The spread of the coronavirus in Stockholm and in six European countries is being tracked through sewage samples by a research group at KTH Royal Institute of Technology in Stockholm, which will report the results at the end of April.
The researchers believe that continuing the measurements will help provide early warning of coronavirus infections, should they return to communities later on.
Italy’s heavily-stricken northern region, as well as Catalonia in Spain, a number of cities in Turkey and India, as well as parts of the Netherlands, are all included in the biweekly sampling, says Zeynep Cetecioglu Gurol, a microbiologist and wastewater engineer at KTH.
Meanwhile the rapid-response effort is quickly generating offers of more water samples, including from Sweden’s second largest city, Gothenburg.
Protocols for preparing the samples, measuring the amount of coronavirus RNA and linking the level readings to an estimate of population infected, will be offered by KTH to all partners in the countries involved.
In addition, sequencing will be applied regularly to follow any potential mutation of COVID-19.
Cetecioglu Gurol says the wastewater authorities outside of Sweden have asked that the researchers to not identify the specific cities where wastewater samples are taken in these countries.
David Nilsson, director of the Water Centre at KTH, which contributed the start-up financing for the project, says that in cooperation with Stockholm’s Water and Wastewater authority, wastewater samples are being collected at two main treatment plans.
He says the concept provides a quick and low cost method for getting a wide angle view of the pandemic’s hold on a community.
“Our method provides significantly shorter detection time. So we can quickly get an early first warning for a second virus wave,” Nilsson says.
The KTH team will provide results to some of the partners, while analysis will also be performed by others.
These include KWR Water Research Institute in Nieuwegein, Netherlands, which recently published the proof of concept paper in Nature showing that coronavirus could be detected in wastewater.
“Everything is going so fast,” Cetecioglu Gurol says. “After we announced the sampling, we have been receiving more samples. And the main point is, this is about the public health, not the research.
“The most important thing is to collaborate and share what we know, and find out what others know. If people want to join our project with samples, or do their own analysis, we are ready to share.”
Cetecioglu Gurol, whose research focuses on recovering biochemical resources from wastewater, says the next step is to prepare the samples in order to concentrate the coronavirus from the sewage.
That’s more difficult to do than finding RNA in bodily fluids. “Wastewater is more complicated. There are a lot of things in it besides the virus,” she says.
The samples then undergo a multi-stage process called reverse-transcription quantitative polymerase chain reaction (RT-qPCR).
This stage basically unwinds the viral RNA and converts it into DNA, which is then further replicated so there are millions of copies – enough for a quantitative PCR instrument to detect the virus.
KTH researchers Anders Andersson and Professor Cecilia Williams are two of the scientists involved in sequencing the virus’ genetic material at the Science for Life Laboratory (SciLifeLab).
The lab is donating hours of work using specifically-optimized diagnostic assays similar those the lab uses for clinical testing.
The final step is to develop the estimating tool, based on simulations with public health data on SARS-CoV-2, coronavirus infection and other data regarding wastewater.
The group is simultaneously sampling sludge, since Sweden and other EU countries allow it to be converted to agricultural fertilizer.
A recent outbreak of novel coronavirus pneumonia (COVID-19) caused by SARS-CoV-2 infection has spread rapidly around the globe, with cases now confirmed in 130 countries worldwide. Although public health authorities are racing to contain the spread of COVID-19 around the world, the situation is still grim.
About 158 111 confirmed cases and 5946 cumulative deaths (81 059 confirmed cases and 3204 cumulative deaths from China) have been reported around the globe as of March 15, 2020. Some clinical cases have found that some carriers of the virus may be asymptomatic, with no fever, and no, or only slight symptoms of infection.
Without the ability to screen these asymptomatic patients quickly and effectively, these unsuspecting carriers have the potential to increase the risk of disease transmission if no early effective quarantine measures are implemented.
Therefore, to trace unknown COVID-19 sources, fast and accurate screening of potential virus carriers and diagnosis of asymptomatic patients is a crucial step for intervention and prevention at the early stage.
It remains a highly challenging logistical exercise for medical professionals to practically and effectively screen suspected infectious cases from individual households. Such a massive undertaking is time-consuming and labor intensive and is constrained by the availability of testing technologies at this extremely critical time.
However, an alternative method utilizing wastewater-based epidemiology (WBE), may provide an effective approach to predict the potential spread of the infection by testing for infectious agents in wastewater, which has been approved as an effective way to trace illicit drugs, and obtain information on health, disease, and pathogens.1
Faeces and urine from disease carriers in the community will contain many biomarkers that can enter the sewer system. A recent study demonstrated that live SARS-CoV-2 was isolated from the faeces and urine of infected people,2 which would then enter the wastewater treatment system.
A further study has shown that SARS-CoV-2 can typically survive for up to several days in an appropriate environment after exiting the human body. There is potential, therefore, that the analysis of SARS-CoV-2 in community wastewater could trace COVID-19 sources through sewage pipe networks and determine whether there are potential SARS-CoV-2 carriers in certain local areas.
If SARS-CoV-2 can be monitored in the community at the early stage through WBE, effective intervention can be taken as early as possible to restrict the movements of that local population, working to minimize the pathogen spread and threat to public health.
Using a WBE approach in developing an early warning system and consequent effective intervention system will require a rapid analytical method for the on-site detection of viruses at the wastewater collection point.
Currently, the most direct method for the detection of SARS-CoV-2 is a nucleic acid–based polymerase chain reaction(PCR) assay, which is also a means for confirmation of COVID-19 patients throughout China.
Although PCR has high sensitivity and specificity, requirements for complicated sample handling in the laboratory, skilled personnel, and a long period of data processing and analysis (4–6 h) are not conducive to real-time and effective monitoring of samples on location.
Therefore, it is critical to develop efficient transportable and robust analytical tools to accurately and quickly trace low-level SARS-CoV-2 sources through WBE to confirm these suspected cases and screen asymptomatic infected cases without centralized laboratories.
Paper analytical devices have emerged as powerful tools for the rapid diagnosis of pathogens and determination of infection transmission.3 The paper-based device is a small analytical tool with different functional areas printed with a wax printer that integrates all processes (extraction, enrichment, purification, elution, amplification, and visual detection) required for nucleic acid testing into an inexpensive paper material.
The whole testing process can be completed through simple folding of a paper-based device in different ways in different steps without a pump or power supply, which overcomes the limitation of PCR and avoids multiple processes. Paper analytical devices enable multiplexed, sensitive assays that rival PCR laboratory assays and provide high-quality, fast precision diagnostics for pathogens.
For example, a recent work has demonstrated that the multiplexed determination of malaria from whole blood using a paper-based device in rural Uganda.4 The test could sensitively analyze multiplexed nucleic acid sequences of pathogens within 50 min, which gave a higher-quality and faster precision diagnosis for malaria than PCR.
In addition, paper analytical devices are easy to stack, store, and transport because they are thin, lightweight, and of different thicknesses. Visual analysis is made simple due to the strong contrast with a colored substrate. Paper-based devices can also be incinerated after use, reducing the risk of further contamination.
Although wastewater is a complex matrix, paper-based devices have shown the potential to detect pathogens in wastewater. We have developed a fast “sample-to-answer” analysis method that can provide quantitative monitoring of nucleic acids and genetic information through the analysis of sewage,5 which was confirmed with a robust electrophoresis and agarose gel image assay, showing promising reliability for wastewater analysis. Additional paper-based devices have also been fabricated for infectious diseases and pathogens determination as shown in Table 1.
Examples of Paper-Based Devices for Infectious Diseases and Pathogens Determination
|ìinfectious diseases/pathogens||characteristics of paper-based devices||detection method|
|malaria||paper device combined vertical flow sample-processing steps||visual UV/lateral flow device|
|rotavirus A||integrated nucleic acid test on a single paper device, including extraction, amplification, and on-site detection||naked eye|
|Zika virus||wax-printed paper devices utilizing isothermal amplification||smartphone|
|human papillomavirus||paper device in a foldable system allowing for fully integrated operation from sample to result||lateral flow device|
|HIV||paper devices fabricated with cellulose paper and flexible plastic plate||electrochemistry|
|Neisseria meningitides||versatile paper devices integrated with isothermal amplification||visual fluorescence|
|Listeria monocytogenes||loop-mediated isothermal amplification (LAMP)-based paper devices||visual fluorescence|
|Cochlodinium polykrikoides||paper devices based on LAMP||visual fluorescence|
|Staphylococcus aureus||self-priming paper devices||visual fluorescence|
|Vibrio parahemolyticus||self-priming paper devices||visual fluorescence|
|Mycobacterium smegmatis||paper devices combined thermal lysis and isothermal amplification into a single step||visual fluorescence|
|Bacillus subtilis||a wax-printed cellulose paper device||colorimetry|
|Salmonella||paper devices integrated with purification, amplification, and on-site detection||colorimetry|
|Escherichia coli||foldable paper devices with the ability of long-term reagents storage||colorimetry|
|paper devices based on isothermal amplification and on-chip detection||visual fluorescence|
|paper machine integrated sample preparation and isothermal amplification with end point detection||visual UV/camera|
|paper devices integrated extraction, purification, amplification and detection||smartphone/naked eye|
|paper devices combined thermal lysis and isothermal amplification||visual fluorescence|
|bovine infectious reproductive diseases||multiplexed and point-of-care paper-analytical device||visual UV/smartphone|
|highly pathogenic strain of porcine reproductive and respiratory syndrome virus (HP-PRRSV)||paper devices fabricated with filter paper and plastic chip||colorimetry|
In summary, the paper-based device has the potential to be used as a small, portable device to detect SARS-CoV-2 in wastewater on site and to track virus carriers in the community. Such an approach could provide near real-time and continuous data and serve as an early warning sensing system to help local governments and agencies make effective interventions to isolate potential virus carriers and prevent the spread of epidemics.
We believe that in the case of asymptomatic infections in the community or people are not sure whether they are infected or not, rapid and real-time community sewage detection through paper analytical devices can determine whether there are SARS-CoV-2 carriers in the area in a timely manner to enable rapid screening, quarantine, and prevention.
The potentially infected patient will also benefit from paper analytical device tracing SARS-CoV-2 sources with WBE, providing information for the correct and timely treatment of COVID-19.
- Yang Z.; Kasprzyk-Hordern B.; Frost C. G.; Estrela P.; Thomas K. V. Community Sewage Sensors for Monitoring Public Health. Environ. Sci. Technol. 2015, 49 (10), 5845–5846. 10.1021/acs.est.5b01434. [PubMed] [CrossRef] [Google Scholar]
- Holshue M. L.; DeBolt C.; Lindquist S.; Lofy K. H.; Wiesman J.; Bruce H.; Spitters C.; Ericson K.; Wilkerson S.; Tural A.; Diaz G.; Cohn A.; Fox L.; Patel A.; Gerber S. I.; Kim L.; Tong S.; Lu X.; Lindstrom S.; Pallansch M. A.; Weldon W. C.; Biggs H. M.; Uyeki T. M.; Pillai S. K. First Case of 2019 Novel Coronavirus in the United States. N. Engl. J. Med. 2020, 382 (10), 929–936. 10.1056/NEJMoa2001191. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Magro L.; Escadafal C.; Garneret P.; Jacquelin B.; Kwasiborski A.; Manuguerra J. C.; Monti F.; Sakuntabhai A.; Vanhomwegen J.; Lafaye P.; Tabeling P. Paper microfluidics for nucleic acid amplification testing (NAAT) of infectious diseases. Lab Chip 2017, 17 (14), 2347–2371. 10.1039/C7LC00013H. [PubMed] [CrossRef] [Google Scholar]
- Reboud J.; Xu G.; Garrett A.; Adriko M.; Yang Z.; Tukahebwa E. M.; Rowell C.; Cooper J. M. Paper-based microfluidics for DNA diagnostics of malaria in low resource underserved rural communities. Proc. Natl. Acad. Sci. U. S. A. 2019, 116 (11), 4834–4842. 10.1073/pnas.1812296116. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- Yang Z.; Xu G.; Reboud J.; Kasprzyk-Hordern B.; Cooper J. M. Monitoring Genetic Population Biomarkers for Wastewater-Based Epidemiology. Anal. Chem. 2017, 89 (18), 9941–9945. 10.1021/acs.analchem.7b02257. [PubMed] [CrossRef] [Google Scholar]