Evaluation of the Rapid Treponemal Point of Care Test introduced in Zambia in 2012. [Original Research]
Yassa Pierre, PhD*, Kwenda SH, PhD*, Hira Rajiv S, MBBS**, Kunda M, MD*, Sarenje K, Bch*, Mwenya D, Msc*, Kantenga, Msc*,Kaswa Jean, MD#, Susan Strasser, PhD***
*University Teaching Hospital, Lusaka, Zambia.
** Emory University-SPH, Atlanta, USA.
*** Country Director, Elizabeth Glaser Pediatric AIDS Foundation.
#CNPP, University of Kinshasa, DR Congo.

[emedpub – International Infectious Diseases: Vol 1:11] [Date of Publication: 07.28.2013]
ISSN 2231-6019

July 28, 2013 at 8:47 PM


Pr Yassa Pierre, University Teaching Hospital, STI Clinic, Lusaka, Zambia.

email: perets31@gmail.com



To evaluate operational characteristics of the rapid treponemal point of care test in terms of its sensitivity and specificity, used among pregnant women attending antenatal clinic in Lusaka, Zambia.


A case control study was conducted


Results of the index test (rapid point of care syphilis test) showed that, of the 121 samples tested, 6 (5.0%) were reactive and 115 non-reactive, where as in the reference standard test (chemiluminescent microparticle immunoassay), 8 were reactive and 113 samples non-reactive. Thus, sensitivity of rapid treponemal test was 75%  and  specificity was 100%. Discriminant Ability = (Sensitivity + Specificity) / 2 =( 75% + 100% ) / 2 = 87.5%


The rapid treponemal point of care test presented low sensitivity (75%) and high specificity (100%). This test still remains effective as a routine screening qualitative rapid test for the detection of antibodies against T.pallidum. Screening for syphilis and its treatment is an important strategy for the prevention of adverse pregnancy outcomes of maternal syphilis.


Elimination of syphilis will require having more people to be tested to meet the World Health Organisation objectives and therefore, accuracy and reliability of the diagnostic test is the key to the success of syphilis elimination programmes (1). Diagnostic tests introduced in the country have to be evaluated to ensure reliability of the results and reduce errors to a minimum level thereby producing quality and acceptable results (2).

Serological tests are the most effective method for the diagnosis of syphilis (3) and most often used are the non-treponemal tests and the treponemal tests. (4). The Rapid Plasma Reagin (RPR) test is the standard test used in Zambia, although simple to use, it requires equipment, training, is difficult to interpret and may produce false positives (1).

New rapid  treponemal point of care tests have been introduced and their performance characteristics have been evaluated in many countries where minimal invasive procedure have been applied to collect whole blood by means of finger prick to detect specific antibodies to syphilis and the results have shown to be highly sensitive and specific (5). The rapid tests have the ability to detect IgM, IgG and IgA antibodies that involve immunochromatographic strips to which one or multiple T. pallidum recombinant antigens (TpN15 and TpN17) are applied to nitrocellulose strips as a capture reagent (5).

The World Health Organisation have compared the performance of eight rapid treponemal point of care tests to a combined reference standard of Treponema Pallidum Haemoagglutination Assay (TPHA) and Treponema Pallidum Particle agglutination Assay (TPPA) and reported sensitivities ranging from 84.8% to 97.7% and specificities from 92.8% to 98% (6). Lower sensitivities have also been reported for whole blood compared with serum or plasma (7,8).

In our study to determine the sensitivity and specificity of the rapid treponemal test among pregnant women attending antenatal clinics, we used the chemiluminescent microparticle immunoassay as the standard reference, instead of the Treponema Pallidum Particle agglutination Assay or the Treponema Pallidum Haemoagglutination Assay  that have been used in other evaluation studies since these were not readily available. The chemiluminescent microparticle immunoassay (CMIA) has been evaluated against the TPPA and TPHA and has been found to be highly sensitive (100%) and specific (100%) and also reported to be able to detect primary syphilis due to TpN47 recombinant antigen (9,10). And as a result of its good performance, the CMIA was used as a reference standard in this study.

The introduction of the rapid treponemal test would greatly increase accessibility to diagnosis and treatment of syphilis in a single visit, patients would no longer have to wait for days to obtain results of their syphilis test, as it may be the case with RPR. Antenatal clinics in rural settings would conduct the rapid test in facilities without the laboratory. Unlike the RPR, reagents of the rapid test can be stored at room temperature, are easy to use and interpret the results (1). Further, early detection of syphilis may reduce the risk of Human Immunoassay Deficiency Virus (HIV) transmission (1).

The objective of the study is to evaluate the operational characteristics of the rapid treponemal point of care test that was introduced in Zambia, in terms of its sensitivity and specificity, used among pregnant women attending antenatal clinics. The assessment of this test would also allow us to consider the appropriateness of the test for clinics in the screening of syphilis in urban and rural clinics that do not have laboratory facilities.

Patients and methods:

Study design: A case control study was conducted to measure the characteristic performance of the rapid treponemal point of care test in terms of its sensitivity and specificity.

Measurement variable: Age at last birth day and marital status were all measured as continuous and categorical variables in relation to the test under evaluation. In a two by two table to assess qualitatively the sensitivity and specificity of the test the following were measured: a = true positive result; b = false positive result; c = false negative result;                                                        d = true negative result.

Study Site: The study was conducted in the Cytology laboratory of the University teaching Hospital (UTH) in Lusaka, Zambia.

Study population: 121 pregnant women attending antenatal clinic at the University Teaching Hospital in Lusaka were recruited for the study from whom a) finger prick whole blood was collected; and b) sera extracted from venous blood for the tests.

Inclusion criteria: Pregnant women over 18 years of age seeking care at the maternity clinic with no previous history of syphilis were recruited. Consent to undergo blood draw via venous puncture in addition to finger prick was obtained from the patients.

Exclusion criteria: Women under 18 years old and/or with previous history of syphilis or positive serology for syphilis were excluded.

Sample size: Using estimate of syphilis prevalence at 7.4%, the calculated minimum sample size was 120.

Standard for use of Rapid Syphilis Tests Operating Procedure

a) Test procedure using the Standard Diagnostics Bioline Syphilis 3.0 Rapid Test Kit.

It was ensured that the expiry date of the kit was checked before using the kit including its intended use as a single reagent assay for the detection of specific antibodies to Treponema pallidum in whole blood.

Principle of the procedure

The recombinant Treponema pallidum – specific antigens are immobilised at the test region of the nitrocellulose strip. The antigens are also linked to colloidal gold and impregnated below the test region of the device.  A narrow band of nitrocellulose membrane is also sensitised as a control region.

During testing, 20µl of whole blood is applied to the sample port, followed by 4 drops (approximately 10µl) of wash buffer and allowed to react with the colloidal gold linked antigens. The antibody peptide-colloidal gold complex moves chromatographically along the membrane through the test and control regions of the test device. A positive reaction is visualised by a pink/red band in the first region of the device. A negative reaction occurs in the absence of human immunoglobulin antibodies to treponemal antigens in the analysed specimen. Consequently no visually detectable band develops in the test region of the device. Excess conjugate forms a second pink/red band in the control region of the device. The appearance of this band indicates completion of the test as well as proper performance of the reagents in the device.

b) Kit Contents

  • 30 Test Devices

Each test device contains colloidal gold labelled with recombinant- Treponema pallidum- specific antigens in the test line, and a control line.

  • Wash Reagents

50mM Tris Hydrochloride (Tris-HCI) buffer containing detergent and preservative (0.02% sodium azide).

  • Disposable capillary Pipettes
  • Instructions for Use
  • Lancets

c) Materials required but not provided with the Kit

  • Timer or stopwatch
  • Blood collection devices (i.e., test tubes)

d) Storage and stability

The Standard Diagnostics Bioline Syphilis 3.0 Rapid Test devices can be stored at 2-30ºC. No kit components should be used after the kit expiry date.

e) Specimen Collection and Storage

Whole blood was collected from finger prick and used immediately for the test.

f) Test Procedure

  1. Removed the test device from the foil pouch, placed it on the flat and dry working bench surface.
  2. Labelled each test with the appropriate patient identification.
  3. Using a capillary pipette, 20µl of whole blood from the finger prick was collected and placed into the sample port.
  4. Then added 4 drops (about 120µl) of assay diluents (Buffer) into the sample port.
  5. As the test begun to work, a purple colour  moved across the result window in the centre of the test device
  6. Allowed 20 minutes for reaction to occur and interpreted the results.

g) Interpretation of Test Results

  1. A colour band would appear in left section of the left window to show that the test is working properly. This is the Control window/Band.
  2. The right section of the result window indicates the test results. If another colour band appears in the right section of the result window, this band is the Test Band.

  1. Positive  result:

The presence of two colour bands (“T” band and “C” band) within the result window, no matter which band appears first, indicates a positive result for TP antibodies.

  1. Negative results:

The presence of only one purple colour band within the result window indicates a negative result.

  1. Invalid Results:
    1. No line appears in both the control and patient areas. Report invalid results and repeat test with a new test device.
    2. No line appears in the control area and one line appears in the patient area. Invalid results. Repeat test with a new test.

h) Quality Control

Good Laboratory Practice requires the use of control specimens to ensure proper device performance at least once daily before the beginning of the tests.

i) Biological Principle of the CMIA procedure

The Abbott ARCHITECT Syphilis TP assay is a two immunoassay (CMIA) for the qualitative detection of antibody to T. pallidum in human serum or plasma based on recombinant antigens TpN15, TpN17, and TpN47. Antibody present in the sample binds to T. pallidum recombinant antigen coated paramagnetic particles. After wash step, murine anti-human IgM acridinium-labelled conjugate is added. Following a further wash step, pre-trigger solution (hydrogen peroxide) and trigger solution (sodium hydroxide) are added. The resulting chemiluminiscent reaction is measured in relative light units (RLUs) which are directly proportional to the amount of anti-pallidum present in the sample. The ARCHTECT System calculates the cut-off (CO) using the mean chemiluminescent signal (RLU) from three replicates of the Calibrator 1 and stores the result.

The ARCHITECT Syphilis TP assay calculates a result based on a cut-off determined by the following calculation.

  • Cu-toff (CO) = Calibrator 1 Mean RLU x 0.20
  • S/CO = Sample RLU / Cut-off RLU
  • The cut-off RLU is stored for each reagent lot calibration.

j) Interpretation of results

  • Specimens with S/CO values < 1.0 are considered nonreactive by the ARCHITECT Syphilis TP assay.
  • Specimens with S/CO values ≥ 1.0 are considered reactive by the ARCHITECT Syphilis TP assay.

Data collection: The data was collected, captured and recorded on the data sheet and finally entered all the variables in the SPSS code book for analysis.

Data analysis : The SPSS statistical package was used to measure the sensitivity and specificity of the rapid treponemal point of care test by cross tabulation. In addition, to test the rapid syphilis test itself, the receiver operator characteristics (ROC) curve was used to measure the area of the test in comparison to the reference test thereby determining its applicability in diagnosis of syphilis upon calculating the discriminant ability. And the frequencies were used to measure the demographic variables. The Pearson Chi-Square was used to determine the relationship between the rapid syphilis test and the chemiluminescent microparticle immunoassay.

The sensitivity and specificity were also analysed by use of a two by two table to assess qualitatively the accuracy of the rapid treponemal POC test in comparison to the reference standard diagnostic (CMIA) test.

Ethical consideration: In seeking informed consent from the subjects participating in the study, an explanation of the purpose of the research, expected duration of the subject’s participation, description of the procedure, any reasonably foreseeable risks or discomfort to the subject, benefits of the research expected from the research were given. In addition, confidentiality of records identifying the subjects were maintained because codes were used instead of names to link particular samples to particular participant or patient to ensure confidentiality. Minimal risk was applied as the test procedure was less invasive when collecting whole blood from the subjects.


This section now provides the answers to the research question: does the rapid point of care test have the sensitivity and specificity to detect antibodies to Treponema pallidum. The marital status of ANC women is presented in Table 1.

Table 1. Marital Status of ANC women.

Status Frequency Percent
Single 27 22.3
Married 94 77.7
Total 121 100.0

Results of the index test (rapid point of care syphilis test) showed that out of the 121 samples tested, 6 (5.0%) were reactive and 115 non-reactive, where as in the reference standard test (chemiluminescent microparticle immunoassay), 8 (6.5%) were reactive and 113 samples non-reactive. Two samples were discordant, the rapid syphilis test recorded that these were non-reactive while the reference standard  test was able to pick them as reactive and were recorded as false negatives as shown in table 2 below.

Sensitivity and specificity

Sensitivity and specificity of the RST test was calculated by using the values obtained from the cross tabulation table, as shown in table 2 below.

Table 2. RST and CMIA Cross Tabulation Results

Reference Test

RST Test

Reactive Non-reactive Total
Reactive 6 0 6
Non-reactive 2 113 115
Total 8 113 121

Sensitivity: 75%

Specificity = 100%

Positive predictive value  = 100%

Negative predictive value = 98.3%

There was a significant difference between the two tests (Chi-square= 89.2;p<0.000).

Discriminant Ability = (Sensitivity + Specificity) / 2

( 75% + 100% ) / 2 = 87.5%

Fig 1: Receiver operator characteristics (ROC) curve.

The receiver operator characteristics curve was found to be 87.5% and lay close to the reference standard in the upper corner of the vertical axis.


In our discussion we focus on the objectives in trying to answer the research question: does the rapid treponemal point of care test have the sensitivity and specificity to detect the antibodies to  T. pallidum of syphilis.

The study conducted at the University Teaching Hospital, Cytology laboratory, to measure the performance characteristics of the SD BIOLINE Syphilis 3.0, presented sensitivity of 75% and specificity of 100%. The discriminant ability, which is the average of sensitivity and specificity, was 87.5%. In addition, the receiver operator characteristics graph that measures the accuracy of an index test found 0.875 or 87.5% as the area against the reference standard test.

It is not clear why the sensitivity of the RST performed in the laboratory was low compared to the relative sensitivity 99.3% (152/153) and relative specificity of 99.5% (209/210) obtained by the manufacturer against the TPHA (10). We can hypothesize that the test in question was unable to pick the antibodies for T. pallidum in the two samples because the infection was in its early primary stage. During this stage of the disease the concentration of antibodies are low to be detected by serology test especially with the use of whole blood (11).

A study conducted by West and others in Gambia, to assess the performance of the RPR and RST screening tests in the diagnosis of syphilis in field conditions of the rural areas against the TPHA as gold standard. A total of 1325 women participated in the study attending antenatal clinic. RST had sensitivity of 75%, similar to our findings, and 95.2% specific. However, RPR sensitivity was 77.5% and specificity 96.4% (West et al., 2002). Our study was almost in agreement or similar to the study by Montoya and colleagues in Mozambique where 4789 women attending their first antenatal visit at one of the six centres reported sensitivity of 79.2% and specificity 99.1%, though the mean in all the six centres recorded 95.3% at reference laboratory and 84.1% at health centres. The sensitivity and the specificity were found to be lower than the TPHA reference tests (12).

Siedner and friends in the study where three rapid syphilis tests were evaluated reported that overall the test performance was poorer on whole blood samples when compared with previous research with serum in the WHO study or with other investigators. They all hypothesized that the reason for lower antibody concentration was responsible for the discrepancy. They explained that the lower antibody concentration could have decreased the sensitivity of the RST test by diminishing the strength of the antigen-antibody interaction that is responsible for producing a positive test (13). Perhaps another contributing factor to discrepancy of the result could be between the whole blood assay and the serum testing, was the presence of ethylenediamintetraacetic acid (EDTA) in the test tubes with which whole blood was collected. It could be possible that the anticoagulant interfered with the antibody-antigen reaction (ibid). This assertion is in agreement with what was observed in our study. Failure by the RST to pick the two samples would be due to the fact that, syphilis infection was in the early primary stage as could be seen in the degree of the chemiluminesce produced by the CMIA of the two samples, which were far lower than the positive samples that could have probably been in their secondary or latent stages of syphilis infection. The other reason could be the fact that since the RST test membrane is only coated with two recombinant antigens (TpN15 and TpN17) to interact with the anti-human antibodies, IgG and IgM as indicated in the operational manual of the test, this could explain why it was unable to detect antibodies in the early stages of the syphilis infection because of lack of the TpN47 antigen which has been reported to be responsible for detection of early syphilis stage, which apparently is present in CMIA (10).

A study by West et al to evaluate the RST hypothesized the discrepancies observed and explained that, like the TPHA, the RST is also based on specific treponemal reactivity, in this case to a recombinant 47 KDa protein antigen. Though this recombinant antigen is specific to T. pallidum, post-translational processing of the protein in vivo or human leukocyte antigens (HLA) halotype restricted responsiveness that could lead to lack of reactivity during infection. Conversely, the large number of treponemal antigens exposed during TPHA test could increase the chance that some cross reaction with other organisms may occur. This may explain some of the discrepancies observed between the two tests. He suggested that perhaps a mixture of several recombinant test antigens might overcome some of these problems.

The RST according to the manual has two treponema pallidum recombinant antigens (TpN15 and TpN17) pre-coated to the membrane strip. The qualitative treponemal CMIA test has apparently three recombinant antigens TpN15, TpN17 and TpN47 used to capture specific antibodies and both the tests work on different principles (10).

The Abbott ARCHITECT Syphilis TP assay used as reference test in this study, work on a two step sandwich chemiluninescent microparticle immunoassay for the qualitative detection of antibody to T. pallidum in human serum or plasma. Antibody present in the sample binds to T. pallidum recombinant antigen coated paramagnetic particles. Several wash steps take place resulting into a chemiluminescent reaction measured in relative light units (RLU) which are directly proportional to the amount of anti-treponmal present in the sample. This test has been reported to be highly sensitive in detecting early primary syphilis (97.5%) and has a specificity (99.1%) (14). In another evaluation, this assay presented 100% sensitivity (121/121) and specificity 100% (500/500) and good performance on reproducibility (9). And recently Wellingausen and others evaluated two automated Chemiluminescent immunoassay (CIA), and LIAISON Treponema screen and the ARCHTECT Syphilis TP in comparison to the Treponemal Pallidum Particle Assay (TPPA) test for laboratory diagnosis of syphilis. 577 sera samples were tested and found sensitivity 100%, 99.8% and 99.6% for LIAISON, ARCHTECT and TPPA (15).

The CMIA was used as a reference standard test because of its high sensitivity and specificity as reported in the previous studies conducted to evaluate its performance against the TPHA and the TPPA as reference tests. And since this particular test has performed well against two reference tests, it therefore, qualified to be used in our study as reference test. TPPA and TPHA tests were not readily available during the time of our study. From the available data it was realized that the two discordant samples were detected by the CMIA because of its versatile mechanism of reaction to capture low concentration antibodies in early primary syphilis  (16), unlike in the  RST when whole blood is used.

It has been reported that highly specific tests rarely miss negative outcomes and so they can be considered reliable when their result is positive. And therefore, a positive result from a test with high specificity means a high probability of the presence of disease. But however, it should always be remembered that no diagnostic test provides absolute assurance that a sample does not contain low levels of antibodies to T. pallidum, such as those present at a very early stage of infection. Therefore, a negative result at any given time does not preclude the possibility of exposure to infection with syphilis. When there is a disagreement between the index test and the reference standard test, the index test is considered wrong and the reference standard test is considered correct (17).

The discriminant ability calculated is a basic measure of how well the index test (RST) discriminates between those with the disease and those who are free of the disease. It provides information regarding the index test compared to the reference test, which by definition provides perfect information (17). And since accuracy of the test depends on how well the test separates  the group being tested into those with or without the disease in question, the receiver operator characteristics (ROC) curve that measures the area discrimination provides information on the ability of the test to correctly classify those with and without the disease (ibid).

The ROC curve therefore, allows us to compare the performance of the RST to the perfect test that lies in the left upper of the ROC curve, shown in figure 1.  The diagonal line that crosses from the lower left to the upper right of the ROC curve indicates zero or no information. And therefore, since the accuracy is measured by the area under the ROC curve, any area below 0.5 represents a worthless test, 0.5 to 0.6 fail, 0.6 to 0.70 poor, 0.7 to 0.8 fair, 0.8 to 0.90 good, and 0.9 to 1 excellent (ibid).

Therefore, according to the ROC, we were able to demonstrate that the RST is a good diagnostic test to use in clinical testing and practice as it fell within acceptable limits for a good diagnostic test (18).

And from this information we can extrapolate  from the results, that the key assumption for the test is that their discriminant ability can remain the same even when the test is applied to new populations with lesser or greater prevalence of the disease. The sensitivity and specificity of the test may be the same regardless of the settings in which it is used (17). However, when applying a test to a population with a very different severity of disease, its discriminant ability may not be the same. Now, to avoid these discrepancies in the discriminant ability, it is therefore advised, that the most cautious extrapolation is to use the clinical populations and clinical situations that are very similar to the ones in the investigation (ibid).

Demographically, in terms of marital status, out of the 121 tested women, seven married women and one single woman were found positive using CMIA, giving prevalence of 6.6%. However, using the RST the prevalence of syphilis was at 5%. This new rapid diagnostic test will further strengthen the landmark study done by Hira SK et al in Zambia in 1990 that demonstrated cost-effectiveness of syphilis screening in reducing the adverse outcomes of syphilis in pregnancy (19). The study was without challenges and limitations.


As demonstrated from this evaluation study, despite the low sensitivity measured, this test still remains effective as a routine screening qualitative rapid test for the detection of antibodies against the T.pallidum that causes syphilis and not as a confirmatory test. The test is easy to use, easy to interpret and does not require refrigeration for storage, the kit can be stored and used  at room temperature and may require little training of laboratory technicians to use the test including nurses and other health personnel.

The rapid treponemal point of care test should be adopted by ministries of health as a screening test due its easy performance and interpretation. With these attributes the test will improve diagnosis and treatment of syphilis due to its turnaround time of less than thirty minutes within which the results could be ready for the patients. Further, the patients will no longer have to wait for days before they have their results and receive treatment. Apart from the test being applied at the primary health care level, the test is also ideal for remote areas where there are no laboratory facilities. This testing strategy will strengthen the operational work established by Hira SK et al in Africa in 1990s that demonstrated cost-effectiveness of syphilis screening in reducing the adverse outcomes of syphilis in pregnancy.

In addition, the rapid plasma reagin test may be retained and integrated as a diagnostic test for syphilis because of its ability to detect active infection since it is able to measure the quantity of antibodies to T. pallidum by means of titration.


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