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Clinical testing has been a vital part of the response to and suppression of the COVID-19 pandemic; however, testing imposes significant burdens on a population. College students had to contend with clinical testing while simultaneously dealing with health risks and the academic pressures brought on by quarantines, changes to virtual platforms, and other disruptions to daily life. The objective of this study was to analyze whether wastewater surveillance can be used to decrease the intensity of clinical testing while maintaining reliable measurements of diseases incidence on campus. Twelve months of human health and wastewater surveillance data for eight residential buildings on a university campus were analyzed to establish how SARS-CoV-2 levels in the wastewater can be used to minimize clinical testing burden on students. Wastewater SARS-CoV-2 levels were used to create multiple scenarios, each with differing levels of testing intensity, which were compared to the actual testing volumes implemented by the university. We found that scenarios in which testing intensity fluctuations matched rise and falls in SARS-CoV-2 wastewater levels had stronger correlations between SARS-CoV-2 levels and recorded clinical positives. In addition to stronger correlations, most scenarios resulted in overall fewer weekly clinical tests performed. We suggest the use of wastewater surveillance to guide COVID-19 testing as it can significantly increase the efficacy of COVID-19 surveillance while reducing the burden placed on college students during a pandemic. Future efforts should be made to integrate wastewater surveillance into clinical testing strategies implemented on college campuses.
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COVID-19 , Aguas Residuales , Humanos , Monitoreo Epidemiológico Basado en Aguas Residuales , Prueba de COVID-19 , Pandemias , Universidades , COVID-19/epidemiología , SARS-CoV-2RESUMEN
Molecular methods have been used to detect human pathogens in wastewater with sampling typically performed at wastewater treatment plants (WWTP) and upstream locations within the sewer system. A wastewater-based surveillance (WBS) program was established at the University of Miami (UM) in 2020, which included measurements of SARS-CoV-2 levels in wastewater from its hospital and within the regional WWTP. In addition to the development of a SARS-CoV-2 quantitative PCR (qPCR) assay, qPCR assays to detect other human pathogens of interest were also developed at UM. Here we report on the use of a modified set of reagents published by the CDC to detect nucleic acids of Monkeypox virus (MPXV) which emerged during May of 2022 to become a concern worldwide. Samples collected from the University hospital and from the regional WWTP were processed through DNA and RNA workflows and analyzed by qPCR to detect a segment of the MPXV CrmB gene. Results show positive detections of MPXV nucleic acids in the hospital and wastewater treatment plant wastewater which coincided with clinical cases in the community and mirrored the overall trend of nationwide MPXV cases reported to the CDC. We recommend the expansion of current WBS programs' methods to detect a broader range of pathogens of concern in wastewater and present evidence that viral RNA in human cells infected by a DNA virus can be detected in wastewater.
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COVID-19 , Mpox , Ácidos Nucleicos , Humanos , Monkeypox virus , Aguas Residuales , Flujo de Trabajo , SARS-CoV-2 , ADN , Hospitales Universitarios , ARN ViralRESUMEN
Wastewater-based surveillance (WBS) is a noninvasive, epidemiological strategy for assessing the spread of COVID-19 in communities. This strategy was based upon wastewater RNA measurements of the viral target, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The utility of WBS for assessing the spread of COVID-19 has motivated research to measure targets beyond SARS-CoV-2, including pathogens containing DNA. The objective of this study was to establish the necessary steps for isolating DNA from wastewater by modifying a long-standing RNA-specific extraction workflow optimized for SARS-CoV-2 detection. Modifications were made to the sample concentration process and included an evaluation of bead bashing prior to the extraction of either DNA or RNA. Results showed that bead bashing reduced detection of RNA from wastewater but improved recovery of DNA as assessed by quantitative polymerase chain reaction (qPCR). Bead bashing is therefore not recommended for the quantification of RNA viruses using qPCR. Whereas for Mycobacterium bacterial DNA isolation, bead bashing was necessary for improving qPCR quantification. Overall, we recommend 2 separate workflows, one for RNA viruses that does not include bead bashing and one for other microbes that use bead bashing for DNA isolation. The experimentation done here shows that current-standing WBS program methodologies optimized for SARS-CoV-2 need to be modified and reoptimized to allow for alternative pathogens to be readily detected and monitored, expanding its utility as a tool for public health assessment.
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COVID-19 , Humanos , SARS-CoV-2/genética , ARN Viral/genética , Aguas Residuales , Monitoreo Epidemiológico Basado en Aguas Residuales , Flujo de TrabajoRESUMEN
The utility of using severe-acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RNA for assessing the prevalence of COVID-19 within communities begins with the design of the sample collection program. The objective of this study was to assess the utility of 24-hour composites as representative samples for measuring multiple microbiological targets in wastewater, and whether normalization of SARS-CoV-2 by endogenous targets can be used to decrease hour to hour variability at different watershed scales. Two sets of experiments were conducted, in tandem with the same wastewater, with samples collected at the building, cluster, and community sewershed scales. The first set of experiments focused on evaluating degradation of microbiological targets: SARS-CoV-2, Simian Immunodeficiency Virus (SIV) - a surrogate spiked into the wastewater, plus human waste indicators of Pepper Mild Mottle Virus (PMMoV), Beta-2 microglobulin (B2M), and fecal coliform bacteria (FC). The second focused on the variability of these targets from samples, collected each hour on the hour. Results show that SARS-CoV-2, PMMoV, and B2M were relatively stable, with minimal degradation over 24-h. SIV, which was spiked-in prior to analysis, degraded significantly and FC increased significantly over the course of 24 h, emphasizing the possibility for decay and growth within wastewater. Hour-to-hour variability of the source wastewater was large between each hour of sampling relative to the variability of the SARS-CoV-2 levels calculated between sewershed scales; thus, differences in SARS-CoV-2 hourly variability were not statistically significant between sewershed scales. Results further provided that the quantified representativeness of 24-h composite samples (i.e., statistical equivalency compared against hourly collected grabs) was dependent upon the molecular target measured. Overall, improvements made by normalization were minimal within this study. Degradation and multiplication for other targets should be evaluated when deciding upon whether to collect composite or grab samples in future studies.
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COVID-19 , SARS-CoV-2 , Humanos , Animales , Aguas Residuales , HecesRESUMEN
Genomic footprints of pathogens shed by infected individuals can be traced in environmental samples, which can serve as a noninvasive method of infectious disease surveillance. The research evaluates the efficacy of environmental monitoring of SARS-CoV-2 RNA in air, surface swabs and wastewater to predict COVID-19 cases. Using a prospective experimental design, air, surface swabs, and wastewater samples were collected from a college dormitory housing roughly 500 students from March to May 2021 at the University of Miami, Coral Gables, FL. Students were randomly screened for COVID-19 during the study period. SARS-CoV-2 concentration in environmental samples was quantified using Volcano 2nd Generation-qPCR. Descriptive analyses were conducted to examine the associations between time-lagged SARS-CoV-2 in environmental samples and COVID-19 cases. SARS-CoV-2 was detected in air, surface swab and wastewater samples on 52 (63.4 %), 40 (50.0 %) and 57 (68.6 %) days, respectively. On 19 (24 %) of 78 days SARS-CoV-2 was detected in all three sample types. COVID-19 cases were reported on 11 days during the study period and SARS-CoV-2 was also detected two days before the case diagnosis on all 11 (100 %), 9 (81.8 %) and 8 (72.7 %) days in air, surface swab and wastewater samples, respectively. SARS-CoV-2 detection in environmental samples was an indicator of the presence of local COVID-19 cases and a 3-day lead indicator for a potential outbreak at the dormitory building scale. Proactive environmental surveillance of SARS-CoV-2 or other pathogens in multiple environmental media has potential to guide targeted measures to contain and/or mitigate infectious disease outbreaks within communities.
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COVID-19 , Humanos , COVID-19/epidemiología , SARS-CoV-2 , Aguas Residuales/análisis , ARN Viral , Estudios ProspectivosRESUMEN
Wastewater, which contains everything from pathogens to pollutants, is a geospatially-and temporally-linked microbial fingerprint of a given population. As a result, it can be leveraged for monitoring multiple dimensions of public health across locales and time. Here, we integrate targeted and bulk RNA sequencing (n=1,419 samples) to track the viral, bacterial, and functional content over geospatially distinct areas within Miami Dade County from 2020-2022. First, we used targeted amplicon sequencing (n=966) to track diverse SARS-CoV-2 variants across space and time, and we found a tight correspondence with clinical caseloads from University students (N = 1,503) and Miami-Dade County hospital patients (N = 3,939 patients), as well as an 8-day earlier detection of the Delta variant in wastewater vs. in patients. Additionally, in 453 metatranscriptomic samples, we demonstrate that different wastewater sampling locations have clinically and public-health-relevant microbiota that vary as a function of the size of the human population they represent. Through assembly, alignment-based, and phylogenetic approaches, we also detect multiple clinically important viruses (e.g., norovirus ) and describe geospatial and temporal variation in microbial functional genes that indicate the presence of pollutants. Moreover, we found distinct profiles of antimicrobial resistance (AMR) genes and virulence factors across campus buildings, dorms, and hospitals, with hospital wastewater containing a significant increase in AMR abundance. Overall, this effort lays the groundwork for systematic characterization of wastewater to improve public health decision making and a broad platform to detect emerging pathogens.
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Wastewater-based epidemiology (WBE) has been utilized to track community infections of Coronavirus Disease 2019 (COVID-19) by detecting RNA of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), within samples collected from wastewater. The correlations between community infections and wastewater measurements of the RNA can potentially change as SARS-CoV-2 evolves into new variations by mutating. This study analyzed SARS-CoV-2 RNA, and indicators of human waste in wastewater from two sewersheds of different scales (University of Miami (UM) campus and Miami-Dade County Central District wastewater treatment plant (CDWWTP)) during five internally defined COVID-19 variant dominant periods (Initial, Pre-Delta, Delta, Omicron and Post-Omicron wave). SARS-CoV-2 RNA quantities were compared against COVID-19 clinical cases and hospitalizations to evaluate correlations with wastewater SARS-CoV-2 RNA. Although correlations between documented clinical cases and hospitalizations were high, prevalence for a given wastewater SARS-CoV-2 level varied depending upon the variant analyzed. The correlative relationship was significantly steeper (more cases per level found in wastewater) for the Omicron-dominated period. For hospitalization, the relationships were steepest for the Initial wave, followed by the Delta wave with flatter slopes during all other waves. Overall results were interpreted in the context of SARS-CoV-2 virulence and vaccination rates among the community.
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Methods of wastewater concentration (electronegative filtration (ENF) versus magnetic bead-based concentration (MBC)) were compared for the analysis of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), beta-2 microglobulin, and human-coronavirus OC43. Using ENF as the concentration method, two quantitative Polymerase Chain Reaction (qPCR) analytical methods were also compared: Volcano 2nd Generation (V2G)-qPCR and reverse transcriptase (RT)-qPCR measuring three different targets of the virus responsible for the COVID-19 illness (N1, modified N3, and ORF1ab). Correlations between concentration methods were strong and statistically significant for SARS-CoV-2 (r=0.77, p<0.001) and B2M (r=0.77, p<0.001). Comparison of qPCR analytical methods indicate that, on average, each method provided equivalent results with average ratios of 0.96, 0.96 and 1.02 for N3 to N1, N3 to ORF1ab, and N1 to ORF1ab and were supported by significant (p<0.001) correlation coefficients (r =0.67 for V2G (N3) to RT (N1), r =0.74 for V2G (N3) to RT (ORF1ab), r = 0.81 for RT (N1) to RT (ORF1ab)). Overall results suggest that the two concentration methods and qPCR methods provide equivalent results, although variability is observed for individual measurements. Given the equivalency of results, additional advantages and disadvantages, as described in the discussion, are to be considered when choosing an appropriate method.
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Background: The concentration and duration of antibodies (Ab) to SARS-CoV-2 infection predicts the severity of the disease and the clinical outcomes. Older people and those with HIV have impaired immune responses, worse outcomes after SARS-CoV-2 infection, and lower antibody responses after viral infection and vaccination. This study evaluated an Ab response to SARS-CoV-2 in people with HIV (PWH) and without HIV (HIV-) and its association with age. Methods: A total of 23 COVID+PWH and 21 COVID+HIV- participants were followed longitudinally for 6 months post-mild COVID-19. Immunoglobin G (IgG) and immunoglobin M (IgM) Ab responses were measured by an in-house developed ELISA. Time points and HIV status interaction were analyzed using Poisson generalized estimating equations, and correlations were analyzed using non-parametric tests. Results: Median age in PWH was 55 years with 28.6% women, while in the HIV- group was 36 years with 60.9% women. The mean time from COVID-19 diagnosis to study enrollment was 16 days for PWH and 11 days for HIV-. The mean CD4+ T-cell count/µl for PWH was 772.10 (±365.21). SARS-CoV-2 IgM and IgG were detected at all time points and Ab response levels did not differ by HIV status (p > 0.05). At entry, age showed a weak direct association with IgG responses (ρ = 0.44, p < 0.05) in HIV- but did not show any association in PWH. Similar associations between age, IgG, and HIV status emerged at day 14 (T1; ρ = 0.50, p < 0.05), 3 months (T3; ρ = 0.50, p < 0.05), and 6 months visit (T4; ρ = 0.78, p < 0.05) in the HIV- group. Conclusion: The Ab responses in the 6-month post-SARS-CoV-2 infection did not differ by HIV status, though a positive association was found between age and Ab response in older PWH. Results suggest that immune protection and vaccine responses are similar for PWH than for those without HIV infection.
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Importance: Genomic footprints of pathogens shed by infected individuals can be traced in environmental samples. Analysis of these samples can be employed for noninvasive surveillance of infectious diseases. Objective: To evaluate the efficacy of environmental surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for predicting COVID-19 cases in a college dormitory. Design: Using a prospective experimental design, air, surface swabs, and wastewater samples were collected from a college dormitory from March to May 2021. Students were randomly screened for COVID-19 during the study period. SARS-CoV-2 in environmental samples was concentrated with electronegative filtration and quantified using Volcano 2 nd Generation-qPCR. Descriptive analyses were conducted to examine the associations between time-lagged SARS-CoV-2 in environmental samples and clinically diagnosed COVID-19 cases. Setting: This study was conducted in a residential dormitory at the University of Miami, Coral Gables campus, FL, USA. The dormitory housed about 500 students. Participants: Students from the dormitory were randomly screened, for COVID-19 for 2-3 days / week while entering or exiting the dormitory. Main Outcome: Clinically diagnosed COVID-19 cases were of our main interest. We hypothesized that SARS-CoV-2 detection in environmental samples was an indicator of the presence of local COVID-19 cases in the dormitory, and SARS-CoV-2 can be detected in the environmental samples several days prior to the clinical diagnosis of COVID-19 cases. Results: SARS-CoV-2 genomic footprints were detected in air, surface swab and wastewater samples on 52 (63.4%), 40 (50.0%) and 57 (68.6%) days, respectively, during the study period. On 19 (24%) of 78 days SARS-CoV-2 was detected in all three sample types. Clinically diagnosed COVID-19 cases were reported on 11 days during the study period and SARS-CoV-2 was also detected two days before the case diagnosis on all 11 (100%), 9 (81.8%) and 8 (72.7%) days in air, surface swab and wastewater samples, respectively. Conclusion: Proactive environmental surveillance of SARS-CoV-2 or other pathogens in a community/public setting has potential to guide targeted measures to contain and/or mitigate infectious disease outbreaks. Key Points: Question: How effective is environmental surveillance of SARS-CoV-2 in public places for early detection of COVID-19 cases in a community?Findings: All clinically confirmed COVID-19 cases were predicted with the aid of 2 day lagged SARS-CoV-2 in environmental samples in a college dormitory. However, the prediction efficiency varied by sample type: best prediction by air samples, followed by wastewater and surface swab samples. SARS-CoV-2 was also detected in these samples even on days without any reported cases of COVID-19, suggesting underreporting of COVID-19 cases.Meaning: SARS-CoV-2 can be detected in environmental samples several days prior to clinical reporting of COVID-19 cases. Thus, proactive environmental surveillance of microbiome in public places can serve as a mean for early detection of location-time specific outbreaks of infectious diseases. It can also be used for underreporting of infectious diseases.
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Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in wastewater has been used to track community infections of coronavirus disease-2019 (COVID-19), providing critical information for public health interventions. Since levels in wastewater are dependent upon human inputs, we hypothesize that tracking infections can be improved by normalizing wastewater concentrations against indicators of human waste [Pepper Mild Mottle Virus (PMMoV), ß-2 Microglobulin (B2M), and fecal coliform]. In this study, we analyzed SARS-CoV-2 and indicators of human waste in wastewater from two sewersheds of different scales: a University campus and a wastewater treatment plant. Wastewater data were combined with complementary COVID-19 case tracking to evaluate the efficiency of wastewater surveillance for forecasting new COVID-19 cases and, for the larger scale, hospitalizations. Results show that the normalization of SARS-CoV-2 levels by PMMoV and B2M resulted in improved correlations with COVID-19 cases for campus data using volcano second generation (V2G)-qPCR chemistry (r s = 0.69 without normalization, r s = 0.73 with normalization). Mixed results were obtained for normalization by PMMoV for samples collected at the community scale. Overall benefits from normalizing with measures of human waste depend upon qPCR chemistry and improves with smaller sewershed scale. We recommend further studies that evaluate the efficacy of additional normalization targets.
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The COVID-19 pandemic has had a profound, detrimental effect on economies and societies worldwide. Where the pandemic has been controlled, extremely high rates of diagnostic testing for the SARS-CoV-2 virus have proven critical, enabling isolation of cases and contact tracing. Recently, diagnostic testing has been supplemented with wastewater measures to evaluate the degree to which communities have infections. Whereas much testing has been done through traditional, centralized, clinical, or environmental laboratory methods, point-of-care testing has proven successful in reducing time to result. As the pandemic progresses and becomes more broadly distributed, further decentralization of diagnostic testing will be helpful to mitigate its spread. This will be particularly both challenging and critical in settings with limited resources due to lack of medical infrastructure and expertise as well as requirements to return results quickly. In this article, we validate the tiny isothermal nucleic acid quantification system (TINY) and a novel loop-mediated isothermal amplification (LAMP)-based assay for the point-of-care diagnosis of SARS-CoV-2 infection in humans and also for in-the-field, point-of-collection surveillance of wastewater. The TINY system is portable and designed for use in settings with limited resources. It can be powered by electrical, solar, or thermal energy and is robust against interruptions in services. These applied testing examples demonstrate that this novel detection platform is a simpler procedure than reverse-transcription quantitative polymerase chain reaction, and moreover, this TINY instrument and LAMP assay combination has the potential to effectively provide both point-of-care diagnosis of individuals and point-of-collection environmental surveillance using wastewater.
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COVID-19 , Humanos , Pandemias , Sistemas de Atención de Punto , ARN Viral , SARS-CoV-2RESUMEN
Standardized protocols for wastewater-based surveillance (WBS) for the RNA of SARS-CoV-2, the virus responsible for the current COVID-19 pandemic, are being developed and refined worldwide for early detection of disease outbreaks. We report here on lessons learned from establishing a WBS program for SARS-CoV-2 integrated with a human surveillance program for COVID-19. We have established WBS at three campuses of a university, including student residential dormitories and a hospital that treats COVID-19 patients. Lessons learned from this WBS program address the variability of water quality, new detection technologies, the range of detectable viral loads in wastewater, and the predictive value of integrating environmental and human surveillance data. Data from our WBS program indicated that water quality was statistically different between sewer sampling sites, with more variability observed in wastewater coming from individual buildings compared to clusters of buildings. A new detection technology was developed based upon the use of a novel polymerase called V2G. Detectable levels of SARS-CoV-2 in wastewater varied from 102 to 106 genomic copies (gc) per liter of raw wastewater (L). Integration of environmental and human surveillance data indicate that WBS detection of 100 gc/L of SARS-CoV-2 RNA in wastewater was associated with a positivity rate of 4% as detected by human surveillance in the wastewater catchment area, though confidence intervals were wide (ß ~ 8.99 ∗ ln(100); 95% CI = 0.90-17.08; p < 0.05). Our data also suggest that early detection of COVID-19 surges based on correlations between viral load in wastewater and human disease incidence could benefit by increasing the wastewater sample collection frequency from weekly to daily. Coupling simpler and faster detection technology with more frequent sampling has the potential to improve the predictive potential of using WBS of SARS-CoV-2 for early detection of the onset of COVID-19.
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COVID-19 , SARS-CoV-2 , Humanos , Pandemias , ARN Viral , Aguas ResidualesRESUMEN
The development of a permissive small animal model for the study of human immunodeficiency virus type (HIV)-1 pathogenesis and the testing of antiviral strategies has been hampered by the inability of HIV-1 to infect primary rodent cells productively. In this study, we explored transgenic rats expressing the HIV-1 receptor complex as a susceptible host. Rats transgenic for human CD4 (hCD4) and the human chemokine receptor CCR5 (hCCR5) were generated that express the transgenes in CD4(+) T lymphocytes, macrophages, and microglia. In ex vivo cultures, CD4(+) T lymphocytes, macrophages, and microglia from hCD4/hCCR5 transgenic rats were highly susceptible to infection by HIV-1 R5 viruses leading to expression of abundant levels of early HIV-1 gene products comparable to those found in human reference cultures. Primary rat macrophages and microglia, but not lymphocytes, from double-transgenic rats could be productively infected by various recombinant and primary R5 strains of HIV-1. Moreover, after systemic challenge with HIV-1, lymphatic organs from hCD4/hCCR5 transgenic rats contained episomal 2-long terminal repeat (LTR) circles, integrated provirus, and early viral gene products, demonstrating susceptibility to HIV-1 in vivo. Transgenic rats also displayed a low-level plasma viremia early in infection. Thus, transgenic rats expressing the appropriate human receptor complex are promising candidates for a small animal model of HIV-1 infection.
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Antígenos CD4 , Modelos Animales de Enfermedad , Infecciones por VIH , VIH-1 , Receptores CCR5 , Animales , Animales Modificados Genéticamente , Antígenos CD4/genética , Antígenos CD4/inmunología , VIH-1/fisiología , Humanos , Macrófagos/inmunología , Ratas , Receptores CCR5/genética , Receptores CCR5/inmunología , Replicación ViralRESUMEN
BACKGROUND: Sterile alpha motif and histidine/aspartic acid domain-containing protein (SAMHD1) is a dNTP triphosphorylase that reduces cellular dNTP levels in non-dividing cells, such as macrophages. Since dNTPs are required for reverse transcription, HIV-2 and most SIVs encode a Vpx protein that promotes proteasomal degradation of SAMHD1. It is unclear how HIV-1, which does not appear to harbor a SAMHD1 escape mechanism, is able to infect macrophages in the face of SAMHD1 restriction. METHODS: To assess whether HIV-1 had a mechanism to negate SAMHD1 activity, we compared SAMHD1 and dNTP levels in macrophages infected by HIV-1 and SIV. We examined whether macrophages infected by HIV-1 still harbored antiviral levels of SAMHD1 by assessing their susceptibility to superinfection by vpx-deleted SIV. Finally, to assess whether HIV-1 reverse transcriptase (RT) has adapted to a low dNTP environment, we evaluated SAMHD1 sensitivity of chimeric HIV-1 and SIV variants in which the RT regions were functionally exchanged. RESULTS: Here, we demonstrate that HIV-1 efficiently infects macrophages without modulating SAMHD1 activity or cellular dNTP levels, and that macrophages permissive to HIV-1 infection remained refractory to superinfection by vpx-deleted SIV. Furthermore, through the use of chimeric HIV/SIV, we demonstrate that the differential sensitivity of HIV-1 and SIV to SAMHD1 restriction is not dictated by RT. CONCLUSIONS: Our study reveals fundamental differences between HIV-1 and SIV in the strategy used to evade restriction by SAMHD1 and suggests a degree of resistance of HIV-1 to the antiviral environment created by SAMHD1. Understanding how these cellular restrictions antagonize viral replication will be important for the design of novel antiviral strategies.