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BackgroundEarly in the COVID-19 pandemic, peak viral loads coincided with symptom onset. We hypothesized that in a highly immune population, symptom onset might occur earlier in infection, coinciding with lower viral loads.MethodsWe assessed SARS-CoV-2 and influenza A viral loads relative to symptom duration in recently-tested adults. Symptomatic participants ≥16y presenting to testing sites in Georgia (4/2022-4/2023; Omicron variant predominant) provided symptom duration. Nasal swab samples were tested by the Xpert Xpress SARS-CoV-2/Flu/RSV assay and Ct values recorded. Nucleoprotein concentrations in SARS-CoV-2 PCR-positive samples were measured by Single Molecule Array. To estimate hypothetical antigen rapid diagnostic test (Ag RDT) sensitivity on each day after symptom onset, percentages of individuals with Ct value ≤30 or ≤25 were calculated.ResultsOf 621 SARS-CoV-2 PCR-positive individuals (64.1% women, median 40.9y), 556/621 (89.5%) had a history of vaccination, natural infection, or both. By both Ct value and antigen concentration measurements, median viral loads rose from the day of symptom onset and peaked on the fourth day. Ag RDT sensitivity estimates were 35.7-71.4% on the first day, 63.9-78.7% on the third day, and 78.6-90.6% on the fourth day of symptoms.In 74 influenza A PCR-positive individuals (55.4% women; median 35.0y), median influenza viral loads peaked on the second day of symptoms.ConclusionsIn a highly immune adult population, median SARS-CoV-2 viral loads peaked on the fourth day of symptoms. Influenza A viral loads peaked soon after symptom onset. These findings have implications for ongoing use of Ag RDTs for COVID-19 and influenza.Key PointsIn a highly immune adult population, median SARS-CoV-2 viral loads by cycle threshold and antigen measurements peaked on the fourth day of symptoms, with implications for testing practice. In contrast, viral loads for influenza A peaked soon after symptom onset.

The Fourth Maastricht Consensus Conference on Thrombosis included the following themes. Theme 1: The “coagulome” as a critical driver of cardiovascular disease. Blood coagulation proteins also play divergent roles in biology and pathophysiology, related to specific organs, including brain, heart, bone marrow, and kidney. Four investigators shared their views on these organ-specific topics. Theme 2: Novel mechanisms of thrombosis. Mechanisms linking factor XII to fibrin, including their structural and physical properties, contribute to thrombosis, which is also affected by variation in microbiome status. Virus infection-associated coagulopathies perturb the hemostatic balance resulting in thrombosis and/or bleeding. Theme 3: How to limit bleeding risks: insights from translational studies. This theme included state-of-the-art methodology for exploring the contribution of genetic determinants of a bleeding diathesis; determination of polymorphisms in genes that control the rate of metabolism by the liver of P2Y12 inhibitors, to improve safety of antithrombotic therapy. Novel reversal agents for direct oral anticoagulants are discussed. Theme 4: Hemostasis in extracorporeal systems: the value and limitations of ex vivo models. Perfusion flow chamber and nanotechnology developments are developed for studying bleeding and thrombosis tendencies. Vascularized organoids are utilized for disease modeling and drug development studies. Strategies for tackling extracorporeal membrane oxygenation-associated coagulopathy are discussed. Theme 5: Clinical dilemmas in thrombosis and antithrombotic management. Plenary presentations addressed controversial areas, i.e., thrombophilia testing, thrombosis risk assessment in hemophilia, novel antiplatelet strategies, and clinically tested factor XI(a) inhibitors, both possibly with reduced bleeding risk. Finally, COVID-19-associated coagulopathy is revisited.

Introduction Pathophysiologic pathways of sickle cell disease (SCD) and air pollution involve inflammation, oxidative stress, and endothelial damage. It is therefore plausible that children with SCD are especially prone to air pollution’s harmful effects. Methods Patient data were collected from a single center, urban/peri-urban cohort of children with confirmed SCD. Daily ambient concentrations of particulate matter (PM ) were collected via satellite-derived remote-sensing technology, and carbon monoxide (CO), nitrogen dioxide (NO ), and ozone from local monitoring stations. We used multivariable regression to quantify associations of pollutant levels and daily counts of emergency department (ED) visits, accounting for weather and time trends. For comparison, we quantified the associations of pollutant levels with daily all-patient (non-SCD) ED visits to our center. Results From 2010-2018, there were 17 731 ED visits by 1740 children with SCD (64.8% HbSS/HbSβ ). Vaso-occlusive events (57.8%), respiratory illness (17.1%), and fever (16.1%) were the most common visit diagnoses. Three-day (lags 0-2) rolling mean PM and CO levels were associated with daily ED visits among those with SCD (PM incident rate ratio (IRR) 1.051 (95% CI 1.010-1.094) per 9.4 µg/m increase; CO 1.088 (1.045-1.132) per 0.5 ppm). NO showed positive associations in secondary analyses; ozone levels were not associated with ED visits. The comparison, all-patient ED visit analyses showed lower IRR for all pollutants. Conclusions Our results suggest short-term air pollution levels as triggers for SCD events and that children with SCD may be more vulnerable to air pollution than those without SCD. Targeted pollution-avoidance strategies could have significant clinical benefits in this population.

Background: At least 5-10% of malignancies occur secondary to an underlying cancer predisposition syndrome (CPS). For patients with a CPS, cancer surveillance is recommended with the goal of identifying malignancy earlier, in a presumably more curable form. Surveillance protocols, including imaging studies, lab work, and procedures, can be complex, differing based on age, gender, and syndrome, which may adversely affect adherence. Mobile health (mHealth) applications have been utilized in the oncology field and could help to facilitate adherence to cancer surveillance protocols. Methods: Applying a user-centered mobile app design approach, patients with a CPS and/or primary caregivers were interviewed to identify current methods for care management and barriers to compliance with recommended surveillance protocols. Broad themes from these interviews informed the design of the mobile app, HomeTown, subsequently evaluated by usability experts. The design was then converted into software code in phases, evaluated by patients and caregivers in an iterative fashion. User population growth and app usage data were assessed. Results: Common themes identified include general distress surrounding surveillance protocol scheduling and results, difficulty remembering medical history, assembling a care team, and seeking resources for self-education. These themes were translated into specific functional app features including push reminders, syndrome-specific surveillance recommendations, ability to annotate visits and results, storage of medical histories, and links to reliable educational resources. Conclusions: Families with CPS demonstrate a desire for mHealth tools to facilitate adherence to cancer surveillance protocols, reduce related distress, relay medical information, and provide educational resources. HomeTown may be a useful tool for engaging this patient population.

Context.— Diagnostic testing for SARS-CoV-2 in symptomatic and asymptomatic children remains integral to care, particularly for supporting return to and attendance in schools. The concordance of SARS-CoV-2 detection in children, using various specimen types, has not been widely studied. Objective.— To compare 3 sample types for SARS-CoV-2 polymerase chain reaction (PCR) testing in children, collected and tested at a single facility. Design.— We prospectively recruited 142 symptomatic and asymptomatic children/young adults into a sample comparison study performed in a single health care system. Each child provided self-collected saliva, and a trained health care provider collected a mid-turbinate nasal swab and nasopharyngeal (NP) swab. Specimens were assayed within 24 hours of collection by using reverse transcription–polymerase chain reaction (RT-PCR) to detect SARS-CoV-2 on a single testing platform. Results.— Concurrently collected saliva and mid-turbinate swabs had greater than 95% positive agreement with NP swabs when obtained within 10 days of symptom onset. Positive agreement of saliva and mid-turbinate samples collected from children with symptom onset >10 days prior, or without symptoms, was 82% compared to NP swab samples. Cycle threshold (Ct) values for mid-turbinate nasal samples more closely correlated with Ct values from NP samples than from saliva samples. Conclusions.— These findings suggest that all 3 sample types from children are useful for SARS-CoV-2 diagnostic testing by RT-PCR, and that concordance is greatest when the child has had symptoms of COVID-19 within the past 10 days. This study provides scientific justification for using sample types other than the NP swab for SARS-CoV-2 testing in pediatric populations.

Traditional cellular and live-virus methods for detection of SARS-CoV-2 neutralizing antibodies (nAbs) are labor- and time-intensive, and thus not suited for routine use in the clinical lab to predict vaccine efficacy and natural immune protection. Here, we report the development and validation of a rapid, high throughput method for measuring SARS-CoV-2 nAbs against native-like trimeric spike proteins. This assay uses a blockade of hACE-2 binding (BoAb) approach in an automated digital immunoassay on the Quanterix HD-X platform. BoAb assays using vaccine and delta variant viral strains showed strong correlation with cell-based pseudovirus and live-virus neutralization activity. Importantly, we were able to detect similar patterns of delta variant resistance to neutralization in samples with paired vaccine and delta variant BoAb measurements. Finally, we screened clinical samples from patients with or without evidence of SARS-CoV-2 exposure by a single-dilution screening version of our assays, finding significant nAb activity only in exposed individuals. In principle, these assays offer a rapid, robust, and scalable alternative to time-, skill-, and cost-intensive standard methods for measuring SARS-CoV-2 nAb levels.

Background Coronavirus disease 2019 (COVID-19) testing policies for symptomatic children attending US schools or daycare vary, and whether isolated symptoms should prompt testing is unclear. We evaluated children presenting for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing to determine if the likelihood of having a positive SARS-CoV-2 test differed between participants with 1 symptom vs ≥2 symptoms, and to examine the predictive capability of isolated symptoms. Methods Participants aged Results Of 602 participants, 21.8% tested positive and 48.7% had a known or suspected close contact. Children reporting 1 symptom (n = 82; odds ratio [OR], 6.00 [95% confidence interval {CI}, 2.70–13.33]) and children reporting ≥2 symptoms (n = 365; OR, 5.25 [95% CI, 2.66–10.38]) were significantly more likely to have a positive COVID-19 test than asymptomatic children (n = 155), but they were not significantly different from each other (OR, 0.88 [95% CI, .52–1.49]). Sensitivity and PPV were highest for isolated fever (33% and 57%, respectively), cough (25% and 32%), and sore throat (21% and 45%); headache had low sensitivity (8%) but higher PPV (33%). Sensitivity and PPV of isolated congestion/rhinorrhea were 8% and 9%, respectively. Conclusions With high Delta variant prevalence, children with isolated symptoms were as likely as those with multiple symptoms to test positive for COVID-19. Isolated fever, cough, sore throat, or headache, but not congestion/rhinorrhea, offered the highest predictive value.

BackgroundCOVID-19 testing policies for symptomatic children attending U.S. schools or daycare vary, and whether isolated symptoms should prompt testing is unclear. We evaluated children presenting for SARS-CoV-2 testing to determine if the likelihood of having a positive SARS-CoV-2 test differed between participants with one versus ≥2 symptoms, and to examine the predictive capability of isolated symptoms.MethodsParticipants ≤ 18 years presenting for clinical SARS-CoV-2 molecular testing in six sites in urban/suburban/rural Georgia (July-October, 2021; delta variant predominant) were queried about individual symptoms. Participants were classified into three groups: asymptomatic, one symptom only, or ≥2 symptoms. SARS-CoV-2 test results and clinical characteristics of the three groups were compared. Sensitivity, specificity, and positive/negative predictive values (PPV/NPV) for isolated symptoms were calculated by fitting a saturated Poisson model.ResultsOf 602 participants, 21.8% tested positive and 48.7% had a known or suspected close contact. Children reporting one symptom (n=82; OR=6.00, 95% CI: 2.70-13.33) and children reporting ≥2 symptoms (n=365; OR=5.25: 2.66-10.38) were significantly more likely to have a positive COVID-19 test than asymptomatic children (n=155), but they were not significantly different from each other (OR=0.88: 0.52-1.49). Sensitivity/PPV were highest for isolated fever (33%/57%), cough (25%/32%), and sore throat (21%/45%); headache had low sensitivity (8%) but higher PPV (33%). Sensitivity/PPV of isolated congestion/rhinorrhea were 8%/9%.ConclusionsWith high delta variant prevalence, children with isolated symptoms were as likely as those with multiple symptoms to test positive for COVID-19. Isolated fever, cough, sore throat, or headache, but not congestion/rhinorrhea, offered highest predictive value.Key pointsIn an area with high community prevalence of the delta variant, children presenting with one symptom were as likely as those with two or more symptoms to test positive for SARS-CoV-2 infection. Isolated symptoms should be considered in testing decisions.

Scientific societies and conference secretariats have recently resumed in-person meetings after a long pause owing to the COVID-19 pandemic. Some safety measures continue to be implemented at these in-person events to limit the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). With increased numbers of waves of infection, caused by the emergence of SARS-CoV-2 variants, additional information is needed to ensure maximal safety at in-person events. The MEX-DART case study was conducted at the in-person Hep-DART 2021 conference, which was held in Los Cabos, Mexico, in December 2021. Many COVID-19 safety measures were implemented, and incidence of SARS-CoV-2 infection during the conference was tested onsite. In this study, we highlight the specific conditions and safety measures set in place at the conference. In addition to vaccination requirements, social distancing, and mask wearing, daily rapid testing was implemented for the duration of the conference. At the end of the 4-day meeting, none of the 166 delegates (and family members attending the conference) had tested antigen positive for SARS-CoV-2. Two delegates tested positive in the week after the conference; the timing of their positive test result suggests that they contracted the virus during their travels home or during postconference vacationing. We believe that this model can serve as a helpful template for organizing future in-person meetings in the era of COVID-19 and any other respiratory virus pandemics of the future. While the outcomes of this case study are encouraging, seasonal surges in respiratory virus infections such as SARS-CoV-2, RSV, and influenza virus incidence suggest that continued caution is warranted.

Microsystems have the potential to make an enormous contribution to biomedical and clinical settings. Fully realizing the capabilities of this established technology lies in designing new robust microsystems capable of answering clinically relevant problems. Here, I discuss the creation of the platelet contraction cytometer, a tool that has led to important insights into our understanding of the biomechanical process of clotting and may even represent a new type of diagnostic based on biophysics. Platelets constantly circulate in the bloodstream and respond to injury to prevent excessive blood loss. At a wound, platelets will aggregate on exposed collagen and undergo muscle-like contraction with nascent fibrin polymers to mechanically stabilize the clot and stem hemorrhage. During clot contraction, platelets can dramatically shrink the fibrin mesh and increase the clot stiffness by an order of magnitude. Previous studies have established that the clot stiffness is linked to disease, as overly softened and stiffened clots are associated with bleeding and thrombotic disorders, respectively. However, defining the reasons that clot mechanical properties change in disease has remained difficult, as clots are inherently complex and heterogeneous, and all existing bulk measurements depend on contributions from fibrin stiffness, fibrin architecture, red blood cell incorporation, and platelet contraction. Moreover, changes to the microenvironmental stiffness, biochemical concentration, and shear are known to alter platelet force or behavior at the single cell level. To that end, we developed a “platelet contraction cytometer (PCC)” that simultaneously measures the contractile force of hundreds of individual adherent platelets across the physiological range of mechanical microenvironments within a clot. Within the PCC, a single platelet attaches, spreads, and applies contractile force to a pair of fibrinogen microdots that are attached to a moveable, spring-like, surface. Since the applied platelet contractile force is directly proportional to the microdot displacement, the force may be calculated from a single fluorescent image of the platelet. As this device is microfabricated, thousands of microdots may be created on a single device to enable high-throughput measurements that are performed in a highly controlled mechanical, biochemical, and shear environment. Using this device, we have defined the range of forces that platelets apply within a clot and shown that mechanical and biochemical cues can synergistically combine to create highly contractile platelets. Moreover, we have been able to probe the pathways involved in contraction and shown which pathways control how platelets apply force in response to local stiffness. This device has even enabled new comparative studies that examine how platelet force varies between humans and other animal species, and may lead to new biological insights into how contractile force is generated and regulated. In addition to enabling mechanistic studies, we have collected data showing that platelet contraction cytometry may represent a new class diagnostic that is based on biophysics. In particular, our studies have focused on immune thrombocytopenia (ITP), a disease which is characterized by low platelet counts in the absence of any other cause. Approximately 10% of these patients will have major bleeding issues, but no existing biomarker can determine which patients are at risk for bleeding and whether the disease will self-resolve or become chronic. In a small cohort of patients, we have found that all patients with bleeding and/or bruising symptoms also have significantly lower platelet contractile forces. By defining an average force cutoff value of 26nN, we found that low forces identified bleeding in ITP with 100% sensitivity and 89.4% specificity. Importantly, since the PCC measures single platelets in high throughput, it also provides information on the distribution of forces applied by platelets and has helped to identify unique subpopulations of platelets in ITP. Healthy individuals have a normal distribution of contractile forces with a single peak, while ITP patients tend to have a bimodal distribution with two prominent peaks, one with a lower contractile force and one with a higher contractile force. Surprisingly, patients with bleeding symptoms are missing the highly contractile peak and suggests that the lack of highly contractile platelets may contribute to bleeding. Taken together, this work highlights the incredible potential for microsystems in solving clinical challenges. Designing a device capable of measuring single platelet forces led to new insights into the basic biophysics of platelets and clotting and may also represent a new type of diagnostic.