Performance evaluation of VITROS syphilis Treponema pallidum agglutination assay in blood donor samples by using centers for disease control and prevention reverse sequence algorithm
Amardeep Pathak1, Manushri Sharma2, Narender Tejwani3, Anurag Mehta4
1 Department of Transfusion Medicine, Rajiv Gandhi Cancer Institute and Research Centre, New Delhi, India
2 Department of Biochemistry, Lady Hardinge Medical College, New Delhi, India
3 Lab Services, Rajiv Gandhi Cancer Institute and Research Centre, New Delhi, India
4 Lab and Transfusion Services, Rajiv Gandhi Cancer Institute and Research Centre, New Delhi, India
|Date of Web Publication||22-Apr-2019|
Dr. Amardeep Pathak
Department of Transfusion Medicine, Rajiv Gandhi Cancer Institute and Research Centre, New Delhi
Source of Support: None, Conflict of Interest: None
Background: VITROS syphilis Treponema pallidum agglutination (VSTPA) assay is a treponemal assay for the detection of antitreponemal antibody in the human serum or plasma using enhanced chemiluminescence technology. Centers for Disease Control and Prevention (CDC) has offered reverse sequence algorithm for screening syphilis. Aim and Objective: This study was undertaken to evaluate the performance of VSTPA assay over the nontreponemal (rapid plasma regain [RPR]) as well as treponemal assay based on immunochromatography principle, which is commonly used in blood transfusion centers, using CDC reverse sequence algorithm. Material and Methods: A total number of 33,367 donor samples were screened for the syphilis infection using either VSTPA assay or syphilis rapid card test and the syphilis reactivity rate was compared between both assays. Results: While screening with syphilis rapid card test, the percentage of reactivity observed was 0.36% whereas with VSTPA assay, the reactivity was increased to 0.87%. As per the CDC reverse sequence algorithm, the seroreactive samples in VSTPA assay were subjected to nontreponemal RPR test. The discordant samples which showed “nonreactive” in RPR assay were sent for syphilis confirmatory test based on fluorescent treponemal antibody absorption (FTA-ABS) test. In the reverse algorithm study, 40% of samples with discordant results were confirmed as “Positive” in FTA-ABS test confirming the superior sensitivity of VSTPA assay over nontreponemal RPR assay as well as treponemal syphilis rapid card test. Conclusion: Automation of syphilis screening using VSTPA assay helps in consolidation of the assay with other transfusion transmittable infections, namely, HIV, HBsAg, and anti-HCV screening assay on chemiluminescence platform which is highly valuable for optimizing workflow, efficiency with excellent sensitivity, and reliable specificity.
Keywords: Centers for Disease Control and Prevention, nontreponemal test, reverse sequence algorithm, syphilis, treponemal test, VITROS Syphilis Treponema pallidum agglutination
|How to cite this article:|
Pathak A, Sharma M, Tejwani N, Mehta A. Performance evaluation of VITROS syphilis Treponema pallidum agglutination assay in blood donor samples by using centers for disease control and prevention reverse sequence algorithm. Glob J Transfus Med 2019;4:69-73
|How to cite this URL:|
Pathak A, Sharma M, Tejwani N, Mehta A. Performance evaluation of VITROS syphilis Treponema pallidum agglutination assay in blood donor samples by using centers for disease control and prevention reverse sequence algorithm. Glob J Transfus Med [serial online] 2019 [cited 2019 Sep 16];4:69-73. Available from: http://www.gjtmonline.com/text.asp?2019/4/1/69/256754
| Introduction|| |
Syphilis is a sexually transmitted (major route) and a transfusion-transmissible (other route) bacterial infection caused by the spirochete Treponema pallidum. It is mandatory to do serological test for syphilis on all donor blood samples. The serological tests for screening syphilis infection currently available are classified according to the antigens used – non treponemal tests, namely, Venereal Disease Research Laboratory (VDRL), rapid plasma reagin (RPR), and TRUST in which cardiolipin-lecithin antigen are used and treponemal tests namely, immunochromatography card test, enzyme-linked immunosorbent assay, chemiluminescence immunoassay, T. pallidum hemagglutination (TPHA), T. pallidum particle agglutination, and fluorescent treponemal antibody-absorption (FTA-ABS) test in which Treponema-specific antigen are used.
The limitations of nontreponemal tests when compared to treponemal tests are its lesser sensitivity and specificity. Further, these tests cannot be automated. Among the treponemal test, a TPHA test has got its own limitations in terms of sensitivity and specificity. TPHA tests are not sensitive enough to detect primary syphilis. Further, the animal erythrocytes used in the assay may show nonspecific agglutination with high titers of heterophilic antibodies.
In addition, batch-to-batch variation was observed from the same manufacturer of TPHA assay. These tests cannot be automated and manual tastings are time-consuming and results interpretation may be subjective. FTA-ABS tests are considered as confirmatory test for syphilis infection and hence they cannot be used for routine screening.
Simple rapid, point-of-care type Treponema-specific card tests based on immunochromatography principle are currently available commercially. However, the rapid card tests have the limitations of sensitivity as well as specificity. Herring et al. in a multicenter evaluation of nine rapid card tests reported the sensitivity of rapid card tests in the range of 84.5%–97.7% and the specificity ranging from 84.5% to 98%. Moreover, the result interpretation in rapid card test was not easy due to the personal subjectivity. In transfusion medicine set up, for transfusion safety, it is very important to use high-sensitive assay for the screening of infectious diseases to minimize the so-called false-negative results, thereby ensure the blood safety.
Enhanced chemiluminescence technology-based syphilis screening assay (VITROS Syphilis TPA assay) for the detection of both immunoglobulin M (IgM) and IgG antibodies against Treponema-specific antigens is currently available which can be automated in VITRO S immunodiagnostics system along with other infectious disease screening assays, namely, HIV, HBsAg, and anti-HCV assay. Enhanced chemiluminescence technology is known for its excellent sensitivity and specificity which helps in detecting the syphilis infection in the early phase as well as in the latent phase of the infection, thereby enhancing the safety of blood and blood components for transfusion.
Centers for Disease Control and Prevention (CDC), Atlanta, United States of America offered reverse sequence algorithm [Figure 1] for the screening of syphilis infection which helps in minimizing biologic false-reactive results. As per this algorithm, Treponema-specific test is used for initial screening to detect early infection as well as treated infection. Nontreponemal test will be performed on the initial reactive samples to detect active infection.
In this study, we adopted a reverse sequence of screening syphilis infection in blood donor samples in which a Treponema-specific assay based on enhanced chemiluminescence technology was performed first followed by testing all the reactive sera using nontreponemal test based on RPR method. Further, we compared the seroreactivity observed when we performed syphilis rapid card test and VSTPA assay and confirm the reactivity of discordant samples with syphilis confirmatory tests syphilis FTA-ABS assay.
| Materials and Methods|| |
Serum samples: This study was conducted in the department of Transfusion Medicine, Rajiv Gandhi Cancer Institute and Research Centre, a Tertiary Care Cancer Hospital, in Delhi. A total number of about 33,367 donor samples collected in the period of Jan 2014 to Dec 2017 were analyzed for the syphilis reactivity, retrospectively. The demographic details of the donor population are shown in [Table 1]. Tests for anti-lipoidal antibodies such as VDRL slide flocculation test and the Kahn standard test were not performed for as they were not the part of CDC syphilis reverse algorithm.
In the Phase I study between Jan 2014 to June 2015, all the donor samples of about 13,660 were screened for the presence of T. pallidum-specific total antibodies (IgM, IgG, and IgA) using SD BIOLINE Syphilis 3.0 (SD Biostandard Diagnostics Private Limited, Gurgaon, Haryana, India), a rapid card test based on immunochromatography principle. The serum samples were separated from the blood samples collected in red-top vacutainer after coagulation by centrifugation at 3500 RPM for about 20 min. After processing the samples as per the manufacturer's recommended protocol, the interpretation of the results was done by two laboratory technologists to minimize the personnel subjectivity. The results interpreted by both the technologists as per the manufacturer's recommendations were recorded in Microsoft Excel sheet and reviewed. If there is any discordant result as per the interpretation by both technologists, the sample was retested and interpreted.
In the Phase 2 study, from July 2015 to December 2017, the donor samples of about 19,707 were screened for the presence of T. pallidum-specific total antibodies using VITROS Syphilis TPA assay based on enhanced chemiluminescence technology as per the manufacturer's recommended protocol in VITRO S ECiQ immunodiagnostics system. All the samples that showed “Initial reactive” or “borderline reactive” (above the cutoff – 1.2 S/Co) were retested after recentrifugation of the sample or using fresh sample to rule out any preanalytical errors. All the results were recorded in the Excel sheet and reviewed.
In the Phase 3 study, according to reverse sequence screening algorithm, samples that are positive by the enzyme immunoassay (EIA)/TPA screening test, but negative by RPR should then be tested by a second treponemal assay for confirmation such as TPHA or FTA-ABS. Hence, from January 2017 to December 2017, all the repeat reactive samples were subjected to nontreponemal RPR test as per the manufacturer's instructions. The obtained results in RPR assay were interpreted by two laboratory technologists to minimize the personnel subjectivity. All the samples with discordant results, that is, reactive by VITROS Syphilis TPA assay and nonreactive in RPR assay were sent for syphilis confirmatory test based on FTA-ABS test to Quest Diagnostics India Pvt. Ltd. The obtained results were recorded in the Excel sheet and reviewed with all the results.
Based on the obtained results as per the CDC – reverse screening algorithm, the sensitivity and specificity of VITROS Syphilis TPA assay was calculated based on the true reactive (reactive in both VITROS Syphilis and Syphilis RPR or Syphilis FTA-ABS test) and false reactive (reactive only in VITRO S Syphilis TPA assay and nonreactive in Syphilis RPR and Syphilis TPA-ABS test).
| Results|| |
In the Phase 1 study, out of 13,660 samples screened, 49 (0.36%) were found reactive by SD Bioline one-step Syphilis 3.0 anti-TP rapid card test.
In the Phase 2 study, out of 19,707 samples screened using VITROS Syphilis TPA assay, 172 (0.87%) were found to be reactive [Table 2]. Among the samples which are found to be “Reactive” for Syphilis antibody, 213 (96%) were from male donors and 08 (3.6%) were from female donors. Further, 66 (29.8%) of reactivity was observed in the first-time donors and 155 (70.2%) of reactivity was observed in repeat donors.
|Table 2: Syphilis reactive rate in SD Bioline syphilis 3.0 rapid card test and VITROS syphilis Treponema pallidum agglutination assay|
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In the Phase 3 study, out of 6831 samples screened between January 2017 and December 2017, 73 (1.06%) were found to be reactive in VITRO S Syphilis TPA assay. All the 73 reactive samples were subjected to nontreponemal RPR assay as per the CDC reverse algorithm. Out of 73 samples screened for anti-lipoidal antibody, 36 samples (49.3%) showed the presence of anti-lipoidal antibodies and about 37 samples (50.7%) showed discordant results.
All these discordant samples (n = 37) were subjected to FTA-ABS test at Quest Diagnostics. In FTA-ABS test, 15 out of 37 discordant samples (40.54%) were confirmed as “Positive” indicating higher sensitivity of VITROS Syphilis TPA assay based on enhanced chemiluminescence technology when compared to RPR assay [Table 3] and [Figure 2]. Further, 22 out of 37 discordant samples (59.46%) were found to be nonreactive in Syphilis FTA-ABS test, which may be false reactive in VITRO S Syphilis TPA assay.
|Figure 2: Results of rapid plasma regain and fluorescent treponemal antibody absorption tests on VITROS Syphilis-Treponema pallidum agglutination-positive samples|
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All those 15 discordant samples which were confirmed as “Positive” by FTA-ABS test were retested in VITRO S Syphilis TPA assay, RPR, and Syphilis rapid card test. All the 15 samples were repeat reactive in VITRO S Syphilis TPA assay and nonreactive in both RPR and Syphilis rapid card test. Based on the CDC – reverse Algorithm study using VITROS Syphilis TPA assay as primary screening test followed by RPR and confirmation of discordant samples by Syphilis FTA-ABS assay, the sensitivity of VITROS Syphilis TPA assay was found to be 100% (all the 51 true reactive samples) and the specificity of VITROS Syphilis TPA assay was found to be 99.68% (nonreactive 6758 out of 6780 samples). In this study, based on the reactivity data, the sensitivity of Syphilis RPR assay was found to be only 70.58% (Reactive 36 out of 51 reactive samples).
| Discussion|| |
Syphilis infection causes significant morbidity and is an important facilitator of HIV transmission. One of the greatest values of serological test for syphilis in blood donor population is it act as a surrogate marker for lifestyle known to be associated with high risk of HIV and HCV infection. Hence, syphilis testing is done as a marker for the detection of preseroconversion window phase infections of HCV, HBV, and HIV. Currently, in pretransfusion testing of blood and blood products, the nontreponemal tests of syphilis are widely used. However, they have several limitations. In early primary disease anti-lipoidal antibodies may not have developed and in late syphilis (late latent and tertiary) up to 30% of individuals may lack anti-lipoidal antibodies., The lack of sensitivity of nontreponemal tests such as RPR in detecting the syphilis infection in both early and late stages of syphilis infections may lead to false-negative reactions in these stages of infection which may impact the safety of blood and blood products for transfusion. In a comparative study between treponemal and nontreponemal assay for the screening of syphilis infection in donor blood samples, Naidu et al. reported that 21.9% of samples were false negative and 56.4% of results were biological false positive in RPR assay. Treponemal antibodies appear earlier than nontreponemal antibodies and usually remain detectable for life, even after resolving infection.
Rapid card tests based on the principle of immunochromatography can qualitatively detect the presence of IgG and IgM class of Treponema-specific antibodies in serum or plasma within 15 min. It is a simple, rapid, point-of-care-type Treponema-specific test suitable for use in primary health-care settings for the diagnosis of syphilis., In our study, in Phase 1, the seroreactivity of syphilis using rapid card test was 0.36%. When the screening for syphilis infection was done with enhanced chemiluminescence technology, the seroreactivity of syphilis was increased to 0.87% may be due to the enhanced sensitivity of VITROS Syphilis TPA assay. When compared to TPHA as reference method, the sensitivity and specificity of rapid card test was reported as 92.86% and 98.28%, respectively. Limitation in the sensitivity of rapid card tests has also been reported earlier and the reason cited for the lower sensitivity was primarily the low levels of antibodies found in the sera of patients with early primary syphilis. In our study, when all the FTA-ABS reactive samples but nonreactive in conventional RPR assay, screened in rapid card tests, all were reported as nonreactive indicating the lower sensitivity of rapid immunochromatography card tests. Because of its limitation in sensitivity, usage of rapid card tests in donor screening for syphilis infection may impact the safety of blood and blood components. Tiwari et al. reported that VITROS Syphilis TPA assay meets the requirements for its use as a screening assay for syphilis antibodies in blood donor serum or plasma samples to enhance the safety of the blood for transfusion.
According to the CDC, if a reverse sequence of screening is used, a specimen positive on EIA/CLIA should be further tested with a quantitative nontreponemal test (e.g., RPR or VDRL). If the subsequent nontreponemal test is negative, then a TP-PA should be used, with a positive result for TP-PA indicating positivity for syphilis infection, either past or present. In our study, instead of TP-PA, we used FTA-ABS test as FTA-ABS test was reported as more sensitive than either the TPHA or the TP-PA and is usually the first serologic test to become reactive, during the primary stage of the disease. In our study, all the reactive samples in VITRO S Syphilis TPA assay were tested in RPR nontreponemal assay. All the samples with discordant results (RPR negative) were subjected to FTA-ABS test. About 40.54% of the samples with discordant results were confirmed as true reactive in FTA-ABS test and these samples were also reported as nonreactive in rapid card test. The reverse algorithm was reported to be more sensitive than the traditional algorithm in detecting cases of latent syphilis, which were missed by the traditional screening algorithm. In populations with a low prevalence of syphilis, the reverse algorithm detected 140 samples with treponemal antibody that went undetected by the traditional algorithm. Our results showed the superior sensitivity of VITROS Syphilis TPA assay over RPR and rapid card test even though there is a limitation in the specificity of the assay. Based on the observed results, the specificity of VITROS Syphilis TPA assay was found to be 99.68%, against the manufacturer reported specificity of 99.98% and the relative sensitivity was found to be 100%.
| Conclusion|| |
VITROS ® Syphilis TPA assay based on enhanced chemiluminescence technology has shown excellent sensitivity in screening for syphilis infection and helps in minimizing false-negative results, thereby enhancing the safety of blood for transfusion. In this study, nontreponemal assays such as RPR assay as well as treponemal assay based on immunochromatography rapid card test showed false-negative results and may impact the safety of blood and blood components for transfusion. Introduction of VITROS ® Syphilis TPA assay in VITRO S ® ECiQ/3600Immunodiagnostics system helps in consolidation of the assay with other transfusion transmittable infections, namely, HIV, HBsAg, and a HCV screening assay on chemiluminescence platform which is highly valuable for optimizing workflow and efficiency.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]