Lab Matters Fall 2024 | Page 73

APHL 2024 POSTER ABSTRACTS resistant to all currently recommended treatments has also been documented . In the United States , GC antibiotic resistance ( AR ) surveillance is overseen by the CDC through several programs : the Gonococcal Isolate Surveillance Project ( GISP ), the “ Enhanced GISP ” ( eGISP ) and the “ Strengthening the U . S . Response to Resistant Gonorrhea ” ( SURRG ). All these AR surveillance programs entail phenotypic testing via agar dilution ( AD ), the gold standard method for determining minimal inhibitory concentrations ( MIC ) values in GC . AD is a very laborious procedure , involving manual evaluation of growth on agar plates and data entry into the laboratory information system ( LIS ). Here we describe the evaluation of the BIOMIC V3 imaging system ( Giles Scientific Inc ) and its AD-specific software module with the goal of improving the efficiency of the AD procedure .

AD for GC is routinely performed in our lab by processing batches of 32 isolates against seven different antibiotics ( ciprofloxacin , penicillin , cefixime , ceftriaxone , tetracycline , azithromycin , gentamicin ) at 9-16 varying concentrations generated by completing 1:2 serial dilutions . 0.5 McFarland suspensions of individual isolates in Mueller Hinton broth are then stamped onto agar plates ( GC medium base containing IsoVitaleX supplement ) containing different antibiotic concentrations using a Steer ’ s replicator . Three quality control strains ( F18 , WHO L , WHO U ) are tested alongside the isolates . The inoculated plates are incubated at 36 º with 5 % CO2 overnight . MIC values from plates read both manually and with the BIOMIC V3 were entered into the LIS and processing time relative to these workflows was measured .

We evaluated 30 isolates including WHO strains and isolates previously tested through GISP , eGISP and SURRG . A total of 83 plates were imaged using the BIOMIC V3 . The AD-specific software was not able to generate an MIC in about 10-20 % of the isolates , indicating that automatic image density scanning is less sensitive than the human eye for the detection of growth . When growth was detected by the software , 85.2-96.3 % of the isolates displayed MICs within one doubling dilution from manual reading . In addition , MICs greater than the manual read were occasionally generated due to the software interpreting bubbles or starch flakes in the agar as growth . This specificity issue occurred while scanning three plates ( 4 % of the total plates imaged ). These performance limitations were bypassed by including a manual review step of the automatically generated MICs , which took under 30 minutes to complete and increased accuracy to 99.9 %. The automated / operator-corrected process allowed us to eliminate the need for having two technicians verifying each other ’ s readings , annotating MICs on worksheets and transcribing them in electronic spreadsheets for upload in the LIS . The overall length of the AD procedure was shortened more than one hour .

This pilot project has led to the development of an AD-specific BIOMIC software and of a workflow that can improve efficiency in labs performing AD . We will be working toward further validating this method and incorporating it into the GISP , eGISP and SURRG testing activities .

Presenter : Bailee Troutman , btroutma @ uccs . edu

Implementation of NG-Test CARBA5 and CHROMagarmSuperCARBA to Streamline Isolate Testing and Colonization Screening of Carbapenemase-producing Organisms at Wisconsin State Laboratory

S . Shama , A . Valley , M . Mamerow , A . Bateman , Wisconsin State Laboratory of Hygiene

Background : Carbapenem antibiotics are the last line of defense against resistant Gram-negative bacteria . Carbapenemases like the Big 5 ( KPC , NDM , VIM , IMP and OXA-48-like ) hydrolyze carbapenem antibiotics , rendering them ineffective for treatment . The Wisconsin State Laboratory of Hygiene ( WSLH ) currently identifies carbapenemase-encoding genes from carbapenem-resistant Enterobacterales ( CRE ) and carbapenem-resistant Pseudomonas aeruginosa ( CRPA ) isolates with the ABI 7500 real-time PCR . For colonization screening at WSLH , Xpert Carba-R is performed . For positive samples on Carba- R , an isolate is attempted to be grown by enriching in MacConkey broth overnight , followed by plating on ESBL agar and PCR from individual colonies . The current workflows are labor and time intensive . NG-Test CARBA 5 ( CARBA 5 ) is a multiplex immunochromatographic assay for the rapid identification of Big 5 carbapenemases from bacterial isolates . The primary goals of this study were to verify and implement CARBA 5 for isolate testing and a streamlined colonization approach : CHROMagar mSuperCARBA ( SuperCarba ) for direct plating of rectal swabs plus CARBA 5 testing from isolates grown on SuperCarba .

Methods : Five separate studies evaluated the utility of CARBA 5 and SuperCarba . A CARBA 5 verification study of 30 AR Bank isolates and isolates sequenced at WSLH was performed . A separate evaluation of 32 isolates was performed to compare three approaches for CRE / CRPA isolate recovery of positive Carba-R samples : our current workflow ( MacConkey broth + ESBL plates ), direct plating to SuperCarba and MacConkey broth enrichment followed by SuperCarba . We also validated CARBA 5 for isolates grown on SuperCarba and validated MALDI-TOF identification from isolates grown on SuperCarba . Lastly , we performed a study for direct testing of rectal swabs on CARBA 5 , to determine if CARBA 5 was sensitive enough to replace Carba-R .

Results : In the verification study , CARBA 5 performed just as well as PCR , with 100 % accuracy , reproducibility and precision . Additionally , isolates with multiples carbapenemases were detected well . Direct plating of rectal swabs on SuperCarba performed better than the current workflow of MacConkey broth + ESBL plates : we found 100 % accuracy and the limit of detection of SuperCarba was found to be better than our current workflow . The CARBA 5 identified all the carbapenemases from isolates grown on SuperCarba with 100 % accuracy in detecting the major resistance mechanisms . For MALDI- TOF validation from pure isolates grown on SuperCarba , accuracy was 100 %. However , direct testing of rectal swabs with CARBA 5 was not sensitive enough : only 40 % of positives by Carba-R were detected by CARBA 5 .

Conclusions : The CARBA 5 test is a low cost , rapid alternative to identify Big 5 carbapenemases in our culture-based colonization screening as well as isolate testing algorithm for CRE / CRPA . The CARBA 5 does not require special instrumentation , maintenance or molecular expertise to perform . CARBA 5 performed well for testing isolates from SuperCarba , which saves additional time and