SARS-CoV-2 Titers in Wastewater Are Higher than Expected from Clinically Confirmed Cases
25 Aug 2020-Vol. 5, Iss: 4
TL;DR: A laboratory protocol to quantify viral titers in raw sewage via qPCR analysis and validate results with sequencing analysis suggests that the number of positive cases estimated from wastewater viral titer is orders of magnitude greater than the numberof confirmed clinical cases and therefore may significantly impact efforts to understand the case fatality rate and progression of disease.
Abstract: Wastewater surveillance represents a complementary approach to clinical surveillance to measure the presence and prevalence of emerging infectious diseases like the novel coronavirus SARS-CoV-2. This innovative data source can improve the precision of epidemiological modeling to understand the penetrance of SARS-CoV-2 in specific vulnerable communities. Here, we tested wastewater collected at a major urban treatment facility in Massachusetts and detected SARS-CoV-2 RNA from the N gene at significant titers (57 to 303 copies per ml of sewage) in the period from 18 to 25 March 2020 using RT-qPCR. We validated detection of SARS-CoV-2 by Sanger sequencing the PCR product from the S gene. Viral titers observed were significantly higher than expected based on clinically confirmed cases in Massachusetts as of 25 March. Our approach is scalable and may be useful in modeling the SARS-CoV-2 pandemic and future outbreaks. IMPORTANCE Wastewater-based surveillance is a promising approach for proactive outbreak monitoring. SARS-CoV-2 is shed in stool early in the clinical course and infects a large asymptomatic population, making it an ideal target for wastewater-based monitoring. In this study, we develop a laboratory protocol to quantify viral titers in raw sewage via qPCR analysis and validate results with sequencing analysis. Our results suggest that the number of positive cases estimated from wastewater viral titers is orders of magnitude greater than the number of confirmed clinical cases and therefore may significantly impact efforts to understand the case fatality rate and progression of disease. These data may help inform decisions surrounding the advancement or scale-back of social distancing and quarantine efforts based on dynamic wastewater catchment-level estimations of prevalence.
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TL;DR: The quantification of SARS-CoV-2 in wastewater affords the ability to monitor the prevalence of infections among the population via wastewater-based epidemiology (WBE) and highlights the viability of WBE for monitoring infectious diseases, such as COVID-19, in communities.
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TL;DR: The detection of the virus RNA in sewage, even when the CO VID-19 prevalence is low, and the correlation between concentration in sewage and reported prevalence of COVID-19, indicate that sewage surveillance could be a sensitive tool to monitor the circulation of theirus in the population.
Abstract: In the current COVID-19 pandemic, a significant proportion of cases shed SARS-Coronavirus-2 (SARS-CoV-2) with their faeces. To determine if SARS-CoV-2 RNA was present in sewage during the emergence...
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TL;DR: The data show the utility of viral RNA monitoring in municipal wastewater for SARS-CoV-2 infection surveillance at a population-wide level and 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.
Abstract: 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. Testing sewage for the novel coronavirus reveals epidemiological trends.
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TL;DR: There is an urgent need for further research to establish methodologies for wastewater surveillance and understand the implications of the presence of SARS-CoV-2 in wastewater.
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TL;DR: In this paper, the authors evaluated the presence of SARS-CoV-2 RNA in stools from COVID-19 patients, and reported the occurrence of the virus in wastewaters worldwide.
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Cites background from "SARS-CoV-2 Titers in Wastewater Are..."
...Furthermore, preliminary studies have reported the detection of SARS-CoV-2 RNA in wastewater in The Netherlands (Medema et al., 2020), France (Wurtzer et al., 2020), USA (Wu et al., 2020), and Australia (Ahmed et al., 2020)....
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...This approach will allow obtaining a rough estimation of the total number of subjects excreting the virus, by integrating - as done by Wu et al. (2020) in samples taken in the United States - the available information on viral shedding rates,WWTPs loads, and virus concentration inwastewaters....
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...Beforemolecular tests, the extracted nucleic acidswere further purified from potential PCR inhibitors using the OneStep PCR Inhibitor Removal Kit (Zymo Research, CA, USA)....
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..., 2020), USA (Wu et al., 2020), and Australia (Ashmed et al....
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...The PCR products were revealed by electrophoresis on 2% agarose gels and were purified using a Montage PCRm96 Microwell Filter Plate (Millipore, Billerica, MA, USA) and then direct sequenced on both strands (BioFab Research, Rome, Italy)....
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References
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TL;DR: Detailed virological analysis of nine cases of coronavirus disease 2019 (COVID-19) provides proof of active replication of the SARS-CoV-2 virus in tissues of the upper respiratory tract.
Abstract: Coronavirus disease 2019 (COVID-19) is an acute infection of the respiratory tract that emerged in late 20191,2. Initial outbreaks in China involved 13.8% of cases with severe courses, and 6.1% of cases with critical courses3. This severe presentation may result from the virus using a virus receptor that is expressed predominantly in the lung2,4; the same receptor tropism is thought to have determined the pathogenicity—but also aided in the control—of severe acute respiratory syndrome (SARS) in 20035. However, there are reports of cases of COVID-19 in which the patient shows mild upper respiratory tract symptoms, which suggests the potential for pre- or oligosymptomatic transmission6–8. There is an urgent need for information on virus replication, immunity and infectivity in specific sites of the body. Here we report a detailed virological analysis of nine cases of COVID-19 that provides proof of active virus replication in tissues of the upper respiratory tract. Pharyngeal virus shedding was very high during the first week of symptoms, with a peak at 7.11 × 108 RNA copies per throat swab on day 4. Infectious virus was readily isolated from samples derived from the throat or lung, but not from stool samples—in spite of high concentrations of virus RNA. Blood and urine samples never yielded virus. Active replication in the throat was confirmed by the presence of viral replicative RNA intermediates in the throat samples. We consistently detected sequence-distinct virus populations in throat and lung samples from one patient, proving independent replication. The shedding of viral RNA from sputum outlasted the end of symptoms. Seroconversion occurred after 7 days in 50% of patients (and by day 14 in all patients), but was not followed by a rapid decline in viral load. COVID-19 can present as a mild illness of the upper respiratory tract. The confirmation of active virus replication in the upper respiratory tract has implications for the containment of COVID-19. Detailed virological analysis of nine cases of coronavirus disease 2019 (COVID-19) provides proof of active replication of the SARS-CoV-2 virus in tissues of the upper respiratory tract.
5,840 citations
TL;DR: This study describes possible transmission of novel coronavirus disease 2019 (COVID-19) from an asymptomatic Wuhan resident to 5 family members in Anyang, a Chinese city in the neighboring province of Hubei.
Abstract: This study describes possible transmission of novel coronavirus disease 2019 (COVID-19) from an asymptomatic Wuhan resident to 5 family members in Anyang, a Chinese city in the neighboring province of Hubei.
3,818 citations
"SARS-CoV-2 Titers in Wastewater Are..." refers result in this paper
...Given the importance of assessing the fraction of SARS-CoV-2 infections that present with symptoms, we note that our results are consistent with the idea that a significant fraction of cases are not detected with current testing algorithms, and that this fraction may include a large number of asymptomatic individuals (21, 22)....
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TL;DR: It is estimated that 86% of all infections were undocumented before the 23 January 2020 travel restrictions, which explains the rapid geographic spread of SARS-CoV-2 and indicates that containment of this virus will be particularly challenging.
Abstract: Estimation of the prevalence and contagiousness of undocumented novel coronavirus [severe acute respiratory syndrome–coronavirus 2 (SARS-CoV-2)] infections is critical for understanding the overall prevalence and pandemic potential of this disease. Here, we use observations of reported infection within China, in conjunction with mobility data, a networked dynamic metapopulation model, and Bayesian inference, to infer critical epidemiological characteristics associated with SARS-CoV-2, including the fraction of undocumented infections and their contagiousness. We estimate that 86% of all infections were undocumented [95% credible interval (CI): 82–90%] before the 23 January 2020 travel restrictions. The transmission rate of undocumented infections per person was 55% the transmission rate of documented infections (95% CI: 46–62%), yet, because of their greater numbers, undocumented infections were the source of 79% of the documented cases. These findings explain the rapid geographic spread of SARS-CoV-2 and indicate that containment of this virus will be particularly challenging.
3,324 citations
"SARS-CoV-2 Titers in Wastewater Are..." refers result in this paper
...Given the importance of assessing the fraction of SARS-CoV-2 infections that present with symptoms, we note that our results are consistent with the idea that a significant fraction of cases are not detected with current testing algorithms, and that this fraction may include a large number of asymptomatic individuals (21, 22)....
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TL;DR: Real-time RT-PCR results of all respiratory and faecal samples from patients with coronavirus disease 2019 (COVID-19) at the Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, China, throughout the course of their illness and obligated quarantine period show associations that should be interpreted with caution because of the possibility of confounding.
1,320 citations
"SARS-CoV-2 Titers in Wastewater Are..." refers background in this paper
...While it is impractical to test every US resident for SARS-CoV-2, the virus has been found in the stool of confirmed COVID-19 patients (1), making it a promising...
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TL;DR: The most abundant fecal virus in this study was pepper mild mottle virus (PMMV), which was found in high concentrations—up to 109 virions per gram of dry weight fecal matter, indicating that this plant virus is prevalent in the human population.
Abstract: The human gut is known to be a reservoir of a wide variety of microbes, including viruses. Many RNA viruses are known to be associated with gastroenteritis; however, the enteric RNA viral community present in healthy humans has not been described. Here, we present a comparative metagenomic analysis of the RNA viruses found in three fecal samples from two healthy human individuals. For this study, uncultured viruses were concentrated by tangential flow filtration, and viral RNA was extracted and cloned into shotgun viral cDNA libraries for sequencing analysis. The vast majority of the 36,769 viral sequences obtained were similar to plant pathogenic RNA viruses. The most abundant fecal virus in this study was pepper mild mottle virus (PMMV), which was found in high concentrations—up to 109 virions per gram of dry weight fecal matter. PMMV was also detected in 12 (66.7%) of 18 fecal samples collected from healthy individuals on two continents, indicating that this plant virus is prevalent in the human population. A number of pepper-based foods tested positive for PMMV, suggesting dietary origins for this virus. Intriguingly, the fecal PMMV was infectious to host plants, suggesting that humans might act as a vehicle for the dissemination of certain plant viruses.
648 citations