It is not possible to systematically screen the environment for rabies virus (RABV) using current approaches. We sought to determine under what conditions RABV is detectable from feces and other accessible samples from infected wildlife to broaden the number of biological samples that could be used to test for RABV. We employed a recently-developed quantitative RT-PCR assay called the "LN34 panlyssavirus real-time RT-PCR assay", which is highly sensitive and specific for all variants of RABV. We harvested and tested brain tissue, fecal, and/or mouth swab samples from 25 confirmed RABV positive bats of six species. To determine if rabies RNA lasts in feces sufficiently long post-defecation to use it as a surveillance tool, we tested fecal samples from 10 bats at the time of sample collection and after 24 hours of exposure to ambient conditions, with an additional test on six bats out to 72 hours. To assess whether we could pool fecal pellets and still detect a positive, we generated dilutions of known positives at 1:1, 1:10, 1:50, and 1:200. For six individuals for which matched brain, mouth swab, and fecal samples were tested, results were positive for 100%, 67%, and 67%, respectively. For the first time test to 24 hours, 63% of feces that were positive at time 0 were still positive after 24 hours, and 50% of samples at 72 hours were positive across all three replicates. Pooling tests revealed that fecal positives were detected at 1:10 dilution, but not at 1:50 or 1:200. Our preliminary results suggest that fecal samples hold promise for a rapid and non-invasive environmental screening system.

A statewide genomic surveillance system for invasive Group A Streptococcus was implemented in Arizona in June 2019, resulting in 1046 isolates being submitted for genomic analysis to characterize emm types and identify transmission clusters. Eleven of the 32 identified distinct emm types comprised >80% of samples, with 29.7% of all isolates being typed as emm49 (and its genetic derivative emm151). Phylogenetic analysis initially identified an emm49 genomic cluster of 4 isolates that rapidly expanded over subsequent months (June 2019 to February 2020). Public health investigations identified epidemiologic links with 3 different long-term care facilities, resulting in specific interventions. Unbiased genomic surveillance allowed for identification and response to clusters that would have otherwise remained undetected.

Background:Most seasonally circulating enteroviruses result in asymptomatic or mildly symptomatic infections. In rare cases, however, infection with some subtypes can result in paralysis or death. Of the 300 subtypes known, only poliovirus is reportable, limiting our understanding of the distribution of other enteroviruses that can cause clinical disease.Objective:The overarching objectives of this study were to: 1) describe the distribution of enteroviruses in Arizona during the late summer and fall of 2022, the time of year when they are thought to be most abundant, and 2) demonstrate the utility of viral pan-assay approaches for semi-agnostic discovery that can be followed up by more targeted assays and phylogenomics.Methods:This study utilizes pooled nasal samples collected from school-aged children and long-term care facility residents, and wastewater from multiple locations in Arizona during July-October of 2022. We used PCR to amplify and sequence a region common to all enteroviruses, followed by species-level bioinformatic characterization using the QIIME 2 platform. For Enterovirus-D68 (EV-D68), detection was carried out using RT-qPCR, followed by confirmation using near-complete whole EV-D68 genome sequencing using a newly designed tiled amplicon approach.Results:In the late summer and early fall of 2022, multiple enterovirus species were identified in Arizona wastewater, with Coxsackievirus A6, EV-D68, and Coxsackievirus A19 composing 86% of the characterized reads sequenced. While EV-D68 was not identified in pooled human nasal samples, and the only reported acute flaccid myelitis case in Arizona did not test positive for the virus, an in-depth analysis of EV-D68 in wastewater revealed that the virus was circulating from August through mid-October. A phylogenetic analysis on this relatively limited dataset revealed just a few importations into the state, with a single clade indicating local circulation.Significance:This study further supports the utility of wastewater-based epidemiology to identify potential public health threats. Our further investigations into EV-D68 shows how these data might help inform healthcare diagnoses for children presenting with concerning neurological symptoms.

Genomic surveillance and wastewater tracking strategies were used to strengthen the public health response to an outbreak of the SARS-CoV-2 Delta AY.25 lineage associated with a university campus in Arizona. Epidemiologic and clinical data routinely gathered through contact tracing were matched to SARS-CoV-2 genomes belonging to an outbreak of AY.25 identified through ongoing phylogenomic analyses. Continued phylogenetic analyses were conducted to further describe the AY.25 outbreak. Wastewater collected twice weekly from sites across campus was tested for SARS-CoV-2 by RT-qPCR, and subsequently sequenced to identify variants. The AY.25 outbreak was defined by a single mutation (C18804T) and comprised 379 genomes from SARS-CoV-2 positive cases associated with the university and community. Several undergraduate student gatherings and congregate living settings on campus likely contributed to the rapid spread of COVID-19 across the university with secondary transmission into the community. The clade defining mutation was also found in wastewater samples collected from around student dormitories a week before the semester began, and 9 days before cases were identified. Genomic, epidemiologic, and wastewater surveillance provided evidence that an AY.25 clone was likely imported into the university setting just prior to the onset of the Fall 2021 semester, rapidly spread through a subset of the student population, and then subsequent spillover occurred in the surrounding community. The university and local public health department worked closely together to facilitate timely reporting of cases, identification of close contacts, and other necessary response and mitigation strategies. The emergence of new SARS-CoV-2 variants and potential threat of other infectious disease outbreaks on university campuses presents an opportunity for future comprehensive One Health genomic data driven, targeted interventions.

Since the reemergence of St. Louis Encephalitis (SLE) Virus (SLEV) in the Southwest United States, identified during the 2015 outbreak in Arizona, SLEV has been seasonally detected within Culex spp. populations throughout the Southwest United States. Previous work revealed the 2015 outbreak was caused by an importation of SLEV genotype III, which had only been detected previously in Argentina. However, little is known about when the importation occurred or the transmission and genetic dynamics since its arrival into the Southwest. In this study, we sought to determine whether the annual detection of SLEV in the Southwest is due to enzootic cycling or new importations. To address this question, we analyzed 174 SLEV genomes (142 sequenced as part of this study) using Bayesian phylogenetic analyses to estimate the date of arrival into the American Southwest and characterize the underlying population structure of SLEV. Phylogenetic clustering showed that SLEV variants circulating in Maricopa and Riverside counties form two distinct populations with little evidence of inter-county transmission since the onset of the outbreak. Alternatively, it appears that in 2019, Yuma and Clark counties experienced annual importations of SLEV that originated in Riverside and Maricopa counties. Finally, the earliest representatives of SLEV genotype III in the Southwest form a polytomy that includes both California and Arizona samples. We propose that the initial outbreak most likely resulted from the importation of a population of SLEV genotype III variants, perhaps in multiple birds, possibly multiple species, migrating north in 2013, rather than a single variant introduced by one bird.

St. Louis Encephalitis Virus (SLEV) has been seasonally detected within the Culex spp . populations within Maricopa County, Arizona and Coachella Valley, California since an outbreak in Maricopa County in 2015. Previous work revealed that the outbreak was caused by an importation of SLEV genotype III, which had only been detected within Argentina in prior years. However, little is known about when the importation occurred or the population dynamics since its arrival into the southwestern United States. In this study, we wanted to determine if the annual detection of SLEV in Maricopa and Riverside counties is due to enzootic cycling or new importations. To address this question, we analyzed 143 SLEV genomes (138 sequenced as part of this study) using the Bayesian phylogenetic analysis software, BEAST, to estimate the date of arrival into the American Southwest and characterize the underlying population structure of SLEV. Phylogenetic clustering showed that SLEV variants circulating in Arizona and California form two distinct populations with little evidence of transmission among the two populations since the onset of the outbreak. Interestingly, the SLEV variants in Coachella Valley appear to be annually imported from a nearby source, whereas the Arizona population is locally sourced each year. Finally, the earliest representatives of SLEV genotype III in the southwestern US formed a polytomy that includes both California and Arizona samples. We propose that the initial outbreak could have resulted from an introductory population of SLEV, perhaps in one or more bird flocks migrating north in 2013, rather than a single variant introduced by one bird.

Dental caries, also known as cavities or dental decay, effects children at a rate five times higher than asthma in the United States. This disease is highly preventable but causes extreme pain and costs millions of dollars to treat every year. The presence of Streptococcus mutansand Streptococcus sobrinusplays a major role in the development and progression of tooth decay; therefore, it is important to establish colonization of these bacteria to assess the risk of developing the disease. Streptococcus bacteria are difficult to grow, extract, and sequence due to strict necessary growth conditions. In this study, we evaluated the performance of different storage and culturing protocols and developed strategies for reducing interference by unwanted bacterial and fungal genomes when sequencing extracted samples. We compared storage conditions of samples at various temperatures, and with and without glycerol. We decided the best storage method was at −80 °C in a specialized solution known as Aimes. When sequencing cultures, we encountered various unwanted bacterial and fungal genomes. To reduce this, we modified our culturing methods by including growth in anaerobic conditions and using serial isolation streaking. These modifications have limited the growth of aerobic specimens and increased culture purity before extraction. With this study, we will be able to better understand the oral microbiome and aim to identify virulence factors in S. mutans and S. sobrinus that contribute to the high rates of dental caries in children.

Staphylococcus aureus is the leading cause of soft tissue and skin infection, but also causes pneumonia, endocarditis, and bone infection. The same infections can be caused by methicillin resistant S. aureus (MRSA), but are much more difficult to treat because MRSA is highly resistant to antibiotics. In ethnic and socioeconomic minority groups, methicillin resistance in S. aureus is 30 % higher than the general population. As a result, methicillin resistance is likely to have higher prevalence in jails and prisons where the demographic is disproportionately represented by ethnic minorities, individuals with lower average socioeconomic status, and less access to healthcare. Jail inmates are an understudied but very important population because diseases acquired in jails can be easily introduced into the general population and vice versa because cycling into and out of jail is very common. The presence of S. aureus is an important determinant of the development of soft tissue and other infections, making the detection of this bacteria and the identification of antibiotic resistance essential in assessing risk of disease. In this study, we identified S. aureus in samples from a United States county jail through bacterial plating and DNA extraction. Methicillin resistance was identified in S. aureus positive samples through PCR and sequencing. Our analysis has found elevated rates of S. aureus in the jail population being studied. This project will determine if rates of S. aureus and methicillin resistance in S. aureus isolated from this demographic are disproportionate to the general population.

West Nile Virus (WNV) has been detected annually in Maricopa County, Arizona, since 2003. With this in mind, we sought to determine if contemporary strains are endemic to the county or are annually imported. As part of this effort, we developed a new protocol for tiled amplicon sequencing of WNV to efficiently attain greater than 99% coverage of 14 WNV genomes collected directly from positive mosquito pools distributed throughout Maricopa County between 2014 and 2017. Bayesian phylogenetic analyses revealed that contemporary genomes fall within two major lineages; NA/WN02 and SW/WN03. We found that all of the Arizona strains possessed an amino acid substitution known to be under positive selection, which has arisen independently at least four times in Arizona. The SW/WN03 strains exhibited transient behavior, with at least 10 separate introductions into Arizona when considering both historical and contemporary strains. However, NA/WN02 strains are geographically differentiated and appear to be endemic in Arizona, with two clades that have been circulating for four and seven years. This establishment in Maricopa County provides the first evidence of local overwintering by a WNV strain over the course of several years in Arizona. Within a national context, the placement of eleven contemporary Arizona strains in the NA/WN02 lineage indicates while WNV first entered the northeastern United States in 1999, the most ancestral extant strains of WNV are now circulating in the American southwest.