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

The process begins at 13 wastewater treatment plants across the Grand Duchy with the first step of an initial basic filtering of sewage is done and 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 are differing levels of concentration.

Step 2

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

When the sample arrives in the laboratory, COVID-19 cannot be immediately detected in it and therefore it has to go through 2 stages before the virus can be identified. The first stage is a “Particle concentration” step. This is to concentrate the virus present in a wastewater sample that is about 120ml. This technique of filtration removes the water and keeps the virus.

This type of filtration step reduces the volume, and when the volume is reduced, the concentration is artificially increased. The result is that the number of 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

The second concentrations stage is a technique known as “ultra-filtration”. The wastewater passes through a membrane and separated from the virus.

Step 5

Once the concentration stages are complete, the next step is to isolate and purify the genetic material of the virus with an RNA (Ribonucleic acid) extraction. This is done because RNA inside the virus can be detected by PCR (Polymerase chain reaction) the method used to detect many viruses including COVID-19. The virus genome is then isolated from the sample.

Step 6

Once the genetic material has been extracted, the PCR method of detection is carried out. To be more specific the process is called RTPCR a system of genome amplification.

Coronavirus is an RNA virus which means the genetic information on RNA molecules have no DNA, a particularity of this virus. Before the step of amplification and purification can happen, the virus molecules have to be “retrotranscribed” from RNA to DNA – so this is called “RT” or reverse transcription. Once the DNA branch has been created it can by amplified with a PCR technique.

Step 7

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

PCR results produce a number of DNA copies by reaction. From there calculations are done to return to the initial sample as a small fractions of the starting sample will be analysed. The volumes need to be taken into consideration when doing calculations as it changes constantly through the many stages of analysis. Then there are data calculations to transform the number of RNA copies by reaction to the number of RNA copies by litre of wastewater.

Step 8

The second part of data calculations relate to comparing data from one wastewater treatment plant to another. The figures need to be standardised 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 9

A calculation known as “in flux” is then made. The quantity of water that enters the wastewater centres daily is deducted. Only a fraction of it is used in the samples, but for example 225,000 square metres can arrive per day. Therefore, this figure is needed to calculate the flux which is important in calculating the final virus infection results.

Rainwater also has to be considered as this can increase the amount of water at the treatment plants and potentially reduce the virus signal. This is taken into account by estimating the number of RNA copies by day in the wastewater treatment plants.

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 10

With all the data accumulated, the information is sent to the government in the form of graphs, and published on the LIST website at this link.

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

The Coronastep programme is now carrying out these analyses three times a week.

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Dr Henry-Michel CAUCHIE
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