A pyrosequencing protocol for rapid identification of SARS-CoV-2 variants.

Next-generation sequencing (NGS) is the primary method used to monitor the distribution and emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants around the world; however, it is costly and time-consuming to perform and is not widely available in low-resourced geographical regions. Pyrosequencing has the potential to augment surveillance efforts by providing information on specific targeted mutations for rapid identification of circulating and emerging variants. The current study describes the development of a reverse transcription (RT)-PCR-pyrosequencing assay targeting >65 spike protein gene (S) mutations of SARS-CoV-2, which permits differentiation of commonly reported variants currently circulating in the United States with a high degree of confidence. Variants typed using the assay included B.1.1.7 (Alpha), B.1.1.529 (Omicron), B.1.351 (Beta), B.1.375, B.1.427/429 (Epsilon), B.1.525 (Eta), B.1.526.1 (Iota), B.1.617.1 (Kappa), B.1.617.2 (Delta), B.1.621 (Mu), P1 (Gamma), and B.1.1 variants, all of which were confirmed by the NGS data. An electronic typing tool was developed to aid in the identification of variants based on mutations detected by pyrosequencing. The assay could provide an important typing tool for rapid identification of candidate patients for monoclonal antibody therapies and a method to supplement SARS-CoV-2 surveillance efforts by identification of circulating variants and novel emerging lineages.

Validation and performance comparison of three SARS-CoV-2 antibody assays.

Serology testing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is increasingly being used during the current pandemic of coronavirus disease 2019 (COVID-19), although its clinical and epidemiologic utilities are still debatable. Characterizing these assays provides scientific basis to best use them. The current study assessed one chemiluminescent assay (Abbott COVID-2 IgG) and two lateral flow assays (STANDARD Q [SQ] IgM/IgG Duo and Wondfo total antibody test) using 113 blood samples from 71 PCR-confirmed COVID-19 hospitalized patients, 119 samples with potential cross-reactions, and 1068 negative controls including 942 pre-pandemic samples. SARS-CoV-2 IgM antibodies became detectable 3-4 days post-symptom onset using SQ IgM test and IgG antibodies were first detected 5-6 days post-onset using SQ IgG. Abbott IgG and Wondfo Total were able to detect antibodies 7 to 8 days post-onset. After 14 days post-symptom onset, the SQ IgG, Abbott IgG and Wondfo Total tests were able to detect antibodies from 100% of the PCR-confirmed patients in this series; 87.5% sensitivity for SQ IgM. Overall agreement was 88.5% between SQ IgM/IgG and Wondfo Total and 94.6% between SQ IgG and Abbott IgG. No cross-reaction due to recent sera with three of the endemic coronaviruses was observed. Viral hepatitis and autoimmune samples were the main source of limited cross-reactions. The specificities were 100% for SQ IgG and Wondfo Total, 99.62% for Abbott IgG, and 98.87% for SQ IgM. These findings demonstrated high sensitivity and specificity of appropriately validated SARS-CoV-2 serologic assays with implications for clinical use and epidemiological seroprevalence studies.

Seroprevalence of SARS-CoV-2 Antibodies in Rhode Island From a Statewide Random Sample.

Objectives. To characterize statewide seroprevalence and point prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Rhode Island.Methods. We conducted a cross-sectional survey of randomly selected households across Rhode Island in May 2020. Antibody-based and polymerase chain reaction (PCR)-based tests for SARS-CoV-2 were offered. Hispanics/Latinos and African Americans/Blacks were oversampled to ensure adequate representation. Seroprevalence estimations accounted for test sensitivity and specificity and were compared according to age, race/ethnicity, gender, housing environment, and transportation mode.Results. Overall, 1043 individuals from 554 households were tested (1032 antibody tests, 988 PCR tests). The estimated seroprevalence of SARS-CoV-2 antibodies was 2.1% (95% credible interval [CI] = 0.6, 4.1). Seroprevalence was 7.5% (95% CI = 1.3, 17.5) among Hispanics/Latinos, 3.8% (95% CI = 0.0, 15.0) among African Americans/Blacks, and 0.8% (95% CI = 0.0, 2.4) among non-Hispanic Whites. Overall PCR-based prevalence was 1.5% (95% CI = 0.5, 3.1).Conclusions. Rhode Island had low seroprevalence relative to other settings, but seroprevalence was substantially higher among African Americans/Blacks and Hispanics/Latinos. Rhode Island sits along the highly populated northeast corridor, making our findings broadly relevant to this region of the country. Continued monitoring via population-based sampling is needed to quantify these impacts going forward.

Low Seroprevalence of SARS-CoV-2 in Rhode Island blood donors during may 2020 as determined using multiple serological assay formats.

BACKGROUND: Epidemic projections and public health policies addressing Coronavirus disease (COVID)-19 have been implemented without data reporting on the seroconversion of the population since scalable antibody testing has only recently become available. METHODS: We measured the percentage of severe acute respiratory syndrome- Coronavirus-2 (SARS-CoV-2) seropositive individuals from 2008 blood donors drawn in the state of Rhode Island (RI). We utilized multiple antibody testing platforms, including lateral flow immunoassays (LFAs), enzyme-linked immunosorbent assays (ELISAs) and high throughput serological assays (HTSAs). To estimate seroprevalence, we utilized the Bayesian statistical method to adjust for sensitivity and specificity of the commercial tests used. RESULTS: We report than an estimated seropositive rate of RI blood donors of approximately 0.6% existed in April-May of 2020. Daily new case rates peaked in RI in late April 2020. We found HTSAs and LFAs were positively correlated with ELISA assays to detect antibodies specific to SARS-CoV-2 in blood donors. CONCLUSIONS: These data imply that seroconversion, and thus infection, is likely not widespread within this population. We conclude that IgG LFAs and HTSAs are suitable to conduct seroprevalence assays in random populations. More studies will be needed using validated serological tests to improve the precision and report the kinetic progression of seroprevalence estimates.

The United States Needs A Better Testing Playbook For Future Public Health Emergencies.

The absence of a comprehensive national playbook for developing and deploying testing has hindered the United States' ability to rapidly suppress recent biological emergencies (for example, the COVID-19 pandemic and outbreaks of mpox). We describe here the Testing Playbook for Biological Emergencies, a national testing playbook we developed. It includes a set of decisions and actions for US officials to take at specific times during infectious disease emergencies to implement testing rapidly and to ensure that available testing meets clinical and public health needs. Although the United States had multiple plans at the federal level for responding to pandemic threats, US leaders were unable to quickly and efficiently operationalize those plans to deploy different types of tests during the COVID-19 pandemic in 2020-21, and again during the US mpox outbreak in 2022. The playbook fills a critical gap by providing the necessary specific and adaptable guidance for decision makers to meet this need.

Mercury, lead, and cadmium in umbilical cord blood.

The study described in this article aimed to determine if measurable levels of mercury, lead, and cadmium are detected in the umbilical cord blood specimens collected in a community hospital in Rhode Island and if prenatal exposure correlates with prematurity or fetal growth indicators. Total mercury, lead, and cadmium concentrations were measured in 538 specimens of cord blood and correlated with demographic characteristics and pregnancy outcomes for each mother-infant pair. Lead concentrations determined in the cord blood of Rhode Island women (geometric mean 0.99 microg/dL) were similar to those reported in U.S. biomonitoring studies. The overall geometric mean for mercury concentration (0.52 microg/L) was slightly lower than in other comparable studies. Cadmium concentrations were generally below the limit of detection. A statistically significant correlation was detected between elevated mercury concentrations and racial and ethnic characteristics of the study participants. Non-Hispanic African-American mothers were 9.6 times more likely to have a mercury concentration > or = 5.8 microg/L compared to women of other racial/ethnic backgrounds. No association was detected between elevated mercury levels and adverse birth outcomes.

Insights from biosurveillance: non-fatal opioid overdoses in Rhode Island 2019-21.

BACKGROUND AND AIMS: Opioids biosurveillance is a new approach to public health surveillance of non-fatal overdoses that relies upon laboratory analysis of residual biospecimens from hospitals treating opioids overdoses. In Rhode Island (RI), USA, hospitals report suspected opioid overdoses to the Department of Health. Residual specimens associated with these overdoses are submitted to the State Health Laboratories for further characterization. This surveillance project aimed to characterize non-fatal overdoses through toxicological testing of urine specimens associated with non-fatal overdoses during the initial 2-year period of biosurveillance implementation in RI to assess the feasibility and public health utility of this approach. METHODS: This study included individuals who presented for treatment for a suspected opioid overdose in 10 RI hospitals between July 2019 and June 2021. Urine samples were received for 1354 unique overdose encounters corresponding to reported overdoses. Some individuals experienced multiple overdoses during this time. Urine samples were extracted and then analyzed by liquid chromatography tandem mass spectrometry with a panel consisting of 1033 opiates, synthetic opioids, fentanyl analogs and select metabolites. Temporal and spatial trends were evaluated for the studied population. RESULTS: A total of 1354 samples were tested for the presence of opioids in urine collected from individuals who experienced a suspected overdose. Fentanyl (and/or norfentanyl) was present in 79% of all samples in which opioids were found (n = 1033). Fentanyl analogs varied in their contribution to these totals, with observations ranging as high as 35% of all opioid-containing samples in August 2019 and May 2021. CONCLUSIONS: Laboratory identification of opioids involved in suspected overdoses shows that fentanyl and its analogs are the main drivers of the opioids overdose epidemic in Rhode Island, USA. The biosurveillance approach is unique in its ability to quantify contributions of novel fentanyl analogs to the burden of overdoses.

SARS-CoV-2 Variants in Rhode Island; May 2022 Update.

BACKGROUND: Genomic surveillance allows identification of circulating SARS-CoV-2 variants. We provide an update on the evolution of SARS-CoV-2 in Rhode Island (RI). METHODS: All publicly available SARS-CoV-2 RI sequences were retrieved from https://www.gisaid.org. Genomic analyses were conducted to identify variants of concern (VOC), variants being monitored (VBM), or non-VOC/non-VBM, and investigate their evolution. RESULTS: Overall, 17,340 SARS-CoV-2 RI sequences were available between 2/2020-5/2022 across five (globally recognized) major waves, including 1,462 (8%) sequences from 36 non-VOC/non-VBM until 5/2021; 10,565 (61%) sequences from 8 VBM between 5/2021-12/2021, most commonly Delta; and 5,313 (31%) sequences from the VOC Omicron from 12/2021 onwards. Genomic analyses demonstrated 71 Delta and 44 Omicron sub-lineages, with occurrence of variant-defining mutations in other variants. CONCLUSION: Statewide SARS-CoV-2 genomic surveillance allows for continued characterization of circulating variants and monitoring of viral evolution, which inform the local health force and guide public health on mitigation efforts against COVID-19.

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.

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.

SARS-CoV-2 Variants in Rhode Island.

COVID-19 is a worldwide public health emergency caused by SARS-CoV-2. Genomic surveillance of SARS-CoV-2 emerging variants is important for pandemic monitoring and informing public health responses. Through an interstate academic-public health partnership, we established Rhode Island's capacity to sequence SARS-CoV-2 genomes and created a systematic surveillance program to monitor the prevalence of SARS-CoV-2 variants in the state. We describe circulating SARS-CoV-2 lineages in Rhode Island; provide a timeline for the emerging and expanding contribution of variants of concern (VOC) and variants of interest (VOI), from their first introduction to their eventual predominance over other lineages; and outline the frequent identification of known adaptively beneficial spike protein mutations that appear to have independently arisen in non-VOC/non-VOI lineages. Overall, the described Rhode Island- centric genomic surveillance initiative provides a valuable perspective on SARS-CoV-2 in the state and contributes data of interest for future epidemiological studies and state-to-state comparisons.

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

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant quickly rose to dominance in mid-2021, displacing other variants, including Alpha. Studies using data from the United Kingdom and India estimated that Delta was 40-80% more transmissible than Alpha, allowing Delta to become the globally dominant variant. However, it was unclear if the ostensible difference in relative transmissibility was due mostly to innate properties of Delta's infectiousness or differences in the study populations. To investigate, we formed a partnership with SARS-CoV-2 genomic surveillance programs from all six New England US states. By comparing logistic growth rates, we found that Delta emerged 37-163% faster than Alpha in early 2021 (37% Massachusetts, 75% New Hampshire, 95% Maine, 98% Rhode Island, 151% Connecticut, and 163% Vermont). We next computed variant-specific effective reproductive numbers and estimated that Delta was 58-120% more transmissible than Alpha across New England (58% New Hampshire, 68% Massachusetts, 76% Connecticut, 85% Rhode Island, 98% Maine, and 120% Vermont). Finally, using RT-PCR data, we estimated that Delta infections generate on average ∼6 times more viral RNA copies per mL than Alpha infections. Overall, our evidence indicates that Delta's enhanced transmissibility could be attributed to its innate ability to increase infectiousness, but its epidemiological dynamics may vary depending on the underlying immunity and behavior of distinct populations.

Rhode Island Unintentional Drug Overdose Death Trends and Ranking - Office of the State Medical Examiners Database.

[Full article available at http://rimed.org/rimedicaljournal-2018-02.asp].

The Use of a Shared Services Model for Mycobacteriology Testing: Lessons Learned.

OBJECTIVES: Public health laboratories (PHLs) provide essential services in the diagnosis and surveillance of diseases of public health concern, such as tuberculosis. Maintaining access to high-quality laboratory testing is critical to continued disease detection and decline of tuberculosis cases in the United States. We investigated the practical experience of sharing tuberculosis testing services between PHLs through the Shared Services Project. METHODS: The Shared Services Project was a 9-month-long project funded through the Association of Public Health Laboratories and the Centers for Disease Control and Prevention during 2012-2013 as a one-time funding opportunity to consortiums of PHLs that proposed collaborative approaches to sharing tuberculosis laboratory services. Submitting PHLs maintained testing while simultaneously sending specimens to reference laboratories to compare turnaround times. RESULTS: During the 9-month project period, 107 Mycobacterium tuberculosis complex submissions for growth-based drug susceptibility testing and molecular detection of drug resistance testing occurred among the 3 consortiums. The median transit time for all submissions was 1.0 day. Overall, median drug susceptibility testing turnaround time (date of receipt in submitting laboratory to result) for parallel testing performed in house by submitting laboratories was 31.0 days; it was 43.0 days for reference laboratories. The median turnaround time for molecular detection of drug resistance results was 1.0 day (mean = 2.8; range, 0-14) from specimen receipt at the reference laboratories. CONCLUSIONS: The shared services model holds promise for specialized tuberculosis testing. Sharing of services requires a balance among quality, timeliness, efficiency, communication, and fiscal costs.

Non-smoking pregnant women and their fetuses are exposed to environmental tobacco smoke as a result of living in multiunit housing.

This study investigates whether pregnant women and their fetuses are exposed to environmental tobacco smoke (ETS) as a result of living in apartments. We measured cotinine concentrations in serum, a biomarker of exposure to ETS, in non-smoking women's umbilical cord blood collected at delivery and in maternal blood drawn shortly after delivering a baby. Concurrently, information was collected regarding the women's housing situation, whether family members or co-workers smoked, and other potential exposure factors. Newborns whose non-smoking mothers lived in an apartment during pregnancy were more than three times (OR 3.17, 95% CI 1.62-6.21) more likely to have detectable levels of cotinine in their cord blood serum than babies whose mothers lived in a detached house. There is a strong association between detectable concentrations of cotinine in cord blood serum and living in an apartment, even after adjusting for confounders, such as exposure at home or at work. A similar association was observed between the detectable levels of cotinine in maternal serum and living in an apartment (OR 1.95, 95% CI 1.03-3.71).

Advancing environmental health surveillance in the US through a national human biomonitoring network.

The United States lacks a comprehensive, nationally-coordinated, state-based environmental health surveillance system. This lack of infrastructure leads to: • varying levels of understanding of chemical exposures at the state & local levels • often inefficient public health responses to chemical exposure emergencies (such as those that occurred in the Flint drinking water crisis, the Gold King mine spill, the Elk river spill and the Gulf Coast oil spill) • reduced ability to measure the impact of public health interventions or environmental policies • less efficient use of resources for cleaning up environmental contamination Establishing the National Biomonitoring Network serves as a step toward building a national, state-based environmental health surveillance system. The Network builds upon CDC investments in emergency preparedness and environmental public health tracking, which have created advanced chemical analysis and information sharing capabilities in the state public health systems. The short-term goal of the network is to harmonize approaches to human biomonitoring in the US, thus increasing the comparability of human biomonitoring data across states and communities. The long-term goal is to compile baseline data on exposures at the state level, similar to data found in CDC's National Report on Human Exposure to Environmental Chemicals. Barriers to success for this network include: available resources, effective risk communication strategies, data comparability & sharing, and political will. Anticipated benefits include high quality data on which to base public health and environmental decisions, data with which to assess the success of public health interventions, improved risk assessments for chemicals, and new ways to prioritize environmental health research.

The Role of the State Health Laboratories in Advancing Health Equity.

While laboratories play an important and recognized role in many public health programs that require surveillance of disease spread or monitoring of environmental conditions, the role of public laboratories in assessing and advancing health equity is not well understood. Yet, public laboratories collect, provide or generate much of the data used to determine health equity status and monitor heath equity trends in multiple settings and disciplines. RI State Health Laboratories, a division of the RI Department of Health, operates programs that help measure and address health disparities. Health equity themes are present in laboratory programs that measure environmental determinants of health and assure equal access to laboratory screening and diagnostic services. This article will review the role of laboratory programs in advancing health equity in the state. Specific examples of laboratory contributions to health equity programs will be provided and examined. Future trends and unmet needs will also be discussed. [Full article available at http://rimed.org/rimedicaljournal-2016-11.asp].

A Model of Shared Mycobacteriology Testing Services: Lessons Learned.

OBJECTIVE: The need for public health laboratories (PHLs) to prioritize resources has led to increased interest in sharing diagnostic services. To address this concept for tuberculosis (TB) testing, the New York State Department of Health Wadsworth Center and the Rhode Island State Health Laboratories assessed the feasibility of shared services for the detection and characterization of Mycobacterium tuberculosis complex (MTBC). METHODS: We assessed multiple aspects of shared services including shipping, testing, reporting, and cost. Rhode Island State Health Laboratories shipped MTBC-positive specimens and isolates to Wadsworth Center. Average turnaround times were calculated and cost analysis was performed. RESULTS: Testing turnaround times were similar at both PHLs; however, the availability of conventional drug susceptibility testing (DST) results for Rhode Island primary specimens and isolates were extended by approximately four days of shipping time. An extended molecular testing panel was performed on every specimen submitted from Rhode Island State Health Laboratories to Wadsworth Center, and the total cost per specimen at Wadsworth Center was $177.12 less than at Rhode Island State Health Laboratories, plus shipping. Following a mid-study review, Wadsworth Center provided testing turnaround times for detection (same day), species determination of MTBC (same day), and molecular DST (2.5 days). CONCLUSION: The collaboration between Wadsworth Center and Rhode Island State Health Laboratories to assess shared services of TB testing highlighted a successful model that may serve as a guideline for other PHLs. The provision of additional rapid testing at a lower cost demonstrated in this study could potentially improve patient management and result in significant cost and resource savings if used in similar models across the country.