Lab Matters Fall 2023 | Page 55

APHL 2023 POSTER ABSTRACTS the case where high-volume testing may be necessary . Here , we describe the process for developing and optimizing a laboratorydeveloped assay ( LDA ) for clade II mpox on the automated Panther Fusion platform and Open Access software . Methods : Various concentrations of reagents in a primer probe mix were tested to optimize the LDA . Then , the LDA was validated using 10 positive and 10 negative human mpox samples previously characterized by the CDC method to test for accuracy , precision across two Panther Fusion machines and two days , sensitivity , and specificity among HSV-1 , HSV-2 , and VZV human samples . Several spiked media extensions ( the Aptima Multitest Swab Transport Media and kit , RTM4 , an in-house Universal Transport Medium , and dry swabs ) were also validated by testing two positive spiked mpox samples and two negative samples across two Panther Fusion machines and two days . Results : The LDA was successfully optimized with respect to lower Ct values , greater precision , and high RFUs for PCR standard curves . The LDA achieved the standards for CLIA validation with 95 % accuracy , 100 % precision , and 100 % specificity among human samples , and the media extensions achieved 98 % accuracy and 100 % precision . Conclusion : The methods outlined in the poster for developing an LDA using the Panther Fusion Open Access system can be useful for public health laboratories to prepare for increased testing volume . The automated extraction saves time and labor costs and increases reproducibility of results . The LDA does not require specimens to be processed under BSL-3 conditions , which improves workflow efficiency . The LDA detects clade II mpox specifically , so samples do not need to be sent to the CDC for confirmation , enhancing convenience and timeliness of results . These methods can be applied to the rapid optimization and development of LDAs for many possible pathogens of public health importance .

Presenter : Rebecca Villa , rvilla @ uiowa . edu

Comparison of Nucleic Acid Extraction Methods for Detection of Candida auris Assessed using RT-PCR and Next Generation Sequencing

L . A . Sealey , E . Zelaya , A . P . Cabello , M . Mann , V . Irani , J . Steyer , W . Bchara , E . Vaughn , J . Doss , A . Murthi , D . Edwards , D . Payne , J . Hauser ; District of Columbia Department of Forensic Sciences , Public Health Laboratory Division

Candida auris is a highly transmissible multi-drug resistant yeast with a high mortality rate in immunocompromised individuals . C . auris outbreaks are of public health concern due to its asymptomatic presentation in infected individuals , infrequent screenings for detection and its propensity to spread in health care settings . Accurate and efficient detection strategies for C . auris are important for coordinating timely treatment and infection control measures . In this study , we compare four nucleic acid extraction methods commonly found in state and local public health laboratories . We compare the suitability of extracted C . auris nucleic acids for use in downstream applications , including real-time PCR and whole genome sequencing . Samples were cultured from lab strains and clinical isolates of C . auris and other fungal species . Nucleic acids were extracted using MagNA Pure 96 , MagNA Pure 24 , QIAcube Connect and bioMérieux EMAG instruments . Nucleic acid yield and purity , cost per sample , tech time and instrument time were compared for each extraction method . Qualitative

PCR analyses were performed targeting the internal transcribed spacer region 2 ( ITS2 ) of C . auris . Ct values were compared across different extraction methods and different strains of C . auris . We also performed long- and short-read next generation sequencing analysis to compare genome coverage , cluster density , sequence length and Q30 scores . While all four extraction methods were able to successfully extract nucleic acids from C . auris , we observed different levels of performance in various metrics . The results contribute to the implementation and verification of the real-time PCR assay and sequencing of C . auris using multiple extraction methods in a public health laboratory . This comparison provides supportive data for the use of multiple methods to process and accurately detect C . auris for rapid screening , thus contributing to existing techniques that can help with surveillance and control in health care facilities . These results will allow public health laboratory leaders to make informed decisions about which methods to use depending upon their desired downstream applications , available instrumentation and staffing levels .

Presenter : Leanna Sealey , leannasealey _@ hotmail . com

Development and Optimization of Candida auris Surveillance Test using a Cartridge-based Assay System

S . Banik 1 , C . Kanna 2 , B . Ozay 3 , M . Trejo 4 , O . Garner 4 , L . Vejar 3 , S . Chakravorty 3 , D . Alland 1 , P . Banada 1 ; 1 Rutgers University - New Jersey Medical School , 2 Rutgers University , 3 Cepheid , 4 Department of Pathology and Laboratory Medicine , University of California Los Angeles

Background : Candida auris , a multidrug resistant fungal pathogen is often misidentified and requires higher technical skills to identify using complex methods . We used a cartridge-based assay system with an integrated sample processing and nucleic acid amplification ( NAA ). Here , we demonstrate development of a rapid , sensitive , and specific PCR-based surveillance assay to detect C . auris in skin swabs , which can be used in near-patient settings . Methods : The assay reagents containing the sample processing buffers , wash buffers and PCR reagents were optimized and built into a sealed , ready-to-use cartridge system . These ready-to-use sealed cartridges were used in subsequent experiments to enumerate dynamic range , limit of detection ( LoD ), inclusivity , and exclusivity . C . auris -negative skin swab matrix ( SSM , confirmed by PCR ) was collected at the Department of Pathology and Laboratory Medicine , the University of California Los Angeles ( UCLA ). The dynamic range of the assay was evaluated in Amies media , by spiking C . auris ( AR0388 ) from 1 CFU / mL through 104 CFU / mL . A 500 µ L of sample was added to the sample chamber of the cartridge and processed for DNA isolation and PCR . The LoD was evaluated by spiking C . auris AR0388 into SSM at concentrations of 0 , 1 , 5 , 10 , 20 and 50 CFU / mL ( N &# 3f20 ) by adding 500 µ L of the spiked SSM into the C . auris assay cartridge . The assay inclusivity was tested at 2-3X LoD with additional strains of C . auris ( N &# 3f8 ) belonging to clades I , II , III , and IV . Exclusivity with non- C . auris strains ( N &# 3f10 ) was also verified . Results : The assay dynamic range showed a linearity of R2 = 0.9887 , demonstrating that the detection was not adversely affected in presence of higher target concentrations . The assay LOD was 10.5 CFU / mL of C . auris AR0388 in SSM . Inclusivity with both aggregative and non-aggregative strains at 2-3X LOD showed 100 % detection . The assay specificity was 100 % when it was tested