Lab Matters Fall 2024 | Page 132

APHL 2024 POSTER ABSTRACTS

The Wisconsin State Laboratory of Hygiene has been conducting wastewater surveillance of SARS-CoV-2 since September of 2020 and has expanded surveillance to other relevant public health disease targets . The success of wastewater testing , in combination with the rise and danger of C . auris , makes a wastewater-based C . auris assay and surveillance program a desirable tool . To determine a robust surveillance method for C . auris from the wastewater two different concentration methods were compared , membrane filtration and use of Microbiome B Nanotrap particles , as well as three PCR assays for sensitivity of detection and quantification : the CDC C . auris PCR assay , GT Molecular ’ s C . auris assay for digital PCR and an internally designed assay . The different methods were assessed for limit of detection and limit of quantification as well as testing of wastewater samples for detection of C . auris . The use of an internal control , NE095 ( NIST ), is also assessed to determine its best practice of use . The ability to sensitively detect C . auris from the wastewater is valuable as wastewater is used in disease surveillance and monitoring , particularly as its use may provide early detection or warning of the infection risk . The additional information gained from wastewater surveillance of C . auris may be useful in aiding facilities in their infection prevention and control strategies .

Presenter : Griffin Knuth , griffin . knuth @ slh . wisc . edu

Finding Value in Sewage : Validation of a Multi-target Assay to Enhance Respiratory Disease Surveillance Using Wastewater Samples in North Carolina .

M . Poole , D . Pettit , B . Bornu , C . Von Dohlen , C . Lopez-Guzman , S . Shone , C . Goforth , North Carolina State Laboratory of Public Health

Wastewater surveillance for respiratory pathogens using digital polymerase chain reaction ( PCR ) is an effective and resourceefficient tool to collect community-level public health information about pathogens circulating within a community . Data derived from this type of surveillance can inform public health actions and messaging . To increase surveillance data available to NC Public Health , the North Carolina State Laboratory of Public Health ( NCSLPH ) validated a multi-target assay for the detection of respiratory viruses in wastewater samples .

Ensuring the quality of wastewater data by limiting false-positives and false-negatives requires strict adherence to best practices . The NCSLPH validated a multiplex assay for the detection of SARS-CoV-2 ( SC2 ), Influenza A and B ( Flu A & B ) and Respiratory syncytial virus ( RSV ) in wastewater based on guidance from the CDC ’ s National Wastewater Surveillance System ( NWSS ). The selected protocol uses GT Molecular ’ s Respiratory Panel Reagents for Wastewater Surveillance . These commercially available GMP reagents were selected to support harmonization between NWSS laboratories .

The validation utilized previously collected wastewater samples from 29 geographically distributed locations across the State of NC . Samples were pooled , pasteurized , autoclaved and then microwaved to minimize biosafety risks and destroy the bulk of existing RNA viruses and intact RNA . This material was then aliquoted and spiked with various concentrations of commercially available QC materials for SC2 , RSV , Flu A & B and a surrogate control ( Bovine Coronavirus ) that allows for an assessment of processing efficiency during all phases of the testing process . In addition , a portion of the aliquots were spiked with viral transport media ( VTM ) from clinical specimens that had previously tested positive for SC2 , RSV and Flu

A & B . The limit of detection ( LOD ) for each target was determined at the onset of the validation and was consistent with previously determined LODs for the N1 and N2 targets that were used in the prior wastewater surveillance panel .

Accuracy was assessed using three levels of commercially available QC material ; previously identified in-house wastewater samples and VTM containing either SC2 , Flu A or B , or RSV . Positive and negative predicative values ( PPV & NPV ) and analytical sensitivity and specificity were calculated and results for total accuracy , PPV , NPV , sensitivity and specificity were all within the acceptance criteria of > 95 % when compared to the known qualitative results .

To assess precision and reproducibility , the aliquots described above were used to conduct testing on different days . Testing personnel changed on different days to ensure that operator variance was assessed . All results met the acceptance criteria of > 95 % when compared to the known qualitative results .

Lastly , frozen sample stability was assessed by evaluating replicate frozen filters stored at -80 ° C (+/ - 10 °) for 1 and 7 days . This multi-target panel provides more robust surveillance data for the State ’ s public health officials to guide actions and messaging during respiratory pathogen season .

Presenter : Chris Goforth , chris . goforth @ dhhs . nc . gov

Flushing the Threat : Tracking Antimicrobial Resistance in Hawaiian Communities through Wastewater-based Epidemiology

D . Di , M . Steadmon , S . Remigio , D . Ornellas , E . Desmond , Hawaii State Department of Health

Antimicrobial resistance ( AR ) poses a growing global threat , with antibiotic-resistant bacteria ( ARB ) becoming more prevalent and impacting communities in terms of public health , healthcare costs and food production . Among the various forms of AR , carbapenem resistance is particularly concerning , as the spread of carbapenem-resistant Enterobacteriaceae ( CRE ) can lead to serious outbreaks , dramatically limiting treatment options . The first fatal case of CRE infection in Hawaii was detected in 2013 in a hospital . While Hawaii still experiences a relatively low incidence of CRE infection , there has been an increase in CRE detection over the last decade , especially among individuals with exposure to healthcare settings . Using wastewater-based epidemiology ( WBE ) as a tool for AR surveillance , the Hawaii State Department of Health ( HDOH ) State Laboratories Division ( SLD ) seeks to understand the temporal distribution and diversity of AR genes in two Hawaiian communities . Both wastewater treatment plants ( WWTPs ) are located in rural areas , serving populations of around 32,000 and 10,000 , respectively . We employed digital PCR ( dPCR ) to quantify five carbapenem-resistant genes ( NDM , IMP , VIM , KPC and OXA-48 ) over a 12-week period across two WWTPs on two separate islands in the State of Hawaii . Additionally , we performed hybrid capture sequencing targeting 2,780 unique AR genes to understand the diversity of AR genes in the two Hawaiian communities . By analyzing data from dPCR and targeted AR sequencing for the detection of carbapenem resistance genes , we aim to better understand the emergence and prevalence of these resistance genes in the communities . AR data from the wastewater samples could also serve as an early warning for any new emergence of AR genes of