Wastewater monitoring of SARS-CoV-2 in on-grid, partially and fully off-grid Bedouin communities in Southern Israel

BackgroundWastewater based epidemiology (WBE) has become an important tool in SARS-CoV-2 surveillance and epidemiology. While WBE measurements generally correlate with observed case numbers in large municipal areas on sewer grids, there are few studies on its utility in communities that are off-grid (non-sewered).Methods and materialsTo explore the applicability of wastewater surveillance in our region, five Bedouin communities along the Hebron Stream in Southern Israel (Negev desert) were sampled. One point (El-Sayed) represents a community with partial connection to the sewer grid system and another point (Um Batin) represents a community with no access to the sewer grid system. The towns of Hura, Lakia, and Tel Al-Sabi/Tel Sheva were on-grid. A total of 87 samples were collected between August 2020 to January 2021 using both grab and composite sampling. RNA was extracted from the raw sewage and concentrated sewage. RT-qPCR was carried out with N1, N2, and N3 gene targets, and findings were compared to human case data from the Israeli Ministry of Health.ResultsSARS-CoV-2 was detected consistently over time in on-grid Bedouin towns (Lakia, Tel Sheva/Tel as-Sabi, and Hura) and inconsistently in smaller, off-grid communities (El-Sayed and Um Batin). The trend in maximum copy number/L appears to be driven by population size. When comparing case numbers normalized to population size, the amount of gene copies/L was inconsistently related to reported case numbers. SARS-CoV-2 was also detected from sewage-impacted environmental waters representing communities with no access to the wastewater grid. When grab sampling and composite sampling data were compared, results were generally comparable however composite sampling produced superior results.ConclusionsThe mismatch observed between detected virus and reported cases could indicate asymptomatic or “silent” community transmission, under-testing within these communities (due to factors like mistrust in government, stigma, misinformation) or a combination therein. While the exact reason for the mismatch between environmental SARS-CoV-2 signals and case numbers remains unresolved, these findings suggest that sewage surveillance, including grab sampling methodologies, can be a critical aspect of outbreak surveillance and control in areas with insufficient human testing and off-grid communities

The Rise and Fall of Omicron BA.1 Variant as Seen in Wastewater Supports Epidemiological Model Predictions

The COVID-19 pandemic caused by the SARS-CoV-2 virus has inflicted significant mortality and morbidity worldwide. Continuous virus mutations have led to the emergence of new variants. The Omicron BA.1 sub-lineage prevailed as the dominant variant globally at the beginning of 2022 but was subsequently replaced by BA.2 in numerous countries. Wastewater-based epidemiology (WBE) offers an efficient tool for capturing viral shedding from infected individuals, enabling early detection of potential pandemic outbreaks without relying solely on community cooperation and clinical testing resources. This study integrated RT-qPCR assays for detecting general SARS-CoV-2 and its variants levels in wastewater into a modified triple susceptible-infected-recovered-susceptible (SIRS) model. The emergence of the Omicron BA.1 variant was observed, replacing the presence of its predecessor, the Delta variant. Comparative analysis between the wastewater data and the modified SIRS model effectively described the BA.1 and subsequent BA.2 waves, with the decline of the Delta variant aligning with its diminished presence below the detection threshold in wastewater. This study demonstrates the potential of WBE as a valuable tool for future pandemics. Furthermore, by analyzing the sensitivity of different variants to model parameters, we are able to deduce real-life values of cross-variant immunity probabilities, emphasizing the asymmetry in their strength

Natural Killer Receptor 1 Dampens the Development of Allergic Eosinophilic Airway Inflammation

<div><p>The function of NCR1 was studied in a model of experimental asthma, classified as a type 1 hypersensitivity reaction, in mice. IgE levels were significantly increased in the serum of OVA immunized NCR1 deficient (<i>NCR1</i>^{<i>gfp/gfp</i>}) mice in comparison to OVA immunized wild type <i>(NCR1</i>^<i>+/+</i>) and adjuvant immunized mice. Histological analysis of OVA immunized <i>NCR1</i>^{<i>gfp/gfp</i>} mice revealed no preservation of the lung structure and overwhelming peribronchial and perivascular granulocytes together with mononuclear cells infiltration. OVA immunized <i>NCR</i>^<i>+/+</i> mice demonstrated preserved lung structure and peribronchial and perivascular immune cell infiltration to a lower extent than that in <i>NCR1</i>^{<i>gfp/gfp</i>} mice. Adjuvant immunized mice demonstrated lung structure preservation and no immune cell infiltration. OVA immunization caused an increase in PAS production independently of NCR1 presence. Bronchoalveolar lavage (BAL) revealed NCR1 dependent decreased percentages of eosinophils and increased percentages of lymphocytes and macrophages following OVA immunization. In the OVA immunized <i>NCR1</i>^{<i>gfp/</i>gfp} mice the protein levels of eosinophils’ (CCL24) and Th2 CD4⁺ T-cells’ chemoattractants (CCL17, and CCL24) in the BAL are increased in comparison with OVA immunized <i>NCR</i>^<i>+/+</i> mice. In the presence of NCR1, OVA immunization caused an increase in NK cells numbers and decreased NCR1 ligand expression on CD11c⁺GR1⁺ cells and decreased NCR1 mRNA expression in the BAL. OVA immunization resulted in significantly increased IL-13, IL-4 and CCL17 mRNA expression in <i>NCR1</i>^<i>+/+</i> and <i>NCR1</i>^{<i>gfp/</i>gfp} mice. IL-17 and TNFα expression increased only in OVA-immunized <i>NCR1</i>^<i>+/+</i>mice. IL-6 mRNA increased only in OVA immunized <i>NCR1</i>^{<i>gfp/gfp</i>} mice. Collectively, it is demonstrated that NCR1 dampens allergic eosinophilic airway inflammation.</p></div

Increased inflammation in the lungs of <i>NCR1</i>^{<i>gfp/gfp</i>} mice compared to <i>NCR1</i>^<i>+/+</i> mice (H&E+PAS x340).

<p>Mice were immunized and samples were process as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160779#pone.0160779.g002" target="_blank">Fig 2</a>. (A) OVA immunized <i>NCR</i>^{<i>gfp/gfp</i>} (H&E+PAS x340). (B) OVA/Alum immunized <i>NCR</i>^<i>+/+</i> (H&E+PAS x340). A thick arrow with a short tail identifies mononuclear cells and thin arrow with a long tail identifies polymorphonuclear cells.</p

NCR1 is involved in eosinophil and macrophage infiltration to the lung.

<p><i>NCR1</i>^<i>+/+</i> (Adjuvant n = 8, OVA n = 10) and <i>NCR1</i>^{<i>gfp/gfp</i>} (adjuvant n = 10, OVA n = 11) mice were immunized with either OVA or adjuvant as described in the Materials and Methods section. (A) The BAL was lavaged from each mouse, stained with PI and analyzed by flow cytometry for live cell count. The bar graph represents normalized number to the adjuvant immunized <i>NCR1</i>^<i>+/+</i> group whose average was considered as 1 of live cells in the BAL. (B-E) The bar graph represents an average percentage of eosinophils (B), macrophages (C), lymphocytes (D), and neutrophils (E) in the lung BAL (percentage of cells ± SEM; two-tailed Student <i>t</i>-test). These are the combined results of 2 experiments performed at different time points. * p < 0.05, **p < 0.01 *** p < 0.001.</p

A decrease in NCR1 mRNA expression and increase of NCR1 ligand expression in the BAL of OVA immunized mice.

<p><i>NCR</i>^<i>+/+</i> and <i>NCR</i>^{<i>gfp/gfp</i>} mice were immunized with either adjuvant alone or with OVA/alum as described in the Material and Methods section. The mice were euthanized and the lung tissue was dissociated into single cells. (A) Total number of live immune cell. (B) NK cell number as determined by anti NK1.1 antibody. (C) qRT PCR performed with <i>NCR1</i> appropriate primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160779#pone.0160779.s006" target="_blank">S1 Table</a>). (D) Five populations of cells carrying or lacking CD11c or GR1 were determined and stained with LY94-Ig fusion protein. *p > 0.05.</p

Increased inflammation in the lungs of <i>NCR1</i>^{<i>gfp/gfp</i>} mice (H&E+PAS x270).

<p>Mice were immunized and samples were process as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160779#pone.0160779.g002" target="_blank">Fig 2</a>. (A) OVA immunized <i>NCR</i>^{<i>gfp/gfp</i>} (H&E+PAS x270). (B) Alum immunized <i>NCR</i>^{<i>gfp/gfp</i>} (H&E+PAS x270).</p

Increased allergic airway inflammation in the lungs of <i>NCR</i>^{<i>gfp/gfp</i>} mice.

<p><i>Ncr1</i>^<i>+/+</i> and <i>Ncr1</i>^{<i>gfp/gfp</i>} <i>C57Bl/6</i> mice were i.p. immunized with either OVA or adjuvant on days 0 and 14. Ten days after the second immunization, mice were challenged twice intranasally with OVA at days 24 and 27. 24 h following the second challenge, BALF was taken from each mouse and used in ELISA to detect levels of (A) CCL24 (n = 8 to 11), (B) CCL17 (n = 9 to 10) and CCL22 (n = 9–10). Results are the summary of 3 independent experiments and were normalized according to the OVA <i>NCR1</i>^<i>+/+</i> ± SEM group average that was considered as 1 in each experiment. **<i>p</i><0.01, ***<i>p<</i>0.001 (two-tailed Student <i>t-</i>test).</p