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Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Dear editor in chief

While the topic of the article is interesting, the study faces limitations due to the constraints in the techniques employed and the study is conducted at a basic and preliminary level. These limitations may affect the reliability and clinical applicability of the findings. To enhance the potential for publication, it is essential for the authors to address these methodological shortcomings and consider incorporating more robust and validated techniques in their research. This would not only strengthen their conclusions but also increase the overall impact of their work in the field. therefore, it seems that it is not appropriate for publication.

Reviewer #2: The work is interesting and done in a careful way. There are some changes or clarifications to be made:

Borrelia burgdorferi sensu lato abbreviate: Bbsl

In the introduction it is written: The causative agent of LB is transmitted to humans by Ixodes ricinus ticks. This should be corrected to: "Ixodes spp: in Europe Ixodes ricinus, in Asia I. persulcatus, in North America I. scapularis and I. pacificus, in North Africa I. inopinatus".

The most common clinical manifestation of LB is erythema migrans (EM), a clinical diagnosis. Replace with "The most common clinical manifestation of LB is erythema migrans (EM), which is present in 75% of cases and allows a clinical diagnosis.

In the sentence: Except for EM, an early localized manifestation of LB, add "characterized by a circular erythema around the tick bite, which does not appear immediately (chemical reaction), but after an incubation period of 4-30 days and enlarges with a greater diameter of 5 cm. In this early localized phase (stage I) antibodies are often still negative."

The Sentence: Borrelia serology is the cornerstone for the diagnosis of LB in routine clinical practice, I would remove "the cornerstone".

After "Diagnosis of LNB requires simultaneous analysis of Borrelia-specific antibodies in serum and cerebrospinal fluid (CSF), I would add the sentence: "In some cases, when neurological manifestations are secondary to ACA, the cerebrospinal fluid may be negative (Ogrink K, Maraspin V. Nervous System Involvement in Lyme Borreliosis. The Open Dermatology Journal, 2016, 10, (Suppl 1: M5) 44-54. DOI: 10.2174/1874372201610010044)"

After the sentence: Furthermore, we do not know if LB serology is used similarly in different counties which follow the same national guidelines from the Swedish authorities [17, 18]. Add: "It should also be noted that serology in the United States and Europe has some differences. In fact, in North America Borrelia burgdorferi sensu stricto (Bbss) is clearly prevalent, while in Europe Lyme Borreliae, which infect humans, are more numerous and serological tests must take into account these different species of Bbsl. Currently, 24 Borreliae belonging to the Lyme Group have been identified (Trevisan G, Cinco M, Trevisini S, di Meo N, Chersi K, Ruscio M, Forgione P and Bonin S. Borreliae Part 1: Borrelia Lyme Group and Echidna‐Reptile Group. Biology 2021, 10, 1036. https://doi.org/10.3390/biology10101036), and the last isolated on the coasts of California and Borrelia maritima (Margos G, Fedorova N, Becker NS, Kleinjan JE, Marosevic D, Krebs S, Hui L, Fingerle V, Lane RS. Borrelia maritima sp. nov., a novel species of the Borrelia burgdorferi sensulato complex, occupying a basal position to North American species. Int J Syst Evol Microbiol. 2020 Feb;70(2):849-856. doi: 10.1099/ijsem.0.003833.).

Interesting are the Polish studies about the chimeric tests in ELISA and Western (or Immuno)-Blot (Grąźlewska W, Holec-Gąsior L, Sołowińska K, Chmielewski T, Fiecek B, Contreras M. Epitope Mapping of BmpA and BBK32 Borrelia burgdorferi Sensu Stricto Antigens for the Design of Chimeric Proteins with Potential Diagnostic Value. ACS Infect Dis. 2023 Nov 10;9(11):2160-2172. doi: 10.1021/acsinfecdis.3c00258. Epub 2023 Oct 6.).

PATIENTS AND METHODS.

After the sentence: The C6 ELISA uses a conserved synthetic peptide derived from the VlsE protein as an antigen, and both IgM and IgG antibodies are detected in the same assay [22], add the sentence: VlsE (Variable major protein like sequence Expressed) is an important and specific antigen for diagnosis, as it is not expressed in culture or in ticks, while it is expressed by Bbsl in human infection, inducing an IgG antibody response.

DISCUSSION

After the sentence: (ii) cerebrospinal fluid (CSF) pleocytosis, "(except for neurological forms secondary to ACA, where there is a spread of Borreliae from the skin to the underlying nerve fibers)"

After the sentence: Twenty-three of these 168 patients were treated with antibiotics for LB, implying that the LB serology may have been misleading, thus delaying the true diagnosis. Add: In fact, serology is reliable in establishing who has had LB, while it is often unable to recognize whether the infection is still microbiologically active. Studies are currently underway in the United States to develop proteomics for Bbsl, with a highly sensitive tandem mass spectrometer and with accurate algorithm development, which allows the identification of microbiologically active forms of Lyme in urine (Cornero R, Irfan SS, Cachaco S, Zhou W, Byne A, Howard M, McIntyre H, Birkaya B, Liotta L, Luchini A. Identification of Unambiguous Borrelia Peptides in Human Urine Using Affinity Capture and Mass Spectrometry. Methods Mol Biol. 2024;2742:105-122. doi: 10.1007/978-1-0716-3561-2_9.). Another promising method is Borrelia Phage PCR (Shan J, Jia Y, Mijatovic T. Use of Specific Borrelia Phages as a New Strategy for Improved Diagnostic Tests. Methods Mol Biol. 2024;2742:99-104. doi: 10.1007/978-1-0716-3561-2_8.).

CONCLUSIONS

After the sentence: The analysis is frequently performed on questionable indications contrary to guidelines, which limits the value and potential of the analysis. Add: "New diagnostic technologies, such as ms/ms proteomics and Bbsl Phage PCR, may improve laboratory diagnostics in the future."

The paper can be accepted with these changes.

Reviewer #3: Review of PONE-D-24-53373

Title: Deficiencies in communication between clinical microbiological laboratories and physicians may impair the diagnosis of Lyme borreliosis: a study of the use and application of serology in three neighbouring counties in Sweden

Conclusions by authors: LB serology is often used for indications it is not intended for, thereby giving the analysis poor clinical value. Notably, the use appears to be different in three neighbouring counties that follow the same national guidelines. Our findings indicate a need for interventions to enable a more rational use of LB serology.

The authors have used an interesting approach of examining medical and laboratory records to determine the relevance of serological testing for LB. The assessment I qualitative by physicians experienced in the field. The authors do not use defined case definitions, but accept the clinical judgement, as is. This choice can be discussed , but in the opinion of this reviewer reflects the clinical reality and is suitable for the present study.

Interesting study qualifying the well-known overuse of Borrelia serology with clinical data.

The use of serology, without spinal fluid examination, for suspected LNB is highlighted.

The use of antibiotic treatment based on positive serology is also quite frequent, even when the clinical diagnosis is uncertain.

Some detailed remarks and suggestions for clarification:

Page 3: It is stated that “and antibiotic therapy may abort serologic response or prevent seroconversion”. This is an interesting theory and often postulated as an established fact. In the opinion of the reviewer this has never truly been documented, this would require a randomized study with weeks delayed treatment in one arm. This is of course unrealistic. There are two possible mechanisms, early treatment may of course abort antibody generation, but on the other hand treatment may also release lots of bacterial antigens and enhance antigenic stimulation. Both scenarios may occur, but which of these two tendencies are most frequent? But as the word “may” is included in the sentence the wording should be OK.

Page 5:

“randomly selected using Microsoft Excel” Somewhat non-specific. Perhaps something like: using a random function in Microsoft Excel.

More importantly also indicate clearly if records from general practice were available? How were the “recommendations made by clinical microbiologists” available. Often telephone calls are not documented systematically, if at all?

Page 6.

The category 2. is difficult to grasp. How do you construct “conclusions in laboratory reports” The laboratory only measured antibody reactivity? “e.g. LB and not LB or not LB and LB.” is not easy to read. Negative serology is not necessarily discordant with the clinical assessment.

Suggest to clarify.

Page 7. Concerning the data in the sentence starting with “The rate found in LNB indication patients was……” and table 2.

Rates of seropositivity are quite high. Was this the first available testing in all patients? Patients were not referred because of already having tested positive? In the methods suggest to explain more clearly how patients were recruited (as also suggested above). Even in a high endemic setting this rate of seropositivity is high. Did you check that the “random selection” was representative for the positivity rate in the total data?

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what does this mean? ). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: Yes:  Prof. Giusto Trevisan University of Trieste

Reviewer #3: Yes:  Ram B. Dessau

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Deficiencies in communication between clinical microbiological laboratories and physicians may impair the diagnosis of Lyme borreliosis: a study of the use and application of serology in three neighbouring counties in Sweden.

PONE-D-24-53373R1

Dear Dr. Johansson,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Pablo Colunga-Salas

Academic Editor

PLOS One

Additional Editor Comments (optional):

Dear Authors,

Based on the comments from two of the three reviewers, as well as the changes made to the manuscript, I appreciate your commitment and contribution to the study of Lyme borreliosis. Therefore, I believe your manuscript is ready for acceptance and to continue with the editorial process.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: (No Response)

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: (No Response)

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: (No Response)

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: (No Response)

Reviewer #2: Yes

Reviewer #3: Yes

**********

Reviewer #1: (No Response)

Reviewer #2: The requested changes have been made correctly. The difference between Borrelia burgdorferi sensu laro and Borrelia burgdorferi sensu stricto has been clarified, and the spread of Lyme borreliosis to other areas of North America and Europe has been clarified. The description of Erythema migrans and its evolution is now thorough, and the neurological and cardiac clinical manifestations are better defined.

The description of the tests has been improved, also with an eye to the future, with new techniques currently under development.

The work can be accepted.

Reviewer #3: No further comments.

However the sentence: Although new diagnostic technologies, such as ms/ms proteomics and Bbsl Phage

PCR, may improve laboratory diagnostics in the future, there is also a need for interventions to enable a more

rational use of LB serology.

Is not really a part of the conclusion - should perhaps appear as a perspective in the discussion. Also these techniques are quite preliminary ideas prestented at conference meetings/publications. Even if borrelia detection is possible clinical sensitivities may be quite low?

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #1: Yes:  Shadi Aghamohammad

Reviewer #2: Yes:  Giusto Trevisan, University of Trieste, Italy

Reviewer #3: Yes:  Ram B. Dessau

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