A framework for integrating wastewater-based epidemiology and public health.

Wastewater-based epidemiology (WBE) is an environmental approach to monitor community health through the analysis of sewage. The COVID-19 pandemic catalyzed scientists and public health professionals to revisit WBE as a tool to optimize resource allocation to mitigate disease spread and prevent outbreaks. Some studies have highlighted the value of WBE programs that coordinate with public health professionals; however, the details necessary for implementation are not well-characterized. To respond to this knowledge gap, this article documents the framework of a successful WBE program in Arizona, titled Wastewater Analysis for Tactical Epidemiological Response Systems (WATERS), detailing the developed structure and methods of communication that enabled public health preparedness and response actions. This communication illustrates how program operations were employed to reduce outbreak severity. The structure outlined here is customizable and may guide other programs in the implementation of WBE as a public health tool.

In vitro small molecule screening to inform novel candidates for use in fluconazole combination therapy in vivo against Coccidioides.

Identifying improved treatments for severe and refractory coccidioidomycosis (Valley fever) is needed. This endemic fungal disease is common in North and South America, and cases have increased substantially over the last 30 years. The current standard of care, oral daily fluconazole, often fails to completely eradicate Coccidioides infection; however, the high cost of identifying new compounds effective in treating Valley fever is a barrier to improving treatment. Therefore, repurposing existing pharmaceutical agents in combination with fluconazole therapy is an attractive option. We screened the Library of Pharmacologically Active Compounds (LOPAC) small molecule library for compounds that inhibited fungal growth in vitro and determined IC50 values for a subset of compounds. Based on these findings, we tested a small subset of these agents to validate the screen, as well as to test the performance of fluconazole in a combination therapy approach, as compared with fluconazole alone, in a murine model. We observed that combination therapy of tamoxifen:fluconazole and sertraline:fluconazole significantly reduced the burden of live fungus in the lung compared with fluconazole alone, and we observed reduced or nonexistent dissemination. These results suggest that tamoxifen and sertraline may be repurposed as adjunctive agents in the treatment of this important fungal disease. IMPORTANCE: Developing new drugs, especially for regional orphan diseases, such as Valley Fever, is a slow and costly endeavor. However, there is a wealth of FDA-approved drugs available for repurposing, offering a more economical and expedited approach to improve treatment. Those existing compounds with antifungal properties can become novel therapies with relative ease: a considerable advantage for patients in need of alternative treatment. Despite the scope of remaining tasks, our comprehensive screening of potential candidates has revealed promising combinations for further exploration. This effort outlines a practical pipeline for Valley fever drug screening and identifies viable drug combinations that could impact patients more rapidly than single drug development pathways.

Sequencing by binding rivals SMOR error-corrected sequencing by synthesis technology for accurate detection and quantification of minor (< 0.1%) subpopulation variants.

BACKGROUND: Detecting very minor (< 1%) subpopulations using next-generation sequencing is a critical need for multiple applications, including the detection of drug resistant pathogens and somatic variant detection in oncology. A recently available sequencing approach termed 'sequencing by binding (SBB)' claims to have higher base calling accuracy data "out of the box." This paper evaluates the utility of using SBB for the detection of ultra-rare drug resistant subpopulations in Mycobacterium tuberculosis (Mtb) using a targeted amplicon assay and compares the performance of SBB to single molecule overlapping reads (SMOR) error corrected sequencing by synthesis (SBS) data. RESULTS: SBS displayed an elevated error rate when compared to SMOR error-corrected SBS and SBB techniques. SMOR error-corrected SBS and SBB technologies performed similarly within the linear range studies and error rate studies. CONCLUSIONS: With lower sequencing error rates within SBB sequencing, this technique looks promising for both targeted and unbiased whole genome sequencing, leading to the identification of minor (< 1%) subpopulations without the need for error correction methods.

Investigation of SARS-CoV-2 Infection among Companion Animals in Households with Confirmed Human COVID-19 Cases.

We aimed to characterize SARS-CoV-2 infection in companion animals living in households with COVID-19-positive people and understand the dynamics surrounding how these animals become infected. Public health investigators contacted households with at least one confirmed, symptomatic person with COVID-19 for study recruitment. Blood, nasal, and rectal swab specimens were collected from pet dogs and cats and a questionnaire was completed. Specimens were tested for SARS-CoV-2 by RT-PCR, and for neutralizing antibodies; genomic sequencing was performed on viral-positive samples. A total of 36.4% of 110 pets enrolled had evidence of infection with SARS-CoV-2. Pets were more likely to test positive if the pet was immunocompromised, and if more than one person in the home was positive for COVID-19. Among 12 multi-pet households where at least one pet was positive, 10 had at least one other pet test positive. Whole-genome sequencing revealed the genomes of viral lineages circulating in the community during the time of sample collection. Our findings suggest a high likelihood of viral transmission in households with multiple pets and when pets had very close interactions with symptomatic humans. Further surveillance studies are needed to characterize how new variants impact animals and to understand opportunities for infection and spillover in susceptible species.

The Hidden Epidemic of Isoniazid-Resistant Tuberculosis in South Africa.

BACKGROUND: Isoniazid-resistant tuberculosis (Hr-TB) is often overlooked in diagnostic algorithms due to reliance on first-line molecular assays testing only for rifampicin resistance. OBJECTIVES: To determine the prevalence, outcomes and molecular mechanisms associated with Hr-TB in the Eastern Cape, South Africa. METHODS: Between April 2016 and October 2017, sputum samples were collected from patients with rifampin-susceptible TB at baseline and at weeks 7 and 23 of drug-susceptible TB treatment. We performed isoniazid phenotypic and genotypic drug susceptibility testing, FluorotypeMTBDR, Sanger sequencing, targeted next-generation sequencing (tNGS), and whole genome sequencing. RESULTS: We analysed baseline isolates from 766 patients with rifampin-susceptible TB. Of 89 patients (11.7%) found to have Hr-TB, 39 (44%) had canonical katG or inhA promoter mutations; 35 (39%) had non-canonical katG mutations (including 5 with underlying large deletions); 4 (5%) had mutations in other candidate genes associated with isoniazid resistance. For 11 (12.4%), no cause of resistance was found. CONCLUSIONS: Among patients with rifampin-susceptible TB diagnosed using first-line molecular TB assays, there is a high prevalence of Hr-TB. Phenotypic DST remains the gold standard. To improve performance of genetic-based phenotyping tests, all isoniazid resistance associated regions should be included, and such tests should have the ability to identify underlying mutations.

DETECTION OF SARS-COV-2 IN A SQUIRREL MONKEY (SAIMIRI SCIUREUS): A ONE HEALTH INVESTIGATION AND RESPONSE.

Through collaborative efforts, One Health partners have responded to outbreaks of COVID-19 among animals, including those in human care at zoos. Zoos have been faced with numerous challenges, including the susceptibility of many mammalian species, and therefore the need to heighten biosecurity measures rapidly. Robust One Health collaborations already exist in Arizona to address endemic and emerging zoonoses, but these have rarely included zoos. The pandemic shed light on this, and Arizona subsequently expanded its SARS-CoV-2 surveillance efforts to include zoo animals. Testing and epidemiologic support was provided to expedite the detection of and response to zoonotic SARS-CoV-2 infection in zoo animals, as well as to understand possible transmission events. Resulting from this program, SARS-CoV-2 was detected from a rectal swab collected from an 8-yr-old squirrel monkey (Saimiri sciureus) from a zoo in Southern Arizona. The animal had rapidly become ill with nonrespiratory symptoms and died in July 2022. Genomic sequencing from the swab revealed mutations consistent with the Omicron (BA.2) lineage. An epidemiologic investigation identified an animal caretaker in close proximity to the affected squirrel monkey who tested positive for COVID-19 the same day the squirrel monkey died. Critical One Health partners provided support to the zoo through engagement of local, state, and federal agencies. Necropsy and pathologic evaluation showed significant necrotizing colitis; the overall clinical and histopathological findings did not implicate SARS-CoV-2 infection alone as a causal or contributing factor in the squirrel monkey's illness and death. This report documents the first identification of SARS-CoV-2 in a squirrel monkey and highlights a successful and timely One Health investigation conducted through multisectoral collaboration.

Sequencing by Binding rivals error-corrected Sequencing by Synthesis technology for accurate detection and quantification of minor (<0.1%) subpopulation variants.

Detecting very minor (< 1%) subpopulations using next-generation sequencing is a critical need for multiple applications including detection of drug resistant pathogens and somatic variant detection in oncology. To enable these applications, wet lab enhancements and bioinformatic error correction methods have been developed for 'sequencing by synthesis' technology to reduce its inherent sequencing error rate. A recently available sequencing approach termed 'sequencing by binding' claims to have higher base calling accuracy data "out of the box." This paper evaluates the utility of using 'sequencing by binding' for the detection of ultra-rare subpopulations down to 0.001%.

Drug resistance and epidemiological success of modern Mycobacterium tuberculosis lineages in western India.

BACKGROUND: Transmission is contributing to the slow decline of tuberculosis (TB) incidence globally. Drivers of TB transmission in India, the country estimated to carry a quarter of the World's burden, are not well studied. We conducted a genomic epidemiology study to compare epidemiological success, host factors and drug resistance (DR) among the four major Mycobacterium tuberculosis (Mtb) lineages (L1-4) circulating in Pune, India. METHODS: We performed whole-genome sequencing (WGS) of Mtb sputum culture-positive isolates from participants in two prospective cohort studies and predicted genotypic susceptibility using a validated random forest model. We used maximum likelihood estimation to build phylogenies. We compared lineage specific phylogenetic and time-scaled metrics to assess epidemiological success. RESULTS: Of the 642 isolates that underwent WGS, 612 met sequence quality criteria. Most isolates belonged to L3 (44.6%). The majority (61.1%) of multidrug-resistant isolates belonged to L2 (P < 0.001). In molecular dating, L2 demonstrated a higher rate and more recent resistance acquisition. We measured higher clustering, and time-scaled haplotypic density (THD) for L4 and L2 compared to L3 and/or L1 suggesting higher epidemiological success. L4 demonstrated higher THD and clustering (OR 5.1 (95% CI 2.3-12.3) in multivariate models controlling for host factors and DR. CONCLUSION: L2 shows a higher frequency of DR and both L2 and L4 demonstrate evidence of higher epidemiological success than L3 or L1 in the study setting. Our findings highlight the need for contact tracing around TB cases, and heightened surveillance of TB DR in India.

Pan-Enterovirus Characterization Reveals Cryptic Circulation of Clinically Relevant Subtypes in Arizona Wastewater.

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.

Large Clusters of Invasive emm49 Group A Streptococcus Identified within and across Arizona Healthcare Facilities through Statewide Genomic Surveillance System, 2019-2021.

A statewide genomic surveillance system for invasive Group A Streptococcus was implemented in Arizona in June 2019, resulting in 1,046 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 four isolates that rapidly expanded over subsequent months (June 2019-February 2020). Public health investigations identified epidemiologic links with three 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.

Understanding the exposure risk of aerosolized Coccidioides in a Valley fever endemic metropolis.

Coccidioides is the fungal causative agent of Valley fever, a primarily pulmonary disease caused by inhalation of fungal arthroconidia, or spores. Although Coccidioides has been an established pathogen for 120 years and is responsible for hundreds of thousands of infections per year, little is known about when and where infectious Coccidioides arthroconidia are present within the ambient air in endemic regions. Long-term air sampling programs provide a means to investigate these characteristics across space and time. Here we present data from > 18 months of collections from 11 air sampling sites across the Phoenix, Arizona, metropolitan area. Overall, prevalence was highly variable across space and time with no obvious spatial or temporal correlations. Several high prevalence periods were identified at select sites, with no obvious spatial or temporal associations. Comparing these data with weather and environmental factor data, wind gusts and temperature were positively associated with Coccidioides detection, while soil moisture was negatively associated with Coccidioides detection. These results provide critical insights into the frequency and distribution of airborne arthroconidia and the associated risk of inhalation and potential disease that is present across space and time in a highly endemic locale.

Lyssa excreta: Defining parameters for fecal samples as a rabies virus surveillance method.

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.

Novel Presentation of Coccidioidomycosis with Myriad Free-Floating Proteinaceous Spheres in the Pericardial Sac of a Southern Sea Otter (Enhydra lutris nereis).

A southern sea otter (Enhydra lutris nereis) stranded dead in central California, USA, with a distended pericardial sac containing thousands of free-floating proteinaceous masses. Serology, fungal culture, PCR, and sequencing confirmed the etiology of this novel lesion as Coccidioides immitis. Range expansion of this zoonotic pathogen is predicted with climate change.

Comparison of carbapenem-susceptible and carbapenem-resistant Enterobacterales at nine sites in the USA, 2013-2016: a resource for antimicrobial resistance investigators.

Carbapenem-resistant Enterobacterales (CRE) are an urgent public health threat. Genomic sequencing is an important tool for investigating CRE. Through the Division of Healthcare Quality Promotion Sentinel Surveillance system, we collected CRE and carbapenem-susceptible Enterobacterales (CSE) from nine clinical laboratories in the USA from 2013 to 2016 and analysed both phenotypic and genomic sequencing data for 680 isolates. We describe the molecular epidemiology and antimicrobial susceptibility testing (AST) data of this collection of isolates. We also performed a phenotype-genotype correlation for the carbapenems and evaluated the presence of virulence genes in Klebsiella pneumoniae complex isolates. These AST and genomic sequencing data can be used to compare and contrast CRE and CSE at these sites and serve as a resource for the antimicrobial resistance research community.

Bedaquiline resistance in patients with drug-resistant tuberculosis in Cape Town, South Africa: a retrospective longitudinal cohort study.

BACKGROUND: Bedaquiline is a life-saving tuberculosis drug undergoing global scale-up. People at risk of weak tuberculosis drug regimens are a priority for novel drug access despite the potential source of Mycobacterium tuberculosis-resistant strains. We aimed to characterise bedaquiline resistance in individuals who had sustained culture positivity during bedaquiline-based treatment. METHODS: We did a retrospective longitudinal cohort study of adults (aged ≥18 years) with culture-positive pulmonary tuberculosis who received at least 4 months of a bedaquiline-containing regimen from 12 drug-resistant tuberculosis treatment facilities in Cape Town, South Africa, between Jan 20, 2016, and Nov 20, 2017. Sputum was programmatically collected at baseline (ie, before bedaquiline initiation) and each month to monitor treatment response per the national algorithm. The last available isolate from the sputum collected at or after 4 months of bedaquiline was designated the follow-up isolate. Phenotypic drug susceptibility testing for bedaquiline was done on baseline and follow-up isolates in MGIT960 media (WHO-recommended critical concentration of 1 µg/mL). Targeted deep sequencing for Rv0678, atpE, and pepQ, as well as whole-genome sequencing were also done. FINDINGS: In total, 40 (31%) of 129 patients from an estimated pool were eligible for this study. Overall, three (8%) of 38 patients assessable by phenotypic drug susceptibility testing for bedaquiline had primary resistance, 18 (47%) gained resistance (acquired or reinfection), and 17 (45%) were susceptible at both baseline and follow-up. Several Rv0678 and pepQ single-nucleotide polymorphisms and indels were associated with resistance. Although variants occurred in Rv0676c and Rv1979c, these variants were not associated with resistance. Targeted deep sequencing detected low-level variants undetected by whole-genome sequencing; however, none were in genes without variants already detected by whole-genome sequencing. Patients with baseline fluoroquinolone resistance, clofazimine exposure, and four or less effective drugs were more likely to have bedaquiline-resistant gain. Resistance gain was primarily due to acquisition; however, some reinfection by resistant strains occurred. INTERPRETATION: Bedaquiline-resistance gain, for which we identified risk factors, was common in these programmatically treated patients with sustained culture positivity. Our study highlights risks associated with implementing life-saving new drugs and shows evidence of bedaquiline-resistance transmission. Routine drug susceptibility testing should urgently accompany scale-up of new drugs; however, rapid drug susceptibility testing for bedaquiline remains challenging given the diversity of variants observed. FUNDING: Doris Duke Charitable Foundation, US National Institute of Allergy and Infectious Diseases, South African Medical Research Council, National Research Foundation, Research Foundation Flanders, Stellenbosch University Faculty of Medicine Health Sciences, South African National Research Foundation, Swiss National Science Foundation, and Wellcome Trust.

"Sniffing" out SARS-CoV-2 in Arizona working dogs: an exploratory serosurvey.

Susceptibility to and infection with SARS-CoV-2 in companion animals has been well-documented throughout the COVID-19 pandemic. Surveillance for the virus in dogs has largely been focused on household pets; however, other canine populations may also be impacted. We partnered with a local veterinary hospital with a high working dog patient volume to conduct viral and neutralizing antibody testing in working dogs and identify potential risk factors in the dog's work and home environments. Surveillance of SARS-CoV-2 in law enforcement and security working dogs in Arizona found 24.81% (32/129) of dogs to be seropositive. Thirteen dogs presenting with clinical signs or with reported exposure to COVID-19 in the 30 days prior to sample collection were also tested by PCR; all samples were negative. 90.7% (n = 117) of dogs were reported to be asymptomatic or have no change in performance at the time of sampling. Two dogs (1.6%) had suspected anosmia as reported by their handlers; one of which was seropositive. Known exposure to the dog's COVID-19 positive handler or household member was identified as a significant risk factor. Demographics factors including sex, altered status, and type of work were not associated with canine seropositivity. Further work is warranted to understand the impact of SARS-CoV-2 and other infectious diseases in working dogs.

Comparing genomic variant identification protocols for Candida auris.

Genomic analyses are widely applied to epidemiological, population genetic and experimental studies of pathogenic fungi. A wide range of methods are employed to carry out these analyses, typically without including controls that gauge the accuracy of variant prediction. The importance of tracking outbreaks at a global scale has raised the urgency of establishing high-accuracy pipelines that generate consistent results between research groups. To evaluate currently employed methods for whole-genome variant detection and elaborate best practices for fungal pathogens, we compared how 14 independent variant calling pipelines performed across 35 Candida auris isolates from 4 distinct clades and evaluated the performance of variant calling, single-nucleotide polymorphism (SNP) counts and phylogenetic inference results. Although these pipelines used different variant callers and filtering criteria, we found high overall agreement of SNPs from each pipeline. This concordance correlated with site quality, as SNPs discovered by a few pipelines tended to show lower mapping quality scores and depth of coverage than those recovered by all pipelines. We observed that the major differences between pipelines were due to variation in read trimming strategies, SNP calling methods and parameters, and downstream filtration criteria. We calculated specificity and sensitivity for each pipeline by aligning three isolates with chromosomal level assemblies and found that the GATK-based pipelines were well balanced between these metrics. Selection of trimming methods had a greater impact on SAMtools-based pipelines than those using GATK. Phylogenetic trees inferred by each pipeline showed high consistency at the clade level, but there was more variability between isolates from a single outbreak, with pipelines that used more stringent cutoffs having lower resolution. This project generated two truth datasets useful for routine benchmarking of C. auris variant calling, a consensus VCF of genotypes discovered by 10 or more pipelines across these 35 diverse isolates and variants for 2 samples identified from whole-genome alignments. This study provides a foundation for evaluating SNP calling pipelines and developing best practices for future fungal genomic studies.

Was Coccidioides a Pre-Columbian Hitchhiker to Southcentral Washington?

Coccidioides immitis, a pathogenic environmental fungus that causes Valley fever (coccidioidomycosis) primarily in the American Southwest and parts of Central and South America, has emerged over the past 12 years in the Columbia River Basin region, near the confluence with the Yakima River, in southcentral Washington state, USA. An initial autochthonous Washington human case was found in 2010, stemming from a wound derived from soil contamination during an all-terrain vehicle crash. Subsequent analysis identified multiple positive soil samples from the park where the crash occurred (near the Columbia River in Kennewick, WA), and from another riverside location several kilometers upstream from the park location. Intensified disease surveillance identified several more cases of coccidioidomycosis in the region that lacked any relevant travel history to known endemic locales. Genomic analysis of both patient and soil isolates from the Washington cases determined that all samples from the region are phylogenetically closely related. Given the genomic and the epidemiological link between case and environment, C. immitis was declared to be a newly endemic fungus in the region, spawning many questions as to the scope of its presence, the causes of its recent emergence, and what it predicts about the changing landscape of this disease. Here, we review this discovery through a paleo-epidemiological lens in the context of what is known about C. immitis biology and pathogenesis and propose a novel hypothesis for the cause of the emergence in southcentral Washington. We also try to place it in the context of our evolving understanding of this regionally specific pathogenic fungus.

Multi-faceted metagenomic analysis of spacecraft associated surfaces reveal planetary protection relevant microbial composition.

The National Aeronautics and Space Administration (NASA) has been monitoring the microbial burden of spacecraft since the 1970's Viking missions. Originally culture-based and then focused 16S sequencing techniques were used, but we have now applied whole metagenomic sequencing to a variety of cleanroom samples at the Jet Propulsion Lab (JPL), including the Spacecraft Assembly Facility (SAF) with the goals of taxonomic identification and for functional assignment. Our samples included facility pre-filters, cleanroom vacuum debris, and surface wipes. The taxonomic composition was carried out by three different analysis tools to contrast marker, k-mer, and true alignment approaches. Hierarchical clustering analysis of the data separated vacuum particles from other SAF DNA samples. Vacuum particle samples were the most diverse while DNA samples from the ISO (International Standards Organization) compliant facilities and the SAF were the least diverse; all three were dominated by Proteobacteria. Wipe samples had higher diversity and were predominated by Actinobacteria, including human commensals Cutibacterium acnes and Corynebacterium spp. Taxa identified by the three methods were not identical, supporting the use of multiple methods for metagenome characterization. Likewise, functional annotation was performed using multiple methods. Vacuum particles and SAF samples contained strong signals of the tricarboxylic acid cycle and of amino acid biosynthesis, suggesting that many of the identified microorganisms have the ability to grow in nutrient-limited environments. In total, 18 samples generated high quality metagenome assembled genomes (MAG), which were dominated by Moraxella osloensis or Malassezia restricta. One M. osloensis MAG was assembled into a single circular scaffold and gene annotated. This study includes a rigorous quantitative determination of microbial loads and a qualitative dissection of microbial composition. Assembly of multiple specimens led to greater confidence for the identification of particular species and their predicted functional roles.

Unique Genomic Epidemiology of COVID-19 in the White Mountain Apache Tribe, April to August 2020, Arizona.

The first case of coronavirus disease 2019 (COVID-19) within the White Mountain Apache Tribe (WMAT) in Arizona was diagnosed almost 1 month after community transmission was recognized in the state. Aggressive contact tracing allowed for robust genomic epidemiology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and subsequent phylogenetic analyses implicated only two virus introductions, which resulted in the spread of two unique viral lineages on the reservation. The phylogenies of these lineages reflect the nature of the introductions, the remoteness of the community, and the extraordinarily high attack rates. The timing and space-limited nature of the outbreaks validate the public health tracing efforts involved, which were illustrated by multiple short transmission chains over a period of several weeks, eventually resulting in extinction of the lineages. Comprehensive sampling and successful infection control efforts are illustrated in both the effective population size analyses and the limited mortality outcomes. The rapid spread and high attack rates of the two lineages may be due to a combination of sociological determinants of the WMAT and a seemingly enhanced transmissibility. The SARS-CoV-2 genomic epidemiology of the WMAT demonstrates a unique local history of the pandemic and highlights the extraordinary and successful efforts of their public health response. IMPORTANCE This article discusses the introduction and spread of two unique viral lineages of SARS-CoV-2 within the White Mountain Apache Tribe in Arizona. Both genomic sequencing and traditional epidemiological strategies (e.g., contract tracing) were used to understand the nature of the spread of both lineages. Beyond providing a robust genomic analysis of the epidemiology of the outbreaks, this work also highlights the successful efforts of the local public health response.