APHL 2019 POSTER ABSTRACTS
Improved Laboratory Workflow Using the BioPlex 2200
Syphilis Total and RPR Assay
W. Zheng, S. Zhou, J. Seiki, R. Walker and R. Kaul, Bio-Rad
Laboratories
Background: Syphilis is a sexually transmitted disease caused by
gram-negative bacterium Treponema pallidum. Serological diagnosis
of syphilis infection requires the combination of treponemal and
non-treponemal test results. The two main testing algorithms
are known as the traditional and reverse sequence algorithms.
Both algorithms have selective advantages and disadvantages.
Historically, non-treponemal assays are manual and labor intensive
with subjective test result interpretation. Bio-Rad Laboratories
has developed a fully automated treponemal and non-treponemal
combo assay that provides both Syphilis Total (IgG and IgM) and
RPR serological test results simultaneously, thereby offering a one-
step universal testing method with reduced turnaround time and
good performance. The BioPlex 2200 Syphilis Total & RPR assay
is highly sensitive and provides on-demand automated RPR titer
determination capability up to 1:2048.
Methods and Materials: 206 samples comprising 108 retrospective
and 98 prospectively collected samples were tested with the
BioPlex 2200 Syphilis Total & RPR assay, Serodia TPPA (Fujirebio
Diagnostics, Malvern, PA), and Macro-Vue RPR Card test (Becton
Dickinson, Franklin Lakes, NJ). Additionally, end point RPR titer
determinations were performed on eight in-house and six American
Proficiency Institute (API) panel members.
Results: Of the 206 samples tested by the BioPlex 2200 Syphilis
Total & RPR assay, 98 (47.6%) tested positive for Syphilis and RPR
indicative of an active infection, 82 (39.8%) tested negative by
both assays suggestive of no syphilis infection, 14/16 Syphilis(+)/
RPR(-) confirmed positive by the TPPA test indicative of previously
treated syphilis infection while 9/10 Syphilis(-)/RPR(+) samples
tested negative by the TPPA test and may be classified as RPR false
positive. Taken together, the new algorithm (co-testing) generated
11/206 (5.3%) discordant samples, compared to 9/108 (8.3%)
using the traditional algorithm and 2/114 (1.8%) using the reverse
algorithm. However, both traditional and reverse algorithms missed
RPR-/Syph+/TPPA+ (N=14) and Syph-/RPR+/TPPA+ (N=1) samples,
a disadvantage with the multi-step testing algorithm. Of note is
the higher diagnostic value of the BioPlex RPR assay since higher
detectable levels of the antibody titers were observed compared to
the BD Macro-Vue RPR card test. When six API RPR positive panel
members and eight in-house acquired RPR positive samples were
subjected to end point RPR titration, 10 generated higherr RPR
titers by at least one dilution factor on the BioPlex 2200 RPR assay.
Conclusions: In summary, the new testing algorithm where syphilis
screen and confirmation is performed simultaneously by running
both treponemal and non-treponemal tests together offers one-step
universal syphilis testing with excellent assay performance coupled
with the higher titer sensitivity of RPR assay.
Presenter: Ravi Kaul, Bio-Rad Laboratories, Hercules, CA,
[email protected]
High-Definition PCR (HDPCR): A Novel and Economic
Multiplexing qPCR Technology for Tickborne Pathogen
Testing
B. Amro, H. Carolan, R. Abanes, J. Nayak, J. Hill, C. Smith, B.
Leatham, A. Estanda, D. Broxterman, A. Schroeder, S. Powell, L.
Jacky and K. Menge, ChromaCode
Background: The US is experiencing an increase in infections from
tickborne diseases and a geographic expansion of tick-borne cases.
There has also been a noticeable increase in the percentage of tick
species carrying multiple disease agents. While access to serologic
and microscopic methods are widespread, there are limited
singleplex PCR tests available and no multiplex PCR tests. This study
describes the development of ChromaCode’s multiplex HDPCR™
Tickborne Panel (TBP) Research Use Only (RUO) and its performance
across a series of analytical studies.
Methods: HDPCR Overview: HDPCR™ is ChromaCode’s novel
multiplexing technology and is the coupling of widely-used, low-
cost chemistries with proprietary data science algorithms. HDPCR
seamlessly integrates onto common real-time and digital PCR
platforms to enhance the multiplexing levels of these instruments
for test applications of 5-50 targets without any hardware changes.
While traditional qPCR multiplexing relies on differentiation of
targets by color, HDPCR enables detection of multiple targets within
a single color channel by differentiating them by signal intensity.
Test Design: TBP is a single well, 4 channel assay: Channel 1 –
Borrelia Group 1 (B. burgdorferi, B. mayonii), Ehrlichia chaffeensis,
Borrelia miyamotoi; Channel 2 – Rickettsia spp., Ehrlichia muris
eauclarensis, Anaplasma phagocytophilum; Channel 4 – Internal
Control; Channel 5 – Borrelia Group 2 (B. hermsii, B. parkeri, B.
turicatae), Babesia microti, Ehrlichia ewingii.
Analytical Studies: Inclusivity, exclusivity, and limit of detection
(LOD) studies were performed to characterize the performance
of TBP. Testing was performed on the ABI 7500 Fast, ViiA 7 and
QuantStudio 7. Results were analyzed on ChromaCode’s cloud-
based software ChromaCode Cloud.
Results: Inclusivity Study: Inclusivity was established for the genus
or group level TBP targets by testing synthetic templates spiked
into TE buffer in triplicate at 3X LOD. In total, Borrelia Group 1 was
inclusive to 6 Borrelia species, Borrelia Group 2 was inclusive to 3
Borrelia species, and Rickettsia spp. was inclusive to 14 Rickettsia
species.
Exclusivity Study: The exclusivity study was performed using a
mix of genomic DNA and synthetic samples. Strains tested were
near-neighbor strains to TBP targets. The samples were tested
in triplicate at 1 million copies/reaction and/or at 1,000 copies/
reaction. A total of 23 unique organisms/strains were shown to be
exclusive to TBP targets.
Limit of Detection: The LOD for each TBP target was established
using synthetic DNA spiked into TE buffer. Each of the nine TBP
targets had a LOD of 10 copies per reaction.
Conclusions: HDPCR is a new multiplex technology that enables
higher multiplexing on existing qPCR instruments. TBP is
ChromaCode’s first HDPCR test and enables multiplex detection of
common tickborne diseases from whole blood.
Presenter: Scott Powell, ChromaCode, Carlsbad, CA,
[email protected]
PublicHealthLabs
@APHL
APHL.org
Summer 2019 LAB MATTERS
71