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Evaluation of a colloidal gold immunochromatography assay in the detection of Treponema pallidum specific IgM antibody in syphilis serofast reaction patients: a serologic marker for the relapse and infection of syphilis
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Diagnostic Microbiology and Infectious Disease 70 (2011) 10 – 16 www.elsevier.com/locate/diagmicrobio
Evaluation of a colloidal gold immunochromatography assay in the detection of Treponema pallidum specific IgM antibody in syphilis serofast reaction patients: a serologic marker for the relapse and infection of syphilis Li-Rong Lina,1 , Man-Li Tonga,b,1 , Zuo-Gen Fua,b , Bing Dana,b , Wei-Hong Zhengc , Chang-Gong Zhangd , Tian-Ci Yanga,⁎, Zhong-Ying Zhanga,⁎ a
Center of Clinical Laboratory, Zhongshan Hospital, Medical College of Xiamen University, Xiamen 361004, China b Medical College of Xiamen University, Xiamen 361005, China c Department of Neurology, Zhongshan Hospital, Medical College of Xiamen University, Xiamen 361004, China d Boson Biotechnology Co., Ltd., Xiamen 361021, China Received 16 July 2010; accepted 25 November 2010
Abstract Syphilis remains as a worldwide public health problem; hence, it is necessary to develop a new diagnostic approach that is easier and faster than conventional tests. A new testing method to detect Treponema pallidum IgM (TP-IgM), named colloidal gold immunochromatography assay (GICA), is presented in place of fluorescent treponemal antibody absorption (FTA-Abs). TP-IgM was detected using GICA developed on syphilis-specific recombinant proteins TPN17 and TPN47. The FTA-Abs IgM test was set as the gold standard. A GICA TP-IgM test was performed to detect syphilis in 1208 patients who received recommended therapy for syphilis for more than 1 year at the Xiamen Center of Clinical Laboratory in China from June 2005 to May 2009. One hundred blood donors were set up as control. The sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, and negative likelihood ratio were 98.21%, 99.04%, 93.75%, 99.73%, 102.3, and 0.018, respectively. Detection on 500 interference specimens indicated that the biological false-positive rate of the GICA test was extremely low and was free from other biological and chemical factors. The patients were divided into the following experimental groups based on the results of toluidine red unheated serum test (TRUST) and treponemal pallidum particle agglutination (TPPA): (1) the syphilis serofast reaction (SSR) group consisted of 411 cases with (+) TRUST and (+) TPPA, which exhibited no clinical manifestations of syphilis after 1 year of recommended syphilis treatment; (2) the serum cure group, which was further subdivided into group A, a group that consisted of 251 cases with (−) TRUST and (+) TPPA, and (3) group B, a group that consisted of 546 cases with (−) TRUST and (−) TPPA; and (4) the blood donor control group, which consisted of 100 healthy persons with (−) ELISA-TP and (−) TPPA. We used the FTA-Abs method and the GICA method to detect TP-IgM; the positive rate of TP-IgM in 411 SSR patients was 34.55% and 36.01%, respectively. However, in serum cure group A, the positive rate of TP-IgM was 10.36% and 11.16%, respectively. The χ2 test revealed that there is a significant difference in the positive rate between these 2 groups (P b 0.01). The TP-IgM positive rate in the same group, as detected by the GICA method and the FTA-Abs method, had no significant difference in statistics. However, as detected by the GICA method and the FTA-Abs method, all the samples in serum cure group B and the control group were negative for TP-IgM. The TPIgM–positive result demonstrated that active T. pallidum remained in the bodies of SSR patients. In summary, the characteristics of GICA TP-IgM correspond to that of FTA-Abs TP-IgM; this can be used as a serologic marker for the relapse and infection of syphilis in place of the conventional FTA-Abs IgM test. © 2011 Elsevier Inc. All rights reserved. Keywords: Colloidal gold immunochromatography assay; Treponema pallidum; TP-IgM; Fluorescent treponemal antibody absorption; Syphilis serofast reaction
⁎ Corresponding authors. Tel.: +86-592-2993046; fax: +86-592-2993043. E-mail addresses: [email protected] (T.-C. Yang), [email protected] (Z.-Y. Zhang). 1 Li-Rong Lin and Man-Li Tong contributed equally to this work. 0732-8893/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2010.11.015
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1. Introduction According to the national sexually transmitted disease surveillance system and sentinel site network of China, primary and secondary syphilis alone affected 5.7 out of 100 000 people in 2005. The rate of congenital syphilis has increased in recent years, from 0.01 per 100 000 live births in 1991 to 19.68 per 100 000 live births in 2005, with an average yearly rise of 71.9% (Chen et al., 2007). In 2008, a total of 9480 babies were born with congenital syphilis in China, with an average of more than one baby per hour; this rate had increased 12-fold during the previous 5 years (Tucker et al., 2010). To date, cases of syphilis in mainland China spread with a speed that has never been witnessed in any other countries; hence, China is encountering greater difficulties and challenges than before. Before syphilis can be successfully controlled by public health measures, the availability of a highly sensitive diagnostic test and a highly effective and affordable treatment must be initially developed. Nontreponemal test antibody titers usually correlate with disease activity. However, in some patients, nontreponemal antibodies can persist for a long time at a low titer; nontreponemal antibodies may even be permanent in some patients. In a previous study, clinical trial data demonstrated that 15% of patients with early syphilis do not achieve 2 dilution declines in nontreponemal titer after undergoing recommended therapy (Rolfs et al., 1997). This response is called syphilis serofast reaction (SSR) (Workowski and Berman, 2006) or sero-resistance (Higuchi et al., 1961). SSR is one of the manifestations of treatment failure; to date, little is known about the characteristics of SSR. Treponema pallidum specific IgM antibody (TP-IgM) first appears in the body affected by syphilis, and its level remains at a certain level with the existence of active T. pallidum (Herremans et al., 2007). At present, there is no standard time division for SSR. In this study, patients who have positive nontreponemal titers without clinical manifestations after 1 year of recommended syphilis therapy were classified to have SSR. In 1971, Faulk and Taylor (1971) first introduced colloid gold into immunochemistry; in 1990, Osikowicz et al. (1990) established gold immunochromatography assay (GICA), which has now become a new clinical diagnosis method because of its many advantages, including the usage of simple, rapid techniques that require no special equipments and complicated processes. This study aims to develop a GICA to detect TP-IgM on syphilis-specific recombinant proteins TPN17 and TPN47. McKevitt et al. (2003) found at least 12 proteins with strong reactogenicity using systematic cloning and expression methods. Three intimal lipoproteins with molecular weights of 47, 17, and 15 kDa showed stronger antigenicity The protein with a molecular weight of 47 kDa was thought to have the highest abundance of T. pallidum in vivo (Akins et al., 1993; Norgard et al., 1986). It is the antigen with the strongest immunity and the best specificity (Lukehart et al., 1986). TPN17 antigen is one of the most important structural
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proteins of T. pallidum, which plays pivotal roles in the immunity of T. pallidum infections. Thus, the TPN47 and TPN17 genes became the first choice for studying a syphilisspecific diagnostic kit (Lin, Fu, and Dan, 2010). It also aims to assess the diagnostic performance, specificity, and sensitivity of GICA TP-IgM for routine laboratory use, as well as to investigate whether GICA TP-IgM can be used as a serologic marker for the relapse and infection of syphilis, in place of the conventional fluorescent treponemal antibody absorption T. pallidum IgM (FTA-Abs IgM) test. 2. Materials and methods 2.1. Sera From June 2005 to May 2009, 1208 cases of syphilis patients with complete patient data were clinically diagnosed with syphilis based on clinical, laboratory, and national guidelines at the Xiamen Center of Clinical Laboratory in China. The following criteria were used in choosing the subjects: i) patients received recommended therapy for syphilis for more than 1 year and the clinical manifestations disappeared (syphilis treatment recommended by the Ministry of Health Expert Advisory Committee on sexually transmitted diseases, China, 2005); (ii) pretreatment syphilis titers (toluidine red unheated serum test [TRUST] and treponemal pallidum particle agglutination [TPPA]) of patients were positive; iii) integrated follow-up data were available; iv) patients had negative HIV antibodies; v) patients who suffered from reinfection were excluded; and vi) patients with diseases associated with autoantibodies were excluded. After treatment, the patients were asked to periodically review their serum TRUST and TPPA every 3 months. The male-to-female gender ratio was 1.29 (681/527), and the average age was 42 years (age range, 1–94 years). Based on the results of TRUST and TPPA, 3 experimental groups (patients groups) + 1 control group (blood donor group) = 4 groups was also set up. Group 1. SSR group: 411 cases (190 males and 221 females) with (+) TRUST and (+) TPPA and without clinical manifestations after 1 year of recommended treatment of syphilis. Group 2. Serum cure group A: 251 cases (163 males and 88 females) with (−) TRUST and (+) TPPA, which originate from 1208 cases. Group 3. Serum cure group B: 546 cases (328 males and 218 females) with (−) TRUST and (−) TPPA, which originate from 1208 cases. Group 4. Blood donor group: 100 sera samples from 50 healthy males and 50 healthy females with an average age of 28 years. The samples were previously screened by ELISA (InTec Products, Xiamen, China) and TPPA (Fujirebio, Tokyo, Japan) at the blood bank in Putian, Fujian Province, China. All serum samples were drawn from patients with empty stomachs and then placed at 4 °C if the patients were tested within 24 h. Otherwise, the samples were divided in small
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aliquots and stored at −70 °C to avoid repeated freezing and thawing. 2.2. Development of a colloidal GICA to detect IgM antibodies to T. pallidum with TPN17 and TPN47 Based on the literature and the production technology on the GICA TP-IgM of the national patent of People's Republic of China (Lin, Fu, and Dan, 2010; Lin, Zhang, and Yang, 2010), we developed the GICA TP-IgM method. The simplified approach is explained in the passages that follow. 2.2.1. Preparation of syphilis recombinant antigen TPN17 and TNP47 The T. pallidum Nichols strain and the pMD18-T vector were from Boson Biotechnology (Xiamen, China). Plasmid pET-22b (+), Escherichia coli DH5α, and BL21 (DE3) were donations from the School of Life Sciences, Xiamen University. The T. pallidum antigen DNA was amplified using the gene clone technology and then expressed in E. coli. The recombinant antigens of T. pallidum TPN17 and TPN47 were prepared and purified (Zhengrong et al., 2000). 2.2.2. Sample application on nitrocellulose membrane The monoclonal antibody to the human μ chain-specific IgM was coated on the “patient area” (T) of the nitrocellulose membrane, whereas the antibody of goat-anti-syphilis antigens (TPN17 and TPN47) was coated on the “control area” (C). The membrane was then dried and kept tightly sealed at room temperature. The concentration of monoclonal antibody to human μ chain-specific IgM was 1 mg/mL, whereas the antibody of goat-anti-syphilis antigens consisted of TPN17-IgG antibody and TPN47-IgG antibody (v/v = 1:1) with a final concentration of 1 mg/mL. The loading sample amount was 1 μL/cm. 2.2.3. Preparation of colloided gold A tri-sodium citrate reduction method was used to produce colloidal gold with a diameter of 25 nm. A solution of 1 mL 1% chloroauric acid (Sigma Resource and Technologies, Inc., Shanghai, China) was diluted with 100 mL deionized double-distilled water to achieve a final concentration of 0.01% and then heated to boiling point in a flask with a condensing unit. A 2 mL 1% sodium citrate solution was added. The mixture was heated until the solution showed a wine-red color. After cooling, the solution was kept in a brown bottle at 4 °C after cooling. 2.2.4. Preparation of colloidal gold-conjugated TPN17 and TPN47 TPN17 was taken as an example. After adjusting the pH to 5.4 with 0.1 mol/L NaOH, 10 mL colloid gold was mixed with TPN17, and 1 mL 5% BSA was added after 5 min. Then, the mixture was centrifuged for 1 h at 4 °C and 10 000 r/min (20 min). The supernatant was decanted and the pellet was dissolved in 10 mL Tris-buffered saline (TBS) buffer. After centrifugation at 4 °C and 10 000 r/min, the pellet was
again dissolved in 1 mL TBS buffer (Bruning et al., 1999; Paek et al., 2000; Reid et al., 2001). The colloidal gold-labeled TPN47 was produced in the same method. Purified colloidal gold-labeled TPN47 and TPN17 (v/v = 1/1) were mixed together, coated on glass fiber paper, and kept sealed after being dried at 37 °C. 2.2.5. Preparation of immunochromatographic test strips An immobilized fibrous membrane, colloidal goldconjugated glass fiber, and absorbent paper were grouped together by a polyvinyl chloride self-adhesive floor and then cut into strips by a slitter. The strips were kept in a tightly sealed container. 2.2.6. GICA TP-IgM test Determination of T. pallidum is based on the reaction between the colloidal gold-conjugated T. pallidum antigen and antitreponemal IgM antibodies present in the patient serum. A 10-μL specimen was dropped at the “patient area” of the immunochromatographic test strips, and the result was observed after adding 100 μL normal saline for 20 min. The T. pallidum results were read blindly by 3 independent observers. An agreement was recorded until all 3 observers interpreted the same result for each sample. A positive result showed 2 red lines on designated strip zones, which represent the “patient area” and the “control area,” respectively. A negative result showed only one red line on the “control area.” The absence of band was considered as an indication of invalid result. Observers also scored their perception about the ease of use of the T. pallidum test, with grades ranging from “low,” “medium,” or “high.” 2.2.7. Cross-reactivity and interference of GICA TP-IgM test To study the cross-reactivity and interference of GICA TP-IgM test, 500 sera samples were collected from patients with other pathologies, as well as from special clinical specimens that may bring false-positive results, by the Xiamen Center of Clinical Laboratory. The detailed composition were as follows: 48 cases had rheumatoid disease; 42 cases had active systemic lupus erythematosus (SLE); 4 cases had human immunodeficiency (HIV); 90 cases had hepatitis B; 40 cases had hepatitis C; 14 cases had herpes simplex virus type II (HSV-II); 10 cases had toxoplasmosis; 12 cases had biological false positives (BFPs); 30 cases were pregnant; and 210 cases were special clinical specimens (70 from hemolytic samples, 70 from lipemic samples, and 70 from icteric samples). 2.3. Serologic tests Laboratory analysis for each sample was performed with the following tests: TRUST (InTec Products), TPPA (Fujirebio), and FTA-Abs IgM (EUROIMMUN Medizinische Labordiagnostika, Schleswig-Holstein, Germany). The tests were carried out according to the instructions of the manufacturers. For TRUST, sera samples were tested at dilutions of 1:1 to 1:32 in a physiological solution (NaCl
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0.9%) to avoid prozone effects and false-negative results. Readings were taken immediately with the naked eye through comparison with negative and positive controls. The sera samples reactive for TRUST were quantified in 2fold serial dilutions until the end was determined. The initial dilution of sera samples in TPPA reactions was 1:80. For FTA-Abs IgM, sera samples were considered reactive with initial dilutions of 1:10 in the sorbent. Sera samples that produced conflicting or inconclusive results for a particular technique were tested in duplicate; the new result was considered as the true result. 2.4. Testimony of diagnosis of syphilis Testimonies were in accordance with the national guidelines (SBTS/MOH, 1996). 2.5. Establishment of GICA gold standard Sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, and negative likelihood ratio were calculated for GICA TP-IgM and compared with FTA-Abs IgM as a gold standard. Thus, for calculation purposes, only samples that tested positive for FTA-Abs IgM were considered as true positives. The gold standard for a negative result was a negative FTA-Abs IgM test. 2.6. Statistical analysis All statistical analyses were conducted using SPSS v13 for Windows. χ2 tests were used to determine the significant differences across groups. P b 0.05 (2-sided) was considered significant. 3. Results 3.1. Reactivity of the TRUST, TPPA, FTA-Abs IgM, and GICA TP-IgM tests among different groups Out of the 1208 patients who received recommended therapy for syphilis, 441 cases were still nontreponemal and treponemal antibody positive in the prospective therapy duration, with a SSR incidence of 34.02%. Detection of TPIgM with FTA-Abs showed that 142 cases in 411 SSR patients (group 1) were positive; hence, that the positive rate was 34.55%. In contrast, only 23 out of 251 cases with (−) TRUST but (+) TPPA (group 2) had (+) TP-IgM test, yielding a positive rate of 10.360%. The positive rates of groups 1 and 2 in the GICA method were 36.01% and 11.16%, respectively. TP-IgM detection by FTA-Abs and GICA both showed significant differences (P b 0.01) in the positive rate between groups 1 and 2. The positive rate of the GICA method was higher than that of the FTA-Abs method; however, the methods were not significantly different from each other. Analysis of the total number of cases showed that 168 out of 1308 cases were FTA-Abs positive; the positive rate was 12.84%, which was lower than that
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detected by the GICA method (13.46%). However, no significant statistical differences exist. Groups 3 and 4 were negative for TP-IgM (Table 1). 3.2. Analysis of sensitivity and specificity of GICA A total of 1308 cases, including syphilis patients and blood donors, underwent syphilis serology test. The number of syphilis-positive serum, as detected by the GICA method and the FTA-Abs method, was 176 and 168, respectively. A total of 165 samples of serum was determined positive using the FTA-Abs IgM test among the 176 samples of GICA TPIgM–positive serum. Among the 1132 GICA TP-IgM– negative specimens, 3 cases were positive and 1129 were negative in the FTA-Abs IgM test. Out of 168 FTA-Abs IgM–positive cases, 165 were determined to be GICA TPIgM–positive, whereas 3 were negative. The T. pallidum GICA TP-IgM test and FTA-Abs IgM test revealed a significant agreement in the overall results. The large number of negative results (86.31%) and positive results (12.61%) caused the significant agreement. The FTA-Abs IgM test is considered as the gold standard in the detection of TP-IgM. The estimated sensitivity and specificity of the FTA-Abs IgM assay as a gold standard was 100%. The parameters of the GICA TP-IgM assay were as follows: sensitivity rate of 98.21% (165/168), specificity rate of 99.04% (1129/1140), positive predictive value of 93.75% (165/176), negative predictive value of 99.73% (1129/1132), positive likelihood ratio of 102.3 (98.21/0.96), and negative likelihood ratio of 0.018 (1.79/99.04) (Table 2). 3.3. Cross-reactivity and interference of GICA TP-IgM A total of 500 sera samples from patients with other pathologies and aberrant specimens were collected by the Xiamen Center of Clinical Laboratory to be tested for reactivity using the GICA TP-IgM test. No cross-reactivity Table 1 Reactivity of the TRUST, TPPA, FTA-Abs IgM, and GICA TP-IgM tests among different experimental groups Total cases Group 1 Group 2 Group 3 Group 4 Total
411 251 546 100 1308
TRUST
411 0 0 0 411
TPPA
411 251 0 0 662
FTA-Abs IgM
GICA TP-IgM
No.
No.
%
148 28 0 0 176
36.01b,c 11.16d 0 0 13.46e
142 26 0 0 168
% a
34.55 10.36 0 0 12.84
a FTA-Abs-IgM positive rate comparison between group 1 and group 2, χ2 = 48.157, P b 0.01. b GICA-IgM positive rate comparison between group 1 and group 2, χ2 = 49.321, P b 0.01. c FTA-Abs-IgM and GICA-IgM positive rate comparison in group 1, χ2 = 0.192, P N 0.05. d FTA-Abs-IgM and GICA-IgM positive rate comparison in group 2, χ2 = 0.083, P N 0.05. e FTA-Abs-IgM and GICA-IgM positive rate comparison in all cases, χ2 = 0.214, P N 0.05.
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Table 2 Comparison of FTA-Abs IgM and the GICA TP-IgM test results GICA TP-IgM
FTA-Abs IgM (gold standard) Positive
Negative
Positive Negative Total
165 (12.61%) 3 (0.23%) 168 (12.84%)
11 (0.85%) 1129 (86.31%) 1140 (87.16%)
Total
176 (13.46%) 1132 (86.54%) 1308 (100%)
was detected in GICA TP-IgM tests with serum from patients with rheumatoid disease, SLE, and toxoplasmosis, or patients who were afflicted with HIV, HBV, HCV, and HSV-II. Hemolytic, lipemic, and icteric samples showed no interferences, except for one hemolytic serum, which showed a red line at the “low” grade (Table 3). 3.4. Stability assay Differences within a group: Strips from the same batch were used to confirm the reactive sera that were used as controls (FTA-Abs-proven TP-IgM–positive serum at high, medium, and low grades). No difference in the color depth of the bands was found on the identical titer, and the negativeserum was proven negative. Differences among groups: Strips from the different batches were used to confirm the reactive sera that were used as controls (FTA-Abs–proven TP-IgM–positive serum at high, medium, and low grades). No difference in the color depth of the bands was found on the identical titer, and the negative-serum was proven negative. To further evaluate the stability of the reagents, we treated the reagent for GICA TP-IgM tests using the following procedure. The reagents were first kept at 4 °C for 18 months. The reagents were tested every 2 months to detect reactive sera that were used as controls (FTAAbs-proven TP-IgM–positive serum at high, medium, and low grades). No difference in the color depth of the bands
Table 3 Reactivity of the GICA TP-IgM test in sera from 500 individuals with interference Category
Rheumatoid disease SLE HIV HBV HCV HSV-II Toxoplasmosis Pregnant women BFP Hemolytic Lipemic Icteric
Gender Male Female
Average No. of GICA TP-IgM age sera tested No. of sera reactive
17 11 3 45 20 4 0 0 4 35 35 35
36 40 34 36 33 28 27 27 28 36 42 37
30 31 1 45 20 10 10 30 8 35 35 35
was found on the identical titer, and the negative-serum was proven negative.
48 42 4 90 40 14 10 30 12 70 70 70
0 0 0 0 0 0 0 0 0 1 0 0
4. Discussion Two criteria define syphilis cure: clinical cure and serum cure. Syphilis patients were classified as clinical cure if the clinical manifestations of syphilis disappeared after the recommended syphilis therapy. Patients who tested also negative in the serum nontreponemal test antibody were classified as serum cure. SSR is one of the manifestations of treatment failure, which is common in clinical treatments. Out of the 1208 patients who received recommended therapy for syphilis, 441 cases were still nontreponemal and treponemal antibody positive in the prospective treatment duration, with an SSR incidence of 34.02%, higher than what was reported by Rolfs on the serum resistance ratio of early syphilis (Rolfs et al., 1997). The reason behind the continuous occurrence of SSR after treatment remains unknown. T. pallidum escapes from the immune response of hosts by impairing the amount of membrane proteins, including their immunogenicity, and finally leading to long-term infection and contamination of hosts (Weigel et al., 1992). In addition, the changes of T. pallidum antigenic structure, as well as the different genotype and the different genetic background in organisms were also related to the occurrence of SSR to some extent. Experiments performed in rabbit models had proven that the Tp rK gene in all subsets of T. pallidum were significant variables, which may help T. pallidum escape from the immune system of hosts and finally lead to chronic infection (LaFond et al., 2006). All the above are important reasons behind the relapse and infection of syphilis. Serologic tests are divided into 2 categories, namely, nontreponemal and treponemal antibody tests. Treponemal antibodies, IgM and IgG, are generated after 2 and 4 weeks, respectively. TP-IgM first appears in the body affected by syphilis, and then its level remains at a certain plane with the existence of active T. pallidum (Herremans et al., 2007). Another nontreponemal antibody is generated after infection for 5–7 weeks (Juarez-Figueroa et al., 2007). Nontreponemal test antibody titers usually correlate with disease activity; thus, it is regarded as basis of verdict for serum cure. The FTA-Abs method and the GICA method were used to detect TP-IgM, and both revealed a TP-IgM positive rate of 34.55% and 36.01% in 411 SSR patients, respectively. The results show that active T. pallidum remains in the body of SSR patients. Although the possibility of infection cannot be excluded completely when TP-IgM is negative, the findings reveal that patients positive for TP-IgM are infectious and can relapse. In serum cure group A, the TPIgM positive rate was both above 10% with the FTA-Abs method and the GICA method, indicating that active T. pallidum still exists in vivo, and proper treatment measures
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should be provided. Apparently, serum cure cannot be properly determined based only on negative nontreponemal antibody test. In contrast, all patients in serum cure group B were negative for TP-IgM. The results of this study also showed that the TP-IgM positive rate is not significantly different among patients with different ages, genders, and clinical phases after 1 year of recommended therapy. To date, the FTA-ABS test is considered as the reference method for TP-IgM detection, as well as the standard confirmatory test for syphilis, except in very early infection. However, the professional training for diagnostic tests, specific equipment, and the detection time of FTAAbs TP-IgM is about 2.5–3.5 h (depending on the skill level of operator), and costly reagents (46 Yuan according the quoted price from EUROIMMUN Medizinische Labordiagnostika in China) affect the prevention and control of syphilis in developing countries, especially in rural areas (Lin, Fu, and Dan, 2010). Therefore, we need not only use specific and precise test methods but also convenient, fast, and cheap reagents to screen patients with syphilis. This can help in the clinical diagnosis and epidemiologic survey of syphilis. After simple training, nonprofessionals could master and operate the GICA test. The result can be directly read with the naked eyes. The detection time is 20 min, and the cost is low. After mass production, the market price of GICA strips must be within 2.5 Yuan (Lin, Fu, and Dan, 2010). We used syphilisspecific recombinant protein as the antigen of the reagents, set FTA-Abs-IgM as the gold standard, and developed rapid test reagents based on GICA technology. This method has 98.21% sensitivity, 99.04% specificity, 93.75% positive predictive value, 99.73% negative predictive value, 102.3 positive likelihood ratio, and 0.018 negative likelihood ratio. Meanwhile, extra weight was given to sera from subjects with pathogenic virus infections, autoimmune diseases, and peculiar clinical specimens to challenge the GICA TP-IgM test. Only one case of hemolytic serum was indicated as a false positive (low reaction) among 500 sera samples collected from patients with other pathologies and peculiar clinical specimens that are free from serum hemolysis, lipemia, jaundice, and so on. In summary, the GICA TP-IgM test is specific and sensitive for routine laboratory use, and its characteristics correspond to that of FTA-Abs IgM (EUROIMMUN Medizinische Labordiagnostika). As rapid treponemal tests emerge because of their enhanced sensitivity, userfriendly qualities, convenience (i.e., no extra equipment), stability at room temperature, and reasonable price, the traditional procedure for laboratory diagnosis of syphilis in developing countries may change (Lin, Fu, and Dan, 2010; Lin, Zhang, and Yang, 2010). Therefore, the GICA TPIgM method can be used as a serologic marker for the relapse and infection of syphilis in place of the conventional FTA-Abs IgM test.
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Acknowledgments This study was supported by the Natural Science Foundation of Fujian Province (grant no. 2009J01200), the Key projects in Fujian Province Science and Technology Program (grant no. 2009D019), and the projects of Xiamen Science and Technology Program (grant no. 3502Z20089015, 3502Z20084019). References Akins DR, Purcell BK, Mitra MM, Norgard MV, Radolf JD (1993) Lipid modification of the 17-kilodalton membrane immunogen of Treponema pallidum determines macrophage activation as well as amphiphilicity. Infect Immun 61:1202–1210. Bruning A, Bellamy K, Talbot D, Anderson J (1999) A rapid chromatographic strip test for the pen-side diagnosis of rinderpest virus. J Virol Methods 81:143–154. Chen ZQ, Zhang GC, Gong XD, Lin C, Gao X, Liang GJ, Yue XL, Chen XS, Cohen MS (2007) Syphilis in China: results of a national surveillance programme. Lancet 369:132–138. Faulk WP, Taylor GM (1971) An immunocolloid method for the electron microscope. Immunochemistry 8:1081–1083. Herremans M, Notermans DW, Mommers M, Kortbeek LM (2007) Comparison of a Treponema pallidum IgM immunoblot with a 19S fluorescent treponemal antibody absorption test for the diagnosis of congenital syphilis. Diagn Microbiol Infect Dis 59:61–66. Higuchi K, Urabe H, Yoshida M (1961) Some problems on seroresistant syphilis. Kyushu J Med Sci 12:283. Juarez-Figueroa L, Uribe-Salas F, Garcia-Cisneros S, Olamendi-Portugal M, Conde-Glez CJ (2007) Evaluation of a rapid strip and a particle agglutination tests for syphilis diagnosis. Diagn Microbiol Infect Dis 59:123–126. LaFond RE, Molini BJ, Van Voorhis WC, Lukehart SA (2006) Antigenic variation of TprK V regions abrogates specific antibody binding in syphilis. Infect Immun 74:6244–6251. Lin LR, Fu ZG, Dan B (2010) Development of a colloidal goldimmunochromatography assay to detect immunoglobulin G antibodies to Treponema pallidum with TPN17 and TPN47. Diagn Microbiol Infect Dis 68:193–200. Lin L-R, Zhang Z-Y, Yang T-C (2010). Preparation methods of a colloidal gold-immunochromatography assay to detect immunoglobulin M antibodies to Treponema pallidum. patent no. 201010179134.6. Lukehart SA, Baker-Zander SA, Sell S (1986) Characterization of the humoral immune response of the rabbit to antigens of Treponema pallidum after experimental infection and therapy. Sex Transm Dis 13:9–15. McKevitt M, Patel K, Smajs D, Marsh M, McLoughlin M, Norris SJ, Weinstock GM, Palzkill T (2003) Systematic cloning of Treponema pallidum open reading frames for protein expression and antigen discovery. Genome Res 13:1665–1674. Norgard MV, Chamberlain NR, Swancutt MA, Goldberg MS (1986) Cloning and expression of the major 47-kilodalton surface immunogen of Treponema pallidum in Escherichia coli. Infect Immun 54:500–506. Osikowicz G, Beggs M, Brookhart P, Caplan D, Ching S, Eck P, Gordon J, Richerson R, Sampedro S, Stimpson D, et al (1990) One-step chromatographic immunoassay for qualitative determination of choriogonadotropin in urine. Clin Chem 36:1586. Paek SH, Lee SH, Cho JH, Kim YS (2000) Development of rapid one-step immunochromatographic assay. Methods 22:53–60. Reid SM, Ferris NP, Bruning A, Hutchings GH, Kowalska Z, Akerblom L (2001) Development of a rapid chromatographic strip test for the
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pen-side detection of foot-and-mouth disease virus antigen. J Virol Methods 96:189–202. Rolfs R, Joesoef M, Hendershot E, Rompalo A, Augenbraun M, Chiu M, Bolan G, Johnson S, French P, Steen E (1997) A randomized trial of enhanced therapy for early syphilis in patients with and without human immunodeficiency virus infection. N Engl J Med 337:307. SBTS/MOH (1996) National standard of the People's Republic of China: diagnostic criteria and management of syphilis (GB 15974-1995). Beijing: Standards Press of China.
Tucker JD, Chen XS, Peeling RW (2010) Syphilis and social upheaval in China. N Engl J Med 362:1658–1661. Weigel LM, Brandt ME, Norgard MV (1992) Analysis of the N-terminal region of the 47-kilodalton integral membrane lipoprotein of Treponema pallidum. Infect Immun 60:1568–1576. Workowski K, Berman S (2006) Sexually transmitted diseases treatment guidelines, 2006. MMWR Recomm Rep 55(RR-11). Zhengrong S, Hao S, Runming L (2000) Cloning and expression of Treponema pallidum and application on the clinical detection. Chin J Microbiol Immunol 02:175–178.
Diagnostic Microbiology and Infectious Disease 70 (2011) 10 – 16 www.elsevier.com/locate/diagmicrobio
Evaluation of a colloidal gold immunochromatography assay in the detection of Treponema pallidum specific IgM antibody in syphilis serofast reaction patients: a serologic marker for the relapse and infection of syphilis Li-Rong Lina,1 , Man-Li Tonga,b,1 , Zuo-Gen Fua,b , Bing Dana,b , Wei-Hong Zhengc , Chang-Gong Zhangd , Tian-Ci Yanga,⁎, Zhong-Ying Zhanga,⁎ a
Center of Clinical Laboratory, Zhongshan Hospital, Medical College of Xiamen University, Xiamen 361004, China b Medical College of Xiamen University, Xiamen 361005, China c Department of Neurology, Zhongshan Hospital, Medical College of Xiamen University, Xiamen 361004, China d Boson Biotechnology Co., Ltd., Xiamen 361021, China Received 16 July 2010; accepted 25 November 2010
Abstract Syphilis remains as a worldwide public health problem; hence, it is necessary to develop a new diagnostic approach that is easier and faster than conventional tests. A new testing method to detect Treponema pallidum IgM (TP-IgM), named colloidal gold immunochromatography assay (GICA), is presented in place of fluorescent treponemal antibody absorption (FTA-Abs). TP-IgM was detected using GICA developed on syphilis-specific recombinant proteins TPN17 and TPN47. The FTA-Abs IgM test was set as the gold standard. A GICA TP-IgM test was performed to detect syphilis in 1208 patients who received recommended therapy for syphilis for more than 1 year at the Xiamen Center of Clinical Laboratory in China from June 2005 to May 2009. One hundred blood donors were set up as control. The sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, and negative likelihood ratio were 98.21%, 99.04%, 93.75%, 99.73%, 102.3, and 0.018, respectively. Detection on 500 interference specimens indicated that the biological false-positive rate of the GICA test was extremely low and was free from other biological and chemical factors. The patients were divided into the following experimental groups based on the results of toluidine red unheated serum test (TRUST) and treponemal pallidum particle agglutination (TPPA): (1) the syphilis serofast reaction (SSR) group consisted of 411 cases with (+) TRUST and (+) TPPA, which exhibited no clinical manifestations of syphilis after 1 year of recommended syphilis treatment; (2) the serum cure group, which was further subdivided into group A, a group that consisted of 251 cases with (−) TRUST and (+) TPPA, and (3) group B, a group that consisted of 546 cases with (−) TRUST and (−) TPPA; and (4) the blood donor control group, which consisted of 100 healthy persons with (−) ELISA-TP and (−) TPPA. We used the FTA-Abs method and the GICA method to detect TP-IgM; the positive rate of TP-IgM in 411 SSR patients was 34.55% and 36.01%, respectively. However, in serum cure group A, the positive rate of TP-IgM was 10.36% and 11.16%, respectively. The χ2 test revealed that there is a significant difference in the positive rate between these 2 groups (P b 0.01). The TP-IgM positive rate in the same group, as detected by the GICA method and the FTA-Abs method, had no significant difference in statistics. However, as detected by the GICA method and the FTA-Abs method, all the samples in serum cure group B and the control group were negative for TP-IgM. The TPIgM–positive result demonstrated that active T. pallidum remained in the bodies of SSR patients. In summary, the characteristics of GICA TP-IgM correspond to that of FTA-Abs TP-IgM; this can be used as a serologic marker for the relapse and infection of syphilis in place of the conventional FTA-Abs IgM test. © 2011 Elsevier Inc. All rights reserved. Keywords: Colloidal gold immunochromatography assay; Treponema pallidum; TP-IgM; Fluorescent treponemal antibody absorption; Syphilis serofast reaction
⁎ Corresponding authors. Tel.: +86-592-2993046; fax: +86-592-2993043. E-mail addresses: [email protected] (T.-C. Yang), [email protected] (Z.-Y. Zhang). 1 Li-Rong Lin and Man-Li Tong contributed equally to this work. 0732-8893/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2010.11.015
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1. Introduction According to the national sexually transmitted disease surveillance system and sentinel site network of China, primary and secondary syphilis alone affected 5.7 out of 100 000 people in 2005. The rate of congenital syphilis has increased in recent years, from 0.01 per 100 000 live births in 1991 to 19.68 per 100 000 live births in 2005, with an average yearly rise of 71.9% (Chen et al., 2007). In 2008, a total of 9480 babies were born with congenital syphilis in China, with an average of more than one baby per hour; this rate had increased 12-fold during the previous 5 years (Tucker et al., 2010). To date, cases of syphilis in mainland China spread with a speed that has never been witnessed in any other countries; hence, China is encountering greater difficulties and challenges than before. Before syphilis can be successfully controlled by public health measures, the availability of a highly sensitive diagnostic test and a highly effective and affordable treatment must be initially developed. Nontreponemal test antibody titers usually correlate with disease activity. However, in some patients, nontreponemal antibodies can persist for a long time at a low titer; nontreponemal antibodies may even be permanent in some patients. In a previous study, clinical trial data demonstrated that 15% of patients with early syphilis do not achieve 2 dilution declines in nontreponemal titer after undergoing recommended therapy (Rolfs et al., 1997). This response is called syphilis serofast reaction (SSR) (Workowski and Berman, 2006) or sero-resistance (Higuchi et al., 1961). SSR is one of the manifestations of treatment failure; to date, little is known about the characteristics of SSR. Treponema pallidum specific IgM antibody (TP-IgM) first appears in the body affected by syphilis, and its level remains at a certain level with the existence of active T. pallidum (Herremans et al., 2007). At present, there is no standard time division for SSR. In this study, patients who have positive nontreponemal titers without clinical manifestations after 1 year of recommended syphilis therapy were classified to have SSR. In 1971, Faulk and Taylor (1971) first introduced colloid gold into immunochemistry; in 1990, Osikowicz et al. (1990) established gold immunochromatography assay (GICA), which has now become a new clinical diagnosis method because of its many advantages, including the usage of simple, rapid techniques that require no special equipments and complicated processes. This study aims to develop a GICA to detect TP-IgM on syphilis-specific recombinant proteins TPN17 and TPN47. McKevitt et al. (2003) found at least 12 proteins with strong reactogenicity using systematic cloning and expression methods. Three intimal lipoproteins with molecular weights of 47, 17, and 15 kDa showed stronger antigenicity The protein with a molecular weight of 47 kDa was thought to have the highest abundance of T. pallidum in vivo (Akins et al., 1993; Norgard et al., 1986). It is the antigen with the strongest immunity and the best specificity (Lukehart et al., 1986). TPN17 antigen is one of the most important structural
11
proteins of T. pallidum, which plays pivotal roles in the immunity of T. pallidum infections. Thus, the TPN47 and TPN17 genes became the first choice for studying a syphilisspecific diagnostic kit (Lin, Fu, and Dan, 2010). It also aims to assess the diagnostic performance, specificity, and sensitivity of GICA TP-IgM for routine laboratory use, as well as to investigate whether GICA TP-IgM can be used as a serologic marker for the relapse and infection of syphilis, in place of the conventional fluorescent treponemal antibody absorption T. pallidum IgM (FTA-Abs IgM) test. 2. Materials and methods 2.1. Sera From June 2005 to May 2009, 1208 cases of syphilis patients with complete patient data were clinically diagnosed with syphilis based on clinical, laboratory, and national guidelines at the Xiamen Center of Clinical Laboratory in China. The following criteria were used in choosing the subjects: i) patients received recommended therapy for syphilis for more than 1 year and the clinical manifestations disappeared (syphilis treatment recommended by the Ministry of Health Expert Advisory Committee on sexually transmitted diseases, China, 2005); (ii) pretreatment syphilis titers (toluidine red unheated serum test [TRUST] and treponemal pallidum particle agglutination [TPPA]) of patients were positive; iii) integrated follow-up data were available; iv) patients had negative HIV antibodies; v) patients who suffered from reinfection were excluded; and vi) patients with diseases associated with autoantibodies were excluded. After treatment, the patients were asked to periodically review their serum TRUST and TPPA every 3 months. The male-to-female gender ratio was 1.29 (681/527), and the average age was 42 years (age range, 1–94 years). Based on the results of TRUST and TPPA, 3 experimental groups (patients groups) + 1 control group (blood donor group) = 4 groups was also set up. Group 1. SSR group: 411 cases (190 males and 221 females) with (+) TRUST and (+) TPPA and without clinical manifestations after 1 year of recommended treatment of syphilis. Group 2. Serum cure group A: 251 cases (163 males and 88 females) with (−) TRUST and (+) TPPA, which originate from 1208 cases. Group 3. Serum cure group B: 546 cases (328 males and 218 females) with (−) TRUST and (−) TPPA, which originate from 1208 cases. Group 4. Blood donor group: 100 sera samples from 50 healthy males and 50 healthy females with an average age of 28 years. The samples were previously screened by ELISA (InTec Products, Xiamen, China) and TPPA (Fujirebio, Tokyo, Japan) at the blood bank in Putian, Fujian Province, China. All serum samples were drawn from patients with empty stomachs and then placed at 4 °C if the patients were tested within 24 h. Otherwise, the samples were divided in small
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aliquots and stored at −70 °C to avoid repeated freezing and thawing. 2.2. Development of a colloidal GICA to detect IgM antibodies to T. pallidum with TPN17 and TPN47 Based on the literature and the production technology on the GICA TP-IgM of the national patent of People's Republic of China (Lin, Fu, and Dan, 2010; Lin, Zhang, and Yang, 2010), we developed the GICA TP-IgM method. The simplified approach is explained in the passages that follow. 2.2.1. Preparation of syphilis recombinant antigen TPN17 and TNP47 The T. pallidum Nichols strain and the pMD18-T vector were from Boson Biotechnology (Xiamen, China). Plasmid pET-22b (+), Escherichia coli DH5α, and BL21 (DE3) were donations from the School of Life Sciences, Xiamen University. The T. pallidum antigen DNA was amplified using the gene clone technology and then expressed in E. coli. The recombinant antigens of T. pallidum TPN17 and TPN47 were prepared and purified (Zhengrong et al., 2000). 2.2.2. Sample application on nitrocellulose membrane The monoclonal antibody to the human μ chain-specific IgM was coated on the “patient area” (T) of the nitrocellulose membrane, whereas the antibody of goat-anti-syphilis antigens (TPN17 and TPN47) was coated on the “control area” (C). The membrane was then dried and kept tightly sealed at room temperature. The concentration of monoclonal antibody to human μ chain-specific IgM was 1 mg/mL, whereas the antibody of goat-anti-syphilis antigens consisted of TPN17-IgG antibody and TPN47-IgG antibody (v/v = 1:1) with a final concentration of 1 mg/mL. The loading sample amount was 1 μL/cm. 2.2.3. Preparation of colloided gold A tri-sodium citrate reduction method was used to produce colloidal gold with a diameter of 25 nm. A solution of 1 mL 1% chloroauric acid (Sigma Resource and Technologies, Inc., Shanghai, China) was diluted with 100 mL deionized double-distilled water to achieve a final concentration of 0.01% and then heated to boiling point in a flask with a condensing unit. A 2 mL 1% sodium citrate solution was added. The mixture was heated until the solution showed a wine-red color. After cooling, the solution was kept in a brown bottle at 4 °C after cooling. 2.2.4. Preparation of colloidal gold-conjugated TPN17 and TPN47 TPN17 was taken as an example. After adjusting the pH to 5.4 with 0.1 mol/L NaOH, 10 mL colloid gold was mixed with TPN17, and 1 mL 5% BSA was added after 5 min. Then, the mixture was centrifuged for 1 h at 4 °C and 10 000 r/min (20 min). The supernatant was decanted and the pellet was dissolved in 10 mL Tris-buffered saline (TBS) buffer. After centrifugation at 4 °C and 10 000 r/min, the pellet was
again dissolved in 1 mL TBS buffer (Bruning et al., 1999; Paek et al., 2000; Reid et al., 2001). The colloidal gold-labeled TPN47 was produced in the same method. Purified colloidal gold-labeled TPN47 and TPN17 (v/v = 1/1) were mixed together, coated on glass fiber paper, and kept sealed after being dried at 37 °C. 2.2.5. Preparation of immunochromatographic test strips An immobilized fibrous membrane, colloidal goldconjugated glass fiber, and absorbent paper were grouped together by a polyvinyl chloride self-adhesive floor and then cut into strips by a slitter. The strips were kept in a tightly sealed container. 2.2.6. GICA TP-IgM test Determination of T. pallidum is based on the reaction between the colloidal gold-conjugated T. pallidum antigen and antitreponemal IgM antibodies present in the patient serum. A 10-μL specimen was dropped at the “patient area” of the immunochromatographic test strips, and the result was observed after adding 100 μL normal saline for 20 min. The T. pallidum results were read blindly by 3 independent observers. An agreement was recorded until all 3 observers interpreted the same result for each sample. A positive result showed 2 red lines on designated strip zones, which represent the “patient area” and the “control area,” respectively. A negative result showed only one red line on the “control area.” The absence of band was considered as an indication of invalid result. Observers also scored their perception about the ease of use of the T. pallidum test, with grades ranging from “low,” “medium,” or “high.” 2.2.7. Cross-reactivity and interference of GICA TP-IgM test To study the cross-reactivity and interference of GICA TP-IgM test, 500 sera samples were collected from patients with other pathologies, as well as from special clinical specimens that may bring false-positive results, by the Xiamen Center of Clinical Laboratory. The detailed composition were as follows: 48 cases had rheumatoid disease; 42 cases had active systemic lupus erythematosus (SLE); 4 cases had human immunodeficiency (HIV); 90 cases had hepatitis B; 40 cases had hepatitis C; 14 cases had herpes simplex virus type II (HSV-II); 10 cases had toxoplasmosis; 12 cases had biological false positives (BFPs); 30 cases were pregnant; and 210 cases were special clinical specimens (70 from hemolytic samples, 70 from lipemic samples, and 70 from icteric samples). 2.3. Serologic tests Laboratory analysis for each sample was performed with the following tests: TRUST (InTec Products), TPPA (Fujirebio), and FTA-Abs IgM (EUROIMMUN Medizinische Labordiagnostika, Schleswig-Holstein, Germany). The tests were carried out according to the instructions of the manufacturers. For TRUST, sera samples were tested at dilutions of 1:1 to 1:32 in a physiological solution (NaCl
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0.9%) to avoid prozone effects and false-negative results. Readings were taken immediately with the naked eye through comparison with negative and positive controls. The sera samples reactive for TRUST were quantified in 2fold serial dilutions until the end was determined. The initial dilution of sera samples in TPPA reactions was 1:80. For FTA-Abs IgM, sera samples were considered reactive with initial dilutions of 1:10 in the sorbent. Sera samples that produced conflicting or inconclusive results for a particular technique were tested in duplicate; the new result was considered as the true result. 2.4. Testimony of diagnosis of syphilis Testimonies were in accordance with the national guidelines (SBTS/MOH, 1996). 2.5. Establishment of GICA gold standard Sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, and negative likelihood ratio were calculated for GICA TP-IgM and compared with FTA-Abs IgM as a gold standard. Thus, for calculation purposes, only samples that tested positive for FTA-Abs IgM were considered as true positives. The gold standard for a negative result was a negative FTA-Abs IgM test. 2.6. Statistical analysis All statistical analyses were conducted using SPSS v13 for Windows. χ2 tests were used to determine the significant differences across groups. P b 0.05 (2-sided) was considered significant. 3. Results 3.1. Reactivity of the TRUST, TPPA, FTA-Abs IgM, and GICA TP-IgM tests among different groups Out of the 1208 patients who received recommended therapy for syphilis, 441 cases were still nontreponemal and treponemal antibody positive in the prospective therapy duration, with a SSR incidence of 34.02%. Detection of TPIgM with FTA-Abs showed that 142 cases in 411 SSR patients (group 1) were positive; hence, that the positive rate was 34.55%. In contrast, only 23 out of 251 cases with (−) TRUST but (+) TPPA (group 2) had (+) TP-IgM test, yielding a positive rate of 10.360%. The positive rates of groups 1 and 2 in the GICA method were 36.01% and 11.16%, respectively. TP-IgM detection by FTA-Abs and GICA both showed significant differences (P b 0.01) in the positive rate between groups 1 and 2. The positive rate of the GICA method was higher than that of the FTA-Abs method; however, the methods were not significantly different from each other. Analysis of the total number of cases showed that 168 out of 1308 cases were FTA-Abs positive; the positive rate was 12.84%, which was lower than that
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detected by the GICA method (13.46%). However, no significant statistical differences exist. Groups 3 and 4 were negative for TP-IgM (Table 1). 3.2. Analysis of sensitivity and specificity of GICA A total of 1308 cases, including syphilis patients and blood donors, underwent syphilis serology test. The number of syphilis-positive serum, as detected by the GICA method and the FTA-Abs method, was 176 and 168, respectively. A total of 165 samples of serum was determined positive using the FTA-Abs IgM test among the 176 samples of GICA TPIgM–positive serum. Among the 1132 GICA TP-IgM– negative specimens, 3 cases were positive and 1129 were negative in the FTA-Abs IgM test. Out of 168 FTA-Abs IgM–positive cases, 165 were determined to be GICA TPIgM–positive, whereas 3 were negative. The T. pallidum GICA TP-IgM test and FTA-Abs IgM test revealed a significant agreement in the overall results. The large number of negative results (86.31%) and positive results (12.61%) caused the significant agreement. The FTA-Abs IgM test is considered as the gold standard in the detection of TP-IgM. The estimated sensitivity and specificity of the FTA-Abs IgM assay as a gold standard was 100%. The parameters of the GICA TP-IgM assay were as follows: sensitivity rate of 98.21% (165/168), specificity rate of 99.04% (1129/1140), positive predictive value of 93.75% (165/176), negative predictive value of 99.73% (1129/1132), positive likelihood ratio of 102.3 (98.21/0.96), and negative likelihood ratio of 0.018 (1.79/99.04) (Table 2). 3.3. Cross-reactivity and interference of GICA TP-IgM A total of 500 sera samples from patients with other pathologies and aberrant specimens were collected by the Xiamen Center of Clinical Laboratory to be tested for reactivity using the GICA TP-IgM test. No cross-reactivity Table 1 Reactivity of the TRUST, TPPA, FTA-Abs IgM, and GICA TP-IgM tests among different experimental groups Total cases Group 1 Group 2 Group 3 Group 4 Total
411 251 546 100 1308
TRUST
411 0 0 0 411
TPPA
411 251 0 0 662
FTA-Abs IgM
GICA TP-IgM
No.
No.
%
148 28 0 0 176
36.01b,c 11.16d 0 0 13.46e
142 26 0 0 168
% a
34.55 10.36 0 0 12.84
a FTA-Abs-IgM positive rate comparison between group 1 and group 2, χ2 = 48.157, P b 0.01. b GICA-IgM positive rate comparison between group 1 and group 2, χ2 = 49.321, P b 0.01. c FTA-Abs-IgM and GICA-IgM positive rate comparison in group 1, χ2 = 0.192, P N 0.05. d FTA-Abs-IgM and GICA-IgM positive rate comparison in group 2, χ2 = 0.083, P N 0.05. e FTA-Abs-IgM and GICA-IgM positive rate comparison in all cases, χ2 = 0.214, P N 0.05.
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Table 2 Comparison of FTA-Abs IgM and the GICA TP-IgM test results GICA TP-IgM
FTA-Abs IgM (gold standard) Positive
Negative
Positive Negative Total
165 (12.61%) 3 (0.23%) 168 (12.84%)
11 (0.85%) 1129 (86.31%) 1140 (87.16%)
Total
176 (13.46%) 1132 (86.54%) 1308 (100%)
was detected in GICA TP-IgM tests with serum from patients with rheumatoid disease, SLE, and toxoplasmosis, or patients who were afflicted with HIV, HBV, HCV, and HSV-II. Hemolytic, lipemic, and icteric samples showed no interferences, except for one hemolytic serum, which showed a red line at the “low” grade (Table 3). 3.4. Stability assay Differences within a group: Strips from the same batch were used to confirm the reactive sera that were used as controls (FTA-Abs-proven TP-IgM–positive serum at high, medium, and low grades). No difference in the color depth of the bands was found on the identical titer, and the negativeserum was proven negative. Differences among groups: Strips from the different batches were used to confirm the reactive sera that were used as controls (FTA-Abs–proven TP-IgM–positive serum at high, medium, and low grades). No difference in the color depth of the bands was found on the identical titer, and the negative-serum was proven negative. To further evaluate the stability of the reagents, we treated the reagent for GICA TP-IgM tests using the following procedure. The reagents were first kept at 4 °C for 18 months. The reagents were tested every 2 months to detect reactive sera that were used as controls (FTAAbs-proven TP-IgM–positive serum at high, medium, and low grades). No difference in the color depth of the bands
Table 3 Reactivity of the GICA TP-IgM test in sera from 500 individuals with interference Category
Rheumatoid disease SLE HIV HBV HCV HSV-II Toxoplasmosis Pregnant women BFP Hemolytic Lipemic Icteric
Gender Male Female
Average No. of GICA TP-IgM age sera tested No. of sera reactive
17 11 3 45 20 4 0 0 4 35 35 35
36 40 34 36 33 28 27 27 28 36 42 37
30 31 1 45 20 10 10 30 8 35 35 35
was found on the identical titer, and the negative-serum was proven negative.
48 42 4 90 40 14 10 30 12 70 70 70
0 0 0 0 0 0 0 0 0 1 0 0
4. Discussion Two criteria define syphilis cure: clinical cure and serum cure. Syphilis patients were classified as clinical cure if the clinical manifestations of syphilis disappeared after the recommended syphilis therapy. Patients who tested also negative in the serum nontreponemal test antibody were classified as serum cure. SSR is one of the manifestations of treatment failure, which is common in clinical treatments. Out of the 1208 patients who received recommended therapy for syphilis, 441 cases were still nontreponemal and treponemal antibody positive in the prospective treatment duration, with an SSR incidence of 34.02%, higher than what was reported by Rolfs on the serum resistance ratio of early syphilis (Rolfs et al., 1997). The reason behind the continuous occurrence of SSR after treatment remains unknown. T. pallidum escapes from the immune response of hosts by impairing the amount of membrane proteins, including their immunogenicity, and finally leading to long-term infection and contamination of hosts (Weigel et al., 1992). In addition, the changes of T. pallidum antigenic structure, as well as the different genotype and the different genetic background in organisms were also related to the occurrence of SSR to some extent. Experiments performed in rabbit models had proven that the Tp rK gene in all subsets of T. pallidum were significant variables, which may help T. pallidum escape from the immune system of hosts and finally lead to chronic infection (LaFond et al., 2006). All the above are important reasons behind the relapse and infection of syphilis. Serologic tests are divided into 2 categories, namely, nontreponemal and treponemal antibody tests. Treponemal antibodies, IgM and IgG, are generated after 2 and 4 weeks, respectively. TP-IgM first appears in the body affected by syphilis, and then its level remains at a certain plane with the existence of active T. pallidum (Herremans et al., 2007). Another nontreponemal antibody is generated after infection for 5–7 weeks (Juarez-Figueroa et al., 2007). Nontreponemal test antibody titers usually correlate with disease activity; thus, it is regarded as basis of verdict for serum cure. The FTA-Abs method and the GICA method were used to detect TP-IgM, and both revealed a TP-IgM positive rate of 34.55% and 36.01% in 411 SSR patients, respectively. The results show that active T. pallidum remains in the body of SSR patients. Although the possibility of infection cannot be excluded completely when TP-IgM is negative, the findings reveal that patients positive for TP-IgM are infectious and can relapse. In serum cure group A, the TPIgM positive rate was both above 10% with the FTA-Abs method and the GICA method, indicating that active T. pallidum still exists in vivo, and proper treatment measures
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should be provided. Apparently, serum cure cannot be properly determined based only on negative nontreponemal antibody test. In contrast, all patients in serum cure group B were negative for TP-IgM. The results of this study also showed that the TP-IgM positive rate is not significantly different among patients with different ages, genders, and clinical phases after 1 year of recommended therapy. To date, the FTA-ABS test is considered as the reference method for TP-IgM detection, as well as the standard confirmatory test for syphilis, except in very early infection. However, the professional training for diagnostic tests, specific equipment, and the detection time of FTAAbs TP-IgM is about 2.5–3.5 h (depending on the skill level of operator), and costly reagents (46 Yuan according the quoted price from EUROIMMUN Medizinische Labordiagnostika in China) affect the prevention and control of syphilis in developing countries, especially in rural areas (Lin, Fu, and Dan, 2010). Therefore, we need not only use specific and precise test methods but also convenient, fast, and cheap reagents to screen patients with syphilis. This can help in the clinical diagnosis and epidemiologic survey of syphilis. After simple training, nonprofessionals could master and operate the GICA test. The result can be directly read with the naked eyes. The detection time is 20 min, and the cost is low. After mass production, the market price of GICA strips must be within 2.5 Yuan (Lin, Fu, and Dan, 2010). We used syphilisspecific recombinant protein as the antigen of the reagents, set FTA-Abs-IgM as the gold standard, and developed rapid test reagents based on GICA technology. This method has 98.21% sensitivity, 99.04% specificity, 93.75% positive predictive value, 99.73% negative predictive value, 102.3 positive likelihood ratio, and 0.018 negative likelihood ratio. Meanwhile, extra weight was given to sera from subjects with pathogenic virus infections, autoimmune diseases, and peculiar clinical specimens to challenge the GICA TP-IgM test. Only one case of hemolytic serum was indicated as a false positive (low reaction) among 500 sera samples collected from patients with other pathologies and peculiar clinical specimens that are free from serum hemolysis, lipemia, jaundice, and so on. In summary, the GICA TP-IgM test is specific and sensitive for routine laboratory use, and its characteristics correspond to that of FTA-Abs IgM (EUROIMMUN Medizinische Labordiagnostika). As rapid treponemal tests emerge because of their enhanced sensitivity, userfriendly qualities, convenience (i.e., no extra equipment), stability at room temperature, and reasonable price, the traditional procedure for laboratory diagnosis of syphilis in developing countries may change (Lin, Fu, and Dan, 2010; Lin, Zhang, and Yang, 2010). Therefore, the GICA TPIgM method can be used as a serologic marker for the relapse and infection of syphilis in place of the conventional FTA-Abs IgM test.
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Acknowledgments This study was supported by the Natural Science Foundation of Fujian Province (grant no. 2009J01200), the Key projects in Fujian Province Science and Technology Program (grant no. 2009D019), and the projects of Xiamen Science and Technology Program (grant no. 3502Z20089015, 3502Z20084019). References Akins DR, Purcell BK, Mitra MM, Norgard MV, Radolf JD (1993) Lipid modification of the 17-kilodalton membrane immunogen of Treponema pallidum determines macrophage activation as well as amphiphilicity. Infect Immun 61:1202–1210. Bruning A, Bellamy K, Talbot D, Anderson J (1999) A rapid chromatographic strip test for the pen-side diagnosis of rinderpest virus. J Virol Methods 81:143–154. Chen ZQ, Zhang GC, Gong XD, Lin C, Gao X, Liang GJ, Yue XL, Chen XS, Cohen MS (2007) Syphilis in China: results of a national surveillance programme. Lancet 369:132–138. Faulk WP, Taylor GM (1971) An immunocolloid method for the electron microscope. Immunochemistry 8:1081–1083. Herremans M, Notermans DW, Mommers M, Kortbeek LM (2007) Comparison of a Treponema pallidum IgM immunoblot with a 19S fluorescent treponemal antibody absorption test for the diagnosis of congenital syphilis. Diagn Microbiol Infect Dis 59:61–66. Higuchi K, Urabe H, Yoshida M (1961) Some problems on seroresistant syphilis. Kyushu J Med Sci 12:283. Juarez-Figueroa L, Uribe-Salas F, Garcia-Cisneros S, Olamendi-Portugal M, Conde-Glez CJ (2007) Evaluation of a rapid strip and a particle agglutination tests for syphilis diagnosis. Diagn Microbiol Infect Dis 59:123–126. LaFond RE, Molini BJ, Van Voorhis WC, Lukehart SA (2006) Antigenic variation of TprK V regions abrogates specific antibody binding in syphilis. Infect Immun 74:6244–6251. Lin LR, Fu ZG, Dan B (2010) Development of a colloidal goldimmunochromatography assay to detect immunoglobulin G antibodies to Treponema pallidum with TPN17 and TPN47. Diagn Microbiol Infect Dis 68:193–200. Lin L-R, Zhang Z-Y, Yang T-C (2010). Preparation methods of a colloidal gold-immunochromatography assay to detect immunoglobulin M antibodies to Treponema pallidum. patent no. 201010179134.6. Lukehart SA, Baker-Zander SA, Sell S (1986) Characterization of the humoral immune response of the rabbit to antigens of Treponema pallidum after experimental infection and therapy. Sex Transm Dis 13:9–15. McKevitt M, Patel K, Smajs D, Marsh M, McLoughlin M, Norris SJ, Weinstock GM, Palzkill T (2003) Systematic cloning of Treponema pallidum open reading frames for protein expression and antigen discovery. Genome Res 13:1665–1674. Norgard MV, Chamberlain NR, Swancutt MA, Goldberg MS (1986) Cloning and expression of the major 47-kilodalton surface immunogen of Treponema pallidum in Escherichia coli. Infect Immun 54:500–506. Osikowicz G, Beggs M, Brookhart P, Caplan D, Ching S, Eck P, Gordon J, Richerson R, Sampedro S, Stimpson D, et al (1990) One-step chromatographic immunoassay for qualitative determination of choriogonadotropin in urine. Clin Chem 36:1586. Paek SH, Lee SH, Cho JH, Kim YS (2000) Development of rapid one-step immunochromatographic assay. Methods 22:53–60. Reid SM, Ferris NP, Bruning A, Hutchings GH, Kowalska Z, Akerblom L (2001) Development of a rapid chromatographic strip test for the
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