Why is one FungiCheck test line dark and the other light?

A question frequently asked by professionals who use FungiCheck is why the test line is sometimes very clearly visible and at other times only shows a faint colour. Does a faint line mean that the result is less reliable? Or even that the patient only has a “little” fungus?

The short answer is: no.

In most cases, the intensity of the test line does not indicate the reliability of the result. To understand why, it is important to know how a lateral flow test, such as FungiCheck, works.

How does a lateral flow test work?

FungiCheck detects specific antigens from dermatophytes (fungi that cause nail and skin infections). When these antigens are present in the sample taken, they bind to antibodies on the test strip. This results in a coloured test line.

The amount of antigen that ultimately reaches the test strip largely determines how clearly the line appears.

This means that a dark line simply indicates that a relatively large amount of detectable antigen was present. A lighter line means that less antigen was present, but as long as the line is visible within the specified reading time, the test is positive.

Why might the FunqiCheck test line appear dark or light?

Once you understand what the recovery process actually involves, the whole experience of the treatment changes. Whereas previously only superficial nail material was taken, the sample often contains fewer fungal antigens. As a result, the test line may appear lighter, even though the patient still has an active fungal infection.

Careful sample collection is therefore essential for optimal test performance.

Not every infection contains the same amount of mould

---

There are also significant differences between patients.

Factors that influence the amount of detectable antigen include:

• the stage of the infection;
• the extent of the infection;
• the type of dermatophyte;
• previous antifungal treatment;
• the amount of actively infected nail tissue.

As a result, two patients who have both tested positive in the FungiCheck test may nevertheless show a marked difference in line intensity.

Before the test can detect the antigen, it must first be released from the nail tissue.

That is why the following steps are also important:

• collect sufficient nail material;
• reduce the sample size sufficiently;
• ensure the correct extraction time is observed;
• Mix the sample thoroughly with the extraction solvent.

When less antigen is released, the test line will often show a less intense colour.

Product variation is minimal

It is sometimes thought that differences in line intensity are caused by variations between test strips. However, for an IVDR-certified medical device, this variation is very limited.

During development and production, the following, amongst other things, are checked:

• the amount of antibody applied;
• the quality of the membrane;
• the properties of the detection particles;
• the flow of fluid across the strip.

The European IVDR sets high standards for reproducibility and consistency. Manufacturers must demonstrate that the performance of different production batches remains within very tight tolerances.

---

A slight weight gain is also a good thing

FungiCheck is a qualitative diagnostic test. This means that the test determines whether or not dermatophyte antigens have been detected.

The test is not designed to measure the amount of mould.

Therefore, the following applies:

Any visible test line that appears within the specified reading time must be interpreted as positive, regardless of the line’s intensity.

A faint line therefore does not mean that the result is less reliable or that the infection is less severe. It simply indicates that there were sufficient dermatophyte antigens present to exceed the test’s detection limit.

Conclusion

The intensity of a FungiCheck test line is primarily determined by the amount of antigen present in the sample. Factors such as the quality of the sample collection, the location from which the nail material was taken, the amount of active fungus and the extraction procedure have a much greater influence on this than the test strip itself.

There is therefore a simple rule for interpreting the results:

If the control line is visible and a test line appears within the specified reading time, however faint it may be, the test is positive.

Correct sample collection and strict adherence to the instructions for use remain the most important prerequisites for a reliable diagnostic result.

How is a test line actually formed?

To understand why a test line is sometimes darker or lighter, it helps to know exactly how a lateral flow test such as FungiCheck works.

Although the test looks simple, a highly precise biological process takes place within a few minutes.

Antibodies: the test’s “detectors”

The most important parts of the test are antibodies. These are proteins that can recognise a single, specific target. You could compare them to a key that fits only one lock.

In FungiCheck, these antibodies are designed to recognise specific dermatophyte antigens to recognise. An antigen is a distinctive molecule produced by the fungus. As soon as an antigen is present, it binds to the corresponding antibody.

This high level of specificity makes lateral flow tests ideal for rapid diagnosis.

Detection particles make the result visible

A bond between an antigen and an antibody is not visible to the naked eye. That is why the antibodies are attached to microscopic particles detection particles.

Depending on the type of test, these may, for example, be gold nanoparticles or coloured latex particles. These particles cause a coloured line to appear at the point where a large number of antigen-antibody complexes are retained.

The more complexes that accumulate, the more intensely the line can change colour.

The membrane: the test’s fast track

At the heart of every lateral flow test is a thin nitrocellulose membrane. This membrane contains millions of microscopic channels through which the liquid is propelled automatically by capillary action.

You could compare it to a sponge that soaks up water all by itself, without needing a pump.

Fixed antibodies are located at various points on this membrane, forming the test line and the control line.