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Dear Editor,
In response to the Rebuttal letter to our paper Zimmermann et al. (2025) titled “The real-time PCR targeting the Phage terminase (terL) is not suitable for diagnostics of human Borrelia infections in Europe”, we would first like to clarify that we do not and did not question the method of targeting the phage terminase large subunit gene (terL) by qPCR per se. We have critically evaluated the method on DNA extracted from the main European Borrelia species and found that it did not produce positive results for all isolates of B. afzelii and B. garinii. This evaluation is independent of DNA purification methods as control PCRs clearly showed that there was DNA of sufficient quality and quantity for PCR in these tests. It also clearly demonstrates that the terL PCR in its published design (Shan et al. 2021) is not suitable for detection of all Borrelia species and isolates. The follow-up publication of Shan et al. 2023 did not provide information on better design of primers and probes for B. burgdorferi sensu lato species, in fact it published primers and probes specific for Borrelia miyamotoi, a relapsing fever species vectored by Ixodes ticks. Further, it was a hypothesis put forward by Shan et al. 2021 that phages are released spontaneously into the bloodstream of infected individuals.
Below are our responses to the specific points that the authors raised:
1. Zimmermann et al. (2025) used the wrong sample type for terL PCRThe authors maintain that the wrong type of sample was used (serum instead of blood). However, we would like to emphasize that we did not use just one type of sample but several.
We used:
1. DNA purified from cultured material of several Borrelia species known to occur in Europe
2. A DNA panel comprising of serial dilutions of DNA of several Borrelia species
3. DNA purified from tissue samples of patients diagnosed with Lyme borreliosis
4. Serum samples of patients diagnosed with Lyme borreliosis
In these samples we found that the real-time PCR designed by Shan et al. 2021 did not produce positive results for all B. afzelii or B. garinii samples tested. Control PCRs showed that the DNA was of sufficient quality and quantity and without PCR inhibiting substances to detect all Borrelia isolates. The serial dilution samples were tested in three laboratories independently with the same results. These results suggest a problem in the terL real-time PCR design and set up itself - independent from the sample type or extraction method.
In their original article (Shan et al. 2021) and in a follow-up review from 2024 (Teulières/Jia et al. 2024) the authors put forward that free phages circulate in the blood and can be detected in higher frequency in blood than in serum samples. However, in their supplementary table they described at least one reactive PCR in 6/13 early, 18/23 healthy and 35/42 late patients (without further defining “early” or “late” or what they would define as a positive result) suggesting that serum can also be used. In addition, in a paper published by Shan et al. 2024 in Methods in Molecular Biology, the authors themselves indicate that human serum can be used for DNA extraction: “Add 300 μL of human serum samples to suitable Eppendorf tubes.”
Furthermore, the proposal to use blood or serum for Borrelia diagnostic using the qPCR targeting the terL locus is based on the assumption that phages are spontaneously released and circulate in the blood stream or body fluids.
However,
this is a hypothesis that was supposedly demonstrated by an artificial in vitro setting (Shan et al. 2021).
To the best of our knowledge,
it has not yet been demonstrated that Borrelia phages are spontaneously induced and circulate free in the blood of infected individuals.
2. Zimmermann et al. (2025) used the wrong DNA extraction method for terL PCRIt may be so that phenol-chloroform extraction might work slightly better for DNA extraction from blood than the methods used in Zimmermann et al. 2025. However, in our evaluation we actually used DNA that was extracted from in vitro cultured bacteria. Quality and quantity of DNA and absence of PCR inhibitors was demonstrated using real-time PCRs that successfully amplified single copy loci of Borrelia.
Thus, the failure of the real-time PCR targeting the terL locus to produce positive results for some B. afzelii and B. garinii isolates could
not be due to using a possible wrong DNA extraction method,
yet due to a problem inherent to the Shan et al. PCR design.
3. Zimmermann et al. fail to acknowledge proof-of-concept context and advances in phage-based methodologiesWe do not fail to acknowledge the potential advantages of a phage-based or blood/serum-based diagnostic procedure for Borrelia in Lyme borreliosis patients and we are aware of potential shortcoming of some diagnostic procedures. However, with our critical evaluation of the terL qPCR we aimed to independently put a new diagnostic test under scientific scrutiny.
Contrary to the authors claim that the 2021 study was a preliminary study and that they “have developed new primers and probes targeting homologous phage genes in B. afzelii, B. garinii, and other genospecies common in Europe“, we did not find any further publications that would support this claim. In fact, a publication in Methods in Molecular Biology by Shan et al. in 2024 reports the same method, same primers, probes and procedures as the 2021 publication. If the study by Shan et al. 2021 and the method published by Shan et al. 2024 were preliminary, it should be clarified so that laboratories wanting to use the method are aware of it. From accessible public information it was not obvious to us that the test had been updated. Thus, “reviewing updated documentation” as suggested in the letter was not possible. None of the references given on the website of R.E.D. Laboratories (as per 25.09.2025: Shan et al. 2021, Targeting Multicopy Prophage gene for increased detection of Borrelia burgdorferi sensu lato (s.l.) the causative agents of Lyme borreliosis, in blood; Shan et al. 2023 Combining citizen science and molecular diagnostic methods to investigate the prevalence of Borrelia burgdorferi s.l. and Borrelia miyamotoi in tick pools across Great Britain; Jia et al. 2023 Overcoming the Challenge of Lyme disease diagnosis: The role of phage-based testing; Jia et al., 2024. Successful diagnosis and treatment of Borrelia miyamotoi in patients with joint and muscle pains, ME/CFS and cognitive dysfuntion following tick bites: a case report) mention a further development of specific primers and probes for B. afzelii or B. garinii or any other Borrelia burgdorferi s.l. species.
Of note, we did not investigate Borrelia miyamotoi or relapsing fever directed approaches in Zimmermann et al. 2025. This “broader diagnostic value” would need to be investigated in an additional study.
4. Zimmermann et al. (2025) failed to follow the scientific practice of faithful protocol reproductionAny diagnostic PCR will need to be sufficiently robust to be performed in different laboratories. We tested the terL qPCR published by Shan et al. 2021
using the same primers, probes, master mixes in combination with DNA of various Borrelia species that did not contain inhibitors.
We tested the PCR in three different laboratories that set up the PCR independently but used identical DNA serial dilutions for comparability.
Each lab used their own control PCR and came to the same conclusion: the terL PCR published by Shan et al. 2021 did not give positive results with some isolates of B. afzelii and B. garinii.
Thus,
again we can only conclude that our data suggest that the lack of sensitivity is inherent in the qPCR protocol published by Shan et al. 2021,
2024.
5. Conclusion:
collaboration,
rigorous replication,
and further developmentWe have published the results of our scientific evaluation of the real-time PCR targeting the terL locus
so that differences or weaknesses can be acknowledged and the scientific and medical community is aware of it.
We very much welcome research into improved diagnostic tests for Lyme borreliosis and independent validation of such tests in other settings and an open,
transparent and professional discussion about these data are crucially important.
Finally, we would also like to point out a commentary that addresses other methodological deficiencies in the study by Shan et al. 2021 (Van de Schoor et al. 2021; Bron
https://www.frontiersin.org/journals/mi ... 02131/full )..
