# The real-time PCR targeting the phage terminase (terL) is not suitable for diagnostics of human Borrelia infections in Europe

Manja Zimmermann, Gabriele Margos, Christine Hartberger, Reto Lienhard, Anna J Henningsson, Malin Lager, Mateusz Markowicz, Anna-Margarita Schötta, Andreas Sing, Benoit Jaulhac, et al.

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Manja Zimmermann, Gabriele Margos, Christine Hartberger, Reto Lienhard, Anna J Henningsson, et al. The real-time PCR targeting the phage terminase (terL) is not suitable for diagnostics of human Borrelia infections in Europe. Ticks and Tick-borne Diseases, 2025, 16 (4), pp.102488. 10.1016/j.ttbdis.2025.102488. hal-05468010

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Original article

# The real-time PCR targeting the phage terminase (terL) is not suitable for diagnostics of human *Borrelia* infections in Europe

<figure><img src="image_3.png" /></figure>

Manja Zimmermann<sup>a</sup>, Gabriele Margos<sup>a,1,*<sup> ID </sup></sup>, Christine Hartberger<sup>a</sup>, Reto Lienhard<sup>b,1</sup>,
Anna J. Henningsson<sup>c,1<sup> ID </sup></sup>, Malin Lager<sup>d</sup>, Mateusz Markowicz<sup>e,1<sup> ID </sup></sup>, Anna-Margarita Schötta<sup>e<sup> ID </sup></sup>,
Andreas Sing<sup>a</sup>, Benoit Jaulhac<sup>f,1</sup>, Per-Eric Lindgren<sup>c,1</sup>, Alje P. van Dam<sup>g,1</sup>,
Joppe W.R. Hovius<sup>g,1</sup>, Volker Fingerle<sup>a,1,*<sup> ID </sup></sup>

## ARTICLE INFO

**Keywords:**

Real-time PCR

Phage terminase

terL

Lyme borreliosis

Lyme disease

Diagnosis

Borrelia burgdorferi sensu lato

## ABSTRACT

Bacteria of the *Borrelia burgdorferi* sensu lato (sl) species complex can cause Lyme borreliosis (LB) in humans. PCR plays an important role in the diagnosis of many infectious diseases but it is used auxiliary in LB diagnostics. Here, we re-analysed a previously published real-time PCR targeting the multicopy gene of the large subunit of phage terminase (terL) in *Borrelia*. We analysed cultured material of *Borrelia burgdorferi* sl species, serum and clinical tissue samples of LB patients. PCR conditions were as previously described by Shan et al. 2021 but we also investigated PCR modifications.

PCR on cultured specimens showed that whilst all samples of *B. burgdorferi* sensu stricto (ss) gave a positive result, not all isolates of *Borrelia* species causing LB in Europe (i.e. *B. afzelii*, *B. garinii*) were detected by the *terL* PCR. Only slight differences in Ct values were detected between PCR runs using the original ZEN/IFBQ double quencher probe compared to other double quencher probes or single quencher probes. Contrary to the hypothesis expressed by the authors of the original paper that the PCR could detect phage DNA in serum, our data show that the *terL* PCR was negative on all tested serum samples of individuals diagnosed with proven LB. Furthermore, using patient's tissue samples not all infections with *B. afzelii* or *B. garinii* were detected, similar to the results obtained with cultured material or serial DNA dilutions of *Borrelia* species. We conclude, that the *terL* PCR in its current form is unsuitable for LB diagnosis in Europe.

## 1. Introduction

PCR plays an important role in the diagnosis of many infectious diseases and it is used supportively in the diagnosis of Lyme borreliosis

(LB), also known as Lyme disease. LB in humans is caused by bacteria of the *Borrelia burgdorferi* sensu lato (sl) species complex. PCR allows detection of DNA of these bacteria in patient samples and is useful to support or confirm other testing methods, such as antibody detection in

<footnote><sup>a</sup> Bavarian Health and Food Safety Authority, German National Reference Center for Borrelia, Veterinärstraße 2, 85764 Oberschleissheim, Germany</footnote>

<footnote><sup>b</sup> Admed Microbiology, CNRT (Swiss Reference Center for Tick transmitted diseases) for Lyme Borreliosis Diagnostic, La Chaux-de-Fonds, Switzerland</footnote>

<footnote><sup>c</sup> Clinical Microbiology, Region Jönköping County, and Division of Inflammation and Infection, Department of Biomedical and Clinical Sciences, Linköping University, Sweden</footnote>

<footnote><sup>d</sup> National Reference Laboratory for Borrelia and Other Tick-Borne Bacteria, Division of Clinical Microbiology, Laboratory Medicine, Region Jönköping County, Sweden</footnote>

<footnote><sup>e</sup> Institute for Medical Microbiology and Hygiene, Department of Vector-Borne Diseases, Austrian Agency for Health and Food Safety (AGES), Vienna, Austria</footnote>

<footnote><sup>f</sup> French National Reference Center for Borrelia, Hôpitaux Universitaires de Strasbourg, Institut de Bactériologie, Fédération de Médecine Translationnelle de Strasbourg University of Strasbourg, UR3073- PHAVI, France</footnote>

<footnote><sup>g</sup> Amsterdam institute for Immunology & Infectious diseases, Department of Internal Medicine, Section of Infectious Diseases & Department of Clinical Microbiology, Amsterdam UMC Multidisciplinary Center for Lyme-borreliosis (AMLC), Amsterdam UMC, Amsterdam, the Netherlands</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: manja.zimmermann@lgl.bayern.de (M. Zimmermann), gabriele.margos@lgl.bayern.de (G. Margos), christine.hartberger@lgl.bayern.de (C. Hartberger), reto.lienhard@ne.ch (R. Lienhard), anna.jonsson.henningsson@rjl.se (A.J. Henningsson), malin.lager@rjl.se (M. Lager), mateusz.markowicz@ages.at (M. Markowicz), anna-margarita.schoetta@ages.at (A.-M. Schötta), andreas.sing@lgl.bayern.de (A. Sing), jaulhac@unistra.fr (B. Jaulhac), per-eric.lindgren@liu.se (P.-E. Lindgren), lyme@amsterdamumc.nl (A.P. van Dam), lyme@amsterdamumc.nl (J.W.R. Hovius), volker.fingerle@lgl.bayern.de (V. Fingerle).</footnote>

<footnote n="1">¹ Members of the ESCMID Study Group for Tick-borne Diseases (ESGBOR).</footnote>
blood, or in early disease before antibodies are detectable (Hofmann et al., 2017; Rauer et al., 2020). By detecting *Borrelia* DNA in tissue samples or body fluids, PCR can help to achieve an early diagnosis and treatment of LB. However, as there may be very few *Borrelia* bacteria in infected individuals and, hence, little DNA in tissue samples, a highly sensitive PCR is needed to provide reliable results.

In 2021, Shan and co-authors (Shan et al., 2021) published a paper suggesting that a multicopy prophage gene, the large terminase subunit (*terL*), would improve diagnostics of LB when used as target in real-time PCR. This PCR is being promoted for diagnostics of LB in humans (R.E.D. Laboratories Belgium, B-1731 Zellik, Belgium; https://www.redlabs.com/p/home.html, access date 07 April 2025). Targeting multicopy genes for diagnostic purposes can be advantageous as it may increase the sensitivity of PCR and, therewith, improve diagnosis. *In silico* analyses suggested that the PCR may be suitable for all *Borrelia* species (Shan et al., 2021). However, in a letter to the editor the reproducibility of the statistical analyses and the specificity of the PCR were questioned (van de Schoor et al., 2021). In a follow up paper (Shan et al., 2023), the usefulness of the approach was highlighted and expanded for detection of *Borrelia* in ticks.

In Europe, the most common *Borrelia* species that cause human LB are *B. afzelii* and *B. garinii* (Hofmann et al., 2017; Rauer et al., 2020), which are also the most prevalent *Borrelia* species found in ticks (Rauter and Hartung, 2005; Strnad et al., 2017). Therefore, a new PCR should detect those species. In this study, we have used the real-time PCR published by Shan et al. (2021) in its original form and in a modified version to re-analyse clinical samples that were diagnosed as *Borrelia* positive in several laboratories using clinical assessment of the patients as well as p41 (Flagellin B (*flaB*)) (Schwaiger et al., 2001; Venczel et al., 2015) and outer surface protein A (*ospA*) (Ivacic et al., 2007; Briciu et al., 2016) as PCR targets. We also used cultured isolates of the *Borrelia* species commonly involved in clinical LB in Europe, i.e. *B. afzelii*, *B. bavariensis*, *B. burgdorferi* sensu stricto (ss), *B. garinii*, and *B. spielmanii* to analyse whether the PCR published by Shan et al. 2021 was suitable for detection of defined amounts of *Borrelia* DNA and to determine its detection limit. Published protocols for PCR on 16S rRNA (Gyllemark et al., 2021), *flaB* (Schwaiger et al., 2001), 5S-23S intergenic spacer (IGS) ((Strube et al., 2010), and unpublished) were used in parallel. Analyses were performed independently by three different laboratories.

## 2. Materials and methods

*Samples included.* DNA purified from in vitro cultures (n = 32) was included in the analyses. These included *B. afzelii* n = 10, *B. burgdorferi* ss (Bbss) n = 8, *B. garinii* n = 6, *B. spielmanii* n = 2, *B. valaisiana* n = 1, *B. Miyamotoi* n = 1, *B. recurrentis* n = 2, *B. hispanica* n = 1, *B. hermsii* n = 1 (Table 1). We further analysed DNA of samples included in a panel that contained serial dilutions of DNA from nine *Borrelia* species (15 isolates) and single concentrations of specificity controls (four species, five isolates) (supplementary material Table S1). In Table 2 a subset of these samples is shown comparing the original *terL* PCR set up (master mix, double quencher probe) with a real-time PCR targeting a different locus (see Table heading for details). DNA dilutions ranged from 1 × 10⁴ to 1 × 10⁻¹ per 5 µl. Pre-treatment serum samples (Table 3a) from 15 patients with proven LB diagnosed by clinical assessment in combination with positive *Borrelia*-specific antibody titres or positive in vitro *Borrelia* culture from tissue biopsy were included. Furthermore, clinical samples (n = 26; Table 3b) of LB patients were included that had previously been diagnosed based on clinical symptoms, patient history and positive PCR using p41 (*flaB*) (Schwaiger et al., 2001; Venczel et al., 2015) and *ospA* (Ivacic et al., 2007; Briciu et al., 2016) as targets.

*DNA purification.* DNA was purified via a Maxwell® 16 using a Maxwell LEV Blood DNA kit (Promega, Germany) as previously described (Margos et al., 2017). 200 µl of serum or 200 µl of tissue of clinical samples was used for purification. DNA of *Borrelia* cultures was purified from approximately 1 – 1.5 ml of cultures. For comparative

purposes, in some experiments DNA was diluted to 1–2 ng/µl and 5 µl were used as template in PCR reactions; DNA concentrations of template DNA solutions are indicated in Table 1.

*PCR and modifications.* Primers and probe for the large subunit of phage terminase (*terL*) were used as previously described by Shan et al. (Shan et al., 2021) and under the same PCR conditions (Annealing temp. 60 °C). The probe (5'FAM/ZEN/3'IBFQ) and master mix (PrimeTime™ One-Step 4x Broad Range Master Mix) as used by Shan et al. 2021 were purchased at IDT, Belgium. In laboratory 1, the Thermocycler BioRad CFX Maestro (BioRad, Germany) was used. In this laboratory, the 2x polymerase master mix Qiagen QuantiTectProbe PCR Kit (200) (cat. No. 204343, Qiagen, Germany) is normally used and was also used for *terL* PCR. As a modification, in laboratory 1 we used a single quencher probe (5'FAM/ 3'BHQ1) and compared the sensitivity of detection to a double quencher probe (5'FAM/abNFQ/3'NFQ). For all PCRs 5 µl template DNA was used in a final reaction volume of 20 µl for all PCRs. The choice of quencher used in the labs was due to availability in the companies where primers and probes were purchased. To evaluate the quality of DNA in samples, control PCRs targeting the 23S rRNA locus (Strube et al., 2010), the *ospA* (Ivacic et al., 2007; Briciu et al., 2016) or the *flaB* gene (Schwaiger et al., 2001; Venczel et al., 2015) were used. In laboratory 2, PCRs were run on a LightCycler 480 (Roche, Switzerland). A single quencher probe (5'FAM/ 3'BHQ1) was employed in combination with the 2x TaqMan Fast Advanced Master mix (Applied Biosystems, 4444556). Ct values were determined using a fit point analysis. The total reaction volume was 20 µl including 4 µl of template DNA. A PCR targeting the 5S-23S rRNA IGS (unpublished) was included as control PCR. In laboratory 3, PCRs were run on a LightCycler 480 (Roche, Switzerland). The master mix Maxima qPCR Probe mix (Thermo Fischer Scientific, Waltham, MA) was used in combination with the double quencher probe 5'FAM/ZEN/IBFQ-3' (Shan et al., 2021) in *terL* PCR. A PCR targeting the 16S rRNA locus (Gyllemark et al., 2021) was used as control for PCR performance with a single quencher probe (5'FAM/3'MGB). For all PCRs in laboratory 3, 5 µl of template DNA was used in a total volume of 20 µl.

## 3. Results

### 3.1. *terL* PCR does not work equally well on all *Borrelia* species

Using cultured material, we found that all samples belonging to the species *B. burgdorferi* ss had positive Ct values using the real-time PCR approach described by Shan et al. (Shan et al., 2021) regardless of master mix, double quencher, or single quencher probes used (Table 1). However, not all *B. afzelii* or *B. garinii* DNA samples from cultures had a positive result in the *terL* real-time PCR. For example, out of 10 *B. afzelii* isolates, only six (60 %) turned out to be positive. Similarly, out of six included *B. garinii* isolates only two (33 %) were positive in *terL* PCR (Table 1). We would like to draw particular attention to the fact that the failure of the *terL* PCR to provide positive results in these samples was not due to low concentrations or low quality of DNA present in the samples. The presence and quality of DNA in these samples were proven by a control PCR targeting the *flaB* gene. For *B. afzelii*, Ct values of the control PCR were in the range of 16.86 to 19.31 when *terL* PCR was either negative or Ct values lay between 35 and 40 (Table 1).

It has been suggested that using a double quencher in real-time PCR probes improves the sensitivity (see https://eu.idtdna.com/pages/pr oducts/qpcr-and-pcr/gene-expression/primetime-qpcr-probes) (Hirotsu et al., 2020). We noted only slight differences in sensitivity using the original ZEN/IFBQ double quencher, double quencher (abNFQ/NFQ) cp32 probes or single quencher (BHQ1) cp32 probes and only in some samples. Direct comparison of *terL* PCR employing a single quencher probe with a double quencher probe on identical template DNA material showed that in some cases using a double quencher probe slight improvements of Ct values were noted (Table 1) (e.g. B31 single quencher Ct value 12.33 – double quencher Ct value 10.53; PBre single
<caption>**Table 1**</caption>

Comparison of cycle threshold (Ct) values using a *terL* probe with single (BHQ1) or double quencher (abNFQ/NFQ, NRZ Borrelia or ZEN/IBFQ, Shan et al., 2021) on cultured material of *Borrelia* species. Comparison of master mixes (MM) used *flagellin B* (*flaB*) PCR. DNA was diluted to 1–2 ng/microl.

<table><thead><tr><th>Species</th><th>Isolate [passage/ tube]</th><th>Ct values (±STD) using terL probe single quencher (BHQ1)</th><th>Ct values using terL probe double quencher (abNFQ/ NFQ)</th><th>Ct values using terL probe double quencher (ZEN/IBFQ)</th><th>Ct values (±STD) flaB (p41) QantiTect MM |* PrimeTime OneStep MM</th><th>DNA conc ng/µl</th></tr></thead><tbody><tr><td>Bbss<sup>a</sup></td><td>B31 [44/1]</td><td>nd</td><td>16.53*</td><td>14.84*</td><td>18.42</td><td>*19.87 0.73</td></tr><tr><td>Bbss</td><td>PBre [61/7]</td><td>nd</td><td>16.36*</td><td>13.86*</td><td>17.07</td><td>*18.27 0.95</td></tr><tr><td>Bbss</td><td>PAbe [33/3]</td><td>nd</td><td>15.64*</td><td>13.55*</td><td>16.38</td><td>*18.42 1.31</td></tr><tr><td>Bbss</td><td>PDri [18/3]</td><td>nd</td><td>17.33*</td><td>16.42*</td><td>18.51</td><td>*18.78 0.338</td></tr><tr><td>Bbss</td><td>PBoe [0]</td><td>nd</td><td>21.53*</td><td>19.13*</td><td>20.41</td><td>*20.04 0.142</td></tr><tr><td>Bbss</td><td>PFra [9/2]</td><td>nd</td><td>20.51*</td><td>18.3*</td><td>22.29</td><td>*23.3 0.134</td></tr><tr><td>Bbss</td><td>PLue [12/74]</td><td>nd</td><td>17.14*</td><td>15.39*</td><td>17.11</td><td>*19.21 1.25</td></tr><tr><td>Bbss</td><td>PFhe II [nd]</td><td>nd</td><td>15.98*</td><td>13.58*</td><td>16.91</td><td>*18.76 1.29</td></tr><tr><td>Bbss</td><td>B31 [44/1]</td><td>12.33</td><td>10.53</td><td>Nd</td><td>17.32</td><td>*nd 0.844</td></tr><tr><td>Bbss</td><td>PBre [61/7]</td><td>9.04</td><td>6.98</td><td>Nd</td><td>18.03</td><td>*nd 1.30</td></tr><tr><td>Bbss</td><td>PAbe [33/3]</td><td>15.24</td><td>14.69</td><td>Nd</td><td>22.96</td><td>*nd 0.486</td></tr><tr><td>Bbss</td><td>PDri [18/3]</td><td>15.91</td><td>15.43</td><td>Nd</td><td>20.4</td><td>*nd 1.45</td></tr><tr><td>Bbss</td><td>PBoe [0]</td><td>22.14</td><td>23.24</td><td>Nd</td><td>25.29</td><td>*nd nd</td></tr><tr><td>Bbss</td><td>PFra [9/2]</td><td>17.61</td><td>19.23</td><td>Nd</td><td>21.60</td><td>*nd 0.20</td></tr><tr><td>Bbss</td><td>PLue [12/74]</td><td>15.7</td><td>15.73</td><td>Nd</td><td>17.23</td><td>*nd 1.36</td></tr><tr><td>Bbss</td><td>PFhe II [nd]</td><td>18.89</td><td>19.80</td><td>Nd</td><td>22.51</td><td>*nd 0.514</td></tr><tr><td>B. afzelii</td><td>PAlt [5/7]</td><td>40.07 (0.29)</td><td>40.84</td><td>Nd</td><td>17.34 (0.05)</td><td>*nd 1.56</td></tr><tr><td>B. afzelii</td><td>PBec [3/3]</td><td>no Ct</td><td>no Ct</td><td>Nd</td><td>17.26 (0.09)</td><td>*nd 2</td></tr><tr><td>B. afzelii</td><td>PFes [20/4]</td><td>no Ct</td><td>no Ct</td><td>Nd</td><td>17.71 (0.03)</td><td>*nd 1.35</td></tr><tr><td>B. afzelii</td><td>PObf [7/2]</td><td>38.36 (0.25)</td><td>37.96</td><td>Nd</td><td>17.26 (0.01)</td><td>*nd 1.96</td></tr><tr><td>B. afzelii</td><td>PMes [10/4]</td><td>no Ct</td><td>no Ct</td><td>Nd</td><td>16.86 (0.11)</td><td>*nd 2.32</td></tr><tr><td>B. afzelii</td><td>PGau [104/19]</td><td>38.38 (0.15)</td><td>35.61</td><td>Nd</td><td>19.31 (0.31)</td><td>*nd 0.43</td></tr><tr><td>B. afzelii</td><td>PHam [15/2]</td><td>35.76 (0.23)</td><td>36.44</td><td>Nd</td><td>18.2 (0.1)</td><td>*nd 0.87</td></tr><tr><td>B. afzelii</td><td>PKo [177/4]</td><td>38.85 (0.38)</td><td>nd</td><td>Nd</td><td>18.41 (0.06)</td><td>*nd 0.82</td></tr><tr><td>B. afzelii</td><td>PKr [15/4]</td><td>36.18 (0.18)</td><td>35.18</td><td>Nd</td><td>18.53 (0.55)</td><td>*nd 0.85</td></tr><tr><td>B. afzelii</td><td>PVPM [4/2]</td><td>no Ct</td><td>no Ct</td><td>Nd</td><td>18.54 (0.09)</td><td>*nd 0.79</td></tr><tr><td>B. garinii 3#</td><td>PBr [30/3]</td><td>no Ct</td><td>no Ct</td><td>Nd</td><td>17.84 (0.14)</td><td>*nd 1.06</td></tr><tr><td>B. garinii 5</td><td>PHei [100/9]</td><td>36.61 (0.25)</td><td>nd</td><td>Nd</td><td>17.85 (0.13)</td><td>*nd 1.71</td></tr><tr><td>B. garinii 6</td><td>PNov [23/3]</td><td>no Ct</td><td>no Ct</td><td>Nd</td><td>17.36 (0.13)</td><td>*nd 1.85</td></tr><tr><td>B. garinii 6</td><td>PWudII [17/2]</td><td>no Ct</td><td>no Ct</td><td>Nd</td><td>18.32 (0.14)</td><td>*nd 1.09</td></tr><tr><td>B. garinii 7</td><td>PRef [2/2]</td><td>no Ct</td><td>no Ct</td><td>Nd</td><td>17.85 (0.1)</td><td>*nd 1.28</td></tr><tr><td>B. garinii 8</td><td>PKi [5/5]</td><td>25.17 (0.1)</td><td>24.93</td><td>Nd</td><td>17.01 (0.12)</td><td>*nd 1.87</td></tr><tr><td>B. spielmanii</td><td>PMai [30/3]</td><td>38.78 (0.44)</td><td>no Ct</td><td>Nd</td><td>26.54 (0.83)</td><td>*nd 0.87</td></tr><tr><td>B. spielmanii</td><td>PMew [33/3]</td><td>38.67 (0.12)</td><td>37.94</td><td>Nd</td><td>22.89 (0.12)</td><td>*nd 2.88</td></tr><tr><td>B. valaisiana</td><td>VS116</td><td>37.29</td><td>no Ct</td><td>Nd</td><td>18.14 (0.15)</td><td>*nd 0.8</td></tr><tr><td>RF species**</td><td>B. miyamotoi</td><td>nd</td><td>39.27</td><td>Nd</td><td>24.33 (0.10)</td><td>*nd 1.26</td></tr><tr><td>B. hispanica</td><td>VM531519</td><td>nd</td><td>no Ct</td><td>Nd</td><td>no Ct</td><td>*nd 0.722</td></tr><tr><td>B. hermsii</td><td>HS1 [27/2]</td><td>nd</td><td>35.76</td><td>Nd</td><td>20.62 (0.10)</td><td>*nd nd</td></tr><tr><td>B. recurrentis</td><td>Pat 614</td><td>nd</td><td>no Ct</td><td>Nd</td><td>33.73 (0.10)</td><td>*nd 1.23</td></tr><tr><td>B. recurrentis</td><td>Pat 619</td><td>nd</td><td>no Ct</td><td>Nd</td><td>35.95 (0.10)</td><td>*nd 1.02</td></tr><tr><td>Bbss</td><td>B31 1:5 [control]</td><td>nd</td><td>34.25/34.36</td><td>Nd</td><td>nd</td><td>*nd</td></tr><tr><td>negative control</td><td>Water</td><td>nd</td><td>no Ct</td><td>Nd</td><td>nd</td><td>*nd 0</td></tr></tbody></table>

a = Bbss = *Borrelia burgdorferi* sensu stricto. Please note that the first 16 samples are duplicates of eight isolates from different DNA purifications.

flaB = flagellin B.

Ct = cycle threshold.

*PCR was run with PrimeTime One Step borad range master mix (MM), IDT, Belgium.
# number refers to ospA type.

RF species ** = relapsing fever species.

nd = no data.

<caption>**Table 2**</caption>

Subset of NRZ DNA Panel from cultured isolates, serial dilutions of DNA, genome equivalents (GE) as indicated, PCR runs made with PrimeTime One Step master mix (MM), primers and probe as described by *Shan et al. (2021)* and controls.

<table><thead><tr><th>Species</th><th>Isolate [passage/tube]</th><th>GE</th><th>Ct values terL PCR, probe (5'FAM/ZEN/ 3'IBFQ), PrimeTime MM</th><th>Ct values control PCR (Strube et al. 2010), PrimeTime MM</th><th>Ct values control PCR (Strube et al. 2010), QuantiTect MM</th><th>No of cp32</th></tr></thead><tbody><tr><td rowspan="7">B. afzelii</td><td rowspan="7">PKo [118/7 + 120/7]</td><td>10<sup>4</sup></td><td>40.8</td><td>23.16</td><td>22.7 (0.1)</td><td rowspan="7">8</td></tr><tr><td>10<sup>3</sup></td><td>no Ct</td><td>26.61</td><td>25.96 (0.07)</td></tr><tr><td>10<sup>2</sup></td><td>no Ct</td><td>29.84</td><td>29.26 (0.12)</td></tr><tr><td>10<sup>1</sup></td><td>no Ct</td><td>33.97</td><td>32.83 (0.19)</td></tr><tr><td>10<sup>0</sup></td><td>no Ct</td><td>35.36</td><td>36.04 (0.44)</td></tr><tr><td>10<sup>-1</sup></td><td>no Ct</td><td>no ct</td><td>no Ct</td></tr><tr><td>10<sup>4</sup></td><td>40.77</td><td>23.62</td><td>22.8 (0.15)</td></tr><tr><td rowspan="7">B. afzelii</td><td rowspan="7">PVPM [34/5]</td><td>10<sup>3</sup></td><td>no Ct</td><td>26.98</td><td>25.91 (0.32)</td><td rowspan="7">8</td></tr><tr><td>10<sup>2</sup></td><td>no Ct</td><td>30.1</td><td>29.38 (0.1)</td></tr><tr><td>10<sup>1</sup></td><td>no Ct</td><td>33.76</td><td>33.02 (0.35)</td></tr><tr><td>10<sup>0</sup></td><td>no Ct</td><td>35.5</td><td>36.48 (0.09)</td></tr><tr><td>10<sup>-1</sup></td><td>no Ct</td><td>no ct</td><td>no Ct</td></tr><tr><td>10<sup>4</sup></td><td>no Ct</td><td>24.13</td><td>23.35 (0.13)</td></tr><tr><td>10<sup>3</sup></td><td>no Ct</td><td>27.72</td><td>27.33 (0.06)</td></tr><tr><td rowspan="7">B. garinii (3)#</td><td rowspan="7">PBr [51/4]</td><td>10<sup>2</sup></td><td>no Ct</td><td>30.79</td><td>30.5 (0.05)</td><td rowspan="7">2</td></tr><tr><td>10<sup>1</sup></td><td>no Ct</td><td>33.69</td><td>33.89 (0.25)</td></tr><tr><td>10<sup>0</sup></td><td>no Ct</td><td>35.82</td><td>37.33 (0.92)</td></tr><tr><td>10<sup>-1</sup></td><td>no Ct</td><td>no ct</td><td>no Ct</td></tr><tr><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

Ct = cycle threshold.

no Ct = no signal in real time PCR; negative.

# = number refers to ospA type.

quencher Ct 9.04 – double quencher Ct 6.98). However, this was not the case in all tested samples (e.g. PBoe single quencher Ct 22.14 – double quencher Ct 23.24; PFra single quencher Ct 17.61 – double quencher 19.23). Similarly, using the PrimeTime master mix in combination with the ZEN/IFBQ double quencher probe slightly improved Ct values in *B. burgdorferi* ss samples (see also Table 1). Importantly, in cases when the single quencher PCR was negative, also the PCR using a double quencher probe turned out to be negative.

Using cultured *Borrelia* isolates, a DNA Panel was generated by the German National Reference Center for Borrelia (NRZ *Borrelia*), Oberschleissheim, Germany. The DNA panel encompasses DNA dilutions that correspond to NRZ genome equivalents (GE) ranging from $10^4 – 10^{-1}$ per 5 µl. It includes serial dilutions of 14 *B. burgdorferi* sl isolates belonging to eight different species and specificity controls. This panel is suited for comparison of PCR methods using different primers, targeting various loci, and using different methods (e.g. real-time PCR or conventional PCR) (unpublished, (Lager et al., 2017)). PCR on samples of the DNA panel with defined numbers of GE ranging from $10^4 – 10^{-1}$ showed that all *B. burgdorferi* ss samples produced positive PCR results targeting the *terL* locus (Table S1). However, this was not the case in *B. afzelii* or *B. garinii*. In *B. afzelii* isolate PKo only the sample with the highest number of GE was positive as it was the case for *B. valaisiana* isolate VS116 (Table S1). Using the ZEN/IFBQ probe in combination with PrimeTime master mix the *B. afzelii* PVPM $10^4$ became positive with a value of 40.77 whilst all other dilutions were negative (Tables 2). Interestingly, the *flaB* PCR performed slightly better in combination with QuantiTect master mix than with PrimeTime master mix (Table 1).

The lack of positivity in *terL* PCR cannot be related to the absence of the phage terminase gene(s) in these *B. afzelii* or *B. garinii* isolates since genome analyses have shown that cp32 plasmids are present in the genome of these isolates and *terL* sequences have been extracted from them (unpublished). In fact, we included the number of cp32 plasmids found in the genomes in Tables 2 and S1 (no. of cp32). There does not seem to be a correlation between presence/number of cp32 and positivity of *terL* PCR since some isolates (PVPM, PKo) that turned out to be negative in the *terL* PCR possess more cp32 ($n = 8$) than isolates (e.g. PBi

cp32 $n = 3$, PSigII cp32 $n = 6$) that were positive in *terL* PCR.

## 3.2. *terL* PCR does not improve diagnostics of serum or tissue samples of *Lyme borreliosis* patients

Using serum of individuals that were diagnosed with LB using standard clinical diagnostic tests and before onset of antibiotic therapy, we found that the *terL* PCR did not give positive results in these samples; none of 15 serum samples had a positive Ct value (Table 3a). The proven *Borrelia* infection in some of these individuals was confirmed by positive in vitro cultures obtained from tissues of these patients.

Similar results as obtained using cultured material were also found in patient tissue material; while samples that were diagnosed as being infected by *B. burgdorferi* ss were positive in *terL* PCR, samples identified as being infected with *B. afzelii* or *B. garinii/B. bavariensis* remained negative in *terL* real-time PCR (Table 3b). Out of 26 tissue samples that were positive in control PCRs (*flaB*, 5S-23S IGS), 15 (57 %) remained negative (no Ct value) using the phage terminase PCR. Species determination revealed that the negative tissue samples belonged to *B. afzelii* ($n = 9$; 34 %), to *B. garinii/B. bavariensis* ($n = 2$; 8 %) and for four samples (15 %) no species designation was available.

# 4. Discussion

## 4.1. *terL* PCR does not consistently detect European human pathogenic *Borrelia* species

Whilst targeting a multicopy locus sounds like a very attractive idea in PCR diagnostic of infectious diseases, much emphasis needs to be applied to the design of the PCR. The authors of the paper that introduced *terL* PCR for *Borrelia* diagnostic (Shan et al., 2021) used *B. burgdorferi* to set up the PCR: design of primers and probe was based on *B. burgdorferi* sl *terL* sequences. In silico analyses suggested that the PCR may be suitable for all *Borrelia* species (Shan et al., 2021). However, our study shows that whilst some isolates of *B. afzelii* or *B. garinii* may be detected using *terL* PCR, this is not the case with all isolates belonging to
**Table 3**

<table><caption>**a and b:** Results of *terL* real-time PCR using serum (3a) and tissue (3b) of patients diagnosed with Lyme Borreliosis using QuantiTec master mix (MM), 5'-FAM/abNFQ/NFQ-3' probe or PrimeTime master mix (MM), primers and probe (5'-FAM/ZEN/IBFQ-3') as published by Shan et al. (2021) and control PCRs.</caption><thead><tr><th>Patient ID</th><th>Material</th><th>Diagnosis/ Species</th><th>Anti-Borrelia antibody</th><th>Ct value (±STD) of terL PCR (abNFQ/NFQ). QuantiTec MM</th><th>Ct value (±STD) of terL PCR (ZEN/IBFQ). PrimeTime MM</th><th>Ct value (±STD) control PCR ospA/ Strube et al. 2010</th></tr></thead><tbody><tr><td>1</td><td>Serum</td><td>Lyme borreliosis/B. afzelii*</td><td>IFT: IgG 8000/IgM&lt; 16<br>Elisa:<br>IgG 15000/IgM 1,015</td><td>No Ct</td><td>No Ct</td><td>No Ct</td></tr><tr><td>2</td><td>Serum</td><td>Arthritis/not determined</td><td>IFT: IgG 1024/IgM 16; ELISA: IgG 2650/IgM 0.24</td><td>No Ct</td><td>No Ct</td><td>No Ct</td></tr><tr><td>3</td><td>Serum</td><td>EM / not determined</td><td>IFT: IgG 16/IgM 64<br>Elisa: IgG negativ/IgM 0,915</td><td>No Ct</td><td>No Ct</td><td>No Ct</td></tr><tr><td>4</td><td>Serum</td><td>EM / not determined</td><td>IFT: IgG&lt;16/IgM 512<br>Elisa: IgG 29/IgM 1,466</td><td>No Ct</td><td>No Ct</td><td>No Ct</td></tr><tr><td>5</td><td>Serum</td><td>EM / not determined</td><td>IFT: IgG 128/IgM 256<br>Elisa: IgG neg/IgM 2,414</td><td>No Ct</td><td>No Ct</td><td>No Ct</td></tr><tr><td>6</td><td>Serum</td><td>Neuroborreliose/ not determined</td><td>ELISA: IgG 1672/IgM borderline</td><td>No Ct</td><td>No Ct</td><td>No Ct</td></tr><tr><td>7</td><td>Serum</td><td>Erythema migrans/B. afzelii*</td><td>Elisa: IgG negativ/IgM 29.8</td><td>nd</td><td>No Ct</td><td>*No Ct</td></tr><tr><td>8</td><td>Serum</td><td>Erythema migrans/B. afzelii*</td><td>Elisa: IgG 12.79/IgM &gt;190</td><td>nd</td><td>No Ct</td><td>*No Ct</td></tr><tr><td>9</td><td>Serum</td><td>Neuroborreliose/ B. garinii/B. bavariensis*</td><td>Elisa: IgG 39.45/IgM 157.7</td><td>nd</td><td>No Ct</td><td>*No Ct</td></tr><tr><td>10</td><td>Serum</td><td>Neuroborreliose relapse/ not determined</td><td>IFT: IgG/IgM negative; Elisa: IgG/IgM negativ</td><td>nd</td><td>No Ct</td><td>*No Ct</td></tr><tr><td>11</td><td>Serum</td><td>ACA<sup>a</sup> / B. afzelii</td><td>IgG_EIA: 48</td><td>nd</td><td>No Ct</td><td>*No Ct</td></tr><tr><td>12</td><td>Serum</td><td>Lyme arthritis/B. afzelii</td><td>IgG_EIA: &lt;240</td><td>nd</td><td>No Ct</td><td>*No Ct</td></tr><tr><td>13</td><td>Serum</td><td>Lyme arthritis/B. afzelii</td><td>IgG_EIA: &lt;240</td><td>nd</td><td>No Ct</td><td>*No Ct</td></tr><tr><td>14</td><td>Serum</td><td>Lyme arthritis/B. afzelii</td><td>IgG_EIA: 65</td><td>nd</td><td>No Ct</td><td>*No Ct</td></tr><tr><td>15</td><td>Serum</td><td>ACA<sup>a</sup> / Bbss</td><td>IgG_EIA: &lt;550</td><td>nd</td><td>No Ct</td><td>*No Ct</td></tr></tbody></table>

<table><thead><tr><th>Patient ID</th><th>Material</th><th>Species/ PCR target</th><th>Ct value (±STD) of terL PCR (abNFQ/NFQ)</th><th>Ct value (±STD) control PCR flaB (P41)</th><th>Elisa CXCL 13</th></tr></thead><tbody><tr><td>16-1</td><td>aspirate knee</td><td>Bbss / hbb</td><td>33.57</td><td>38.46 (0.29)</td><td>nd</td></tr><tr><td>16-2</td><td>aspirate knee</td><td>Bbss / hbb</td><td>37.47</td><td>38.01</td><td>nd</td></tr><tr><td>17-1</td><td>synovia right knee</td><td>Bbss / hbb</td><td>39.08</td><td>No Ct (Ct 23S rRNA 37)</td><td>nd</td></tr><tr><td>17-2</td><td>synovia right knee</td><td>Bbss / hbb</td><td>37.06 (0.66)</td><td>no sample left</td><td>nd</td></tr><tr><td>18</td><td>synovial liquid</td><td>Bbss / hbb</td><td>34.47 (0.18)</td><td>37.62 (0.09)</td><td>nd</td></tr><tr><td>19-1</td><td>aspirate right knee</td><td>B. afzelii / hbb</td><td>No Ct</td><td>35.34 (0.36)</td><td>nd</td></tr><tr><td>19-2</td><td>aspirate right knee</td><td>B. afzelii / hbb</td><td>No Ct</td><td>no sample left (original 35.34)</td><td>nd</td></tr><tr><td>20</td><td>Skin</td><td>B. afzelii / nd</td><td>No Ct</td><td>37.60 (0.4)</td><td>nd</td></tr><tr><td>21</td><td>elbow. fibroid knot</td><td>B. bavariensis or B. garinii / hbb</td><td>No Ct</td><td>35</td><td>nd</td></tr><tr><td>22</td><td>joint aspirate</td><td>Bbss / hbb</td><td>34.25</td><td>No Ct</td><td>nd</td></tr><tr><td>23</td><td>shoulder tissue</td><td>B. afzelii / hbb</td><td>41.39 (2.34)</td><td>38.05 [1.07]</td><td>nd</td></tr><tr><td>24</td><td>aspirate knee</td><td>negative</td><td>36.34 (0.34)</td><td>39.30 (0.56)</td><td>nd</td></tr><tr><td>25</td><td>joint aspirate</td><td>B. afzelii / hbb</td><td>41.29 (3.08)</td><td>38.54 (0.31)</td><td>nd</td></tr><tr><td>26</td><td>tissue</td><td>B. afzelii / hbb</td><td>No Ct</td><td>34.76 (0.15)</td><td>nd</td></tr><tr><td>27</td><td>aspirate right knee</td><td>B. afzelii / hbb</td><td>No Ct</td><td>39.16 (0.02)</td><td>nd</td></tr><tr><td>28</td><td>aspirate</td><td>B. afzelii / hbb</td><td>36.53 (0.18)</td><td>34.76 (0.24)</td><td>nd</td></tr><tr><td>29</td><td>skin tissue</td><td>B. afzelii / hbb</td><td>No Ct</td><td>38.28 (0.52)</td><td>nd</td></tr><tr><td>30</td><td>aspirate knee</td><td>B. afzelii / hbb</td><td>No Ct</td><td>38.57 (0.5)</td><td>nd</td></tr><tr><td>31 / F10</td><td>aspirate knee</td><td>Bbss / MLST</td><td>30.17</td><td>33.33</td><td>nd</td></tr><tr><td>32/HU3</td><td>aspirate knee</td><td>B. afzelii / MLST</td><td>No Ct</td><td>31.02</td><td>nd</td></tr><tr><td>33/FB4</td><td>aspirate knee</td><td>Bbss / MLST</td><td>31.12</td><td>34.08</td><td>nd</td></tr><tr><td>34/RL6</td><td>aspirate knee</td><td>B. afzelii / MLST</td><td>No Ct</td><td>37.92</td><td>nd</td></tr><tr><td>35/AA7</td><td>aspirate knee</td><td>B. afzelii / MLST</td><td>No Ct</td><td>34.68</td><td>nd</td></tr><tr><td>36/NN8</td><td>aspirate knee</td><td>B. garinii / MLST</td><td>No Ct</td><td>No Ct</td><td>nd</td></tr><tr><td>37/CB9</td><td>aspirate knee</td><td>B. garinii / MLST</td><td>34.27</td><td>34.22</td><td>Nd</td></tr><tr><td>38</td><td>CSF</td><td>nd/nd</td><td>No Ct</td><td>No Ct</td><td>249 pg/ml</td></tr><tr><td>39</td><td>CSF</td><td>nd/nd</td><td>No Ct</td><td>No Ct</td><td>6570 pg/ml</td></tr><tr><td>40</td><td>CSF</td><td>nd/nd</td><td>No Ct</td><td>No Ct</td><td>4410 pg/ml</td></tr></tbody></table>

(continued on next page)
<table><caption>**Table 3 (continued)**</caption><thead><tr><th colspan="7">3b</th></tr><tr><th>Patient ID</th><th>Material</th><th>Species/ PCR target</th><th>Ct value (±STD) of <i>terL</i> PCR (abNFQ/NFQ)</th><th>Ct value (±STD) control PCR <i>flaB</i> (P41)</th><th>Elisa CXCL 13</th></tr></thead><tbody><tr><td>41</td><td>CSF</td><td>nd/nd</td><td>No Ct</td><td>No Ct</td><td>24920 pg/ml</td></tr><tr><td>B31 control</td><td>B31 1:1000</td><td></td><td>21.78</td><td>nd</td><td>nd</td></tr><tr><td>Extra control</td><td>No Ct</td><td></td><td>No Ct</td><td>nd</td><td>nd</td></tr><tr><td>Negative control</td><td>No Ct</td><td></td><td>No Ct</td><td>nd</td><td>nd</td></tr></tbody></table>

Ct = cycle threshold.

* = positive in vitro culture or positive PCR from patient tissue (not serum)

# real-time PCR according to (Strube et al., 2010)

nd = no data

ELISA, EIA = enzyme-linked immunosorbent assay

IFT = immuno fluorescent test

§ borderline

§ ACA = Acrodermatitis chronica atrophicans

Bbss = *Borrelia burgdorferi* sensu stricto

x -1,x -2 = samples from the same patient, repeat DNA extraction

hbb = product belongs to a family of histone binding proteins; nucleotide sequence similarity to HU/integration host factor (IHF) family of DNA accessory factors (Portnoi et al., 2006)

MLST = multilocus sequence typing (MLST) housekeeping genes (Margos et al., 2008); sequences of 2-8 genes were used for species determination

CSF = cerebrospinal fluid

CXCL13 = chemokine ligand CXCL13 ELISA (Rupprecht et al., 2018)

these species. Thus, whilst the *terL* PCR may well be suited for regions where *B. burgdorferi* ss is the main pathogen causing human LB, it will likely produce false negatives in regions where other *Borrelia* species are the cause of human LB, i.e. Europe or Asia.

## 4.2. *terL* PCR does not detect phage DNA in serum of patients with *Lyme borreliosis*

In individuals with *B. burgdorferi* sl infection the bacteria are found in the blood only transiently (Wormser, 2006). In a murine model it was shown that *B. burgdorferi* ss bacteria are rapidly cleared from the bloodstream (Liang et al., 2020). Therefore, blood or serum samples have been considered challenging for use in diagnostic PCR approaches (Santino et al., 2009; Branda et al., 2020) and are not routinely used for *B. burgdorferi* sl diagnostics. One of the major advantages suggested by Shan et al. (Shan et al., 2021) was the use of serum samples for LB diagnostic. The authors hypothesized that *Borrelia* prophages may spontaneously be activated in the patient and thus, be released into the blood circulation (Shan et al., 2021). An artificial setup using cell-free filtrates of *Borrelia* cultures 36 h post-incubation was used. Polyethylene glycol purified culture supernatant taken at that time (i.e. 36 h post-incubation) yielded positive results for *terL* PCR but negative results for a PCR targeting the *Borrelia* 16S rRNA locus. The authors present this as evidence that prophages may be spontaneously released in the blood stream of patients and that the *terL* PCR is able to pick up the signal. However, spontaneous release of prophage by *Borrelia* is not that readily observed in cultures and may vary depending on the isolate used (Eggers and Samuels, 1999). Induction of prophages may be increased chemically in culture but this (i) requires the presence of bacteria, which in vivo are only transiently in the blood stream and (ii) may vary between species or strains (Eggers and Samuels, 1999). Notably, these authors did not observe spontaneous release or induction of prophages in *B. afzelii* or *B. garinii* cultures.

Although an interesting idea, it remains an open question whether or not phages may be released and disseminated unhindered in a human patient. Shan et al. (Shan et al., 2021) reported positive results not only in sera of people with diagnosed LB but also in control sera of individuals that had no symptoms of LB. The authors explain this with undetected *Borrelia* infections in apparently healthy people. However, in our study no serum of individuals diagnosed with proven LB and before therapy was *terL* PCR positive suggesting a problem in specificity of the described PCR (Shan et al. 2021). Thus, whether the *terL* PCR will be

valuable for *Borrelia* diagnostic on serum samples is as yet unproven. In fact, our results indicate otherwise as in none of the 15 serum samples of patients with untreated, active *Borrelia* infection the *terL* PCR became positive.

## 4.3. PCR intrinsic and extrinsic parameter only marginally affect *terL* PCR

Shan et al. (Shan et al., 2021) emphasize in their paper that the DNA purification process plays a major role in the quality of DNA used for PCR. They suggest the use of phenol-chloroform DNA purification is superior to other methods using column or magnetic bead purification. Whilst this may be true, the use of phenol-chloroform has been replaced in many laboratories because of health and safety issues. Regardless of the DNA purification method used, we show here that the *terL* PCR in its current form does not work on DNA purified from cultured *Borrelia* samples that became positive using other established PCR protocols. This failure is not due to the DNA purification protocol used or the absence of the *terL* locus in these samples, but appears to be inherent to the PCR setup.

We have not attempted to precisely quantify the qPCR results in our study. We are aware that Ct values in real-time PCR are influenced by many factors, such as the master mix or PCR machines used. Therefore, each participating laboratory in our study included a control PCR that was conducted under identical conditions in their labs as the *terL* PCR (in terms of master mix and PCR machine), making Ct values within laboratories more comparable. In addition, we have used a DNA panel consisting of serial DNA dilutions of cultured isolates belonging to eight different *B. burgdorferi* sl species, five of which are considered human pathogenic and two that are potentially human pathogenic. This provided identical DNA dilutions that were used in the three laboratories making the PCR results comparable as far as template DNA is concerned. Indeed, although some differences were noticed in limits of detection of the employed PCRs, the results obtained for the *terL* PCR are congruent in all three laboratories. Isolates of *B. afzelii* and *B. garinii* that were negative in *terL* PCR were negative in all three laboratories, strongly supporting the notion that the *terL* PCR in its current form is not suited for LB diagnostic in Europe.

In conclusion, use of the current form of the *terL* PCR assay cannot be recommended for the diagnosis of LB in patients in Europe and Asia. Furthermore, it is not suitable for detecting *B. burgdorferi* sl DNA in serum samples from infected individuals.
## Author agreement statement

I the undersigned declare that this manuscript is original, has not been published before and is not currently being considered for publication elsewhere. I confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed.

## Funding

The National Reference Center for Borrelia, Oberschleissheim, Germany is funded via the Robert Koch-Institute, Berlin, Germany. Sweden National Reference Laboratory for Borrelia and Other Tick-Borne Bacteria and The Research Council of Southeast Sweden (FORSS) (grant no: FORSS-994295). The contribution of the Austrian Agency for Health and Food Safety was co-funded by the European Union (grant no: 101132974).

Supplementary material: Table S1 NRZ DNA Panel from cultured isolates, serial dilutions of DNA, genome equivalents (GE) as indicated tested using *terL* PCR independently in three different laboratories.

## CRediT authorship contribution statement

**Manja Zimmermann:** Writing – review & editing, Writing – original draft, Investigation, Formal analysis. **Gabriele Margos:** Writing – review & editing, Writing – original draft, Supervision, Formal analysis, Conceptualization. **Christine Hartberger:** Writing – review & editing, Investigation, Formal analysis, Data curation. **Reto Lienhard:** Writing – review & editing, Resources. **Anna J. Henningsson:** Writing – review & editing, Supervision, Resources. **Malin Lager:** Writing – review & editing, Investigation, Formal analysis. **Mateusz Markowicz:** Writing – review & editing, Supervision, Resources. **Anna-Margarita Schötta:** Writing – review & editing, Investigation, Formal analysis. **Andreas Sing:** Writing – review & editing, Supervision. **Benoit Jaulhac:** Writing – review & editing, Supervision, Resources, Formal analysis. **Per-Eric Lindgren:** Writing – review & editing, Supervision. **Alje P. van Dam:** Writing – review & editing, Supervision, Resources. **Joppe W.R. Hovius:** Writing – review & editing, Supervision, Resources. **Volker Fingerle:** Writing – review & editing, Writing – original draft, Supervision, Resources, Conceptualization.

## Declaration of competing interest

MZ, CH, GM, RL, AMS. AS, ML, BJ declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. MM discloses receiving honoraria for invited talks from Diasorin and Mikrogen. PL, senior external scientific advisor Pfizer Inc, Novo-Nordisk A/S, Valneva GmbH. VF discloses research support: RKI/BMG, ESCMID, ECDC, StMGP/StMUG, INSTAND; lecturing activities (fees, travel expenses): DIAMEDIS, Diasorin, Mikrogen, Seramun, Siemens, HLR, Pfizer; Consultancy, round robin tests (no fees): QCMD, Pfizer, INSTAND, ECDC. JWRH, research grants: Pfizer, Moderna, Abbott, Biorad and Zeus; advisory boards: Pfizer, Tarsus; patent on LB diagnostic antigens; Steering committee National Dutch Lyme borreliosis expertise center; scientific board member: INFECT-NL Foundation; Editor: Ticks and Tick-borne Diseases

## Acknowledgements

None.

## Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.tttdis.2025.102488.

## Data availability

No data was used for the research described in the article.

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