Development of an amplicon-based sequencing approach in response to the global emergence of mpox.

The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.

Antibodies in healthcare personnel following severe acute respiratory syndrome coronavirus virus 2 (SARS-CoV-2) infection.

In a prospective cohort of healthcare personnel (HCP), we measured severe acute respiratory syndrome coronavirus virus 2 (SARS-CoV-2) nucleocapsid IgG antibodies after SARS-CoV-2 infection. Among 79 HCP, 68 (86%) were seropositive 14-28 days after their positive PCR test, and 54 (77%) of 70 were seropositive at the 70-180-day follow-up. Many seropositive HCP (95%) experienced an antibody decline by the second visit.

Bone marrow necrosis and fat embolism syndrome in sickle cell disease during COVID-19 infection treated successfully with sequential red cell and plasma exchange.

Fat embolism syndrome (FES) is a rare life-threatening condition that is particularly seen in milder forms of sickle cell disease (SCD). Widespread systemic fat emboli are generated in the context of extensive bone marrow necrosis. Multi-organ failure with a high morbidity and mortality may quickly develop. Infection with Parvovirus B19 is a common precipitant. Here, the authors report the case of a 35-year-old Afro-Caribbean man with HbSC disease who presented with FES having tested positive for SARS-COV-2. He rapidly became critically ill and required admission to the intensive care unit for organ support. He was treated with red cell exchange and plasma exchange and made a good recovery to leave hospital at week 7.

Personal protective equipment use among dental healthcare personnel during the coronavirus disease 2019 (COVID-19) pandemic and the impact of an educational video in clinical practice.

OBJECTIVE: Dental healthcare personnel (DHCP) are at high risk of exposure to coronavirus disease 2019 (COVID-19). We sought to identify how DHCP changed their use of personal protective equipment (PPE) as a result of the COVID-19 pandemic, and to pilot an educational video designed to improve knowledge of proper PPE use. DESIGN: The study comprised 2 sets of semistructured qualitative interviews. SETTING: The study was conducted in 8 dental clinics in a Midwestern metropolitan area. PARTICIPANTS: In total, 70 DHCP participated in the first set of interviews; 63 DHCP participated in the second set of interviews. METHODS: In September-November 2020 and March-October 2021, we conducted 2 sets of semistructured interviews: (1) PPE use in the dental community during COVID-19, and (2) feedback on the utility of an educational donning and doffing video. RESULTS: Overall, 86% of DHCP reported having prior training. DHCP increased the use of PPE during COVID-19, specifically N95 respirators and face shields. DHCP reported real-world challenges to applying infection control methods, often resulting in PPE modification and reuse. DHCP reported double masking and sterilization methods to extend N95 respirator use. Additional challenges to PPE included shortages, comfort or discomfort, and compatibility with specialty dental equipment. DHCP found the educational video helpful and relevant to clinical practice. Fewer than half of DHCP reported exposure to a similar video. CONCLUSIONS: DHCP experienced significant challenges related to PPE access and routine use in dental clinics during the COVID-19 pandemic. An educational video improved awareness and uptake of appropriate PPE use among DHCP.

Bone marrow necrosis and fat embolism syndrome in sickle cell disease during COVID‐19 infection treated successfully with sequential red cell and plasma exchange

Fat embolism syndrome (FES) is a rare life‐threatening condition that is particularly seen in milder forms of sickle cell disease (SCD). Widespread systemic fat emboli are generated in the context of extensive bone marrow necrosis. Multi‐organ failure with a high morbidity and mortality may quickly develop. Infection with Parvovirus B19 is a common precipitant. Here, the authors report the case of a 35‐year‐old Afro‐Caribbean man with HbSC disease who presented with FES having tested positive for SARS‐COV‐2. He rapidly became critically ill and required admission to the intensive care unit for organ support. He was treated with red cell exchange and plasma exchange and made a good recovery to leave hospital at week 7.

High-depth sequencing characterization of viral dynamics across tissues in fatal COVID-19 reveals compartmentalized infection.

SARS-CoV-2 distribution and circulation dynamics are not well understood due to challenges in assessing genomic data from tissue samples. We develop experimental and computational workflows for high-depth viral sequencing and high-resolution genomic analyses from formalin-fixed, paraffin-embedded tissues and apply them to 120 specimens from six subjects with fatal COVID-19. To varying degrees, viral RNA is present in extrapulmonary tissues from all subjects. The majority of the 180 viral variants identified within subjects are unique to individual tissue samples. We find more high-frequency (>10%) minor variants in subjects with a longer disease course, with one subject harboring ten such variants, exclusively in extrapulmonary tissues. One tissue-specific high-frequency variant was a nonsynonymous mutation in the furin-cleavage site of the spike protein. Our findings suggest adaptation and/or compartmentalized infection, illuminating the basis of extrapulmonary COVID-19 symptoms and potential for viral reservoirs, and have broad utility for investigating human pathogens.

Geographic variability of antibiotic prescribing for acute respiratory tract infections within a direct-to-consumer telemedicine practice.

In this retrospective cohort study of patients presenting to a national direct-to-consumer medical practice, we found that provider geographic location is a stronger driver of antibiotic prescribing than patient location. Physicians in the Northeast and South are significantly more likely than physicians in the West to prescribe antibiotics for upper respiratory infection and bronchitis.

Antibodies in healthcare personnel following severe acute respiratory syndrome coronavirus virus 2 (SARS-CoV-2) infection

In a prospective cohort of healthcare personnel (HCP), we measured severe acute respiratory syndrome coronavirus virus 2 (SARS-CoV-2) nucleocapsid IgG antibodies after SARS-CoV-2 infection. Among 79 HCP, 68 (86%) were seropositive 14–28 days after their positive PCR test, and 54 (77%) of 70 were seropositive at the 70–180-day follow-up. Many seropositive HCP (95%) experienced an antibody decline by the second visit.

Assessment of dental health care personnel protocol deviations and self-contamination during personal protective equipment donning and doffing.

BACKGROUND: Dental health care personnel (DHCP) may be at increased risk of exposure to severe acute respiratory syndrome coronavirus 2, the virus that causes COVID-19, as well as other clinically important pathogens. Proper use of personal protective equipment (PPE) reduces occupational exposure to pathogens. The authors performed an assessment of PPE donning and doffing practices among DHCP, using a fluorescent marker as a surrogate for pathogen transmission. METHODS: Participants donned PPE (that is, disposable gown, gloves, face mask, and eye protection) and the fluorescent marker was applied to their palms and abdomen. DHCP then doffed PPE according to their usual practices. The donning and doffing processes were video recorded, areas of fluorescence were noted, and protocol deviations were assessed. Statistical analyses included frequency, type, and descriptions of protocol deviations and factors associated with fluorescence. RESULTS: Seventy DHCP were enrolled. The donning and doffing steps with the highest frequency of protocol deviations were hand hygiene (66% of donning and 78% of doffing observations involved a deviation) and disposable gown use (63% of donning and 60% of doffing observations involved a deviation). Fluorescence was detected on 69% of DHCP after doffing, most frequently on hands. An increasing number of protocol deviations was significantly associated with increased risk of fluorescence. DHCP with a gown doffing deviation, excluding doffing out of order, were more likely to have fluorescence detected. CONCLUSIONS: DHCP self-contamination was common with both donning and doffing PPE. PRACTICAL IMPLICATIONS: Proper use of PPE is an important component of occupational health.

Benchmark datasets for SARS-CoV-2 surveillance bioinformatics.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), has spread globally and is being surveilled with an international genome sequencing effort. Surveillance consists of sample acquisition, library preparation, and whole genome sequencing. This has necessitated a classification scheme detailing Variants of Concern (VOC) and Variants of Interest (VOI), and the rapid expansion of bioinformatics tools for sequence analysis. These bioinformatic tools are means for major actionable results: maintaining quality assurance and checks, defining population structure, performing genomic epidemiology, and inferring lineage to allow reliable and actionable identification and classification. Additionally, the pandemic has required public health laboratories to reach high throughput proficiency in sequencing library preparation and downstream data analysis rapidly. However, both processes can be limited by a lack of a standardized sequence dataset. Methods: We identified six SARS-CoV-2 sequence datasets from recent publications, public databases and internal resources. In addition, we created a method to mine public databases to identify representative genomes for these datasets. Using this novel method, we identified several genomes as either VOI/VOC representatives or non-VOI/VOC representatives. To describe each dataset, we utilized a previously published datasets format, which describes accession information and whole dataset information. Additionally, a script from the same publication has been enhanced to download and verify all data from this study. Results: The benchmark datasets focus on the two most widely used sequencing platforms: long read sequencing data from the Oxford Nanopore Technologies platform and short read sequencing data from the Illumina platform. There are six datasets: three were derived from recent publications; two were derived from data mining public databases to answer common questions not covered by published datasets; one unique dataset representing common sequence failures was obtained by rigorously scrutinizing data that did not pass quality checks. The dataset summary table, data mining script and quality control (QC) values for all sequence data are publicly available on GitHub: https://github.com/CDCgov/datasets-sars-cov-2. Discussion: The datasets presented here were generated to help public health laboratories build sequencing and bioinformatics capacity, benchmark different workflows and pipelines, and calibrate QC thresholds to ensure sequencing quality. Together, improvements in these areas support accurate and timely outbreak investigation and surveillance, providing actionable data for pandemic management. Furthermore, these publicly available and standardized benchmark data will facilitate the development and adjudication of new pipelines.

The Penis, the Vagina and HIV Risk: Key Differences (Aside from the Obvious).

Globally, most Human Immunodeficiency Virus type 1 (HIV) transmission occurs through vaginal-penile sex (heterosexual transmission). The local immune environment at the site of HIV exposure is an important determinant of whether exposure during sex will lead to productive infection, and the vaginal and penile immune milieus are each critically shaped by the local microbiome. However, there are key differences in the microbial drivers of inflammation and immune quiescence at these tissue sites. In both, a high abundance of anaerobic taxa (e.g., Prevotella) is associated with an increased local density of HIV target cells and an increased risk of acquiring HIV through sex. However, the taxa that have been associated to date with increased risk in the vagina and penis are not identical. Just as importantly, the microbiota associated with comparatively less inflammation and HIV risk-i.e., the optimal microbiota-are very different at the two sites. In the vagina, Lactobacillus spp. are immunoregulatory and may protect against HIV acquisition, whereas on the penis, "skin type" flora such as Corynebacterium are associated with reduced inflammation. Compared to its vaginal counterpart, much less is known about the dynamics of the penile microbiome, the ability of clinical interventions to alter the penile microbiome, or the impact of natural/induced microbiome alterations on penile immunology and HIV risk.

Analysis of 6.4 million SARS-CoV-2 genomes identifies mutations associated with fitness.

Repeated emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with increased fitness underscores the value of rapid detection and characterization of new lineages. We have developed PyR0, a hierarchical Bayesian multinomial logistic regression model that infers relative prevalence of all viral lineages across geographic regions, detects lineages increasing in prevalence, and identifies mutations relevant to fitness. Applying PyR0 to all publicly available SARS-CoV-2 genomes, we identify numerous substitutions that increase fitness, including previously identified spike mutations and many nonspike mutations within the nucleocapsid and nonstructural proteins. PyR0 forecasts growth of new lineages from their mutational profile, ranks the fitness of lineages as new sequences become available, and prioritizes mutations of biological and public health concern for functional characterization.

Comparative transmissibility of SARS-CoV-2 variants Delta and Alpha in New England, USA.

The SARS-CoV-2 Delta variant rose to dominance in mid-2021, likely propelled by an estimated 40%-80% increased transmissibility over Alpha. To investigate if this ostensible difference in transmissibility is uniform across populations, we partner with public health programs from all six states in New England in the United States. We compare logistic growth rates during each variant's respective emergence period, finding that Delta emerged 1.37-2.63 times faster than Alpha (range across states). We compute variant-specific effective reproductive numbers, estimating that Delta is 63%-167% more transmissible than Alpha (range across states). Finally, we estimate that Delta infections generate on average 6.2 (95% CI 3.1-10.9) times more viral RNA copies per milliliter than Alpha infections during their respective emergence. Overall, our evidence suggests that Delta's enhanced transmissibility can be attributed to its innate ability to increase infectiousness, but its epidemiological dynamics may vary depending on underlying population attributes and sequencing data availability.

SARS-CoV-2 surveillance and outbreak response on an urban American college campus.

OBJECTIVE: The George Washington University (GW) in Washington, D.C., USA established the Public Health Laboratory and Campus COVID-19 Support Team (CCST) to develop and implement its SARS-CoV-2 surveillance testing and outbreak response for the 2020-2021 academic year. PARTICIPANTS AND METHODS: Approximately 4,000 GW members had access to campus for living accommodations, limited in-person instruction, athletics, research, and university operations. The outbreak response included daily risk assessment surveys, weekly surveillance testing, symptomatic and voluntary testing, case investigation, and contact tracing. RESULTS: Between August 17 - November 24, 2020, 42,350 SARS-CoV-2 PCR tests were performed, and 194 (0.46%) of tests were positive. Surveillance testing identified 59 (30.4%); voluntary testing 97 (50%); and symptomatic testing 30 (15.5%) of the cases, respectively. CONCLUSIONS: Robust testing of asymptomatic people and rapid isolation and quarantine of members who are exposed or infected effectively limited the spread of SARS-CoV-2 during the Fall 2020 semester.

Future-proofing and maximizing the utility of metadata: The PHA4GE SARS-CoV-2 contextual data specification package.

BACKGROUND: The Public Health Alliance for Genomic Epidemiology (PHA4GE) (https://pha4ge.org) is a global coalition that is actively working to establish consensus standards, document and share best practices, improve the availability of critical bioinformatics tools and resources, and advocate for greater openness, interoperability, accessibility, and reproducibility in public health microbial bioinformatics. In the face of the current pandemic, PHA4GE has identified a need for a fit-for-purpose, open-source SARS-CoV-2 contextual data standard. RESULTS: As such, we have developed a SARS-CoV-2 contextual data specification package based on harmonizable, publicly available community standards. The specification can be implemented via a collection template, as well as an array of protocols and tools to support both the harmonization and submission of sequence data and contextual information to public biorepositories. CONCLUSIONS: Well-structured, rich contextual data add value, promote reuse, and enable aggregation and integration of disparate datasets. Adoption of the proposed standard and practices will better enable interoperability between datasets and systems, improve the consistency and utility of generated data, and ultimately facilitate novel insights and discoveries in SARS-CoV-2 and COVID-19. The package is now supported by the NCBI's BioSample database.

Multiplexed CRISPR-based microfluidic platform for clinical testing of respiratory viruses and identification of SARS-CoV-2 variants.

The coronavirus disease 2019 (COVID-19) pandemic has demonstrated a clear need for high-throughput, multiplexed and sensitive assays for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other respiratory viruses and their emerging variants. Here, we present a cost-effective virus and variant detection platform, called microfluidic Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (mCARMEN), which combines CRISPR-based diagnostics and microfluidics with a streamlined workflow for clinical use. We developed the mCARMEN respiratory virus panel to test for up to 21 viruses, including SARS-CoV-2, other coronaviruses and both influenza strains, and demonstrated its diagnostic-grade performance on 525 patient specimens in an academic setting and 166 specimens in a clinical setting. We further developed an mCARMEN panel to enable the identification of 6 SARS-CoV-2 variant lineages, including Delta and Omicron, and evaluated it on 2,088 patient specimens with near-perfect concordance to sequencing-based variant classification. Lastly, we implemented a combined Cas13 and Cas12 approach that enables quantitative measurement of SARS-CoV-2 and influenza A viral copies in samples. The mCARMEN platform enables high-throughput surveillance of multiple viruses and variants simultaneously, enabling rapid detection of SARS-CoV-2 variants.

Synthetic DNA spike-ins (SDSIs) enable sample tracking and detection of inter-sample contamination in SARS-CoV-2 sequencing workflows.

The global spread and continued evolution of SARS-CoV-2 has driven an unprecedented surge in viral genomic surveillance. Amplicon-based sequencing methods provide a sensitive, low-cost and rapid approach but suffer a high potential for contamination, which can undermine laboratory processes and results. This challenge will increase with the expanding global production of sequences across a variety of laboratories for epidemiological and clinical interpretation, as well as for genomic surveillance of emerging diseases in future outbreaks. We present SDSI + AmpSeq, an approach that uses 96 synthetic DNA spike-ins (SDSIs) to track samples and detect inter-sample contamination throughout the sequencing workflow. We apply SDSIs to the ARTIC Consortium's amplicon design, demonstrate their utility and efficiency in a real-time investigation of a suspected hospital cluster of SARS-CoV-2 cases and validate them across 6,676 diagnostic samples at multiple laboratories. We establish that SDSI + AmpSeq provides increased confidence in genomic data by detecting and correcting for relatively common, yet previously unobserved modes of error, including spillover and sample swaps, without impacting genome recovery.

Epidemiology and Seasonality of Endemic Human Coronaviruses in South African and Zambian Children: A Case-Control Pneumonia Study.

Endemic human coronaviruses (HCoV) are capable of causing a range of diseases from the common cold to pneumonia. We evaluated the epidemiology and seasonality of endemic HCoVs in children hospitalized with clinical pneumonia and among community controls living in countries with a high HIV burden, namely South Africa and Zambia, between August 2011 to October 2013. Nasopharyngeal/oropharyngeal swabs were collected from all cases and controls and tested for endemic HCoV species and 12 other respiratory viruses using a multiplex real-time PCR assay. We found that the likelihood of detecting endemic HCoV species was higher among asymptomatic controls than cases (11% vs. 7.2%; 95% CI: 1.2-2.0). This was however only observed among children > 6 months and was mainly driven by the Betacoronavirus endemic species (HCoV-OC43 and -HKU1). Endemic HCoV species were detected through the year; however, in Zambia, the endemic Betacoronavirus species tended to peak during the winter months (May-August). There was no association between HIV status and endemic HCoV detection.

Outbreak of SARS-CoV-2 Infections, Including COVID-19 Vaccine Breakthrough Infections, Associated with Large Public Gatherings - Barnstable County, Massachusetts, July 2021.

During July 2021, 469 cases of COVID-19 associated with multiple summer events and large public gatherings in a town in Barnstable County, Massachusetts, were identified among Massachusetts residents; vaccination coverage among eligible Massachusetts residents was 69%. Approximately three quarters (346; 74%) of cases occurred in fully vaccinated persons (those who had completed a 2-dose course of mRNA vaccine [Pfizer-BioNTech or Moderna] or had received a single dose of Janssen [Johnson & Johnson] vaccine ≥14 days before exposure). Genomic sequencing of specimens from 133 patients identified the B.1.617.2 (Delta) variant of SARS-CoV-2, the virus that causes COVID-19, in 119 (89%) and the Delta AY.3 sublineage in one (1%). Overall, 274 (79%) vaccinated patients with breakthrough infection were symptomatic. Among five COVID-19 patients who were hospitalized, four were fully vaccinated; no deaths were reported. Real-time reverse transcription-polymerase chain reaction (RT-PCR) cycle threshold (Ct) values in specimens from 127 vaccinated persons with breakthrough cases were similar to those from 84 persons who were unvaccinated, not fully vaccinated, or whose vaccination status was unknown (median = 22.77 and 21.54, respectively). The Delta variant of SARS-CoV-2 is highly transmissible (1); vaccination is the most important strategy to prevent severe illness and death. On July 27, CDC recommended that all persons, including those who are fully vaccinated, should wear masks in indoor public settings in areas where COVID-19 transmission is high or substantial.* Findings from this investigation suggest that even jurisdictions without substantial or high COVID-19 transmission might consider expanding prevention strategies, including masking in indoor public settings regardless of vaccination status, given the potential risk of infection during attendance at large public gatherings that include travelers from many areas with differing levels of transmission.