Lab Matters Fall 2024 | Page 76

APHL 2024 POSTER ABSTRACTS

40.4 %), Enterobacter spp . ( n = 54 , 9.5 %) and three less common genera ( n = 23 , 4.1 %). In 2022 , 6,209 CP-CRE were detected , 1,968 ( 31.7 %) of which were identified as NDM-CRE . The 2022 NDM-CRE iso-lates were Klebsiella spp . ( n = 1,147 , 58.3 %), E . coli ( n = 511 , 26.0 %), Enterobacter spp . ( n = 234 , 11.9 %) and six less common genera ( n = 76 , 3.9 %).

NDM variant analysis conducted on 275 NDM-CRE from 2017 – 2018 and 1,752 NDM-CRE from 2022 showed notable differences in the frequency of blaNDM-1 ( 41.5 % vs 46.1 %), blaNDM-5 ( 49.8 % vs 41.6 %) and blaNDM-7 ( 4.4 % vs 10.7 %) variants , respectively . These differences in variant detection frequencies were explained in part by relative differences in organism frequency ( e . g ., decreased NDM- CRE E . coli carrying NDM-5 ) and geography ( e . g ., clusters of patients with NDM-1 in 2022 ).

Our findings detail an increasing overall frequency of NDM-CRE detected by the AR Lab Network . Analysis of changes in the geography and microbiology of NDM-CRE , including the organisms carrying these genes and specific variants , will provide novel insight into the epidemiology of NDM-CRE emergence in the US . Understanding these genotypic and phenotypic characteristics can help public health officials assess containment and prevention priorities and testing strategies .

Presenter : Trevor Darby , tqg0 @ cdc . gov

New Approach for Coronavirus Infection Identification : Targeting Negative RNA Strand as a Replacement for Positive RNA-based qPCR Detection

D . Davis , H . Ullah , Howard University

Amid the ongoing global challenge of the COVID-19 pandemic , caused by a virus in the Coronaviridae family , the scientific community is contending with evolving variants that undermine existing pharmaceutical responses by evading immune detection . This situation underscores the urgency for identifying viral components critical for its survival , thereby thwarting the development of new mutation-driven variants . Targeting such components impedes virus proliferation and offers potential therapeutic avenues . One such target is the 5 ’ -polyU tract of the antigenome , a feature absent in host cell transcripts , making it a unique and virus-specific target . Here , we show that replication of a mouse coronavirus can be inhibited by targeting the 5 ’ -polyU on the negative strand with a simple polyA oligonucleotide . In addition , recent advancements have made significant progress in our detection capabilities . Traditional positive RNA-based detection systems have struggled distinguishing between active and inactive viral particles . This has created a challenge for quarantine measures and therapeutic strategies , particularly for virulent strains . However , new methodologies focusing on the virus ’ s negative strand show promise in providing a more precise assessment of viral spread . This precision is vital in implementing effective public health policies and managing the pandemic more effectively . These breakthroughs represent a critical step forward in our ongoing battle against COVID-19 and similar viral threats .

Presenter : Darnell Davis , darnellddavis86 @ gmail . com

New Real-time PCR Assay for Detection of Candida auris and Echinocandin Resistance-associated Mutations

C . Filippis 1 , L . Opitz 1 , J . Zapf 1 , M . Veling 2 , K . Wilke 1 , S . Stallmann 1 , C . Mazzochi 3 , S . Saschenbrecker 1 , U . Steller 1 , Institute for Experimental Immunology , affiliated to EUROIMMUN Medizinische Labodiagnostika AG 1 , Revvity Health Sciences 2 , EUROIMMUN US 3

Introduction : As reflected in growing numbers of reported cases , Candida auris infections have become a major health challenge that is of particular concern as many strains are resistant to common antifungals , with an increasing occurrence of multidrug- and panresistant strains . According to susceptibility profiles , about 90 % of C . auris isolates are resistant to fluconazole , 30 % to amphotericin B and 5 % to echinocandins ( ECH ). Limited treatment options , tolerance to disinfectants and heat as well as the ability to colonize skin and smooth surfaces support the spread of C . auris in hospitals and intensive care units , causing nosocomial infections with high mortality rates . This study evaluates a new real-time polymerase chain reaction ( PCR ) test for the molecular detection of C . auris and of the most relevant mutations associated with ECH resistance .

Methods : The EURORealTime C . auris ECHR ( EUROIMMUN ) is based on primers / probes targeting [ i ] the C . auris multicopy ITS gene ( intended for sensitive detection of any C . auris strain ) or [ ii ] mutations coding for S639F / Y / P in the FKS1 HS1 gene ( intended for detection of ECH resistance markers as well as C . auris ). In silico analysis was performed using BLASTn of the primer sequences against C . auris genomes available in public databases . In addition , analytical sensitivity and specificity were examined in vitro using synthetic plasmid DNA constructs containing relevant C . auris gene sections or genomic DNA purified from laboratory culture strains .

Results : According to in silico analysis , the FKS1 HS1-specific primers and amplification control probes matched 100 % to the target gene in all 2,834 C . auris strains analyzed . 88 genome sequences contained ECH resistance markers ( 22xS369F , 19xS639Y , 47xS639P ), all of which matched 100 % to the respective mutation-specific probe sequence . ITS-specific primers / probes were predicted to detect strains of all five clades , except for 10 Lebanese strains , which were however detectable via FKS1 HS1 . No relevant cross-reactivity was predicted in silico for FKS HS1 and ITS primers / probes .

The limit of detection was 30 copies / reaction for both targets . Using DNA extracted from C . auris suspensions , 1800 CFU / ml ( FKS1 HS1 ) and 600 CFU / ml ( ITS ) were necessary for detection rates of 100 %. Specificity analysis using synthetic FKS1 HS1 wild-type ( wt ) targets revealed a very weak cross signal of the mutation-specific probes only for wt S639 . In contrast , all plasmids containing mutations coding for S639F / Y / P generated strong positive signals , allowing clear distinction from wt . Out of 23 C . auris culture strains covering all five clades , the only ECH-resistant strain ( S639F ) was correctly identified , while all 22 wt strains tested negative for the respective mutations . All 23 strains were detected via the ITS target . No crossreactivity was observed with closely related Candida species and other yeasts .

Conclusion : The new EURORealTime C . auris ECH-R assay allows both sensitive screening for C . auris and detection of the pathogen ’ s antifungal susceptibility . Mutations conferring ECH resistance are reliably determined with highly specific probes without crossreactivity to wt mutations or related yeasts . Compared to standard