Lab Matters Fall 2024 | Page 69

APHL 2024 POSTER ABSTRACTS days . The specificity of the assay was determined by testing over 60 fungal pathogens closely and distantly related to Bd , Bg and Hc . Finally , the accuracy was assessed by performing a blinded panel of 50 fungal DNA including Bd , Bg , Hc and other fungal species .

Results : The multiplex rt-PCR assay was highly sensitive with detection limit of 1 pg gDNA / PCR reaction at 45 PCR cycles for all three organisms . The assay was highly reproducible as it produced reproducible cycle threshold ( Ct ) for same amount of DNA on same and different days of testing . The assay was highly specific and did not cross react with both closely and distantly related fungal pathogens .

Conclusion : The preliminary data of multiplex rt-PCR assay for culture identification of Bd , Bg and Hc is promising and provides a sound platform for further validation with primary samples , including blood , sputum and bronchial aspirate . The rapid and highly specific molecular assay will help in patient care as well as in further understanding the ecology and epidemiology of blastomycosis and histoplasmosis .

Presenter : Brittany O ’ Brien , Brittany . obrien @ health . ny . gov

Development of a Luciferase-based Assay for the Detection of Mpox Neutralizing Antibody in Human Serum

S . Rodriguez , L . Priyamvada , T . Smith , M . Townsend , P . Satheshkumar , Centers for Disease Control & Prevention

The incidence of mpox disease , caused by mpox virus ( MPXV ), was unprecedented in 2022 . Over 80,000 cases were identified globally from May 2022 to May 2023 . Mpox disease resulted in mild to severe morbidity with rare mortality events occurring in immunocompromised individuals . To improve our understanding of how the immune system responds to mpox infections , we sought to adapt a high-throughput vaccinia virus luciferase-based antibody neutralization assay , published to be comparable to traditional plaque reduction neutralization testing ( PRNT ). We developed a recombinant MPXV clade IIa isolate ( from the United States 2003 outbreak ) expressing firefly luciferase under the early / late promoter . Using human sera collected from mpox vaccinees , as well as confirmed cases from the 2022 outbreak , we compared the MPXVluciferase neutralization assays to PRNT . Herein , we report on our findings validating this luciferase-based neutralization assay for mpox .

Presenter : Sergio Rodriguez , pze7 @ cdc . gov

Digital PCR Assay for Identification of Critical β-lactamases Associated with Carbapenem- and ESBL-producing Bacteria

K . Hecker , C . Cambridge , S . Cossette , C . Connelly , Streck

Post-pandemic monitoring of emerging infectious diseases has reemphasized the importance of surveillance for antimicrobial resistant ( AR ) bacteria . Often labeled as the “ silent pandemic ,” AR bacteria are responsible for an estimated 700,000 deaths annually . To address the gaps in national laboratory testing capacity , including AR mechanism testing , the CDC has organized the Antibiotic Resistance Laboratory Network , which released a technical appendix that establishes Streck ARM-D kits as a method for AR mechanism testing . Streck ARM-D kits are qPCR-based methods that test for 21 different β-lactamase gene families and over 1000 different allelic variants . Recently the CDC has expanded the scope for AR genetic mechanism surveillance to include wastewater through the National Wastewater Surveillance System . CDC guidance for wastewater testing defines prioritized AR mechanisms and recommended methods for wastewater testing , including use of digital PCR as the preferred testing technology . Consequently , this study describes the compatibility of Streck ARM-D kits with the QIAGEN QIAcuity dPCR system and expanded coverage of ARM-D Kit OXA gene families to support detection of new AMR mechanisms of interest . Advancement of the ARM-D kit technology to comply with new CDC surveillance strategies permits use of the same commercial product for the expanded applications , allowing public health labs to use the same test with multiple sample types and technologies for testing these medically relevant resistance mechanisms .

Extracts from bacterial isolates and wastewater samples , previously characterized as AR positive or negative by qPCR using the Streck ARM-D kits , were reevaluated using the Streck ARM-D Kit , β-Lactamase with the QIAGEN QIAcuity dPCR System . Reactions were prepared using the ARM-D Kit 10X PCR Mixes paired with the QIAGEN QIAcuity OneStep Advanced Probe Mix on 26k QIAGEN QIAcuity nanoplates . As external positive controls , extracts from 5 AR Bank Isolates were also evaluated for concordance to whole genome sequencing and ARM-D kit qPCR data .

Following ARM-D kit protocol optimization for compatibility with QIAGEN ’ s digital PCR system , positive and negative agreement for qualitative detection of AR mechanisms , detected by qPCR and dPCR , were concordant across 40 total extracts obtained from either culture isolates or wastewater samples as well as the 5 AR bank isolates .

Data presented in this study demonstrate the ARM-D Kits can be optimized to support digital PCR testing when combined with manufacturer reagents . As AR spreads , it is crucial to collaborate with the public health sector to promote continued monitoring of emerging antibiotic resistance mechanisms and to support expanded initiatives , such as wastewater testing for AMR using digital PCR , by ensuring that tests are updated to detect these critical mechanisms of interest .

Presenter : Chris Connelly , cconnelly @ streck . com

Dual dPCR Platform Validation of Multiplex Assays for Wastewater-based Epidemiology for SARS-CoV-2 , Influenza A / B , RSV , Mpox and Internal Process Control

M . Gilbert , E . Keim , R . Mwashite , G . Mascarinas , C . Carlson , J . Raney , N . Flack , J . Lahti , Washington State Public Health Laboratories

Implementation of wastewater-based epidemiology through the CDC NWSS program since the outbreak of the 2020 COVID-19 pandemic has provided valuable insight into local and regional trends of SARS- CoV-2 prevalence . These are especially useful when epidemiologists lack clinical data and community level demographics and access to care varies . To expand the wastewater-based epidemiology program to include additional infectious disease targets , Washington State Public Health Laboratories developed and validated two triplex assays . The target analytes included SARS-CoV-2 , influenzas A / B , respiratory syncytial virus ( RSV ), mpox and an internal process control betacoronavirus OC43 ( HCoV-OC43 ), which were all tested across Qiagen ’ s QIAcuity 8 and Bio-Rad ’ s QXONE digital PCR