APHL 2022 POSTER ABSTRACTS
Methods : Antibody detection in the VITROS HIV Combo Assay is achieved using recombinant transmembrane antigens specific to HIV-1 ( group M and O ) and to HIV-2 . The p24 antigen detection is accomplished using monoclonal antibodies ( mAb ). The antigens and p24 mAb are coated onto the well . Sample is added to the wells in the first stage of the reaction and HIV Ag / Ab from the sample is captured . After washing , HRP conjugated antigens and p24 mAb are added . Following a final wash , bound HRP conjugates are detected using the VITROS signal reagent . Specificity was assessed using 6003 samples from low-risk populations . Sensitivity was evaluated using 2074 antibody positive samples ( 1845 HIV-1 , including 413 from various Group M and O subtypes and 229 HIV-2 ). In addition , 52 samples with various HIV-1 group M antigen genotypes were tested . Seroconversion sensitivity was assessed by testing 32 commercially available panels on both the VITROS HIV Combo assay and a commercially available Ag / Ab assay . Assay reproducibility was assessed at three sites using three reagent lots with a 14-member panel . Antigen sensitivity was determined by testing serial dilutions of the NIBSC and the AFSSAPS HIV-1 p24 Ag standards across two reagent lots .
Results : The specificity of the VITROS HIV Combo Assay for the low-risk population was 99.6 % ( 5934 / 5959 ) [ 95 % exact CI ( 99.4-99.7 %)] ( 44 reactive samples were confirmed positive by supplemental testing ). The sensitivity for HIV-1 and HIV-2 antibody positive samples was 100.0 % ( 2074 / 2074 ) [ exact 95 % CI ( 99.8- 100.0 %)]. All 413 HIV-1 group M and O antibody positive subtypes were reactive . 50 of 52 HIV-1 group M antigen genotypes were reactive with the VITROS HIV Combo assay with 49 of 52 reactive on the commercially available Ag / Ab assay . For seroconversion panels , the VITROS HIV Combo assay was reactive at the same panel member as the commercially available Ag / Ab assay for 25 of the 32 panels , was reactive one panel member earlier for 6 panels , and was reactive later for 1 panel . For the reproducibility study the observed precision for the 12 panel members positioned near the assay cutoff ranged from 9.1 to 17.8 % CV . The overall sensitivity of the VITROS HIV Combo test for the NIBSC HIV-1 p24 Antigen Standard ( 90 / 636 ) was ≤0.46 IU / mL and for the AFSSAPS HIV-1 p24 Antigen Standard was ≤12.8 pg / mL .
Conclusion : The VITROS HIV Combo Assay demonstrates acceptable clinical and analytical performance in the simultaneous detection of antibodies to HIV-1 ( group M and O ), HIV-2 and HIV p24 antigen .
Presenter : Paul Contestable , Ortho Clinical Diagnostics , paul . contestable @ orthoclinicaldiagnostics . com
A Streamlined Workflow For High-Throughput , Multiplexed HiFi Sequencing Of Microbial Genomes
M Ashby , A Wenger , J Wilson , S Chakraborty , D Lee , C Dunn , I Sovic , Z Kronenberg , H Ferrao and S Dillon , Pacific Biosciences
The SARS-CoV-2 global pandemic has highlighted the utility of pathogen surveillance pipelines that provide comprehensive genomic information , giving public health scientists a more complete view of the spread and characteristics of circulating pathogens . Beyond COVID-19 , there is great interest in public health to expand high resolution surveillance to other infectious diseases . Highly accurate , long HiFi reads produced by the PacBio Sequel IIe System have brought new levels of contiguity , completeness and accuracy to large genome assembly . HiFi reads are similarly beneficial for microbial genome assembly , as higher quality assemblies enhance our ability to investigate foodborne illnesses and monitor antimicrobial resistance . However , obstacles in library preparation workflow , cost and recovery of small plasmids have limited use in public health . Here , we introduce a new library prep workflow and assembly algorithm based on HiFi reads that enables a high throughput , end-to-end solution for microbial genome assembly . The new workflow combines steps , eliminates the need for strict size selection , shortens the total time to six hours and enables library prep automation . The assembly algorithm uses strict read-to-read overlaps enabled by HiFi read accuracy to resolve repeats . It uses a two-stage approach to first assemble chromosomes and then recover short , high-coverage plasmids . To evaluate the method , a pool of HiFi libraries with 96 microbial samples and total genome size of 375 Mb was generated . The protocol was evaluated with microbes relevant to pathogen surveillance including common foodborne pathogens ( Listeria , Salmonella ) and species often seen in hospital settings ( Klebsiella , Staphylococcus ). The microbes represent a range of genome sizes , assembly complexity , GC content , chromosome counts and plasmid content . DNA samples were sheared to 7 kb - 10 kb , prepared as barcoded libraries , pooled and sequenced on one SMRT Cell 8M on the Sequel IIe System . Reference quality de novo microbial assemblies with 5 contigs or fewer were achieved for all samples . Typical chromosome assembly quality was Q50 , measured as concordance to reference assemblies . Nearly all plasmids were recovered , including those shorter than 5 kb which are often lost in workflows with strict size selection . Taken together , the new method provides a high-throughput , cost-effective approach suitable to routinely generate reference quality microbial genomes in a public health environment as part of a pathogen surveillance program .
Presenter : Meredith Ashby , Pacific Biosciences , mashby @ pacificbiosciences . com
Rapid and Quantitative Enumeration of Live Legionella using Multiplexed Viability qPCR
N Feirer 1 , C Shi 2 , T Kirkland 2 , M Scurria 2 , S Goueli 1 , J Cali 1 , S Mondal 1 ; 1 Promega Corporation , 2 Promega Biosciences
PCR-based bacterial enumeration often leads to an overestimation of viable cells due to the amplification of DNA from dead cells . To overcome this disadvantage , DNA-intercalating crosslinking dyes such as ethidium monoazide ( EMA ) or propidium monoazide ( PMA ) have been used in combination with qPCR to discriminate between viable and non-viable cells . Drawbacks of using PMA and EMA include inhibition of photocrosslinking in turbid samples , lack of consistent photocrosslinking protocols and a requirement of large amplicon sizes for robust results . The need for large amplicon size has encouraged the use of SYBR green dye-based qPCR which is not amenable to multiplexing or the inclusion of appropriate qPCR controls . To overcome these challenges and simplify the PCR workflow we synthesized a novel viability PCR crosslinker , which is composed of a live cell-impermeable DNA crosslinking dye that spontaneously crosslinks and prevents dead cell DNA amplification without the need for photoactivation . We optimized probe-hydrolysis qPCR assays for large amplicon sizes , allowing multiplexing for Legionella pneumophila and Legionella spp . detection . We also
Infectious Diseases