The National Wastewater Surveillance System (NWSS): From inception to widespread coverage, 2020-2022, United States.

Wastewater surveillance is a valuable tool that can be used to track infectious diseases in a community. In September 2020, the Centers for Disease Control and Prevention (CDC) established the National Wastewater Surveillance System (NWSS) to coordinate and build the nation's capacity to detect and quantify concentrations of SARS-CoV-2 RNA in U.S. wastewater. This is the first surveillance summary of NWSS, covering September 1, 2020 to December 31, 2022. Through partnerships with state, tribal, local, and territorial health departments, NWSS became a national surveillance platform that can be readily expanded and adapted to meet changing public health needs. Beginning with 209 sampling sites in September 2020, NWSS rapidly expanded to >1500 sites by December 2022, covering ≈47 % of the U.S. population. As of December 2022, >152,000 unique wastewater samples have been collected by NWSS partners, primarily from wastewater treatment plants (WWTPs). WWTPs participating in NWSS tend to be larger than the average U.S. WWTP and serve more populated communities. In December 2022, ≈8 % of the nearly 16,000 U.S. WWTPs were participating in NWSS. NWSS partners used a variety of methods for sampling and testing wastewater samples; however, progress is being made to standardize these methods. In July 2021, NWSS partners started submitting SARS-CoV-2 genome sequencing data to NWSS. In October 2022, NWSS expanded to monkeypox virus testing, with plans to include additional infectious disease targets in the future. Through the rapid implementation and expansion of NWSS, important lessons have been learned. Wastewater surveillance programs should consider both surge and long-term capacities when developing an implementation plan, and early standardization of sampling and testing methods is important to facilitate data comparisons across sites. NWSS has proven to be a flexible and sustainable surveillance system that will continue to be a useful complement to case-based surveillance for guiding public health action.

Early Detection and Surveillance of the SARS-CoV-2 Variant BA.2.86 - Worldwide, July-October 2023.

Early detection of emerging SARS-CoV-2 variants is critical to guiding rapid risk assessments, providing clear and timely communication messages, and coordinating public health action. CDC identifies and monitors novel SARS-CoV-2 variants through diverse surveillance approaches, including genomic, wastewater, traveler-based, and digital public health surveillance (e.g., global data repositories, news, and social media). The SARS-CoV-2 variant BA.2.86 was first sequenced in Israel and reported on August 13, 2023. The first U.S. COVID-19 case caused by this variant was reported on August 17, 2023, after a patient received testing for SARS-CoV-2 at a health care facility on August 3. In the following month, eight additional U.S. states detected BA.2.86 across various surveillance systems, including specimens from health care settings, wastewater surveillance, and traveler-based genomic surveillance. As of October 23, 2023, sequences have been reported from at least 32 countries. Continued variant tracking and further evidence are needed to evaluate the full public health impact of BA.2.86. Timely genomic sequence submissions to global public databases aided early detection of BA.2.86 despite the decline in the number of specimens being sequenced during the past year. This report describes how multicomponent surveillance and genomic sequencing were used in real time to track the emergence and transmission of the BA.2.86 variant. This surveillance approach provides valuable information regarding implementing and sustaining comprehensive surveillance not only for novel SARS-CoV-2 variants but also for future pathogen threats.

MycoSNP: A Portable Workflow for Performing Whole-Genome Sequencing Analysis of Candida auris.

Candida auris is an urgent public health threat characterized by high drug-resistant rates and rapid spread in healthcare settings worldwide. As part of the C. auris response, molecular surveillance has helped public health officials track the global spread and investigate local outbreaks. Here, we describe whole-genome sequencing analysis methods used for routine C. auris molecular surveillance in the United States; methods include reference selection, reference preparation, quality assessment and control of sequencing reads, read alignment, and single-nucleotide polymorphism calling and filtration. We also describe the newly developed pipeline MycoSNP, a portable workflow for performing whole-genome sequencing analysis of fungal organisms including C. auris.

Rapid Implementation of High-Frequency Wastewater Surveillance of SARS-CoV-2.

There have been over 507 million cases of COVID-19, the disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulting in 6 million deaths globally. Wastewater surveillance has emerged as a valuable tool in understanding SARS-CoV-2 burden in communities. The National Wastewater Surveillance System (NWSS) partnered with the United States Geological Survey (USGS) to implement a high-frequency sampling program. This report describes basic surveillance and sampling statistics as well as a comparison of SARS-CoV-2 trends between high-frequency sampling 3-5 times per week, referred to as USGS samples, and routine sampling 1-2 times per week, referred to as NWSS samples. USGS samples provided a more nuanced impression of the changes in wastewater trends, which could be important in emergency response situations. Despite the rapid implementation time frame, USGS samples had similar data quality and testing turnaround times as NWSS samples. Ensuring there is a reliable sample collection and testing plan before an emergency arises will aid in the rapid implementation of a high-frequency sampling approach. High-frequency sampling requires a constant flow of information and supplies throughout sample collection, testing, analysis, and data sharing. High-frequency sampling may be a useful approach for increased resolution of disease trends in emergency response.

Skin Metagenomic Sequence Analysis of Early Candida auris Outbreaks in U.S. Nursing Homes.

Candida auris is a human fungal pathogen classified as an urgent threat to the delivery of health care due to its extensive antimicrobial resistance and the high mortality rates associated with invasive infections. Global outbreaks have occurred in health care facilities, particularly, long-term care hospitals and nursing homes. Skin is the primary site of colonization for C. auris. To accelerate research studies, we developed microbiome sequencing protocols, including amplicon and metagenomic sequencing, directly from patient samples at health care facilities with ongoing C. auris outbreaks. We characterized the skin mycobiome with a database optimized to classify Candida species and C. auris to the clade level. While Malassezia species were the predominant skin-associated fungi, nursing home residents also harbored Candida species, including C. albicans, and C. parapsilosis. Amplicon sequencing was concordant with culturing studies to identify C. auris-colonized patients and provided further resolution that distinct clades of C. auris are colonizing facilities in New York and Illinois. Shotgun metagenomic sequencing from a clinical sample with a high fungal bioburden generated a skin-associated profile of the C. auris genome. Future larger scale clinical studies are warranted to more systematically investigate the effects of commensal microbes and patient risk factors on the colonization and transmission of C. auris. IMPORTANCE Candida auris is a human pathogen of high concern due to its extensive antifungal drug resistance and high mortality rates associated with invasive infections. Candida auris skin colonization and persistence on environmental surfaces make this pathogen difficult to control once it enters a health care facility. Residents in long-term care hospitals and nursing homes are especially vulnerable. In this study, we developed microbiome sequencing protocols directly from surveillance samples, including amplicon and metagenomic sequencing, demonstrating concordance between sequencing results and culturing.

Integrated genomic, epidemiologic investigation of Candida auris skin colonization in a skilled nursing facility.

Candida auris is a fungal pathogen of high concern due to its ability to cause healthcare-associated infections and outbreaks, its resistance to antimicrobials and disinfectants and its persistence on human skin and in the inanimate environment. To inform surveillance and future mitigation strategies, we defined the extent of skin colonization and explored the microbiome associated with C. auris colonization. We collected swab specimens and clinical data at three times points between January and April 2019 from 57 residents (up to ten body sites each) of a ventilator-capable skilled nursing facility with endemic C. auris and routine chlorhexidine gluconate (CHG) bathing. Integrating microbial-genomic and epidemiologic data revealed occult C. auris colonization of multiple body sites not targeted commonly for screening. High concentrations of CHG were associated with suppression of C. auris growth but not with deleterious perturbation of commensal microbes. Modeling human mycobiome dynamics provided insight into underlying alterations to the skin fungal community as a possible modifiable risk factor for acquisition and persistence of C. auris. Failure to detect the extensive, disparate niches of C. auris colonization may reduce the effectiveness of infection-prevention measures that target colonized residents, highlighting the importance of universal strategies to reduce C. auris transmission.

Positive Correlation Between Candida auris Skin-Colonization Burden and Environmental Contamination at a Ventilator-Capable Skilled Nursing Facility in Chicago.

BACKGROUND: Candida auris is an emerging multidrug-resistant yeast that contaminates healthcare environments causing healthcare-associated outbreaks. The mechanisms facilitating contamination are not established. METHODS: C. auris was quantified in residents' bilateral axillary/inguinal composite skin swabs and environmental samples during a point-prevalence survey at a ventilator-capable skilled-nursing facility (vSNF A) with documented high colonization prevalence. Environmental samples were collected from all doorknobs, windowsills and handrails of each bed in 12 rooms. C. auris concentrations were measured using culture and C. auris-specific quantitative polymerase chain reaction (qPCR) The relationship between C. auris concentrations in residents' swabs and associated environmental samples were evaluated using Kendall's tau-b (τ b) correlation coefficient. RESULTS: C. auris was detected in 70/100 tested environmental samples and 31/57 tested resident skin swabs. The mean C. auris concentration in skin swabs was 1.22 × 105 cells/mL by culture and 1.08 × 106 cells/mL by qPCR. C. auris was detected on all handrails of beds occupied by colonized residents, as well as 10/24 doorknobs and 9/12 windowsills. A positive correlation was identified between the concentrations of C. auris in skin swabs and associated handrail samples based on culture (τ b = 0.54, P = .0004) and qPCR (τ b = 0.66, P = 3.83e-6). Two uncolonized residents resided in beds contaminated with C. auris. CONCLUSIONS: Colonized residents can have high C. auris burdens on their skin, which was positively related with contamination of their surrounding healthcare environment. These findings underscore the importance of hand hygiene, transmission-based precautions, and particularly environmental disinfection in preventing spread in healthcare facilities.

Candida auris Whole-Genome Sequence Benchmark Dataset for Phylogenomic Pipelines.

Candida auris is a multidrug-resistant pathogen that represents a serious public health threat due to its rapid global emergence, increasing incidence of healthcare-associated outbreaks, and high rates of antifungal resistance. Whole-genome sequencing and genomic surveillance have the potential to bolster C. auris surveillance networks moving forward. Laboratories conducting genomic surveillance need to be able to compare analyses from various national and international surveillance partners to ensure that results are mutually trusted and understood. Therefore, we established an empirical outbreak benchmark dataset consisting of 23 C. auris genomes to help validate comparisons of genomic analyses and facilitate communication among surveillance networks. Our outbreak benchmark dataset represents a polyclonal phylogeny with three subclades. The genomes in this dataset are from well-vetted studies that are supported by multiple lines of evidence, which demonstrate that the whole-genome sequencing data, phylogenetic tree, and epidemiological data are all in agreement. This C. auris benchmark set allows for standardized comparisons of phylogenomic pipelines, ultimately promoting effective C. auris collaborations.

Clade-specific chromosomal rearrangements and loss of subtelomeric adhesins in Candida auris.

Candida auris is an emerging fungal pathogen of rising concern due to global spread, the ability to cause healthcare-associated outbreaks, and antifungal resistance. Genomic analyses revealed that early contemporaneously detected cases of C. auris were geographically stratified into four major clades. While Clades I, III, and IV are responsible for ongoing outbreaks of invasive and multidrug-resistant infections, Clade II, also termed the East Asian clade, consists primarily of cases of ear infection, is often susceptible to all antifungal drugs, and has not been associated with outbreaks. Here, we generate chromosome-level assemblies of twelve isolates representing the phylogenetic breadth of these four clades and the only isolate described to date from Clade V. This Clade V genome is highly syntenic with those of Clades I, III, and IV, although the sequence is highly divergent from the other clades. Clade II genomes appear highly rearranged, with translocations occurring near GC-poor regions, and large subtelomeric deletions in most chromosomes, resulting in a substantially different karyotype. Rearrangements and deletion lengths vary across Clade II isolates, including two from a single patient, supporting ongoing genome instability. Deleted subtelomeric regions are enriched in Hyr/Iff-like cell-surface proteins, novel candidate cell wall proteins, and an ALS-like adhesin. Cell wall proteins from these families and other drug-related genes show clade-specific signatures of selection in Clades I, III, and IV. Subtelomeric dynamics and the conservation of cell surface proteins in the clades responsible for global outbreaks causing invasive infections suggest an explanation for the different phenotypes observed between clades.

Riddles in the cold: Antarctic endemism and microbial succession impact methane cycling in the Southern Ocean.

Antarctica is estimated to contain as much as a quarter of earth's marine methane, however we have not discovered an active Antarctic methane seep limiting our understanding of the methane cycle. In 2011, an expansive (70 m × 1 m) microbial mat formed at 10 m water depth in the Ross Sea, Antarctica which we identify here to be a high latitude hydrogen sulfide and methane seep. Through 16S rRNA gene analysis on samples collected 1 year and 5 years after the methane seep formed, we identify the taxa involved in the Antarctic methane cycle and quantify the response rate of the microbial community to a novel input of methane. One year after the seep formed, ANaerobic MEthane oxidizing archaea (ANME), the dominant sink of methane globally, were absent. Five years later, ANME were found to make up to 4% of the microbial community, however the dominant member of this group observed (ANME-1) were unexpected considering the cold temperature (-1.8°C) and high sulfate concentrations (greater than 24 mM) present at this site. Additionally, the microbial community had not yet formed a sufficient filter to mitigate the release of methane from the sediment; methane flux from the sediment was still significant at 3.1 mmol CH4 m-2 d-1. We hypothesize that this 5 year time point represents an early successional stage of the microbiota in response to methane input. This study provides the first report of the evolution of a seep system from a non-seep environment, and reveals that the rate of microbial succession may have an unrealized impact on greenhouse gas emission from marine methane reservoirs.

Evaluation of nine surface disinfectants against Candida auris using a quantitative disk carrier method: EPA SOP-MB-35.

We tested 9 disinfectants against Candida auris using the quantitative disk carrier method EPA-MB-35-00: 5 products with hydrogen peroxide or alcohol-based chemistries were effective and 4 quaternary ammonium compound-based products were not. This work supported a FIFRA Section 18 emergency exemption granted by the US Environmental Protection Agency to expand disinfectant guidance for C. auris.

Tracing the Evolutionary History and Global Expansion of Candida auris Using Population Genomic Analyses.

Candida auris has emerged globally as a multidrug-resistant yeast that can spread via nosocomial transmission. An initial phylogenetic study of isolates from Japan, India, Pakistan, South Africa, and Venezuela revealed four populations (clades I, II, III, and IV) corresponding to these geographic regions. Since this description, C. auris has been reported in more than 30 additional countries. To trace this global emergence, we compared the genomes of 304 C. auris isolates from 19 countries on six continents. We found that four predominant clades persist across wide geographic locations. We observed phylogeographic mixing in most clades; clade IV, with isolates mainly from South America, demonstrated the strongest phylogeographic substructure. C. auris isolates from two clades with opposite mating types were detected contemporaneously in a single health care facility in Kenya. We estimated a Bayesian molecular clock phylogeny and dated the origin of each clade within the last 360 years; outbreak-causing clusters from clades I, III, and IV originated 36 to 38 years ago. We observed high rates of antifungal resistance in clade I, including four isolates resistant to all three major classes of antifungals. Mutations that contribute to resistance varied between the clades, with Y132F in ERG11 as the most widespread mutation associated with azole resistance and S639P in FKS1 for echinocandin resistance. Copy number variants in ERG11 predominantly appeared in clade III and were associated with fluconazole resistance. These results provide a global context for the phylogeography, population structure, and mechanisms associated with antifungal resistance in C. aurisIMPORTANCE In less than a decade, C. auris has emerged in health care settings worldwide; this species is capable of colonizing skin and causing outbreaks of invasive candidiasis. In contrast to other Candida species, C. auris is unique in its ability to spread via nosocomial transmission and its high rates of drug resistance. As part of the public health response, whole-genome sequencing has played a major role in characterizing transmission dynamics and detecting new C. auris introductions. Through a global collaboration, we assessed genome evolution of isolates of C. auris from 19 countries. Here, we described estimated timing of the expansion of each C. auris clade and of fluconazole resistance, characterized discrete phylogeographic population structure of each clade, and compared genome data to sensitivity measurements to describe how antifungal resistance mechanisms vary across the population. These efforts are critical for a sustained, robust public health response that effectively utilizes molecular epidemiology.

Candida auris: A Review of Recommendations for Detection and Control in Healthcare Settings.

Candida auris is an emerging multidrug-resistant fungal pathogen. Since first reported in 2009, C. auris has caused healthcare outbreaks around the world, often involving high mortality. Identification of C. auris has been a major challenge as many common conventional laboratory methods cannot accurately detect it. Early detection and implementation of infection control practices can prevent its spread. The aim of this review is to describe recommendations for the detection and control of C. auris in healthcare settings.

Insights into the Unique Nature of the East Asian Clade of the Emerging Pathogenic Yeast Candida auris.

The emerging yeast Candida auris can be highly drug resistant, causing invasive infections, and large outbreaks. C. auris went from an unknown pathogen a decade ago to being reported in over thirty countries on six continents. C. auris consists of four discrete clades, based on where the first isolates of the clade were reported, South Asian (clade I), East Asian (clade II), African (clade III), and South American (clade IV). These clades have unique genetic and biochemical characteristics that are important to understand and inform the global response to C. auris Clade II has been underrepresented in the literature despite being the first one discovered. In this issue of the Journal of Clinical Microbiology, Y. J. Kwon et al. (J Clin Microbiol 57:e01624-18, 2019, https://doi.org/10.1128/JCM.01624-18) describe the largest collection of clinical isolates from Clade II, which is also the longest-running span of clinical cases, 20 years, from any single region to date. Clade II appears to have a propensity for the ear that is uncharacteristic of the other clades, which typically cause invasive infections and large-scale outbreaks. This study provides new information on an understudied lineage of C. auris and has important implications for future surveillance.

Molecular Epidemiology of Candida auris in Colombia Reveals a Highly Related, Countrywide Colonization With Regional Patterns in Amphotericin B Resistance.

Background: Candida auris is a multidrug-resistant yeast associated with hospital outbreaks worldwide. During 2015-2016, multiple outbreaks were reported in Colombia. We aimed to understand the extent of contamination in healthcare settings and to characterize the molecular epidemiology of C. auris in Colombia. Methods: We sampled patients, patient contacts, healthcare workers, and the environment in 4 hospitals with recent C. auris outbreaks. Using standardized protocols, people were swabbed at different body sites. Patient and procedure rooms were sectioned into 4 zones and surfaces were swabbed. We performed whole-genome sequencing (WGS) and antifungal susceptibility testing (AFST) on all isolates. Results: Seven of the 17 (41%) people swabbed were found to be colonized. Candida auris was isolated from 37 of 322 (11%) environmental samples. These were collected from a variety of items in all 4 zones. WGS and AFST revealed that although isolates were similar throughout the country, isolates from the northern region were genetically distinct and more resistant to amphotericin B (AmB) than the isolates from central Colombia. Four novel nonsynonymous mutations were found to be significantly associated with AmB resistance. Conclusions: Our results show that extensive C. auris contamination can occur and highlight the importance of adherence to appropriate infection control practices and disinfection strategies. Observed genetic diversity supports healthcare transmission and a recent expansion of C. auris within Colombia with divergent AmB susceptibility.

Phenotypic switching in newly emerged multidrug-resistant pathogen Candida auris.

Candida auris is an emerging, multidrug-resistant yeast that can spread rapidly in healthcare settings. Phenotypic switching has been observed in other Candida species and can potentially interfere with correct identification. The aim of this study is to address misidentification of C. auris by describing alternate phenotypes after broth enrichment and subculturing on CHROMagar Candida. Each isolate displayed different frequencies of phenotypic switching, suggesting a strain to strain variability. Increased knowledge of the multiple phenotypes of C. auris increases the chance of isolating and identifying C. auris by reducing the risk of discarding false negative alternate colony morphologies.

Direct Detection of Emergent Fungal Pathogen Candida auris in Clinical Skin Swabs by SYBR Green-Based Quantitative PCR Assay.

The recent emergence of the multidrug-resistant and pathogenic yeast Candida auris continues to cause public health concern worldwide. C. auris is alarming because it causes health care-associated outbreaks and can establish invasive infections with high mortality rates. Transmission between patients is facilitated by the ability of C. auris to persistently colonize multiple body sites, including the skin, and survive for weeks on surfaces in health care settings. Rapid identification of colonized patients is needed to implement timely infection control measures. Currently, CDC laboratories use an enrichment culture-based approach that can take up to 2 weeks to identify C. auris from composite swabs from the bilateral axillae and groin. A rapid SYBR green quantitative PCR (qPCR) assay that can identify C. auris in a single day was recently described. In this study, we developed the SYBR green qPCR assay further by incorporating a DNA extraction procedure for skin swabs and by including an internal amplification control based on the distinguishable melt curve of a lambda DNA amplicon. The assay was conducted using 103 clinical axilla/groin skin swab samples. Using the enrichment culture-based approach as a gold standard, we determined that the SYBR green C. auris qPCR has a sensitivity of 0.93 and specificity of 0.96. Overall, we found that the SYBR green C. auris qPCR assay can be successfully applied for rapid and accurate detection of C. auris in patient skin swabs, thereby increasing diagnostic options for this emerging pathogen.

Evaluation of a new T2 Magnetic Resonance assay for rapid detection of emergent fungal pathogen Candida auris on clinical skin swab samples.

Candida auris is a multidrug-resistant pathogenic yeast whose recent emergence is of increasing public-health concern. C. auris can colonise multiple body sites, including patients' skin, and survive for weeks in the health care environment, facilitating patient-to-patient transmission and fueling health care-associated outbreaks. Rapid and accurate detection of C. auris colonisation is essential for timely implementation of infection control measures and to prevent transmission. Currently, axilla/groin composite swabs, used to assess colonisation status, are processed using a culture-based method that is sensitive and specific but requires 14 days. This delay limits the opportunity to respond and highlights the need for a faster alternative. The culture-independent T2 Magnetic Resonance (T2MR) system is a rapid diagnostic platform shown to detect target pathogens of interest from unprocessed blood samples in <5 hours. In this study, a new C. auris-specific T2 assay was evaluated for screening of the skin surveillance samples. Inclusivity and limit of detection of the T2 C.  auris assay were assessed with spiked samples in a representative skin flora background. The T2 C. auris assay recognised isolates from each of the 4 known clades of C. auris and consistently detected cells at 5 CFU/mL. Finally, 89 clinical axilla/groin swab samples were processed with the T2 C. auris assay. The culture-based diagnostic assay was used as a gold standard to determine performance statistics including sensitivity (0.89) and specificity (0.98). Overall, the T2 C. auris assay performed well as a rapid diagnostic and could help expedite the detection of C. auris in patient skin swabs.