Nucleocapsid antibody assays can be used to estimate SARS-CoV-2 infection prevalence in regions implementing spike-based COVID-19 vaccines. However, poor sensitivity of nucleocapsid antibody assays in detecting infection after vaccination has been reported. We derived a lower cutoff for identifying previous infections in a large blood donor cohort (N = 142,599) by using the Ortho VITROS Anti-SARS-CoV-2 Total-N Antibody assay, improving sensitivity while maintaining specificity >98%. We validated sensitivity in samples donated after self-reported swab-confirmed infections diagnoses. Sensitivity for first infections in unvaccinated donors was 98.1% (95% CI 98.0-98.2) and for infection after vaccination was 95.6% (95% CI 95.6-95.7) based on the standard cutoff. Regression analysis showed sensitivity was reduced in the Delta compared with Omicron period, in older donors, in asymptomatic infections, <30 days after infection, and for infection after vaccination. The standard Ortho N antibody threshold demonstrated good sensitivity, which was modestly improved with the revised cutoff.

Group testing is the process of testing items as an amalgamation, rather than separately, to determine the binary status for each item. Its use was especially important during the COVID-19 pandemic through testing specimens for SARS-CoV-2. The adoption of group testing for this and many other applications is because members of a negative testing group can be declared negative with potentially only one test. This subsequently leads to significant increases in laboratory testing capacity. Whenever a group testing algorithm is put into practice, it is critical for laboratories to understand the algorithm's operating characteristics, such as the expected number of tests. Our paper presents the binGroup2 package that provides the statistical tools for this purpose. This R package is the first to address the identification aspect of group testing for a wide variety of algorithms. We illustrate its use through COVID-19 and chlamydia/gonorrhea applications of group testing.

BACKGROUND: Alpha-gal syndrome (AGS) is an allergy to galactose-α-1,3-galactose (alpha-gal), a carbohydrate found in most mammals. Evidence indicates that AGS develops following a tick bite, and in the United States, AGS is most associated with bites from Amblyomma americanum (lone star tick); however, not all persons bitten by ticks develop clinical AGS. OBJECTIVE: This study investigated intrinsic risk factors associated with the development of AGS. METHODS: We performed a case-control study among adults presenting for diagnosis or management of AGS at an allergy clinic in North Carolina during 2019-2020 and compared them to controls enrolled from two nearby internal medicine clinics. A questionnaire gathered epidemiologic and tick exposure data and blood was obtained for alpha-gal specific IgE (sIgE) and other testing. RESULTS: The 82 enrolled case patients and 191 controls did not differ significantly by age or sex. Case patients were more likely than controls to have A or O blood types (non-B-antigen), have experienced childhood allergies, and have a family history of AGS and other food allergies. Case patients were also more likely to report experiencing long healing times for insect bites or stings and a family history of allergy to stinging or biting insects. CONCLUSION: This study suggests that intrinsic factors contribute to risk of developing AGS. Some traits are genetic, but common behaviors among households and family units likely also contribute. Identification of these risk factors can inform personal risk, aid healthcare providers in understanding susceptible populations, and contribute to ongoing understanding of AGS epidemiology.

California serogroup viruses (CSGVs) of medical importance in the United States include La Crosse virus, Jamestown Canyon virus (JCV), California encephalitis virus, and snowshoe hare virus. Current diagnosis of CSGVs relies heavily on serologic techniques for detecting immunoglobulin M (IgM), an indication of a recent CSGV infection. However, human-positive control sera reactive to viruses in the serogroup are scarce because detection of recent infections is rare. Here, we describe the development of new murine monoclonal antibodies (MAbs) reactive to CSGVs and the engineering of a human-murine chimeric antibody by combining the variable regions of the broadly CSGV cross-reactive murine MAb, 3-3B6/2-3B2 and the constant region of the human IgM. MAb 3-3B6/2-3B2 recognizes a tertiary epitope on the Gn/Gc heterodimer, and epitopes important in JCV neutralization were mapped to the Gc glycoprotein. This engineered human IgM constitutively expressed in a HEK-293 stable cell line can replace human-positive control sera in diagnostic serological techniques such as IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA). Compared to the parent murine MAbs, the human-chimeric IgM antibody had identical serological activity to CSGVs in ELISA and demonstrated equivalent reactivity compared to human immune sera in the MAC-ELISA.IMPORTANCEOrthobunyaviruses in the California serogroup cause severe neurological disease in children and adults. While these viruses are known to circulate widely in North America, their occurrence is rare. Serological testing for CSGVs is hindered by the limited availability and volumes of human-positive specimens needed as controls in serologic assays. Here, we described the development of a murine monoclonal antibody cross-reactive to CSGVs engineered to contain the variable regions of the murine antibody on the backbone of human IgM. The chimeric IgM produced from the stably expressing HEK293 cell line was evaluated for use as a surrogate human-positive control in a serologic diagnostic test.

SARS-CoV-2 serosurveys can estimate cumulative incidence for monitoring epidemics but require characterization of employed serological assays performance to inform testing algorithm development and interpretation of results. We conducted a multi-laboratory evaluation of 21 commercial high-throughput SARS-CoV-2 serological assays using blinded panels of 1,000 highly-characterized blood-donor specimens. Assays demonstrated a range of sensitivities (96%-63%), specificities (99%-96%) and precision (IIC 0.55-0.99). Durability of antibody detection in longitudinal samples was dependent on assay format and immunoglobulin target, with anti-spike, direct, or total Ig assays demonstrating more stable, or increasing reactivity over time than anti-nucleocapsid, indirect, or IgG assays. Assays with high sensitivity, specificity and durable antibody detection are ideal for serosurveillance. Less sensitive assays demonstrating waning reactivity are appropriate for other applications, including characterizing antibody responses after infection and vaccination, and detection of anamnestic boosting by reinfections and vaccine breakthrough infections. Assay performance must be evaluated in the context of the intended use.Competing Interest StatementThe authors have declared no competing interest.Funding StatementThis work was supported by research contracts from the Centers for Disease Control and Prevention (CDC Contract 75D30120C08170).Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:All blood donors consented to use of de-identified, residual specimens for further research purposes. UCSF IRB provided explicit approval for VRI self-certification that use of the de-identified CCP donations in this study does not meet the criteria for human subjects research. CDC investigators reviewed and relied on this determination as consistent with applicable federal law and CDC policy (45 C.F.R. part 46, 21 C.F.R. part 56; 42 U.S.C. Sect. 241(d); 5 U.S.C. Sect. 552a; 44 U.S.C. Sect. 3501).All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesThe analytic data set is available upon request.

Emerging epidemics are challenging to track. Only a subset of cases is recognized and reported, as seen with the Zika virus (ZIKV) epidemic where large proportions of infection were asymptomatic. However, multiple imperfect indicators of infection provide an opportunity to estimate the underlying incidence of infection. We developed a modeling approach that integrates a generic Time-series Susceptible-Infected-Recovered epidemic model with assumptions about reporting biases in a Bayesian framework and applied it to the 2016 Zika epidemic in Puerto Rico using three indicators: suspected arboviral cases, suspected Zika-associated Guillain-Barré Syndrome cases, and blood bank data. Using this combination of surveillance data, we estimated the peak of the epidemic occurred during the week of August 15, 2016 (the 33rd week of year), and 120 to 140 (50% credible interval [CrI], 95% CrI: 97 to 170) weekly infections per 10,000 population occurred at the peak. By the end of 2016, we estimated that approximately 890,000 (95% CrI: 660,000 to 1,100,000) individuals were infected in 2016 (26%, 95% CrI: 19% to 33%, of the population infected). Utilizing multiple indicators offers the opportunity for real-time and retrospective situational awareness to support epidemic preparedness and response.Competing Interest StatementThe authors have declared no competing interest.Funding StatementThe author(s) received no specific funding for this work.Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:Exemption was obtained from the CDC Human Subjects Research Office as the data were collected as part of regular surveillance activities.All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesAll relevant data are within the manuscript and its Supporting Information files.

SARS-CoV-2 seroprevalence studies are instrumental in monitoring epidemic activity and require well-characterized, high-throughput assays, and appropriate testing algorithms. The U.S. Nationwide Blood Donor Seroprevalence Study performed monthly cross-sectional serological testing from July 2020 to December 2021, implementing evolving testing algorithms in response to changes in pandemic activity. With high vaccine uptake, anti-Spike (S) reactivity rates reached >80% by May 2021, and the study pivoted from reflex Roche anti-nucleocapsid (NC) testing of Ortho S-reactive specimens to parallel Ortho S/NC testing. We evaluated the performance of the Ortho NC assay as a replacement for the Roche NC assay and compared performance of parallel S/NC testing on both platforms. Qualitative and quantitative agreement of Ortho NC with Roche NC assays was evaluated on preselected S/NC concordant and discordant specimens. All 190 Ortho S+/Roche NC+ specimens were reactive on the Ortho NC assay; 34% of 367 Ortho S+/Roche NC- specimens collected prior to vaccine availability and 43% of 37 Ortho S-/Roche NC+ specimens were reactive on the Ortho NC assay. Performance of parallel S/NC testing using Ortho and Roche platforms was evaluated on 200 specimens collected in 2019 and 3,903 study specimens collected in 2021. All 200 pre-COVID-19 specimens tested negative on the four assays. Cross-platform agreement between Roche and Ortho platforms was 96.4% (3,769/3,903); most discordant results had reactivity close to the cutoffs on the alternate assays. These findings, and higher efficiency and throughput, support the use of parallel S/NC testing on either Roche or Ortho platforms for large serosurveillance studies. IMPORTANCE Seroprevalence studies like the U.S. Nationwide Blood Donor Seroprevalence Study (NBDS) have been critical in monitoring SARS-CoV-2 epidemic activity. These studies rely on serological assays to detect antibodies indicating prior infection. It is critical that the assays and testing algorithms used in seroprevalence studies have adequate performance (high sensitivity, high specificity, ability to discriminate vaccine-induced and infection-induced antibodies, etc.), as well as appropriate characteristics to support large-scale studies, such as high throughput and low cost. In this study we evaluated the performance of Ortho's anti-nucleocapsid assay as a replacement for the Roche anti-nucleocapsid assay and compared performance of parallel anti-spike and anti-nucleocapsid testing on both platforms. These data demonstrate similar performance of the Ortho and Roche anti-nucleocapsid assays and that parallel anti-spike and anti-nucleocapsid testing on either platform could be used for serosurveillance applications.

I congratulate the authors on an interesting, instructive, and timely paper. Certainly, including the testing for SARS‐CoV‐2 example in a paper on a topic—group testing—of wide interest during the current COVID‐19 pandemic emphasizes the relevance of the topic in public health and diagnostic medicine generally. But the thoughtful inclusion of the remaining examples—HIV, HPV, and cancer biomarker detection—illustrates that the issues considered are important even in less urgent times. In this note of discussion I will comment on two particular aspects in the paper that I found particularly informative, and then I will comment on two other, related areas that I believe the conclusions inform.

In May 2019, the Food and Drug Administration issued approval for Dengvaxia (Sanofi Pasteur), a live-attenuated, chimeric tetravalent dengue vaccine (1). In June 2021, the Advisory Committee on Immunization Practices (ACIP) recommended vaccination with Dengvaxia for children and adolescents aged 9–16 years with laboratory confirmation of previous dengue virus infection and who live in areas with endemic dengue transmission, such as the U.S. Virgin Islands (USVI)† (2). Confirming previous dengue virus infection before vaccine administration (prevaccination screening) is important because 1) although Dengvaxia decreases hospitalization and severe disease from dengue among persons with a previous infection, it increases the risk for these outcomes among persons without a previous infection; 2) many dengue virus infections are asymptomatic; and 3) many patients with symptomatic infections do not seek medical attention or receive appropriate testing (3). Sufficient laboratory evidence of previous dengue virus infection includes a history of laboratory-confirmed dengue§ or a positive serologic test result that meets ACIP-recommended performance standards for prevaccination screening, defined as high specificity (≥98%) and sensitivity (≥75%). A seroprevalence of 20% in the vaccine-eligible population (corresponding to a positive predictive value of ≥90% for a test with minimum sensitivity of 75% and minimum specificity of 98%) is recommended to maximize vaccine safety and minimize the risk for vaccinating persons without a previous dengue virus infection (2).

In 2020, Montana, USA, reported a large increase in Colorado tick fever (CTF) cases. To investigate potential causes of the increase, we conducted a case-control study of Montana residents who tested positive or negative for CTF during 2020, assessed healthcare providers' CTF awareness and testing practices, and reviewed CTF testing methods. Case-patients reported more time recreating outdoors on weekends, and all reported finding a tick on themselves before illness. No consistent changes were identified in provider practices. Previously, only CTF serologic testing was used in Montana. In 2020, because of SARS-CoV-2 testing needs, the state laboratory sent specimens for CTF testing to the Centers for Disease Control and Prevention, where more sensitive molecular methods are used. This change in testing probably increased the number of CTF cases detected. Molecular testing is optimal for CTF diagnosis during acute illness. Tick bite prevention measures should continue to be advised for persons doing outdoor activities.

BACKGROUND: The disaccharide galactose-α-1,3-galactose (alpha-gal) is expressed in mammals other than humans, apes, and old-world monkeys. In humans, elevated immunoglobulin E (IgE) antibodies specific for alpha-gal can result in allergic hypersensitivity known as alpha-gal syndrome (AGS). Case reports and series suggest that tick bites can induce alpha-gal-specific IgE (sIgE) antibodies. OBJECTIVE: To evaluate tick exposure as a risk factor for AGS and elevated alpha-gal sIgE level. METHODS: We conducted a case-control study comparing patients with AGS from a North Carolina allergy clinic with controls who were patients at a nearby internal medicine clinic. Cases and controls were administered a questionnaire to obtain information about demographics, home environment, outdoor activities, and recollection of tick bite. Serum samples taken at the time of enrollment were tested for total IgE, alpha-gal sIgE, and antibodies to other tick-borne pathogens. RESULTS: The patients with AGS were more likely to recall finding a tick on themselves (odds ratio [OR], 11.20; 95% confidence interval [CI], 4.97-25.15), live near wooded forest (OR, 2.27; 95% CI, 0.92-5.55), and spend 17 or more hours per week outdoors in wooded areas (OR, 5.58; 95% CI, 2.56-12.19). The patients with AGS were also more likely to report 4 or more tick bites (OR, 33.05; 95% CI, 9.92-155.12) and reactions at the site of tick bites (OR, 7.93; 95% CI, 3.74-16.80). Furthermore, elevated alpha-gal sIgE level was observed in 33% of the controls and was associated with tick exposure in the controls (OR, 4.25; 95% CI, 2.21-8.18). CONCLUSION: The results define tick bite as a risk factor for AGS and elevated alpha-gal sIgE level.

BACKGROUND: Current isolation techniques for spotted fever group Rickettsia from clinical samples are laborious and are limited to tissue, blood and blood derivatives with volumes ideally greater than 1 mL. We validated the use of simplified methodologies for spotted fever group Rickettsia culture isolation that overcome sample volume limitations and provide utility in clinical diagnostics and research studies. METHODOLOGY/PRINCIPAL FINDINGS: A modified cell culture method is evaluated for the isolation of Rickettsia ssp. from human diagnostic samples. Culture sampling method, culture platform, and growth phase analysis were evaluated to determine best practices for optimal culture isolation conditions. Rickettsial isolates (R. conorii, R. rickettsii, and R. parkeri) were grown in Vero E6 cells over a course of 5 to 7 days at low inoculum treatments (~40 bacterial copies) to standardize the sampling strategy at a copy number reflective of the bacteremia in acute diagnostic samples. This methodology was verified using small volumes (50 μL) of 25 unprocessed clinical whole blood, plasma, and serum samples from acute samples of patients suspected of having Rocky Mountain Spotted Fever, of which 10 were previously confirmed positive via the PanR8 qPCR assay, 13 had no detectable Rickettsia DNA by the PanR8 qPCR assay, and 2 were not previously tested; these samples resulted in the cultivation of 7 new R. rickettsii isolates. CONCLUSIONS/SIGNIFICANCE: We observed that rickettsial isolate growth in culture is reproducibly identified by real-time PCR testing of culture media within 72 hours after inoculation. Additionally, specimen sedimentation prior to isolation to remove red blood cells was found to decrease the amount of total viable organism available in the inoculum. A small volume culture method was established focusing on comparative qPCR detection rather than bacterial visualization, taking significantly shorter time to detect, and requiring less manipulation compared to traditional clinical isolate culture methods.

BACKGROUND: Alpha-gal syndrome (AGS) is an IgE-mediated allergy to galactose-alpha-1,3-galactose. Clinical presentation ranges from hives to anaphylaxis; episodes typically occur 2-6 hours after exposure to alpha-gal-containing products. In the United States, lone star tick bites are associated with development of AGS. To characterize features of AGS, we evaluated a cohort of patients presenting for care at the University of North Carolina, focusing on symptoms, severity, and identifying features unique to specific alpha-gal-containing product exposures. METHODS: We performed a chart review and descriptive analysis of 100 randomly selected patients with AGS during 2010-2019. RESULTS: Median age at onset was 53years, 56% were female, 95% reported White race, 86% reported a history of tick bite, and 75% met criteria for anaphylaxis based on involvement of 2 organ systems. Those reporting dairy reactions were significantly less likely to report isolated mucocutaneous symptoms (3% vs 24%; ratio [95% CI]: 0.1 [0.1, 0.3]) than those who tolerated dairy, and were more likely to report gastrointestinal symptoms (79% vs 59%; ratio [95% CI]: 1.3 [0.7, 2.6]), although this difference was not statistically significant. Dairy-tolerant patients demonstrated higher alpha-gal sIgE titers (as a percentage of total IgE) than dairy-reactive patients (GM 4.1[95% CI: 2.7, 6.1] vs. GM 2.5 [95% CI: 1.3, 4.8], respectively; ratio - 1.6 [95% CI: -1.0, 3.9]). CONCLUSION: While tick exposure is common in the southern United States, nearly all AGS patients reported a tick bite. Gastrointestinal symptoms were prominent among those reporting reactions to dairy. Anaphylaxis was common, underscoring the severity and need to raise awareness of AGS among patients and providers.

BACKGROUND: Previous SARS-CoV-2 infection and coronavirus disease 2019 (COVID-19) vaccination, independently and combined ("hybrid immunity"), result in partial protection from subsequent infection and strong protection from severe disease. Proportions of the U.S. population that have been infected, vaccinated, or with hybrid immunity remain unclear, posing a challenge for assessing effective pandemic mitigation strategies. METHODS: In this serial cross-sectional study, nationwide blood donor specimens collected during January-December 2021 were tested for spike and nucleocapsid antibodies, and donor COVID-19 vaccination history of ≥1 dose was collected. Monthly seroprevalence induced from SARS-CoV-2 infection, COVID-19 vaccination, or both, were estimated. Estimates were weighted to account for demographic differences from the general population, and were compared temporally and by demographic factors. RESULTS: Overall, 1,123,855 blood samples were assayed. From January to December 2021, the weighted percentage of donations with seropositivity due to: vaccination without previous infection increased from 3.5% (95% CI, 3.4%-3.7%) to 64.0%, (95% CI, 63.5%-64.5%); previous infection without vaccination decreased from 15.6% (95% CI, 15.2%-16.0%) to 11.7% (95% CI, 11.4%-12.0%); hybrid immunity increased from 0.7% (95% CI, 0.6%-0.7%) to 18.9% (95% CI, 18.5%-19.3%); and from infection, vaccination, or both increased from 19.8% (95% CI (19.3-20.2) to 94.5% (95% CI, 93.5%-94.0% 0.1%). Infection- and vaccination-induced antibody responses varied significantly by age, race-ethnicity, and region, but not by gender. CONCLUSIONS: Our results indicate substantial increases in population humoral immunity from SARS-CoV-2 infection, COVID-19 vaccination, and hybrid immunity during 2021. These findings are important to consider in future COVID-19 studies and long-term pandemic mitigation efforts.

Flaviviruses are important human pathogens worldwide. Diagnostic testing for these viruses is difficult because many of the pathogens require specialized biocontainment. To address this issue, we generated 39 virus-like particle (VLP)- and nonstructural protein 1 (NS1)-secreting stable cell lines in HEK-293 cells of 13 different flaviviruses, including dengue, yellow fever, Japanese encephalitis, West Nile, St. Louis encephalitis, Zika, Rocio, Ilheus, Usutu, and Powassan viruses. Antigen secretion was stable for at least 10 cell passages, as measured by enzyme-linked immunosorbent assays and immunofluorescence assays. Thirty-five cell lines (90%) had stable antigen expression over 10 passages, with three of these cell lines (7%) increasing in antigen expression and one cell line (3%) decreasing in antigen expression. Antigen secretion in the HEK-293 cell lines was higher than in previously developed COS-1 cell line counterparts. These antigens can replace current antigens derived from live or inactivated virus for safer use in diagnostic testing. IMPORTANCE Serological diagnostic testing for flaviviral infections is hindered by the need for specialized biocontainment for preparation of reagents and assay implementation. The use of previously developed COS-1 cell lines secreting noninfectious recombinant viral antigen is limited due to diminished antigen secretion over time. Here, we describe the generation of 39 flaviviral virus-like particle (VLP)- and nonstructural protein 1 (NS1)-secreting stable cell lines in HEK-293 cells representing 13 medically important flaviviruses. Antigen production was more stable and statistically higher in these newly developed cell lines than in their COS-1 cell line counterparts. The use of these cell lines for production of flaviviral antigens will expand serological diagnostic testing of flaviviruses worldwide.

Lyme disease and other tick-borne diseases are a major public health threat in the Upper Midwestern United States, including Michigan, Minnesota, and Wisconsin. To prevent tick bites and tick-borne diseases, public health officials commonly recommend personal protective measures and property management techniques. Adoption of tick-borne disease prevention behaviors and practices by individuals are, however, highly variable. We aimed to characterize current tick-borne disease knowledge, attitudes, and prevention behaviors (KAB) practiced by the public in these states, as well as their willingness to use specific tick control methods. We conducted a population-based survey in summer 2019 in 48 high-risk counties (those having a five-year average (2013-2017) Lyme disease incidence of ≥ 10 cases per 100,000 persons per year), in Michigan, Minnesota, and Wisconsin. A total of 2713 surveys were analyzed; survey weights were used to account for household selection probability and post-stratified to match county-level joint age and sex population distributions in population-level inference. An estimated 98% of the population had heard of Lyme disease, with most perceiving it as very or extremely serious (91%); however, only an estimated 25% perceived tick-borne diseases as very or extremely common in their community. Among those who spent time in places with ticks from April through October, an estimated 68% check themselves thoroughly for ticks most of the time or always and 43% use bug repellent on skin or clothing most of the time or always. An estimated 13% of the population had ever treated their property with a pesticide to kill ticks, and 3% had ever used devices that apply pesticide to rodents to kill ticks on their property. Willingness to practice tick bite prevention behaviors, however, was estimated to be much higher; with 82% being willing to perform tick checks at least once a day, and more than 60% willing to use bug repellent, tick control products on pets, or to bathe within two hours of being outdoors. We found that residents would likely be willing to support a county-wide tick control program to reduce the risk of tick-borne disease in their community (81%) or to apply tick control products to their property to reduce the risk of tick-borne disease in their household (79%). Tick checks were more likely to be practiced among participants who perceived tick-borne diseases to be highly prevalent in their community, if they or a household member had been previously diagnosed with a tick-borne disease?, or if they perceived tick exposure to be likely around their home, cabin, or vacation home. In addition, property-based tick control methods were associated with perceived risk of encountering ticks around the home, cabin, or vacation home. Participants who had seen information from state health departments were also more likely to practice preventive measures. The most common reported barriers to using any of these methods were forgetfulness, safety concerns, and lack of awareness. Our survey findings shed light on how residents from these Upper Midwest states may adopt tick control and tick bite prevention measures and how public health outreach may be most effective for this population.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serosurveys can estimate cumulative incidence for monitoring epidemics, requiring assessment of serologic assays to inform testing algorithm development and interpretation of results. We conducted a multilaboratory evaluation of 21 commercial high-throughput SARS-CoV-2 serologic assays using blinded panels of 1,000 highly characterized specimens. Assays demonstrated a range of sensitivities (96%-63%), specificities (99%-96%), and precision (intraclass correlation coefficient 0.55-0.99). Durability of antibody detection was dependent on antigen and immunoglobulin targets; antispike and total Ig assays demonstrated more stable longitudinal reactivity than antinucleocapsid and IgG assays. Assays with high sensitivity, specificity, and durable antibody detection are ideal for serosurveillance, but assays demonstrating waning reactivity are appropriate for other applications, including correlation with neutralizing activity and detection of anamnestic boosting by reinfections. Assay performance must be evaluated in context of intended use, particularly in the context of widespread vaccination and circulation of SARS-CoV-2 variants.

BACKGROUND: Lyme disease incidence is increasing, despite current prevention options. New Lyme disease vaccine candidates are in development, however, investigation of the acceptability of a Lyme disease vaccine among potential consumers is needed prior to any vaccine coming to market. We conducted a population-based, cross-sectional study to estimate willingness to receive a potential Lyme disease vaccine and factors associated with willingness. METHODS: The web-based survey was administered to a random sample of Connecticut, Maryland, Minnesota, and New York residents June-July 2018. Survey-weighted descriptive statistics were conducted to estimate the proportion willing to receive a potential Lyme disease vaccine. Multivariable multinomial logistic regression models were used to quantify the association of sociodemographic characteristics and Lyme disease vaccine attitudes with willingness to be vaccinated. RESULTS: Surveys were completed by 3313 respondents (6% response rate). We estimated that 64% of residents were willing to receive a Lyme disease vaccine, while 30% were uncertain and 7% were unwilling. Compared to those who were willing, those who were uncertain were more likely to be parents, adults 45-65 years old, non-White, have less than a bachelor's degree, or have safety concerns about a potential Lyme disease vaccine. Those who were unwilling were also more likely to be non-White, have less than a bachelor's degree, or have safety concerns about a potential Lyme disease vaccine. In addition, the unwilling had low confidence in vaccines in general, had low perceived risk of contracting Lyme disease, and said they would not be influenced by a positive recommendation from a healthcare provider. DISCUSSION: Overall, willingness to receive a Lyme disease vaccine was high. Effective communication by clinicians regarding safety and other vaccine parameters to those groups who are uncertain will be critical for increasing vaccine uptake and reducing Lyme disease incidence.

BACKGROUND: SARS-CoV-2 virus testing among first responders and healthcare personnel who participated in a May-August 2020 serosurvey which assessed spike protein antibodies (S1 region) provided an opportunity to assess reinfection. METHODS: Serology survey data were merged with virus testing results from Rhode Island (March 1, 2020-February 17, 2021) and New York City (March 10-December 14, 2020). Participants with a positive virus test ≥14 days before their serology test were included. Reinfection was defined as a second positive SARS-CoV-2 test result ≥90 days after the first positive test. The association between serostatus and reinfection was assessed with a proportional hazards model adjusting for demographics, exposures, and virus testing frequency. RESULTS: Among 1,572 previously infected persons, 40 (2.5%) were reinfected. Reinfection differed by serostatus: 8.4% among seronegative versus 1.9% among seropositive participants (p<0.0001). Most reinfections occurred among Rhode Island nursing home and corrections (RINHC) personnel (n=30) who were most frequently tested (mean 30.3 tests versus 4.6 for other Rhode Island and 2.3 for New York City participants). The adjusted hazard ratio (aHR) for reinfection in seropositive versus seronegative persons was 0.41 (95% CI 0.20, 0.81). Exposure to a household member with COVID-19 before the serosurvey was also protective (aHR 0.34, 95% CI 0.13, 0.89). CONCLUSIONS: Reinfections were uncommon among previously infected persons over a 9-month period that preceded widespread variant circulation. Seropositivity decreased reinfection risk. Lower reinfection risk associated with exposure to a household member with COVID-19 before the serosurvey may reflect subsequently reduced household transmission among members of previously infected households.

OBJECTIVES: The number of SARS-CoV-2 infections is underestimated in surveillance data. Various approaches to assess the seroprevalence of antibodies to SARS-CoV-2 have different resource requirements and generalizability. We estimated the seroprevalence of antibodies to SARS-CoV-2 in Denver County, Colorado, via a cluster-sampled community survey. METHODS: We estimated the overall seroprevalence of antibodies to SARS-CoV-2 via a community seroprevalence survey in Denver County in July 2020, described patterns associated with seroprevalence, and compared results with cumulative COVID-19 incidence as reported to the health department during the same period. In addition, we compared seroprevalence as assessed with a temporally and geographically concordant convenience sample of residual clinical specimens from a commercial laboratory. RESULTS: Based on 404 specimens collected through the community survey, 8.0% (95% CI, 3.9%-15.7%) of Denver County residents had antibodies to SARS-CoV-2, an infection rate of about 7 times that of the 1.1% cumulative reported COVID-19 incidence during this period. The estimated infection-to-reported case ratio was highest among children (34.7; 95% CI, 11.1-91.2) and males (10.8; 95% CI, 5.7-19.3). Seroprevalence was highest among males of Black race or Hispanic ethnicity and was associated with previous COVID-19-compatible illness, a previous positive SARS-CoV-2 test result, and close contact with someone who had confirmed SARS-CoV-2 infection. Testing of 1598 residual clinical specimens yielded a seroprevalence of 6.8% (95% CI, 5.0%-9.2%); the difference between the 2 estimates was 1.2 percentage points (95% CI, -3.6 to 12.2 percentage points). CONCLUSIONS: Testing residual clinical specimens provided a similar seroprevalence estimate yet yielded limited insight into the local epidemiology of COVID-19 and might be less representative of the source population than a cluster-sampled community survey. Awareness of the limitations of various sampling strategies is necessary when interpreting findings from seroprevalence assessments.

We estimated the distributions of duration of SARS-CoV-2 nucleic acid shedding and time to reinfection among 137 persons with at least two positive nucleic acid amplification test (NAAT) results from March to September 2020. We analyzed gaps of varying length between subsequent positive and negative NAAT results and estimated a mean duration of nucleic acid shedding of 30.1 (95% CI 26.3, 34.5) days. The mean time to reinfection was 89.1 (95% CI 75.3, 103.5) days. Together, these indicate that a 90-day period between positive NAAT results can reliably define reinfection in immunocompetent persons although reinfection can occur at shorter intervals.

IMPORTANCE: People who have been infected with or vaccinated against SARS-CoV-2 have reduced risk of subsequent infection, but the proportion of people in the US with SARS-CoV-2 antibodies from infection or vaccination is uncertain. OBJECTIVE: To estimate trends in SARS-CoV-2 seroprevalence related to infection and vaccination in the US population. DESIGN, SETTING, AND PARTICIPANTS: In a repeated cross-sectional study conducted each month during July 2020 through May 2021, 17 blood collection organizations with blood donations from all 50 US states; Washington, DC; and Puerto Rico were organized into 66 study-specific regions, representing a catchment of 74% of the US population. For each study region, specimens from a median of approximately 2000 blood donors were selected and tested each month; a total of 1 594 363 specimens were initially selected and tested. The final date of blood donation collection was May 31, 2021. EXPOSURE: Calendar time. MAIN OUTCOMES AND MEASURES: Proportion of persons with detectable SARS-CoV-2 spike and nucleocapsid antibodies. Seroprevalence was weighted for demographic differences between the blood donor sample and general population. Infection-induced seroprevalence was defined as the prevalence of the population with both spike and nucleocapsid antibodies. Combined infection- and vaccination-induced seroprevalence was defined as the prevalence of the population with spike antibodies. The seroprevalence estimates were compared with cumulative COVID-19 case report incidence rates. RESULTS: Among 1 443 519 specimens included, 733 052 (50.8%) were from women, 174 842 (12.1%) were from persons aged 16 to 29 years, 292 258 (20.2%) were from persons aged 65 years and older, 36 654 (2.5%) were from non-Hispanic Black persons, and 88 773 (6.1%) were from Hispanic persons. The overall infection-induced SARS-CoV-2 seroprevalence estimate increased from 3.5% (95% CI, 3.2%-3.8%) in July 2020 to 20.2% (95% CI, 19.9%-20.6%) in May 2021; the combined infection- and vaccination-induced seroprevalence estimate in May 2021 was 83.3% (95% CI, 82.9%-83.7%). By May 2021, 2.1 SARS-CoV-2 infections (95% CI, 2.0-2.1) per reported COVID-19 case were estimated to have occurred. CONCLUSIONS AND RELEVANCE: Based on a sample of blood donations in the US from July 2020 through May 2021, vaccine- and infection-induced SARS-CoV-2 seroprevalence increased over time and varied by age, race and ethnicity, and geographic region. Despite weighting to adjust for demographic differences, these findings from a national sample of blood donors may not be representative of the entire US population.

West Nile virus (WNV) and Zika virus (ZIKV) are mosquito-borne viruses in the family Flaviviridae. Residents in, and travelers to, areas where the viruses are circulating are at risk for infection, and both viruses can cause an acute febrile illness. Given known cross-reactivity in flavivirus serologic assays, it is possible a patient with acute WNV infection could be misdiagnosed as having ZIKV infection if appropriate testing is not conducted. To understand how frequently persons with WNV infection have detectable cross-reactive ZIKV immunoglobulin M (IgM) antibody, we used archived serum samples from patients in the United States with recent WNV infection confirmed by a microsphere-based immunoassay test for IgM antibody and neutralizing antibody testing. Samples were tested for ZIKV IgM antibody with the Centers for Disease Control and Prevention (CDC) ZIKV IgM antibody capture enzyme-linked immunosorbent assay. Among 153 sera from patients with acute WNV infection, the ZIKV IgM antibody result was positive in 56 (37%; 95% confidence interval [CI] 29-44%) and equivocal in 28 (18%; 95% CI 13-25%). With 55% of samples having cross-reactive antibodies, it is important for health care providers to request appropriate testing based on the most likely cause of a patient's possible arboviral infection considering their clinical symptoms and signs, travel history, and place of residence. For cases where the epidemiology does not support the preliminary IgM findings, confirmatory neutralizing antibody testing should be performed. These measures will avoid an incorrect diagnosis of ZIKV infection, based on cross-reactive antibodies, in a person truly infected with WNV.

Tickborne diseases are an increasing public health problem in the northeastern USA. Bait boxes that apply acaricide to rodents have been shown in small field studies to significantly reduce abundance of Ixodes scapularis ticks as well as their pathogen infection rates in treated areas. The effectiveness of this intervention for preventing human tickborne diseases (TBDs) has not been demonstrated. During 2012-2016, TickNET collaborators conducted a randomized, blinded, placebo-controlled trial among 622 Connecticut households. Each household received active (containing fipronil wick) or placebo (empty) bait boxes in their yards over two consecutive years. Information on tick encounters and TBDs among household members was collected through biannual surveys. Nymphal ticks were collected from a subset of 100 properties during spring at baseline, during treatment, and in the year post-intervention. Demographic and property characteristics did not differ between treatment groups. There were no significant differences post-intervention between treatment groups with respect to tick density or pathogen infection rates, nor for tick encounters or TBDs among household members. We found no evidence that rodent-targeted bait boxes disrupt pathogen transmission cycles or significantly reduce household risk of tick exposure or TBDs. The effectiveness of this intervention may depend on scale of use or local enzootic cycles.

Dengue viruses (DENV) and Zika virus (ZIKV) are related mosquito-borne flaviviruses with similar disease manifestations, vector ecology and geographic range. The ability to differentiate these viruses serologically is vital due to the teratogenic nature of ZIKV and the potential confounding of pre-existing cross-reactive anti-DENV antibodies. Here we illustrate the kinetics of the IgM neutralizing antibody (NAb) response using longitudinal samples ranging from acute ZIKV infection to late convalescence from individuals with evidence of prior DENV infection. By serially depleting antibody isotypes prior to neutralization assay, we determined that IgM contributes predominantly to ZIKV neutralization, and it is less cross-reactive than the IgG NAb. The IgM NAb peaked around 14 days (95% CI: 13-15) with a median duration of 257 days (95% CI: 133-427). These results demonstrate the persistence of IgM NAb after ZIKV infections and imply its potential role in diagnosis, vaccine evaluation, serosurveillance, and research of flaviviral-host interactions.

BACKGROUND: Nasopharyngeal specimens (NPS) are commonly used for SARS-CoV-2 testing but can be uncomfortable for patients. Self-collected saliva or anterior nasal specimens (ANS) for SARS-CoV-2 detection are less invasive but the sensitivity of these specimen types has not been thoroughly evaluated. METHODS: During September-November 2020, 730 adults undergoing SARS-CoV-2 testing at community testing events and homeless shelters in Denver provided self-collected saliva and ANS specimens before NPS collection and answered a short survey about symptoms and specimen preference. Specimens were tested for SARS-CoV-2 by rRT-PCR; viral culture was performed on a subset of specimens positive by rRT-PCR. Sensitivity of saliva and ANS for SARS-CoV-2 detection by rRT-PCR was measured against NPS. Subgroup analyses included test outcomes by symptom status and culture results. RESULTS: Sensitivity for SARS-CoV-2 detection by rRT-PCR appeared higher for saliva than for ANS (85% vs. 80%) and among symptomatic participants than among those without symptoms (94% vs. 29% for saliva; 87% vs. 50% for ANS). Among participants with culture-positive SARS-CoV-2 by any specimen type, sensitivity of saliva and ANS by rRT-PCR was 94% and 100%, respectively. Saliva and ANS were equally preferred by participants; most would undergo NPS again despite being least preferred. CONCLUSIONS: Saliva was slightly more sensitive than ANS for SARS-CoV-2 detection by rRT-PCR. Both saliva and ANS reliably detected SARS-CoV-2 among participants with symptoms. Self-collected saliva and ANS offer practical advantages, are preferred by patients, and might be most useful for testing people with COVID-19 symptoms.

BACKGROUND: Zika virus (ZIKV) can be transmitted sexually, but the risk of sexual transmission remains unknown. Most evidence of sexual transmission is from partners of infected travelers returning from areas with ZIKV circulation. METHODS: We used data from the U.S. national arboviral disease surveillance system (ArboNET) on travel- and sexually-acquired ZIKV disease cases during 2016-2017 to develop individual-level simulations for estimating risk of male-to-female, male-to-male, and female-to-male sexual transmission of ZIKV via vaginal and/or anal intercourse. We specified parametric distributions to characterize individual-level variability of parameters for ZIKV persistence and sexual behaviors. RESULTS: Using ZIKV RNA persistence in semen/vaginal fluids to approximate infectiousness duration, male-to-male transmission had the highest estimated probability [1.3% (95% CI: 0.4-6.0) per anal sex act], followed by male-to-female and female-to-male transmission [0.4% (95% CI: 0.3-0.6) per vaginal/anal sex act and 0.1% (95% CI:0-0.8) per vaginal sex act, respectively]. Models using viral isolation in semen vs. RNA detection to approximate infectiousness duration predicted greater risk of sexual transmission. CONCLUSIONS: While likely insufficient to maintain sustained transmission, the estimated risk of ZIKV transmission through unprotected sex is not trivial and is especially important for pregnant women, as ZIKV infection can cause severe congenital disorders.

Cat-associated Bartonella species, which include B. henselae, B. koehlerae, and B. clarridgeiae, can cause mild to severe illness in humans. In the present study, we evaluated 1362 serum samples obtained from domestic cats across the U.S. for seroreactivity against three species and two strain types of Bartonella associated with cats (B. henselae type 1, B. henselae type 2, B. koehlerae, and B. clarridgeiae) using an indirect immunofluorescent assay (IFA). Overall, the seroprevalence at the cutoff titer level of ≥1:64 was 23.1%. Seroreactivity was 11.1% and 3.7% at the titer level cutoff of ≥1:128 and at the cutoff of ≥1:256, respectively. The highest observation of seroreactivity occurred in the East South-Central, South Atlantic, West North-Central, and West South-Central regions. The lowest seroreactivity was detected in the East North-Central, Middle Atlantic, Mountain, New England, and Pacific regions. We observed reactivity against all four Bartonella spp. antigens in samples from eight out of the nine U.S. geographic regions.

Emerging epidemics are challenging to track. Only a subset of cases is recognized and reported, as seen with the Zika virus (ZIKV) epidemic where large proportions of infection were asymptomatic. However, multiple imperfect indicators of infection provide an opportunity to estimate the underlying incidence of infection. We developed a modeling approach that integrates a generic Time-series Susceptible-Infected-Recovered epidemic model with assumptions about reporting biases in a Bayesian framework and applied it to the 2016 Zika epidemic in Puerto Rico using three indicators: suspected arboviral cases, suspected Zika-associated Guillain-Barré Syndrome cases, and blood bank data. Using this combination of surveillance data, we estimated the peak of the epidemic occurred during the week of August 15, 2016 (the 33rd week of year), and 120 to 140 (50% credible interval [CrI], 95% CrI: 97 to 170) weekly infections per 10,000 population occurred at the peak. By the end of 2016, we estimated that approximately 890,000 (95% CrI: 660,000 to 1,100,000) individuals were infected in 2016 (26%, 95% CrI: 19% to 33%, of the population infected). Utilizing multiple indicators offers the opportunity for real-time and retrospective situational awareness to support epidemic preparedness and response.

In the estimation of proportions by pooled testing, the MLE is biased. Hepworth and Biggerstaff (JABES, 22:602–614, 2017) proposed an estimator based on the bias correction method of Firth (Biometrika 80:27–38, 1993) and showed that it is almost unbiased across a range of pooled testing problems involving no misclassification. We now extend their work to allow for imperfect testing. We derive the estimator, provide a Newton–Raphson iterative formula for its computation and test it in situations involving equal or unequal pool sizes, drawing on problems encountered in plant disease assessment and prevalence estimation of mosquito-borne viruses. Our estimator is highly effective at reducing the bias for prevalences consistent with the pooled testing procedure employed. © 2020, This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.