Wei-Fang Zhu, Shui-Ying Lei and Lan-Juan Li

Hangzhou, China

Hepatitis C virus infection and biological falsepositive syphilis test: a single-center experience

Wei-Fang Zhu, Shui-Ying Lei and Lan-Juan Li

Hangzhou, China

BACKGROUND:Rapid plasma regain positive and/or treponema pallidum hemagglutination negative [RPR(+)/TPHA(-)] results were designated as biologic false-positive (BFP). There are limited data about BFP reactions against syphilis in patients with hepatitis C virus (HCV) infection. This study aimed to determine the prevalence of BFP reactions for syphilis in patients with HCV infection in a large sample and assess the relationship between BFP reactions and HCV infection.

METHODS:A total of 2656 patients with positive anti-HCV and 5600 healthy control subjects were enrolled in this study. Hepatitis C serology was determined by a second generation ELISA test for HCV antibody. Syphilis serology was determined by the RPR test. Those subjects with reactive RPR positive underwent the TPHA test. Demographics and laboratory data were collected by trained clinicians.

RESULTS:Among 2656 patients, 111 (4.2%) had a reactive RPR test. Of the 111 patients who were subjected to reactive RPR test, 30 (27.0%) showed HCV(+)/RPR(+). Of 5600 healthy controls, 80 (1.4%) had a reactive RPR test. Fourteen (17.5%) controls with HCV(-)/RPR(+) had a non-reactive TPHA test. These represented 1.1% of all HCV-positive and 0.3% of all HCV-negative subjects (P<0.001). A significantly increased prevalence shown by false-positive tests for syphilis was observed in elderly HCV-seropositive patients. BFP-HCV positive group had a higher prevalence of eosinophilia. The eosinophil abnormality was compared between the patients and controls (66.7% vs 21.4%,P=0.0043). No significant results were observed in antinuclear antibodies, antiphospholipid and complement (C3, C4) (P>0.05).

CONCLUSIONS:The data of this study demonstrate that HCV infection is associated with a false-positive RPR test. In this study BFPs were significantly more common in HCV positive patients compared to HCV-negative ones. Eosinophil abnormality can be considered as a predictor for BFP. Excessive BFPs must be considered in assessing the frequency of syphilis in a HCV-positive population and the importance of the treponemal specific serologic test should be emphasized for a diagnosis of syphilis in such population.

(Hepatobiliary Pancreat Dis Int 2011; 10: 399-402)

hepatitis C virus; biological false-positive; syphilis

Introduction

The diagnosis of syphilis requires a two-step serological test. Not infrequently, sensitive screening is reactive, but it is not confirmed by a more specific test yielding a biological false-positive (BFP).[1]The rapid plasma reagin (RPR) test for syphilis screening may, under certain circumstances, yield positive results in patients not infected by Treponemapallidum. Occasional BFP may occur in any of the serological tests for syphilis. In general, BFP is more likely to be seen in autoimmune disease, HIV infection, pregnancy, and intravenous drug abuse.[2-5]The nonrecognition of BFP reaction for syphilis may have negative social and economic implications.

An array of infectious and noninfectious conditions has been reported to cause BFP reaction.[2-5]Hepatitis C virus (HCV) infection has been associated with mixed cryoglobulinemia and immune complex glomerulonephriris,[6,7]but has rarely been investigated as a cause of BFP reactions in a large sample. This study aimed to describe the prevalence of BFP in a large HCV-infected or non-infected population.

Methods

A total of 2656 patients with HCV infection (patientsgroup) and 5600 healthy outpatients (control group) were enrolled in this study. The patients had been treated at the First Affiliated Hospital, Zhejiang University School of Medicine from December 1, 2008 to April 1, 2011.

Healthy controls of comparable age and gender were recruited from our hospital. They had no history of cardiovascular, liver or kidney diseases. Clinical examination and laboratory test (liver and kidney function, hepatitis B surface antigen, HCV antibody and HIV antibody) were normal. The main exclusion criteria of both groups should included incomplete clinical data, current pregnancy, pre-existing hepatitis B, and HIV infection or connective tissue diseases.

HCV antibody of all enrollees was determined by a second generation ELISA (Ortho Diagnostics) test. Syphilis detection was done using the RPR test; those with reactive RPR positive underwent the treponema pallidum hemagglutination (TPHA) test. Autoantibodies [antinuclear antibodies (ANA), antiphospholipid (aPL)] were detected in sera by indirect immunofluorescence. Eosinophil count was determined by an automated blood cells counter (Sysmex XS800i). The levels of complement (C3, C4) were measured by the photometry immuno-turbidimetric test (BECKMAN). The levels of γ-globulin were measured by cellulose acetate membrane electrophoresis (INTERLAB).

Clinical and laboratory data were collected from records through an Excel spreadsheet (version 2003). Stratified analysis was made using the chi-square test or Fisher's exact test. A P value less than 0.05 was considered statistically significant.

Results

Of the 2656 HCV seropositive patients, 1100 were female and 1556 male, with an average age of 37 (range 20-69) years. Among the 5600 healthy subjects, 2101 were female and 3499 male, with an average age of 35 (range 19-71) years (Table 1). No significance was observed in gender and age between the two groups.

One hundred and eleven (4.2%) patients with HCV seropositive and 80 (1.4%) controls with HCV seronegative were RPR reactive (P<0.0001). Thirty (27.0%) patients were HCV(+)/RPR(+) and 14 (17.5%) controls were HCV(-)/RPR(+). All of them had a nonreactive TPHA test and represented 1.1% of HCV-positive and 0.3% of HCV-negative subjects (P<0.001) (Fig. 1).

Laboratory data were compared between the 30 BFP-HCV positive patients and 14 BFP-HCV negative controls (Table 2). False-positive tests for syphilis showed a significantly increased prevalence in elderly HCV seropositive patients (≥60 years old). The BFP-HCV positive group had a higher prevalence of eosinophilia. The eosinophil abnormality was compared between theBFP-HCV positive group and BFP-HCV negative group (66.7% vs 21.4%, P=0.0043). To investigate whether there is an association between BFP and immunological abnormality, we analyzed the results of ANA, aPL and complement (C3, C4). Seven (23.3%) HCV-positive BFP patients were ANA positive. Two (1.4%) patients with HCV-negative BFP were ANA positive. Four (13.3%) HCV-positive BFP patients were aPL positive. One (7.1%) of the HCV-negative BFP patients were aPL positive. Four (1.4%) of the HCV-negative BFP patients were aPL positive. But no significant results were observed in ANA, aPL and complement (C3, C4) (P>0.05, Fig. 2).

Table 1. Demographic and laboratory characteristics of the patients and control groups

Table 2. Demographic characteristics of the BFP group by HCV serostatus

Fig. 1. BFP results as a proportion of reactive syphilis serological tests by HCV serostatus. *: A significantly increased prevalence in RPR reactive HCV between seropositive and controls with HCV seronegative. #: Significance was noted between HCV seropositive patients and HCV seronegative controls.

Fig. 2. Laboratory findings of the BFP group by HCV serostatus. *: P<0.05, BFP-HCV positive group vs BFP-HCV negative group.

We measured γ-globulin in 23 HCV-positive BFP patients and 10 HCV-negative BFP. Six (26.1%) of the HCV-positive BFP patients had a high level of serum γ-globulin. Two (20.0%) of the HCV-negative BFP patients had an abnormal level of serum γ-globulin.

Discussion

BFP serological test for syphilis has long been recognized. Reviewing the pertinent literature revealed that BFP may be associated with vaccinations, infections (infectious mononucleosis, measles, typhoid, varicella, influenza, lymphogranuloma venereum, malaria, etc), pregnancy, connective tissue diseases, multiple blood transfusions.[3,8-11]But the association of BFP reactions for syphilis with HCV has rarely been discussed. HCV infection associated with false-positive RPR test has been found mainly in patients with sexually transmitted disease.[12,13]In African-American women, Augenbraun et al[14]found that patients with HCV infection were 4-4.5 times more likely to have a false-positive reaction for syphilis than anti-HCV-negative patients. However, in this study we investigated HCV infection and BFP in Chinese including males and females in a local center. The HCV-positive group was 5 times more likely to have BFP for syphilis than the HCV-negative control group; this finding is similar to the results reported elsewhere.[12-14]

BFP is known to be associated with autoimmune disease, HIV infection, pregnancy, and hepatitis B.[12,15-17]In our study, we excluded such populations in design. Thus we can explain why there was no difference in the prevalence of autobodies (ANA, aPL) and the level of complement (C3, C4) between the two groups. We observed that the BFP-HCV positive group had a higher prevalence of eosinophil abnormality (P=0.0043). The association with HCV and eosinophilia abnormality in the BFP group was not reported. This association has not been elucidated. We infer that eosinophilia may be associated with active hepatitis, but this needs further investigation. Studies[5,6,12]have confirmed the association of immunoglobulin production disorder with HCV infection. In this study, we also found that a higher prevalence of serum γ-globulin abnormality in the BFP-HCV positive patients compared with the BFPHCV negative patients.

The proposed mechanism by which infectious pathogens cause BFP reactions includes coincidental cross-reactivity of an idiotype of the infecting pathogen with nontreponemal antigens and nonspectifically increased production of antibodies associated with infectious diseases.[16]HCV may be conclusively considered a cause of BFP. It may make an already nonspecific treponemal test less specific. HCV might alter the operational parameters of other treponemal-specific serological tests or serological tests used for other nonsyphilis clinical conditions. In our study, BFP may result from the cross-reactivity of HCV-induced antibodies with nontreponemal antigens in the RPR test.

According to these data, RPR positive in hepatitis C may be BFP with regard to the reaginic tests. Therefore, therapeutic measures should not be initiated without confirmation with a TPHA test.

This study has drawbacks. We have small positive BFP samples because of the lower prevalence rate of BFP in China. Traditionally BFP reactions were classified as acute and chronic ones. We couldn’t get the result because we failed to follow up further.

In conclusion, the data of this study demonstrate that HCV infection is associated with false-positive RPR test results. BFP was more common in HCV-positive patients than in HCV-negative patients. Eosinophil abnormality can be considered as a predictor for BFP. Excessive BFP must be considered in assessing the frequency of syphilis in HCV-positive population as well as the importance of the specific serologic TPHA test in confirming a diagnosis of syphilis in such population.

Acknowledgement

We thank to Dr. Da-Gan Yang and Shu-Juan Song for their technical assistance.

Funding:None.

Ethical approval:Not needed.

Contributors:ZWF and LSY contributed equally to this study. ZWF proposed the study. ZWF and LSY wrote the first draft. All authors contributed to the design and interpretation of the study and to further drafts. LLJ is the guarantor.

Competing interest:No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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Received May 21, 2011

Accepted after revision July 13, 2011

Author Affiliations: State Key Laboratory for Diagnosis and Treatment of Infectious Diseases; Key Laboratory of Infectious Diseases, Zhejiang Province; Department of Infectious Diseases, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China (Zhu WF, Lei SY and Li LJ)

Lan-Juan Li, MD, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases; Key Laboratory of Infectious Diseases, Zhejiang Province; Department of Infectious Diseases, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China (Tel: 86-571-87236340; Email: ljli@zju.edu.cn)

? 2011, Hepatobiliary Pancreat Dis Int. All rights reserved.