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.

Evaluation of the Access Bio CareStart rapid SARS-CoV-2 antigen test in asymptomatic individuals tested at a community mass-testing program in Western Massachusetts.

Point-of-care antigen-detecting rapid diagnostic tests (RDTs) to detect Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) represent a scalable tool for surveillance of active SARS-CoV-2 infections in the population. Data on the performance of these tests in real-world community settings are paramount to guide their implementation to combat the COVID-19 pandemic. We evaluated the performance characteristics of the CareStart COVID-19 Antigen test (CareStart) in a community testing site in Holyoke, Massachusetts. We compared CareStart to a SARS-CoV-2 reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) reference, both using anterior nasal swab samples. We calculated the sensitivity, specificity, and the expected positive and negative predictive values at different SARS-CoV-2 prevalence estimates. We performed 666 total tests on 591 unique individuals. 573 (86%) were asymptomatic. There were 52 positive tests by RT-qPCR. The sensitivity of CareStart was 49.0% (95% Confidence Interval (CI) 34.8-63.4) and specificity was 99.5% (95% CI 98.5-99.9). Among positive RT-qPCR tests, the median cycle threshold (Ct) was significantly lower in samples that tested positive on CareStart. Using a Ct ≤ 30 as a benchmark for positivity increased the sensitivity of the test to 64.9% (95% CI 47.5-79.8). Our study shows that CareStart has a high specificity and moderate sensitivity. The utility of RDTs, such as CareStart, in mass implementation should prioritize use cases in which a higher specificity is more important, such as triage tests to rule-in active infections in community surveillance programs.

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.

A Test-to-Stay Modified Quarantine Program for COVID-19 in Schools.

BACKGROUND AND OBJECTIVES: Mandatory quarantine upon exposure to coronavirus disease 2019 (COVID-19) results in a substantial number of lost days of school. We hypothesized that implementation of a state-wide test-to-stay (TTS) program would allow more students to participate in in-person learning, and not cause additional clusters of COVID-19 cases due to in-school transmission. METHODS: For the 2020-2021 academic year, Massachusetts implemented an opt-in TTS program, in which students exposed to COVID-19 in school are tested each school day with a rapid antigen test. If negative, students may participate in school-related activities that day. Testing occurs daily for a duration of 7 calendar days after exposure. Here, we report the results from the first 13 weeks of the program. RESULTS: A total of 2298 schools signed up for TTS, and 504 167 individuals out of a total population of 860 457 consented. During the first 13 weeks with complete data, 1959 schools activated the program at least once for 102 373 individual, exposed students. Out of 328 271 tests performed, 2943 positive cases were identified (per person positivity rate, 2.9%, 95% confidence interval, 2.8-3.0). A minimum of 325 328 and a maximum of 497 150 days of in-person school were saved through participation in the program. CONCLUSIONS: Daily, rapid on-site antigen testing is a safe and feasible alternative to mandatory quarantine and can be used to maximize safe in-person learning time during the pandemic.

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.

Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) From Asymptomatic and Presymptomatic Individuals in Healthcare Settings Despite Medical Masks and Eye Protection.

We describe 3 instances of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission despite medical masks and eye protection, including transmission despite the source person being masked, transmission despite the exposed person being masked, and transmission despite both parties being masked. Whole genome sequencing confirmed perfect homology between source and exposed persons' viruses in all cases.

Phylogenetic analysis of SARS-CoV-2 in Boston highlights the impact of superspreading events.

Analysis of 772 complete severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from early in the Boston-area epidemic revealed numerous introductions of the virus, a small number of which led to most cases. The data revealed two superspreading events. One, in a skilled nursing facility, led to rapid transmission and significant mortality in this vulnerable population but little broader spread, whereas other introductions into the facility had little effect. The second, at an international business conference, produced sustained community transmission and was exported, resulting in extensive regional, national, and international spread. The two events also differed substantially in the genetic variation they generated, suggesting varying transmission dynamics in superspreading events. Our results show how genomic epidemiology can help to understand the link between individual clusters and wider community spread.

Implementation of SARS-CoV2 Screening in K-12 Schools Using In-School Pooled Molecular Testing and Deconvolution by Rapid Antigen Test.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) testing is one component of a multilayered mitigation strategy to enable safe in-person school attendance for the K-12 school population. However, costs, logistics, and uncertainty about effectiveness are potential barriers to implementation. We assessed early data from the Massachusetts K-12 public school pooled SARS-CoV2 testing program, which incorporates two novel design elements: in-school "pod pooling" for assembling pools of dry anterior nasal swabs from 5 to 10 individuals and positive pool deconvolution using the BinaxNOW antigen rapid diagnostic test (Ag RDT), to assess the operational and analytical feasibility of this approach. Over 3 months, 187,597 individual swabs were tested across 39,297 pools from 738 schools. The pool positivity rate was 0.8%; 98.2% of pools tested negative and 0.2% inconclusive, and 0.8% of pools submitted could not be tested. Of 310 positive pools, 70.6% had an N1 or N2 probe cycle threshold (CT) value of ≤30. In reflex testing (performed on specimens newly collected from members of the positive pool), 92.5% of fully deconvoluted pools with an N1 or N2 target CT of ≤30 identified a positive individual using the BinaxNOW test performed 1 to 3 days later. However, of 124 positive pools with full reflex testing data available for analysis, 32 (25.8%) of BinaxNOW pool deconvolution testing attempts did not identify a positive individual, requiring additional reflex testing. With sufficient staffing support and low pool positivity rates, pooled sample collection and reflex testing were feasible for schools. These early program findings confirm that screening for K-12 students and staff is achievable at scale with a scheme that incorporates in-school pooling, primary testing by reverse transcription-PCR (RT-PCR), and Ag RDT reflex/deconvolution testing.

Cross-Sectional Assessment of SARS-CoV-2 Viral Load by Symptom Status in Massachusetts Congregate Living Facilities.

Transmission of coronavirus disease 2019 (COVID-19) from people without symptoms confounds societal mitigation strategies. From April to June 2020, we tested nasopharyngeal swabs by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) from 15 514 staff and 16 966 residents of nursing homes and assisted living facilities in Massachusetts. Cycle threshold (Ct) distributions were very similar between populations with (n = 739) and without (n = 2179) symptoms at the time of sampling (mean Ct, 25.7 vs 26.4; ranges 12-38). However, as local cases waned, those without symptoms shifted towards higher Ct. With such similar viral load distributions, existing testing modalities should perform comparably regardless of symptoms, contingent upon time since infection.

Performance and Operational Evaluation of the Access Bio CareStart Rapid Antigen Test in a High-Throughput Drive-Through Community Testing Site in Massachusetts.

BACKGROUND: To facilitate deployment of point-of-care testing for severe acute respiratory syndrome coronavirus 2, we evaluated the Access Bio CareStart COVID-19 Antigen test in a high-throughput, drive-through, free community testing site using anterior nasal (AN) swab reverse-transcription polymerase chain reaction (RT-PCR) for clinical testing. METHODS: Consenting symptomatic and asymptomatic children (≤18 years) and adults received dual AN swabs. CareStart testing was performed with temperature/humidity monitoring. All tests had 2 independent reads to assess interoperator agreement. Patients with positive CareStart results were called and instructed to isolate pending RT-PCR results. The paired RT-PCR result was the reference for sensitivity and specificity calculations. RESULTS: Of 1603 participants, 1245 adults and 253 children had paired RT-PCR/CareStart results and complete symptom data. Eighty-three percent of adults and 87% of children were asymptomatic. CareStart sensitivity/specificity were 84.8% (95% confidence interval [CI], 71.1-93.7)/97.2% (95% CI, 92.0-99.4) and 85.7% (95% CI, 42.1-99.6)/89.5% (95% CI, 66.9-98.7) in adults and children, respectively, within 5 days of symptoms. Sensitivity/specificity were 50.0% (95% CI, 41.0-59.0)/99.1% (95% CI, 98.3-99.6) in asymptomatic adults and 51.4% (95% CI, 34.4-68.1)/97.8% (95% CI, 94.5-99.4) in asymptomatic children. Sensitivity in all 234 RT-PCR-positive people was 96.3% with cycle threshold (Ct) ≤25, 79.6% with Ct ≤30, and 61.4% with Ct ≤35. All 21 false-positive CareStart tests had faint but normal bands. Interoperator agreement was 99.5%. Operational challenges included identification of faint test bands and inconsistent swab elution volumes. CONCLUSIONS: CareStart had high sensitivity in people with Ct ≤25 and moderate sensitivity in symptomatic people overall. Specificity was unexpectedly lower in symptomatic versus asymptomatic people. Excellent interoperator agreement was observed, but operational challenges indicate that operator training is warranted.

Performance and Implementation Evaluation of the Abbott BinaxNOW Rapid Antigen Test in a High-Throughput Drive-Through Community Testing Site in Massachusetts.

Rapid diagnostic tests (RDTs) for SARS-CoV-2 antigens (Ag) that can be performed at point of care (POC) can supplement molecular testing and help mitigate the COVID-19 pandemic. Deployment of an Ag RDT requires an understanding of its operational and performance characteristics under real-world conditions and in relevant subpopulations. We evaluated the Abbott BinaxNOW COVID-19 Ag card in a high-throughput, drive-through, free community testing site in Massachusetts using anterior nasal (AN) swab reverse transcriptase PCR (RT-PCR) for clinical testing. Individuals presenting for molecular testing in two of seven lanes were offered the opportunity to also receive BinaxNOW testing. Dual AN swabs were collected from symptomatic and asymptomatic children (≤18 years of age) and adults. BinaxNOW testing was performed in a testing pod with temperature/humidity monitoring. One individual performed testing and official result reporting for each test, but most tests had a second independent reading to assess interoperator agreement. Positive BinaxNOW results were scored as faint, medium, or strong. Positive BinaxNOW results were reported to patients by phone, and they were instructed to isolate pending RT-PCR results. The paired RT-PCR result was the reference for sensitivity and specificity calculations. Of 2,482 participants, 1,380 adults and 928 children had paired RT-PCR/BinaxNOW results and complete symptom data. In this study, 974/1,380 (71%) adults and 829/928 (89%) children were asymptomatic. BinaxNOW had 96.5% (95% confidence interval [CI], 90.0 to 99.3) sensitivity and 100% (95% CI, 98.6 to 100.0) specificity in adults within 7 days of symptoms and 84.6% (95% CI, 65.1 to 95.6) sensitivity and 100% (95% CI, 94.5 to 100.0) specificity in children within 7 days of symptoms. Sensitivity and specificity in asymptomatic adults were 70.2% (95% CI, 56.6 to 81.6) and 99.6% (95% CI, 98.9 to 99.9), respectively, and in asymptomatic children, they were 65.4% (95% CI, 55.6 to 74.4) and 99.0% (95% CI, 98.0 to 99.6), respectively. By cycle threshold (CT ) value cutoff, sensitivity in all subgroups combined (n = 292 RT-PCR-positive individuals) was 99.3% with CT values of ≤25, 95.8% with CT values of ≤30, and 81.2% with CT values of ≤35. Twelve false-positive BinaxNOW results (out of 2,308 tests) were observed; in all 12, the test bands were faint but otherwise normal and were noted by both readers. One invalid BinaxNOW result was identified. Interoperator agreement (positive versus negative BinaxNOW result) was 100% (n = 2,230/2,230 double reads). Each operator was able to process 20 RDTs per hour. In a separate set of 30 specimens (from individuals with symptoms ≤7 days) run at temperatures below the manufacturer's recommended range (46 to 58.5°F), sensitivity was 66.7% and specificity 95.2%. BinaxNOW had very high specificity in both adults and children and very high sensitivity in newly symptomatic adults. Overall, 95.8% sensitivity was observed with CT values of ≤30. These data support public health recommendations for use of the BinaxNOW test in adults with symptoms for ≤7 days without RT-PCR confirmation. Excellent interoperator agreement indicates that an individual can perform and read the BinaxNOW test alone. A skilled laboratorian can perform and read 20 tests per hour. Careful attention to temperature is critical.

A SARS-CoV-2 Cluster in an Acute Care Hospital.

BACKGROUND: Little is known about clusters of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in acute care hospitals. OBJECTIVE: To describe the detection, mitigation, and analysis of a large cluster of SARS-CoV-2 infections in an acute care hospital with mature infection control policies. DESIGN: Descriptive study. SETTING: Brigham and Women's Hospital, Boston, Massachusetts. PARTICIPANTS: Patients and staff with cluster-related SARS-CoV-2 infections. INTERVENTION: Close contacts of infected patients and staff were identified and tested every 3 days, patients on affected units were preemptively isolated and repeatedly tested, affected units were cleaned, room ventilation was measured, and specimens were sent for whole-genome sequencing. A case-control study was done to compare clinical interactions, personal protective equipment use, and breakroom and workroom practices in SARS-CoV-2-positive versus negative staff. MEASUREMENTS: Description of the cluster, mitigation activities, and risk factor analysis. RESULTS: Fourteen patients and 38 staff members were included in the cluster per whole-genome sequencing and epidemiologic associations. The index case was a symptomatic patient in whom isolation was discontinued after 2 negative results on nasopharyngeal polymerase chain reaction testing. The patient subsequently infected multiple roommates and staff, who then infected others. Seven of 52 (13%) secondary infections were detected only on second or subsequent tests. Eight of 9 (89%) patients who shared rooms with potentially contagious patients became infected. Potential contributing factors included high viral loads, nebulization, and positive pressure in the index patient's room. Risk factors for transmission to staff included presence during nebulization, caring for patients with dyspnea or cough, lack of eye protection, at least 15 minutes of exposure to case patients, and interactions with SARS-CoV-2-positive staff in clinical areas. Whole-genome sequencing confirmed that 2 staff members were infected despite wearing surgical masks and eye protection. LIMITATION: Findings may not be generalizable. CONCLUSION: SARS-CoV-2 clusters can occur in hospitals despite robust infection control policies. Insights from this cluster may inform additional measures to protect patients and staff. PRIMARY FUNDING SOURCE: None.