Lab Matters Summer 2019 - Page 73

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, ravi_kaul@bio-rad.com 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, spowell@chromacode.com PublicHealthLabs @APHL APHL.org Summer 2019 LAB MATTERS 71