Lab Matters Fall 2023 | Page 61

APHL 2023 POSTER ABSTRACTS

Validating a PCR Test for the Detection of Legionella in Clinical Specimens at the New Jersey Public Health Laboratory

M . Eickhoff , S . Delaney , P . Patel , M . Orsini-Lidicker , M . Ellethy , R . Siderits , T . Kirn ; New Jersey Department of Health Public Health and Environmental Laboratories

Approximately 250-350 cases of Legionnaires ’ disease , a severe pneumonia caused by Legionella bacteria , are reported to the New Jersey Department of Health ( NJDOH ) each year . Legionella are found naturally in freshwater environments , however the risk for humans is increased when Legionella contaminate human-made waters systems . Legionnaires ’ disease is contracted by inhaling or aspirating water containing Legionella . Thus , outbreaks of Legionnaires ’ disease are associated with contaminated water systems that create aerosolized droplets such as cooling towers , hot tubs , and fountains . When Legionnaires ’ disease cases are reported , public health laboratory testing is crucial to rapidly identify a potential outbreak and prevent additional cases . Clinical testing is required to diagnose and connect cases within an outbreak , and environmental testing is required to identify the contaminated source . Currently , the NJDOH Public Health and Environmental Laboratories ( PHEL ) uses culture-based methods to perform environmental Legionella testing of potable and non-potable water samples . The NJDOH PHEL has not previously performed Legionella testing for clinical specimens . Here , we report the optimization of a qPCR test for the detection and differentiation of Legionella species , Legionella pneumophila , and L . pneumophila serogroup 1 in clinical respiratory specimens . For this diagnostic test , DNA is extracted from sputum using either the Roche High Pure PCR Template Preparation Kit or the Qiagen EZ1 Advanced XL system . The extracted DNA is used as template in a multiplex qPCR reaction using previously published primers and probes ( Benitez and Winchell , 2013 ). To analyze the test ’ s performance , a validation study was conducted by testing sputum specimens that were spiked with L . pneumophila serogroup 1 , L . pneumophila serogroup 3 , or Fluoribacter bozemanae ( previously Legionella bozemanae ). The test detected the expected species and serogroup of Legionella spiked into 40 sputum specimens with 100 % sensitivity and specificity . The limit of detection of the method was ~ 250 CFU / mL in sputum . Lastly , a stability study was conducted in which sputum specimens stored at either 4 ° C or -20 ° C were repeatedly tested over 14 days . Specimen handling requirements for the test were modified to account for our finding that Ct values for Legionella positive specimens remained relatively constant over a two-week period . By validating this qPCR test , PHEL now has the capability to detect Legionella within both environmental and clinical specimens . An additional validation study has been conducted so that Legionella can be cultured from clinical specimens . With these increased testing capabilities , the future goal of the laboratory is to perform whole-genome sequencing of both environmental and clinical Legionella isolates to rapidly identify and connect outbreaks of Legionnaires ’ disease .

Presenter : Mohammed Ellethy , mohammed . ellethy @ doh . nj . gov

Validation of High-Throughput Screen for Carbapenemase Gene Variants

N . Olivarez , M . Tran , P . Dykema ; Washington State Public Health Laboratories

Increasing throughput and minimizing result turnaround times are critical to improving detection methods for large screens of patient samples to identify carbapenemase gene variants in multi-drug resistant , carbapenem-producing organisms ( CPOs ) in healthcare facilities . CPOs are serious threats to public health that have been identified in increasing numbers of healthcare-acquired bacterial infections . Rapid detection of CPOs from patient rectal swabs is key for health professionals to prevent the spread of these antimicrobial resistant phenotypes within the health care system . The currently approved CPO screening method begins with the Cepheid GeneXpert Carba-R PCR detection method , which is only able to detect the presence of five antimicrobial resistance genes ( IMP , KPC , NDM , OXA-48 , and VIM ). Positive samples are then processed by culture-based methods followed by CPO gene target detection by Real-Time PCR , all of which takes 5-10 days . The Cepheid method cannot detect the presence of clinically relevant variants like IMP- 14 , IMP-18 , IMP-27 OXA-23 , OXA-24 / 40 , OXA-58 , or OXA-235 that are validated for detection by the Real-Time PCR . In addition to detecting more variants than the Cepheid-based method , directly detecting variants from patient rectal swabs is one-tenth the cost / sample compared to the Cepheid-based method .

Here we combined the reliable detection method of cabapenemase gene variants by RT-PCR with an automated DNA extraction system to screen for a broad set of carbapenemase gene variants directly from rectal swabs , eliminating the need for culture-based methods , allowing for same day turnaround time of results . Validation of this method allows healthcare providers to respond quickly to prevent the spread of harmful CPOs in acute and long-term healthcare facilities , while minimizing costs and labor for public health laboratory analysts process patient samples .

Presenter : Nicholas Olivarez , nicholas . olivarez @ doh . wa . gov

Validation of MagNA Pure 24 Instruments for Downstream use of DNA in CLIA-regulated and Nonregulated Sequencing

M . Martin 1 , A . Mooney 2 , A . Bateman 2 ; 1 US Centers for Disease Control and Prevention , 2 Wisconsin State Laboratory of Hygiene

Background : Whole genome sequencing is a powerful analytical tool that allows public health laboratories to rapidly identify and characterize pathogens and determine genetic relatedness among organisms . The Wisconsin State Laboratory of Hygiene ( WSLH ) previously used the MagNA Pure LC 2.0 ( Roche ) to extract DNA for sequencing ; the instrument was discontinued at the end of 2022 and WSLH chose to replace it with the MagNA Pure 24 ( MP24 ) system ( Roche ). Onboarding new automated laboratory instruments requires validation before use , but validation requirements differ based on how data will be used . Data used for patient results or to guide care must meet Clinical Laboratory Improvement Amendments ( CLIA ) regulatory requirements ; whereas results used for surveillance purposes are not regulated . Using both instruments , we extracted DNA from a panel of organisms representative of CLIA-regulated and nonregulated tests and compared quality and