How’s it done? Coronastep by step guide

Published on 19/11/2020

LIST’s Coronastep programme detecting COVID-19 traces in wastewater has received a lot of attention recently as it can show the presence of the virus and its concentration around Luxembourg.

However, while the principal of collecting samples from wastewater treatment plants across the country is an understandable principal, how does it actually work? What are the steps that need to be taken before final data can be analysed and the graphs produced?

Below is a step by step guide of how LIST’s Coronastep wastewater analysis programme is carried out.

Step 1 – Wastewater Sampling

The process begins at 13 wastewater treatment plants across the Grand Duchy where wastewater samples are taken by workers at each plant.

However to obtain a sample that is truly representative for an entire day, small samples are taken at 5 minute or 15 minute intervals, depending on the treatment plant, over a 24 hour period and mixed to give a true picture over an entire day. This is important as the amount of wastewater and what is in it, fluctuates throughout that day. For example, nearly everyone goes to the toilet in the morning, or has a shower, so throughout the day there differing levels of concentration.

Step 2 – Wastewater sample collection

A representative from LIST collects the samples, representative of the previous 24 hours from the 13 wastewater treatment plants two or three times a weeks, across the country located in; Schifflange, Pétange, Beggen, Bettembourg, Hespérange, Mersch, Boevange-sur-Attert, Echternach, Uebersyren, Grevenmacher, Bleesbruck, Wiltz and Troisvierges. The samples are taken to LIST laboratory located at the site Belvaux.

Step 3 – Wastewater sample pre-treatment

When the sample arrives in the laboratory, SARS-CoV-2 cannot be immediately detected in it and therefore it has to go through 2 stages before the virus can be identified. This is called the concentration step.

The first stage of the concentration step is a clarification. By centrifugation, the suspended matters encountered in wastewater sample is removed, as they can interfere with subsequent steps in the detection process.

The second stage of the concentration step is a technique known as “ultra-filtration”. The wastewater passes through a membrane and separated from the virus. This is to concentrate the virus present in a wastewater sample that is about 120ml. Simply, this technique of ultrafiltration removes the water and keeps the virus.

This stage reduces the volume, and when the volume is reduced, the virus concentration is artificially increased. The result is that the number of viral particles by volume unit makes the virus detectable. An original sample of 120ml reduces down to just 2 or 3ml giving concentration factor of around 60, ready for the next stage.

Step 4 – SARS-CoV-2 genetic material isolation from wastewater

Once the concentration step is complete, the next step is to isolate and purify the genetic material of the virus with a RNA (Ribonucleic acid) extraction. This is done because RNA inside the virus can be detected by RT-PCR (Reverse Transcriptase - Polymerase Chain Reaction) the method used to detect many viruses including SARS-CoV-2.

Step 5 – SARS-CoV-2 detection by RT-qPCR

Once the genetic material has been extracted, the PCR detection method, i.e. amplification of the viral genome, is applied to the samples. To be more precise, the process is in reality an RT-PCR and not PCR, because SARS-CoV-2 is an RNA virus and not a DNA virus.

Coronaviruses are RNA viruses which means the genetic information is carried by an RNA molecule and not by DNA molecule, a particularity of these viruses. Before the step of amplification can happen, the genetic material of the virus must be “retrotranscribed” from RNA to DNA – so this is called “RT” or Reverse Transcription. Once the DNA strand has been created it can by amplified with a PCR technique.

Step 6 – Raw data analysis

With the physical lab analysis and extraction results complete, it is time to look at the date and begin the calculations phase.

RT-PCR provides a number of RNA copies per reaction. From there, calculations are made to return to the original sample, as only a small fraction of the original sample is analysed. The volumes analysed throughout the process must be taken into account when making calculations; they change constantly during the many stages of analysis. This results in a number of RNA copies per litre of wastewater.

Step 7 – Data normalisation

The second part of data treatment relates to comparing data from one wastewater treatment plant to another. The figures need to be normalised because the wastewater treatment plants are not the same; some are bigger some are smaller, and the number of people connected differ substantially from one to the other. 

Step 8 – Flux calculation

A so-called "flow" calculation is then carried out. For this, the quantity of water that enters the wastewater treatment centres on a daily basis must be taken into account. In fact, only a fraction of this is analysed, but more than 2225,000 cubic metres can arrive per day. At the end, an estimation of the number of SARS-CoV-2 RNA copies by day in each wastewater treatment plants is obtained.

This flow calculation allows us to take into account the quantity of rainwater that arrives at the wastewater treatment plant. This can drastically increase the amount of water in the treatment plant and potentially reduce the virus signal.

The calculation is then made to make it equivalent to 100,000 inhabitants in relation to the size of the centre and number of people connected to it.

Step 9 - Reporting

With all the data accumulated, the information is sent to the government in the form of synthetic report presenting the dynamics of SARS-CoV-2 in wastewater using graphs and published on the LIST website at this link.

From the moment the samples arrive in the lab at LIST, to getting results, the whole process can take between six and eight hours. If you take into consideration the samples that take 24 hours to be sampled and collected, the whole process takes about two days.

The Coronastep programme is now carrying out this analysis three times a week.

 

 

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Dr Henry-Michel CAUCHIE
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