Respiratory disease is a major cause of morbidity and mortality; however, surveillance for circulating respiratory viruses is passive and biased. Wastewater-based epidemiology has been used to understand SARS-CoV-2, influenza A, and respiratory syncytial virus (RSV) infection rates at a community level but has not been used to investigate other respiratory viruses. We aimed to use wastewater-based epidemiology to understand community viral respiratory infection occurrence.
Methods
A retrospective wastewater-based epidemiology surveillance study was carried out at a large wastewater treatment plant located in California, USA. Using droplet digital RT-PCR, we measured RNA concentrations of influenza A and influenza B viruses, RSV A and RSV B, parainfluenza (1–4) viruses, rhinovirus, seasonal coronaviruses, and metapneumovirus in wastewater solids three times per week for 17 months (216 samples) between Feb 1, 2021, and June 21, 2022. Novel probe-based RT-PCR assays for non-influenza viral targets were developed and validated. We compared viral RNA concentrations to positivity rates for viral infections from clinical specimens submitted to California Sentinel Clinical Laboratories (sentinel laboratories) to assess concordance between the two datasets.
Findings
We detected RNA from all tested viruses in wastewater solids. Human rhinovirus (median concentration 4300 [0–9500] copies per gram dry weight) and seasonal human coronaviruses (35 000 [17 000–56 000]) were found at the highest concentrations. Concentrations of viral RNA correlated significantly and positively with positivity rates of associated viral diseases from sentinel laboratories (tau 0·32–0·57, p<0·0009); the only exceptions were influenza B and RSV A, which were rarely detected in wastewater solids. Measurements from wastewater indicated coronavirus OC43 dominated the seasonal human coronavirus infections whereas parainfluenza 3 dominated among parainfluenza infections during the study period. Concentrations of all tested viral RNA decreased noticeably after the omicron BA.1 surge suggesting a connection between changes in human behaviour during the surge and transmission of all respiratory viruses.
Interpretation
Wastewater-based epidemiology can be used to obtain information on circulation of respiratory viruses at a localised, community level without the need to test many individuals because a single sample of wastewater represents the entire contributing community. Results from wastewater can be available within 24 h of sample collection, generating real time information to inform public health responses, clinical decision making, and individual behaviour modifications.
Funding
CDC Foundation.
Introduction
Acute respiratory illnesses are defined as symptomatic infections of the lower or upper respiratory tract and can also result in systemic symptoms and secondary infections. They are consistently the top causes of illness and mortality globally in children younger than 5 years, after the neonatal period, and represent a large burden of infectious diseases.
Global, regional, national, and selected subnational levels of stillbirths, neonatal, infant, and under-5 mortality, 1980–2015: a systematic analysis for the Global Burden of Disease Study 2015.
Viral acute respiratory illnesses are most commonly caused by SARS-CoV-2, seasonal coronavirus, parainfluenza, metapneumovirus, influenza, respiratory syncytial virus, rhinovirus, adenoviruses, bocaviruses, and non-rhinovirus enteroviruses.
Advances in the laboratory diagnosis of viral respiratory disease.
Respiratory disease surveillance in the USA is passive and draws from many sources, often relying on institutions to identify specific diseases through keywords in clinical records and death certificates, and testing of clinical specimens. The passive respiratory disease surveillance system is voluntary and biased towards identifying infections in individuals with comorbidities or for whom symptoms are severe and patients require clinical care.
Human coronavirus circulation in the United States 2014–2017.
Since many acute respiratory illnesses are self-limiting, diagnostic testing of individuals with respiratory illness is uncommon. A lack of robust, unbiased data for disease occurrence and the resulting lack of knowledge on occurrence and trends in circulating respiratory diseases limits public health responses, institutional awareness that can guide clinical decision making regarding testing and treatment, and efforts to understand disease epidemiology.
Research in context
Evidence before this study
Wastewater is a composite biological sample from the contributing community containing various human excretions. Wastewater-based epidemiology uses measured concentrations of pathogen targets in wastewater to understand occurrence and trends in infectious diseases. It has been applied in the past to understand circulation of poliovirus, as well as enteric viruses and bacteria. During the COVID-19 pandemic, wastewater-based epidemiology has been extensively used to understand the occurrence of COVID-19; to our knowledge, this work represents the first time wastewater-based epidemiology has been used to understand respiratory disease occurrence. On Aug 1, 2022, we searched PubMed and Google Scholar, for papers published since database inception, using the terms: wastewater AND (influenza OR metapneumovirus OR rhinovirus OR parainfluenza OR respiratory syncytial virus OR RSV OR “coronavirus OC43” OR “coronavirus HKU-1” OR “coronavirus NL63” OR “coronavirus 229E”); study language was not restricted to English and studies had to relate to wastewater concentrations of these viruses to respiratory disease occurrence. We found that the extension of wastewater-based epidemiology to other respiratory viruses was limited to two studies. One of those studies showed that concentrations of a respiratory syncytial virus (RSV) genomic RNA target in wastewater solids at two wastewater treatment plants correlated to a measure of RSV community infections, whereas the other showed that concentrations of an influenza A genomic RNA target in wastewater solids on two university campuses correlated well with active surveillance data of influenza A incidence in students. There was no other study that presented concentrations of other respiratory viruses in wastewater.
Added value of this study
This study fills the knowledge gaps identified in the literature review by providing measurements of concentrations of various respiratory viruses in wastewater solids including human rhinovirus, parainfluenza, metapneumovirus, influenza A and influenza B, RSV A and RSV B, and seasonal coronaviruses, and showing that their concentrations are associated with traditional measures of disease occurrence in the community. The work illustrates the use of wastewater-based epidemiology for understanding the occurrence of respiratory viral diseases beyond COVID-19.
Implications of all the available evidence
Viral respiratory infections cause substantial morbidity and mortality. In the USA, surveillance for respiratory infections is passive and voluntary, biased to only testing individuals with severe illness, subject to reporting delays of weeks, and temporally and spatially coarse. Wastewater measurements can be available within 24 h of sample collection and are inclusive of the entire contributing community, including those who are asymptomatic or unwilling or unable to seek clinical care, therefore allowing a rapid assessment of the presence of infections and infection trends. Such data can inform local clinical decision making including the prescription of drugs and testing, availability of community testing resources, and vaccination or behaviour-change campaigns; and can be communicated to the public so the community, particularly vulnerable individuals, can make informed decisions about behaviours that limit their risk. Clinical testing remains important for individualised care particularly for patients with severe disease.
Before the COVID-19 pandemic, viral respiratory infections largely circulated seasonally with predictable dynamics.
The changing pattern of common respiratory and enteric viruses among outpatient children in Shanghai, China: two years of the COVID-19 pandemic.
If and when respiratory disease dynamics will return to those observed pre-pandemic is uncertain. More active approaches to disease surveillance are needed to better understand and respond to atypical respiratory disease dynamics.
Wastewater-based epidemiology uses information from wastewater to gain insights into infectious disease occurrence in contributing communities. Wastewater represents a composite biological sample containing bodily excretions including urine, faeces, sputum, and mucus. Excretions of individuals who are infected can contain markers of infectious disease (including live and dead organisms, proteins, and nucleic acids) and methods have been developed to detect and quantify these markers in the wastewater matrix. Wastewater-based epidemiology does not require that individuals who are infected receive medical care or testing or even have symptoms to be represented in the community-level data source. Wastewater-based epidemiology, however, cannot discern information about individual disease severity.
Wastewater-based epidemiology has been shown to be useful for understanding community circulation of poliovirus,
Retrospective surveillance of wastewater to examine seasonal dynamics of enterovirus infections.
but it has not been widely used for disease surveillance outside of polio in endemic regions. The application of wastewater-based epidemiology to respiratory diseases was prompted by the COVID-19 pandemic, during which concentrations of SARS-CoV-2 RNA in wastewater were shown to correlate to incident COVID-19.
High-frequency, high-throughput quantification of SARS-CoV-2 RNA in wastewater settled solids at eight publicly owned treatment works in Northern California shows strong association with COVID-19 incidence.
Wastewater-based detection of two influenza outbreaks.
showed that respiratory syncytial virus (RSV) and influenza A RNA concentrations, respectively, in wastewater solids were associated with disease occurrence in associated sewer sheds.
In this study, we aimed to test the use of wastewater-based epidemiology for a range of viral respiratory diseases.
Results
Results are reported as suggested in the Environmental Microbiology Minimal Information guidelines (appendix pp 4, 13).
The Environmental Microbiology Minimum Information (EMMI) Guidelines: qPCR and dPCR quality and reporting for environmental microbiology.
Extraction and PCR negative and positive controls performed as expected (ie, negative and positive, respectively) with the exception of a positive control that did not amplify for a HCoV run including samples collected before Feb 26, 2021; HCoV results before this date were therefore omitted. The median PMMoV concentration across the samples was 1·6 × 109 copies per gram, similar to measurements previously reported for the plant.
High-frequency, high-throughput quantification of SARS-CoV-2 RNA in wastewater settled solids at eight publicly owned treatment works in Northern California shows strong association with COVID-19 incidence.
PMMoV concentrations were stable across samples (IQR 1·3 × 109–2·0 × 109 copies per gram), suggesting consistent faecal strength and RNA extraction efficiency. Additionally, comparisons of PMMoV concentrations to those measured in the same samples with no storage indicates sample storage and freeze–thaw cycles had a limited effect on PMMoV RNA concentrations, and we assume that the effect was similarly minimal for the other RNA targets (appendix pp 3–4).
In-silico analysis indicated no cross reactivity of the novel RT-PCR probe-based assays (table 1) with sequences deposited in NCBI. The novel assays for HPIV, HMPV, HRV, and the HCoV were tested in vitro against non-target viral gRNA as well as target gRNA (appendix pp 7–9). No cross reactivity was observed (appendix p 15).
Table 1Forward and reverse primer and probe sequences for the detection of the respiratory viral RNA used in this study
Additional details of the target region of the genomes are in the appendix (pp 7–8). Primers and probes were purchased from Integrated DNA Technologies (Coralville, IA, USA). All probes contained fluorescent molecules and quenchers (5′ FAM and or HEX/ZEN/3′ IBFQ); FAM, 6-fluorescein amidite; HEX, hexachloro-fluorescein; ZEN, a proprietary internal quencher from Intrgrated DNA Technologies (Coralville, IA, USA); and IBFQ, Iowa Black FQ. The appendix (p 2) indicates whether hexachloro-fluorescein or 6-fluorescein amidite molecules were used. The size of the product generated by the primers is provided in brackets in the first column. All assays run at an annealing temperature of 59°C (appendix pp 2–3).
RNA from all viruses were detected in wastewater samples (figure 1). Concentrations varied from a median of 0 to more than a million copies per gram. In order of the highest to lowest, median (IQR) concentrations in units of copies per gram were 35 000 (17 000–56 000) for HCoV, 4300 (0–9500) for HRV, 3500 (1400–6300) for HPIV, 1700 (0–5400) for RSV B, 760 (0–2000) for HMPV, 0 (0–660) for IAV, 0 (0–0) for IBV, and 0 (0–0) for RSV A, for which 0 represents non-detects (table 2). Although median IAV, IBV, and RSV A were non-detects, viral RNA from IAV was detected in 60 (28%) of 216 samples, as was IBV in 12 (6%) of 216 samples and RSV A in 30 (14%) of 216 samples. For context, median concentration for SARS-CoV-2 was 48 000 (25 000–130 000) copies per gram. In general, concentrations were lowest at the beginning of the study (February, 2021), and increased until January, 2022, at which time RNA concentrations for all viruses showed a steep drop. After the steep drop, viral RNA concentrations began to increase until the end of the time series with the exception of RSV B for which we did not observe a rebound. HCoV concentrations rebounded first, when all other viral RNA concentrations, including those of SARS-CoV-2, were still relatively low (figure 2).
Figure 1State-aggregated positivity rate and viral RNA in wastewater solids
Grey symbols represent measurements; error bars are SD. Black lines represent Morbidity and Mortality Weekly Report weekly median wastewater measurements. Asterisks indicate dates for which a point is off scale. (A) RSV positivity rates include RSV A and RSV B. (B) SARS-CoV-2 wastewater results and state-aggregated positivity rate are from all laboratories in the state (not just sentinel laboratories) and are shown in red to distinguish from other clinical data. (C) HCoV is the sum of all four seasonal HCoVs (ie, OC43, HKU-1, 229E, and NL63); three measurements were located off scale (1·1 × 106 copies per gram on April 9, 2021, 3·6 × 105 on June 24, 2021, and 5·1 × 105 on March 3, 2022). (D) HPIV is the sum of HPIVs (1–4); one measurement was located off scale (3·6 × 104 copies per gram on April 14, 2022). (E) HRV includes HRV A, B, and C; one measurement was located off scale (7·6 × 104 copies per gram on April 14, 2022). (G) Three IAV measurements were located off scale (2·1 × 104 copies per gram on Sept 30, 2021, 7·5 × 104 on Oct 31, 2021, and 1·5 × 104 on April 14, 2022). RSV=respiratory syncytial virus. HCoV=human coronaviruses. HPIV=human parainfluenza viruses. HRV=human rhinovirus. HMPV=human metapneumovirus. IAV=influenza A virus. IBV=influenza B virus.
Table 2Summary statistics of measurements of viral RNA in wastewater solids
Viral RNA concentration is reported in copies per gram dry weight of wastewater solids. Human coronaviruses is the sum of all four non-severe acute respiratory syndrome human coronaviruses (OC43, HKU-1, 229E, and NL63). Human parainfluenza virus is the sum of human parainfluenza viruses 1–4. Human rhinovirus is the sum of human rhinovirus A, B, and C. SARS-CoV-2 data are included for context.
Standardised concentrations of viral RNA for all viruses except for RSV A and influenza B virus, which were rarely detected, to allow for clear visualisation of concordance in the reduction of virus concentrations after the omicron BA.1 surge, and the rise of HCoV concentrations shortly thereafter. The grey shaded backgrounds indicate periods of time that indoor masking mandates were in effect locally. The red SARS-CoV-2 line is labelled with the dominating circulating variant at the time. Standardised concentrations were calculated using the following formula: Cst(t) = (C(t) – Cmin)/(Cmax– Cmin), where Cst(t) is the standardised concentration at a given week t, C(t) is the Morbidity and Mortality Weekly Report weekly median concentration shown in figure 1 at week t, Cmin is the minimum concentration, and Cmax is the maximum concentration. HMPV=human metapneumovirus. IAV=influenza A virus. RSV=respiratory syncytial virus. HPIV=human parainfluenza viruses. HRV=human rhinovirus. HCoV=human coronavirus.
To assess which HCoV were circulating during the study, we measured concentrations of OC43, HKU-1, 229E, and NL63 RNA in 18 samples (figure 3). Most of the total HCoV RNA was OC43 RNA in 15 of 18 samples. For the three remaining samples collected in April, May, and early June, 2022, most of the total HCoV RNA was 229E. HKU-1 RNA and NL63 RNA were each detected in six samples (concentrations ranging from 600 to 14 000 copies per gram for HKU-1 RNA and 500 and 4000 copies per gram for NL63 RNA) and never represented the majority of HCoV RNA.
Figure 3Concentrations of viral RNA in wastewater solids and comparison to sentinel laboratory percent positivity data
(A) Concentration of total HCoV RNA (weekly medians from figure 1 shown as grey filled areas) and individual HCoVs. (B) Concentrations of total concentration of HPIV RNA (weekly medians from figure 1 shown as grey filled areas) and individual HPIVs. (C) State-aggregated percent positivity from sentinel laboratories for tests for individual HCoVs. (D) State-aggregated percent positivity from sentinel laboratories for tests for individual HPIVs. Percent positivity for unknown HCoV and unknown HPIV from the sentinel laboratories surveillance system are not shown in panels C and D. Error bars on individual HCoV and HPIV are SDs and in some cases are smaller than the symbol. HCoV=human coronavirus. HPIV=human parainfluenza virus.
Similarly, we measured RNA concentrations of each HPIV (1, 2, 3, 4A, and 4B) in 18 samples. RNA from all five HPIVs were present; HPIV 3 dominated in most samples, although HPIV 2 and HPIV 4B each dominated in one of the 18 tested samples. HPIV 1 and HPIV 4A contributed the least to total HPIV (figure 3).
We tested whether state-aggregated positivity rates for respiratory viral infections were associated with the wastewater concentrations of viral RNA. Sentinel laboratories tested a median (IQR) of 5918 (2321–6874) clinical specimens per week for influenza (IAV and IAB), 2419 (1171–6065) for RSV (RSV A and RSV B), 1084 (848–1363) for HPIV, HMPV, and HRV, and 931 (746–1165) for HCoV. The associations between wastewater RNA concentrations and positivity rates were significant and positive for all viruses except for IBV. Kendall's tau between positivity rates and wastewater concentration were 0·47 (p<10–7) for HRV, 0·45 (p<10–5) for IAV, 0·52 (p<10–9) for HPIV, 0·57 (p<10–10) for RSV (using RSV B wastewater concentrations as RSV A was mostly non-detect), 0·32 (p=0·0002) for HMPV, 0·32 (p=0·0001) for HCoV, and –0·010 (p=0·92) for IBV. Wastewater concentrations and positivity rates aggregated across viruses were positively correlated (tau=0·43, p <10–15; appendix p 16). Results using county-aggregated positivity rates, when available, are similar to those using state-aggregated rates (appendix pp 4, 17). All the tests of association were repeated with wastewater respiratory virus RNA concentrations normalised by PMMoV RNA concentrations and the results were unchanged.
State-aggregated positivity rates for individual HCoV infections are similar to observations of the relative occurrence of their RNA in wastewater solids (figure 3). OC43 had the highest positivity relative to other HCoVs, in agreement with the wastewater measurements. Wastewater indicates that 229E dominated at the end of the study (spring 2022), but this finding is not reflected in the state-aggregated clinical data. Positivity rates for individual HPIV suggest HPIV 3 dominated HPIV infections throughout the study period; HPIV 2 and 4 also circulated with lower positivity rates. HPIV 1 was rarely detected in clinical specimens. This finding agrees with the wastewater data that indicated HPIV 3 dominated with HPIV 2 and HPIV 4B also present at relatively high levels, and HPIV 1 was rarely detected (figure 3). Wastewater concentrations and positivity rates aggregated across individual HCoV and HPIV were positively correlated (tau 0·49 for HCoV and 0·66 and HPIV, both p<10–6; appendix p 18).
Viral RNA concentrations were associated with each other, as well as with SARS-CoV-2 N gene concentrations available from a regional monitoring programme at the plant (appendix p 11). HPIV, HRV, HMPV, RSV B, and SARS-CoV-2 RNA were significantly correlated with each other (p<0·0009) with tau from 0·16 (SARS-CoV-2 and HRV) to 0·58 (HRV and HMPV). Given the low occurrence of IAV, IBV, and RSV A RNA, the fact that they were not consistently correlated with other viral RNA concentrations is unsurprising. HCoV RNA concentrations were not correlated to concentrations of other viral targets.
Discussion
We detected RNA from all tested respiratory viruses in wastewater solids, including HRV, HPIV, HMPV, influenza, RSV, and seasonal coronaviruses. Additionally, we detected four seasonal HCoVs (OC43, 229E, NL63, and HKU-1), and five HPIVs (1, 2, 3, 4A, and 4B). Non-influenza respiratory viral RNA targets were measured using novel assays; these assays were found to be specific to the intended viral genomic targets when tested against a range of viruses. Previous studies have documented SARS-CoV-2,
High-frequency, high-throughput quantification of SARS-CoV-2 RNA in wastewater settled solids at eight publicly owned treatment works in Northern California shows strong association with COVID-19 incidence.
Wastewater-based detection of two influenza outbreaks.
RNA in wastewater, but to our knowledge, no study to date has showed concentrations of other respiratory viruses.
RSV A RNA was rarely detected in wastewater, yet RSV B RNA concentrations were more than 10 000 copies per gram at times, and RSV B wastewater trends mirrored RSV clinical surveillance data. This finding suggests limited local cocirculation of the two RSV groups. Changes in dominant RSV strains have been documented to occur on continental
Influence of respiratory syncytial virus strain differences on pathogenesis and immunity.
The association between viral RNA in wastewater solids and the clinical specimen positivity rates for respective respiratory viruses, available through passive disease surveillance, suggests that wastewater data are representative of respiratory disease occurrence in the community. The available clinical data were aggregated across the entire state, which could obfuscate local disease occurrence patterns. For example, deviation in HMPV wastewater and state-aggregated positivity rate trends suggest localised patterns in HMPV infections. Additionally, state-aggregated HCoV case positivity suggests OC43 infections dominated HCoV infections, yet wastewater measurements suggest 229E dominated locally in the spring of 2022. Similarly, based on wastewater measurements, HPIV 2 and HPIV 4 could have contributed to more HPIV infections locally than on average across the state.
The analysis between wastewater data and clinical data is limited by the use of passive surveillance data and no local data matching the sewershed on respiratory disease occurrence. The state-aggregated clinical data used serves as a proxy for sewershed clinical data; it is a valid proxy if state-wide disease occurrence trends mimic those at the local scale. Limited county-aggregated sentinel laboratory positivity rates generally match state-aggregated positivity rates (appendix p 17) and associations between county-aggregated clinical data (as available) and wastewater are consistent with results obtained with state-aggregated clinical data (appendix pp 4–5). Clinical surveillance data are also biased towards those receiving medical care and not all specimens are tested for all viruses, making estimating disease occurrence from positivity rate data difficult. Positivity rates might not be associated with disease occurrence in the same manner for all viruses.
There are additional uncertainties associated with understanding disease occurrence using wastewater testing. Little information is available on concentrations of viral RNA excreted into wastewater from individuals who are infected, despite evidence showing that shedding does occur for viruses included in this study.
Detection of respiratory viruses in sputum from adults by use of automated multiplex PCR.
Given the current state of knowledge, knowing how inputs of viral RNA to the wastewater system differ between individuals infected with different viruses and with different disease severities is difficult. Wastewater cannot be used to infer specifics about the severity of illness, or characteristics of infected subpopulations. Wastewater captures contributions from anyone using sewers in the sewershed, including transient populations.
Despite these limitations and uncertainties, the association between viral RNA in wastewater solids and case positivity holds when data from the measured viruses are combined, suggesting that the relative concentrations of viruses in wastewater solids are related to their different rates of disease occurrence. HCoV and HRV concentrations were highest during the study period, suggesting seasonal HCoV and HRV infections were most common. Conversely, IBV and RSV A concentrations were lowest, suggesting limited circulation of those viruses. Future work is needed to document time varying patterns of respiratory viral RNA inputs from individuals who are infected to the wastewater system to enable modelling of the number of individuals who are infected within a sewershed from wastewater viral RNA concentrations,
Wastewater-based estimation of the effective reproductive number of SARS-CoV-2.
Viral RNA concentrations in wastewater solids, including those of SARS-CoV-2, generally followed similar trends over time, with the exception of HCoV. Evident in the trends is a notable drop-off in concentrations after the omicron BA.1 surge in January, 2022. The drop-off might suggest that disease mitigation measures practiced by the community in response to the surge (eg, isolation due to illness, working remotely, and wearing masks) reduced the spread of all respiratory viruses.
Face masks to prevent transmission of COVID-19: a systematic review and meta-analysis.
Local indoor mask mandates ended shortly after the omicron surge (March 2, 2022), and this change was followed by an increase in concentrations of respiratory viruses in wastewater (figure 2). HCoVs, specifically OC43, were the first viruses to reappear in wastewater after the omicron surge. HCoVs are highly transmissible with R0 values higher, on average, than influenza and HRV,
Transmissibility and transmission of respiratory viruses.
Infection with omicron also possibly reduced susceptibility to other respiratory viruses and contributed to the decrease in all viral activity; antibody cross immunity has been suggested to control temporal patterns of some respiratory viruses.
Cross-immunity between strains explains the dynamical pattern of paramyxoviruses.
Data generated from wastewater can be available within 24 h of sample collection, and samples represent the entire contributing population—even those with asymptomatic infections—thereby overcoming various biases and the inherent reporting delays of the passive clinical surveillance system. Results suggest that wastewater surveillance for multiple respiratory viruses that commonly circulate can provide information on a community scale on causes of acute respiratory illnesses. Additional studies from diverse geographical locations are needed to further confirm these results. The acute respiratory illnesses caused by the viruses included in this study have differing levels of likely severity and associated prevention measures, treatments, and complications. Therefore, more information about circulating viruses, obtained from wastewater, can inform local clinical, public health, and individual decision making. Medical doctors can use information on circulating viruses to aid in differential diagnosis and making decisions about patient specimen testing that could influence the use of therapeutics. Hospital directors can use the information to inform hospital staffing, and the stocking of therapeutics and prevention supplies in anticipation of needs when outbreaks begin. Wastewater data can be used to guide the provision of community testing resources and targeting of vaccination or behavioural change campaigns by public health departments. Finally, wastewater data can also be communicated to the public, similar to a weather report, so the community and particularly individuals who are considered to be vulnerable can make informed decisions about behaviours that could put them at risk.
ABB contributed to the conceptualisation of the study, the resources, data curation, writing of the original draft, reviewing and editing of subsequent drafts, visualisation, supervision, and funding acquisition. BH and DD contributed to the investigation, data curation, and reviewing and editing of manuscript drafts. VC-H and AB contributed to the investigation and reviewing and editing of manuscript drafts. MKW contributed to the conceptualisation of the study and the reviewing and editing of manuscript drafts. BJW contributed to the conceptualisation of the study, reviewing and editing of manuscript drafts, and supervision. All authors had full access to all the data in the study and had final responsibility for the decision to submit for publication. ABB and BH verified the underlaying data of the study.
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Update: Each purchase of Breathing+ will now include three machine washable mouthpieces. Previous buyers will be supplied with those by their country representatives but will have to cover shipping costs. Please be patient while we arrange distribution. https://www.breathinglabs.com/latest-news/announcement-breathing-mouthpieces-for-clinical-and-professional-use-are-now-available/
Update: We moved servers + relocated all our games to our servers, please be patient while google reviews all that (showing unsafe website atm). Use duckduckgo or non-chromium browsers to reach our pages in the meantime. Everything ok + new product addons coming out in a month!
Registration and all functionalities at http://breathinglabs.com (and in our iOS and Android games) are fixed and fully working. If you find any issues -> [email protected]
We are back in stock with Breathing+, currently searching for VR supplier, and setting up mass production for toys and tens stimulation + in November we will be signing up new erasmus traineeships, research projects, bilateral, FP(eu), and asia-pacific ->[email protected]
Notice to b2b partners: we are running late with some minor upgrade-> briefly running out of stock -> retail and b2b sale is closed until early october. To get a list of partners with stock to sell contact us at [email protected] Thanks, we'll go strong again in winter 💪
