Lab Matters Fall 2024 | Page 80

APHL 2024 POSTER ABSTRACTS

FOOD SAFETY AND SECURITY

A Tale of Two Pathotypes : Increasing Surveillance for Enteroaggregative and Enterotoxigenic Escherichia coli in Minnesota

E . Verbrugge , E . Cebelinski , D . Gerlach , S . Buuck , C . Medus , J . Haan , Minnesota Department of Health

Background : Enteroaggregative Escherichia coli ( EAEC ) and Enterotoxigenic Escherichia coli ( ETEC ) are a common cause of traveler ’ s diarrhea and have been implicated in foodborne disease outbreaks . Increasingly , EAEC and ETEC are being diagnosed in clinical laboratories through cul-ture-independent diagnostic tests like the FilmArray ® GI Panel ( Biofire Diagnostics ). In Minnesota , infections of these two pathotypes of diarrheagenic E . coli are reportable and culture confirmation is imperative for public health surveillance and identification of outbreaks .

Methods : To improve EAEC and ETEC laboratory surveillance , the Minnesota Department of Health Public Health Laboratory ( MDH-PHL ) began routinely isolating and sequencing these pathotypes . Stool or rectal swab specimens were cultured on Sorbitol MacConkey agar and culture sweeps were used to create crude extracts for PCR . Extracts were tested for the presence of either eltB and estA ( ETEC ) or aatA ( EAEC ) using SYBR Green PCR . Isolation was attempted on all positive specimens using isolate PCR . Isolated colonies were sequenced on the Illuminia MiSeq platform and were analyzed using PulseNet ’ s BioNumerics 7.6 and the Center for Genomic Epidemiology ’ s VirulenceFinder . In Minnesota , all diarrheagenic E . coli pathotype cases are interviewed with a standard questionnaire .

Results : From February 2023 to January 2024 , MDH-PHL sequenced 395 EAEC isolates and 76 ETEC isolates . For the duration of this study , the isolation rate was 74.8 % ( 395 / 528 ) for EAEC and 64.9 % ( 76 / 119 ) for ETEC . When culture sweeps positive for EAEC or ETEC had a cycle threshold ( CT ) value less than 29 , isolation rate was 84.4 %. Contrarily , when the CT value was greater than or equal to 29 , isolation rate for EAEC and ETEC was at 29.0 % and 41.3 %, respectively . ETEC and EAEC isolates were found to be both lactose fermenting and non-lactose fermenting on MacConkey agar . The most common serotype assigned to isolates after sequencing was O86 : H2 for EAEC and O25 : H16 for ETEC . EAEC isolates contained 90 different serotypes and ETEC isolates contained 26 different serotypes . Analysis utilizing the VirulenceFinder service from the Center for Genomic Epidemiology enhanced the detection of virulence gene variants for ETEC . Eight ( 53 %) of 15 EAEC sequencing clusters and 6 ( 86 %) of seven ETEC sequencing clusters were likely travel-associated . In addition , an outbreak of norovirus and ETEC was identified at a long-term care facility ( sequencing helped confirm the ETEC etiology ). Routine surveillance of EAEC and ETEC identified an outbreak associated with a juice bar .

Conclusions : Isolation rates could be improved through the validation of new PCR assays , which is currently in the works at MDH-PHL . Lactose fermentation differed between strains of EAEC and ETEC , describing the need for laboratorians to select a variation of morphologies during isolation . There was also variation among serotypes present for both EAEC and ETEC . Additionally , utilizing the most recent version of VirulenceFinder is recommended to detect any virulence gene variants among ETEC isolates . The epidemiology of EAEC and ETEC cases in Minnesota shows that these pathotypes are a cause of domestic as well as travel-associated illness . This can be further assessed by linking cases through epidemiological surveillance as well as sequencing data .

Presenter : Emily Verbrugge , Emily . Verbrugge . Contractor @ state . mn . us

A Look at the Genotype Prevalence of Cryptosporidium in Texas

D . Gehlbach , R . Tuladhar , C . Wang , C . Schroeder , Texas Department of State Health Services

Introduction : There are an estimated 750,000 Cryptosporidiosis cases each year in the United States , with Texas having an annual average of 597 reported cases from 2011 to 2020 . Cryptosporidium is distributed worldwide and transmits from contaminated water and food or from infected people and / or animals . Symptoms include prolonged diarrhea that can result in severe dehydration . The disease may be fatal if left untreated , with immunocompromised individuals being at the most risk . There is indication that particular species and subtypes result in differing symptoms and severity ; therefore , it is important to speciate and genotype Cryptosporidium . Sanger sequencing analyses of gp60 and 18S rRNA are widely used in Cryptosporidium subtyping . Here we review the incidence of Cryptosporidium genotype prevalence identified by the Texas Department of State Health Services ( TX DSHS ).

Results : From May 2023 to December 2023 , the TX DSHS had 50 requests for Cryptosporidium analysis from stool specimens , resulting in an 86 % positivity rate . Through our validated modified CDC procedure using nested polymerase chain reaction ( PCR ) detection and Sanger sequencing for subtyping Cryptosporidium , we found C . parvum ( 38 %), C . hominis ( 28 %), C . felis ( 8 %), C . meleagridis ( 6 %), C . canis ( 4 %) and C . skunk ( 2 %). For specimens identified as C . parvum , we found 10 specific subtypes . Likewise , there were four subtypes identified for C . hominis . The microscopy result for Cryptosporidium was compared with the PCR assay . Here , we found an 82 % correlation , 16 % were negative via microscopy but PCR positive and 2 % microscopy positive but PCR negative . We found Texas Public Health Regions 3 , 8 and 6 had the highest rates of Cryptosporidium infections ( 23.8 %, 15.9 % and 11.1 %, respectively ).

Conclusion : The TX DSHS analyzed genotyping prevalence data from submitted stool samples for Cryptosporidium testing . In particular , we found C . parvum subtype IIaA17G2R1 as the most predominate species and subtype , respectively , in Texas . For C . hominis subtype IfA12G1R5 was the most predominant in Texas . While there was a strong correlation between microscopy and PCR for positivity , the use of a molecular assay demonstrated a higher sensitivity . Understanding the parasite ’ s distribution in Texas is critical with the development of Texas public health prevention strategies .

Presenter : Deanne Gehlbach , deanne . gehlbach @ dshs . texas . gov