Lab Matters Fall 2024 | Page 54

APHL 2024 POSTER ABSTRACTS

to analyze SARS-CoV-2 Omicron spike ( B . 1.1.529 ) as compared to a G614D revertant . Proteins were labeled for 0.5 , 3.5 , 24.5 and 171.5 minutes , digested using a 1x5.0 mm C18 column and analyzed on a Thermo Orbitrap Eclipse TM mass spectrometer using datadependent EThcD & HCD MS2 acquisition . Peptide identification and deuteration analysis were performed using Byos ® HDX 5.3.44 .

Results : We analyzed the structure of Omicron spike and G614D revertant through pull-downs with dimeric ACE2 and monoclonal antibodies directed at the receptor binding domain ( RBD ) and S2 domain . The anti-RBD and -S2 antibodies immunoprecipitated less G614D than Omicron spike , demonstrating reduced binding of the revertant . However , binding to ACE2 was equivalent . We also assessed G614D revertant structure as compared to Omicron spike with HDX-MS . Increased deuteration of G614D as compared to Omicron spike was interpreted as increased flexibility , while decreased deuteration was interpreted as decreased flexibility . We observed reduced dynamic motion in the C-terminal end of RBD but little difference in the area surrounding the RBD / ACE2 binding site , correlating with our antibody and ACE2 binding data . We also observed large differences in the mobility of the S2 domain , including reduced mobility beginning at K854 , evidence that the salt bridge between D614 and K854 has reformed in G614D .

Conclusions : Our findings show that the D614G mutation continues to contribute to the structure and function of SARS-CoV-2 spike . We show that HDX-MS can detect structural differences caused by a single amino acid change in spike , which correlate to ACE2 and antibody binding data . HDX-MS may be useful in predicting immune escape mutations in emerging SARS-CoV-2 variants .

The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention . Use of trade names and commercial sources are for identification only and do not imply endorsement by the CDC .

Presenter : Lindsay Nyhoff , uqo8 @ cdc . gov

An Automated High-throughput Extraction and Real-time PCR Solution for Rapid Candida auris Detection

M . Veling 1 , P . Xie 2 , C . Filippis 3 , K . Wilke 3 , Y . Tong 2 , Revvity Health Sciences 1 , Revvity 2 , Institute for Experimental Immunology , affiliated to EUROIMMUN Medizinische Labodiagnostika AG 3

Candida auris is an emerging fungal pathogen that presents a global health threat . It can cause severe illness and spread easily among patients in healthcare facilities . Recently , multidrug-resistant strains of C . auris have been identified , making it a high priority pathogen to be monitored . Real-time PCR is a rapid method for tracking the emergence of C . auris . Here , we present an automated extraction workflow together with the Candida auris Detection Real-time PCR Reagents ( RUO ) for qualitative detection of C . auris . This workflow is suitable with samples collected from human skin swabs , environmental surface swabs , or laboratory cultures .

The extraction reagents are optimized for fungal pathogen extraction . It can be used in manual process or automated with the chemagicTM 360 extraction system ( up to 96 rxn / run ). The PCR reagents utilize sequence-specific primer and Taqman™ probe to amplify the ribosomal RNA ( rRNA ) gene and the partial genetic region of the internal transcribed spacer ( ITS ) 1 & 2 of the fungus for C . auris identification . A primer / probe set to detect the internal control ( must be spiked-in to samples prior to extraction ) is included for extraction and PCR process monitoring . For samples collected from human skin swabs , an additional primer / probe set to detect endogenous hu-man gene ( RNase P ) is included for sample quality check . The reagents also use a dUTP / UNG carryover prevention system to avoid contamination of PCR products and are compatible with commercial PCR instruments with available FAM™ , VIC ®/ HEX™ and CY5™ channels .

Our studies have revealed that the analytical sensitivity using ddPCR quantified synthetic C . auris plasmid spike-in to the PCR reagents was 30 copies / rxn . Limit-of-detection of the entire workflow from extraction with contrived positive samples in liquid amies to PCR was 600 CFU / mL . Inclusivity analysis by in silico predicted all five C . auris clades were predicted to be detectable by the reagents , with exception to the 10 strains identified in Lebanon in 2021 . Exclusivity analysis by in silico predicted no cross-reactivity to a total of 103 non-C . auris microorganisms that either from the Candida family or those often found on skin infections .

( For research use only . Not for use in diagnostic procedures .) Presenter : Peishan Xie , Peishan . Xie @ revvity . com

Antimicrobial Susceptibility Test Profile of Klebsiella pneumoniae Carbapenemase Positive Enterobacterales Over Five-year Period

B . Ryan , A . Herrera , M . Udoye , D . Barker , R . Tuladhar , C . Wang , C . Schroeder , Texas Department of State Health Services

Introduction : Carbapenem-resistant Enterobacterales ( CRE ) are a prevalent and persistent risk to public health , being designated by the CDC as an urgent threat . These bacteria possess carbapenemase genes that produce enzymes capable of hydrolyzing broad-spectrum carbapenem antibiotics . First characterized in 2001 , the Klebsiella pneumoniae carbapenemase ( KPC ) gene is the most prevalent in the US . Since joining the CDC Antimicrobial Resistance Lab Network ( AR Lab Network ) in 2017 , the Texas Department of State Health Services ( TX DSHS ) has performed antimicrobial susceptibility testing ( AST ) on KPC-positive CRE isolates . Here , we analyzed 773 isolates , confirmed via an adapted CDC RT-PCR assay , collected between 2019 and 2023 and detail their AST profile .

Results : With few exceptions , the AST profile of KPC-positive CRE has remained mostly unchanged . Of samples resistant to aztreonam , cefotaxime , ceftazidime , piperacillin / tazobactam and ticarcillin / clavulanic acid , a ≤5 % variation has been observed . Aminoglycosides and tetracyclines resistance remained at < 50 %. Cefepime resistance peaked in 2020 at 86.3 % of isolates , steadily decreasing to 69 %. Similarly , tobramycin ( 70.5 %) and ciprofloxacin ( 95.4 %) resistance declined to 38.8 % and 78.3 %, respectively . Interestingly , trimethoprim / sulfamethoxazole resistance declined from 66 % to 49 % in 2021 but rose to 64 % in 2023 . Of notable exception , doxycycline resistance increased from 32 % to 41 %.

Conclusions : The resistance profile of KPC positive CRE isolates has remained stable over the last 5 years , with three antimicrobials showing an increased susceptibility and doxycycline having increased resistance . This antibiogram provides clinicians additional information in predicting antimicrobial susceptibility for CRE