Lab Matters Fall 2024 | Page 79

APHL 2024 POSTER ABSTRACTS using an automated workflow starting with nucleic acid derived from either saliva ( extraction free ) or extracted nasopharyngeal swabs . The use of nanoliter-scale microfluidic reactions conserves precious reagents while reducing plastic waste and enabling sustainable lab operations . Following preparation of sample and assay mixes , which are dispensed into a microfluidic chip that is subsequently loaded onto the Biomark™ X9 System for processing , results are available in approximately two hours without manual intervention .

The current list of targeted pathogens includes influenza A , influenza B , respiratory syncytial virus and SARS-CoV-2 and can be further expanded and customized using the open architecture of the microfluidic chip . Each of the chip ’ s 48 assay inlets connects with an independent reaction chamber , which enables use of all assays in a single-plex reaction and thermal profile while preventing assayto-assay interference associated with multiplex reactions .

Additional studies are planned to expand the targets in the respiratory pathogen panel , apply the same workflow to new pathogen targets ( for example , gastrointestinal and vector-borne pathogens ) and evaluate performance using alternate formats of the microfluidic chip .

Presenter : Brad Mire , brad . mire @ standardbio . com

Validation of a Heat-inactivation Protocol For Mycobacterium tuberculosis Identification by MALDI-TOF Mass Spectrometry

L . Franco 1 , K . Chambers 2 , B . Hull 2 , H . Khan 1 , K . Gavina 1 , J . Lavik 1 , C . Emergy 1 , R . Relich 1 , Indiana University School of Medicine 1 , Indiana University Health 2

Mycobacterium tuberculosis ( MTB ) complex bacteria can cause severe respiratory and disseminated diseases and they caused an estimated 10.6 million new cases globally in 2022 . Microbiological diagnosis of MTB infection is essential for patient treatment , infection control precaution implementation and contact tracing . Previously , the Indiana University Health Division of Clinical Microbiology used DNA hybridization probes to identify or rule out MTB in cultures , but because these probes were discontinued by their manufacturer , we pursued matrix-assisted laser desorption / ionization time-of-flight ( MALDI-TOF ) mass spectrometry as an alternative . Analysis of MTB by MALDI-TOF in biosafety level-2 laboratories requires that cultures be inactivated prior to removal from high containment . For this study , we evaluated a heat-inactivation protocol to ensure complete inactivation of MTB . Briefly , colonies from 7H10 agar and broth aliquots from mycobacterial growth indicator tubes ( MGIT ) were harvested from 14 Mycobacterium spp . cultures comprising MTB and non-tuberculous mycobacteria ( NTM ) species . Isolates were incubated in a 95 ° C water bath for 30 minutes followed by protein extraction using a formic acid and acetonitrile protocol . Extracts were split in half ; one half was filtered through a 0.1- µ m centrifuge filter and the remainder was unfiltered . All extracts were inoculated into MGIT tubes and incubated for six weeks to assess viability . Growth controls of each test isolate were incubated concurrently . No growth was detected in the heat-inactivated samples , both filtered and unfiltered , indicating that this simple protocol was sufficient to completely inactivate the tested mycobacteria . Growth was detected in all positive controls . Further , we validated that the heat-inactivated samples could be identified using the Bruker MALDI Biotyper Sirius instrument equipped with the Bruker MBT Mycobacteria RUO Module . We evaluated three spotting protocols for aliquoting heat-inactivated and extracted material to MALDI-ToF target plates and used a score value of 1.8 as a cut-off for species-level identification . For protocol one , 1 µ l of sample was overlaid with 1 µ l of matrix ; for protocol two , 2 µ l of sample was overlaid with 2 µ l of matrix ; and for protocol three , 2 µ l of sample was spotted and allowed to dry and then an additional 2 µ l of sample followed by 2 µ l of matrix were added . Forty-four percent , 85 % and 96 % of isolates were identified with a score value greater than 1.8 , using protocol one , two and three , respectively . This study details a relatively simple protocol for heatinactivation of MTB and NTM , with optimized isolate identification by MALDI-ToF mass spectrometry .

Presenter : Lauren Franco , laurencfranco @ gmail . com

Validation of a Molecular Workflow to Identify the Diphtheria Exotoxin Gene in Corynebacterium Species

A . Chia , M . Tran , N . Olivarez , P . Dykema , Washington State Department of Health

Toxin producing Corynebacterium diphtheriae is the causative agent of diphtheria , a historically devastating communicable disease that can result in long-term disability and death if left untreated . However , vaccination campaigns and antibiotic treatment regimens have limited these infection-related outcomes in developed settings . Emerging non-toxin producing variants of C . diphtheriae have become pathogens of interest as strain-related cases continue to be reported on the west coast of North America . This is especially concerning for members of vulnerable populations , such as the homeless , as they are at greater risk for life-threatening outcomes when infected . A culture-based detection assay ( the Elek test ) is conventionally used , but the growing number of Seattle-area cases necessitates a higher-throughput diagnostic method . Previously , the Pertussis and Diphtheria laboratory at the CDC has developed a real-time polymerase chain reaction ( RT-PCR ) assay that targets the diphtheria exotoxin gene found in C . diphtheriae , Corynebacterium ulcerans and Corynebacterium pseudotuberculosis . This assay has allowed for faster phenotype identification required for timely treatment . Here we describe the validation and verification of the CDC ’ s RT-PCR assay for use by the Washington State Department of Health , as well as a streamlined workflow that uses both matrixassisted laser desorption and ionization ( MALDI ) mass spectrometry and RT-PCR to identify pathogens more effectively . Our revised pipeline increases the accuracy of identifying toxin genotype for Corynebacterium species , while simultaneously lowering cost and improving diagnostic turnaround time .

Presenter : Andy Chia , andy . chia @ doh . wa . gov