RDTs were selected to include locally commercially available tests approved by the Colombian regulatory authority (Instituto Nacional de Vigilancia de Medicamentos y Alimentos [INVIMA]): SD Bioline Syphilis 3.0 (Standard Diagnostics Inc, Kyonggi-do, Korea) and Alere Determine Syphilis TP (Abbott Diagnostics Medical Co, Ltd). Both tests use an immunochromatographic platform with a strip showing a colored test line if treponemal antibodies are detected in the specimen and a colored control line if the test is working properly. RDTs were run according to manufacturer’s instructions, read after 20 minutes and were considered positive if both the test and control lines were colored, even if the test line was faint. They were considered negatives if only the control line was colored and invalid when only the test or neither of the lines were colored.
RDTs were performed at the point-of-care using capillary blood (CB) from a finger prick and at a reference laboratory using serum. A volume of 20μL of CB or 10μL of serum with its corresponding buffer for Bioline and 50μL of CB with its corresponding buffer or serum without buffer for Determine were used. RDTs on CB were read by one of 5 operators at the point-of-care and, on sera at the reference laboratory by three evaluators, two microbiologists and one physician, RDTs on sera were considered positive or negative when at least two evaluators agreed on the assessment of the test and were considered invalid when at least one evaluator considered the test invalid. Invalid tests were repeated once.
Diagnosis of syphilis is challenging due to the lack of a reliable reference standard; however, serology tests remains as the most widely implemented tests for syphilis [21 (link), 22 (link)]. Considering that the index tests under study detect treponemal antibodies, we decided to use two TT as reference standard: Treponema pallidum haemagglutination test (TPHA) and enzyme linked immunoassay (ELISA) for syphilis. TPHA (Human Gesellschaft für Biochemica und Diagnostica mbH, Wiesbaden, Germany) and T. pallidum ELISA (Human Gesellschaft für Biochemica und Diagnostica mbH, Wiesbaden, Germany) were performed using 10 μL of serum for each test. As with index tests, selection of the reference standard kits was based on availability and approval by INVIMA. We decided to use composite reference standard due to the limitations of each individual test such as higher false positive rate of immunoassays and lower sensitivity of agglutination assays during latent syphilis [23 (link)]. The reference standard was considered positive when both tests were positive, negative when both tests were negative and undetermined when the two results were discordant or one of the tests was invalid.
RDTs were performed at the point-of-care using capillary blood (CB) from a finger prick and at a reference laboratory using serum. A volume of 20μL of CB or 10μL of serum with its corresponding buffer for Bioline and 50μL of CB with its corresponding buffer or serum without buffer for Determine were used. RDTs on CB were read by one of 5 operators at the point-of-care and, on sera at the reference laboratory by three evaluators, two microbiologists and one physician, RDTs on sera were considered positive or negative when at least two evaluators agreed on the assessment of the test and were considered invalid when at least one evaluator considered the test invalid. Invalid tests were repeated once.
Diagnosis of syphilis is challenging due to the lack of a reliable reference standard; however, serology tests remains as the most widely implemented tests for syphilis [21 (link), 22 (link)]. Considering that the index tests under study detect treponemal antibodies, we decided to use two TT as reference standard: Treponema pallidum haemagglutination test (TPHA) and enzyme linked immunoassay (ELISA) for syphilis. TPHA (Human Gesellschaft für Biochemica und Diagnostica mbH, Wiesbaden, Germany) and T. pallidum ELISA (Human Gesellschaft für Biochemica und Diagnostica mbH, Wiesbaden, Germany) were performed using 10 μL of serum for each test. As with index tests, selection of the reference standard kits was based on availability and approval by INVIMA. We decided to use composite reference standard due to the limitations of each individual test such as higher false positive rate of immunoassays and lower sensitivity of agglutination assays during latent syphilis [23 (link)]. The reference standard was considered positive when both tests were positive, negative when both tests were negative and undetermined when the two results were discordant or one of the tests was invalid.
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