Question
How well do the RPR and TPPA assays perform for routine syphilis serodiagnosis among Ethiopian factory workers participating in a cohort study on HIV-1 infection?

Design
A longitudinal cohort was screened for syphilis every 6 months using either the T. pallidum particle agglutination assay (TPPA) or the rapid plasma reagin test (RPR).  The results of participants with a minimum of 6 follow-up visits were interpreted over time and samples from participants with inconsistent results were retested to determine the rate of false positive results.  

Participants
Five hundred forty men and women participating in a cohort study on HIV-1 infection incidence and progression in a factory in the suburbs of Addis-Ababa, Ethiopia, with six or more study visits, were included in the study.  The mean age at enrollment was 35 years (range = 18-44 years), 54% were male, and 8.3% were HIV positive at enrollment.  The total number of blood samples tested was 4376, the mean number per subject was 8.3.   

Description of Tests and Diagnostic Standard
Blood for syphilis serology was drawn during follow-up visits every 6 months.  Screening for syphilis was performed with a TPPA test (Serodia-TP, Fujirebio Inc., Tokyo, Japan) for the first 2 years of the study and positive TPPA tests were tested by RPR (RPR Slide test, Biomerieux, Marcy l´Etoile, France).  During the last 2 years of the study, screening was performed with the RPR (RPR-Nosticon II, Organon Teknika, Boxtel, Netherlands) and positive RPR tests were tested by TPPA.  All tests were performed according to the instructions of the manufacturers.  Plasma samples were tested undiluted for RPR and diluted 1:80 for TPPA.  Weak reactive RPR results were interpreted as positive.  Subjects were classified into 3 categories based on results from all visits: a) no syphilis, tests negative on all visits, b) biological false positive (BFP) test, one or more positive RPR tests but negative TPPA results, c) syphilis or inconclusive diagnosis, at least one positive TPPA result, with or without a positive RPR result.

Samples from some subjects in category c were retested by the same assay if they showed inconsistent sequential results including: a positive TPPA among several negative TPPA results, or a negative RPR among several positive RPR results (with positive TPPA results), or a positive RPR (with positive TPPA result) among several negative RPR results.  Repeat tests were performed independently by three technicians who were blinded to the previous results, and the result obtained by at least two of the three was used.   Based on revised test results, the subjects in category c were classified as having past, prevalent, incident, or no syphilis.  Definitions used were: past syphilis-positive TPPA without positive RPR; prevalent syphilis-positive RPR and TPPA; incident syphilis-sequential RPR seroconversion with or without TPPA seroconversion; and no syphilis-tests on all visits negative.  

Main Outcome Measures
For all samples with a positive TPPA screening result during the first 2 years of the study, the percentage of sample mix-ups was calculated.  For all samples screened with the RPR test in the last 2 years of the study, the percentage of BFP results was calculated.  

Main Results
The number of subjects placed into each of the syphilis diagnostic categories before and after retesting is shown in the table.  One hundred fifty-two samples from 93 (53%) of the 176 subjects in category c were retested by TPPA (n = 18), by RPR (n = 100), or by both (n = 34).  The same result was obtained by all 3 technicians for all 52 of the samples retested by TPPA.  For the 134 samples retested by RPR, all 3 technicians agreed on the result for 99 (74%).  After retesting, 17 subjects in category c were syphilis negative.  Based on the results of retesting, 9 (1.2%) of the 785 samples screened and positive by TPPA were regarded as false positive due to sample mix-ups, while 190 (8.2%) of the 2325 samples screened by RPR were determined to be BFP.  The BFP rate was 3.2% if weak positive results (n = 115) were excluded.  No chronic false positive reactions were seen.  HIV seropositivity was not correlated with false positive RPR results.  The prevalence of syphilis increased with age (p<0.001).  

Syphilis diagnosis of 540 Ethiopian factory workers based on the results of screening at least 6 sequential blood samples by TPPA or RPR classified before and after retesting 152 samples from 93 subjects in category c

Authors' Conclusions
A high percentage of BFP RPR results, inconsistent inter-individual interpretation of the non-treponemal RPR test, and sample mix-ups were observed.  The use of the RPR test as a single screening assay for syphilis is questionable.  The data gave no indication that the syphilis serological tests were influenced by HIV infection. 

Source of funding:  The Ethio-Netherlands AIDS Research Project, Dutch Ministry for Development Co-operation and the Ethiopian Ministry of Health

For correspondence:  J. W. Dorigo-Zetsma, Ethio-Netherlands AIDS Research Project, Ethiopian Health and Nutrition Research Institute, PO Box 1242, Addis-Ababa, Ethiopia.  E-mail address:  moud@gggd.amsterdam.nl.