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.

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.

A CRISPR-enhanced metagenomic NGS test to improve pandemic preparedness.

The lack of preparedness for detecting and responding to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogen (i.e., COVID-19) has caused enormous harm to public health and the economy. Testing strategies deployed on a population scale at day zero, i.e., the time of the first reported case, would be of significant value. Next-generation sequencing (NGS) has such capabilities; however, it has limited detection sensitivity for low-copy-number pathogens. Here, we leverage the CRISPR-Cas9 system to effectively remove abundant sequences not contributing to pathogen detection and show that NGS detection sensitivity of SARS-CoV-2 approaches that of RT-qPCR. The resulting sequence data can also be used for variant strain typing, co-infection detection, and individual human host response assessment, all in a single molecular and analysis workflow. This NGS work flow is pathogen agnostic and, therefore, has the potential to transform how large-scale pandemic response and focused clinical infectious disease testing are pursued in the future.

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.

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.

Genomic Epidemiology Linking Nonendemic Coccidioidomycosis to Travel.

Coccidioidomycosis is a fungal infection endemic to hot, arid regions of the western United States, northern Mexico, and parts of Central and South America. Sporadic cases outside these regions are likely travel-associated; alternatively, an infection could be acquired in as-yet unidentified newly endemic locales. A previous study of cases in nonendemic regions with patient self-reported travel history suggested that infections were acquired during travel to endemic regions. We sequenced 19 Coccidioides isolates from patients with known travel histories from that earlier investigation and performed phylogenetic analysis to identify the locations of potential source populations. Our results show that those isolates were phylogenetically linked to Coccidioides subpopulations naturally occurring in 1 of the reported travel locales, confirming that these cases were likely acquired during travel to endemic regions. Our findings demonstrate that genomic analysis is a useful tool for investigating travel-related coccidioidomycosis.

One health genomic surveillance and response to a university-based outbreak of the SARS-CoV-2 Delta AY.25 lineage, Arizona, 2021.

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.

Phylogenomic Placement of American Southwest-Associated Clinical and Veterinary Isolates Expands Evidence for Distinct Cryptococcus gattii VGVI.

Whole-genome sequencing has advanced our understanding of the population structure of the pathogenic species complex Cryptococcus gattii, which has allowed for the phylogenomic specification of previously described major molecular type groupings and novel lineages. Recently, isolates collected in Mexico in the 1960s were determined to be genetically distant from other known molecular types and were classified as VGVI. We sequenced four clinical isolates and one veterinary isolate collected in the southwestern United States and Argentina from 2012 to 2021. Phylogenomic analysis groups these genomes with those of the Mexican VGVI isolates, expanding VGVI into a clade and establishing this molecular type as a clinically important population. These findings also potentially expand the known Cryptococcus ecological range with a previously unrecognized endemic area.

Molecular type distribution and fluconazole susceptibility of clinical Cryptococcus gattii isolates from South African laboratory-based surveillance, 2005-2013.

As is the case globally, Cryptococcus gattii is a less frequent cause of cryptococcosis than Cryptococcus neoformans in South Africa. We performed multilocus sequence typing (MLST) and fluconazole susceptibility testing of 146 isolates randomly selected from 750 South African patients with C. gattii disease identified through enhanced laboratory surveillance, 2005 to 2013. The dominant molecular type was VGIV (101/146, 70%), followed by VGI (40/146, 27%), VGII (3/146, 2%) and VGIII (2/146, 1%). Among the 146 C. gattii isolates, 99 different sequence types (STs) were identified, with ST294 (14/146, 10%) and ST155 (10/146, 7%) being most commonly observed. The fluconazole MIC50 and MIC90 values of 105 (of 146) randomly selected C. gattii isolates were 4 µg/ml and 16 µg/ml, respectively. VGIV isolates had a lower MIC50 value compared to non-VGIV isolates, but these values were within one double-dilution of each other. HIV-seropositive patients had a ten-fold increased adjusted odds of a VGIV infection compared to HIV-seronegative patients, though with small numbers (99/136; 73% vs. 2/10; 20%), the confidence interval (CI) was wide (95% CI: 1.93-55.31, p = 0.006). Whole genome phylogeny of 98 isolates of South Africa's most prevalent molecular type, VGIV, identified that this molecular type is highly diverse, with two interesting clusters of ten and six closely related isolates being identified, respectively. One of these clusters consisted only of patients from the Mpumalanga Province in South Africa, suggesting a similar environmental source. This study contributed new insights into the global population structure of this important human pathogen.

Genotypic, proteomic, and phenotypic approaches to decipher the response to caspofungin and calcineurin inhibitors in clinical isolates of echinocandin-resistant Candida glabrata.

BACKGROUND: Echinocandin resistance represents a great concern, as these drugs are recommended as first-line therapy for invasive candidiasis. Echinocandin resistance is conferred by mutations in FKS genes. Nevertheless, pathways are crucial for enabling tolerance, evolution, and maintenance of resistance. Therefore, understanding the biological processes and proteins involved in the response to caspofungin may provide clues indicating new therapeutic targets. OBJECTIVES: We determined the resistance mechanism and assessed the proteome response to caspofungin exposure. We then evaluated the phenotypic impact of calcineurin inhibition by FK506 and cephalosporine A (CsA) on caspofungin-resistant Candida glabrata isolates. METHODS: Twenty-five genes associated with caspofungin resistance were analysed by NGS, followed by studies of the quantitative proteomic response to caspofungin exposure. Then, susceptibility testing of caspofungin in presence of FK506 and CsA was performed. The effects of calcineurin inhibitor/caspofungin combinations on heat stress (40°C), oxidative stress (0.2 and 0.4 mM menadione) and on biofilm formation (polyurethane catheter) were analysed. Finally, a Galleria mellonella model using blastospores (1 × 109 cfu/mL) was developed to evaluate the impact of the combinations on larval survival. RESULTS: F659-del was found in the FKS2 gene of resistant strains. Proteomics data showed some up-regulated proteins are involved in cell-wall biosynthesis, response to stress and pathogenesis, some of them being members of calmodulin-calcineurin pathway. Therefore, the impact of calmodulin inhibition was explored. Calmodulin inhibition restored caspofungin susceptibility, decreased capacity to respond to stress conditions, and reduced biofilm formation and in vivo pathogenicity. CONCLUSIONS: Our findings confirm that calmodulin-calcineurin-Crz1 could provide a relevant target in life-threatening invasive candidiasis.

Normalization of SARS-CoV-2 viral load via RT-qPCR provides higher-resolution data for comparison across time and between patients.

The 2020 pandemic has transformed the world and elicited thousands of studies to better understand the SARS-CoV-2 virus. Viral load has been a common measure to monitor treatment therapies and associate viral dynamics with patient outcomes; however, methods associated with viral load have varied across studies. These variations have the potential to sacrifice the accuracy of findings as they often do not account for inter-assay variation or variation across samples. In a retrospective study of nasopharyngeal samples, we found a significant amount of variation within the DNA and RNA targets; for example, across time within a single patient, there was an average of a 32-fold change. Further, we explore the impacts of host normalization on 94 clinical samples using the TGen Quantitative SARS-CoV-2 assay, finding that without host normalization samples with the same viral concentration can have up to 100-fold variation in the viral load.

Genomic investigation of a household SARS-CoV-2 disease cluster in Arizona involving a cat, dog, and pet owner.

Arizona's COVID-19 and Pets Program is a prospective surveillance study being conducted to characterize how SARS-CoV-2 impacts companion animals living in households with SARS-CoV-2-positive individuals. Among the enrolled pets, we identified a SARS-CoV-2-infected cat and dog from the same household; both animals were asymptomatic but had close contact with the symptomatic and SARS-CoV-2-positive owner. Whole genome sequencing of animal and owner specimens revealed identical viral genomes of the B.1.575 lineage, suggesting zoonotic transmission of SARS-CoV-2 from human to at least one pet. This is the first report of the B.1.575 lineage in companion animals. Genetically linking SARS-CoV-2 between people and animals, and tracking changes in SARS-CoV-2 genomes is essential to detect any cross-species SARS-CoV-2 transmission that may lead to more transmissible or severe variants that can affect humans. Surveillance studies, including genomic analyses of owner and pet specimens, are needed to further our understanding of how SARS-CoV-2 impacts companion animals.

SaQuant: a real-time PCR assay for quantitative assessment of Staphylococcus aureus.

BACKGROUND: Molecular assays are important tools for pathogen detection but need to be periodically re-evaluated with the discovery of additional genetic diversity that may cause assays to exclude target taxa or include non-target taxa. A single well-developed assay can find broad application across research, clinical, and industrial settings. Pathogen prevalence within a population is estimated using such assays and accurate results are critical for formulating effective public health policies and guiding future research. A variety of assays for the detection of Staphylococcus aureus are currently available. The utility of commercial assays for research is limited, given proprietary signatures and lack of transparent validation. RESULTS: In silico testing of existing peer-reviewed assays show that most suffer from a lack of sensitivity and specificity. We found no assays that were specifically designed and validated for quantitative use. Here we present a qPCR assay, SaQuant, for the detection and quantification of S. aureus as might be collected on sampling swabs. Sensitivity and specificity of the assay was 95.6 and 99.9 %, respectively, with a limit of detection of between 3 and 5 genome equivalents and a limit of quantification of 8.27 genome equivalents. The presence of DNA from non-target species likely to be found in a swab sample, did not impact qualitative or quantitative abilities of the assay. CONCLUSIONS: This assay has the potential to serve as a valuable tool for the accurate detection and quantification of S. aureus collected from human body sites in order to better understand the dynamics of prevalence and transmission in community settings.

Applying Genomic Epidemiology to Characterize a COVID-19 Outbreak in a Developmentally Disabled Adult Group Home Setting, Arizona.

Individuals living in congregate settings, including those in group homes, have been disproportionately impacted by COVID-19 and may be at increased risk of exposure or infection due to underlying illness. In mid-May 2020, local public health officials responded to an outbreak of COVID-19 among staff and residents associated with a multi-residential group home that provides care for adults with intellectual and developmental disabilities. Samples were collected at 16 of the homes. In four of the homes all the residents tested positive, and in the remaining 12 houses where samples were collected, all residents tested negative. Of the 152 individuals tested, 15/58 (25.9%) residents and 27/94 (28.7%) staff were positive for SARS-CoV-2, including eight hospitalizations and four deaths. Phylogenetic analysis of genomes from this outbreak in the context of genomes from Northern Arizona shows that very few mutations separate the samples from this outbreak. A potential transmission network was developed to illustrate person-place epidemiologic linkages and further demonstrates the dynamic connections between staff and residents with respect to each group home location. Epidemiologic and genomic evidence correlate, and suggest that asymptomatic infected staff likely introduced and spread COVID-19 in this setting. Implementation of public health prevention measures alongside rapid genomic analysis can help guide policy development and guide management efforts to prevent and mitigate future outbreaks.

Genotype, Antifungal Susceptibility, and Virulence of Clinical South African Cryptococcus neoformans Strains from National Surveillance, 2005-2009.

In South Africa, Cryptococcus neoformans is the most common cause of adult meningitis. We performed multi locus sequence typing and fluconazole susceptibility testing of clinical C. neoformans isolates collected from 251 South African patients with cryptococcosis through national surveillance from 2005 to 2009. We examined the association between clinical characteristics of patients and genotype, and the effect of genotype on in-hospital mortality. We performed whole genome phylogenetic analysis of fifteen C. neoformans isolates with the molecular type VNB and tested their virulence in a Galleria mellonella model. Most isolates had the molecular type VNI (206/251, 82%), followed by VNII (25/251, 10%), VNB (15/251, 6%), and VNIV (5/251, 2%); 67 sequence types were identified. There were no differences in fluconazole minimum inhibitory concentration (MIC) values among molecular types and the majority of strains had low MIC values (MIC50 of 1 µg/mL and MIC90 of 4 µg/mL). Males were almost twice as likely of being infected with a non-VNI genotype (adjusted odds ratio [OR]: 1.65, 95% confidence interval [CI]: 0.25-10.99; p = 0.61). Compared to patients infected with a VNI genotype, those with a non-VNI genotype had a 50% reduced adjusted odds of dying in hospital (95% CI: 0.03-7.57; p = 0.62). However, for both these analyses, our estimates had wide confidence intervals spanning 1 with large p-values. Fifteen VNB strains were not as virulent in a G. mellonella larval model as the H99 reference strain. A majority of these VNB strains belonged to the VNBII clade and were very closely related by phylogenetic analysis.

Methods for sequencing the pandemic: benefits of rapid or high-throughput processing.

Genomic epidemiology has proven successful for real-time and retrospective monitoring of small and large-scale outbreaks. Here, we report two genomic sequencing and analysis strategies for rapid-turnaround or high-throughput processing of metagenomic samples. The rapid-turnaround method was designed to provide a quick phylogenetic snapshot of samples at the heart of active outbreaks, and has a total turnaround time of <48 hours from raw sample to analyzed data. The high-throughput method was designed for semi-retrospective data analysis, and is both cost effective and highly scalable. Though these methods were developed and utilized for the SARS-CoV-2 pandemic response in Arizona, U.S, and we envision their use for infectious disease epidemiology in the 21 st Century.

Novel Odoribacter splanchnicus Strain and Its Outer Membrane Vesicles Exert Immunoregulatory Effects in vitro.

Odoribacter splanchnicus, belonging to the order Bacteroidales, is a common, short-chain fatty acid producing member of the human intestinal microbiota. A decreased abundance of Odoribacter has been linked to different microbiota-associated diseases, such as non-alcoholic fatty liver disease, cystic fibrosis and inflammatory bowel disease (IBD). The type strain of O. splanchnicus has been genome-sequenced, but otherwise very little is known about this anaerobic bacterium. The species surfaces in many microbiota studies and, consequently, comprehension on its interactions with the host is needed. In this study, we isolated a novel strain of O. splanchnicus from a healthy fecal donor, identified it by genome sequencing and addressed its adhesive, epithelium reinforcing and immunoregulatory properties. Our results show that O. splanchnicus strain 57 is non-adherent to enterocytes or mucus, does not reinforce nor compromise Caco-2 monolayer integrity and most likely harbors penta-acylated, less endotoxic lipid A as part of its lipopolysaccharide (LPS) structure based on the lack of gene lpxM and in vitro results on low-level NF-κB activity. The studies by transmission electron microscopy revealed that O. splanchnicus produces outer membrane vesicles (OMV). O. splanchnicus cells, culture supernatant i.e., spent medium or OMVs did not induce interleukin-8 (IL-8) response in HT-29 enterocyte cells suggesting a very low proinflammatory capacity. On the contrary, the treatment of HT-29 cells with O. splanchnicus cells, spent medium or OMVs prior to exposure to Escherichia coli LPS elicited a significant decrease in IL-8 production as compared to E. coli LPS treatment alone. Moreover, O. splanchnicus spent supernatant induced IL-10 production by immune cells, suggesting anti-inflammatory activity. Our in vitro findings indicate that O. splanchnicus and its effector molecules transported in OMVs could potentially exert anti-inflammatory action in the gut epithelium. Taken together, O. splanchnicus seems to be a commensal with a primarily beneficial interaction with the host.

Coyotes as Reservoirs for Onchocerca lupi, United States, 2015-2018.

The Onchocerca lupi nematode infects dogs, cats, and humans, but whether it can be spread by coyotes has been unknown. We conducted surveillance for O. lupi nematode infection in coyotes in the southwestern United States. We identified multiple coyote populations in Arizona and New Mexico as probable reservoirs for this species.

Bacterial Genome Wide Association Studies (bGWAS) and Transcriptomics Identifies Cryptic Antimicrobial Resistance Mechanisms in Acinetobacter baumannii.

Antimicrobial resistance (AMR) in the nosocomial pathogen, Acinetobacter baumannii, is becoming a serious public health threat. While some mechanisms of AMR have been reported, understanding novel mechanisms of resistance is critical for identifying emerging resistance. One of the first steps in identifying novel AMR mechanisms is performing genotype/phenotype association studies; however, performing these studies is complicated by the plastic nature of the A. baumannii pan-genome. In this study, we compared the antibiograms of 12 antimicrobials associated with multiple drug families for 84 A. baumannii isolates, many isolated in Arizona, USA. in silico screening of these genomes for known AMR mechanisms failed to identify clear correlations for most drugs. We then performed a bacterial genome wide association study (bGWAS) looking for associations between all possible 21-mers; this approach generally failed to identify mechanisms that explained the resistance phenotype. In order to decrease the genomic noise associated with population stratification, we compared four phylogenetically-related pairs of isolates with differing susceptibility profiles. RNA-Sequencing (RNA-Seq) was performed on paired isolates and differentially-expressed genes were identified. In these isolate pairs, five different potential mechanisms were identified, highlighting the difficulty of broad AMR surveillance in this species. To verify and validate differential expression, amplicon sequencing was performed. These results suggest that a diagnostic platform based on gene expression rather than genomics alone may be beneficial in certain surveillance efforts. The implementation of such advanced diagnostics coupled with increased AMR surveillance will potentially improve A. baumannii infection treatment and patient outcomes.