Survey of white-footed mice in Connecticut, USA reveals low SARS-CoV-2 seroprevalence and infection with divergent betacoronaviruses (preprint)

Diverse mammalian species display susceptibility to and infection with SARS-CoV-2. Potential SARS-CoV-2 spillback into rodents is understudied despite their host role for numerous zoonoses and human proximity. We assessed exposure and infection among white-footed mice (Peromyscus leucopus) in Connecticut, USA. We observed 1% (6/540) wild-type neutralizing antibody seroprevalence among 2020-2022 residential mice with no cross-neutralization of variants. We detected no SARS-CoV-2 infections via RT-qPCR, but identified non-SARS-CoV-2 betacoronavirus infections via pan-coronavirus PCR among 1% (5/468) of residential mice. Sequencing revealed two divergent betacoronaviruses, preliminarily named Peromyscus coronavirus-1 and -2. Both belong to the Betacoronavirus 1 species and are ~90% identical to the closest known relative, Porcine hemagglutinating encephalomyelitis virus. Low SARS-CoV-2 seroprevalence suggests white-footed mice may not be sufficiently susceptible or exposed to SARS-CoV-2 to present a long-term human health risk. However, the discovery of divergent, non-SARS-CoV-2 betacoronaviruses expands the diversity of known rodent coronaviruses and further investigation is required to understand their transmission extent.

Multi-site validation of an amplicon-based sequencing approach for human monkeypox virus (preprint)

The 2022 multi-country monkeypox outbreak concurrent with the ongoing COVID-19 pandemic has further highlighted the need for genomic surveillance and pathogen whole genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early human monkeypox virus infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the current outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there is an urgent need for a more sensitive and broadly applicable sequencing approach. Amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for SARS-CoV-2. Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented during the COVID-19 pandemic. We sequenced clinical samples that tested presumptive positive for monkeypox virus with amplicon-based and metagenomic sequencing approaches. Upon comparison, we found notably higher genome coverage across the virus genome, particularly in higher PCR cycle threshold (lower DNA titer) samples, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach. By sending out primer pool aliquots to laboratories across the United States and internationally, we validated the primer scheme in 12 public health laboratories with their established Illumina or Oxford Nanopore Technologies sequencing workflows and with different sample types across a range of Ct values. Our findings suggest that amplicon-based sequencing increases the success rate across a wider range of viral DNA concentrations, with the PCR Ct value threshold at which laboratories were able to achieve 80% genome coverage at 10X ranging between Ct 25-33. Therefore, it increases the number of samples where near-complete genomes can be generated and it provides a cost-effective and widely applicable alternative to metagenomics for continued human monkeypox virus genomic surveillance. Importantly, we show that the human monkeypox virus primer scheme can be used with currently implemented amplicon-based SARS-CoV-2 sequencing workflows, with minimal change to the protocol.

Insights into the limited global spread of the immune evasive SARS-CoV-2 variant Mu (preprint)

SARS-CoV-2 Variants of Concern (VOCs) continue to reshape the trajectory of the COVID-19 pandemic. However, why some VOCs, like Omicron, become globally dominant while the spread of others is limited is not fully understood. To address this question, we investigated the VOC Mu, which was first identified in Colombia in late 2020. Our study demonstrates that, although Mu is less sensitive to neutralization compared to variants that preceded it, it did not spread significantly outside of South and Central America. Additionally, we find evidence that the response to Mu was impeded by reporting delays and gaps in the global genomic surveillance system. Our findings suggest that immune evasion alone was not sufficient to outcompete highly transmissible variants that were circulating concurrently with Mu. Insights into the complex relationship between genomic and epidemiological characteristics of previous variants should inform our response to variants that are likely to emerge in the future.

Rapid emergence of SARS-CoV-2 Omicron variant is associated with an infection advantage over Delta in vaccinated persons (preprint)

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continues to shape the coronavirus disease 2019 (Covid-19) pandemic. The detection and rapid spread of the SARS-CoV-2 Omicron variant (lineage B.1.1.529) in Botswana and South Africa became a global concern because it contained 15 mutations in the spike protein immunogenic receptor binding domain and was less neutralized by sera derived from vaccinees compared to the previously dominant Delta variant. To investigate if Omicron is more likely than Delta to cause infections in vaccinated persons, we analyzed 37,877 nasal swab PCR tests conducted from 12-26 December 2021 and calculated the test positivity rates for each variant by vaccination status. We found that the positivity rate among unvaccinated persons was higher for Delta (5.2%) than Omicron (4.5%). We found similar results in persons who received a single vaccine dose. Conversely, our results show that Omicron had higher positivity rates than Delta among those who received two doses within five months (Omicron = 4.7% vs. Delta = 2.6%), two doses more than five months ago (4.2% vs. 2.9%), and three vaccine doses (2.2% vs. 0.9%). Our estimates of Omicron positivity rates in persons receiving one or two vaccine doses were not significantly lower than unvaccinated persons but were 49.7% lower after three doses. In comparison, the reduction in Delta positivity rates from unvaccinated to 2 vaccine doses was 45.6-49.6% and to 3 vaccine doses was 83.2%. Despite the higher positivity rates for Omicron in vaccinated persons, we still found that 91.2% of the Omicron infections in our study occurred in persons who were eligible for 1 or more vaccine doses at the time of PCR testing. In conclusion, escape from vaccine-induced immunity likely contributed to the rapid rise in Omicron infections.

Comparison of infectious SARS-CoV-2 from the nasopharynx of vaccinated and unvaccinated individuals (preprint)

The frequency of SARS-CoV-2 breakthrough infections in fully vaccinated individuals increased with the emergence of the Delta variant, particularly with longer time from vaccine completion. However, whether breakthrough infections lead to onward transmission remains unclear. Here, we conducted a study involving 125 patients comprised of 72 vaccinated and 53 unvaccinated individuals, to assess the levels of infectious virus in vaccinated and unvaccinated individuals. Quantitative plaque assays showed no significant differences in the titers of virus between these cohorts. However, the proportion of nasopharyngeal samples with culturable virus was lower in the vaccinated patients relative to unvaccinated patients (21% vs. 40%). Finally, time-to-event analysis with Kaplan-Myer curves revealed that protection from culturable infectious virus waned significantly starting at 5 months after completing a 2-dose regimen of mRNA vaccines. These results have important implications in timing of booster dose to prevent onward transmission from breakthrough cases.

Immunogenicity of heterologous BNT162b2 booster in fully vaccinated individuals with CoronaVac against SARS-CoV-2 variants Delta and Omicron: the Dominican Republic Experience (preprint)

The recent emergence of the SARS-CoV-2 Omicron variant is raising concerns because of its increased transmissibility and by its numerous spike mutations with potential to evade neutralizing antibodies elicited by COVID-19 vaccines. The Dominican Republic was among the first countries in recommending the administration of a third dose COVID-19 vaccine to address potential waning immunity and reduced effectiveness against variants. Here, we evaluated the effects of a heterologous BNT162b2 mRNA vaccine booster on the humoral immunity of participants that had received a two-dose regimen of CoronaVac, an inactivated vaccine used globally. We found that heterologous CoronaVac prime followed by BNT162b2 booster regimen induces elevated virus-specific antibody levels and potent neutralization activity against the ancestral virus and Delta variant, resembling the titers obtained after two doses of mRNA vaccines. While neutralization of Omicron was undetectable in participants that had received a two-dose regimen of CoronaVac vaccine, BNT162b2 booster resulted in a 1.4-fold increase in neutralization activity against Omicron, compared to two-dose mRNA vaccine. Despite this increase, neutralizing antibody titers were reduced by 6.3-fold and 2.7-fold for Omicron compared to ancestral and Delta variant, respectively. Surprisingly, previous SARS-CoV-2 infection did not affect the neutralizing titers for Omicron in participants that received the heterologous regimen. Our findings have immediate implications for multiples countries that previously used a two-dose regimen of CoronaVac and reinforce the notion that the Omicron variant is associated with immune escape from vaccines or infection-induced immunity, highlighting the global need for vaccine boosters to combat the impact of emerging variants.

Comparative transmissibility of SARS-CoV-2 variants Delta and Alpha in New England, USA (preprint)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant quickly rose to dominance in mid-2021, displacing other variants, including Alpha. Studies using data from the United Kingdom and India estimated that Delta was 40-80% more transmissible than Alpha, allowing Delta to become the globally dominant variant. However, it was unclear if the ostensible difference in relative transmissibility was due mostly to innate properties of Deltas infectiousness or differences in the study populations. To investigate, we formed a partnership with SARS-CoV-2 genomic surveillance programs from all six New England US states. By comparing logistic growth rates, we found that Delta emerged 37-163% faster than Alpha in early 2021 (37% Massachusetts, 75% New Hampshire, 95% Maine, 98% Rhode Island, 151% Connecticut, and 163% Vermont). We next computed variant-specific effective reproductive numbers and estimated that Delta was 58-120% more transmissible than Alpha across New England (58% New Hampshire, 68% Massachusetts, 76% Connecticut, 85% Rhode Island, 98% Maine, and 120% Vermont). Finally, using RT-PCR data, we estimated that Delta infections generate on average [~]6 times more viral RNA copies per mL than Alpha infections. Overall, our evidence indicates that Deltas enhanced transmissibility could be attributed to its innate ability to increase infectiousness, but its epidemiological dynamics may vary depending on the underlying immunity and behavior of distinct populations.

Variant abundance estimation for SARS-CoV-2 in wastewater using RNA-Seq quantification (preprint)

Effectively monitoring the spread of SARS-CoV-2 variants is essential to efforts to counter the ongoing pandemic. Wastewater monitoring of SARS-CoV-2 RNA has proven an effective and efficient technique to approximate COVID-19 case rates in the population. Predicting variant abundances from wastewater, however, is technically challenging. Here we show that by sequencing SARS-CoV-2 RNA in wastewater and applying computational techniques initially used for RNA-Seq quantification, we can estimate the abundance of variants in wastewater samples. We show by sequencing samples from wastewater and clinical isolates in Connecticut U.S.A. between January and April 2021 that the temporal dynamics of variant strains broadly correspond. We further show that this technique can be used with other wastewater sequencing techniques by expanding to samples taken across the United States in a similar timeframe. We find high variability in signal among individual samples, and limited ability to detect the presence of variants with clinical frequencies <10%; nevertheless, the overall trends match what we observed from sequencing clinical samples. Thus, while clinical sequencing remains a more sensitive technique for population surveillance, wastewater sequencing can be used to monitor trends in variant prevalence in situations where clinical sequencing is unavailable or impractical.

Impact of circulating SARS-CoV-2 variants on mRNA vaccine-induced immunity in uninfected and previously infected individuals (preprint)

The emergence of SARS-CoV-2 variants with mutations in major neutralizing antibody-binding sites can affect humoral immunity induced by infection or vaccination (1-6). We analysed the development of anti-SARS-CoV-2 antibody and T cell responses in previously infected (recovered) or uninfected (naive) individuals that received mRNA vaccines to SARS-CoV-2. While previously infected individuals sustained higher antibody titers than uninfected individuals post-vaccination, the latter reached comparable levels of neutralization responses to the ancestral strain than previously infected individuals 7 days after the second vaccine dose. T cell activation markers measured upon spike or nucleocapsid peptide in vitro stimulation showed a progressive increase after vaccination in the time-points analysed. Comprehensive analysis of plasma neutralization using 16 authentic isolates of distinct locally circulating SARS-CoV-2 variants revealed a range of reduction in the neutralization capacity associated with specific mutations in the spike gene: lineages with E484K and N501Y/T (e.g., B.1.351 and P.1) had the greatest reduction, followed by lineages with L452R (e.g., B.1.617.2) or with E484K (without N501Y/T). While both groups retained neutralization capacity against all variants, plasma from previously infected vaccinated individuals displayed overall better neutralization capacity when compared to plasma from uninfected individuals that also received two vaccine doses, pointing to vaccine boosters as a relevant future strategy to alleviate the impact of emerging variants on antibody neutralizing activity.

Combining genomic and epidemiological data to compare the transmissibility of SARS-CoV-2 lineages (preprint)

Emerging SARS-CoV-2 variants have shaped the second year of the COVID-19 pandemic and the public health discourse around effective control measures. Evaluating the public health threat posed by a new variant is essential for appropriately adapting response efforts when community transmission is detected. However, this assessment requires that a true comparison can be made between the new variant and its predecessors because factors other than the virus genotype may influence spread and transmission. In this study, we develop a framework that integrates genomic surveillance data to estimate the relative effective reproduction number (Rt) of co-circulating lineages. We use Connecticut, a state in the northeastern United States in which the SARS-CoV-2 variants B.1.1.7 and B.1.526 co-circulated in early 2021, as a case study for implementing this framework. We find that the Rt of B.1.1.7 was 6-10% larger than that of B.1.526 in Connecticut in the midst of a COVID-19 vaccination campaign. To assess the generalizability of this framework, we apply it to genomic surveillance data from New York City and observe the same trend. Finally, we use discrete phylogeography to demonstrate that while both variants were introduced into Connecticut at comparable frequencies, clades that resulted from introductions of B.1.1.7 were larger than those resulting from B.1.526 introductions. Our framework, which uses open-source methods requiring minimal computational resources, may be used to monitor near real-time variant dynamics in a myriad of settings.

Sequencing SARS-CoV-2 Genomes from Saliva (preprint)

Genomic sequencing is crucial to understanding the epidemiology and evolution of SARS-CoV-2. Often, genomic studies rely on remnant diagnostic material, typically nasopharyngeal swabs, as input into whole genome SARS-CoV-2 next-generation sequencing pipelines. Saliva has proven to be a safe and stable specimen for the detection of SARS-CoV-2 RNA via traditional diagnostic assays, however saliva is not commonly used for SARS-CoV-2 sequencing. Using the ARTIC Network amplicon-generation approach with sequencing on the Oxford Nanopore MinION, we demonstrate that sequencing SARS-CoV-2 from saliva produces genomes comparable to those from nasopharyngeal swabs, and that RNA extraction is necessary to generate complete genomes from saliva. In this study, we show that saliva is a useful specimen type for genomic studies of SARS-CoV-2.

A stem-loop RNA RIG-I agonist confers prophylactic and therapeutic protection against acute and chronic SARS-CoV-2 infection in mice (preprint)

As SARS-CoV-2 continues to cause morbidity and mortality around the world, there is an urgent need for the development of effective medical countermeasures. Here, we assessed the antiviral capacity of a minimal RIG-I agonist, stem-loop RNA 14 (SLR14), in viral control, disease prevention, post-infection therapy, and cross-variant protection in mouse models of SARS-CoV-2 infection. A single dose of SLR14 prevented viral replication in the lower respiratory tract and development of severe disease in a type I interferon (IFN-I) dependent manner. SLR14 demonstrated remarkable protective capacity against lethal SARS-CoV-2 infection when used prophylactically and retained considerable efficacy as a therapeutic agent. In immunodeficient mice carrying chronic SARS-CoV-2 infection, SLR14 elicited near-sterilizing innate immunity by inducing IFN-I responses in the absence of the adaptive immune system. In the context of infection with variants of concern (VOC), SLR14 conferred broad protection and uncovered an IFN-I resistance gradient across emerging VOC. These findings demonstrate the therapeutic potential of SLR14 as a host-directed, broad-spectrum antiviral for early post-exposure treatment and for treatment of chronically infected immunosuppressed patients.

Longitudinal immune profiling of a SARS-CoV-2 reinfection in a solid organ transplant recipient (preprint)

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 patients 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.

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

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 2500 COVID-19 cases associated with this variant have been detected in the US since December 2020, but the extent of establishment is relatively unknown. Using travel, genomic, and diagnostic 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. 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.

Evidence for SARS-CoV-2 Spike Protein in the Urine of COVID-19 patients (preprint)

SARS-CoV-2 infection has so far affected over 42 million people worldwide, causing over 1.1 million deaths. With the large majority of SARS-CoV-2 infected individuals being asymptomatic, major concerns have been raised about possible long-term consequences of the infection. We developed an antigen capture assay to detect SARS-CoV-2 spike protein in urine samples from COVID-19 patients whose diagnosis was confirmed by PCR from nasopharyngeal swabs (NP-PCR+). The study used a collection of 233 urine samples from 132 participants from Yale New Haven Hospital and the Childrens Hospital of Philadelphia obtained during the pandemic (106 NP-PCR+ and 26 NP-PCR-) as well as a collection of 20 urine samples from 20 individuals collected before the pandemic. Our analysis identified 23 out of 91 (25%) NP-PCR+ adult participants with SARS-CoV-2 spike S1 protein in urine (Ur-S+). Interestingly, although all NP-PCR+ children were Ur-S-, 1 NP-PCR- child was found to be positive for spike protein in urine. Of the 23 Ur-S+ adults, only 1 individual showed detectable viral RNA in urine. Our analysis further showed that 24% and 21% of NP-PCR+ adults have high levels of albumin and cystatin C in urine, respectively. Among individuals with albuminuria (>0.3 mg/mg of creatinine) statistical correlation could be found between albumin and spike protein in urine. Together, our data showed that 1 of 4 of SARS-CoV-2 infected individuals develop renal abnormalities such as albuminuria. Awareness about the long-term impact of these findings is warranted.

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

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 in vitro. 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.

Saliva viral load is a dynamic unifying correlate of COVID-19 severity and mortality (preprint)

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.

Diverse Functional Autoantibodies in Patients with COVID-19 (preprint)

COVID-19 manifests with a wide spectrum of clinical phenotypes that are characterized by exaggerated and misdirected host immune responses1-8. While pathological innate immune activation is well documented in severe disease1, 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.

Pooling saliva to increase SARS-CoV-2 testing capacity (preprint)

Expanding testing capabilities is integral to managing the further spread of SARS-CoV-2 and developing reopening strategies, particularly in regards to identifying and isolating asymptomatic and pre-symptomatic individuals. Central to meeting testing demands are specimens that can be easily and reliably collected and laboratory capacity to rapidly ramp up to scale. We and others have demonstrated that high and consistent levels of SARS-CoV-2 RNA can be detected in saliva from COVID-19 inpatients, outpatients, and asymptomatic individuals. As saliva collection is non-invasive, extending this strategy to test pooled saliva samples from multiple individuals could thus provide a simple method to expand testing capacity. However, hesitation towards pooled sample testing arises due to the dilution of positive samples, potentially shifting weakly positive samples below the detection limit for SARS-CoV-2 and thereby decreasing the sensitivity. Here, we investigated the potential of pooling saliva samples by 5, 10, and 20 samples prior to RNA extraction and RT-qPCR detection of SARS-CoV-2. Based on samples tested, we conservatively estimated a reduction of 7.41%, 11.11%, and 14.81% sensitivity, for each of the pool sizes, respectively. Using these estimates we modeled anticipated changes in RT-qPCR cycle threshold to show the practical impact of pooling on results of SARS-CoV-2 testing. In tested populations with greater than 3% prevalence, testing samples in pools of 5 requires the least overall number of tests. Below 1% however, pools of 10 or 20 are more beneficial and likely more supportive of ongoing surveillance strategies.

Simply saliva: stability of SARS-CoV-2 detection negates the need for expensive collection devices (preprint)

Most currently approved strategies for the collection of saliva for COVID-19 diagnostics require specialized tubes containing buffers promoted for the stabilization of SARS-CoV-2 RNA and virus inactivation. Yet many of these are expensive, in limited supply, and not necessarily validated specifically for viral RNA. While saliva is a promising sample type as it can be reliably self-collected for the sensitive detection of SARS-CoV-2, the expense and availability of these collection tubes are prohibitive to mass testing efforts. Therefore, we investigated the stability of SARS-CoV-2 RNA and infectious virus detection from saliva without supplementation. We tested RNA stability over extended periods of time (2-25 days) and at temperatures representing at-home storage and elevated temperatures which might be experienced when cold chain transport may be unavailable. We found SARS-CoV-2 RNA in saliva from infected individuals is stable at 4{degrees}C, room temperature (~19{degrees}C), and 30{degrees}C for prolonged periods and found limited evidence for viral replication in saliva. This work demonstrates that expensive saliva collection options involving RNA stabilization and virus inactivation buffers are not always needed, permitting the use of cheaper collection options. Affordable testing methods are urgently needed to meet current testing demands and for continued surveillance in reopening strategies.