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Abstract
Objectives Although the burden of syphilis is slightly increasing worldwide, there are a limited number of rapid, simple-to-use, accurate and cost-effective diagnostic tools available. Thus, we aimed to determine the diagnostic performance of the Treponema pallidum particle agglutination (TPPA) test (hereafter called index test) against an electrochemiluminescence immunoassay (ECLIA) (hereafter called reference test). We selected the available treponemal reference test (ECLIA) to evaluate the index test (TPPA) which is not currently in use in Ethiopia.
Methods We conducted a multicentre cross-sectional study to evaluate the diagnostic performance of the index test against the reference test. We enrolled 581 syphilis-suspected cases from the five selected health facilities in Addis Ababa in this study. We collected data on sociodemographic and clinical characteristics and whole blood from each participant. We estimated the sensitivity, specificity and positive and negative predictive values of the index test.
Results Of the 581 participants, 380 (65.4%) were female. The mean age of the participants was 39.7 years (±SD 16.6), with an age range of 18–94 years. The sensitivity of the index test was 99.4% (95% CI 96.4% to 100%), while the specificity was 100%. The positive predictive value of the index test was 100%, and the negative predictive value was 99.8% (95% CI 98.4% to 100%). There was no invalid test result.
Conclusions The diagnostic performance of TPPA for syphilis was equivalent to that of ECLIA. Thus, TPPA can be used for the diagnosis of syphilis at the health facility level equivalent to a reference test.
- INFECTIOUS DISEASES
- SYPHILIS
- Bacterial Infections
- Clinical Laboratory Techniques
- Communicable Diseases
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Introduction
Syphilis continues to be a global public health problem, with an increasing number of new cases each year.1 A recent estimated incidence that is 8 million new cases occurred across the world in 2022.1 The African region of the WHO is highly affected by syphilis.2 A report on the implementation of a global strategy for the prevention and control of sexually transmitted infections (STIs) from 2006 to 2015 highlighted an increased prevalence of syphilis in pregnant women.3 Ethiopia is a country that is highly affected by STIs.4 5 A recently reported study indicates that 18.2% of university students developed STIs.5 In addition, 30% of people who visited STI clinics were infected with Treponema pallidum.4
Moreover, national-level antenatal care-based surveillance has shown a 1.2% seroprevalence of syphilis.6 Previous studies reported in different parts of Ethiopia also indicate variable burdens of syphilis among pregnant women, ranging from 1.4% to 11.8%.7 8 For example, a study reported in southwest Ethiopia shows the highest (11.8%) prevalence of syphilis among pregnant women.8 A study from southwest Ethiopia reported a 1.4% prevalence of syphilis among pregnant women.7 Thus, appropriate diagnostic tools are required to diagnose syphilis. The use of rapid tests is increasing globally, and low-sensitivity and low-specificity diagnostic tools are out of use because high-performance tools are available for the diagnosis of syphilis on the market.
There are many commercially available simple and cost-effective Treponema-specific rapid diagnostic tests (RDTs) on the market. The TPPA is among the Treponema-specific RDTs that have been evaluated in different countries against different reference tests.9 10 It has also been tested against electrochemiluminescence immunoassay (ECLIA) and has shown high performance.9 10 However, to the best of our knowledge, the performance of TPPA against ECLIA has not been tested in the Ethiopian setting, although the performance of diagnostic tools could vary in different set-ups due to several factors. Moreover, although there are several alternative RDTs for the diagnosis of syphilis, the available tests are not sufficient to provide diagnostic services for suspected patients in resource-limited countries.
Clinical history and non-treponemal test (RPR) are currently used to diagnose syphilis in Ethiopia. Thus, TPPA could be used as a treponemal initial screening test in reverse algorithm implementation. Different scientists have been using ECLIA and TPPA as index and reference tests interchangeably in evaluation studies. In Ethiopia, ECLIA is in use in some health facilities, whereas TPPA is not. Thus, it needs to be verified in the country before use at the health facilities level. Therefore, we selected the available treponemal reference test (ECLIA) to evaluate the index test (TPPA). While the ECLIA test necessitates electric utilities, the TPPA rapid diagnostic tool is suitable, particularly for primary healthcare settings in regions with limited electricity infrastructure. Thus, we aimed to evaluate the diagnostic performance of TPPA against ECLIA among people visiting sexual and reproductive clinics and women attending antenatal care in selected health facilities in Addis Ababa, Ethiopia.
Materials and methods
Study design and site
We conducted a multicentre cross-sectional study in five selected public (three) and private (two) health facilities in Addis Ababa to evaluate the diagnostic performance of TPPA from November 2021 to February 2022.
Study population
All patients visiting sexual and reproductive health clinics and pregnant women attending antenatal care in selected health facilities of Addis Ababa were the study population.
Inclusion and exclusion criteria
Patients who have current clinical symptoms of syphilis and a history of syphilis, and pregnant women with first antenatal visit and follow-up visit were included in the study. Some participants included were laboratory-tested positive with RPR. However, severely sick individuals, individuals who were participating in another study, vulnerable groups (under 18 years of age, mentally incapable and prisoners) and individuals who have received treatment for syphilis or other antibiotics that could affect the study outcomes in the last 30 days were excluded from this study.
Sample size and sampling method
We used the Clinical and Laboratory Standards Institute (CLSI) User Protocol for Evaluation of Qualitative Test Performance guidelines to determine the sample size for this study. The guidelines state that:
The options available to the evaluator are to test specimens until the desired number of positive and negative results with the comparative method is obtained, or to test specimens until a desired number of specimens with positive results is obtained with the comparative method, using all accumulated specimens with negative results in the analysis. As a minimum guideline, testing should continue until results from at least 50 positive specimens are obtained with both the new test and reference methods.11
CLSI also recommended using a higher sample size to narrow the CI. Thus, to obtain sufficient positive specimens and include more pregnant women in the study, we screened 581 eligible patients with suspected syphilis or who had a history of syphilis. We included more pregnant women as they are at a higher risk for syphilis than the general population and to help prevent congenital diseases that could lead to severe health outcomes for infants. All the screened participants were included in the analysis as per the recommendation of CLSI. We selected the study sites purposely based on their syphilis case loads at reproductive and antenatal clinics of the health facilities to obtain the required sample size in the set study period.
Laboratory test methods
Reference and index tests were employed in this study. The ECLIA technique was used for the reference test, which was performed with a Cobas 6000 instrument system. It is an immunoassay used for the in vitro qualitative determination of total antibodies developed against T. pallidum in human serum and plasma.12 The decision on the results (either positive or negative) was made based on a standard cut-off index <1.00 for negative and >1.00 for positive.12 This test method was selected due to its demonstrated 100% sensitivity and specificity.
TPPA is a qualitative gelatin particle agglutination assay intended to be used for the detection of T. pallidum antibodies in human serum or plasma for the diagnosis of syphilis.13 In the microplate wells, the serum samples were serially diluted with sample diluent, and sensitised gelatin particles were added to each well while mixing with a tray mixer. The test mixture was incubated for 2 hours to facilitate the incubation of specific antibodies and antigens, which led to the formation of mat agglutinated particles. The negative reactions were determined by compact particles at the bottom of the well.13 This treponemal test is compared against another treponemal test, a reference test.
Data and specimen collection
We used a structured questionnaire to collect demographic and clinical data. Trained health professionals collected the data. Whole blood was collected in serum separator tubes with gel which forms an effective barrier between serum and blood cells. The specimen was collected by trained and experienced health professionals working at selected health facilities. The serum was separated after whole blood centrifugation and transferred into a Nunc tube, packed in triple packaging containers with ice at the study sites and finally transported to the National Clinical Chemistry Reference Laboratory at the Ethiopian Public Health Institute for testing. The serum samples were stored at 4°C and tested within 2 days of collection.
Evaluation process (laboratory investigation)
Laboratory tests were conducted by trained and experienced professionals blinded to specimen status. The specimens were tested for T. palladium antibodies by reference test. The same specimens were tested by index test by independent laboratory personnel blinded to reference test results. Reference test and index test results were interpreted following manufacturer-specific instructions. Samples were tested following the algorithm presented in figure 1.
Schematic representation of testing algorithm in this study. ECLIA, electrochemiluminescence immunoassay; TPPA, Treponema pallidum particle agglutination.
Data analysis
Data analysis was performed using SPSS V.27. Descriptive statistics such as frequency, per cent, mean with SD or median with IQR were used to summarise the data. The diagnostic performance of TPPA against ECLIA was assessed by calculating sensitivity, specificity, positive predictive value and negative predictive value using MedCalc statistical software. All the performance indicators were estimated with 95% CIs.
Ethical consideration
The study was conducted based on the Helsinki Declaration on Human Subject Research. Informed consent was obtained from all study participants included in the study. Both questionnaire data and specimen collection were conducted after providing explanations of the study’s objectives, procedures and benefits. Positive results were communicated promptly to the participants through healthcare workers.
Results
Sociodemographic and clinical characteristics
A total of 581 participants were included in this performance evaluation study. Of the total participants, two-thirds (65.4%) were female. The mean age of the participants was 39.7 years (±SD 16.6), with an age range of 18–94 years. The majority (45.3%) of the participants were aged 26–35 years. Among the 380 female participants, 77 (20.3%) were pregnant.
Clinical performance of the TPPA test
A total of 581 specimens were tested by both index test and reference test. According to the results of the reference test, 151 specimens were positive (table 1). The sensitivity of the index test against the reference test was 99.4% (95% CI 96.4% to 100%), while the specificity was 100% (95% CI 99.2% to 100%) (table 1). The positive predictive value of the index test compared with that of the reference test was 100% (95% CI 97.6% to 100%), while the negative predictive value was 99.8% (95% CI 98.4% to 100%) (table 1). There were 0% invalid test results.
Performance characteristics of the index test compared with those of the reference test
Analysis of discordant results
Among the 581 participants, one test was discordant between the reference test and index test methods. An alternative method such as western blotting or immunoblotting is recommended to investigate the discrepant results. However, we do not have these methods in our country. Thus, to explore this discrepancy, the participant had a history of syphilis infection with no notable current symptom which seems latent syphilis, and further testing was conducted by repeating both the reference test and index test. The repeated test result confirmed the discordant result in which the index test yielded a false negative result for a single sample.
Discussion
The current study aimed to determine the diagnostic performance of TPPA against ECLIA. The sensitivity of the index test was 99.4%, while the specificity was 100%. The positive predictive value of the index test was 100%, and the negative predictive value was 99.8%. Moreover, 0% of the test results were invalid.
In the present study, the index test could detect 100% of true negatives and 99.4% of true positives for diagnosing syphilis which favours the use index test equivalent to a reference test. This finding is similar to those of a previous study in which the agreement between the two methods was high (100%).10 In different studies, ECLIA was compared against TPPA and reported high agreement in both sensitivity and specificity between the two methods.9 14 15 Of course, in some of these studies, TPPA was considered a reference test in which high agreement between TPPA and ECLIA was reported. This finding is comparable with the findings of the current study, in which the sensitivity and specificity of TPPA were high (99.4% sensitivity and 100% specificity), which is highly comparable with the reference test ECLIA in our case. Therefore, TPPA is an ideal diagnostic tool for syphilis with high-performance characteristics compared with the reference test.
A previous study reported six invalid test results for TPPA that were positive for ECLIA.10 This result is inconsistent with the findings of the present study, in which no invalid results were observed. This discrepancy between the current and previous studies regarding the validity of the results could be due to sample volume, testing procedures and technician competency.
The traditional algorithms (non-treponemal test followed by confirmatory treponemal test) and/or reverse (initial screening by treponemal followed by non-treponemal test) screening algorithms for syphilis diagnosis are used in different parts of the world. Totten et al reported that the sensitivity of the reverse algorithm was 98.3%, whereas the traditional algorithm was 72.9%, which indicates reverse algorithm is better than the traditional algorithm.16 Moreover, Nah et al showed patients diagnosed with latent syphilis could be diagnosed by the reverse screening algorithm but not by the traditional algorithm.17
In general, the current study evaluated the performance of the index test TPPA against the reference test. It is already known that TPPA is a treponemal test mostly used as a confirmatory test. It is often employed when initial screening tests, such as non-treponemal tests, are positive. However, this test is not in use in Ethiopia and there was a need to evaluate it in-country before use. Therefore, the only treponemal test we have in use and can be used as confirmatory is ECLIA. Keeping in mind the TPPA can be used as a confirmatory test, we evaluated it in an Ethiopian setting to generate evidence of the performance characteristics of the index test. The results of the present study suggest that TPPA can be used as equivalent to the ECLIA. This helps to ensure accurate and reliable results in the diagnosis of syphilis in low-income countries particularly where there is no electric utility.
Our study demonstrated that the diagnostic performance of the TPPA test is comparable to that of the ECLIA for the detection of syphilis. Thus, TPPA could be used as an equivalent to ECLIA and an equally effective alternative and cost-effective tool for syphilis diagnosis.
Ethics statements
Patient consent for publication
Ethics approval
This study involves human participants and was approved by the Scientific and Ethical Review Committee of EPHI (approval number: EPHI-IRB-377-2021). Participants gave informed consent to participate in the study before taking part.
Acknowledgments
The authors acknowledge the data collection sites, the National Clinical Chemistry Reference Laboratory, Ethiopian Public Health Institute, for their support during specimen collection and laboratory tests. The authors also appreciate all healthcare workers who supported the data and specimen collection, and all participants for their cooperation during data collection.
Footnotes
Handling editor Apostolos Beloukas
Contributors Conceptualisation of the study: TL. Methodology: TL, HHT. Study validation: TL, DB, CB. Formal analysis: TL, HHT. Investigation: YT, MM, TG, WH, GA. Resources arrangement: DMD, AKA, GA. Data curation: TL, FC. Manuscript draft preparation: TL. Manuscript review and editing: TL, HHT, GA, DMD, FC. Supervision: DB. Project administration: TL. TL was responsible for the overall content of the study. All authors have read and agreed to the submitted version of the manuscript.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.