Bacteriophage therapy for the treatment of Mycobacterium tuberculosis infections in humanized mice.

The continuing emergence of new strains of antibiotic-resistant bacteria has renewed interest in phage therapy; however, there has been limited progress in applying phage therapy to multi-drug resistant Mycobacterium tuberculosis (Mtb) infections. In this study, we tested three bacteriophage strains for their Mtb-killing activities and found that two of them efficiently lysed Mtb H37Rv in 7H10 agar plates. However, only phage DS6A efficiently killed H37Rv in liquid culture and in Mtb-infected human primary macrophages. In subsequent experiments, we infected humanized mice with aerosolized H37Rv, then treated these mice with DS6A intravenously to test its in vivo efficacy. We found that DS6A treated mice showed increased body weight and improved pulmonary function relative to control mice. Furthermore, DS6A reduced Mtb load in mouse organs with greater efficacy in the spleen. These results demonstrated the feasibility of developing phage therapy as an effective therapeutic against Mtb infection.

Genetic Diversity and Evolutionary Convergence of Cryptic SARS-CoV-2 Lineages Detected Via Wastewater Sequencing

Wastewater-based epidemiology (WBE) is an effective way of tracking the appearance and spread of SARS-COV-2 lineages through communities. Beginning in early 2021, we implemented a targeted approach to amplify and sequence the receptor binding domain (RBD) of SARS-COV-2 to characterize viral lineages present in sewersheds. Over the course of 2021, we reproducibly detected multiple SARS-COV-2 RBD lineages that have never been observed in patient samples in 9 sewersheds located in 3 states in the USA. These cryptic lineages contained between 4 to 24 amino acid substitutions in the RBD and were observed intermittently in the sewersheds in which they were found for as long as 14 months. Many of the amino acid substitutions in these lineages occurred at residues also mutated in the Omicron variant of concern (VOC), often with the same substitution. One of the sewersheds contained a lineage that appeared to be derived from the Alpha VOC, but the majority of the lineages appeared to be derived from pre-VOC SARS-COV-2 lineages. Specifically, several of the cryptic lineages from New York City appeared to be derived from a common ancestor that most likely diverged in early 2020. While the source of these cryptic lineages has not been resolved, it seems increasingly likely that they were derived from immunocompromised patients or animal reservoirs. Our findings demonstrate that SARS-COV-2 genetic diversity is greater than what is commonly observed through routine SARS-CoV-2 surveillance. Wastewater sampling may more fully capture SARS-CoV-2 genetic diversity than patient sampling and could reveal new VOCs before they emerge in the wider human population. Author SummaryDuring the COVID-19 pandemic, wastewater-based epidemiology has become an effective public health tool. Because many infected individuals shed SARS-CoV-2 in feces, wastewater has been monitored to reveal infection trends in the sewersheds from which the samples were derived. Here we report novel SARS-CoV-2 lineages in wastewater samples obtained from 3 different states in the USA. These lineages appeared in specific sewersheds intermittently over periods of up to 14 months, but generally have not been detected beyond the sewersheds in which they were initially found. Many of these lineages may have diverged in early 2020. Although these lineages share considerable overlap with each other, they have never been observed in patients anywhere in the world. While the wastewater lineages have similarities with lineages observed in long-term infections of immunocompromised patients, animal reservoirs cannot be ruled out as a potential source.

The prevalence of SARS-CoV-2 infection and uptake of COVID-19 antiviral treatments during the BA.2/BA.2.12.1 surge, New York City, April-May 2022

BackgroundRoutine case surveillance data for SARS-CoV-2 are incomplete, unrepresentative, missing key variables of interest, and may be increasingly unreliable for both timely surge detection and understanding the burden of infection and access to treatment. MethodsWe conducted a cross-sectional survey of a representative sample of 1,030 New York City (NYC) adult residents [≥]18 years on May 7-8, 2022, when BA.2.12.1 comprised 47% of reported cases per genomic surveillance. We estimated the prevalence of SARS-CoV-2 infection during the preceding 14-day period (April 23-May 8), weighted to represent the 2020 NYC adult population. Respondents were asked about SARS-CoV-2 testing (including at-home rapid antigen tests), testing outcomes, COVID-like symptoms, and contact with SARS-CoV-2 cases. Based on responses, we classified individuals into three mutually exclusive categories of SARS-CoV-2 infection according to a hierarchical case definition as follows: confirmed (positive test with a provider), probable (positive at home rapid test), and possible (COVID-like symptoms and close contact with a confirmed/probable case). SARS-CoV-2 prevalence estimates were age- and sex-adjusted to the 2020 US population. Individuals with SARS-CoV-2 were asked about awareness/use of antiviral medications. We triangulated survey-based prevalence estimates with NYCs official SARS-CoV-2 metrics on cases, hospitalizations, and deaths, as well as SARS-CoV-2 concentrations in wastewater for the same time period. ResultsAn estimated 22.1% (95%CI 17.9%-26.2%) of respondents had SARS-CoV-2 infection during the two-week study period, corresponding to [~]1.5 million adults (95%CI 1.3-1.8 million). The official SARS-CoV-2 case count during the study period was 51,218. This 22.1% prevalence estimate included 11.4%, 6.5%, and 4.3% who met the confirmed, probable, and possible criteria of our case definition, respectively. Prevalence was estimated at 34.9% (95%CI 26.9%-42.8%) among individuals with co-morbidities, 14.9% (95% CI 11.0%-18.8%) among those 65+ years, and 18.9% (95%CI 10.2%-27.5%) among unvaccinated persons. Hybrid immunity (i.e., history of both vaccination and prior infection) was 66.2% (95%CI 55.7%-76.7%) among those with COVID and 46.3% (95%CI 40.2-52.2) among those without. Among individuals with COVID, 44.1% (95%CI 33.0%-55.1%) were aware of the antiviral nirmatrelvir/ritonavir (Paxlovid), and 15.1% (95%CI 7.1%-23.1%) reported receiving it. Deaths and hospitalizations increased, but remained well below the levels of the BA.1 surge. SARS-CoV-2 virus concentrations in wastewater surveillance showed only a modest signal in comparison to that of the BA.1 surge. Conclusions and RelevanceThe true magnitude of NYCs BA.2/BA.2.12.1 surge may have been vastly underestimated by routine SARS-CoV-2 case counts and wastewater surveillance. Hybrid immunity, bolstered by the recent BA.1 surge, likely limited the impact of the BA.2/BA.2.12.1 surge on severe outcomes. Representative surveys are needed as part of routine surveillance for timely surge detection, and to estimate the true burden of infection, hybrid immunity, and uptake of time-sensitive treatments among those most vulnerable to severe COVID. Short abstractChanges in testing practices and behaviors, including increasing at-home rapid testing and decreasing provider-based testing make it challenging to assess the true prevalence of SARS-CoV-2. We conducted a population-representative survey of adults in New York City to estimate the prevalence of SARS-CoV-2 infection during the BA.2./BA.2.12.1 surge in late April/early May 2022. We triangulated survey-based SARS-CoV-2 prevalence estimates with contemporaneous city-wide SARS-CoV-2 metrics on diagnosed cases, hospitalizations, deaths, and SARS-CoV-2 concentration in wastewater. Survey-based prevalence estimates were nearly 30 times higher than official case counts, and estimates of recently acquired hybrid immunity among those with active infection were high. We conclude that no single data source provides a complete or accurate assessment of the epidemiologic situation. Taken together, however, our results suggest that the magnitude of the BA.2/BA.2.12.1 surge was likely significantly underestimated, and high levels of hybrid immunity likely prevented a major surge in BA.2/BA.2.12.1-associated hospitalizations/deaths.

Monitoring SARS-CoV-2 in wastewater during New York City's second wave of COVID-19: Sewershed-level trends and relationships to publicly available clinical testing data

New York Citys ongoing wastewater monitoring program tracked trends in sewershed-level SARS-CoV-2 loads starting in the fall of 2020, just before the start of the Citys second wave of the COVID-19 outbreak. During a five-month study period, from November 8, 2020 to April 11, 2021, viral loads in influent wastewater from each of New York Citys 14 wastewater treatment plants were measured and compared to new laboratory-confirmed COVID-19 cases for the populations in each corresponding sewershed, estimated from publicly available clinical testing data. We found significant positive correlations between viral loads in wastewater and new COVID-19 cases. The strength of the correlations varied depending on the sewershed, with Spearmans rank correlation coefficients ranging between 0.38 and 0.81 (mean = 0.55). Based on a linear regression analysis of a combined data set for New York City, we found that a 1 log10 change in the SARS-CoV-2 viral load in wastewater corresponded to a 0.6 log10 change in the number of new laboratory-confirmed COVID-19 cases/day in a sewershed. An estimated minimum detectable case rate between 2 - 8 cases/day/100,000 people was associated with the method limit of detection in wastewater. This work offers a preliminary assessment of the relationship between wastewater monitoring data and clinical testing data in New York City. While routine monitoring and method optimization continue, information on the development of New York Citys ongoing wastewater monitoring program may provide insights for similar wastewater-based epidemiology efforts in the future.

Tracking Cryptic SARS-CoV-2 Lineages Detected in NYC Wastewater

Tracking SARS-CoV-2 genetic diversity is strongly indicated because diversifying selection may lead to the emergence of novel variants resistant to naturally acquired or vaccine-induced immunity. To monitor New York City (NYC) for the presence of novel variants, we amplified regions of the SARS-CoV-2 Spike protein gene from RNA acquired from all 14 NYC wastewater treatment plants (WWTPs) and ascertained the diversity of lineages from these samples using high throughput sequencing. Here we report the detection and increasing frequencies of novel SARS-CoV-2 lineages not recognized in GISAIDs EpiCoV database. These lineages contain mutations rarely observed in clinical samples, including Q493K, Q498Y, H519N and T572N. Many of these mutations were found to expand the tropism of SARS-CoV-2 pseudoviruses by allowing infection of cells expressing the human, mouse, or rat ACE2 receptor. In addition, pseudoviruses containing the Spike amino acid sequence of these lineages were found to be resistant to many different classes of receptor binding domain (RBD) binding neutralizing monoclonal antibodies. We offer several hypotheses for the anomalous presence of these mutations, including the possibility of a non-human animal reservoir. Although wastewater sampling cannot provide direct inference of SARS-CoV-2 clinical sequences, our research revealed several lineages that could be relevant to public health and they would not have been discovered if not for wastewater surveillance.

Detection of Mutations Associated with Variants of Concern Via High Throughput Sequencing of SARS-CoV-2 Isolated from NYC Wastewater

Monitoring SARS-CoV-2 genetic diversity is strongly indicated because diversifying selection may lead to the emergence of novel variants resistant to naturally acquired or vaccine-induced immunity. To date, most data on SARS-CoV-2 genetic diversity has come from the sequencing of clinical samples, but such studies may suffer limitations due to costs and throughput. Wastewater-based epidemiology may provide an alternative and complementary approach for monitoring communities for novel variants. Given that SARS-CoV-2 can infect the cells of the human gut and is found in high concentrations in feces, wastewater may be a valuable source of SARS-CoV-2 RNA, which can be deep sequenced to provide information on the circulating variants in a community. Here we describe a safe, affordable protocol for the sequencing of SARS-CoV-2 RNA using high-throughput Illumina sequencing technology. Our targeted sequencing approach revealed the presence of mutations associated with several Variants of Concern at appreciable frequencies. Our work demonstrates that wastewater-based SARS-CoV-2 sequencing can inform surveillance efforts monitoring the community spread of SARS-CoV-2 Variants of Concern and detect the appearance of novel emerging variants more cheaply, safely, and efficiently than the sequencing of individual clinical samples. IMPORTANCEThe SARS-CoV-2 pandemic has caused millions of deaths around the world as countries struggle to contain infections. The pandemic will not end until herd immunity is reached, that is, when most of the population has either recovered from SARS-CoV-2 infection or is vaccinated against SARS-CoV-2. However, the emergence of new SARS-CoV-2 variants of concern threatens to erase gains. Emerging new variants may re-infect persons who have recovered from COVID-19 or may evade vaccine-induced immunity. However, scaling up SARS-CoV-2 genetic sequencing to monitor Variants of Concern in communities around the world is challenging. Wastewater-based sequencing of SARS-CoV-2 RNA can be used to monitor the presence of emerging variants in large communities to enact control measures to minimize the spread of these variants. We describe here the identification of alleles associated with several variants of concern in wastewater obtained from NYC watersheds.

Protocol for Safe, Affordable, and Reproducible Isolation and Quantitation of SARS-CoV-2 RNA from Wastewater

The following protocol describes our workflow for processing wastewater with the goal of detecting the genetic signal of SARS-CoV-2. The steps include pasteurization, virus concentration, RNA extraction, and quantification by RT-qPCR. We include auxiliary steps that provide new users with tools and strategies that will help troubleshoot key steps in the process. This protocol is one of the safest, cheapest, and most reproducible approaches for the detection of SARS-CoV-2 RNA in wastewater. Furthermore, the RNA obtained using this protocol, minus the pasteurization step, can be sequenced both using a targeted approach sequencing specific regions or the whole genome. The protocol was adopted by the New York City Department of Environmental Protection in August 2020 to support their efforts in monitoring SARS-CoV-2 prevalence in wastewater in all five boroughs of the city. Owing to a pasteurization step, it is safe for use in a BSL1+ facility. This step also increases the genetic signal of the virus while making the protocol safe for the personnel involved. This protocol could be used to isolate a variety of other clinically relevant viruses from wastewater and serve as a foundation of a wastewater surveillance strategy for monitoring community spread of known and emerging viral pathogens.