How is the testing of monkeypox samples carried out in the Health Board’s Communicable Diseases Laboratory?

A person goes to their family physician complaining of itchy bumps. The doctor looks at it and says it's like chicken pox, but maybe it's monkeypox. What next? Who will take the sample, and how will it get to the Health Board’s Communicable Diseases Laboratory?

The WHO has provided guidelines to follow when taking a sample. The symptoms of monkeypox are similar to those of smallpox, but milder: they include a fever, severe headache, muscle aches, backache, a swelling of the lymph nodes, and fatigue as the initial symptoms. The characteristic skin rash occurs between 1-3 days after the onset of fever. Initial experience shows that family physicians tend to confuse the signs of chickenpox with those of monkeypox. In general, adults suffer more severely from chickenpox than children, and cases so far have mainly been chickenpox. In collaboration with the Communicable Diseases Department, instructions were prepared in good time, to which we added instructions from the laboratory both for taking samples and for sending them to the laboratory. In cases of suspected infection, patient sampling is carried out either by the family nurse or by a hospital-based sampler. A very specific sampling device is used, which is described in detail in the instructions and is accompanied by a photograph.

So a sample is taken at your family physician or hospital and is sent to the Health Board's laboratory. What does the laboratory do with this sample?

This is in fact a common second biohazard class assay which extracts nucleic acid, or DNA. And then a PCR reaction mixture is mixed together (PCR being a polymerase chain reaction, ie. the synthesis of copies of the viral DNA in large replicates), and then a real-time PCR (RT-PCR) is carried out. The entire procedure takes a total of three hours before a response can be issued.

What is included in the RT-PCR reagent kit which is ordered by the laboratory and how many samples can be tested with one kit?

It depends on the kit. If there are commercial kits available, they usually have all this information contained within them. We have a test kit which can handle fifty test analyses. It includes a buffer for the PCR reaction, as well as primers which allow a specific target region of viral DNA to be copied during PCR, and an enzyme which carries out the synthesis reaction. The reagent kit also includes fluorescent molecules, which allow the RT-PCR reaction to be monitored in real time to see whether or not a positive signal is forming. For example, if the DNA of the virus which causes monkeypox is present in the sample, the signal will fluoresce and be visible, and if it is not, there is no signal.

In the laboratory, we prefer to use a ready-to-use reagent kit, which eliminates the need for labour-intensive and resource-intensive reaction optimisation and subsequent validation to implement the method there.

Are all the necessary liquids already inside?

They certainly are. And the test also comes with precise method instructions, so that we simply open the box and mix. It also includes all the necessary checks. For example, it includes an additional control which amplifies a small region of the human genome. If this human gene signal is positive, we know that the reaction most certainly contains material from a human sample.

So if somebody sends a sample containing apple juice, for example, no human genome will be detected?

Yes, there will be no positive response.

Now that you've carried out all your procedures in terms of the monkeypox test, will the answer appear on the computer screen?

Yes. The equipment software provides visualised results. The same is true for coronavirus, so it's pretty similar. But while coronavirus is an RNA-based virus, the monkeypox virus is a DNA-based virus.

Is the laboratory organised in such a way, if a sample arrives with a suspected case of monkeypox, so that it will ensure that there will not be a long queue and that we can tell within three hours whether the sample is positive or not?

Yes. Everything you need has already been organised. The Communicable Diseases Department has sent instructions to Estonian Family Physicians Association along with a referral form. All of the necessary information has also been published on the Health Board’s website. Precisely the same information has been provided to hospitals.

The answers were provided by Janne Pullat, head of the Infectious Diseases Laboratory at the National Health Board, and Irina Eero, senior laboratory specialist.