Lab Matters Fall 2024 | Page 78

APHL 2024 POSTER ABSTRACTS

kit is a commercially available Research-Use Only ( RUO ) multiplex PCR assay that detects three clinically relevant groups of extendedspectrum β-lactamases ( ESBLs ) ( blaTEM , blaSHV , blaGES ). In this study , the performance of the Streck ARM-D ® TEM / SHV / GES kit was evaluated against whole genome sequencing ( WGS ) for the detection of three ESBL genes ( TEM / SHV / GES ) using a set of wellcharacterized bacterial isolates from the CDC & FDA Antimicrobial Resistance Isolate Bank ( AR Bank ).

Methods : Fifty-six isolates comprised of Pseudomonas aeruginosa and four species of Enterobacterales from the AR Bank were used to assess the performance of the Streck ARM-D ® TEM / SHV / GES kit . Among the 56 isolates , 41 harbored at least one of the targeted genes including three variants of blaTEM , 14 variants of blaSHV and five variants of blaGES ; 14 isolates carried more than one of these β-lactamase genes . A subset of 27 isolates also carried additional β-lactamase genes including 18 blaCTX-M , three blaKPC , 1one blaNDM , 17 blaOXA and four blaVIM . Fifteen isolates were negative for the targeted genes but carried other β-lactamase genes including 15 blaCTX-M and 3 blaOXA . The Streck ARM-D ® assay was performed per the manufacturer ’ s instructions for use ( IFU ) with the following modifications . Instead of using a commercial DNA extraction kit , bacterial cells were treated with 0.1 N NaOH at 95 ° C for 10 minutes in Tris-HCl buffered molecular grade water and centrifuged to extract DNA . Lysates were stored at -20 ° C until use . Each lysate was evaluated by the Streck ARM-D ® TEM / SHV / GES kit using the Applied Biosystems 7500 Fast instrument and protocol . Results from the Streck ARM-D ® assay were compared to previously generated WGS data by either PacBio RS2 ( Pacific Biosciences , Menlo Park , CA ) or HiSeq 3000 ( Illumina , San Diego , CA ) platform analyzed using Resfinder and Antibiotic Resistance Gene-ANNOTation databases with thresholds set at 99 % identity and 100 % coverage .

Results : The Streck ARM-D ® assay showed 100 % sensitivity and specificity for all β-lactamase gene variants evaluated . The β-lactamase gene variants expected to be detected by the Streck assay were identified correctly 55 / 55 times . The Streck ARM-D ® kit was able to simultaneously detect more than one of the targeted genes in a single reaction . No cross-reactivity was observed against other β-lactamase genes present in the isolates .

Conclusions : The Streck ARM-D ® TEM / SHV / GES kit demonstrated accurate β-lactamase gene detection across a diverse set of Gram-negative isolates . For the genes and variants evaluated , the Streck assay represents an accurate and rapid tool that clinical laboratories , following proper validation studies , can implement for the detection of clinically relevant β-lactamase resistance genes . Moreover , detection of these genes can support surveillance efforts needed to combat the recent rise in infections caused by ESBLproducing Gram-negative bacteria in healthcare settings and the community .

Presenter : Justina Ilutsik , nkl9 @ cdc . gov

Rapid Detection of Carbapenemase Activity in Enterobacterales , Pseudomonas aeruginosa and Acinetobacter baumannii using MALDI-TOF MS

C . Lu , M . Theophilus , A . Sy , H . Berman-Watson , J . Lau , R . Mejia , Z . Berrada , P . Zhang , California Department of Public Health

Background : The increasing emergence of carbapenem-resistant bacteria , including Enterobacterales ( CRE ), Pseudomonas aeruginosa ( CRPA ) and Acinetobacter baumannii ( CRAB ), is a major public health threat . The modified carbapenem inactivation method ( mCIM ) is a reference method that is widely used in clinical and public health laboratories to detect carbapenemase production ; however , it is time-consuming and not suitable for detecting carbapenemases in A . baumannii . New methods that can rapidly detect an extended carbapenemase-producing bacterial species are essential for effective infection prevention and control . We evaluated the performance of the MBT STAR ® -Carba IVD assay ( Bruker Daltonics ) to detect carbapenemase-producing organisms ( CPO ) from bacterial isolates using matrix-assisted laser desorption ionization-time of flight mass spectrometry ( MALDI-TOF MS ).

Methods : We analyzed 165 Gram-negative bacteria strains , including 102 CPO strains with various carbapenemase gene variants ( blaKPC , blaNDM , blaVIM , blaIMP , blaOXA , blaSME , blaIMI and blaSPM ) that are non-susceptible to at least one carbapenem and 57 non-CPO . We also analyzed six carbapenem non-resistant A . baumannii isolates carrying a single blaOXA gene ( OXA-66 , 69 , 91 or 500 ) encoding a weak carbapenemase . Samples were prepared with the MBT STAR ® -Carba kit and imipenem hydrolysis by the potential carbapenemase was analyzed using the MBT STAR-BL module ( Bruker Daltonics ) on MALDI-TOF MS .

Results : The assay detected all carbapenemase-producing Enterobacterales ( n = 40 ), P . aeruginosa ( n = 15 ) and A . baumannii ( n = 47 ) with 100 % sensitivity . The specificity of the assay was 96.8 %. Of 57 non-CPO isolates tested , 55 isolates tested negative for carbapenemase production as expected and two isolates ( one Enterobacterales and one P . aeruginosa ) produced inconclusive results . The six A . baumannii isolates encoding weak carbapenemases tested negative for carbapenemase production as expected .

Conclusion : The MBT STAR ® -Carba IVD assay is a highly accurate and rapid method for the detection of carbapenemase activity not only for CRE and CRPA , but also for CRAB .

Presenter : Joseph Lau , joseph . lau @ cdph . ca . gov

Two-Hour Microfluidics-based Workflow for Concurrent Detection and Identification of Multiple Common Upper Respiratory Pathogens

B . Mire , N . Ramalingam , B . Hunt , L . Stewart , Standard BioTools

Timely detection and accurate identification of infectious pathogens are key in developing tools used by public health programs to conduct outbreak surveillance and management . This can help shorten outbreak duration and support identification of the causative agent ( s ) so that appropriate corrective measures can be designed and implemented . Molecular methods such as polymerase chain reaction ( PCR ) and next-generation sequencing ( NGS ) routinely contribute to the effort by allowing multiple pathogens to be genomically profiled at the same time and , in some cases , without a dependency on isolate culture . This effectively reduces the time and testing needed to identify pathogens present in samples collected from an outbreak .

In this study , we demonstrate proof of concept and describe a microfluidics-based protocol designed to detect and identify four upper respiratory pathogens in up to 48 samples from a single run