1. Rapid Diagnostic Tests for Syphilis A Preliminary Review of the U. S
Rapid Diagnostic Tests for Syphilis A Preliminary Review of the U. S. Clinical Data
M Sutton, S Zackery, C Ciesielski, M Zajackowski, M Santana, C Langley, L Bernard, V Pope, M Fears, R Johnson, L Markowitz
Good afternoon, everyone. Today I am very excited to share with you some preliminary data for an ongoing clinical evaluation of rapid diagnostics for syphilis in the United States. I’d like to thank my co-authors who are listed here.

2. Background
Syphilis is diagnosed in the U.S. by a nontreponemal screening test and a treponemal confirmatory test.
on-site RPR plus offsite treponemal test, OR
RPR and treponemal tests are sent offsite (turn-around time up to 1 week or more).
Rapid immunochromatographic (ICS) diagnostic tests:
treponemal-based tests
can provide results in 15 minutes
may potentially be of use in non-traditional settings
Rapid ICS tests for syphilis are not FDA-cleared for use in the US.
A U.S. based study was conceived to evaluate these rapid diagnostics for syphilis.
Syphilis is routinely diagnosed serologically in this country by using a 2-tiered system of a nontreponemal screening test followed by a confirmatory treponemal test. The two most commonly used methods in U.S. clinical settings are 1—an on-site RPR plus an offsite treponemal test, and 2—sending both the RPR and treponemal test to offsite laboratories. Sending specimens offsite can delay treatment by as much as 1 week, and if a patient is unable to be located and brought back in for treatment, efforts to prevent further transmission of untreated syphilis can be hindered. A possible solution to facilitating more timely diagnosis and treatment of syphilis are rapid immunochromatographic (ICS) diagnostic tests. They are treponemal-based tests and can provide results in under 15 minutes. Their ease of use makes them a potential asset for syphilis prevention and control in both traditional clinical as well as non-traditional, non-clinical settings. Although several rapid ICS tests for syphilis have been used internationally for many years, these tests are not yet cleared by the FDA for use in this country. To learn more about these rapid syphilis tests and evaluate their possible use in the U. S. as a syphilis intervention tool, a clinical trial was started in areas with high rates of disease.

3. Cross-sectional study
Multi-site study of rapid diagnostic syphilis assays of persons attending STD clinics in high syphilis morbidity areas
Cross-sectional study
3 ICS strips included vary by which Treponema pallidum antigens are present on the test band region
Two U.S. sites are currently evaluating the performance of 3 rapid diagnostic strips
This ongoing clinical trial is a cross-sectional study enrolling persons attending STD clinics in areas with high rates of disease. It began enrollment in 2001 and includes three manufacturers’ ICS tests. The 3 ICS tests being evaluated vary by which Treponema pallidum antigens are present on the test band region. Two U.S. cities with high rates of syphilis received grant funding to conduct this study. The study team members at each site were trained on how to perform and interpret the ICS tests based on the manufacturers’ recommendations prior to beginning the study. These sites are currently enrolling patients and evaluating the performance of these 3 ICS tests.

4. ICS Strip
Very little equipment is needed to perform this test, and I want to show you that here. Here is a photo of an ICS test, an alcohol pad and a lancet. Other items needed are about 3 drops of buffer and a bandaid if needed for the patient. To perform this test, the alcohol pad is used prior to doing the finger stick with the lancet. The drop of blood from the finger stick is applied to this sample pad portion of the ICS so that the entire pad is covered. Buffer is then added to the sample pad region, and 15 minutes are allowed for the sample to wick up the strip past the control line.

5. Immunochromatographic Strip (ICS) Rapid Diagnostics for Syphilis
Interpretation of Results
I I
Membrane strip based immunoassay
Test procedure
Recombinant T. pallidum antigen is coated on the test band region.
Sample is placed on application pad and reacts with antigen-colloidal gold conjugate.
If T. pallidum antibody is present an antigen-antibody-antigen gold complex will form (evident by line in the test area).
Line should always appear in control region.
Application Test Control Absorbent
Pad Line Line Pad
Positive Sample
I I
Negative Sample
This slide has a schematic of the ICS tests just decribed, all of which work by the same lateral flow technique. The various antigens that the 3 tests use are coated in the test band region. The specimen, whether fingerstick, whole blood, serum, or plasma, is placed here on the application pad and reacts with the antigen colloidal gold conjugate. If Treponema pallidum antibody is present in the person’s sample, an antigen-antibody complex will form and a line will be evident in the test area. The conjugate then continues migrating up to the control region where a second line should appear. Two lines are indicative of a positive sample, one line in the control region only indicates a negative sample, and no line in the control region indicates an inconclusive result. The study team personnel were instructed the presence of any line, even a faint one, is indicative of a positive result, based on the manufacturers’ guidelines. I
Inconclusive

6. Objectives
To evaluate ICS test performance using finger stick, whole blood, serum, and plasma specimens compared to RPR and TPPA
To compare ICS test performance with serum at local sites and CDC
To evaluate ICS test performance by syphilis stage of disease
For this preliminary review of the data, the objectives are as follows: 1—To evaluate ICS test performance using finger stick, whole blood, serum, and plasma specimens compared to RPR and TPPA; 2---To compare ICS test performance for serum at local test sites and the CDC laboratory, and 3---To evaluate ICS test performance by syphilis stage of disease.

7. Methods I Enrollment- consenting adults age 18 or older
Finger stick done for point-of-care testing on 1 ICS test
Blood drawn for whole blood, serum, and plasma ICS testing at local sites and serum for CDC testing for 3 ICS tests
Data collection questionnaire includes clinical assessment of syphilis stage of disease, if applicable
Persons tested and treated for syphilis if needed according to established standard of care.
ICS tests are for investigational purposes only.
Slide To meet these objectives, our methods were as follows: Enrollment consists of consenting adults ages 18 and older. For each person enrolled, a fingerstick specimen is obtained for a point-of-care test with one of the three ICS tests. Next, venipuncture blood is obtained for the whole blood, serum, and plasma specimens, and each of these specimens is evaluated on each of the 3 ICS tests at the local site. Due to lack of specimen stability for whole blood and plasma, only a serum specimen is sent to CDC, where the specimen is again tested using all 3 ICS tests. The data collection questionnaire provides demographics and other clinical information, including presumed stage of disease. Because the ICS tests are for investigational purposes only, persons are tested and treated for syphilis according to the established standard of care (RPR with TPPA).

8. Methods II Data analyzed using Epi-Info and SAS
Results reported by comparing to clinical standard of both RPR and TP-PA results
Sensitivity= positives/ RPR (+) TP-PA (+)
Specificity= negatives/RPR (-) TP-PA (-)
Syphilis case definitions
Once at CDC, the data are analyzed using Epi-Info and SAS. Results are reported by comparing the ICS tests to our best clinical measure of disease, both RPR and TPPA. Sensitivity and specificity equations used are shown here. Case definitions for syphilis are as per the 1997 MMWR standard case definitions and study protocol.

9. Results (Demographics)
N=383
64% male
47% African-American
32% White
18% Latino
Reactive RPRs=24
Titers < 1:8=8 (33%)
Titers ≥ 1:8=16 (67%)
TP-PA R NR
TOTAL 20 4 24 21
338
359 41
342
383 R
RPR
This slide shows the overall demographics of those included in this preliminary analysis enrollees were included in this analysis, 64% of them were male. This sample includes a diverse group, with African-Americans, Whites, and Latinos represented. 24 persons in this group had reactive RPRs, and two-thirds had titers of greater than or equal to 1:8. A few persons who did not have each ICS test performed or had no sample available for a particular test were excluded in an effort to make the analyses more comparable. NR
TOTAL

10. Results: Test performance (sensitivity) of ICS at local sites, by specimen type, compared to RPR and TP-PA
Specimen Type
Sensitivity (%)
Test 1
Test 2
Test 3
Whole Blood
100
(17/17)
47.1
(8/17)
82.4
(14/17)
Serum
76.5
(13/17)
88.2
(15/17)
Plasma 75
(12/16)
First, I’ll review the venipuncture specimen results. This table shows the sensitivities of each of the 3 ICS tests in whole blood, serum ,and plasma at the local study sites compared to reactive RPRs and TPPAs. The bold numbers here are the percentages, and under those are the numerators and denominators in parentheses. Here Test 1 was 100% sensitive in each of the three specimen types. Test 2 was less sensitive in whole blood than in serum and plasma, but the range was only 47-76%. Test 3 was most sensitive in plasma.

11. Results: Test performance (specificity) of ICS at local sites, by specimen type, compared to RPR and TP-PA
Specimen Type
Specificity (%)
Test 1
Test 2
Test 3
Whole Blood
97.7
(334/342)
99.7
(336/337)
(340/341)
Serum
93.3
(319/342)
96.2
(327/340)
99.1
(339/342)
Plasma
93.2
(313/336)
97.9
(322/329)
(331/334)
This table shows the specificities of each of the 3 ICS tests in each specimen type, whole blood, serum ,and plasma at the local sites compared to RPR and TPPA. Test 1 was most specific, or identified the most true negatives, in whole blood specimens. Test 2 had specificities ranging from 96 to over 99% in all specimen types, and Test 3 had specificities of over 99% in each of the 3 specimens.

12. Results: Test Performance on Sera, Local Sites vs
Results: Test Performance on Sera, Local Sites vs. CDC Laboratory, compared to RPR and TPPA
As stated in the methods, the local study sites and CDC have only the serum specimens in common. This graph compares the test performance of serum specimens tested locally with serum specimens tested at CDC. This comparison allows us to look at real-world local study site scenarios and the CDC laboratory setting using the only specimen type that both settings had in common. Overall, the test performance is very similar in these settings for test specificity. For sensitivity, Tests 1 and 3 perform similarly, but Test 2 performs slightly better at the CDC laboratory.
Sensitivity
Specificity

13. Results: Point-of-care ICS (Finger stick) compared to RPR and TP-PA
Overall (any RPR titer and reactive TP-PA)
Sensitivity=17/17 (100%)
Specificity=324/327 (99.1%)
Clinical Diagnosis of Syphilis
Number of Cases
Primary 1
Secondary 7
Latent 9
Previously treated
(TP-PA (+) & RPR (-)) 10
Now we’ve discussed the venipuncture specimen results, so now I’ll turn to the finger stick or point-of-care results performed by the clinicians. Again, only 1 of the 3 ICS tests was used as the point-of-care test, mainly because of the desire to avoid multiple finger sticks. Overall, the point-of-care ICS was able to detect 100% of syphilis cases and the specificity was 99.1%. This table shows how the cases enrolled were categorized by stage of disease. Based on our standard case definitions, 1 person was classified as primary, 7 as secondary, and 9 as latent, for a total of 17 cases, all detected by the finger stick ICS. It is also important to point out these 10 persons who had reactive treponemal tests, with negative RPRs, most of whom had been previously treated. As the ICS tests are treponemal-based tests, they were also effective at picking up these previously treated patients with reactive TPPAs.

14. Conclusions
There is a broad range of sensitivities and specificities for the 3 ICS tests in whole blood, serum, and plasma.
Test performance for sera locally is very comparable to test performance at CDC.
The ICS point-of-care (finger stick) data show sensitivities of 100% and specificities of over 99%.
ICS tests are performing well to date with various stages of disease.
In conclusion, for the ICS tests included in this evaluation, there is a wide range of sensitivities and specificities in whole blood, serum, and plasma. Test performance at the local study sites is comparable to test performance at CDC. The point-of-care ICS data show 100% sensitivity and specificity of over 99%. With the stages of disease that are in our sample to date, the point-of-care ICS test is performing well.

15. Limitations
Treponemal tests are unable to differentiate between active and previously treated syphilis.
Interval analysis only; more data needed.
However, it is important to note that these ICS tests are treponemal tests and are unable to differentiate between active and previously treated cases of syphilis. As such, it could lead to overtreatment of some persons if eventually used as a screening test. Another limitation is that as this is an interval analysis only, numbers are small and more data are needed.

16. Summary & Future Steps
Some rapid diagnostic tests show promising performance as screening tests for syphilis in the United States.
These ICS tests require few supplies and can potentially be used in both traditional clinical settings as well as non-traditional settings.
Data collection is ongoing.
In summary, the data to date are promising, and some rapid ICS tests for syphilis show promising performance as screening tests in this country, with specificities and sensitivities of greater than 99%. Also, these ICS tests require few supplies and have the potential to be used in both traditional clinical settings as well as non-traditional, non-clinical settings to effect more timely diagnosis and possibly treatment of persons with syphilis. To help reach sample size goals, and to continue learning about these products, enrollment and data collection is ongoing.

17. Acknowledgments S Zackery C Ciesielski M Zajackowski M Santana
C Langley
L Bernard
V Pope
M Fears
R Johnson
L Markowitz
J Braxton
J Chapin
J Lewis
S Berman
J Douglas
B Litchfield
CDC DASTLR
Local Public Health Departments
Finally, I’d like to thank everyone on this list who helped move this study forward. Thank you for your attention.