|Year : 2017 | Volume
| Issue : 1 | Page : 29-33
Evaluation of nonspecific treponemal test rapid plasma reagin in comparison with specific treponemal test immunochromatographic assay for screening healthy blood donors
Aseem K Tiwari, Devi Prasad Acharya, Ravi C Dara, Dinesh Arora, Geet Aggarwal, Ganesh Singh Rawat
Department of Transfusion Medicine, Medanta - The Medicity, Gurgaon, Haryana, India
|Date of Web Publication||22-Mar-2017|
Aseem K Tiwari
Department of Transfusion Medicine, Medanta - The Medicity, Gurgaon, Haryana
Source of Support: None, Conflict of Interest: None
Introduction: Serological tests for syphilis (STS) contributed greatly to the detection of Treponema pallidum infection in blood donors and especially in those who were not identified during the medical selection. Most of the blood centers use nontreponemal tests due to lesser cost, ease of performance, and their ability to pick up early primary stage infection, despite reported high false positivity. However, increasingly large number of blood centers have begun testing with the treponemal tests such as immuno-chromatographic assay (ICA) which is possibly easier and more objective than rapid plasma reagin (RPR), albeit slightly expensive. It is with this background that we undertook a head-to-head comparison of nontreponemal test RPR with treponemal test ICA in over 10,000 consecutive blood donor samples with confirmation of all reactive and discordant samples by fluorescent treponemal antibody absorption assay (FTA-ABS) as the gold standard. Materials and Methods: The study was conducted in the department of transfusion medicine in a large tertiary care hospital in India. Consecutive blood donors from July 2014 to January 2015 were evaluated simultaneously for antitreponemal antibodies by solid phase ICA (SD BIOLINE Syphilis 3.0, Alere Medical Pvt. Ltd., USA), RPR (Immutrep RPR, Omega Diagnostics, Scotland, UK) and FTA-ABS (Biocientifica SA, FTA-ABS, Argentina). Performances of both the assays were evaluated in statistical terms. Results: A total of 10,200 donor samples were evaluated. There were 10,124 confirmed concordant negatives. Thirty-eight samples were concordant positive. Thirty-eight samples (thirty samples were ICA positive and RPR negative and eight samples were RPR positive and ICA negative) were discordant. The sensitivity of ICA was higher (93%) as compared to that of RPR (66%), whereas the specificity of RPR was higher (83%) than that of ICA (66%). Positive predictive value (PPV) of both the tests was similar while negative predictive value (NPV) of ICA was higher (86%) than that of RPR (60%). Youden's index was 0.81 for ICA whereas it was 0.62 for RPR. Conclusion: ICA has a better sensitivity than the RPR and thus seems to be more appropriate test for screening in blood donors.
Keywords: Immunochromatographic assay, rapid plasma reagin test, screening test for blood donors, serological test for syphilis
|How to cite this article:|
Tiwari AK, Acharya DP, Dara RC, Arora D, Aggarwal G, Rawat GS. Evaluation of nonspecific treponemal test rapid plasma reagin in comparison with specific treponemal test immunochromatographic assay for screening healthy blood donors. Glob J Transfus Med 2017;2:29-33
|How to cite this URL:|
Tiwari AK, Acharya DP, Dara RC, Arora D, Aggarwal G, Rawat GS. Evaluation of nonspecific treponemal test rapid plasma reagin in comparison with specific treponemal test immunochromatographic assay for screening healthy blood donors. Glob J Transfus Med [serial online] 2017 [cited 2020 Aug 6];2:29-33. Available from: http://www.gjtmonline.com/text.asp?2017/2/1/29/202717
| Introduction|| |
Syphilis is an infectious venereal disease caused by the spirochete Treponema pallidum (TP). Serological tests for syphilis (STS) have contributed greatly to the detection of TP infection in blood donors and especially in those who were not identified during the medical history screening. The STS is generally based on the detection of antibodies against TP antigens in blood using either specific or nonspecific reagents. In nonspecific treponemal tests, tests measure antibodies against a product (cardiolipin-lecithin-cholesterol) that is produced when TP interacts with human tissue. The most commonly used nontreponemal antibody tests are the rapid plasma reagin (RPR) and venereal disease research laboratory (VDRL) assay. Nontreponemal antibody tests can give false-positive tests since they are directed against cardiolipin-lecithin-cholesterol antigen. Any process in the body that produces this antigen can result in a positive test. The more specific or the treponemal tests measure antibodies directed at the pathogen, TP. The detection of specific treponemal antigens is possible using methods employing passive agglutination, such as TP hemagglutination (TPHA) assay or the TP particle agglutination assay; indirect immunofluorescence as the fluorescent treponemal antibody absorption (FTA-ABS) assay or enzyme immunoassay for the detection of specific immunoglobulin (Ig) G and IgM or total Ig. Qualitative detection of antibodies of all isotypes (IgG, IgM, and IgA) against TP can also be performed using recombinant TP antigen in immunochromatographic assay (ICA).
Most blood centers of our country routinely use nontreponemal tests for screening of blood donors due to cost-effectiveness and ease of performance. However, with nontreponemal tests, false-positive reactions can occur for many reasons, the most common of which is other infections, both viral and bacterial. Furthermore, these tests may show false negative result when the patient's antibody titer is very high due to a hook effect (also called a prozone effect). Thus, confirmation with a second treponemal test that is specific for TP antibodies is recommended. Increasingly large number of blood centers have begun testing with the specific treponemal tests such as ICA which is as easy or possibly easier as RPR, albeit slightly expensive. It is with this background that we decided to do a head-to-head comparison of nontreponemal test, RPR with treponemal test ICA in over 10,000 consecutive blood donor samples , with FTA-ABS as the gold standard to resolve the discordant samples.,
| Materials and Methods|| |
This was a prospective study conducted on consecutive blood donors from July 2014 to January 2015 in the department of transfusion medicine at a large tertiary care hospital in North India. All blood donors who met the criteria for whole blood donation laid down in Drugs and Cosmetic Act and provided the informed written consent for blood donation were included in the study. Any donor who failed to fulfill the criteria was deferred with appropriate counseling.
For the purpose of analysis, donors were classified on the basis of:
- Gender: Male or female
- Nature of donation: Voluntary (donor who is not acquainted or related to any patient and has donated the blood on his/her own free will) or replacement (donor who is family member or friend)
- Frequency of donation: Regular (donor who has donated at least twice in his/her lifetime and donated at least once in preceding 12 months) or occasional (either first time donor, or donor who has donated once before in his/her lifetime, or more than once but not in preceding 12 months).
Mandatory screening for transfusion-transmitted infection
All donors underwent mandatory testing for transfusion-transmitted infection (HIV, hepatitis B virus, hepatitis C virus [HCV], syphilis, and malaria). Anti-HIV, hepatitis B surface antigen, and anti-HCV were performed on VITROS 3600 (Ortho Clinical Diagnostics, USA) using enhanced chemiluminescence technique. Test for malaria (Microgene Diagnostics, India) and syphilis (Alere Diagnostics, USA) was done by solid phase ICA.
Study protocol for syphilis testing and confirmation
In addition to ICA, all consecutive blood donor samples were tested for antitreponemal antibodies by RPR also. All reactive (concordant) samples and any discordance between these two tests (ICA and RPR) was confirmed with FTA-ABS which was considered as the gold standard for this study. Concordant samples were those samples which were reactive for both the tests, that is, RPR and ICA. Discordant samples were those samples in which only one test was reactive and the other test was nonreactive.
ICA (SD BIOLINE Syphilis 3.0, Alere Medical Pvt. Ltd, USA) - SD BIOLINE Syphilis 3.0 test was a membrane-based solid phase ICA for the qualitative detection of antibodies of all isotypes (IgG, IgM, and IgA) against TP in serum, plasma, or whole blood.
RPR (Immutrep RPR, Omega Diagnostics, UK) - Immutrep RPR is nontreponemal flocculation test for the qualitative and semi-quantitative determination of reagin antibodies in serum or plasma. No visual flocculation indicated a negative result.
FTA-ABS (Biocientifica Inmunofluor FTA-ABS, Argentina): TP subspecies pallidum was fixed on a microscope slide. Serum from patients suspected of having syphilis because of previous positive ICA or RPR test was diluted 1:5 in sorbent (to reduce nonspecific cross-reactivity) and was layered on that slide. If the patient's serum contains antibody, the antibody would coat the treponeme. Next, fluorescein isothiocyanate-labeled antihuman Ig was added; this combines with the patient's IgG and IgM antibodies that were adhering to TP and results in a visible test reaction when examined by fluorescence microscopy.
The analysis included profiling of healthy donors on different demographic parameters. ICA and RPR were evaluated and compared with FTA-ABS for sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and Youden's index. Additional 107 known negative samples (concordant negative; i.e., negative on both RPR and ICA) were also tested with FTA-ABS for appropriate statistical evaluation. P< 0.05 was considered statistically significant. SPSS software, version 22.0 (IBM, New York, USA), was used for analysis.
| Results|| |
A total of 10,200 donor samples were simultaneously evaluated for syphilis by RPR and ICA as screening test. Out of 10,200 donor samples, 76 samples (38 concordant and 38 discordant) were reactive.
[Table 1] describes the demographic details in terms of gender, frequency of donation and nature of donation of all donors, nonreactive donors, and reactive donors. [Table 2a] compares ICA and FTA-ABS, and [Table 2b] compares RPR and FTA-ABS with respect to reactivity in form of two-by-two table. [Table 3] compares ICA and RPR in terms of different statistical parameters.
|Table 2a: Comparison between immunochromatographic assay and fluorescent treponemal antibody-absorption|
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|Table 2b: Comparison between rapid plasma regain and fluorescent treponemal antibody-absorption|
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|Table 3: Comparison between immunochromatographic assay and rapid plasma reagin on different statistical parameters|
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| Discussion|| |
Treponemal tests detect antibodies specific to TP. In addition to TP, which causes syphilis, other treponemal subspecies (e.g., T. pertenue, which causes yaws, and T. carateum, which causes pinta) also can produce reactive results to treponemal tests, but these subspecies are rare and rarely pose challenge in screening process. A reactive treponemal test result indicates that treponemal infection has occurred at some point in the past but cannot distinguish between treated and untreated infections. Treponemal tests can produce reactive results for life, even after adequate treatment for syphilis.
Nontreponemal tests, such as the RPR test and VDRL test, detect antibodies to cardiolipin and are not specific for treponemal infection. Nontreponemal tests are more likely than treponemal tests to produce nonreactive results after treatment; therefore, reactive results from nontreponemal tests are more reliable indicators of untreated infection. Quantitative nontreponemal tests are also used to monitor responses to treatment or to indicate new infections. However, this nontreponemal test may give false-positive results in several cases.
In our study, we can make an inference that ICA is more sensitive than RPR and more suitable for screening. The PPV of both the tests was similar while NPV of ICA is more than RPR. The specificity of RPR is more than ICA. Higher specificity indicates a lower false-positive rate which is contrary to the present belief that nonspecific syphilis tests have higher false-positive rate. It may be due to the fact that the study was conducted among healthy voluntary blood donors. ICA in comparison to RPR as screening test for blood donors is quite effective because it has superior sensitivity as compared to RPR (91.9% vs. 67.7%) without losing out much on specificity (90.8% vs. 95%). In a study conducted by Montoya et al. for WHO Health Bulletin, compared diagnostic accuracy of RPR and rapid ICA for antenatal screening found that the diagnostic accuracy of ICA is comparable to that of their gold standard (TPHA). The sensitivity, specificity, PPV, and NPV were comparable to our study. Similarly, in another study conducted by Sato et al., the assessment of ICA found a sensitivity of 93.6%, specificity of 92.5%, and PPV and NPV of 95.2% and 93.7%, respectively, for rapid ICA. They also observed that the ICA technique obviates the need of equipment and its diagnostic features demonstrate that it may be applicable as an alternative assay for syphilis screening under some emergency conditions or for patients living in remote localities. In a letter to editor, Goel et al. concluded that ICA is a simple, rapid, point-of-care type treponemal specific test suitable for use in primary health-care settings for the diagnosis of syphilis. Evaluating the performance of rapid tests, their utility in a disease control program and acceptability to patients and health-care providers will improve the diagnosis of syphilis in primary health-care settings in developing countries and reduce overtreatment.
However, the main concern lies in the fact that ICA is unable differentiate past infection from present one. Therefore, the higher positive samples may be due to the past infection or receiving treatment. RPR as a screening test for voluntary donors is quite effective but higher false-positive results in many other diseases and physiological conditions add to its limited use. In a letter to editor, Arora et al. mentioned that RPR as a screening test for blood donors gives high false-positive rate. They also raised questions about continuation of RPR as a screening assay in their institution due to the high false-positive rate.
| Conclusion|| |
The statistical analysis of the study shows that ICA is a better screening test for blood donors as compared to RPR. Furthermore, the ease of performing ICA and better result interpretation makes it a preferred choice among blood banking professionals.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2a], [Table 2b], [Table 3]