1. Recommendation 10, which was also reflected in the previous directive effective from 28.02.2020 and was also taken verbatim in the new directive: "Direct microscopic examination with or without enrichment, detection from tissues is not suitable for verifying/excluding Lb... (D)", and for some reason the authors considered it important to repeat it under Recommendation 25: "Direct microscopic examination, detection from tissues, is not suitable for verifying/excluding Lb. (D)"

We also support this clause. There is still a scientific debate about the applicability of direct microscopic examinations in the diagnosis of Lyme borreliosis (1) (2). Given the many false results, the developers of DualDur represent the opinion in this debate that in the case of direct blood testing, it is very difficult to avoid the formation of artifacts and superinfection of the sample from the skin surface. Therefore, a professionally trained investigator would be required to find the Borrelia burgdorferi pathogen, which is otherwise present in 1-3 orders of magnitude less density than other Borreliosis infections, separately from artificial products. The manufacturer of DualDur reports on this in detail on its website, with the presentation of scientific publications.

Direct examination, i.e. unprepared, usually from a drop of blood, is worrying for several reasons: spirochetes on the surface of the skin can superinfect the sample. Furthermore, the so-called pseudo-spirochetes, which are formed from the blood cells, are deceptively similar to a Borrelia, but their movement is rather passive, so in theory a careful observer could distinguish them (3) (4). However, the density of Borrelia burgdorferi in the blood usually does not exceed the limit of 10-100 thousand pathogens / ml (5) (6) (7), which would mean that in the usually 2 microliters of blood examined, we would often see one pathogen in every 50 or more fields of view under the microscope, if it is not covered by the cellular elements of the blood. That is why cultivation is used to increase the number of Borrelia, for which "enriching mediums" are used. ("medium = a mixture, a mixture that satisfies the nutrient needs of bacteria and provides favourable conditions for their reproduction") (8).

Borrelia can also be cultured (enriched) from blood plasma, which has been published in several publications by Éva Sápi (9) and Eva Ruzic (10). In these studies, Borrelia could be detected microscopically and specifically identified by PCR or other testing. Although the publications show reliable results, this methodology cannot be used in routine diagnostics due to its time-consuming and costly nature. (So it is not true that the method is unsuitable, rather impractical).

The DualDur method chooses a third way: a larger amount of venous blood, 4 ml, is taken into a "cell technology medium" (see patent number 231425: "The object of the invention is a procedure for the direct detection of tick-borne infections. The procedure is characterized by the fact that the human/animal body fluid sample, preferably a blood sample, is taken onto a specific amount of cell technology medium providing a semi-hostile environment,...") (11) and then its pathogen content is concentrated (12). DualDur is therefore not an enrichment procedure, it is not a culture medium, and it is not a "direct microscopic" examination, as the blood is not examined directly, but after preparation.

In summary, DualDur is not a direct microscopic or enrichment method, although it is a category D qualification ("In the absence of reliable scientific evidence or international consensus, or if these are not applicable to the own population or domestic environment, the determination of the domestic "best practice" is based on the experience of the members of the policy development team or professional feedback obtained through consultation. Research results may modify this.") it is not suitable for discrediting direct microscopic procedures, especially with regard to the numerous publications on the success of culture (enrichment) procedures – a research result that does indeed modify the category D "opinion".

2. As a new element in the 2024 recommendation, the reference to a new method appears: "The same applies to the microscopic test, which is called a world-famous Hungarian discovery. After adding a so-called cellular technology medium and centrifugation, it examines blood samples with a dark-field microscope and finds formulas that are considered to be borrelia. Until the scientific evidence of the procedure is published in a prestigious international journal, it cannot be used in routine diagnostics. (44)."

The quoted excerpt refers, below all scientific categories, to a Youtube video that is no longer available, which does not contain any scientific references, and cannot be taken as an appropriate reference due to its scientific errors of magnitude. Nevertheless, in terms of the claim, it is recognizable as a reference to non-scientific press coverage of the DualDur process. Given that this recommendation has been listed as recommendation 11, the authors also point out that DualDur does not fall under either recommendation 10 or recommendation 25. On the other hand, such a procedure can be used in routine diagnostics provided that "its scientific evidence is published in a prestigious international journal". DualDur's early publications include two prestigious journals (SJR-listed, impact factor journals) (12) (6), so the DualDur procedure can be used in routine diagnostics without any further ado. „tudományos bizonyítékai rangos nemzetközi folyóiratban publikálásra kerülnek”. A DualDur korai publikációi között két rangos folyóirat is szerepel (SJR szerint jegyzett, impakt faktoros folyóiratok) (12) (6), így a DualDur eljárás minden további nélkül alkalmazható a rutindiagnosztikában.

We note that many of the procedures considered to be followed in the recommendation have not been published in prestigious journals, and articles have been published in several prestigious journals about the poor performance of the methods, e.g. serological tests, two-step serological procedure.

However, the use of a procedure in laboratory diagnostics is not ensured by a professional recommendation, but by the so-called IVD CE registration procedure, which requires a performance assessment test (e.g. clinical research), which many Lyme diagnostic tests do not have, and which does not oblige the manufacturer to publish the results of the research, but only to send the results in the research report to the Authority and the Ethics Committee. In the case of DualDur, the research report was formally adopted by the authority and unanimously adopted by the ethics committee. And the IVD CE registration was made on the basis of this report.

Finally, the Hungarian recommendation did not implement the European Parliament's resolution, published in 2018 and maintained several times since then, that "the medical profession often follows outdated recommendations for Lyme disease that do not sufficiently take into account research results" and "called for further funding of methods for diagnosing and treating Lyme disease" and "also calls on the Member States to extend the use of clinical symptom-based diagnosis so that doctors can diagnose Lyme disease even if serological tests are negative', and notes that 'Lyme disease, despite being a disease known to medical science, is still often unrecognised, in particular because of the difficulty of detecting symptoms and the lack of adequate diagnostic tests' and mentions, inter alia, DualDur, which was then in the research phase (13).