APHL 2022 POSTER ABSTRACTS
Rapid Molecular Detection of Echinocandin-Resistance in Candida auris
YC Zhu , K Hager , V Chaturvedi and S Chaturvedi , Wadsworth Center , New York State Department of Health
Background : Candida auris is a multidrug-resistant yeast pathogen causing outbreaks in healthcare facilities worldwide and emergence of echinocandin-resistant C . auris is a concern . Currently used CLSI and commercial antifungal susceptibility tests are phenotype-based , slow and not scalable , limiting their effectiveness in the surveillance of C . auris . There is an urgent need for accurate and rapid assessment of resistance to echinocandin-class antifungals , which are the current first line drugs of choice . We report the development of a TaqMan chemistry probe-based fluorescence melt curve analysis ( FMCA ) following asymmetric PCR to assess mutations within the hot spot one ( HS1 ) region of FKS1 , encoding 1,3-beta-Dglucan synthase , a target for echinocandins .
Methods : C . auris isolates exhibiting echinocandin resistance by broth microdilution method were obtained from our repository . These were cultured , followed by DNA extraction , PCR amplification , and Sanger sequencing of FKS1 . Multiple alignments of FKS1 were done using Geneious 9.1.8 software . Primers and probes were designed from HS1 for FMCA on LightCycler ® 480 . Assay validation was performed using pure C . auris cultures , crude DNA extraction in PBS-BSA , and testing five microliters of extracted DNA in duplicate for assay reproducibility , limit of detection , specificity , blinded verification and melting temperature ( Tm ) range .
Results : Twenty-five C . auris isolates were sequenced using ten primer pairs to produce full-length FKS1 (~ 5600-bp ). Multiple alignments revealed several mutations within the ~ 590-bp of FKS1 comprising HS1 but not HS2 region . Additional 39 isolates were sequenced for HS1 with three primer sets . Of 64 isolates , 25 were wildtype and 39 were mutants . Of mutants , one each was F635del and D642H / R645T , 4 were F635Y , 9 were F635C , 3 were S639P , 7 were S639F and 14 were S639Y . All wildtype isolates were susceptible while all mutants were resistant to echinocandins except D642H / R645T by broth microdilution . FMCA correctly identified wildtype , F635C , F635Y , S639P and S639F mutations with distinct Tm . FMCA also produced unique but identical Tm for S639F and S639Y but could not identify F635del or D642H / R645T . Results showed FMCA is highly specific and reproducible for identifying mutations in FKS1 for C . auris isolates , conferring echinocandin resistance . Additionally , we found excellent concordance between FMCA , Sanger sequencing and antifungal susceptibility testing .
Conclusions : Echinocandin-resistant C . auris pose a high risk for outbreaks worldwide . There is an urgent need for accurate and rapid assessment of echinocandin resistance as that is the drug of choice for patient management , infection control and prevention programs . The FMCA assay developed in this study will aid in the shortcomings of the turnaround time faced with current phenotypic assays used for resistant detection .
Presenter : Yan Chun Zhu , Wadsworth Center , New York State Department of Health , yanchun . zhu @ health . ny . gov
WSLH Response to the Measles Outbreak in Afghan Refugees at Ft . McCoy in September 2021
E Hanson , R Griesser , K Guenther , T Davis , T Danz , A Sterkel and A Bateman , Wisconsin State Laboratory of Hygiene
On August 29 , 2021 , the United States government started an effort to resettle Afghan refugees from Afghanistan to the US . Many of the refugees were temporarily housed at military bases , where they were given medical care and screenings before being resettled in communities across country . Roughly 13,000 refugees were housed at Ft . McCoy in Wisconsin . A 17-year-old Afghan evacuee arrived at Ft . McCoy on September 4 and shortly after presented with a fever of 107.6 º F and a maculopapular rash . Specimens were collected for measles testing and sent to the Wisconsin State Laboratory of Hygiene ( WSLH ) for testing . Active , wild-type measles was confirmed by real-time reverse-transcriptase polymerase chain reaction ( rRT-PCR ) on September 5 and molecular genotyping using Sanger sequencing revealed a genotype of B3 . WSLH immediately started working to validate a higher throughput extraction platform to better respond to a potential large scale measles outbreak . Over the course of the outbreak , 50 measles specimens were tested with both the CDC-developed pan-measles PCR and the CDCdeveloped measles vaccine PCR assay , which is able to differentiate wild-type from vaccine-related measles infections . A total of 28 specimens were positive for pan-measles PCR and 22 of those specimens were wild-type . All of these specimens genotyped using Sanger sequencing as B3 , which is the genotype most common in Afghanistan . In response to the measles positives at Ft . McCoy and other bases , incoming flights of refugees were temporarily paused on September 10th and a mass vaccination campaign was undertaken . By September 24 , > 99 % of eligible evacuees were vaccinated with MMR and Varicella vaccines . The last positive measles case tested at WSLH from Ft . McCoy was on October 5 . In addition to measles specimens , Ft . McCoy also sent specimens to WSLH for a variety of other viral detection assays including 1 rubella , 1 mumps and 7 Varicella Zoster Virus ( VZV ). Three of the VZV specimens were positive for wild-type VZV using the CDC VZV Bi-allelic Strain Typing assay ; all three specimens were further genotyped using Sanger sequencing as Clade 5 . In summary , the availability of PCR testing for measles at a Wisconsin laboratory allowed for the rapid identification and typing of a measles specimen over Labor Day weekend . The confirmation of wild-type measles in this Afghan refugee was instrumental in launching a swift public health response and led to the eventual containment of the measles outbreak at Ft . McCoy .
Presenter : Erika Hanson , Wisconsin State Laboratory of Hygiene , erika . hanson @ slh . wisc . edu
Identifying Carbapenemase Genes using Whole Genome Sequencing
L Patterson , A Shockey , A Mooney , A Valley , A Bateman and K Florek , Wisconsin State Laboratory of Hygiene
The rise in carbapenem-resistant organisms ( CRO ) and carbapenemase-producing organisms ( CPO ) necessitates new detection and intervention strategies to maintain the effectiveness of these critical antibiotics . Currently , detection of CRO / CPO in clinical laboratories involves a combination of antimicrobial susceptibility testing ( AST ), phenotypic or biochemical assays
Infectious Diseases / Informatics