Coast-to-Coast Spread of SARS-CoV-2 during the Early Epidemic in the United States.

The novel coronavirus SARS-CoV-2 was first detected in the Pacific Northwest region of the United States in January 2020, with subsequent COVID-19 outbreaks detected in all 50 states by early March. To uncover the sources of SARS-CoV-2 introductions and patterns of spread within the United States, we sequenced nine viral genomes from early reported COVID-19 patients in Connecticut. Our phylogenetic analysis places the majority of these genomes with viruses sequenced from Washington state. By coupling our genomic data with domestic and international travel patterns, we show that early SARS-CoV-2 transmission in Connecticut was likely driven by domestic introductions. Moreover, the risk of domestic importation to Connecticut exceeded that of international importation by mid-March regardless of our estimated effects of federal travel restrictions. This study provides evidence of widespread sustained transmission of SARS-CoV-2 within the United States and highlights the critical need for local surveillance.

Diverse functional autoantibodies in patients with COVID-19.

COVID-19 manifests with a wide spectrum of clinical phenotypes that are characterized by exaggerated and misdirected host immune responses1-6. Although pathological innate immune activation is well-documented in severe disease1, the effect of autoantibodies on disease progression is less well-defined. Here we use a high-throughput autoantibody discovery technique known as rapid extracellular antigen profiling7 to screen a cohort of 194 individuals infected with SARS-CoV-2, comprising 172 patients with COVID-19 and 22 healthcare workers with mild disease or asymptomatic infection, for autoantibodies against 2,770 extracellular and secreted proteins (members of the exoproteome). We found that patients with COVID-19 exhibit marked increases in autoantibody reactivities as compared to uninfected individuals, and show a high prevalence of autoantibodies against immunomodulatory proteins (including cytokines, chemokines, complement components and cell-surface proteins). We established that these autoantibodies perturb immune function and impair virological control by inhibiting immunoreceptor signalling and by altering peripheral immune cell composition, and found that mouse surrogates of these autoantibodies increase disease severity in a mouse model of SARS-CoV-2 infection. Our analysis of autoantibodies against tissue-associated antigens revealed associations with specific clinical characteristics. Our findings suggest a pathological role for exoproteome-directed autoantibodies in COVID-19, with diverse effects on immune functionality and associations with clinical outcomes.

Sex differences in immune responses that underlie COVID-19 disease outcomes.

There is increasing evidence that coronavirus disease 2019 (COVID-19) produces more severe symptoms and higher mortality among men than among women1-5. However, whether immune responses against severe acute respiratory syndrome coronavirus (SARS-CoV-2) differ between sexes, and whether such differences correlate with the sex difference in the disease course of COVID-19, is currently unknown. Here we examined sex differences in viral loads, SARS-CoV-2-specific antibody titres, plasma cytokines and blood-cell phenotyping in patients with moderate COVID-19 who had not received immunomodulatory medications. Male patients had higher plasma levels of innate immune cytokines such as IL-8 and IL-18 along with more robust induction of non-classical monocytes. By contrast, female patients had more robust T cell activation than male patients during SARS-CoV-2 infection. Notably, we found that a poor T cell response negatively correlated with patients' age and was associated with worse disease outcome in male patients, but not in female patients. By contrast, higher levels of innate immune cytokines were associated with worse disease progression in female patients, but not in male patients. These findings provide a possible explanation for the observed sex biases in COVID-19, and provide an important basis for the development of a sex-based approach to the treatment and care of male and female patients with COVID-19.

Longitudinal analyses reveal immunological misfiring in severe COVID-19.

Recent studies have provided insights into the pathogenesis of coronavirus disease 2019 (COVID-19)1-4. However, the longitudinal immunological correlates of disease outcome remain unclear. Here we serially analysed immune responses in 113 patients with moderate or severe COVID-19. Immune profiling revealed an overall increase in innate cell lineages, with a concomitant reduction in T cell number. An early elevation in cytokine levels was associated with worse disease outcomes. Following an early increase in cytokines, patients with moderate COVID-19 displayed a progressive reduction in type 1 (antiviral) and type 3 (antifungal) responses. By contrast, patients with severe COVID-19 maintained these elevated responses throughout the course of the disease. Moreover, severe COVID-19 was accompanied by an increase in multiple type 2 (anti-helminths) effectors, including interleukin-5 (IL-5), IL-13, immunoglobulin E and eosinophils. Unsupervised clustering analysis identified four immune signatures, representing growth factors (A), type-2/3 cytokines (B), mixed type-1/2/3 cytokines (C), and chemokines (D) that correlated with three distinct disease trajectories. The immune profiles of patients who recovered from moderate COVID-19 were enriched in tissue reparative growth factor signature A, whereas the profiles of those with who developed severe disease had elevated levels of all four signatures. Thus, we have identified a maladapted immune response profile associated with severe COVID-19 and poor clinical outcome, as well as early immune signatures that correlate with divergent disease trajectories.

Serological profiling of pneumococcal proteins reveals unique patterns of acquisition, maintenance and waning of antibodies throughout life.

Streptococcus pneumoniae is a leading cause of morbidity and mortality in children and older adults. Yet knowledge on the development of pneumococcal protein-specific antibody responses throughout life is limited. To investigate this, we measured serum IgG levels to 55 pneumococcal proteins in 11-month old infants (n=73), 24-month old children (n=101), parents (n=99), adults without children <6 years of age (n= 99) and older adults aged >60 years (n=100). Our findings revealed low IgG levels in infancy, with distinct development patterns peaking in adults. A decrease in levels was observed for 27 antigens towards older age. Adults and older adults had increased IgG levels during pneumococcal carriage and at increased exposure risk to S. pneumoniae. Carriage was a stronger predictor than exposure or age for antibody responses. These findings highlight the dynamic nature of naturally acquired humoral immunity to pneumococcal proteins throughout life, offering insights for age-targeted interventions.

Association of Upper Respiratory Streptococcus pneumoniae Colonization With Severe Acute Respiratory Syndrome Coronavirus 2 Infection Among Adults.

BACKGROUND: Streptococcus pneumoniae interacts with numerous viral respiratory pathogens in the upper airway. It is unclear whether similar interactions occur with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: We collected saliva specimens from working-age adults undergoing SARS-CoV-2 molecular testing at outpatient clinics and via mobile community-outreach testing between July and November 2020 in Monterey County, California. After bacterial culture enrichment, we tested for pneumococci by means of quantitative polymerase chain reaction targeting the lytA and piaB genes, and we measured associations with SARS-CoV-2 infection using conditional logistic regression. RESULTS: Analyses included 1278 participants, with 564 enrolled in clinics and 714 enrolled through outreach-based testing. The prevalence of pneumococcal carriage was 9.2% (117 of 1278) among all participants (11.2% [63 of 564] in clinic-based testing and 7.6% [54 of 714] in outreach-based testing). The prevalence of SARS-CoV-2 infection was 27.4% (32 of 117) among pneumococcal carriers and 9.6% (112 of 1161) among noncarriers (adjusted odds ratio [aOR], 2.73 [95% confidence interval (CI): 1.58-4.69). Associations between SARS-CoV-2 infection and pneumococcal carriage were enhanced in the clinic-based sample (aOR, 4.01 [95% CI: 2.08-7.75]) and among symptomatic participants (3.38 [1.35-8.40]), compared with findings within the outreach-based sample and among asymptomatic participants. The adjusted odds of SARS-CoV-2 coinfection increased 1.24-fold (95% CI: 1.00-1.55-fold) for each 1-unit decrease in piaB quantitative polymerase chain reaction cycle threshold value among pneumococcal carriers. Finally, pneumococcal carriage modified the association of SARS-CoV-2 infection with recent exposure to a suspected coronavirus disease 2019 case (aOR, 7.64 [95% CI: 1.91-30.7] and 3.29 [1.94-5.59]) among pneumococcal carriers and noncarriers, respectively). CONCLUSIONS: Associations of pneumococcal carriage detection and density with SARS-CoV-2 suggest a synergistic relationship in the upper airway. Longitudinal studies are needed to determine interaction mechanisms between pneumococci and SARS-CoV-2.

Saliva as an alternative sample type for detection of pneumococcal carriage in young children.

For children, the gold standard for the detection of pneumococcal carriage is conventional culture of a nasopharyngeal swab. Saliva, however, has a history as one of the most sensitive methods for surveillance of pneumococcal colonization and has recently been shown to improve carriage detection in older age groups. Here, we compared the sensitivity of paired nasopharyngeal and saliva samples from PCV7-vaccinated 24-month-old children for pneumococcal carriage detection using conventional and molecular detection methods. Nasopharyngeal and saliva samples were collected from 288 24-month-old children during the autumn/winter, 2012/2013. All samples were first processed by conventional diagnostic culture. Next, DNA extracted from all plate growth was tested by qPCR for the presence of the pneumococcal genes piaB and lytA and a subset of serotypes. By culture, 161/288 (60 %) nasopharyngeal swabs tested positive for pneumococcus, but detection was not possible from saliva due to abundant polymicrobial growth on culture plates. By qPCR, 155/288 (54 %) culture-enriched saliva samples and 187/288 (65 %) nasopharyngeal swabs tested positive. Altogether, 219/288 (76 %) infants tested positive for pneumococcus, with qPCR-based carriage detection of culture-enriched nasopharyngeal swabs detecting significantly more carriers compared to either conventional culture (P<0.001) or qPCR detection of saliva (P=0.002). However, 32/219 (15 %) carriers were only positive in saliva, contributing significantly to the overall number of carriers detected (P=0.002). While testing nasopharyngeal swabs by qPCR proved most sensitive for pneumococcal detection in infants, saliva sampling could be considered as complementary to provide additional information on carriage and serotypes that may not be detected in the nasopharynx and may be particularly useful in longitudinal studies, requiring repeated sampling of study participants.

Real-time public health communication of local SARS-CoV-2 genomic epidemiology.

Genomic epidemiology can provide a unique, real-time understanding of SARS-CoV-2 transmission patterns. Yet the potential for genomic analyses to guide local policy and community-based behavioral decisions is limited because they are often oriented towards specially trained scientists and conducted on a national or global scale. Here, we propose a new paradigm: Phylogenetic analyses performed on a local level (municipal, county, or state), with results communicated in a clear, timely, and actionable manner to strengthen public health responses. We believe that presenting results rapidly, and tailored to a non-expert audience, can serve as a template for effective public health response to COVID-19 and other emerging viral diseases.

Detection of SARS-CoV-2 RNA by multiplex RT-qPCR.

The current quantitative reverse transcription PCR (RT-qPCR) assay recommended for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing in the United States requires analysis of 3 genomic targets per sample: 2 viral and 1 host. To simplify testing and reduce the volume of required reagents, we devised a multiplex RT-qPCR assay to detect SARS-CoV-2 in a single reaction. We used existing N1, N2, and RP primer and probe sets by the Centers for Disease Control and Prevention, but substituted fluorophores to allow multiplexing of the assay. The cycle threshold (Ct) values of our multiplex RT-qPCR were comparable to those obtained by the single assay adapted for research purposes. Low copy numbers (≥500 copies/reaction) of SARS-CoV-2 RNA were consistently detected by the multiplex RT-qPCR. Our novel multiplex RT-qPCR improves upon current single diagnostics by saving reagents, costs, time, and labor.

Longitudinal Immune Profiling of a Severe Acute Respiratory Syndrome Coronavirus 2 Reinfection in a Solid Organ Transplant Recipient.

BACKGROUND: The underlying immunologic deficiencies enabling severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfection are currently unknown. We describe deep longitudinal immune profiling of a transplant recipient hospitalized twice for coronavirus disease 2019 (COVID-19). METHODS: A 66-year-old male renal transplant recipient was hospitalized with COVID-19 March 2020 then readmitted to the hospital with COVID-19 233 days after initial diagnosis. Virologic and immunologic investigations were performed on samples from the primary and secondary infections. RESULTS: Whole viral genome sequencing and phylogenetic analysis revealed that viruses causing both infections were caused by distinct genetic lineages without evidence of immune escape mutations. Longitudinal comparison of cellular and humoral responses during primary SARS-CoV-2 infection revealed that this patient responded to the primary infection with low neutralization titer anti-SARS-CoV-2 antibodies that were likely present at the time of reinfection. CONCLUSIONS: The development of neutralizing antibodies and humoral memory responses in this patient failed to confer protection against reinfection, suggesting that they were below a neutralizing titer threshold or that additional factors may be required for efficient prevention of SARS-CoV-2 reinfection. Development of poorly neutralizing antibodies may have been due to profound and relatively specific reduction in naive CD4 T-cell pools. Seropositivity alone may not be a perfect correlate of protection in immunocompromised patients.

Saliva-based methods for SARS-CoV-2 testing in low- and middle-income countries.

As the coronavirus disease 2019 (COVID-19) continues to disproportionately affect low- and middle-income countries, the need for simple, accessible and frequent diagnostic testing grows. In lower-resource settings, case detection is often limited by a lack of available testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To address global inequities in testing, alternative sample types could be used to increase access to testing by reducing the associated costs. Saliva is a sensitive, minimally invasive and inexpensive diagnostic sample for SARS-CoV-2 detection that is appropriate for asymptomatic surveillance, symptomatic testing and at-home collection. Saliva testing can lessen two major challenges faced by lower- and middle-income countries: constrained resources and overburdened health workers. Saliva sampling enables convenient self-collection and requires fewer resources than swab-based methods. However, saliva testing for SARS-CoV-2 diagnostics has not been implemented on a large scale in low- and middle-income countries. While numerous studies based in these settings have demonstrated the usefulness of saliva sampling, there has been insufficient attention on optimizing its implementation in practice. We argue that implementation science research is needed to bridge this gap between evidence and practice. Low- and middle-income countries face many barriers as they continue their efforts to provide mass COVID-19 testing in the face of substantial inequities in global access to vaccines. Laboratories should look to replicate successful approaches for sensitive detection of SARS-CoV-2 in saliva, while governments should act to facilitate mass testing by lifting restrictions that limit implementation of saliva-based methods.

La maladie à coronavirus 2019 (COVID-19) continue à affecter les pays à revenu faible et intermédiaire de manière disproportionnée, accentuant le besoin en tests diagnostiques simples, accessibles et fréquents. Dans les endroits disposant de ressources limitées, la détection des cas se heurte souvent au manque de tests disponibles pour le syndrome respiratoire aigu sévère (SARS-CoV-2). Afin de lutter contre les inégalités mondiales en la matière, d'autres types d'échantillons pourraient être exploités, dans le but d'améliorer l'accès au dépistage tout en diminuant les frais qu'il engendre. Les échantillons de salive offrent une méthode de diagnostic fiable, peu invasive et peu coûteuse pour détecter le SARS-CoV-2. Cette méthode est compatible avec le suivi des personnes asymptomatiques, le dépistage des personnes symptomatiques et la collecte d'échantillons à domicile. Les tests salivaires permettent d'atténuer deux problèmes majeurs rencontrés par les pays à revenu faible et intermédiaire: une pénurie de ressources et des soignants surmenés. En outre, les patients peuvent effectuer le prélèvement eux-mêmes et cette méthode nécessite moins de moyens que celle reposant sur l'écouvillonnage. Pourtant, les tests salivaires de détection du SARS-CoV-2 n'ont pas été déployés à grande échelle dans les pays à revenu faible et intermédiaire. Malgré les nombreuses études démontrant l'utilité des tests salivaires dans ces régions, les perspectives d'optimisation de leur mise en œuvre n'ont suscité que peu d'attention. Dans le présent document, nous affirmons que des recherches scientifiques sur leur exécution sont requises pour combler ce fossé entre les faits et la pratique. Les pays à revenu faible et intermédiaire sont confrontés à une multitude d'obstacles dans leurs efforts de dépistage massif de la COVID-19. Et ce, en dépit des profondes inégalités qu'ils subissent dans le monde en matière d'accès aux vaccins. Les laboratoires devraient tenter de reproduire les approches les plus efficaces pour détecter le SARS-CoV-2 dans la salive, tandis que les gouvernements devraient prendre des mesures favorisant un dépistage de masse en levant les restrictions qui entravent le déploiement des tests salivaires.

A medida que la enfermedad por coronavirus de 2019 (COVID-19) sigue afectando de manera desproporcionada a los países de ingresos bajos y medios, crece la necesidad de realizar pruebas de diagnóstico sencillas, accesibles y frecuentes. En entornos de bajos recursos, la detección de casos suele estar limitada por la falta de pruebas disponibles para diagnosticar el coronavirus del síndrome respiratorio agudo grave de tipo 2 (SARS-CoV-2). Para abordar las desigualdades globales en las pruebas, se podrían utilizar tipos de muestra alternativos para aumentar el acceso a las pruebas reduciendo los costes asociados. La saliva es una muestra de diagnóstico sensible, poco invasiva y económica para la detección del SARS-CoV-2 que es apropiada para la vigilancia asintomática, las pruebas sintomáticas y la obtención en el hogar. Las pruebas de saliva pueden reducir dos de los principales problemas a los que se enfrentan los países de ingresos bajos y medios: la escasez de recursos y la sobrecarga de trabajo del personal sanitario. La toma de muestras de saliva permite realizar fácilmente la obtención por cuenta propia y requiere menos recursos que los métodos con hisopos. Sin embargo, las pruebas de saliva para el diagnóstico del SARS-CoV-2 no se han aplicado a gran escala en los países de ingresos bajos y medios. Aunque varios estudios realizados en estos entornos han demostrado la utilidad del muestreo de saliva, no se ha prestado suficiente atención a la optimización de su aplicación en la práctica. En este sentido, se considera que la investigación científica sobre la implementación es necesaria para subsanar esta deficiencia entre la evidencia y la práctica. Los países de ingresos bajos y medios se enfrentan a muchas dificultades en sus esfuerzos por realizar pruebas masivas en relación con la COVID-19, a pesar de las grandes desigualdades en el acceso global a las vacunas. Los laboratorios deberían intentar reproducir los enfoques que han tenido éxito para la detección sensible de la infección por el SARS-CoV-2 en la saliva, mientras que los gobiernos deberían actuar para facilitar las pruebas masivas eliminando las restricciones que limitan la aplicación de los métodos de diagnóstico salival.

Evaluation of saliva self-collection devices for SARS-CoV-2 diagnostics.

BACKGROUND: There is an urgent need to expand testing for SARS-CoV-2 and other respiratory pathogens as the global community struggles to control the COVID-19 pandemic. Current diagnostic methods can be affected by supply chain bottlenecks and require the assistance of medical professionals, impeding the implementation of large-scale testing. Self-collection of saliva may solve these problems, as it can be completed without specialized training and uses generic materials. METHODS: We observed 30 individuals who self-collected saliva using four different collection devices and analyzed their feedback. Two of these devices, a funnel and bulb pipette, were used to evaluate at-home saliva collection by 60 individuals. SARS-CoV-2-spiked saliva samples were subjected to temperature cycles designed to simulate the conditions the samples might be exposed to during the summer and winter seasons and sensitivity of detection was evaluated. RESULTS: All devices enabled the safe, unsupervised self-collection of saliva. The quantity and quality of the samples received were acceptable for SARS-CoV-2 diagnostic testing, as determined by human RNase P detection. There was no significant difference in SARS-CoV-2 nucleocapsid gene (N1) detection between the freshly spiked samples and those incubated with the summer and winter profiles. CONCLUSION: We demonstrate inexpensive, generic, buffer free collection devices suitable for unsupervised and home saliva self-collection.

Serotype Patterns of Pneumococcal Disease in Adults Are Correlated With Carriage Patterns in Older Children.

BACKGROUND: The importance of specific serotypes causing invasive pneumococcal disease (IPD) differs by age. Data on pneumococcal carriage in different age groups, along with data on serotype-specific invasiveness, could help explain these age-related patterns and their implications for vaccination. METHODS: Using pneumococcal carriage and disease data from Israel, we evaluated the association between serotype-specific IPD in adults and serotype-specific carriage prevalence among children in different age categories, while adjusting for serotype-specific invasiveness. We estimated carriage prevalence using different age groupings that were selected a priori. The Deviance Information Criterion was used to determine which age groupings of carriage data best fit the adult IPD data. Serotype-specific disease patterns were further evaluated by stratifying IPD data by comorbidity status. RESULTS: The relative frequency of serotypes causing IPD differed between adults and children, and also differed between older and younger adults and between adults with and without comorbidities. Serotypes overrepresented as causes of IPD in adults were more commonly carried in older children compared with younger children. In line with this, the serotype-specific frequency of carriage in older children, rather than infants, best correlated with serotype-specific IPD in adults. CONCLUSIONS: These analyses demonstrate that the serotype patterns in carriage in older children, rather than infants, are best correlated with disease patterns in adults. This might suggest these older children are more influential for disease patterns in adults. These insights could help in optimizing vaccination strategies to reduce disease burden across all ages.

Increased SARS-CoV-2 Testing Capacity with Pooled Saliva Samples.

We analyzed feasibility of pooling saliva samples for severe acute respiratory syndrome coronavirus 2 testing and found that sensitivity decreased according to pool size: 5 samples/pool, 7.4% reduction; 10 samples/pool, 11.1%; and 20 samples/pool, 14.8%. When virus prevalence is >2.6%, pools of 5 require fewer tests; when <0.6%, pools of 20 support screening strategies.

Stability of SARS-CoV-2 RNA in Nonsupplemented Saliva.

The expense of saliva collection devices designed to stabilize severe acute respiratory syndrome coronavirus 2 RNA is prohibitive to mass testing. However, virus RNA in nonsupplemented saliva is stable for extended periods and at elevated temperatures. Simple plastic tubes for saliva collection will make large-scale testing and continued surveillance easier.

Acute encephalopathy with elevated CSF inflammatory markers as the initial presentation of COVID-19.

BACKGROUND: COVID-19 is caused by the severe acute respiratory syndrome virus SARS-CoV-2. It is widely recognized as a respiratory pathogen, but neurologic complications can be the presenting manifestation in a subset of infected patients. CASE PRESENTATION: We describe a 78-year old immunocompromised woman who presented with altered mental status after witnessed seizure-like activity at home. She was found to have SARS-CoV-2 infection and associated neuroinflammation. In this case, we undertake the first detailed analysis of cerebrospinal fluid (CSF) cytokines during COVID-19 infection and find a unique pattern of inflammation in CSF, but no evidence of viral neuroinvasion. CONCLUSION: Our findings suggest that neurologic symptoms such as encephalopathy and seizures may be the initial presentation of COVID-19. Central nervous system inflammation may associate with neurologic manifestations of disease.

Variation of growth characteristics of pneumococcus with environmental conditions.

BACKGROUND: Pneumococcus is exposed to a variety of temperature and oxygen levels in the upper respiratory tract and as it invades the lung, tissues, and blood. We sought to determine the effect of environmental variability on growth in vitro and to assess variability between strains. We evaluated the effect of temperature and oxygen on the growth of 256 isolates representing 53 serotypes, recovered from healthy carriers and disease patients. Strains were grown at a range of temperatures, anaerobically or in ambient air with catalase, and were monitored by reading the optical density. Regression models evaluated variation in the characteristics of the growth curves. RESULTS: Most isolates grew to the maximal density at low temperatures (~33C) and under aerobic conditions. There was considerable variability between strains, and some of this variability was linked to serotype. However, capsule-switch experiments suggest that the production of different capsules might not be sufficient to explain this variation, suggesting there could be interactions between the capsule and genetic background. CONCLUSIONS: Pneumococcal strains vary in how they respond to environmental variations, some of this variation can be explained by the capsule type being produced, but capsule production itself is not sufficient to explain the variability. This variability could help to explain why different lineages of pneumococcus are more common in carriage or disease.