2023-03-31

Whispers of Viruses and Bacteria in Wastewater

Professor Marlene Wolfe, one of the leaders of the US Sewer Coronavirus Alert Network (SCAN) team, said in an interview with the British Medical Journal: We can take that sample, which is less than a gram of wastewater solids, from communities all across the country: that small sample can represent up to 4 million people in some cases.”.

The whisper of SARS-CoV-2 was loud in less than a gram of wastewater sample. At the end of 2022, more than 3,800 monitoring stations in at least 70 countries around the world are watching the wind and listening to the whisper of SARS-CoV-2 in the water.


Figure 1: History of Wastewater Science Terminology

History of Wastewater Science Terminology

The origin of the detected wastewater can be traced back to the Broad Street cholera outbreak in Soho, London in 1854. At that time, in a public well and cesspool near a house where many people died of cholera, feces were found to leak bacteria and contaminate the water supply system of the pump, causing an epidemic. 

  • In the mid-1950s, "Wastewater Tracing or Tracking" appeared successively after the research on the wastewater infected by snail schistosomiasis in South Africa. 
  • Around 1970s, "wastewater monitoring" and "wastewater surveillance, "WWS)" or "Wastewater-Based Surveillance (WBS)" are commonly used in industrial wastewater detection research.
  • In the 2000s, issues such as tracking heroin and other illicit drugs emerged as "sewage epidemiology" and "wastewater-based epidemiology (WBE)". The mixed use of subject terms mainly describes the scientific based on the premise that the substances excreted by humans in wastewater can be used to calculate the initial concentration.

However, by 2014, the term "wastewater epidemiology" gradually replaced the term "sewage epidemiology". Although the terms sewage and wastewater are still commonly used interchangeably, the recent official documents of the World Health Organization (WHO) mainly use the term "wastewater surveillance". Scholars also suggested the standardization of related science and technology, and supported the use of "wastewater surveillance (WWS)" and "wastewater epidemiology (WBE)" as common terms.

If we examine the differences in definitions, we can distinguish them from the perspective of public health (Figure 1):

  • wastewater tracing or tracking is mainly to identify the source of pathogens or toxins;
  • wastewater monitoring is an action to ensure that waste water discharge does not cause public health risks ;
  • wastewater surveillance emphasizes systematic, continuous testing of wastewater for the benefit of public health and may be relevant for public health policy;
  • wastewater epidemiology (WBE) is the scientific field that links pathogens and chemicals found in wastewater to population health.

Which infectious human pathogens were studied in wastewater before COVID-19?

Wastewater surveillance (WWS) has been used to assess waterborne and fecal-oral pathogens that cause diarrhea-related diseases. The well-studied pathogens of human infectious diseases include picornaviridae, caliciviridae and reoviridae, etc. (indicated by * in Fig. 2). Epidemics of international concern such as coronavirus, Ebola virus, Zika fever, and polio/poliomyelitis virus (indicated by *** in Figure 2) have provided information on public health actions or policies, but influenza is rare founded in WWS literature.

Figure 2: Wastewater Surveillance (WWS) of known infectious diseases before the COVID-19 pandemic: virus classification by family/genus
 

 

 

 

 

 

 

 

 

 

 

 

If the coronavirus is the subject, the early coronavirus wastewater monitoring is in view of the emergence of new viruses with high epidemic potential, which usually involves complex dynamic effects on animals, humans and the environment. Therefore, since the 1970s, environmental monitoring has been implemented by monitoring surface water, mud and biosolids to understand the status of such viruses in the water cycle, ex. human coronavirus (HCoV), human coronavirus 229E (CoV-229E), HKU1 and severe acute respiratory syndrome coronavirus (SARS-CoV); zoonotic coronaviruses such as Middle East respiratory syndrome coronavirus (MERS-CoV) and animal coronaviruses: such as bovine coronavirus (BCoV), mouse hepatitis virus (MHV), etc. This stage focuses on coronavirus, the survival status of viruses in wastewater and the efficiency of virus recovery, etc., but the overall knowledge is still very scarce and fragmented.

After the COVID-19 pandemic

Figure 3: Overview of Wastewater Surveillance (WWS) after COVID-19

The COVID-19 pandemic has seen extensive adaptation of global wastewater surveillance (WWS), and wastewater can also be an effective potential application for surveillance of respiratory-transmitted pathogens. Technologies for detecting SARS-CoV-2 and new variants through wastewater are becoming more sophisticated. 

Figure 3 generally illustrates the current WWS in addition to early development of chemicals such as drugs, cleaning agents, industrial pollutants or pathogens like antibiotic-resistant bacteria, etc., as well as human infectious disease pathogens (described in Figure 2).

After COVID-19, in addition to the new monitoring of SARS-CoV-2, monkeypox and influenza RSV have recently become emergencies of international concern, and such pathogens have also become the focus of WWS, especially when most people cannot receive RSV clinical test, wastewater data can fill the gaps. 

Meanwhile, the progress in wastewater genome monitoring technology has solved the problem of multiple virus strains in wastewater. As shown in Figure 3, according to the WHO classification of coronavirus variant strains, new variants can be found in wastewater samples 14 days in advance, and clinical monitoring can be determined instances of virus transmission that cannot be captured, for high-risk groups such as student dormitories, airports, hospitals, nursing homes and other long-term care facilities. In addition to providing early warnings, it can also help contain and mitigate virus outbreaks. In the long run, WWS is more an important tool for tracking the dynamics of viral lineages in combination with dominance.

Further analysis from the perspective of wastewater epidemiology (WBE), in resource-rich countries is mainly assessed in sewers and sewage systems, but in resource-poor environments, most residents are not connected to centralized wastewater treatment plants, using pit toilets, septic or open defecation, so the WWS process varies depending on the wastewater system.

Overall, as shown in Figure 4, WBE includes the process of sampling, sampling methods, virus concentration and concentration techniques, use of control viruses in the control process, virus isolation, RNA extraction, virus detection and quantitative sequencing, and finally completes epidemiological modeling to analyze epidemic trends.

However, WBE still faces many challenges, such as sampling control: daily changes in water flow, differences in wastewater systems, weather factors, temperature, sedimentation rate, and virus shedding and other factors. In terms of virus recovery and concentration: concentration method efficiency, RNA extraction, purification efficiency and storage of RNA, etc. Plus virus detection and/or quantification. Challenges such as RNA quality, RNA quantity, PCR inhibitors, and normalization of data will all affect COVID-19 wastewater surveillance, which faces shortcomings such as low recovery rate and long processing time.

Figure 4: Analysis Process of COVID-19 Wastewater Epidemiology (WBE)

WWS is intended to complement, not replace clinical tests

SARS-CoV-2 wastewater surveillance differs from clinical diagnostic testing in the design and interpretation of community-scale sampling programs, as well as in the different assays which attempted to concentrate and extract RNA from wastewater and environmental water samples.

Because viral RNA can be discharged into wastewater before symptoms and diagnostic testing, SARS-CoV-2 wastewater surveillance can help document trends in high-prevalence cases of COVID-19, and in cases of low prevalence or lack of clinical testing evidence.

Early warning can be provided, while wastewater viral load can be used to monitor the impact of public health social measures, including increased or relaxed restrictions, as well as enhanced risk communication, warning the community about the (re)emergence of the virus, and advising the community about testing, quarantine, isolation, as well as suggest actions such as vaccinations and seeking health care.

In sum, WWS advantages include providing objective metrics that are less susceptible to biases inherent in clinical testings, such as 

  • health-seeking behaviour, 
  • disease severity (including symptomatic and asymptomatic), 
  • healthcare and testing accessibility, 
  • physician and individual response to testing propensity, 
  • cost and reporting constraints, etc.

We take the interactive COVID-19 WWS of the New Zealand Institute of Environmental Research and Science as an example (Figure 5). This dashboard has been launched in July 2022 to allow the public to track the footprints of SARS-CoV-2 and view national and regional epidemics, etc. latest trends.

It provides a spatial-temporal visual map; it presents wastewater statistics and provides functions such as regional options, search, and comparison of trends in different periods. 

  • From Figure 5(a), we can observe that Wellington shows that the wastewater data and the corresponding spikes of confirmed cases are different in three time periods (green dotted circles): during the peak period of the epidemic, due to sufficient clinical testing, the confirmed cases and wastewater data are about the same.
  • In the low epidemic period, the peak of wastewater data far exceeds the confirmed status due to factors such as people feeling tired (or thinking it is unnecessary) to rub their noses, the overall detection capacity slows down, or the willingness to report decreases. 
  • The public can also check the latest SARS-CoV-2 variant virus. Figure 5 (b) and (c) show the ratio and trend information respectively.

In general, the wastewater data not only supplements clinical monitoring, but also provides background information on the public’s epidemic prevention environment base. The dashboard shows that wastewater data covers 73% of the population in New Zealand. A small sample of less than one gram only needs a few expensive PCRs.

Compared with the costs of tests and costs for clinical PCR, yes, using WWS to listen to the muttering sounds from all directions in the wastewater is cheaper and wider. Plus, virus and bacteria of various kinds can be identified, warned and tracked broadly. It can also assist the public in preventing and managing risks by them selves, easily in their daily life and sustainably for the whole country health.

Figure 5:SARS-CoV-2 wastewater monitoring network of the Institute of Environmental Science and Research (ESR) in New Zealand
COVID-19 Wastewater Surveillance Dashboard: https://esr-cri.shinyapps.io/wastewater


Reference

[1] Nelson, Bryn. What poo tells us: wastewater surveillance comes of age amid covid, monkeypox, and polio. BMJ 378 (2022).

[2] COVIDPoops19: https://ucmerced.maps.arcgis.com/apps/dashboards/c778145ea5bb4daeb58d31afee389082

[3] Kilaru P, Hill D, Anderson K, Collins MB, Green H, Kmush BL, Larsen DA. Wastewater Surveillance for Infectious Disease: A Systematic Review. Am J Epidemiol. (2022) Oct 13.

[4] Larsen, David A., et al. Wastewater monitoring, surveillance and epidemiology: a review of terminology for a common understanding. FEMS microbes 2 (2022). 2021-08-19

[5] Gonçalves, José, et al. Centralized and decentralized wastewater-based epidemiology to infer COVID-19 transmission–A brief review. One Health (2022): 100405.

[6] World Health Organization. Environmental surveillance for SARS-COV-2 to complement public health surveillance: interim guidance, 14 April 2022. No. WHO/HEP/ECH/WSH/2022.1. WHO (2022).

[7] [3]

[8] Carducci, Annalaura, et al. Making waves: coronavirus detection, presence and persistence in the water environment: state of the art and knowledge needs for public health. Water Research 179 (2020): 115907.

[9] McPhillips, D and Howard,J (CNN), The RSV surge didnt come out of nowhere, but gaps in data made it tougher to predict, October 27, 2022. https://edition.cnn.com/2022/10/27/health/virus-surveillance-data-gaps/index.html

[10] Karthikeyan, Smruthi, et al. Wastewater sequencing reveals early cryptic SARS-CoV-2 variant transmission. Nature 609.7925 (2022): 101-108.

[11] Aguiar-Oliveira, Maria de Lourdes, et al. Wastewater-based epidemiology (WBE) and viral detection in polluted surface water: A valuable tool for COVID-19 surveillance—A brief review. International journal of environmental research and public health 17.24 (2020): 9251.

[12] [6] 


medium URI: https://medium.com/@andreahuang2019/whispers-of-viruses-and-bacteria-in-wastewater-95a7776a31b3

 

廢水中病毒細菌的嘀咕(初稿)

國下水道冠狀病毒警報網(The Sewer Coronavirus Alert Network, SCAN)團隊領導人之一Marlene Wolfe教授接受英國醫學雜誌訪問時談到 [1] :從全國各地社區採集不到一克的廢水固體樣本:在某些情況下,這個小樣本可代表多達400萬人。」嚴重急性呼吸道症候群冠狀病毒II(新冠病毒,SARS-CoV-2)的嘀咕聲在不到一克廢水樣本中,很大聲。2022年末此刻,全球至少70個國家、超過3800監測站正在細看風中聆聽水中SARS-CoV-2的嘀咕聲 [2] 

廢水科學術語的歷史

檢測廢水起源可追溯到1854年倫敦蘇荷區的寬街霍亂爆發事件。當時在一座有多人霍亂死亡的房屋附近公井和污水池,發現糞便洩漏細菌並汙染到泵的供水系統而引發流行病。1950年代中期「廢水追蹤或跟蹤(Wastewater Tracing or Tracking)」自南非大壩蝸牛血吸蟲感染的廢水相關研究後陸續出現,至1970前後「廢水偵測(wastewater monitoring)」與「廢水監測(wastewater surveillance, WWS) 」或「基於廢水的監測(Wastewater-Based Surveillance, WBS) 」常見於工業廢水檢測研究中。到了2000年代研究追蹤海洛因和其他非法藥物等議題出現了「污水流行病學(sewage epidemiology)」和「基於廢水的流行病學(wastewater-basedepidemiology, WBE)(中文經常簡稱廢水流行病學)學科用語的混用,主要描述以廢水中穩定人類排泄的物質,可用於計算初始濃度為前提的科學領域。

但至2014年「廢水流行病學」漸漸取代「污水流行病學」一詞,雖然汙水和廢水二詞仍常見互用,但世界衛生組織(WHO)近期官方文件主要使用「廢水監測」一詞學者亦建議標準化相關科學技術,支持使用「廢水監測(WWS)」和「廢水流行病學(WBE)」作為共通用詞。若檢視定義的差別,我們可自公共衛生視角區分(圖一附表): 廢水追蹤或跟蹤,主要是為識別病原體或毒物來源; 廢水偵測是為確保廢水排放不會造成公共衛生風險的行動; 廢水監測則強調系統地、持續測試廢水以造福公眾健康可能與公共衛生政策相關; 廢水流行病學(WBE)即是將廢水中發現的病原體和化學物質與人口健康聯繫起來的科學領域[3][4][5][6]

COVID-19前廢水研究了哪些傳染性人類病原體? 

歷來廢水監測(WWS)用於評估引起腹瀉相關疾病的水傳播和糞口傳播病原體,研究較為成熟的人類傳染病病原體包括微小核糖核酸病毒科、杯狀病毒科和呼腸孤病毒科等(圖二*表示)。國際關注流行病如冠狀病毒、伊波拉病毒、茲卡熱以及已提供公共衛生行動或政策資訊的小兒麻痺/脊髓灰白質炎病毒(圖二***表示)為代表,但文獻中罕見流感和愛滋病毒在WWS中出現[7]

圖二: 新冠大流行前已知的傳染病廢水監測(WWS) : 病毒以科/屬分類

若專以冠狀病毒為對象,早期的冠狀病毒廢水檢測鑒於具有高流行潛力的新病毒出現,通常涉及動物、人類和環境的複雜動態影響,因此環境監測自1970年代起,透過監測地表水、廢水、泥漿和生物固體中的冠狀病毒來了解這類病毒在水迴圈中的狀態,其種類包括人類冠狀病毒(HCoV)、人類冠狀病毒229E (CoV-229E)HKU1和嚴重急性呼吸道症候群冠狀病毒(SARS-CoV;人畜共患的β冠狀病毒如中東呼吸症候群冠狀病毒(MERS-CoV)以及動物冠狀病毒:如牛冠狀病毒(BCoV)、小鼠肝炎病毒 (MHV)等,此階段著重冠狀病毒在廢水中的生存狀態與病毒回收效率等,但整體知識仍非常稀缺和零碎[8]

COVID-19大流行後

圖三: 新冠大流行後廢水監測(WWS)概況
新冠疫情見證了全球廢水監測(WWS)的廣泛調整,以及廢水也能成為呼吸道傳播病原體監測的有效潛在應用。透過廢水檢測SARS-CoV-2和新變種的技術變得越來越成熟。圖三總體說明目前WWS除早期發展的化學物如藥物、清潔劑、工業污染物或病原體中抗生素抗藥性細菌等以及圖二描述的人類傳染病病原體。COVID-19後,新增監測SARS-CoV-2外,近期猴痘病毒與流感RSV病毒成為國際關注的突發公共衛生事件,此類病原體亦成為WWS關注的對象,尤其是當多數人未能接受RSV臨床檢測,廢水資料即填補了傳統監測的空白[9]

與此同時,廢水基因組監測技術的新進展,解決了廢水中多種病毒株問題,如圖三中根據WHO分類新冠病毒變種病毒株,能在廢水樣本中提前 14 天發現新變種,並確定臨床監測未能捕獲的病毒傳播實例,這對高風險人群如學生宿舍,機場、醫院、養老院等長期護理設施,除提供早期揭示預警外,亦能輔助遏制和緩解病毒的爆發,長期而言WWS更是追蹤病毒譜系動態綜合優勢的重要工具[10]

進一步就廢水流行病學(WBE)角度分析,在資源豐富的國家主要在下水道和污水系統進行評估,但在資源匱乏的環境中,大部分居民沒有連接到廢水集中處理廠,使用坑式廁所、化糞池或露天排便,因此根據廢水系統的差異,WWS過程亦有所不同。總體而言如圖四所示,包括採樣、採樣方法、病毒濃度濃縮技術、控制過程使用對照病毒、病毒分離、RNA提取、病毒檢測和定量測序,最後完成流行病學建模分析流行趨勢。不過WBE目前仍面臨許多挑戰,包括在採樣控制方面中: 水流日常變化、廢水系統差異、天氣因素、溫度、沉澱率、以及病毒脫落等因素。在病毒復原和濃縮方面: 濃縮方法效率、核糖核酸(RNA)萃取和純化效率、以及RNA的儲存等。病毒檢測和/或定量方面: RNA品質、RNA數量、PCR抑製劑、以及資料正歸化等這些挑戰均會影響新冠廢水監測面臨回收率低和處理時間長等缺點[11]

圖四: 新冠廢水流行病學(WBE)分析過程


WWS是用於補充而不是取代基於個人診斷測試的環境監測

與臨床診斷測試相比,SARS-CoV-2廢水監測的不同處在於社區規模採樣計劃的設計和解釋,以及從廢水和環境水樣中濃縮和提取RNA所嘗試不同的檢測方法。由於病毒RNA可在症狀出現和診斷測試前排放到廢水中,在新冠高流行情況下SARS-CoV-2廢水監測有助於記錄趨勢,而在低流行情況或缺乏臨床測試證據情況下,則可提供早期預警,同時廢水病毒載量可用於監測公共衛生社會措施的影響,包括增加或放寬限制,以及加強風險溝通,警告社區有關病毒(重新)出現,並建議社區有關檢測、檢疫、隔離、疫苗接種和尋求醫療保健等行為。綜合來說,其優點包括提供客觀指標,不易受到診斷測試中固有偏見影響,如尋求健康的行為、疾病嚴重程度(包括有症狀無症狀)、醫療保健和測試可及性、醫生和個人對測試的傾向以及成本和報告的限制等[12]

我們以紐西蘭環境研究與科學研究所的互動式新冠廢水監測網作為實例(圖五),該儀錶板於20227月上線提供民眾追蹤SARS-CoV-2足跡,查看全國、區域疫情等最新趨勢。一方面提供時空視覺化地圖,另一方面呈現廢水統計資料提供區域選項及搜尋、比較不同時段趨勢等功能。自圖五(a)我們可觀察到威靈頓在三個時段(綠色虛線圈)顯示廢水資料與確診病例相應尖峰的不同: 疫情高峰時期由於臨床檢測充足,確診病例與廢水資料約呈一致,而低流行時期或因人們對搓鼻子感到疲憊(或認為不需要)、整體檢測能量趨緩,亦或是通報意願下降等因素出現廢水資料高峰遠超過確診狀況。民眾亦可檢視最新的SARS-CoV-2變種病毒,在圖五(b)(c)分別呈現比例與升降趨勢資訊,綜合來說,廢水資料不僅補充了臨床監測,也提供民眾防疫環境背景資訊基礎。儀錶板顯示廢水資料涵蓋全紐西蘭73%的人群,不到一克的小樣本只需少數昂貴PCR,相對百萬人每人臨床PCR的大量檢驗與成本,是的,SARS-CoV-2在廢水中水聽八方的嘀咕聲實在非常廣,滴滴咕咕,可識別可預警可追蹤還可輔助民眾防疫自主時,做好自我風險再評估。

圖五: 紐西蘭環境研究與科學研究所(Institute of Environmental Science and Research, ESR)新冠廢水監測網儀錶板
COVID-19 Wastewater Surveillance Dashboard: https://esr-cri.shinyapps.io/wastewater

參考書目


[1] Nelson, Bryn. What poo tells us: wastewater surveillance comes of age amid covid, monkeypox, and polio. BMJ 378 (2022).

[2] COVIDPoops19: https://ucmerced.maps.arcgis.com/apps/dashboards/c778145ea5bb4daeb58d31afee389082

[3] Kilaru P, Hill D, Anderson K, Collins MB, Green H, Kmush BL, Larsen DA. Wastewater Surveillance for Infectious Disease: A Systematic Review. Am J Epidemiol. (2022) Oct 13.

[4] Larsen, David A., et al. Wastewater monitoring, surveillance and epidemiology: a review of terminology for a common understanding. FEMS microbes 2 (2022). 2021-08-19

[5] Gonçalves, José, et al. Centralized and decentralized wastewater-based epidemiology to infer COVID-19 transmission–A brief review. One Health (2022): 100405.

[6] World Health Organization. Environmental surveillance for SARS-COV-2 to complement public health surveillance: interim guidance, 14 April 2022. No. WHO/HEP/ECH/WSH/2022.1. WHO (2022).

[7] [3]

[8] Carducci, Annalaura, et al. Making waves: coronavirus detection, presence and persistence in the water environment: state of the art and knowledge needs for public health. Water Research 179 (2020): 115907.

[9] McPhillips, D and Howard,J (CNN), The RSV surge didnt come out of nowhere, but gaps in data made it tougher to predict, October 27, 2022. https://edition.cnn.com/2022/10/27/health/virus-surveillance-data-gaps/index.html

[10] Karthikeyan, Smruthi, et al. Wastewater sequencing reveals early cryptic SARS-CoV-2 variant transmission. Nature 609.7925 (2022): 101-108.

[11] Aguiar-Oliveira, Maria de Lourdes, et al. Wastewater-based epidemiology (WBE) and viral detection in polluted surface water: A valuable tool for COVID-19 surveillance—A brief review. International journal of environmental research and public health 17.24 (2020): 9251.

[12] [6] 

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: 本文之圖均作者自製,許多影像改製來源為Wikimedia Commons (https://commons.wikimedia.org/)中公眾領域(Public domain)授權圖片。唯圖五是網站截圖再製:https://esr-cri.shinyapps.io/wastewater

 

 

medium URI: https://medium.com/@andreahuang2019/廢水中病毒細菌的嘀咕-初稿-7603328d1896