Epidemiological investigations of the foodborne parasitic illness cyclosporiasis can be aided by molecular techniques that enable the identification of genetically related clusters of Cyclospora isolates. At the Centers for Disease Control and Prevention (CDC), routine Cyclospora genotyping for the purpose of informing epidemiological outbreak investigations has occurred since 2018 using clinical stool specimens from case patients diagnosed with cyclosporiasis. This approach involves targeted amplicon deep sequencing of eight genotyping markers, followed by bioinformatic processing through a custom clustering algorithm. However, not all stool specimens submitted to the CDC for genotyping successfully amplify for at least five of the eight genotyping markers, the minimum required to be bioinformatically processed through the clustering algorithm. In this study, we utilized information from clinical stool specimens sent to the CDC from the years 2019 to 2023 to assess if the type of preservative, the age of the specimen, or the method used to diagnose the patient influenced the probability of successfully genotyping parasites from a fecal specimen. Additionally, we assessed the analytical specificity of the Cyclospora genotyping workflow by analyzing samples positive for other intestinal parasites, including closely related non-human infecting Cyclospora species and other coccidia. We found that stool specimens stored in preservatives had a greater likelihood of sequencing success over time relative to specimens without preservatives or those stored in non-nutritive transport media. Additionally, stool specimens from case patients diagnosed via microscopy-based methods were more likely to yield DNA of sufficient quality and quantity for genotyping compared to PCR or multiplex panels. Lastly, we determined that the genotyping workflow has an analytical specificity of 100%, as no non-human-infecting Cyclospora or other parasites yielded sequence data at >1 of the genotyping markers. This knowledge will help strengthen the quality of Cyclospora genotyping data produced in the future, improving the utility of this data for supporting epidemiological investigations.IMPORTANCEDetermining the genetic relatedness among parasites causing foodborne illness, such as Cyclospora, is a valuable tool to complement outbreak investigations. However, this molecular genotyping approach is limited by the quality and quantity of genetic data obtained from the samples being investigated. In this study, we demonstrate that the storage conditions of clinical stool specimens are correlated to the quality of sequence data produced for Cyclospora genotyping. Our insights can be used to guide storage recommendations for stool specimens, which can improve the quality of foodborne illness outbreak investigations conducted in the future. Additionally, we showed that the current Cyclospora genotyping tool used by the Centers for Disease Control (CDC) is highly specific to human-infecting Cyclospora parasites; this valuable information indicates that the CDC's Cyclospora investigations are not negatively impacted by false-positive detections.

Cyclospora cayetanensis is a foodborne parasite that causes cyclosporiasis, an enteric illness in humans. Genotyping methods are used to genetically discriminate between specimens from cyclosporiasis cases and can complement source attribution investigations if the method is sufficiently sensitive for application to food items. A very sensitive targeted amplicon sequencing (TAS) assay for genotyping C. cayetanensis encompassing 52 loci was recently designed. In this study, we analyzed 66 genetically diverse clinical specimens to assess the change in phylogenetic resolution between the TAS assay and a currently employed eight-marker scheme. Of the 52 markers, ≥50 were successfully haplotyped for all specimens, and these results were used to generate a hierarchical cluster dendrogram. Using a previously described statistical approach to dissect hierarchical trees, the 66 specimens resolved into 24 and 27 distinct genetic clusters for the TAS and an 8-loci scheme, respectively. Although the specimen composition of 15 clusters was identical, there were substantial differences between the two dendrograms, highlighting the importance of both inclusion of additional genome coverage and choice of loci to target for genotyping. To evaluate the ability to genetically link contaminated food samples with clinical specimens, C. cayetanensis was genotyped from DNA extracted from raspberries inoculated with fecal specimens. The contaminated raspberry samples were assigned to clusters with the corresponding clinical specimen, demonstrating the utility of the TAS assay for traceback efforts.

Human-infecting Cyclospora was recently characterized as three species, two of which (C. cayetanensis and C. ashfordi) are currently responsible for all known human infections in the USA, yet much remains unknown about the genetic structure within these two species. Here, we investigate Cyclospora genotyping data from 2018 through 2022 to ascertain if there are temporal patterns in the genetic structure of Cyclospora parasites that cause infections in US residents from year to year. First, we investigate three levels of genetic characterization: species, subpopulation, and strain, to elucidate annual trends in Cyclospora infections. Next, we determine if shifts in genetic diversity can be linked to any of the eight loci used in our Cyclospora genotyping approach. We observed fluctuations in the abundance of Cyclospora types at the species and subpopulation levels, but no significant temporal trends were identified; however, we found recurrent and sporadic strains within both C. ashfordi and C. cayetanensis. We also uncovered major shifts in the mitochondrial genotypes in both species, where there was a universal increase in abundance of a specific mitochondrial genotype that was relatively abundant in 2018 but reached near fixation (was observed in over 96% of isolates) in C. ashfordi by 2022. Similarly, this allele jumped from 29% to 82% relative abundance of isolates belonging to C. cayetanensis. Overall, our analysis uncovers previously unknown temporal-genetic patterns in US Cyclospora types from 2018 through 2022 and is an important step to presenting a clearer picture of the factors influencing cyclosporiasis outbreaks in the USA. © 2023

It is becoming increasingly likely that rodents will drive future disease epidemics with the continued expansion of cities worldwide. Though transmission risk is a growing concern, relatively little is known about pathogens carried by urban rats. Here, we assess whether the diversity and prevalence of Bartonella bacteria differ according to the (co)occurrence of rat hosts across New Orleans, LA (NO), where both Norway (Rattus norvegicus) and roof rats (Rattus rattus) are found, relative to New York City (NYC) which only harbors Norway rats. We detected human pathogenic Bartonella species in both NYC and New Orleans rodents. We found that Norway rats in New Orleans harbored a more diverse assemblage of Bartonella than Norway rats in NYC and that Norway rats harbored a more diverse and distinct assemblage of Bartonella compared to roof rats in New Orleans. Additionally, Norway rats were more likely to be infected with Bartonella than roof rats in New Orleans. Flea infestation appears to be an important predictor of Bartonella infection in Norway rats across both cities. These findings illustrate that pathogen infections can be heterogeneous in urban rodents and indicate that further study of host species interactions could clarify variation in spillover risk across cities.