Publications by authors named "Nathan D Grubaugh"

88 Publications

Diverse functional autoantibodies in patients with COVID-19.

Nature 2021 May 19. Epub 2021 May 19.

Center for Outcomes Research and Evaluation, Yale-New Haven Hospital, New Haven, CT, USA.

COVID-19 manifests with a wide spectrum of clinical phenotypes that are characterized by exaggerated and misdirected host immune responses. Although pathological innate immune activation is well-documented in severe disease, 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 profiling 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.
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http://dx.doi.org/10.1038/s41586-021-03631-yDOI Listing
May 2021

Lying in wait: the resurgence of dengue virus after the Zika epidemic in Brazil.

Nat Commun 2021 05 11;12(1):2619. Epub 2021 May 11.

Department of Epidemiology of Microbial Diseases, Yale School of Public Health, Yale University, New Haven, CT, USA.

After the Zika virus (ZIKV) epidemic in the Americas in 2016, both Zika and dengue incidence declined to record lows in many countries in 2017-2018, but in 2019 dengue resurged in Brazil, causing ~2.1 million cases. In this study we use epidemiological, climatological and genomic data to investigate dengue dynamics in recent years in Brazil. First, we estimate dengue virus force of infection (FOI) and model mosquito-borne transmission suitability since the early 2000s. Our estimates reveal that DENV transmission was low in 2017-2018, despite conditions being suitable for viral spread. Our study also shows a marked decline in dengue susceptibility between 2002 and 2019, which could explain the synchronous decline of dengue in the country, partially as a result of protective immunity from prior ZIKV and/or DENV infections. Furthermore, we performed phylogeographic analyses using 69 newly sequenced genomes of dengue virus serotype 1 and 2 from Brazil, and found that the outbreaks in 2018-2019 were caused by local DENV lineages that persisted for 5-10 years, circulating cryptically before and after the Zika epidemic. We hypothesize that DENV lineages may circulate at low transmission levels for many years, until local conditions are suitable for higher transmission, when they cause major outbreaks.
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http://dx.doi.org/10.1038/s41467-021-22921-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8113494PMC
May 2021

Maternal respiratory SARS-CoV-2 infection in pregnancy is associated with a robust inflammatory response at the maternal-fetal interface.

Med (N Y) 2021 May 30;2(5):591-610.e10. Epub 2021 Apr 30.

Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale School of Medicine, New Haven, CT, USA.

Background: Pregnant women are at increased risk for severe outcomes from coronavirus disease 2019 (COVID-19), but the pathophysiology underlying this increased morbidity and its potential effect on the developing fetus is not well understood.

Methods: We assessed placental histology, ACE2 expression, and viral and immune dynamics at the term placenta in pregnant women with and without respiratory severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

Findings: The majority (13 of 15) of placentas analyzed had no detectable viral RNA. ACE2 was detected by immunohistochemistry in syncytiotrophoblast cells of the normal placenta during early pregnancy but was rarely seen in healthy placentas at full term, suggesting that low ACE2 expression may protect the term placenta from viral infection. Using immortalized cell lines and primary isolated placental cells, we found that cytotrophoblasts, the trophoblast stem cells and precursors to syncytiotrophoblasts, rather than syncytiotrophoblasts or Hofbauer cells, are most vulnerable to SARS-CoV-2 infection . To better understand potential immune mechanisms shielding placental cells from infection , we performed bulk and single-cell transcriptomics analyses and found that the maternal-fetal interface of SARS-CoV-2-infected women exhibited robust immune responses, including increased activation of natural killer (NK) and T cells, increased expression of interferon-related genes, as well as markers associated with pregnancy complications such as preeclampsia.

Conclusions: SARS-CoV-2 infection in late pregnancy is associated with immune activation at the maternal-fetal interface even in the absence of detectable local viral invasion.

Funding: NIH (T32GM007205, F30HD093350, K23MH118999, R01AI157488, U01DA040588) and Fast Grant funding support from Emergent Ventures at the Mercatus Center.
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http://dx.doi.org/10.1016/j.medj.2021.04.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8084634PMC
May 2021

Divergent and self-reactive immune responses in the CNS of COVID-19 patients with neurological symptoms.

Cell Rep Med 2021 May 3;2(5):100288. Epub 2021 May 3.

Department of Internal Medicine, Section of Infectious Diseases, Yale School of Medicine, New Haven, CT, USA.

Individuals with coronavirus disease 2019 (COVID-19) frequently develop neurological symptoms, but the biological underpinnings of these phenomena are unknown. Through single-cell RNA sequencing (scRNA-seq) and cytokine analyses of cerebrospinal fluid (CSF) and blood from individuals with COVID-19 with neurological symptoms, we find compartmentalized, CNS-specific T cell activation and B cell responses. All affected individuals had CSF anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies whose target epitopes diverged from serum antibodies. In an animal model, we find that intrathecal SARS-CoV-2 antibodies are present only during brain infection and not elicited by pulmonary infection. We produced CSF-derived monoclonal antibodies from an individual with COVID-19 and found that these monoclonal antibodies (mAbs) target antiviral and antineural antigens, including one mAb that reacted to spike protein and neural tissue. CSF immunoglobulin G (IgG) from 5 of 7 patients showed antineural reactivity. This immune survey reveals evidence of a compartmentalized immune response in the CNS of individuals with COVID-19 and suggests a role of autoimmunity in neurologic sequelae of COVID-19.
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http://dx.doi.org/10.1016/j.xcrm.2021.100288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8091032PMC
May 2021

COVID-19 one year into the pandemic: from genetics and genomics to therapy, vaccination, and policy.

Hum Genomics 2021 05 10;15(1):27. Epub 2021 May 10.

Australian Institute of Tropical Health and Medicine, James Cook University, Smithfield, Queensland, 4878, Australia.

COVID-19 has engulfed the world and it will accompany us all for some time to come. Here, we review the current state at the milestone of 1 year into the pandemic, as declared by the WHO (World Health Organization). We review several aspects of the on-going pandemic, focusing first on two major topics: viral variants and the human genetic susceptibility to disease severity. We then consider recent and exciting new developments in therapeutics, such as monoclonal antibodies, and in prevention strategies, such as vaccines. We also briefly discuss how advances in basic science and in biotechnology, under the threat of a worldwide emergency, have accelerated to an unprecedented degree of the transition from the laboratory to clinical applications. While every day we acquire more and more tools to deal with the on-going pandemic, we are aware that the path will be arduous and it will require all of us being community-minded. In this respect, we lament past delays in timely full investigations, and we call for bypassing local politics in the interest of humankind on all continents.
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http://dx.doi.org/10.1186/s40246-021-00326-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8107019PMC
May 2021

Multiplex qPCR discriminates variants of concern to enhance global surveillance of SARS-CoV-2.

PLoS Biol 2021 05 7;19(5):e3001236. Epub 2021 May 7.

Murphy Medical Associates, Greenwich, Connecticut, United States of America.

With the emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants that may increase transmissibility and/or cause escape from immune responses, there is an urgent need for the targeted surveillance of circulating lineages. It was found that the B.1.1.7 (also 501Y.V1) variant, first detected in the United Kingdom, could be serendipitously detected by the Thermo Fisher TaqPath COVID-19 PCR assay because a key deletion in these viruses, spike Δ69-70, would cause a "spike gene target failure" (SGTF) result. However, a SGTF result is not definitive for B.1.1.7, and this assay cannot detect other variants of concern (VOC) that lack spike Δ69-70, such as B.1.351 (also 501Y.V2), detected in South Africa, and P.1 (also 501Y.V3), recently detected in Brazil. We identified a deletion in the ORF1a gene (ORF1a Δ3675-3677) in all 3 variants, which has not yet been widely detected in other SARS-CoV-2 lineages. Using ORF1a Δ3675-3677 as the primary target and spike Δ69-70 to differentiate, we designed and validated an open-source PCR assay to detect SARS-CoV-2 VOC. Our assay can be rapidly deployed in laboratories around the world to enhance surveillance for the local emergence and spread of B.1.1.7, B.1.351, and P.1.
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http://dx.doi.org/10.1371/journal.pbio.3001236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8133773PMC
May 2021

Delayed production of neutralizing antibodies correlates with fatal COVID-19.

Nat Med 2021 May 5. Epub 2021 May 5.

Department of Medicine, Section of Infectious Diseases, Yale University School of Medicine, New Haven, CT, USA.

Recent studies have provided insights into innate and adaptive immune dynamics in coronavirus disease 2019 (COVID-19). However, the exact features of antibody responses that govern COVID-19 disease outcomes remain unclear. In this study, we analyzed humoral immune responses in 229 patients with asymptomatic, mild, moderate and severe COVID-19 over time to probe the nature of antibody responses in disease severity and mortality. We observed a correlation between anti-spike (S) immunoglobulin G (IgG) levels, length of hospitalization and clinical parameters associated with worse clinical progression. Although high anti-S IgG levels correlated with worse disease severity, such correlation was time dependent. Deceased patients did not have higher overall humoral response than discharged patients. However, they mounted a robust, yet delayed, response, measured by anti-S, anti-receptor-binding domain IgG and neutralizing antibody (NAb) levels compared to survivors. Delayed seroconversion kinetics correlated with impaired viral control in deceased patients. Finally, although sera from 85% of patients displayed some neutralization capacity during their disease course, NAb generation before 14 d of disease onset emerged as a key factor for recovery. These data indicate that COVID-19 mortality does not correlate with the cross-sectional antiviral antibody levels per se but, rather, with the delayed kinetics of NAb production.
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http://dx.doi.org/10.1038/s41591-021-01355-0DOI Listing
May 2021

Intrahost speciations and host switches played an important role in the evolution of herpesviruses.

Virus Evol 2021 Jan 18;7(1):veab025. Epub 2021 Mar 18.

Department of Life Sciences, Imperial College London, South Kensington Campus. London SW7 2AZ, UK.

In times when herpesvirus genomic data were scarce, the cospeciation between these viruses and their hosts was considered to be common knowledge. However, as more herpesviral sequences were made available, tree reconciliation analyses started to reveal topological incongruences between host and viral phylogenies, indicating that other cophylogenetic events, such as intrahost speciation and host switching, likely played important roles along more than 200 million years of evolutionary history of these viruses. Tree reconciliations performed with undated phylogenies can identify topological differences, but offer insufficient information to reveal temporal incongruences between the divergence timing of host and viral species. In this study, we performed cophylogenetic analyses using time-resolved trees of herpesviruses and their hosts, based on careful molecular clock modelling. This approach enabled us to infer cophylogenetic events over time and also integrate information on host biogeography to better understand host-virus evolutionary history. Given the increasing amount of sequence data now available, mismatches between host and viral phylogenies have become more evident, and to account for such phylogenetic differences, host switches, intrahost speciations and losses were frequently found in all tree reconciliations. For all subfamilies in , under all scenarios we explored, intrahost speciation and host switching were more frequent than cospeciation, which was shown to be a rare event, restricted to contexts where topological and temporal patterns of viral and host evolution were in strict agreement.
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http://dx.doi.org/10.1093/ve/veab025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8062258PMC
January 2021

Early introductions and transmission of SARS-CoV-2 variant B.1.1.7 in the United States.

Cell 2021 05 3;184(10):2595-2604.e13. Epub 2021 Apr 3.

Connecticut State Department of Public Health, Rocky Hill, CT 06067, USA; Centers for Disease Control and Prevention, Atlanta, GA 30329, USA.

The emergence and spread of SARS-CoV-2 lineage B.1.1.7, first detected in the United Kingdom, has become a global public health concern because of its increased transmissibility. Over 2,500 COVID-19 cases associated with this variant have been detected in the United States (US) since December 2020, but the extent of establishment is relatively unknown. Using travel, genomic, and diagnostic data, we highlight that the primary ports of entry for B.1.1.7 in the US were in New York, California, and Florida. Furthermore, we found evidence for many independent B.1.1.7 establishments starting in early December 2020, followed by interstate spread by the end of the month. Finally, we project that B.1.1.7 will be the dominant lineage in many states by mid- to late March. Thus, genomic surveillance for B.1.1.7 and other variants urgently needs to be enhanced to better inform the public health response.
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http://dx.doi.org/10.1016/j.cell.2021.03.061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8018830PMC
May 2021

Case Study: Longitudinal immune profiling of a SARS-CoV-2 reinfection in a solid organ transplant recipient.

medRxiv 2021 Mar 26. Epub 2021 Mar 26.

Prior to the emergence of antigenically distinct SARS-CoV-2 variants, reinfections were reported infrequently - presumably due to the generation of durable and protective immune responses. However, case reports also suggested that rare, repeated infections may occur as soon as 48 days following initial disease onset. The underlying immunologic deficiencies enabling SARS-CoV-2 reinfections are currently unknown. Here we describe a renal transplant recipient who developed recurrent, symptomatic SARS-CoV-2 infection - confirmed by whole virus genome sequencing - 7 months after primary infection. To elucidate the immunological mechanisms responsible for SARS-CoV-2 reinfection, we performed longitudinal profiling of cellular and humoral responses during both primary and recurrent SARS-CoV-2 infection. We found that the patient responded to the primary infection with transient, poor-quality adaptive immune responses. The patient's immune system was further compromised by intervening treatment for acute rejection of the renal allograft prior to reinfection. Importantly, we also identified the development of neutralizing antibodies and the formation of humoral memory responses prior to SARS-CoV-2 reinfection. However, these neutralizing antibodies failed to confer protection against reinfection, suggesting that additional factors are required for efficient prevention of SARS-CoV-2 reinfection. Further, we found no evidence supporting viral evasion of primary adaptive immune responses, suggesting that susceptibility to reinfection may be determined by host factors rather than pathogen adaptation in this patient. In summary, our study suggests that a low neutralizing antibody presence alone is not sufficient to confer resistance against reinfection. Thus, patients with solid organ transplantation, or patients who are otherwise immunosuppressed, who recover from infection with SARS-CoV-2 may not develop sufficient protective immunity and are at risk of reinfection.
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http://dx.doi.org/10.1101/2021.03.24.21253992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010761PMC
March 2021

PCR assay to enhance global surveillance for SARS-CoV-2 variants of concern.

medRxiv 2021 Mar 12. Epub 2021 Mar 12.

Connecticut State Department of Public Health, Rocky Hill, CT 06067, USA.

With the emergence of SARS-CoV-2 variants that may increase transmissibility and/or cause escape from immune responses , there is an urgent need for the targeted surveillance of circulating lineages. It was found that the B.1.1.7 (also 501Y.V1) variant first detected in the UK could be serendipitously detected by the ThermoFisher TaqPath COVID-19 PCR assay because a key deletion in these viruses, spike Δ69-70, would cause a "spike gene target failure" (SGTF) result. However, a SGTF result is not definitive for B.1.1.7, and this assay cannot detect other variants of concern that lack spike Δ69-70, such as B.1.351 (also 501Y.V2) detected in South Africa and P.1 (also 501Y.V3) recently detected in Brazil . We identified a deletion in the ORF1a gene (ORF1a Δ3675-3677) in all three variants, which has not yet been widely detected in other SARS-CoV-2 lineages. Using ORF1a Δ3675-3677 as the primary target and spike Δ69-70 to differentiate, we designed and validated an open source PCR assay to detect SARS-CoV-2 variants of concern . Our assay can be rapidly deployed in laboratories around the world to enhance surveillance for the local emergence spread of B.1.1.7, B.1.351, and P.1.
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http://dx.doi.org/10.1101/2021.01.28.21250486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7987060PMC
March 2021

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

Emerg Infect Dis 2021 04;27(4)

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.
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http://dx.doi.org/10.3201/eid2704.204200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8007323PMC
April 2021

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

Emerg Infect Dis 2021 04;27(4):1146-1150

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.
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http://dx.doi.org/10.3201/eid2704.204199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8007305PMC
April 2021

Tracking smell loss to identify healthcare workers with SARS-CoV-2 infection.

PLoS One 2021 3;16(3):e0248025. Epub 2021 Mar 3.

Department of Medicine, Section of Infectious Diseases, Yale School of Medicine, New Haven, Connecticut, United States of America.

Introduction: Healthcare workers (HCW) treating COVID-19 patients are at high risk for infection and may also spread infection through their contact with vulnerable patients. Smell loss has been associated with SARS-CoV-2 infection, but it is unknown whether monitoring for smell loss can be used to identify asymptomatic infection among high risk individuals. In this study we sought to determine if tracking smell sensitivity and loss using an at-home assessment could identify SARS-CoV-2 infection in HCW.

Methods And Findings: We performed a prospective cohort study tracking 473 HCW across three months to determine if smell loss could predict SARS-CoV-2 infection in this high-risk group. HCW subjects completed a longitudinal, behavioral at-home assessment of olfaction with household items, as well as detailed symptom surveys that included a parosmia screening questionnaire, and real-time quantitative polymerase chain reaction testing to identify SARS-CoV-2 infection. Our main measures were the prevalence of smell loss in SARS-CoV-2-positive HCW versus SARS-CoV-2-negative HCW, and timing of smell loss relative to SARS-CoV-2 test positivity. SARS-CoV-2 was identified in 17 (3.6%) of 473 HCW. HCW with SARS-CoV-2 infection were more likely to report smell loss than SARS-CoV-2-negative HCW on both the at-home assessment and the screening questionnaire (9/17, 53% vs 105/456, 23%, P < .01). 6/9 (67%) of SARS-CoV-2-positive HCW reporting smell loss reported smell loss prior to having a positive SARS-CoV-2 test, and smell loss was reported a median of two days before testing positive. Neurological symptoms were reported more frequently among SARS-CoV-2-positive HCW who reported smell loss compared to those without smell loss (9/9, 100% vs 3/8, 38%, P < .01).

Conclusions: In this prospective study of HCW, self-reported changes in smell using two different measures were predictive of SARS-CoV-2 infection. Smell loss frequently preceded a positive test and was associated with neurological symptoms.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0248025PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7928484PMC
March 2021

MOG-associated encephalitis following SARS-COV-2 infection.

Mult Scler Relat Disord 2021 May 23;50:102857. Epub 2021 Feb 23.

Department of Neurology, Yale University School of Medicine, 333 Cedar St. New Haven, CT, 06510, USA.

A variety of neurologic manifestations of COVID-19 infections have been reported. Here, we present a case of steroid-responsive MOG-antibody associated encephalitis, characterized by cognitive decline, headaches, fever, unilateral FLAIR-hyperintensities, and leptomeningeal enhancement, that occurred in the setting of recent COVID-19 infection.
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http://dx.doi.org/10.1016/j.msard.2021.102857DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900751PMC
May 2021

Early introductions and community transmission of SARS-CoV-2 variant B.1.1.7 in the United States.

medRxiv 2021 Feb 12. Epub 2021 Feb 12.

The emergence and spread of SARS-CoV-2 lineage B.1.1.7, first detected in the United Kingdom, has become a national public health concern in the United States because of its increased transmissibility. Over 500 COVID-19 cases associated with this variant have been detected since December 2020, but its local establishment and pathways of spread are relatively unknown. Using travel, genomic, and diagnostic testing data, we highlight the primary ports of entry for B.1.1.7 in the US and locations of possible underreporting of B.1.1.7 cases. New York, which receives the most international travel from the UK, is likely one of the key hubs for introductions and domestic spread. Finally, we provide evidence for increased community transmission in several states. Thus, genomic surveillance for B.1.1.7 and other variants urgently needs to be enhanced to better inform the public health response.
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http://dx.doi.org/10.1101/2021.02.10.21251540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7885932PMC
February 2021

Public health actions to control new SARS-CoV-2 variants.

Cell 2021 03 29;184(5):1127-1132. Epub 2021 Jan 29.

Division of Infection and Global Health Research, School of Medicine, University of St Andrews, Fife, UK.

Recent reports suggest that some SARS-CoV-2 genetic variants, such as B.1.1.7, might be more transmissible and are quickly spreading around the world. As the emergence of more transmissible variants could exacerbate the pandemic, we provide public health guidance for increased surveillance and measures to reduce community transmission.
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http://dx.doi.org/10.1016/j.cell.2021.01.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7846239PMC
March 2021

Usability of saliva collection devices for SARS-CoV-2 diagnostics.

medRxiv 2021 Feb 4. Epub 2021 Feb 4.

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 because it can be completed without specialized training and uses generic materials. In this study, we observed thirty individuals who self-collected saliva using four different collection devices and analyzed their feedback. These devices enabled the safe collection of saliva that was acceptable for SARS-CoV-2 diagnostic testing.
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http://dx.doi.org/10.1101/2021.02.01.21250946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7872382PMC
February 2021

Saliva for Detection of SARS-CoV-2. Reply.

N Engl J Med 2021 03 3;384(9):e31. Epub 2021 Feb 3.

Yale School of Public Health, New Haven, CT

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http://dx.doi.org/10.1056/NEJMc2032165DOI Listing
March 2021

SARS-CoV-2 infection in pregnancy is associated with robust inflammatory response at the maternal-fetal interface.

medRxiv 2021 Jan 26. Epub 2021 Jan 26.

Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale School of Medicine, New Haven, CT, USA.

Pregnant women appear to be at increased risk for severe outcomes associated with COVID-19, but the pathophysiology underlying this increased morbidity and its potential impact on the developing fetus is not well understood. In this study of pregnant women with and without COVID-19, we assessed viral and immune dynamics at the placenta during maternal SARS-CoV-2 infection. Amongst uninfected women, ACE2 was detected by immunohistochemistry in syncytiotrophoblast cells of the normal placenta during early pregnancy but was rarely seen in healthy placentas at full term. Term placentas from women infected with SARS-CoV-2, however, displayed a significant increase in ACE2 levels. Using immortalized cell lines and primary isolated placental cells, we determined the vulnerability of various placental cell types to direct infection by SARS-CoV-2 . Yet, despite the susceptibility of placental cells to SARS-CoV-2 infection, viral RNA was detected in the placentas of only a subset (∼13%) of women in this cohort. Through single cell transcriptomic analyses, we found that the maternal-fetal interface of SARS-CoV-2-infected women exhibited markers associated with pregnancy complications, such as preeclampsia, and robust immune responses, including increased activation of placental NK and T cells and increased expression of interferon-related genes. Overall, this study suggests that SARS-CoV-2 is associated with immune activation at the maternal-fetal interface even in the absence of detectable local viral invasion. While this likely represents a protective mechanism shielding the placenta from infection, inflammatory changes in the placenta may also contribute to poor pregnancy outcomes and thus warrant further investigation.
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http://dx.doi.org/10.1101/2021.01.25.21250452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7852242PMC
January 2021

SalivaDirect: A simplified and flexible platform to enhance SARS-CoV-2 testing capacity.

Med (N Y) 2021 Mar 26;2(3):263-280.e6. Epub 2020 Dec 26.

Department of Pathology, Yale University School of Medicine, New Haven, CT 06510, USA.

Background: Scaling SARS-CoV-2 testing to meet demands of safe reopenings continues to be plagued by assay costs and supply chain shortages. In response, we developed SalivaDirect, which received Emergency Use Authorization (EUA) from the U.S. Food and Drug Administration (FDA).

Methods: We simplified our saliva-based diagnostic test by (1) not requiring collection tubes with preservatives, (2) replacing nucleic acid extraction with a simple enzymatic and heating step, and (3) testing specimens with a dualplex qRT-PCR assay. Moreover, we validated SalivaDirect with reagents and instruments from multiple vendors to minimize supply chain issues.

Findings: From our hospital cohort, we show a high positive agreement (94%) between saliva tested with SalivaDirect and nasopharyngeal swabs tested with a commercial qRT-PCR kit. In partnership with the National Basketball Association (NBA) and National Basketball Players Association (NBPA), we tested 3,779 saliva specimens from healthy individuals and detected low rates of invalid (0.3%) and false-positive (<0.05%) results.

Conclusions: We demonstrate that saliva is a valid alternative to swabs for SARS-CoV-2 screening and that SalivaDirect can make large-scale testing more accessible and affordable. Uniquely, we can designate other laboratories to use our sensitive, flexible, and simplified platform under our EUA (https://publichealth.yale.edu/salivadirect/

Funding: This study was funded by the NBA and NBPA (N.D.G.), the Huffman Family Donor Advised Fund (N.D.G.), a Fast Grant from Emergent Ventures at the Mercatus Center at George Mason University (N.D.G.), the Yale Institute for Global Health (N.D.G.), and the Beatrice Kleinberg Neuwirth Fund (A.I.K.). C.B.F.V. is supported by NWO Rubicon 019.181EN.004.
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http://dx.doi.org/10.1016/j.medj.2020.12.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7836249PMC
March 2021

Saliva viral load is a dynamic unifying correlate of COVID-19 severity and mortality.

medRxiv 2021 Jan 10. Epub 2021 Jan 10.

While several clinical and immunological parameters correlate with disease severity and mortality in SARS-CoV-2 infection, work remains in identifying unifying correlates of coronavirus disease 2019 (COVID-19) that can be used to guide clinical practice. Here, we examine saliva and nasopharyngeal (NP) viral load over time and correlate them with patient demographics, and cellular and immune profiling. We found that saliva viral load was significantly higher in those with COVID-19 risk factors; that it correlated with increasing levels of disease severity and showed a superior ability over nasopharyngeal viral load as a predictor of mortality over time (AUC=0.90). A comprehensive analysis of immune factors and cell subsets revealed strong predictors of high and low saliva viral load, which were associated with increased disease severity or better overall outcomes, respectively. Saliva viral load was positively associated with many known COVID-19 inflammatory markers such as IL-6, IL-18, IL-10, and CXCL10, as well as type 1 immune response cytokines. Higher saliva viral loads strongly correlated with the progressive depletion of platelets, lymphocytes, and effector T cell subsets including circulating follicular CD4 T cells (cTfh). Anti-spike (S) and anti-receptor binding domain (RBD) IgG levels were negatively correlated with saliva viral load showing a strong temporal association that could help distinguish severity and mortality in COVID-19. Finally, patients with fatal COVID-19 exhibited higher viral loads, which correlated with the depletion of cTfh cells, and lower production of anti-RBD and anti-S IgG levels. Together these results demonstrated that viral load, as measured by saliva but not nasopharyngeal, is a dynamic unifying correlate of disease presentation, severity, and mortality over time.
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http://dx.doi.org/10.1101/2021.01.04.21249236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7805468PMC
January 2021

Asynchronicity of endemic and emerging mosquito-borne disease outbreaks in the Dominican Republic.

Nat Commun 2021 01 8;12(1):151. Epub 2021 Jan 8.

Instituto de Medicina Tropical & Salud Global, Universidad Iberoamericana, Santo Domingo, Dominican Republic.

Mosquito-borne viruses threaten the Caribbean due to the region's tropical climate and seasonal reception of international tourists. Outbreaks of chikungunya and Zika have demonstrated the rapidity with which these viruses can spread. Concurrently, dengue fever cases have climbed over the past decade. Sustainable disease control measures are urgently needed to quell virus transmission and prevent future outbreaks. Here, to improve upon current control methods, we analyze temporal and spatial patterns of chikungunya, Zika, and dengue outbreaks reported in the Dominican Republic between 2012 and 2018. The viruses that cause these outbreaks are transmitted by Aedes mosquitoes, which are sensitive to seasonal climatological variability. We evaluate whether climate and the spatio-temporal dynamics of dengue outbreaks could explain patterns of emerging disease outbreaks. We find that emerging disease outbreaks were robust to the climatological and spatio-temporal constraints defining seasonal dengue outbreak dynamics, indicating that constant surveillance is required to prevent future health crises.
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http://dx.doi.org/10.1038/s41467-020-20391-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7794562PMC
January 2021

Kinetics of antibody responses dictate COVID-19 outcome.

medRxiv 2020 Dec 22. Epub 2020 Dec 22.

Recent studies have provided insights into innate and adaptive immune dynamics in coronavirus disease 2019 (COVID-19). Yet, the exact feature of antibody responses that governs COVID-19 disease outcomes remain unclear. Here, we analysed humoral immune responses in 209 asymptomatic, mild, moderate and severe COVID-19 patients over time to probe the nature of antibody responses in disease severity and mortality. We observed a correlation between anti-Spike (S) IgG levels, length of hospitalization and clinical parameters associated with worse clinical progression. While high anti-S IgG levels correlated with worse disease severity, such correlation was time-dependent. Deceased patients did not have higher overall humoral response than live discharged patients. However, they mounted a robust, yet delayed response, measured by anti-S, anti-RBD IgG, and neutralizing antibody (NAb) levels, compared to survivors. Delayed seroconversion kinetics correlated with impaired viral control in deceased patients. Finally, while sera from 89% of patients displayed some neutralization capacity during their disease course, NAb generation prior to 14 days of disease onset emerged as a key factor for recovery. These data indicate that COVID-19 mortality does not correlate with the cross-sectional antiviral antibody levels , but rather with the delayed kinetics of NAb production.
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http://dx.doi.org/10.1101/2020.12.18.20248331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7781347PMC
December 2020

Diverse Functional Autoantibodies in Patients with COVID-19.

medRxiv 2020 Dec 12. Epub 2020 Dec 12.

Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT, USA.

COVID-19 manifests with a wide spectrum of clinical phenotypes that are characterized by exaggerated and misdirected host immune responses . While pathological innate immune activation is well documented in severe disease , the impact of autoantibodies on disease progression is less defined. Here, we used a high-throughput autoantibody discovery technique called Rapid Extracellular Antigen Profiling (REAP) to screen a cohort of 194 SARS-CoV-2 infected COVID-19 patients and healthcare workers for autoantibodies against 2,770 extracellular and secreted proteins (the "exoproteome"). We found that COVID-19 patients exhibit dramatic increases in autoantibody reactivities compared to uninfected controls, with 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 signaling and by altering peripheral immune cell composition, and found that murine surrogates of these autoantibodies exacerbate disease severity in a mouse model of SARS-CoV-2 infection. Analysis of autoantibodies against tissue-associated antigens revealed associations with specific clinical characteristics and disease severity. In summary, these findings implicate a pathological role for exoproteome-directed autoantibodies in COVID-19 with diverse impacts on immune functionality and associations with clinical outcomes.
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http://dx.doi.org/10.1101/2020.12.10.20247205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7743105PMC
December 2020

Epidemiological hypothesis testing using a phylogeographic and phylodynamic framework.

Nat Commun 2020 11 6;11(1):5620. Epub 2020 Nov 6.

Department of Microbiology, Immunology and Transplantation, Rega Institute, KU Leuven, Herestraat 49, 3000, Leuven, Belgium.

Computational analyses of pathogen genomes are increasingly used to unravel the dispersal history and transmission dynamics of epidemics. Here, we show how to go beyond historical reconstructions and use spatially-explicit phylogeographic and phylodynamic approaches to formally test epidemiological hypotheses. We illustrate our approach by focusing on the West Nile virus (WNV) spread in North America that has substantially impacted public, veterinary, and wildlife health. We apply an analytical workflow to a comprehensive WNV genome collection to test the impact of environmental factors on the dispersal of viral lineages and on viral population genetic diversity through time. We find that WNV lineages tend to disperse faster in areas with higher temperatures and we identify temporal variation in temperature as a main predictor of viral genetic diversity through time. By contrasting inference with simulation, we find no evidence for viral lineages to preferentially circulate within the same migratory bird flyway, suggesting a substantial role for non-migratory birds or mosquito dispersal along the longitudinal gradient.
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http://dx.doi.org/10.1038/s41467-020-19122-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7648063PMC
November 2020

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

PLoS Biol 2020 10 7;18(10):e3000867. Epub 2020 Oct 7.

Department of Immunobiology, Yale School of Medicine, New Haven, Connecticut, United States of America.

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.
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http://dx.doi.org/10.1371/journal.pbio.3000867DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7571696PMC
October 2020

Two Sides of a Coin: a Zika Virus Mutation Selected in Pregnant Rhesus Macaques Promotes Fetal Infection in Mice but at a Cost of Reduced Fitness in Nonpregnant Macaques and Diminished Transmissibility by Vectors.

J Virol 2020 11 23;94(24). Epub 2020 Nov 23.

University of California, Davis, School of Veterinary Medicine, Department of Pathology, Microbiology and Immunology, Davis, California, USA

Although fetal death is now understood to be a severe outcome of congenital Zika syndrome, the role of viral genetics is still unclear. We sequenced Zika virus (ZIKV) from a rhesus macaque fetus that died after inoculation and identified a single intrahost substitution, M1404I, in the ZIKV polyprotein, located in nonstructural protein 2B (NS2B). Targeted sequencing flanking position 1404 in 9 additional macaque mothers and their fetuses identified M1404I at a subconsensus frequency in the majority (5 of 9, 56%) of animals and some of their fetuses. Despite its repeated presence in pregnant macaques, M1404I has occurred rarely in humans since 2015. Since the primary ZIKV transmission cycle is human-mosquito-human, mutations in one host must be retained in the alternate host to be perpetuated. We hypothesized that ZIKV I1404 increases viral fitness in nonpregnant macaques and pregnant mice but is less efficiently transmitted by vectors, explaining its low frequency in humans during outbreaks. By examining competitive fitness relative to that of ZIKV M1404, we observed that ZIKV I1404 produced lower viremias in nonpregnant macaques and was a weaker competitor in tissues. In pregnant wild-type mice, ZIKV I1404 increased the magnitude and rate of placental infection and conferred fetal infection, in contrast to ZIKV M1404, which was not detected in fetuses. Although infection and dissemination rates were not different, mosquitoes transmitted ZIKV I1404 more poorly than ZIKV M1404. Our data highlight the complexity of arbovirus mutation-fitness dynamics and suggest that intrahost ZIKV mutations capable of augmenting fitness in pregnant vertebrates may not necessarily spread efficiently via mosquitoes during epidemics. Although Zika virus infection of pregnant women can result in congenital Zika syndrome, the factors that cause the syndrome in some but not all infected mothers are still unclear. We identified a mutation that was present in some ZIKV genomes in experimentally inoculated pregnant rhesus macaques and their fetuses. Although we did not find an association between the presence of the mutation and fetal death, we performed additional studies with ZIKV with the mutation in nonpregnant macaques, pregnant mice, and mosquitoes. We observed that the mutation increased the ability of the virus to infect mouse fetuses but decreased its capacity to produce high levels of virus in the blood of nonpregnant macaques and to be transmitted by mosquitoes. This study shows that mutations in mosquito-borne viruses like ZIKV that increase fitness in pregnant vertebrates may not spread in outbreaks when they compromise transmission via mosquitoes and fitness in nonpregnant hosts.
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http://dx.doi.org/10.1128/JVI.01605-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7925200PMC
November 2020

Measurement of SARS-CoV-2 RNA in wastewater tracks community infection dynamics.

Nat Biotechnol 2020 10 18;38(10):1164-1167. Epub 2020 Sep 18.

Department of Epidemiology of Microbial Disease, School of Public Health, Yale University, New Haven, CT, USA.

We measured severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA concentrations in primary sewage sludge in the New Haven, Connecticut, USA, metropolitan area during the Coronavirus Disease 2019 (COVID-19) outbreak in Spring 2020. SARS-CoV-2 RNA was detected throughout the more than 10-week study and, when adjusted for time lags, tracked the rise and fall of cases seen in SARS-CoV-2 clinical test results and local COVID-19 hospital admissions. Relative to these indicators, SARS-CoV-2 RNA concentrations in sludge were 0-2 d ahead of SARS-CoV-2 positive test results by date of specimen collection, 0-2 d ahead of the percentage of positive tests by date of specimen collection, 1-4 d ahead of local hospital admissions and 6-8 d ahead of SARS-CoV-2 positive test results by reporting date. Our data show the utility of viral RNA monitoring in municipal wastewater for SARS-CoV-2 infection surveillance at a population-wide level. In communities facing a delay between specimen collection and the reporting of test results, immediate wastewater results can provide considerable advance notice of infection dynamics.
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http://dx.doi.org/10.1038/s41587-020-0684-zDOI Listing
October 2020

Tracking Smell Loss to Identify Healthcare Workers with SARS-CoV-2 Infection.

medRxiv 2020 Sep 10. Epub 2020 Sep 10.

Department of Medicine, Section of Infectious Diseases, Yale School of Medicine, New Haven, CT.

Background: Healthcare workers (HCW) treating COVID-19 patients are at high risk for infection and may also spread infection through their contact with vulnerable patients. Smell loss has been associated with SARS-CoV-2 infection, but it is unknown whether monitoring for smell loss can be used to identify asymptomatic infection among high risk individuals, like HCW.

Methods: We performed a prospective cohort study, tracking 473 HCW across three months to determine if smell loss could predict SARS-CoV-2 infection in this high-risk group. HCW subjects completed a longitudinal, novel behavioral at-home assessment of smell function with household items, as well as detailed symptom surveys that included a parosmia screening questionnaire, and RT-qPCR testing to identify SARS-CoV-2 infection.

Results: SARS-CoV-2 was identified in 17 (3.6%) of 473 HCW. Among the 17 infected HCW, 53% reported smell loss, and were more likely to report smell loss than COVID-negative HCW on both the at-home assessment and the screening questionnaire (P < .01). 67% reported smell loss prior to having a positive SARS-CoV-2 test, and smell loss was reported a median of two days before testing positive. Neurological symptoms were reported more frequently among COVID-positive HCW who reported smell loss (P < .01).

Conclusions: In this prospective study of HCW, self-reported changes in smell using two different measures were predictive of COVID-19 infection. Smell loss frequently preceded a positive test and was associated with neurological symptoms.
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http://dx.doi.org/10.1101/2020.09.07.20188813DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7491536PMC
September 2020