Lab Matters Fall 2023 | Page 108

APHL 2023 POSTER ABSTRACTS

processes used to perform risk assessment , SOP writing , correlation and validations of the various testing methodologies , and the necessary steps to integrate such testing into our LIMS point-of-care module , as well as pilot deployment data . This program has shown that not only are these mobile laboratories useful in the COVID-19 response but are also an asset to other departments within the State of New Jersey . The FDLs are opening the road to more testing avenues for vulnerable populations .

Presenter : Lisa Kimball , lisa . kimball @ doh . nj . gov

TheiaEuk : Bioinformatics Workflow for Species-Agnostic Characterization of Fungal Pathogens

F . Ambrosio 1 , M . Scribner 1 , D . Hess 2 , A . Gorzalski 2 , S . Wright 1 , M . Pandori 2 , K . Libuit 1 , J . Sevinsky 1 ; 1 Theiagen Genomics LLC , 2 Nevada State Public Health Laboratory

Clinical incidence of antifungal-resistant fungal infection has increased dramatically in recent years leading to the CDC ’ s Antimicrobial Resistance Laboratory Networks ( ARLN ) adding drug-resistant Candida auris , other drug-resistant Candida species , and azole-resistant Aspergillus fumigatus to the AR Threats report in 2019 . These pathogens are capable of colonizing various body cavities as well as causing life-threatening bloodstream infections . There does not exist a comprehensive laboratory assay for the characterization of fungal pathogens or the detection of antifungal resistance biomarkers . Here we introduce TheiaEuk _ PE , a cloud-native bioinformatics workflow which accepts paired end next generation sequencing data as an input , performs de novo genome assembly , taxon identification , clade-typing for C . auris , and for several fungal species it will report any mutations found in genes known to contain mutations that confer resistance to any antifungals . The Shovill genome assembly package is used to perform de novo assembly with SKESA set as the default assembler . This workflow addresses the issue of taxon identification using GAMBIT , with a custom fungal database containing over 5000 unique genomes representing 245 species . Snippy is used to call variants relative to a reference genome , and the variant call format file is queried for genes known to contain mutations associated with antifungal resistance . This workflow is open source and available on the Theiagen Genomic github page . The workflow was designed using dockerized components and written in the workflow description language to facilitate deployment on the cloud-based bioinformatics platform Terra . bio .

Presenter : Frank Ambrosio , frank . ambrosio @ theiagen . com

Validation and Implementation of Sanger Sequencing for Cryptosporidium Subtyping in the State of Texas

J . Cen , M . Sciulli , R . Tuladhar , C . Wang ; Texas Department of State Health Services

Introduction : The protozoan parasite Cryptosporidium is an important cause of diarrheal disease ( cryptosporidiosis ) in humans and animals . Between 2011-2020 , an average of 597 cryptosporidiosis cases per year were reported in Texas , with 2019 alone having 12 outbreaks resulting in 1,244 reported cases . Due to the sexual lifecycle , large-scale genetic recombination may occur within a host , potentially confounding epidemiological analysis . Previous studies indicate that different Cryptosporidium species and subtypes have varying clinical manifestations and severity of virulence . Therefore , it is important to speciate and genotype Cryptosporidium at the subtype level . Sanger sequencing analyses of gp60 and 18S rRNA are widely used in Cryptosporidium subtyping . Texas validated a modified CDC procedure for the subtyping of Cryptosporidium from fecal specimens by using nested polymerase chain reaction ( PCR ) detection and Sanger sequencing . Method : The CDC provided a panel of fecal samples for this validation study . Following DNA extraction using Fast DNA SPIN Kit for Soil , nested PCRs were performed for either gp60 or 18S rRNA using GoTaq Hot Start Master Mix . Utilizing the BigDye Terminator v3.1 Cycle Sequencing kit , Sanger sequencing was employed for subtype identification , and subsequent sequences were analyzed with Geneious Prime software . Identified species and / or subtypes were reported to the CDC and compared with their analyses . To optimize workflow efficiency , modifications were made to the current CDC protocol . These include the use of 1 ) a commercial mastermix for nested PCR , 2 ) a quick gel running system , 3 ) PCR and sequencing reaction optimization , 4 ) new PCR and sequencing clean up kits , and 5 ) a 96-well plate format to facilitate future large scale , high throughput applications . Results and Conclusion : The panel provided by CDC was successfully validated with the abovementioned modifications . Upon comparing results , there was 100 % agreement with the CDC on accuracy , precision , sensitivity , and specificity . The successful implementation of Sanger sequencing for Cryptosporidium subtyping in Texas allows for rapid and accurate identification of these infections as well as contamination sources . Further efforts have been made on recruitment and implementation of Cryptosporidium samples for sequencing by using the DSHS listserv , laboratory webpage , and recruitment letter .

Presenter : Joyce Cen , joyce . cen @ dshs . texas . gov

Validation and Implementation of Antimicrobial Susceptibility Testing to Monitor Trends in Antimicrobial Resistant Neisseria gonorrhoeae in the State of Texas

N . Niyah , N . Gray , T . Baldwin , C . Wang , C . Schroeder ; Texas Department of State Health Services

Introduction : Neisseria gonorrhoeae ( GC ) is the etiological agent of gonorrhea , a worldwide prevalent sexually transmitted infection . Individuals with untreated GC infections can develop pelvic inflammatory disease , prostatitis and infertility . Antimicrobial resistance ( AR ) within GC has risen sharply , creating an urgent public health concern . Surveillance of AR gonorrhea is a means to understanding the spread of AR and determining antimicrobial susceptibility patterns in a given population . Currently , only one Texas county performs routine GC antimicrobial susceptibility testing ( AST ). The implementation of GC AST at the DSHS Austin Laboratory will be a valuable tool in tracking AR GC prevalence across the state . Results : Using 50 GC specimens from the Antimicrobial Resistance Isolate Bank and the Gonococcal Isolate Surveillance Project ( CDC & FDA ), we report successful validation of the Biomerieux ETEST ® Gradient Diffusion Method for four antibiotics : azithromycin , cefixime , ceftriaxone , and ciprofloxacin . The overall accuracy , precision , sensitivity , and specificity of the ETEST ® were found to be 98 %, 100 %, 100 % and 97.2 %, respectively . Further , three commercial specimen collection and transport kits were validated for organism recovery . These were : 1 ) Copan TransPorter Amies gel collection kit without Charcoal , 2 ) Copan E-swabTM Regular-Sized nylon flocked swab kit with liquid Amies , and 3 ) Copan E-swabTM Minitip nylon flocked swab kit with liquid Amies . GC was successfully