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DPP SYPHILIS SCREEN and
Confirm Assay
®
A Unique Point of Care, Single Use Rapid Diagnostic
Rapid Tests for Earlier Treatment
CHEMBIO DPP® SYPHILIS
SCREEN and CONFIRM
Background
T
he cause of Syphilis is Treponema pallidum, a
spirochete. The Syphilis infection is limited to
the human host and is generally transmitted by
sexual contact. The disease is spread widely throughout
the world. Low concentrations of penicillin can kill
T. pallidum, and resistance to penicillin has not been
demonstrated.
Approximately 70,000 new Syphilis cases are reported
in the U.S. annually; however, the actual number of
infections is estimated at 500,000.
The disease manifests itself in infectious lesions on the
skin or mucous membranes of the genitalia. In almost
20% of the cases, the primary lesion is intrarectal,
perianal or oral, suggesting that T.pallidum can
penetrate the intact mucous membranes and skin.
Later in the disease, the bacteria multiply at the site
of entry, spread to nearby lymph nodes, and reach the
bloodstream. In 2 to 10 weeks, a papule develops at the
site of infection and breaks down to form an ulcer with
a clean, hard base (“hard chancre”). This primary lesion
always heals spontaneously, but 2 to 10 weeks later,
the secondary lesions appear. Syphilitic meningitis,
chorioretinitis, hepatitis, nephritis (immune complex
type) or periostitis frequently occur in Syphilis. The
Syphilitic infection may remain subclinical and the
patient may pass through primary and secondary
stages without symptoms and yet develop tertiary
lesions. About 30% of cases are cured, 30% remain
latent, and 40% progress to the tertiary stage,
characterized by granulomatous lesions in skin, bones
and liver, degenerative changes in the central nervous
system or cardiovascular lesions. However, tertiary
Syphilis is not usually infectious.
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Syphilis can be transmitted
to the fetus through the
placenta during the first
and second trimesters
of gestation. As a result,
some infected fetuses
can die and miscarry,
but some are stillborn
at birth, while others are
born live, but develop
congenital Syphilis or
other anomalies.
Public health officials in
many countries are attempting to modify current
control methods to better identify and treat Syphilis
infected prostitutes, drug users, and their sexual
contacts based on a dramatic increase of Syphilis rates
in many areas.
Syphilis Serological Testing
The recommended serological screening algorithm
for syphilis involves two types of serologic tests
(nontreponemal and treponemal).
Non-Treponemal Tests
Among the non-treponemal tests commonly used are
the Venereal Disease Research Lab (VDRL) and rapid
plasma reagin (RPR) tests. These tests are based on
the fact that particles of the lipid antigen (beef heart
cardiolipins) remain dispensed with normal serum, but
form visible clumps when combined with reagin. The
results are available in a few minutes and the tests are
inexpensive, yet very labor intensive and technique
dependent.
sensitivity for Syphilis antibodies. In the European
Union (EU) the treponemal test is used as the first
screen for syphilis. This test, however, cannot be used
to judge the efficacy of Syphilis treatment. In addition,
the use of treponemal tests alone may also result
in overtreatment because they do not differentiate
between adequately treated and previous infections.
Treponemal tests have long been considered a
confirmatory test that have followed a positive nontreponemal screening test. Positive screening tests are
then typically referred for a non-treponemal titer for
treatment surveillance and epidemiology purposes.
A Typical RPR Card Test
Positive VDRL and RPR tests revert to negative in 6 to
18 months after effective treatment. The VDRL can be
performed on Cerebral Spinal Fluid (CSF).
Non-treponemal tests are subject to false positives
(due to malaria, leprosy, measles, infectious mononucleosis, vaccinations, SLE, rheumatoid arthritis
and numerous other conditions), and false negative
in tertiary Syphilis. The use of a nontreponemal test
alone, such as the rapid plasma reagin (RPR) test, may
result in some overtreatment (treatment of uninfected
persons) owing to biological false-positive reactions.
Algorithms
We will consider three algorithms and their pluses
and minuses: the Traditional Algorithm, The Alternate
or Reverse Sequence Algorithm and the Rapid Point
of Care Algorithm. As mentioned earlier, traditional
algorithm, in the USA, has been a specimen received
Common patterns of serological reactivity in syphilis patients
Treponemal Tests
Among the most commonly performed treponemal
tests are Fluorescent Treponema Antibody Test (FTAABS) and Treponema Pallidum Particle Agglutination
Test (TPPA). Newer enzyme immune assays (EIA) and
chemiluminescence immunoassay (CLIA) treponemal
antibody tests have been introduced in both microplate format, for large volume lab testing (blood bank)
as well as a rapid point of care test (POCT).
is screened with a non-treponemal assay, most
commonly the RPR, with negatives reported out as
non-reactive. Reactive results reflex to a treponemal
confirmation, generally the TPPA or EIA.
The treponemal tests show excellent specificity and
The advantages of the traditional algorithm is the
the R Peeling et al WHO 2004
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majority of tests are normally non-reactive and are
culled out with a relatively low cost assay in RPR. The
disadvantages to the traditional algorithm are in the
labor issues and the methodology issues including
specificity challenges. The RPR is a manual method
that requires serum separation and heat inactivation,
a technician to manually pipette and disburse sample
and add reagent, and after a mixing period, read a
very subjective result. The RPR procedure should be
run under very controlled environmental and lighting
conditions that can result in reproducibility issues,
technician performance variability, and reading errors.
An additional issue, most important to high volume
testing labs, is the labor issue. The RPR being so labor
intensive, requires a significant amount of trained
technician time as compared to a fully automated assay
procedure. There is a large problem with few qualified
technicians choosing the laboratory field today as
well as many current technicians retiring or moving
on to other fields. The result is finding technicians for
the lab (at higher labor cost) and the actual tech time
involved in RPR testing, has become more expensive
and in many cases outweighs the advantage of the less
expensive, lower cost RPR assay.
life of a one time infected individual. Thus a positive
treponemal test does not indicate an active infection,
which can result in misdiagnosis if results are released
prior to, or in the absence of, a non-treponemal follow
up. This can result in over treatment of antibiotic and
patient angst.
The newest arrival is a Rapid Point of Care (POC)
Algorithm. With the approval of rapid point of care
single use tests, a new paradigm must be considered.
The current syphilis POC tests are simple lateral flow
devices that utilize a treponemal component. The test
upside is that a sample can be run with the patient
present, in 15 to 30 minutes, allowing a physician
to determine if the patient has been infected with
syphilis. The downside of the test is that it does not
indicate that a positive test is an active infection and
must be reflexed to a traditional nontreponemal assay
such as RPR. This would allow a screening of negatives
and involves the traditional blood draw, lab submission
and extended time for any positive result.
Figure. CDC recommended algorithm for reverse sequence syphilis screening
(treponemal sets screening followed by nontreponemal test confirmation*
EIA or CIA
Recently in the USA an Alternate or Reverse Sequence
Screening algorithm is being reviewed by several
federal agencies and has been instituted in some labs.
As mentioned earlier, this is the preferred algorithm in
the EU. This alternate algorithm involves performing
the Treponemal assay first then reflexing reactive
specimens to a non-treponemal assay for confirmation,
thus sometimes referred to as the “reverse algorithm”.
The clear advantage of the reverse algorithm is the
labor savings. The screening of the samples is done
with automated equipment eliminating the bulk of the
labor cost.
The drawback on the reverse assay is it determines
treponemal immunity status which can appear for the
EIA / CIA
+
Quantitative RPR
or other
nontreponemal
test
RPR
+
Syphilis
RPR
–
(past or present)5
TP-PA
TP-PA
+
Syphilis
(past or present)5
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EIA / CIA
_Ɨ
TP-PA
–
Syphilis unlikely1
A
B
Fig. 1. Structure of the Chembio dual POC test for syphilis showing the locations of the antigen lines.
A. Dissected view following testing of reactive serum; B, complete cassette following testing of reactive serum.
New DPP® Rapid Screen & Confirm Paradigm
A new point of care, rapid, single use syphilis test has
been developed by Chembio Diagnostic Systems
which employs both a Treponemal and
Nontreponemal component on their proprietary Dual Path
Platform (DPP®).
The Chembio DPP® Syphilis Screen & Confirm Assay
IS NOT CURRENTLY CLEARED OR AVAILABLE FOR
CLINICAL USE IN THE US.
The Chembio DPP® Syphilis Screen & Confirm Assay IS
CE MARKED AND AVAILABLE FOR SALE AND CLINICAL
USE OUTSIDE THE US WHERE INDICATED.
The DPP® Syphilis Screen and Confirm Assay (DPPSC)
provides a rapid (20 minutes), accurate visual reading at
room temperature allowing for the utilization of either
the traditional algorithm or the reverse algorithm. The
treponemal marker (line) indicates a history of syphilis
infection detecting IgG to T. palladium. The nontreponemal marker was developed at the US Center
for Disease Control and has been licensed for use in the
DPP® format by Chembio Diagnostic Systems.
Considering the presence of both the treponemal
and nontreponemal markers on the dual syphilis POC
assay will permit the clinician to initiate treatment
and partner notification activities at the consultation
site. Statistics have shown that in outreach and clinical
settings as many as 40 to 50 percent of those testing for
STDs never return for test results that traditionally have
taken from 24 to 48 hours to turn around. A rapid dual
syphilis test could greatly improve efforts to control
and eradicate syphilis.
In addition to clinical and outreach use the DPP®
Syphilis Screen and Confirm will be of utility in the lab
setting as a follow up to a syphilis positive screening.
In either the traditional or alternate algorithm the
suggested next step is to run the complementary assay
to the Treponemal or non treponemal screen. The
DPPSC will allow for that follow-up step and a repeat
of the screening result effectively and efficiently. This
will be a welcome addition to most small, medium
and large laboratories that currently only offer non
treponemal screens, or routinely send out specimens
for syphilis serology. In addition, the rapid and simple
performance characteristics of DPPSC outweigh
the cost and labor of running current or traditional
methods for Syphilis confirmation.
The DPP® dual POC test exhibits remarkable
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Fig. 2. Dual POC syphilis tests showing patterns of reactivity and their interpretations
performance characteristics compared to the standard
laboratory-based testing algorithm. When sera have
RPR titers of 1:8, the dual POC test has, in preclinical
studies, a sensitivity of 99.7%.
Table 1. Performance characteristics of the dual POC syphilis test (nontreponemal line) compared to the rapid plasma reagin (RPR) test.
Dual test
Non-treponemal Line
Positive
Number of samples tested with RPR
Negative
Positive
739
11
Negative
95*
756
834
767
Sensitivity 88.6%, Specificity 98.6%
*Of the 95 samples missed with the dual POC test had a RPR titer of 1:1 with RPR titer ≥ 1:2 the sensitivity was 98.7%
Total
750
851
1601
Table 2. Performance characteristics of the dual POC syphilis test (treponemal line) compared to the comparator Treponema pallidum
passive particle agglutination (TP-PA) test.
Dual test
Treponemal Line
Positive
Positive
Negative
Sensitivity 96.5%, Specificity 95.5%
972
35
1007
Number of samples tested with TP-PA
Negative
27
567
594
Total
999
602
1601
Table 3. Documented cases of syphilis tested with the dual DPP-POC test and the comparator rapid plasma reagin (RPR) and the
Treponema pallidum passive particle agglutination (TP-PA) tests.
Number of serum samples reactive (Sensitivity)
Dual rapid test
Comparator
Syphilis category
Primary untreated
Primary treated
Secondary untreated
Secondary treated
Latent untreated
Latent treated
Total
Non-Trep, Nontreponemal Line
a
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Total
7
14
6
28
5
45
105
Non-Trepa
7 (100%)
9 (100%)
6 (100%)
22 (84.6%)
3 (100%)
35 (85.4%)
82
Treponemal Line
b
Trepb
7 (100%)
13 (100%)
6 (100%)
28 (100%)
4 (80%)
44 (97.8%)
102
RPR
7
9
6
26
3
41
92
TP-PA
7
13
6
28
5
45
104
World Health Organization 2011 New Syphilis Cases Yearly Estimates
Access to syphilis diagnostics is limited in regions of high disease burden
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edition. American Public Health Association, Washington, DC.
Rapid Tests for Rapid
Earlier Treatment
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Chembio Diagnostic Systems, Inc.
3661 Horseblock Road
Medford, NY 11763 USA
info@chembio.com • www.chembio.com