Lab Matters Fall 2023 | Page 58

APHL 2023 POSTER ABSTRACTS

2-8 ° C ) for samples collected for HCV RNA testing . This requirement can be difficult for providers to meet due to limited access to courier services . Additionally , to align with the recommended CDC testing algorithm , the NH PHL sought to offer automatic reflexing to HCV RNA quantitative testing for samples testing positive for HCV antibody . This service was previously unmanageable due to the short storage requirements for HCV RNA samples . Therefore , the NH PHL evaluated specimen stability of samples submitted for quantitative HCV RNA testing . This study was funded by APHL . Methods : Specimens submitted to the NH PHL for routine quantitative HCV RNA testing were used ; positive HCV RNA specimens were stored at < 70 ° C . 30 specimens were selected for the study . They were thawed , aliquoted , and stored at 2-8 ° C . Samples were tested four times ; Day 0 , 3 , 7 , and 10 after thawing ( Day 0 representing the day of thawing , used to control for loss of virus while frozen ). Day 3 represented the maximum time at 2-8 ° C and established the “ true ” viral load for each specimen . This was then compared to results obtained from testing after 7 and 10 days at 2-8 ° C . Results : For our stability validation , the acceptable level of variation across time points was defined as the three day time point detectable viral load , +/ - 0.31 logs . This acceptable level of variation was established by a review of College of American Pathologists ( CAP ) proficiency test ( PT ) results . Peer group results for HCV viral load quantitative testing using the CAP / CTM v2.0 for the years 2018-2019 were analyzed . The acceptable variation in score for peer group samples during this period ranged from a low of the sample peer group mean +/ - 0.23 logs to a high of the sample peer group mean +/ - 0.45 logs . We chose +/ - 0.31 logs as our acceptable range as it represented the mean of the acceptable low and high ranges . All samples met our criteria for acceptability of results on Day 7 and Day 10 , and all samples that displayed a detectable viral load on Day 0 retained positivity through Day 10 ( the acceptability range could not be calculated for all samples on Day 3 ). Discussion : This study demonstrates that HCV RNA in serum specimens may be stable for up to 10 days when stored at 2-8 ° C . We found this to be true for specimens with viral loads ranging from 2.68 to 7.41 logs . A limitation of this study is the relatively low number of samples , particularly samples with very low viral loads . Only one sample with viral load of < 1.0 log was available in sufficient quantity to use in this study , and that sample yielded a result of “ Not Detected ” across all time points . Inclusion of more samples with low starting levels of viral RNA would bolster these results .

Presenter : Jessica Alexander , jessica . l . alexander @ dhhs . nh . gov

Implementation and Validation of a Method for Legionella Detection and Enumeration using the QIAcuity Digital Polymerase Chain Reaction Platform

B . Bornu , K . Plemmons , D . Newborn , M . Poole , D . Pettit , S . Shone , C . Goforth ; North Carolina State Laboratory of Public Health

Legionella , a genus of pathogenic gram-negative bacteria , that contains the species L . pneumophila , is responsible for legionellosis , which encompasses illnesses including Legionnaires ’ disease and Pontiac fever . With over 50 species recognized , it is widespread throughout many water sources . Transmission of these microorganisms are not person to person but are often the result of poorly maintained cooling towers , decorative fountains , hot tubs , as well as potable water sources . This procedure utilizes a digital PCR assay for the detection of Legionella spp ., Legionella pneumophila serogroups 1-15 , and specifically L . pneumophila serogroup 1 from environmental water samples . Using digital PCR allows for DNA quantification , which can assist in determining the effectiveness of Legionella remediation strategies . Validation included distinct filtering methods for potable and non-potable water sources , followed by a Kingfisher DNA extraction . The QIAcuity digital PCR system was validated using the 26K Nanoplate with multiplex analysis . This method utilizes three sets of oligonucleotide primers and probes that amplify unique target sequences within the Legionella genome . For Legionella spp ., a target was selected which amplifies a portion of the 23S rRNA gene that is thought to detect all species and serogroups of Legionella . The macrophage infectivity potentiator ( MIP ) gene target is used to detect L . pneumophila serogroups 1-15 . L . pneumophila serogroup 1 is identified via a region of the wzm gene . A bicoid inhibitor control is also implemented to identify sources of inhibition . This method , as with other PCR methods , is beneficial due to the possibility of a oneday turnaround time for a presumptive positive result , as opposed to several days for cultural methods .

Presenter : Burabari Bornu , burabari . bornu @ dhhs . nc . gov

Molecular Detection of OXA β-Lactamase Genes in Antimicrobial-Resistant Bacteria

E . Bui , M . Eickhoff , P . Patel , M . Ellethy , R . Siderits , T . Kirn ; New Jersey Department of Health Public Health and Environmental Laboratories

Antimicrobial resistance ( AMR ) is a rapidly increasing global health threat driven by the spread of antibiotic resistance genes in bacteria . The CDC estimates that 2.8 million infections and 35,000 deaths in the United States are due to antimicrobial-resistant pathogens annually . Public health laboratory testing is crucial for detecting outbreaks caused by antimicrobial-resistant pathogens and for surveillance of AMR threats . Of particular concern is the emergence of multidrug-resistant microorganisms for which few or no treatment options are available . Carbapenems are a class of β-lactam antibiotics that are typically reserved as antibiotics of last resort due to their potency against both Gram-negative and Gram-positive bacteria and their ability to resist hydrolysis by most β-lactamases . For this reason , the spread of carbapenemase genes which confer carbapenem resistance in bacteria is an urgent public health threat . The New Jersey Department of Health ( NJDOH ) Public Health and Environmental Laboratories ( PHEL ) surveils for AMR threats in clinical bacterial isolates by performing antimicrobial susceptibility testing , a modified carbapenem inactivation method ( mCIM ) for the detection of carbapenemase production , and real-time qPCR testing for surveillance of nine β-lactamase gene families . Except for OXA-48 , testing is not currently performed by the NJDOH to detect OXA-family β-lactamase genes encoding class D carbapenemases called oxacillinases . OXA genes can confer resistance to penicillins and some cephalosporin and carbapenem antibiotics . OXA genes are most often identified among isolates of Acinetobacter baumannii , a Gram-negative opportunistic pathogen that is a major cause of hospital-acquired infections . In this study , we tested the performance of a commercially available real-time qPCR kit for the detection of 6 OXA gene families in antibiotic resistant bacteria : OXA-23 , OXA-24 / 40 , OXA-48 , OXA-51 , OXA-58 , and OXA-143 . Bacterial isolates known to harbor OXA genes were provided by the CDC and FDA Antibiotic Resistance Isolate Bank and the Antibiotic Resistance Laboratory Group ( ARLG ) and were tested