Lab Matters Fall 2023 | Page 59

APHL 2023 POSTER ABSTRACTS using the Streck ARM-D ® OXA real-time qPCR kit . The isolates tested in this study included 47 A . baumannii , four Acinetobacter pittii , and six Klebsiella pneumoniae isolates . The performance of the test was validated at the NJDOH PHEL by measuring the accuracy , precision , and specificity of the test in which PCR results were compared to the known genotype of each bacterial isolate . The addition of the realtime qPCR test for OXA-family genes at the NJDOH PHEL increases the AMR surveillance capabilities of the laboratory , particularly for the detection and genotyping of carbapenem-resistant A . baumannii isolates which pose a significant threat in healthcare settings .

Presenter : Eagle Bui , eagle . bui @ doh . nj . gov

Optimization of Multiplex RT-PCR Assay for Detection of Respiratory Syncytial Virus , Enterovirus , Human Parechovirus , and Adenovirus using the Panther Fusion System

E . Roloff , K . Patel , P . Raj , J . Hauser ; District of Columbia Department of Forensic Sciences

Many viral pathogens present the opportunity for influenza-like illness in infants and immunologically compromised adults , including respiratory syncytial virus ( RSV ), enterovirus ( EV ), parechovirus ( HPeV ), and adenovirus ( HAdV ). Rapid identification of these diseases could assist clinical providers with appropriate therapies to alleviate severity of illness , as well as provide outbreak surveillance data to local health departments . These pathogens can be identified using conventional PCR ; however , it can be costly , labor intensive , and inefficient to test samples for one viral target at a time . The Open Access feature within Hologic ’ s Panther Fusion System allows users to fully automate laboratory developed tests ( LDTs ) for nucleic acid amplification and real-time PCR ( RT-PCR ). The DC PHL aims to optimize a multiplex RT-PCR LDT to detect respiratory syncytial virus types A and B , adenovirus type 14 , pan-human parechovirus , and pan-enterovirus in human respiratory samples . To assess assay performance , nasopharyngeal , oropharyngeal , and nasal swabs will be spiked with positive reference material for each target of interest . Samples will be tested for accuracy , precision , analytical sensitivity ( limit of detection ) and specificity . Variables to be optimized for this assay include concentration of primers , probes , MgCl2 , KCl , and Tris . Primers and probe sequences used for detection were obtained from published literature and produced by Integrated DNA Technologies . Human RNAse P will be used as a positive extraction control .

Presenter : Erika Roloff , roloff @ gwmail . gwu . edu

Piloting Whole-Genome Sequencing to Identify Mycobacterium tuberculosis Complex and Associated Antimicrobial Resistance : The North Carolina State Laboratory of Public Health ’ s Experience

A . Yaya , T . Lawson , K . Chase , W . Glover , S . Shone ; North Carolina State Laboratory of Public Health

Introduction : The North Carolina State Laboratory of Public Health ’ s ( NCSLPH ) experience with whole genome sequencing ( WGS ) of Mycobacterium tuberculosis Complex ( MTBC ) started with an APHL- CDC pilot to generate quality genomic sequences from Mycobacteria Growth Indicator Tubes ( MGIT ). The developed methods were applied to another pilot project to evaluate the Wadsworth Center ,

New York State Department of Health ’ s ( NYSDOH ) NGS TB Pipeline Service for which historical sequencing data from the CDC and the Michigan Department of Health and Human Services ( MDHHS ) were also used . Method : For the APHL-CDC pilot , NCSLPH extracted and sequenced DNA from 50 MTBC MGITs using the Qiagen EZ1 instrument . WGS library preparation was performed following the laboratory Standard Operation Procedure for PulseNet Nextera DNA Flex Library Preparation . The Wadsworth Center , NYSDOH ’ s NGS TB Pipeline Service , was used for bioinformatics data analysis . Subsequently , 112 MGIT and solid media ( Lowenstein Jensen ( LJ ) and 7H10 ) local North Carolina ( NC ) MTBC isolates were processed and sequenced using the optimized DNA extraction and sequencing protocol identified through the APHL-CDC pilot . These sequences , along with 263 historical local NC sequences from the MDHHS were analyzed using the Wadsworth Center , NYSDOH ’ s NGS TB Pipeline Service . Overall , 425 sequences composed of 350 MTBC and 75 Nontuberculous Mycobacterium ( NTM ) across 287 patients ( 266 MTBC and 21 NTM ) were analyzed . Results : The 425 analyzed sequences yielded 13 resistance-associated genes or noncoding regions with 21 unique high-confidence single nucleotide polymorphisms ( SNPs ) predicting resistance to Rifampin , Isoniazid , Pyrazinamide , Ethionamide , Streptomycin , and Fluoroquinolones . All the mutations identified by WGS were confirmed phenotypically using MGIT ( for first-line antimicrobials ) or agar proportion ( for second-line antimicrobials ) based Drug Susceptibility Testing ( DST ). The bioinformatics analysis also yielded a significant number of unknown and neutral mutations that are yet to be reported in the literature . Discussion : These pilots yielded results that proved the value of WGS integration in the NCSLPH diagnostic testing workflow . Our short-term goal is to sequence additional specimens to gain a perspective of currently circulating resistance markers within the NC population . The long-term objective is to develop a validation set composed of a panel of isolates with diverse high-confidence resistant mutation markers . This could be a collaborative effort with the CDC and is critical to jurisdictions with limited resistant strains diversity . During this process , it will be crucial to develop a plan to catalog neutral and unknown mutations that might be of interest for future use . Finally , NCSPHL will be evaluating the workflow algorithm to determine the revision of DST testing .

Presenter : Al-Mounawara Yaya , dr . yayamouna @ gmail . com

Rapid , Point of Care Testing using Surface-enhanced Raman Spectroscopy and Deep Learning

T . Spikes 1 , H . Naikare 1 , Y . Zhao 2 , Y . Yang 2 , Y . Mosley 1 , L . Whittington 1 , D . Baughman 1 , R . Cox 1 , Allen Kalantari 1 ; 1 Tifton Veterinary Diagnostic & Investigational Laboratory , 2 University of Georgia

Laboratory based services have been the norm for diagnostic testing procedures . While the results of these diagnostic tests are reliable , the time from when the samples are submitted to when the results are obtained can range anywhere from a few hours to a few days or longer . Herein lies an ever-present issue in diagnostic testing : the ability to obtain accurate results while minimizing turnaround time . This issue was notable during the peak COVID-19 pandemic as many labs became overwhelmed due to the high volume of tests received . This , along with the numerous restrictions and testing guidelines put in place , has increased the need for rapid , point of care testing . One method that may prove to be a viable option would be a portable surface – enhanced Raman spectroscopy ( SERS ) sensor combining with deep learning for rapid SARS-CoV-2 RNA