Clinical Biochemistry 46 (2013) 834–837
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Short Communication
Comparisons of fully automated syphilis tests with conventional VDRL and FTA-ABS tests Seung Jun Choi, Yongjung Park, Eun Young Lee, Sinyoung Kim, Hyon-Suk Kim ⁎ Department of Laboratory Medicine, Severance Hospital, Yonsei University College of Medicine, Seoul, Republic of Korea
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Article history: Received 19 August 2012 Received in revised form 12 February 2013 Accepted 16 February 2013 Available online 27 February 2013 Keywords: Fluorescent treponemal antibody absorption (FTA-ABS) Rapid plasma regain (RPR) Syphilis Treponema pallidum particle agglutination (TPPA) Venereal Disease Research Laboratory (VDRL)
a b s t r a c t Background: Serologic tests are widely used for the diagnosis of syphilis. However, conventional methods require well-trained technicians to produce reliable results. We compared automated nontreponemal and treponemal tests with conventional methods. Methods: The HiSens Auto Rapid Plasma Reagin (AutoRPR) and Treponema Pallidum particle agglutination (AutoTPPA) tests, which utilize latex turbidimetric immunoassay, were assessed. A total of 504 sera were assayed by AutoRPR, AutoTPPA, conventional VDRL and FTA-ABS. Among them, 250 samples were also tested by conventional TPPA. Results: The concordance rate between the results of VDRL and AutoRPR was 67.5%, and 164 discrepant cases were all VDRL reactive but AutoRPR negative. In the 164 cases, 133 showed FTA-ABS reactivity. Medical records of 106 among the 133 cases were reviewed, and 82 among 106 specimens were found to be collected from patients already treated for syphilis. The concordance rate between the results of AutoTPPA and FTA-ABS was 97.8%. The results of conventional TPPA and AutoTPPA for 250 samples were concordant in 241 cases (96.4%). AutoRPR showed higher specificity than that of VDRL, while VDRL demonstrated higher sensitivity than that of AutoRPR regardless of whether the patients had been already treated for syphilis or not. Both FTA-ABS and AutoTPPA showed high sensitivities and specificities greater than 98.0%. Conclusions: Automated RPR and TPPA tests could be alternatives to conventional syphilis tests, and AutoRPR would be particularly suitable in treatment monitoring, since results by AutoRPR in cases after treatment became negative more rapidly than by VDRL. © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Syphilis is a disease caused by Treponema pallidum infection. Tests for detecting syphilis remain important, particularly in testing for blood donors and before invasive procedures or surgery, but there is no single definitive test for diagnosing syphilis. Therefore, serologic tests are still commonly used as standard methods. Recently, the US Centers for Diseases Control and Prevention and the Association of Public Health Laboratories released an updated algorithm for syphilis testing. This algorithm suggests that in countries of low prevalence, samples may be screened using a treponemaspecific test, with positive samples being assayed with nontreponemal tests to assess the phases of disease and treatment status. The conventional syphilis tests still require well-trained laboratory technicians to perform manual procedures and to interpret the results. Recently, some commercially available assays for the detection
⁎ Corresponding author at: Department of Laboratory Medicine, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Republic of Korea. Fax: + 82 2 364 1583. E-mail address:
[email protected] (H.-S. Kim).
of antibodies specific to T. pallidum using various methods have been evaluated and compared with conventional tests [1–3]. However, few reports have assessed the performance of automated nontreponemal tests. We aimed to compare the results of automated rapid plasma reagin (RPR) and T. pallidum particle agglutination (TPPA) tests utilizing latex turbidimetric immunoassay with the results of other conventional syphilis tests.
Materials and methods HiSens AutoRPR and Auto TP The HiSens AutoRPR (AutoRPR) and Auto TP (AutoTPPA) (HBI, Anyang, Korea) are latex turbidimetric immunoassays for the quantitative detection of syphilis-related antibodies in serum. All specimens were tested by an automated clinical chemistry analyzer CA 400 (Furuno electric, Hyogo, Japan). For AutoRPR, the autoanalyzer calculates the ratio between the sample relative light unit (RLU) and cutoff, and samples with a ratio less than 1.0 were regarded as negative. In the AutoTPPA assay, the samples with concentrations of antibodies to T. pallidum less than 10 U/mL were considered negative.
0009-9120/$ – see front matter © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clinbiochem.2013.02.008
S.J. Choi et al. / Clinical Biochemistry 46 (2013) 834–837
Statistical analysis
Table 1 Comparison of the results by VDRL and AutoRPRa tests. AutoRPR
Positive Negative Total
VDRL Reactive
Nonreactive
Total
140 164 304
0 200 200
140 364 504
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Concordance rate (%)
k (95% CI)
p value
67.5
0.40 (0.34 to 0.46)
b0.0001
Abbreviations: VDRL, Venereal Disease Research Laboratory test; RPR, rapid plasma reagin test; CI, confidence interval. a Automated tests by the HiSens AutoRPR LTIA.
Conventional tests Venereal Disease Research Laboratory (VDRL) tests were carried out using BD VDRL Antigen kits (Becton Dickinson, Sparks, MD). When an undiluted sample was reactive for VDRL, semiquantitative tests were performed with a serial dilution of the sample to a 1:32 ratio. Fluorescent treponemal antibody absorption (FTA-ABS) tests were performed by using commercially available glass slides (Zeus Scientific, Raritan, NJ). Positive results were graded on a scale of 1+ to 4 + or minimally reactive, and all minimally reactive or 1 + samples were repeatedly tested. Conventional TPPA tests were performed using SERODIA-TP·PA kits (Fujirebio Inc., Tokyo, Japan).
Specimens A total of 504 serum samples, requested for VDRL tests were collected from subjects who were suspected of having syphilis or who underwent a medical examination at Severance Hospital of Yonsei University. All specimens were aliquoted, and one of the aliquots was assayed for VDRL, and the rest was stored at −70 °C until assayed by other tests. FTA-ABS, AutoRPR and AutoTPPA tests were performed for all specimens, and randomly selected 250 sera were also assayed by conventional TPPA test.
All statistical analyses were performed by using the Analyse-it Method Evaluation edition software, version 2.27 (Analyse-it Software, Leeds, UK). The k coefficients were calculated to estimate the agreement between different tests. Multiple pairwise comparisons among the results by the AutoRPR according to the groups classified by the results of VDRL tests were conducted using Kruskal–Wallis test with Bonferroni correction. When the results of the VDRL and AutoRPR were concordant, they were regarded as true-positive or -negative, while the true diagnosis was determined by retrospective blind review of the medical records, when there was discrepancy between the VDRL and AutoRPR. Receiver operating characteristic (ROC) curve was plotted for RLUs by AutoRPR test to assess its performance for differentiating current syphilis infections from treated syphilis cases. The best cut-off RLU was determined as the value when the sum of sensitivity and specificity was maximized. Results Comparison of VDRL and AutoRPR The concordance rate between the results of VDRL and AutoRPR assays was 67.5% (k = 0.40, p b 0.0001). All 164 (32.5%) discrepant results among 504 were VDRL-reactive and AutoRPR-negative (Table 1). FTA-ABS was reactive in 133 cases among the 164 discrepant results, and the other 31 cases showed FTA-ABS nonreactivity. Medical records of 106 subjects among the 133 cases could be reviewed. Those 106 cases included 82 already-treated syphilis, 22 late latent syphilis, one primary syphilis, and one suspected of having congenital syphilis. In the 31 cases which showed FTA-ABS nonreactivity, only one case had a history of already-treated syphilis. In a total of 504 specimens, the RLUs of AutoRPR were significantly lower in VDRL-nonreactive cases than in all VDRL-reactive groups (p b 0.0001) (Fig. 1A). There were also significant differences in the AutoRPR RLUs among all the groups classified by the VDRL titers (p b 0.0001). The median AutoRPR RLU was less than 0.01 for the VDRL-nonreactive group (n = 200), 0.10 for the 1:1 weakly reactive group (n = 65), 0.50 for the 1:1 reactive group (n = 111), 2.20 for
Fig. 1. The relative light units (RLUs) of automated rapid plasma reagin (AutoRPR) test according to the reactivity of the Venereal Disease Research Laboratory (VDRL). A, The RLUs were lower in the VDRL nonreactive group than in all other VDRL reactive groups. There were also significant differences in the AutoRPR RLUs among all the groups according to the VDRL titers in a total of 504 cases. B, The RLUs of the AutoRPR were significantly different between the 1:1 weakly and the 1:1 reactive groups, and between the 1:1 weakly reactive and the 1:2 to 1:4 reactive groups in the 82 treated cases. Inner lines, upper and lower ends of boxes indicate median values, and upper and lower quartiles, respectively. Whiskers denote maximum and minimum values, and circles represent individual values. NR, nonreactive; w, weakly reactive.
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the 1:2 reactive group (n = 62), and 8.30 for the group with VDRL reactivity of equal or greater than 1:4 (n = 66). A total of 82 treated syphilis cases among the 133 AutoRPR negative and VDRL reactive showed FTA-ABS reactivity. The RLUs of the AutoRPR assay were significantly different between the 1:1 weakly reactive group (n = 29) and the 1:1 reactive group (n = 38) (p = 0.0001), and between the 1:1 weakly reactive group and the 1:2 to 1:4 reactive group (n = 15) (p = 0.0208). However, there was no significant difference between the 1:1 reactive group and the 1:2 to 1:4 reactive group (p = 1.0000) (Fig. 1B). Comparison among other tests The results by FTA-ABS agreed with those by AutoTPPA in 97.8% of cases (k = 0.96) (p b 0.0001). The concordance rate between the results of the VDRL and FTA-ABS test was 91.9% (k = 0.83, p b 0.0001), and that between the AutoRPR and FTA-ABS tests was 71.6% (k = 0.45, p b 0.0001). The conventional TPPA and AutoTPPA showed high concordance, 96.4% (k = 0.80, p b 0.0001). Sensitivities and specificities of the tests When the treated syphilis cases were regarded as true positive for the syphilis infection, the sensitivities of AutoRPR and VDRL were 56.7% and 100.0%, while the specificities of AutoRPR and VDRL were 100.0% and 87.0%, respectively. However, the sensitivity of AutoRPR was 84.8%, and that of VDRL was 100.0%, when the treated syphilis cases were considered as true negative. In the same condition, the specificities of AutoRPR and VDRL were 100.0% and 64.1%. In addition, the sensitivity and specificity of FTA-ABS were 99.6% and 99.1%, and those of AutoTPPA were 98.5% and 98.3%. Cut-off RLU by AutoRPR in treatment monitoring The medical records of all 304 VDRL reactive cases were reviewed, and 31 cases with insufficient medical records and 50 VDRL false positive cases were excluded from the analysis. The remaining 223 subjects were divided into 2 groups, current syphilis (n = 73) and treated syphilis (n = 150). The area under the ROC curve plotted with RLUs by AutoRPR in predicting successful treatment was 0.71 (95% CI = 0.63 to 0.79, p b 0.0001). When the cases with AutoRPR RLUs less than 4.7 were considered as successfully treated, the sensitivity and specificity were 88.0% and 56.2%. Discussion In our study, a total of 164 cases showed discrepancy between AutoRPR and VDRL tests, and all were VDRL reactive but AutoRPR negative. In those cases, 133 showed FTA-ABS reactivity and the remaining 31 were nonreactive for FTA-ABS. Medical records of 106 among the 133 cases showing FTA-ABS reactivity were able to be reviewed, and 82 of 106 specimens were collected from patients who were already treated. In addition, the RLUs of the AutoRPR were not significantly different between the 1:1 reactive and the 1:2 to 1:4 reactive groups in the 82 treated cases. These results could imply that the results of AutoRPR could be more rapidly changed to be negative after treatment than those of VDRL. Medical records of the 31 cases that showed FTA-ABS nonreactivity were reviewed, and only one case had a history of already treated syphilis. Therefore, cases presented with VDRL reactivity but AutoRPR and FTA-ABS nonreactivity could be caused by false-positivity of the VDRL. In patients treated for primary or secondary syphilis, the VDRL titers declined two dilutions at three months and three dilutions at six months [4]. However, the serologic response is often slower in patients with late or reinfected syphilis [5]. The risk of serologic
treatment failure is generally greater in males, patients during the late stages of syphilis, and individuals with HIV infection [6]. Among the 82 samples mentioned above, 27 specimens were collected from patients with late latent syphilis and one was HIV-positive patient. Since the results of AutoRPR converted to negative in these cases but VDRL remained positive after treatment and the RLU values of the AutoRPR were significantly different among all the groups divided by VDRL reactivity in all 504 cases, AutoRPR test would be more useful for treatment monitoring, particularly in late latent syphilis. Adjusting cutoff value or establishing an “intermediate range” for RLUs on the AutoRPR might be needed, because the median RLU of the AutoRPR in the VDRL 1:1 reactive group was 0.50, which is lower than the cutoff suggested by the manufacturer. AutoTPPA test showed high concordance rates with conventional treponemal tests in our study, but the concordance rate between the results of AutoRPR and FTA-ABS was lower than that between the results of VDRL and FTA-ABS. These discrepancies could be caused by rapid changes of results by AutoRPR after treatment. The sensitivity of AutoRPR was lower than VDRL, while the specificity of VDRL was lower than AutoRPR. Therefore, it would be difficult to determine which of the two methods is useful, and each method could be selected based on the aim of testing in each clinical laboratory. In a previous study, the sensitivities and specificities of RPR and VDRL according to different syphilis stages were similar with each other method [7]. However, it was controversial that RPR test can replace VDRL in the diagnosis of neurosyphilis with cerebrospinal fluid specimens [8,9]. Therefore, it can be hard to conclude which test is preferable in the diagnosis of syphilis according to different stages. In our results, sensitivity and specificity of AutoTPPA were greater than 98.0%, and the concordance rates between the results of AutoTPPA and those of conventional treponemal tests were also high. In these contexts, AutoTPPA could be also used for syphilis screening according to the reverse sequence syphilis screening algorithm. Unfortunately, we could not obtain follow-up samples from the same subjects enrolled in our study, thus, we could not determine the cut-off for the decrease in the RLUs in a same patient to predict whether a patient is successfully treated or not. Instead, we have performed ROC analysis to identify the usefulness of the RLUs by AutoRPR. In our results, the sensitivity and specificity for predicting successful treatment were 88.0% and 56.2%, when the cases with AutoRPR RLUs less than 4.7 were considered as successfully treated. Further large-scaled studies are needed to determine the most useful cut-off RLU in patient follow-up. In conclusion, Automated RPR would be suitable in treatment monitoring, especially for patients with late latent syphilis, although sensitivity of automated RPR might be lower than conventional nontreponemal tests. Automated TPPA test evaluated in this study showed comparable results to conventional FTA-ABS test. Further evaluations on sensitivities of these methods as well as cutoff values of automated RPR seem to be required. Acknowledgement All authors have nothing to declare. References [1] Park Y, Joo SY, Park MH, Kim HS. Evaluation of a fully automated treponemal test and comparison with conventional VDRL and FTA-ABS tests. Am J Clin Pathol 2011;136(5):705–10. [2] Binnicker MJ, Jespersen DJ, Rollins LO. Treponema-specific tests for serodiagnosis of syphilis: comparative evaluation of seven assays. J Clin Microbiol 2011;49(4): 1313–7. [3] Onoe T, Honda M, Matsuo K, Sasaki H, Sawamura M, Onoe Y, et al. Examination of the correlation between the manual and automated serological testing methods for syphilis. J Dermatol 2011. http://dx.doi.org/10.1111/j.1346-8138.2011.01341.x. [4] Brown ST, Zaidi A, Larsen SA, Reynolds GH. Serological response to syphilis treatment. A new analysis of old data. JAMA 1985;253(9):1296–9.
S.J. Choi et al. / Clinical Biochemistry 46 (2013) 834–837 [5] Romanowski B, Sutherland R, Fick GH, Mooney D, Love EJ. Serologic response to treatment of infectious syphilis. Ann Intern Med 1991;114(12):1005–9. [6] Gonzalez-Lopez JJ, Guerrero ML, Lujan R, Tostado SF, de Gorgolas M, Requena L. Factors determining serologic response to treatment in patients with syphilis. Clin Infect Dis 2009;49(10):1505–11. [7] Larsen SA, Steiner BM, Rudolph AH. Laboratory diagnosis and interpretation of tests for syphilis. Clin Microbiol Rev 1995;8(1):1–21.
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