Lab Matters Fall 2024 | Page 67

APHL 2024 POSTER ABSTRACTS

Comparison of KingFisher and Qiagen DSP Manual Extraction Methods and Efficiency Using Influenza A and B Subtypes / Lineages

B . Parchment , J . Alexander , New Hampshire Public Health Laboratories

Background : At the New Hampshire Public Health Laboratories ( NH PHL ), the ThermoFisher KingFisher extraction method was implemented during 2020 to create a high throughput method of extracting large numbers of SARS-CoV-2 samples that were submitted to the lab for testing . Following this , the dual-purpose CDC Influenza SARS-CoV-2 Multiplex assay was introduced to test for both influenza positive and COVID positives samples . The detection of these influenza positive samples then allowed for further determination of influenza A subtypes or influenza B lineages using the CDC Human Influenza Diagnostic Panel . Outside of emergency usage , the KingFisher extraction method was never officially validated for use with the influenza subtyping assay . As such , we undertook the task of doing a formal validation of the KingFisher extraction method by comparing subtyping results from that to subtyping results obtained via manual extraction with the previously approved Qiagen DSP Viral RNA Mini Kit .

Methods : Twenty-four samples ( 10 Influenza A , 10 Influenza B and four negative VTMs ) were utilized for the validation . Positive influenza samples were created from viral stocks obtained from the International Reagent Resource ( IRR ), which were then diluted accordingly to create five serial dilutions for each subtype / lineage . These dilutions were then extracted side-by-side using both the KingFisher and Qiagen DSP Viral RNA Mini Kit . Following extraction , the samples underwent real-time RT-PCR analysis to determine and compare the resulting Ct values for all of them .

Results : Influenza A results have been relatively consistent between the two extraction methods . Ct values tended to be higher across the board at the highest dilution factor of 1:10000 , which was anticipated compared to the lower values obtained at lower dilution factors . KingFisher extracted samples tended to have minimally lower Ct value cutoffs than DSP extracted samples , indicating the overall higher sensitivity of this method .

Discussion : Overall , we hope to validate the Kingfisher as a viable extraction method for direct influenza sample subtyping . As of this abstract , the validation process is still underway for Influenza B samples . We aim to have compiled results by the time APHL 2024 commences in May .

Presenter : Briah Parchment , briah . m . parchment @ dhhs . nh . gov

Detecting Antimicrobial-resistant Genes in Raw Nanopore Reads

E . Young , J . Hergert , J . Wagner , K . Oakeson , Utah Public Health Laboratory

Detecting genes associated with Antimicrobial Resistance ( AR ) traditionally occurs post-assembly , driven by the limitations posed by the short Illumina sequencing reads . The constrained length of Illumina reads often falls short of capturing the entirety of a typical AR gene . Nanopore reads are significantly longer , potentially spanning the entire gene sequence and mitigating the issue of fragmented sequences . We explored the potential of using raw Nanopore reads for AR gene detection . This approach eliminates the need for assembly steps previously essential with Illumina data . In a dataset of 124 isolates and 15 species , we were able to identify beta-lactamase GES , KPC , NDM and OXA families of genes with similar accuracy to Illumina sequencing of the same isolate . By capitalizing on the extended read lengths provided by Nanopore sequencing , AR gene detection could be performed in real-time .

Presenter : Erin Young , eriny @ utah . gov

Developing High-Throughput RT-PCR Testing for Candida auris on the Hologic Panther Fusion Open Access System .

B . Schumitsch-Jewell , Wisconsin State Laboratory of Hygiene

Candida auris is an emerging drug resistant opportunistic pathogen common to healthcare settings . This yeast can colonize the skin of people as well as surfaces with which they come into contact . Due to the growing prevalence and increasing severity of C . auris infections , it has become a priority to identify colonized individuals and track the prevalence of antifungal resistance . This has led to a large burden of testing for public health laboratories that often screen entire facilities at one time .

The Wisconsin State Laboratory of Hygiene has recently developed an assay on the Hologic Panther Fusion Open Access platform to aid in high throughput identification of C . auris colonization from skin swabs . Using comparative analysis with other real-time polymerase chain reaction testing methods ( primarily the BDMax / BioGX assay ) we determined that this was at least as sensitive and much more hands off when included in existing testing algorithms . Combined with culturing of patient specimens and other molecular assays can accurately detect colonization . This new method has greatly improved the processing speed of C . auris specimens and greatly decreased the workload of C . auris testing on laboratory staff .

Within this poster , we will present how we went about developing and validating an assay on the Hologic Panther Fusion Open Access platform in order to achieve an improved workflow .

Presenter : Brandon Schumitsch-Jewell , Brandon . SchumitschJewell @ slh . wisc . edu

Developing Threat Agnostic Sequencing Capabilities and the Utility in Public Health Laboratories

T . Wolford 1 , M . Mauldin 2 , J . Schiffer 2 , C . Hanigan 1 , B . Merrit 3 , P . Thielen 3 , Association of Public Health Laboratories 1 , Centers for Disease Control and Prevention 2 , The Johns Hopkins University Applied Physics Laboratory 3

Next-generation sequencing ( NGS ) technologies are widespread across the public health landscape . These technologies are typically used to characterize pathogen genomes , investigate out-breaks and rarely to sequence specimens from clinical patients with unknown etiologies for ag-nostic pathogen identification . However , use of NGS technologies in threat agnostic surveillance has potential to significantly impact national and international early warning , detection and public health action systems . Additionally , use of metagenomic sequencing in clinical settings under the Centers for Medicare and Medicaid Services ( CMS ) Clinical Laboratory Improvement Amendments ( CLIA ) standards is in its nascency . A handful of agnostic assays have been devel-oped for highly specific specimen types ( e . g ., cerebrospinal fluid , plasma ).