Lab Matters Fall 2024 | Page 44

APHL 2024 POSTER ABSTRACTS

Optimization and Utilization of an RT-PCR Based Method for the Detection of West Nile Virus in Sentinel Chickens

P . Dumoulin 1 , E . Kopp 2 , T . Locksmith 2 , M . Castaneda 2 , A . LaCrue 2 , L . Heberlein 2 , A . Cannons 2 , Centers for Disease Control and Prevention 1 , Florida Department of Health 2

Background : Arboviral surveillance programs seek to identify circulating pathogens along with geographic and temporal transmission risks . One facet of arboviral surveillance in Florida is monitoring sentinel chickens to detect transmission of circulating arboviruses , including West Nile virus ( WNV ), before clinical presentation in humans . Sentinel chickens are seronegative when placed in field locations . Their serum is collected weekly and initially screened using a hemagglutination inhibition assay ( HAI ) to identify broad reactivity to either the flavivirus ( e . g ., WNV ) or alphavirus families . To identify viral specific reactivity , HAI-positive samples are reflexed to a confirmatory enzyme-linked immunosorbent assay ( ELISA ) and , if inconclusive by ELISA , a plaque reduction neutralization test ( PRNT ). During peak WNV transmission season in Florida ( i . e ., mid-July though late October ), sentinel chicken confirmatory serologic results are delayed because of limited testing capacity . More rapid confirmation of WNV infections in sentinel chickens would allow county mosquito control partners to implement interventions to prevent WNV transmission across Florida . Because prior work demonstrated that experimentally infected chickens exhibit low level transient (< 7 days ) WNV viremia , the Bureau of Public Health Laboratories in Tampa , Florida , investigated utility of adapting an RT-PCR assay originally designed for use with human sera as a reflex test for confirmation of WNV infections in sentinel chickens .

Methods : Using HAI , we identified chickens with immunologic reactivity to flavivirus antigen . Using samples collected one week prior and stored , we then tested the same chickens by RT-PCR . Because chicken serum is not a validated matrix for our nucleic acid extraction methods , an MS2 bacteriophage process control was included before extraction to evaluate performance . Extraction methods tested included silica-based spin columns , TRIzolchloroform phase separation and magnetic bead separation . After nucleic acid extraction , samples were tested by RT-PCR to measure relative extraction efficiencies ( i . e ., comparison of MS2 Ct values ) and presence of WNV .

Results : Using RT-PCR we tested nine chickens with confirmed WNV ; three ( 33 %) had detectable WNV RNA . However , the MS2 bacteriophage process control identified highly variable RNA extraction efficiencies ( Ct range : 25.45 – 19.27 ) when silica-based spin columns were used for extraction . Poor RNA extraction and high variability coupled with low WNV viremia prompted further optimization to minimize probability of false negative results . Using extraction optimization , we discovered that a TRIzol-chloroform phase separation before downstream processing greatly increased RNA yield ( increased yield range : 1.5x – 2863x ).

Conclusions : We established that an RT-PCR assay adapted for use with chicken serum detects local transmission of WNV in sentinel chickens in Florida . Because of expected transiency of WNV viremia in chickens , combined with a weekly cadence of sera collection , RT-PCR did not identify WNV in all chickens with HAI evidence of serologic conversion . Confirmation of WNV transmission by RT-PCR will greatly reduce , but not eliminate required confirmatory reflex testing .

Presenter : Peter Dumoulin , tqa9 @ cdc . gov

Optimization of Fish Homogenization Procedures for EPA Method 1633 to Measure Per- and Polyfluoroalkyl Substance ( PFAS ) in Fish

S . Shaw 1 , C . Xu 1 , D . Wene 1 , S . O ’ Leary 1 , T . Fan 2 , New Jersey Public Health Environmental Laboratories 1 , New Jersey Department of

Health 2

Per- and polyfluoroalkyl substances ( PFAS ) are man-made , aliphatic compounds that persist in the environment due to their stability and resistance to degradation . These forever chemicals can have adverse effects on health outcomes such as liver damage , reduced immune response and increases in certain types of cancer . Bioaccumulation and biomagnification in fish , particularly those inhabiting local contaminated water sources , exacerbate the PFAS contamination risk to humans .

In addressing this concern , EPA Method 1633 employs liquid chromatography / mass spectrometry ( LC-MS / MS ) to analyze 40 distinct short and long-chained PFAS in various matrices , including tissue samples . The New Jersey Department of Health ( NJDOH ) endeavors to implement this method for monitoring PFAS levels in fish from the Delaware River .

This poster focuses on the optimization of homogenization procedures as a pivotal initial step in tissue sample preparation . The objective is to ensure sample consistency and enhance extraction efficiency . This is achieved through the assessment of different grinders / technologies and the optimization of homogenization parameters / conditions . The experiments involve screening grinding devices / technologies , comparing temperatures ( with or without dry ice ) and selecting grinding parameters . Evaluation of experiment results adopts a combined qualitative and quantitative approach , considering sample homogeneity , cartridge clogging , ease of operation and cleanup , capacity and safety considerations , as well as contamination and matrix effect assessment .

This optimization process is anticipated to improve the overall method accuracy and precision .

Presenter : Shenelle Shaw , shenelle . shaw @ doh . nj . gov

Overview of a Novel HRAM Orbitrap Workflow for Combined 40 PFAS Targeted Testing and Profiling of 40,000 + PFAS Compounds in Samples

C . Grim 1 , R . Cochran 1 , K . McHale 1 , L . Ferguson 2 , T . Astill 1 , Thermo Fisher Scientific 1 , Duke University , Department of Civil and Environmental Engineering 2

Per- and polyfluoroalkyl substances ( PFAS ) are a class of persistent chemicals that are widely utilized in industrial and consumer products . Given their persistence and potential negative health effects , it is essential to comprehensively understand the presence of PFAS compounds in environmental and food safety matrices .

This poster will showcase the utilization of the Thermo Scientific™ Orbitrap™ Exploris™ 240 , in conjunction with Thermo Scientific™ Compound Discoverer™ software , as a robust tool for non-targeted analysis of PFAS compounds in APHL stakeholders ’ laboratories . This straightforward approach enables the detection and identification of over 40,000 + PFAS compounds in a single analysis by leveraging fine signature fragment and FluoroMatch Suite databases , along with other specific libraries , thereby providing a