Lab Matters Fall 2024 | Page 97

APHL 2024 POSTER ABSTRACTS reconfirmed using PHoeNIx . We performed cgSNP analysis to identify the relatedness of samples using the fl _ cg _ snp pipeline developed by the Florida Public Health Laboratory .

The blaNDM genes have been detected in 18 NDM-positive Enterobacter hormaechei samples . Of the 18 samples , 8 had blaNDM-7 ; 6 had blaNDM-1 ; and 4 had blaNDM-5 gene detected . The PHoeNIx and MGE pipelines showed that all 6 of the blaNDM-1 positive samples had the In-cHI2 / 2A plasmid and that 5 of them had a mcr-9 gene . cgSNP analysis demonstrated that 4 of the 6 blaNDM-1 positive samples were closely related . The mcr-9.2 gene variant was detected in 3 of these related samples . In this presentation , we discuss the epidemiological significance of these findings as well as the potential relationships between the blaNDM-1 gene , mcr-9.2 gene and the IncHI2 / 2A plasmids .

Presenter : Srimadhav Nallani , Srimadhav . Nallani @ msdh . ms . gov

Development of an Enzyme Linked Immunosorbent Assay ( ELISA ) for Detection of B . pseudomallei Specific Antibody in Human Sera

X . Tang 1 , D . Boulay 1 , E . David 2 , L . Jia 1 , Y . Li 3 , M . Glass Elrod 3 , R . Stoddard 1 , Z . Weiner 1 , S . Vishwanathan 1 , Centers for Disease Control and Prevention ( CDC ) 1 , CFD Research 2 , IHRC 3

Background : Melioidosis is an emerging infectious disease caused by Burkholderia pseudomallei , a Tier 1 select agent in the United States . Worldwide , 10-50 % of melioidosis cases result in death . Although melioidosis is considered predominantly a tropical disease , especially in Southeast Asia and northern Australia where it is widespread , B . pseudomallei was isolated in 2022 in the environment along the Gulf Coast of Mississippi in US and linked to three human melioidosis cases . Now that melioidosis is considered endemic in the southern continental US and a nationally notifiable disease , enhanced domestic surveillance and a strong public health response entailing prompt diagnosis and treatment is needed . The standard culture diagnosis method requires BSL-3 facilities to reduce occupational exposure and the commonly used indirect hemagglutination assay ( IHA ) has low sensitivity and specificity . Recent studies have suggested that hemolysin coregulation protein 1 ( Hcp1 ), a B . pseudomallei virulence factor , is a promising target antigen for serodiagnosis of melioidosis . In this study , we developed an improved ELISA to detect specific antibody against Hcp1 in human sera , optimizing reagents and procedures to improve assay sensitivity and specificity .

Methods : Using purified recombinant Hcp1 protein , we determined antibody levels in two sets of sera : 66 heat-treated positive sera from melioidosis patients confirmed by culture or IHA and 191 negative sera from healthy donors . We optimized 96-well plates , antigen concentration ( titration range : 0.5 to 5.0 µ g / mL ), serum dilution , blocking buffer , substrate and stop solution . Briefly , 96-well plates were coated overnight with Hcp1 at 2-8 ° C . After washing with PBST ( 1XPBS with 0.1 % Tween-20 ), the plates were blocked for one hour at 37 ° C and washed with PBST . Sera at selected dilutions were incubated for 1 hour at 37 ° C , washed with PBST and incubated with HRP-mouse anti-human IgG for one hour at 37 ° C . After washing with PBST , plates were developed by ABTS or TMB . Absorbance was measured at 405 , 450 or 650 nm on a BioTek microplate reader . Diagnostic specificity ( DSP ) and sensitivity ( DSN ) were calculated for reactivity thresholds ( RT ) ranging from 0.10 to 0.40 ( OD650 ) using SAS 9.4 software .

Results : After comparison of three types of high binding plates including Corning , MaxiSorp and Immulon 2HB , we identified Corning 96-well EIA plate as optimal and cost-effective . Antigen concentration of 2 µ g / mL was found to be the optimum and the coated plates were determined to be stable for seven days at 2-8 ° C . 5 % skim milk in PBST was a better blocking buffer than BSA diluent . Serum dilution at 1:100 and the HRP conjugate at 1:4,000 dilution were optimal . TMB substrate and BlueSTOP ( 650nm ) were more sensitive than TMB and TMB stop solution ( 450nm ) or ABTS substrate and ABTS peroxidase stop solution ( 405nm ). We determined that the optimal RT was 0.16 at 650nm , with a sensitivity of 93.94 % and a specificity of 98.43 %, yielding high values for DSN and DSP while minimizing the disparity between them .

Conclusions : The optimized ELISA using Hcp1 with high DSP and DSN is a reliable method for serodiagnosis of melioidosis . Compared to previously published methods , this protocol can be performed in BSL2 labs with a short turnaround time and hence better positioned to fulfill national and global public health needs .

Presenter : Xiaoling Tang , CDC , Email : gqi3 @ cdc . gov

Development of a Protocol and Data Set for Validation of Neisseria gonorrhoeae Genome Profiler and Typing Tool

K . Hebrank , N . Burg , E . Tran , A . Clemons , N . Shen , J . Reimche , S . St Cyr , M . Schmerer , K . Gernert , E . Kersh , S . Joseph , Centers for Disease Control and Prevention

Background : Whole genome sequence ( WGS ) analysis is an essential tool for genomic epidemiology , surveillance of strain types and identifying antimicrobial resistant ( AMR ) determinants of Neisseria gonorrhoeae ( Ng ), the causative agent of gonorrhea . High accuracy is required for genome-based epidemiological surveillance , yet there is a dearth of validated analytical tools for public health laboratory use .

Methods : Using the CDC ’ s Next Generation Sequencing ( NGS ) Quality Initiative ’ s ( QI ) template , we developed a dataset and protocol for validating a customized bioinformatics pipeline , Ng Genome Profiler and Typing Tool , for the analysis of Ng WGS data . The pipeline intakes Illumina sequences , performs quality control assessments , completes de novo assembly , extracts the chromosomal and plasmid-based antimicrobial resistance ( AR ) markers based on 26 gene loci and performs sequence typing ( Multilocus Sequence Typing ( MLST ), Ng Multi-Antigen Sequence Typing ( NGMAST ) and Ng Sequence Typing for Antimicrobial Resistance ( NGSTAR )). We defined our reference set as 57 Ng closed genomes , sequenced using the long-read PacBio platform . True reference values for AR markers and loci calls were obtained for these 57 genomes via PubMLST and manual curation . We defined our test dataset as the corresponding Illumina reads for the 57 reference isolates . In addition , 15 reference isolates were sequenced independently at least three separate times by 4-5 separate laboratories ; and were designated as technical replicates to assess the reproducibility ( n = 206 ). We processed the 57 test isolates through the Ng Genome Profiler and Typing Tool ( versions 2.9.2 and 3.0.0 ) and examined 16 single nucleotide polymorphisms ( SNPs ), three allele calls and two sequence typing methods ( MLST , ngMAST ) for accuracy . Genomic calls were compared against the results obtained from reference genomes and statistical analyses of accuracy , sensitivity and specificity were completed .