Tracing the origin of SARS-CoV-2 Omicron-like Spike sequences detected in wastewater

ImportanceThe origin of highly divergent "cryptic" SARS-CoV-2 Spike sequences, which appear in wastewater but not clinical samples, is unknown. These wastewater sequences have harbored many of the same mutations that later emerged in Omicron variants. If these enigmatic sequences are human-derived and transmissible, they could both be a source of future variants and a valuable tool for forecasting sequences that should be incorporated into vaccines and therapeutics. ObjectiveTo determine whether enigmatic SARS-CoV-2 lineages detected in wastewater have a human or non-human (i.e., animal) source. DesignOn January 11, 2022, an unusual Spike sequence was detected in municipal wastewater from a metropolitan area. Over the next four months, more focused wastewater sampling resolved the source of this variant. SettingThis study was performed in Wisconsin, United States, which has a comprehensive program for detecting SARS-CoV-2 in wastewater. ParticipantsComposite wastewater samples were used for this study; therefore, no individuals participated. Main Outcome(s) and Measure(s)The primary outcome was to determine the host(s) responsible for shedding this variant in wastewater. Both human and non-human hosts were plausible candidates at the studys outset. ResultsThe presence of the cryptic virus was narrowed from a municipal wastewater sample (catchment area >100,000 people) to an indoor wastewater sample from a single facility (catchment area [~]30 people), indicating the human origin of this virus. Extraordinarily high concentrations of viral RNA ([~]520,000,000 genome copies / L and [~]1,600,000,000 genome copies / L in June and August 2022, respectively) were detected in the indoor wastewater sample. The virus sequence harbored a combination of fixed nucleotide substitutions previously observed only in Pango lineage B.1.234, a variant that circulated at low levels in Wisconsin from October 2020 to February 2021. Conclusions and RelevanceHigh levels of persistent SARS-CoV-2 shedding from the gastrointestinal tract of an infected individual likely explain the presence of evolutionarily advanced "cryptic variants" observed in some wastewater samples. Key points QuestionWhat is the source of unusual SARS-CoV-2 Omicron-like Spike variants detected in wastewater but not in clinical samples? FindingsWe identified a cryptic SARS-CoV-2 lineage in wastewater collected at a central wastewater treatment facility and traced its source to a single wastewater outlet serving six restrooms. The virus in this sample resembled a 2020-2021 lineage except for the Spike protein, in which Omicron-like variants were observed. MeaningProlonged shedding from the human gastrointestinal tract is the most likely source for evolutionarily advanced SARS-CoV-2 variant sequences found in wastewater.

Evolution of a globally unique SARS-CoV-2 Spike E484T monoclonal antibody escape mutation in a persistently infected, immunocompromised individual.

Prolonged infections in immunocompromised individuals may be a source for novel SARS-CoV-2 variants, particularly when both the immune system and antiviral therapy fail to clear the infection, thereby promoting adaptation. Here we describe an approximately 16-month case of SARS-CoV-2 infection in an immunocompromised individual. Following monotherapy with the monoclonal antibody Bamlanivimab, the individuals virus was resistant to this antibody via a globally unique Spike amino acid variant (E484T) that evolved from E484A earlier in infection. With the emergence and spread of the Omicron Variant of Concern, which also contains Spike E484A, E484T may arise again as an antibody-resistant derivative of E484A.

Anti-membrane and anti-spike antibodies are long-lasting and together discriminate between past COVID-19 infection and vaccination

The consequences of past COVID-19 infection for personal health and long-term population immunity are only starting to be revealed. Unfortunately, detecting past infection is currently a challenge, limiting clinical and research endeavors. Widely available anti-SARS-CoV-2 antibody tests cannot differentiate between past infection and vaccination given vaccine-induced anti-spike antibodies and the rapid loss of infection-induced anti-nucleocapsid antibodies. Anti-membrane antibodies develop after COVID-19, but their long-term persistence is unknown. Here, we demonstrate that anti-membrane IgG is a sensitive and specific marker of past COVID-19 infection and persists at least one year. We also confirm that anti-receptor binding domain (RBD) Ig is a long-lasting, sensitive, and specific marker of past infection and vaccination, while anti-nucleocapsid IgG lacks specificity and quickly declines after COVID-19. Thus, a combination of anti-membrane and anti-RBD antibodies can accurately differentiate between distant COVID-19 infection, vaccination, and naive states to advance public health, individual healthcare, and research goals.

Vaccinated and unvaccinated individuals have similar viral loads in communities with a high prevalence of the SARS-CoV-2 delta variant

The SARS-CoV-2 Delta Variant of Concern is highly transmissible and contains mutations that confer partial immune escape. The emergence of Delta in North America caused the first surge in COVID-19 cases after SARS-CoV-2 vaccines became widely available. To determine whether individuals infected despite vaccination might be capable of transmitting SARS-CoV-2, we compared RT-PCR cycle threshold (Ct) data from 20,431 test-positive anterior nasal swab specimens from fully vaccinated (n = 9,347) or unvaccinated (n=11,084) individuals tested at a single commercial laboratory during the interval 28 June - 1 December 2021 when Delta variants were predominant. We observed no significant effect of vaccine status alone on Ct value, nor when controlling for vaccine product or sex. Testing a subset of low-Ct (<25) samples, we detected infectious virus at similar rates, and at similar titers, in specimens from vaccinated and unvaccinated individuals. These data indicate that vaccinated individuals infected with Delta variants are capable of shedding infectious SARS-CoV-2 and could play a role in spreading COVID-19.

SARS-CoV-2 transmission in intercollegiate athletics not fully mitigated with daily antigen testing

BackgroundHigh frequency, rapid turnaround SARS-CoV-2 testing continues to be proposed as a way of efficiently identifying and mitigating transmission in congregate settings. However, two SARS-CoV-2 outbreaks occurred among intercollegiate university athletic programs during the fall 2020 semester despite mandatory directly observed daily antigen testing. MethodsDuring the fall 2020 semester, athletes and staff in both programs were tested daily using Quidels Sofia SARS Antigen Fluorescent Immunoassay (FIA), with positive antigen results requiring confirmatory testing with real-time reverse transcription polymerase chain reaction (RT-PCR). We used genomic sequencing to investigate transmission dynamics in these two outbreaks. ResultsIn Outbreak 1, 32 confirmed cases occurred within a university athletics program after the index patient attended a meeting while infectious despite a negative antigen test on the day of the meeting. Among isolates sequenced from Outbreak 1, 24 (92%) of 26 were closely related, suggesting sustained transmission following an initial introduction event. In Outbreak 2, 12 confirmed cases occurred among athletes from two university programs that faced each other in an athletic competition despite receiving negative antigen test results on the day of the competition. Sequences from both teams were closely related and unique from strains circulating in the community, suggesting transmission during intercollegiate competition. ConclusionsThese findings suggest that antigen testing alone, even when mandated and directly observed, may not be sufficient as an intervention to prevent SARS-CoV-2 outbreaks in congregate settings, and highlights the importance of supplementing serial antigen testing with appropriate mitigation strategies to prevent SARS-CoV-2 outbreak in congregate settings. SummaryHigh frequency, rapid turnaround SARS-CoV-2 testing continues to be proposed as a way of efficiently identifying and mitigating transmission in congregate settings. However, here we describe two SARS-CoV-2 outbreaks occurred among intercollegiate university athletic programs during the fall 2020 semester.

Fever, Diarrhea, and Severe Disease Correlate with High Persistent Antibody Levels against SARS-CoV-2

Lasting immunity will be critical for overcoming the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, factors that drive the development of high titers of anti-SARS-CoV-2 antibodies and how long those antibodies persist remain unclear. Our objective was to comprehensively evaluate anti-SARS-CoV-2 antibodies in a clinically diverse COVID-19 convalescent cohort at defined time points to determine if anti-SARS-CoV-2 antibodies persist and to identify clinical and demographic factors that correlate with high titers. Using a novel multiplex assay to quantify IgG against four SARS-CoV-2 antigens, a receptor binding domain-angiotensin converting enzyme 2 inhibition assay, and a SARS-CoV-2 neutralization assay, we found that 98% of COVID-19 convalescent subjects had anti-SARS-CoV-2 antibodies five weeks after symptom resolution (n=113). Further, antibody levels did not decline three months after symptom resolution (n=79). As expected, greater disease severity, older age, male sex, obesity, and higher Charlson Comorbidity Index score correlated with increased anti-SARS-CoV-2 antibody levels. We demonstrated for the first time that COVID-19 symptoms, namely fever, abdominal pain, diarrhea and low appetite, correlated consistently with higher anti-SARS-CoV-2 antibody levels. Our results provide new insights into the development and persistence of anti-SARS-CoV-2 antibodies.

Oil Immersed Lossless Total Analysis System (OIL-TAS): Integrated RNA Extraction and Detection for SARS-CoV-2 Testing

The coronavirus disease 2019 (COVID-19) pandemic exposed difficulties in scaling current quantitative PCR (qPCR)-based diagnostic methodologies for large-scale infectious disease testing. Bottlenecks include the lengthy multi-step process of nucleic acid extraction followed by qPCR readouts, which require costly instrumentation and infrastructure, as well as reagent and plastic consumable shortages stemming from supply chain constraints. Here we report a novel Oil Immersed Lossless Total Analysis System (OIL-TAS), which integrates RNA extraction and detection onto a single device that is simple, rapid, cost effective, uses minimal supplies and requires reduced infrastructure to perform. We validated the performance of OIL-TAS using contrived samples containing inactivated SARS-CoV-2 viral particles, which show that the assay can reliably detect an input concentration of 10 copies/L and sporadically detect down to 1 copy/L. The OIL-TAS method can serve as a faster, cheaper, and easier-to-deploy alternative to current qPCR-based methods for infectious disease testing.

Optimizing direct RT-LAMP to detect transmissible SARS-CoV-2 from primary patient samples

SARS-CoV-2 testing is crucial to controlling the spread of this virus, yet shortages of nucleic acid extraction supplies and other key reagents have hindered the response to COVID-19 in the US. Several groups have described loop-mediated isothermal amplification (LAMP) assays for SARS-CoV-2, including testing directly from nasopharyngeal swabs and eliminating the need for reagents in short supply. Here we describe a fluorescence-based RT-LAMP test using direct nasopharyngeal swab samples and show consistent detection in clinically confirmed samples, albeit with approximately 100-fold lower sensitivity than qRT-PCR. We demonstrate that adding lysis buffer directly into the RT-LAMP reaction improves the sensitivity of some samples by approximately 10-fold. Overall, the limit of detection (LOD) of RT-LAMP using direct nasopharyngeal swab or saliva samples without RNA extraction is 1x105-1x106 copies/ml. This LOD is sufficient to detect samples from which infectious virus can be cultured. Therefore, samples that test positive in this assay contain levels of virus that are most likely to perpetuate transmission. Furthermore, purified RNA achieves a similar LOD to qRT-PCR. These results indicate that high-throughput RT-LAMP testing could augment qRT-PCR in SARS-CoV-2 screening programs, especially while the availability of qRT-PCR testing and RNA extraction reagents is constrained.

Initial evaluation of a mobile SARS-CoV-2 RT-LAMP testing strategy

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) control in the United States remains hampered, in part, by testing limitations. We evaluated a simple, outdoor, mobile, colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay workflow where self-collected saliva is tested for SARS-CoV-2 RNA. From July 16 to November 19, 2020, 4,704 surveillance samples were collected from volunteers and tested for SARS-CoV-2 at 5 sites. A total of 21 samples tested positive for SARS-CoV-2 by RT-LAMP; 12 were confirmed positive by subsequent quantitative reverse-transcription polymerase chain reaction (qRT-PCR) testing, while 8 were negative for SARS-CoV-2 RNA, and 1 could not be confirmed because the donor did not consent to further molecular testing. We estimated the RT-LAMP assays false-negative rate from July 16 to September 17, 2020 by pooling residual heat-inactivated saliva that was unambiguously negative by RT-LAMP into groups of 6 or less and testing for SARS-CoV-2 RNA by qRT-PCR. We observed a 98.8% concordance between the RT-LAMP and qRT-PCR assays, with only 5 of 421 RT-LAMP negative pools (2,493 samples) testing positive in the more sensitive qRT-PCR assay. Overall, we demonstrate a rapid testing method that can be implemented outside the traditional laboratory setting by individuals with basic molecular biology skills and can effectively identify asymptomatic individuals who would not typically meet the criteria for symptom-based testing modalities.

Distinct patterns of SARS-CoV-2 transmission in two nearby communities in Wisconsin, USA

Evidence-based public health approaches that minimize the introduction and spread of new SARS-CoV-2 transmission clusters are urgently needed in the United States and other countries struggling with expanding epidemics. Here we analyze 247 full-genome SARS-CoV-2 sequences from two nearby communities in Wisconsin, USA, and find surprisingly distinct patterns of viral spread. Dane County had the 12th known introduction of SARS-CoV-2 in the United States, but this did not lead to descendant community spread. Instead, the Dane County outbreak was seeded by multiple later introductions, followed by limited community spread. In contrast, relatively few introductions in Milwaukee County led to extensive community spread. We present evidence for reduced viral spread in both counties, and limited viral transmission between counties, following the statewide "Safer at Home" public health order, which went into effect 25 March 2020. Our results suggest that early containment efforts suppressed the spread of SARS-CoV-2 within Wisconsin.

Determining the source of transmission of SARS-CoV-2 infection in a healthcare worker

BackgroundHealthcare workers (HCWs) are at the frontlines of the COVID-19 pandemic and are at risk of exposure to SARS-CoV-2 infection from their interactions with patients and in the community (1, 2). Limited availability of recommended personal protective equipment (PPE), in particular N95 respirators, has fueled concerns about whether HCWs are adequately protected from exposure while caring for patients. Understanding the source of SARS-CoV-2 infection in a HCW - the community or the healthcare system - is critical for understanding the effectiveness of hospital infection control and PPE practices. In Dane County, Wisconsin, community prevalence of SARS-CoV-2 is relatively low (cumulative prevalence of ~0.06% - positive cases / total population in Dane county as of April 17). Although SARS-CoV-2 infections in HCWs are often presumed to be acquired during the course of patient care, there are few reports unambiguously identifying the source of acquisition. ObjectiveTo determine the source of transmission of SARS-CoV-2 in a healthcare worker.