Comparing the performance of traditional non-treponemal tests on syphilis and non-syphilis serum samples

Abstract

The goal of the present study was to determine the performance of two traditional non-treponemal tests for syphilis. Syphilis sera (n = 209) included different stages of disease, and control sera (n = 247) were from patients with tumours, leprosy, systemic lupus erythematosus, hepatitis, pregnant women and healthy individuals. Treponema pallidum ELISA, Treponema pallidum particle agglutination and rapid treponema-specific tests were used as gold standards. Rapid plasma reagin or toluidine red unheated serum test had a sensitivity and specificity of over 95%. False-negative reactions of rapid plasma reagin and toluidine red unheated serum test were observed mainly in primary and latent syphilis cases, and false-positive reactions were present in systemic lupus erythematosus, hepatitis-infected patients. Overall, both non-treponemal tests had high sensitivities and specificities making the assays attractive as screening tests for syphilis. When examined on WHO reference serum samples and based on lower limits of detection, non-treponemal tests were less sensitive than treponema-specific tests.

Keywords

syphilis diagnosis diagnostic test serology Rapid plasma reagin RPR toluidine red unheated serum test TRUST sensitivity specificity lower limit of detection

Introduction

Syphilis is a multistage chronic infection caused by Treponema pallidum subsp. pallidum, namely primary, secondary, latent and late syphilis.¹ Diagnosis of syphilis largely relies on serologic tests, which fall into two categories of non-treponemal and treponemal tests.^2–4 Non-treponemal tests measure antiphospholipid antibodies formed by the host in response to lipoidal material released by damaged host cells early in infection and lipid from the cell surfaces of the treponeme itself and is often used for initial screening of sera such as the Venereal Disease Research Laboratory (VDRL), rapid plasma reagin (RPR) or toluidine red unheated serum test (TRUST). The antibody titres of non-treponemal tests are low in the early stage of primary infections and decline in the late latent stage or post-treatment of infections.⁴ Treponemal tests use T. pallidum or its components as the antigen and are often used as confirmatory assays, such as enzyme immunoassays (EIAs), T. pallidum particle agglutination (TPPA), T. pallidum haemagglutination assay (TPHA) and fluorescent treponemal antibody absorption (FTA-ABS). Treponemal tests usually remain reactive for life regardless of treatment and correlate poorly with disease activity.⁴

The sensitivity and specificity of serologic tests vary depending on the type of assays and stage of syphilis.³ For example, the sensitivity of RPR for primary, secondary, latent and late syphilis is around 78%, 100%, 95% and 73%, respectively; and the specificity of RPR is 98%. TRUST has similar sensitivity and specificity to RPR. The treponemal tests are highly sensitive for diagnosis of secondary and latent syphilis (97%–100%). The specificity of treponemal tests is almost 100% (94%–100%), which deems these methods suitable for confirmatory assays.²

Recently, several rapid treponema-specific tests have been developed as “point-of -care tests” (POCTs) for syphilis diagnosis because they are easy to perform and widely available.^2,5–8 The sensitivities of POCTs range between 70% and 100% depending on different reports using various reference standard tests, and their specificity is from 97% to 100%.^8–10 However, treponema-specific tests are designed to detect treponemal antibodies, which persist for many years after treatment. Thus, the tests cannot be used to distinguish active syphilis from past treated infections, and non-treponemal tests are required to confirm active infections.¹⁰

This study aimed to compare the performance of two non-treponemal tests (RPR and TRUST) for diagnosis of syphilis. We reported high sensitivity and specificity of RPR and TRUST as compared to the treponema-specific reference tests. Using a WHO reference serum sample, we also compared the lower limit of detection (LLD) for the treponema-specific and non-treponemal tests.

Materials and methods

Serum samples from syphilis patients

Two hundred and nine serum samples (n = 209) from active syphilis patients were retrieved from the syphilis serum bank at the Shanghai Skin Disease Hospital (SSDH). These samples were selected from collections during the nine-month period between October 2009 and June 2010. Based on syphilis diagnosis, all samples were first stratified into categories of primary, secondary, latent syphilis and neurosyphilis. Approximately 20% samples from each category were then randomly selected into this study.

Serum samples were confirmed to be reactive by both EIA and TPPA (TPPA, Fujirebio Inc. Japan; EIA, Phoenix Bio-Tech Corp., New York, USA). The reactivity of the sera in treponema-specific tests was also confirmed using two treponema-specific POCTs. The two POCT kits (POCT1 and POCT2) were purchased from Shanghai Chemtron Biotech Co., Ltd., Shanghai, China (Lot # RD20101001, http://www.chemtronbio.com/en/, last accessed on April 2, 2012), and Inverness Medical Japan Co., Ltd., Japan (Lot # 09631 K 101, http://www.alere.com/EN_US/brands/our-brands/determine/index.jsp, accessed 2 April 2012), respectively.

Patient information was retrieved from the hospital database, including age, gender and diagnosis with staging of syphilis. The 209 syphilis patients comprised primary (n = 30), secondary (n = 92), latent (n = 39) and neurosyphilis (n = 44); four cases were from systemic lupus erythematosus (SLE) patients with a syphilis history. Those patients had active syphilis except for the four SLE patients with a syphilis history. There were 112 male patients and 97 female patients. The mean age was 42 years (ranging from 17 to 82).

Criteria to differentiate stages of syphilis were those described by the Ministry of Health China.¹¹ In all cases, TPPA and EIA tests were positive. Briefly, criteria for primary syphilis were recent unprotected sex, chancres or non-painful ulcers, enlargement of lymph nodes, dark-field microscopy positive and/or non-treponemal serologic tests positive (RPR or TRUST). Criteria for secondary syphilis included unprotected sex, having primary syphilis in the past 2 years, skin/mucosal characteristic lesions or other organ damage suggestive of syphilis, dark-field microscopy positive and non-treponemal serologic tests positive. Latent syphilis was suggested when a patient had history of primary or secondary syphilis and currently without any clinical signs and symptoms, treponemal serologic tests positive and normal cerebrospinal fluid (CSF), a non-treponemal antibody titre higher than 1:8 in patients without a syphilis history, or in patients with a past syphilis history, conversion of non-treponemal tests from negative to positive or with at least a four-fold increase in antibody titres. Neurosyphilis was diagnosed when a patient presented with syphilis signs and symptoms, including central nervous system involvement, CSF-VDRL positive, abnormal CSF (WBC ≥ 10 × 10⁶/L, protein concentration >500 mg/L), which could not be explained by other illness, and positive serologic non-treponemal or treponemal tests.

Serum samples of the syphilis-negative groups

There were 247 serum samples from non-STD clinic patients. Syphilis was ruled out in these patients by negative results of EIA, TPPA and POCTs and by patient history. Patients included tumours (n = 48), leprosy (n = 20), systemic lupus erythematosus (SLE) (n = 48), hepatitis B (n = 35), hepatitis C (n = 15), pregnant women (n = 50) and patients with no disease (n = 31). The diagnosis of these diseases was determined according to the standard criteria of infectious diseases or internal medicine reference books. These samples were provided from SSDH and the ZhongShan Hospital (http://www.zs-hospital.sh.cn/e/index.asp) in Shanghai.

Serology tests

Before testing, all serum samples were numerically coded; thus technicians were blinded from sample identity. Sample codes were broken for results analysis. TPPA, EIA and two POCTs were used as reference tests for syphilis serologic reactivity of these panels of serum samples and were performed according to the manufacturers’ instructions. All sera were then examined for reactivity for two non-treponemal tests (RPR and TRUST). The WHO quality control sera (09/B585-01, 09/B559-03) were used as testing controls (http://www.nibsc.ac.uk/products.aspx, last accessed on April 2, 2012). Un-diluted sera were used for qualitative assays, and in quantitative assays sera were serially diluted two-fold. Assays were conducted according to the manufacturers’ instructions.

Two RPR kits were purchased from the Shanghai Kehua Bio-Engineering Co., Ltd. Shanghai, China (RPR-1, Lot # 20100801, http://www.skhb.com/news/english/index.shtml, last accessed on April 2, 2012) and from the Stanbio Laboratory, Texas, USA (RPR-2, Lot # 102381LE, http://www.stanbio.com/, last accessed on April 2, 2012), respectively.. Two TRUST kits were purchased from the Shanghai Rongsheng Biotech Co., Ltd, Shanghai, China (TRUST-1, Lot # 20100701, http://www.rsbio.com/aboutus.asp, last accessed on April 2, 2012) and from the New Horizons Diagnostics Corporation, Columbia, Maryland, USA (TRUST-2, Lot # 196 P, http://www.nhdiag.com/index.htm, last accessed on April 2, 2012), respectively.

LLD

To determine the LLD, a WHO international standard (IS) serum (Lot# 05/132, National Institute for Biological Standards and Control, http://www.nibsc.ac.uk/products.aspx, last accessed on April 2, 2012) was serially diluted and tested for reactions in the TPPA, EIA, POCT1, POCT2, RPR-1, RPR-2, TRUST-1 and TRUST-2 assays. The WHO IS serum contains three international units (IU) per mL upon reconstitution.

Statistical analysis

Data were analyzed using the SPSS software (http://www-01.ibm.com/software/analytics/spss/?Country_ID = 1, last accessed on April 5, 2012). Chi-square tests were used to determine significant difference between percentages. The geometric mean (GM) model¹² with repeated measures was used to examine significant differences on detectable titres of the syphilis serum samples for the non-trepanemal serologic tests. The GM values represented log₂(x + 1) of antibody titers. A p value of <0.05 was significant in difference.

Ethics approval was obtained from the Ethics Board of the SSDH.

Results

Comparison of two non-treponemal tests (RPR and TRUST)

Using TPPA, EIA and treponema-specific POCTs as reference tests, the sensitivities of the non-treponemal tests (RPR-1, RPR-2, TRUST-1 and TRUST-2) were 95.7%–99.0% (Table 1). The specificities of non-treponemal serologic tests were 96.8%–98.0%. There was no significant difference in sensitivity or specificity between any tests (p > 0.05).

Table 1.

Sensitivity and specificity of RPR and TRUST on 209 syphilis-positive and 247 syphilis-negative serum samples.

Assay	Sensitivity % (95% CI)	Specificity % (95% CI)
RPR-1	98.7 (95.9–99.5)	96.8 (93.7–98.4)
RPR-2	95.7 (92.0–97.7)	97.6 (94.8–98.9)
TRUST–1	99.0 (96.6–99.7)	98.0 (95.4–99.1)
TRUST–2	96.7 (93.3–98.4)	96.8 (93.7–98.4)

CI: Confidence interval; RPR: rapid plasma reagin; TRUST: toluidine red unheated serum test.

Serologic non-reactivity in non-treponemal tests

A small percentage of the sera from the syphilis patients (TPPA, EIA and POCTs reactive) exhibited non-reactions in the non-reactivity tests (Table 2, 1%–4.3%). Most of the non-reactions were observed for sera of primary syphilis, accounting for 6.7%–10% (n = 30). Non-reactions were also occasionally observed in sera from latent or neurosyphilis patients.

Table 2.

Serologic non-reactions of 209 serum samples from different stages of syphilis.

Stage of syphilis	RPR-1 N (%)	RPR-2 N (%)	TRUST-1 N (%)	TRUST-2 N (%)
Primary (n = 30)	2 (6.7)	3 (10.0)	2 (6.7)	2 (6.7)
Secondary (n = 92)	0	1 (1.1)	0	0
Latent (n = 39)	1 (2.6)	1 (2.6)	0	2 (5.1)
Neurosyphilis (n = 44)	0	2 (4.5)	0	2 (4.5)
Past Syphilis (n = 4)^a	0	2 (50.0)	0	1 (25.0)
Total (n = 209)	3 (1.4)	9 (4.3)	2 (1.0)	7 (3.3)

CI: Confidence interval; RPR: rapid plasma reagin; TRUST: toluidine red unheated serum test.

No information about disease stages was available. Two cases also had systemic lupus erythematosus.

Cross reactivity in non-treponemal tests

A small portion of the control sera exhibited positive reactions in the non-treponemal tests (Table 3, 2%–3.2%). Cross reactions were mainly observed in SLE (4.2%–10.4%) and occasionally in the serum samples from leprosy and hepatitis C patients (Table 3).

Table 3.

Cross-reactions of RPR and TRUST in 247 syphilis negative sera.

Syphilis negative serum	RPR-1 N (%)	RPR-2 N (%)	TRUST-1 N (%)	TRUST-2 N (%)
Tumour (n = 48)	1 (2.1)	0	0	0
Leprosy (n = 20)^a	1 (5.0)	1 (5.0)	1 (5.0)	1 (5.0)
SLE (n = 48)^b	5 (10.4)	2 (4.2)	3 (6.3)	5 (10.4)
Hepatitis B (n = 35)	0	0	0	0
Hepatitis C (n = 15)^c	1 (6.7)	1 (6.7)	1 (6.7)	1 (6.7)
Pregnant women (n = 50)	0	2 (4.0)	0	0
Normal (n = 31)	0	0	0	1 (3.2)
Total (n = 247)	8 (3.2)	6 (2.4)	5 (2.0)	8 (3.2)

RPR: rapid plasma reagin; SLE: systemic lupus erythematosus; TRUST: toluidine red unheated serum test.

False-positive of the four tests was from one single leprosy patient.

Two patients gave false positive results in all of the four tests.

False-positive of the four tests was from one single Hepatitis C patient.

Serologic titres determined by non-treponemal tests

To compare the sensitivities of the four non-treponemal assays for syphilis, syphilis serum samples were serially diluted two-fold and were tested for their reactivity. The GM of titers (95% confidence intervals, CI) are shown in Figure 1, which were 20.0 (95% CI: 17.5–22.6), 18.8 (95% CI: 16.5–21.3), 20.68 (95% CI: 18.2–23.3), 20.0 (95% CI: 17.7–22.4), for RPR1, RPR2, TRUST1 and TRUST2, respectively. A general comparison of the four non-treponemal tests revealed significant differences of titres (Pillai's Trace F value = 9.41, p < 0.001). The GM of titres of RPR2 was significantly lower than that of RPR1, TRUST1 or TRUST2 (p ≤ 0.001). There were no significant differences for the other two-by-two comparisons (p > 0.05).

Figure 1.

Comparison of detectable titres in non-treponemal tests. Titres were converted to log₂(x + 1) units. Statistical analysis was performed according to the General Linear Model with repeated measures.¹⁴ Diamond: GM; Square: upper limit of 95% confidence interval GM; Triangle: lower limit of 95% confidence interval GM. GM: geometric mean.

LLD

The LLDs were 60 and 1.87 mIU/mL for TPPA and EIA, respectively. Both POCT1 and POCT2 had an LLD of 15 mIU/mL, respectively, whereas RPR or TRUST assays had an LLD of 112--225 mIU/mL (150 for RPR1, 225 for RPR2, 112 for TRUST1, 187 for TRUST2).

Discussion

Serologic diagnosis is the hallmark in syphilis control. The commonly used syphilis serologic tests in Shanghai include RPR and TRUST for non-treponemal tests and rapid treponemal specific POCTs. In this study, using the treponemal-specific tests (EIA, TPPA and POCTs) as gold standards, we compared the performance of the traditional non-treponemal tests RPR and TRUST. We demonstrated that RPR and TRUST had similar sensitivity and specificity (p > 0.05), which were similar to previous reports by others.³ We found that there were cross reactions and false-negative reactions in these non-treponemal tests, confirming that treponema-specific tests are required in diagnosis of syphilis.

Serologic non-reactivity by non-treponemal tests was observed, particularly in primary or latent syphilis.¹³ Among primary syphilis cases, 26% (224/863) were non-reactive on initial RPR.¹³ According to the US Centers for Disease Control and Prevention, non-reactive rates could be as high as 23% for RPR and TRUST in primary syphilis cases.³ Another study showed that non-reactive rates of RPR in primary syphilis cases were over 14%.¹⁴ RPR non-reactivity was observed in 39% (512/1303), 5% or 0%, respectively, in latent syphilis cases.^3,13,14 In our study, we observed serologic non-reactive rates for RPR or TRUST ranging from 1% to 4.3% of all syphilis cases, 6.7%–10% in primary syphilis cases and up to 5% in latent syphilis cases, respectively (Table 2). The discrepancy of non-reactive rates of non-treponemal tests in various studies may reflect the variations in case selection, as it has been certain that there is a seroconversion window between initial infection and seroconversion.^1–3 Non-reactive rates of RPR in secondary syphilis cases are lower, less than 1% or 4%.^3,14 In our study, all secondary syphilis cases (n = 92) exhibited RPR or TRUST positivity, except for one case with a negative RPR2 (Table 2).

In a population-based multiple centre study, Yin et al.¹⁵ reported that a dual POCT treponema-specific test displayed, when compared to TPPA, a sensitivity of over 94% and a specificity of over 99% for the results of Treponemal Line 1, for various populations and specimen types (whole blood, fingerprick and plasma). Zhuang et al.¹⁶ used the Determine test to examine STD clinic patients and reported that, when compared to TPPA, the Determine TP assay had a sensitivity of 97% and specificity of 99%. Our findings of nearly 100% sensitivity and specificity in the treponemal-specific POCTs are concordant with these two studies, suggesting that these tests may be used in syphilis diagnosis in various populations and different types of specimens.

Despite the high specificity of RPR and TRUST tests, false-positive reactions were observed in non-syphilis controls in our study (Table 3). Previous studies by others have demonstrated that several conditions can cause biological false-positive (BFP) reactivity.^17–20 Antilipoidal antibodies increase due to tissue damage from tumours, infectious diseases such as hepatitis and leprosy or autoimmune diseases such as SLE; these antibodies can cause a false-positive RPR, VDRL or TRUST test. Because autoantibodies increase as a result of aging or pregnancy, elderly people and pregnant women are also at risk of a false-positive result in non-treponemal serologic tests.¹ In the present study, we recruited patients with these disease conditions in our control groups. BFP of VDRL was observed in 11.8% of SLE patients and 0.72% of pregnant women.¹⁸ BFP of RPR was detected in 4% of hepatitis C-infected individuals.²⁰ BFP percentages in SLE and rheumatoid arthritis were 14.6% (6/41) and 4.3% (1/23), respectively.²¹ BFP has also been observed in high percentages of injecting drug users (8.7%, 143/1646).²² In the present study, BFP of non-treponemal serologic tests was observed in patients with leprosy and hepatitis C, and BFP was present in up to 10% of SLE patients (4.2%–10.4%, n = 48). Our observations confirmed previous reports by others, indicating that BFP of non-treponemal serologic tests must be ruled out, especially in patients with autoimmune disorders, drug users and in some viral infections.

Changes in antibody titres in non-treponemal tests are indicators of re-infections or syphilis treatment.^1–3 In the present study, we evaluated the maximal detectable titre for the four non-treponemal tests. The percent distributions of qualitative detection of syphilis sera were not significantly different (data not shown), however, the GM titers for RPR2 were significantly lower than those of RPR1, TRUST1 or TRUST2 (Figure 1). Reagent variations might affect the performance, indicating that quality assurance of reagents by producers is critical.

We also demonstrated that, based on LLDs, non-treponemal tests are less sensitive than treponema-specific tests. Laboratory methods have many performance characteristics that must be understood and assessed for their appropriate use. The first is the smallest amount that the method can reliably detect to determine presence or absence of an analyte. This is the limit of detection (LoD). The second characteristic is the smallest amount the method can reliably measure quantitatively. This is the limit of quantitation (LoQ). The limits of detection and quantitation are critical because detecting extremely small amounts of an analyte can be necessary to define disease states, screen for disease, identify significant exposure or reveal infectious agents.²³

Traditional syphilis testing algorithms use non-treponemal tests as screening methods, and reactive non-treponemal tests are confirmed by treponema-specific tests.¹⁰ As the treponema-specific tests have been developed to be easy to perform and widely available, reverse syphilis testing algorithms have been recommended, using the treponema-specific tests as a screening method.⁹ However, treponema-specific tests are designed to detect treponemal antibodies that persist for many years after treatment. Thus, the tests cannot be used to distinguish active syphilis from past treated infections, and non-treponemal tests are required to confirm active infections.¹⁰

Our study used highly selected serum samples for both patients and controls. The predictive values and cost-effectiveness of the tests could not be assessed. Use of the two algorithms (traditional and reverse) was not compared in the present study. However, selection of algorithms in syphilis laboratory diagnosis shall be discriminated based on high- or low-prevalence populations.⁹ Indeed, reverse screening may identify a higher number of patients with false-positive results than the traditional algorithm. These false-positive results may lead to unnecessary follow-up, treatment, patient anxiety and higher costs.⁹ Non-treponemal tests are required to confirm the POCT-reactive samples. Our study revealed that two RPR and TRUST tests have similar sensitivity and specificity and that non-treponemal tests are less sensitive than treponema-specific tests for LLDs. Further population-based evaluations on selection of screening tests is warranted.

Footnotes

Acknowledgements

This work was supported by the Shanghai Nature Science Fund, Shanghai, China (#09DZ1907104). We are grateful for Dr. Mingmin Liao’s (Vaccine and Infectious Disease Organization, University of Saskatchewan, Canada) contributions to the preparation of, editing and critiques on the manuscript. We thank Dr Yue Chen (University of Ottawa, Canada) for reviewing statistical analysis of this manuscript. The authors would like to thank Lilin Xu (Shanghai Changning Maternity and Infant Health Hospital, Shanghai, China) for assistance in data analysis.

Conflicts of interest

The authors declare no conflict of interest.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

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