PLoS ONE plos plosone PLOS ONE 1932-6203 Public Library of Science San Francisco, CA USA 10.1371/journal.pone.0287586 PONE-D-23-05893 Research Article Medicine and health sciencesHealth careBlood donors Medicine and health sciencesMedical conditionsInfectious diseasesBacterial diseasesBorrelia infectionLyme disease Medicine and health sciencesDiagnostic medicine Medicine and health sciencesMedical conditionsInfectious diseasesBacterial diseasesBorrelia infection Biology and life sciencesBiochemistryBiomarkers Biology and life sciencesOrganismsBacteriaBorrelia Biology and life sciencesMicrobiologyMedical microbiologyMicrobial pathogensBacterial pathogensBorrelia Medicine and health sciencesPathology and laboratory medicinePathogensMicrobial pathogensBacterial pathogensBorrelia Biology and life sciencesPhysiologyImmune physiologyAntibodies Biology and life sciencesImmunologyImmune system proteinsAntibodies Medicine and health sciencesImmunologyImmune system proteinsAntibodies Biology and life sciencesBiochemistryProteinsImmune system proteinsAntibodies Research and analysis methodsImmunologic techniquesImmunoassaysEnzyme-linked immunoassays Identification of potential biomarkers in active Lyme borreliosis A novel laboratory approach to discriminate active Lyme borreliosis from previous Borrelia exposure https://orcid.org/0000-0001-8657-2496 Tjernberg Ivar Conceptualization Data curation Formal analysis Funding acquisition Methodology Project administration Supervision Writing – original draft Writing – review & editing 1 2 * Lager Malin Data curation Investigation Resources Writing – review & editing 3 Furset Jensen Guro Data curation Investigation Resources Writing – review & editing 4 5 Eikeland Randi Data curation Investigation Methodology Resources Writing – review & editing 6 7 8 Nyman Dag Data curation Methodology Resources Writing – review & editing 8 9 Brudin Lars Data curation Formal analysis Investigation Methodology Writing – review & editing 10 11 Henningsson Anna J. Data curation Funding acquisition Investigation Methodology Resources Writing – review & editing 8 12 13 Department of Clinical Chemistry and Transfusion Medicine, Region Kalmar County, Kalmar, Sweden Department of Biomedical and Clinical Sciences, Division of Inflammation and Infection, Linköping University, Linköping, Sweden National Reference Laboratory for Borrelia, Department of Clinical Microbiology in Jönköping, Region Jönköping County, Linköping University, Linköping, Sweden National Reference Laboratory for Borrelia, Department of Clinical Microbiology, Sørlandet Hospital Trust, Kristiansand, Norway Department of Clinical Microbiology, Sørlandet Hospital Health Enterprise, Kristiansand, Norway National Advisory Unit on Tick Borne Diseases, Sørlandet Hospital Trust, Kristiansand, Norway Faculty of Health and Sports Science, University of Agder, Grimstad, Norway ESCMID Study Group for Lyme Borreliosis—ESGBOR, Part of the European Society for Clinical Microbiology and Infectious Diseases, Basel, Switzerland The Åland Group for Borrelia Research, Mariehamn, Finland Department of Clinical Physiology, Region Kalmar County, Kalmar, Sweden Department of Health, Medicine and Caring Sciences, Linköping University, Linköping, Sweden National Reference Laboratory for Borrelia and Other Tick-Borne Bacteria, Department of Clinical Microbiology in Jönköping, Region Jönköping County, Linköping University, Linköping, Sweden Department of Biomedical and Clinical Sciences, Linköping University, Linköping, Sweden Chegou Novel Njweipi Editor Stellenbosch University, SOUTH AFRICA

I have read the journal’s policy and the authors of this manuscript have the following competing interests: IT reports participation in advisory board and personal fees from Pfizer Inc outside the submitted work. AJH has a collaborative research agreement with Abbott Laboratories, Chicago, USA and Reagena Oy, Toivala, Finland. Remaining authors report no conflict of interest.

 * E-mail: ivar.tjernberg@liu.se 26 6 2023 2023 18 6 e0287586 3 3 2023 8 6 2023 2023 Tjernberg et al This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Objectives

Lyme serology does not readily discriminate an active Lyme borreliosis (LB) from a previous Borrelia infection or exposure. Here, we aimed to investigate a large number of immunological protein biomarkers to search for an immunological pattern typical for active LB, in contrast to patterns found in healthy blood donors, a proportion of whom were previously exposed to Borrelia.

 Methods

Serum samples from well-characterised adult patients with ongoing LB and healthy blood donors were included and investigated using a proximity extension assay (provided by Olink®) by which 92 different immune response-related human protein biomarkers were analysed simultaneously.

 Results

In total, 52 LB patients and 75 healthy blood donors were included. The blood donors represented both previously Borrelia exposed (n = 34) and not exposed (n = 41) based on anti-Borrelia antibody status. Ten of the examined 92 proteins differed between patients and blood donors and were chosen for further logistic regression (p<0.1). Six proteins were statistically significantly different between LB patients and blood donors (p<0.05). These six proteins were then combined in an index and analysed using receiver-operating-characteristic curve analysis showing an area under the curve of 0.964 (p<0.001).

 Conclusions

The results from this study suggest that there is an immunological protein pattern that can distinguish a present Borrelia infection from a previous exposure as well as anti-Borrelia antibody negative blood donors. Although this method is not adapted for routine clinical use at this point, the possibility is interesting and may open new diagnostic opportunities improving the laboratory diagnostics of LB.

 Region Kalmar County (IT), Futurum-Academy for Healthcare and Division of Medical Diagnostics, Region Jönköping County, Interreg IVA Program ScandTick 167226 Interreg V program ScandTick Innovation project ID. 20200422, reference no. 2015-29 000167 The Medical Research Council of Southeast Sweden FORSS-475511 the foundation for medical research of the Åland cultural foundation Wilhelm and Else Stockmann Foundation Henningsson Anna J. This study was supported by university grants to Region Kalmar County (IT), Futurum-Academy for Healthcare and Division of Medical Diagnostics, Region Jönköping County, Interreg IVA Program ScandTick (grant no. 167226) and Interreg V program ScandTick Innovation (project ID. 20200422, reference no. 2015-29 000167), The Medical Research Council of Southeast Sweden (FORSS-475511), the foundation for medical research of the Åland cultural foundation and the Wilhelm and Else Stockmann Foundation (AJH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data Availability There are restrictions in place that prevent public sharing of original and raw data according to the regional ethical permissions for the study. Data contain potentially identifying and sensitive patient information and public sharing is not allowed according to the consents signed by the study participants. For sharing of restricted data, please contact the corresponding author Ivar Tjernberg at ivar.tjernberg@liu.se or Linköping University at registrator@liu.se. Introduction

Lyme borreliosis (LB) is considered the most common tick-borne disease in both Europe and North America [1–3]. The disease, caused by bacteria belonging to the Borrelia burgdorferi sensu lato (s.l.) complex, may give rise to a number of different clinical manifestations including erythema migrans, Lyme neuroborreliosis, acrodermatitis chronica atrophicans, Lyme arthritis, and other more rare manifestations [2]. However, Borrelia exposure may also pass sub-clinically in a significant proportion of exposed individuals [3–5]. In the routine clinical setting, diagnosis of erythema migrans relies on patient history and physical examination, whereas in the remaining manifestations laboratory support is also required in the diagnostic workup. Although much effort has been invested in the development of direct detection by molecular methods, these are not widely used due to low sensitivity in some materials, e.g. cerebrospinal fluid. In clinical practice, indirect methods, essentially antibody detection by serology, remains the gold standard for the laboratory diagnosis of LB [2, 6]. However, there are challenges in the interpretation of anti-Borrelia antibody results that must be considered in the diagnosis of LB. The serological results must be interpreted in relation to the variable clinical presentation of LB including the dissemination of the infection. It is important to recognise that a single positive serological result does not discriminate an active infection from a previous symptomatic or subclinical exposure [6, 7]. Furthermore, the local epidemiological situation needs to be considered. In highly endemic areas, the seroprevalence may reach high figures in the healthy population thus significantly reducing the positive predictive value of a positive test result [8, 9]. In addition, test cross-reactions may also render false positive serological results [6, 10, 11]. However, the most important limitation of Borrelia serology in the routine clinical setting in endemic areas is that the population is highly seropositive after previous exposure to Borrelia. Additional tests to discriminate an active LB from a previous Borrelia infection would improve the diagnostic process considerably and limit the risk for overdiagnosing and thereby counteract overuse of antibiotics with associated risks for side-effects for patients as well as the negative effects on the environment including antibiotic resistance.

Using proximity extension assay, large numbers of proteins may be detected and measured simultaneously in the same patient sample. Predefined analytical panels of different sizes and combinations of biomarkers are available through Olink®, e.g. neurology, cardiovascular and inflammation panels. For the purpose of this study, the immune response panel was chosen based on the hypothesis that the immune response shows different signatures of biomarkers during an active LB as opposed to a previous Borrelia infection.

Thus, in this study, we wanted to investigate serum samples from clinically well characterised LB patients and healthy blood donors examining a large number of immunological protein biomarkers in order to possibly distinguish active LB from previous Borrelia exposure.

 Material and methods Lyme borreliosis patients and blood donors

Serum samples from a previous prospective study performed 2013–2017 of adult LB patients with ongoing infection and blood donors were included in the present study. The original study included patients from Jönköping County in Sweden, Kristiansand in Norway and on the Åland Islands, and blood donors from Jönköping County, Sweden [12]. In the original study 59 patients with various manifestations of LB were included, of whom seven patients were considered possible Lyme neuroborreliosis. Only the 52 LB patients with a definite diagnosis were included in this study. The 52 samples represented patients with different ongoing manifestations of LB. Serum samples were obtained from the LB patients at the time of diagnostic investigation, thus with ongoing symptoms/complaints, but before treatment initiation, thereby considered active/ongoing LB. Classification and confirmation of LB diagnosis in the patients were based on clinical information including laboratory results and review of medical records by experienced physicians in the field. Patients with Lyme neuroborreliosis fulfilled the European case definition for definite Lyme neuroborreliosis [13]. Patients with Lyme arthritis and/or acrodermatitis chronica atrophicans showed clinical signs compatible with the conditions and were all confirmed with detection of Borrelia-specific DNA. Patients with erythema migrans reported recent tick-bite and presented with a typical skin rash of >5 cm in diameter assessed by a physician [12].

Furthermore, a random subset (n = 80) of the original 201 blood donor samples were included as a healthy comparison group, thus considered not having ongoing LB. Blood donors were all healthy at the time of sampling and only included after an approved health declaration without current symptoms or signs of disease. The health declaration includes questions regarding potential fever, infection, other diseases, medical examinations, treatments, medications. Information regarding possible previous LB was gathered from the blood donors. Both blood donors with and without anti-Borrelia antibodies were included in order to also cover individuals with a previous exposure to B. burgdorferi s.l. The blood donors were screened for anti-Borrelia antibodies using Enzygnost Borrelia Lyme IgM/IgG (Siemens/DADE Behring, Marburg, Germany); for IgG based on a mix of native Borrelia antigens from B. afzelii strain PKo and recombinant VlsE obtained from genospecies the B. burgdorferi sensu stricto, B. garinii and B. afzelii. These 80 blood donors comprised 37 IgM and/or IgG anti-Borrelia antibody positive (34 IgG-positive and three IgM and IgG-positive) and 43 anti-Borrelia antibody negative subjects. The IgG antibody reactivities in these 37 samples were also confirmed by positive findings in at least one additional and independent Borrelia serological assay including Liaison Borrelia IgG (DiaSorin, IgG-analysis based on recombinant Borrelia-specific VlsE antigens from B. garinii strain PBi) and the Immunetics C6 LYME ELISA (IgM/IgG analysis based on a synthetic 25 amino acid sequence derived from IR6 of VlsE B. burgdorferi strain B31). All dilution steps in the antibody assays were performed according to the respective manufacturer’s instructions. After classification of LB patients and blood donors data was anonymised. Further details and definitions of the study subjects have been described in a previous publication [12].

 Laboratory analyses

Serum samples were all investigated using a proximity extension assay provided by the Clinical Biomarkers facility, Science for Life Laboratory, Uppsala University, Sweden. Detailed description on the principle of the method has been published previously [14]. In short, the 92 human immune system biomarkers in the chosen panel are detected in parallel by individually paired matched antibodies labelled with unique strands of DNA oligonucleotides that may hybridize when in proximity with each other, allowing for subsequent amplification, e.g. using quantitative PCR, in which the results are related to the initial concentration of each target. The panel used was the immune response panel v.3202 covering 92 immune response-related human protein biomarkers. A comprehensive list of analytes covered in the panel may be found at the Olink website [15]. Proteins in this publication have been referenced by their respective gene as well as their unique protein identification number (UniProt ID) according to the Universal Protein Resource (www.uniprot.org).

 Data analysis and statistics

Results for all parameters were presented as normalised protein expression values on a logarithmic scale with base 2 (log2) based on the respective analytical cycle threshold values.

The lowest concentration possible to detect (LOD) for each of the proteins is given. Individual values below LOD are replaced by LOD-1 which represents 50% of LOD. The number of values below LOD (%) per protein are presented in the S1 Table. Parameters with at least 50% values within the detectable range were chosen for the subsequent analysis. The further analyses were as follows: 1) The database was randomly divided in two halves, equally for both LB patients and blood donors, and the difference between the two groups within each half analysed separately with Mann-Whitney’s U-test. This was done in order to reduce random statistical effects. 2) Secondly, the differences between LB patients and blood donors, with a significance level of <0.1 simultaneously for the two groups were qualified for successive analyses with multivariate logistic regression with p<0.05 as a criterion. These were then subjected to form an index using a linear equation with the log(OR) as weights for the various proteins, then presented in receiver-operating characteristic (ROC) analysis. Furthermore, the possibility of an age and/or sex impact on the index was investigated on the odds ratios. Statistical analyses were performed in Statistica version 13.

 Ethics

The study was approved by the respective regional ethical review boards in Sweden, Åland and Norway. For Sweden 2013/238-31, 2014/326-32, 2016/211-31, 2019–03832, for Åland the approval was given at the regional ethical review board meeting no 2/2014 and for Norway 2014/1100 and additionally September 13, 2019 with reference no 9463. All study participants agreed to take part in the study and signed a written informed consent form.

 Results

A complete set of laboratory results with approved quality control results were obtained from all 52 LB patients and 75 of the original 80 blood donors. Thus, five blood donors with incomplete or failed results were excluded from the study (three anti-Borrelia antibody positive and two anti-Borrelia antibody negative). Basic demographics of the LB patients and the remaining blood donors are shown in Table 1.

10.1371/journal.pone.0287586.t001 Demographic characteristics of Lyme borreliosis patients (n = 52) and blood donors (n = 75).
Clinical group Lyme borreliosis patients (n = 52) Blood donors (n = 75)
  Definite LNB (n = 41) ACA/LA (n = 7) Erythema migrans (n = 4) Blood donors AB+ (n = 34) Blood donors AB− (n = 41)
Sex, female/male (% female) 25/16 (70) 4/3 (57) 3/1 (75) 6/28 (18) 13/28 (32)
Median age in years (range) 61 (21–85) 48 (27–55) 51 (45–69) 44 (20–68) 48 (21–66)
Self-reported previous LB (%) N/A N/A N/A 6 (18) 2 (5)

LNB, Lyme neuroborreliosis; ACA, Acrodermatitis chronica atrophicans; LA, Lyme arthritis; AB+, anti-Borrelia antibody positive; AB−, anti-Borrelia antibody negative; LB, Lyme borreliosis

N/A, not available

The LB patients (n = 52) consisted of the following manifestations; Lyme neuroborreliosis (n = 41), acrodermatitis chronica atrophicans and/or Lyme arthritis (n = 7) and erythema migrans (n = 4). Further details regarding the subjects may be found in the publication by Lager et al. [12]. Thus, LB patients were all sampled during an ongoing manifestation of LB, while the blood donors were all healthy at sampling. Median normalised protein expression values in log2 scale together with quartile one-three results for the included 92 parameters from the remaining 52 LB patients and 75 blood donors (34 anti-Borrelia antibody positive and 41 anti-Borrelia antibody negative) are shown in S1 Table. In total, ten of the 92 variables showed a difference with p<0.1, further included in a logistic regression model in which six (PPP1R9B, PRDX5, ITM2A, EIF4GI, DDX58, ITGB6) of the ten protein markers remained (p<0.05), Table 2. Furthermore, an index was created using the individual results for these six protein markers: Index=PPP1R9B*(‐1.962)+PRDX5*(2.307)+ITM2A*(‐2.632)+EIF4G1*(‐2.477)+DDX58*(2.295)+ITGB6*(‐2.121)

10.1371/journal.pone.0287586.t002 Median (Q1-Q3) of the ten proteins qualified for the successive multivariate logistic regression.

OR is odds ratio with 95% confidence limits where control is reference.

Lyme borreliosis patients Blood donors
Protein Uniprot_ID n = 52 n = 75 p-value* Odds ratio (95% CL) p-value
CKAP4 Q07065 4.10 (3.96–4.33) 3.93 (3.72–4.15) 0.071
DDX58 O95786 2.43 (2.00–2.89) 1.95 (1.75–2.40) 0.037 9.93 (1.49–66.21) 0.018
EIF4G1 Q04637 2.30 (1.84–2.84) 2.59 (2.25–3.03) 0.087 0.08 (0.01–0.58) 0.013
HEXIM1 O94992 3.27 (2.95–3.46) 3.39 (3.10–3.85) 0.052
IL6 P05231 3.09 (2.51–3.55) 2.29 (1.87–2.73) 0.004
ITGB6 P18564 2.66 (2.37–2.98) 3.03 (2.76–3.23) 0.004 0.12 (0.01–0.97) 0.046
ITM2A O43736 3.06 (2.67–3.45) 3.75 (3.41–4.03) 0.012 0.07 (0.01–0.41) 0.003
PPP1R9B Q96SB3 2.12 (1.09–2.62) 2.59 (2.29–2.98) 0.076 0.14 (0.04–0.51) 0.003
PRDX5 P30044 5.57 (4.93–6.17) 4.77 (4.24–5.26) 0.016 10.0 (2.0–50.6) 0.006
SH2B3 Q9UQQ2 0.98 (0.98–2.22) 2.33 (0.98–2.93) 0.008    

*) p-values from Mann-Whitneys U-test (<0.1), necessary for inclusion into the succeeding logistic regression (for details see "Methods").

For comparison between LB patients and blood donors, this index was used in a subsequent ROC curve analysis shown in Fig 1. A significant difference (p<0.001) was shown with an area under curve (AUC) of 0.964. In addition, two further ROC curve analyses of subgroups of patients and blood donors were performed as follows: a) LB patients (n = 52) and the subgroup of anti-Borrelia antibody positive blood donors (n = 34) showed an AUC of 0.973 and b) Lyme neuroborreliosis patients (n = 41) and all blood donors (n = 75) resulting in an AUC of 0.962. No statistically significant differences were found comparing these three AUCs.

10.1371/journal.pone.0287586.g001 Receiver operating characteristic curve displaying the diagnostic power in the six protein index discriminating Lyme borreliosis patients from blood donors.

AUC, area under curve.

Using an index criterion value of >−8.8178 to discriminate LB patients from blood donors in the original ROC curve analysis, a sensitivity of 86% and a specificity of 92% could be achieved. As shown by the index calculation, high values of PRDX5 and DDX58 in combination with low values for the remaining markers were typical for the LB patients, while the opposite pattern was characteristic for the blood donors. Age and sex had no impact on the odds ratio of the index, p-values for odds ratios were for age 0.68 and for sex 0.73.

 Discussion

We here show the possibility to successfully distinguish active ongoing LB from blood donors with and without previous exposure to Borrelia using a multiplex biochemical approach independent from Borrelia serology. The results in this study imply that a number of immune-related serum protein biomarkers may serve this purpose.

In detail, the results show that six proteins are indicated, namely the gene products corresponding to PPP1R9B, PRDX5, ITM2A, EIF4GI, DDX58, and ITGB6. These proteins serve different functions in the human body briefly described here. PPP1R9B, protein phosphatase 1 regulatory subunit 9B, also named neurabin2 and spinophilin seems to be involved in multiple signalling pathways as a scaffold protein. Furthermore, it has been reported to play a significant role as a link between actin cytoskeleton and the plasma membrane at synaptic junctions as well as a G-protein signalling regulator. It also appears that this protein is of special importance in the nervous system [16, 17]. PRDX5, peroxiredoxin-5, also known as PMP20 is a thiol-specific peroxidase that catalyses reduction of organic hydroperoxides and hydrogen peroxide thereby playing a role in cell protection against oxidative stress [17, 18]. ITM2A, integral membrane protein 2A, has been reported to play a role in regulating the development and function of T cells, but additional data on the function of this protein is lacking [19]. EIF4GI, eukaryotic translation initiation factor 4 gamma 1, constitutes a part of the protein complex eIF4F, central for ribosome protein translation initiation and its function thereby regulates protein synthesis. Several viruses have been reported to modify the function of EIF4G by proteolytic cleavage resulting in down-regulation of cellular translation initiation [20], thus a target protein that may be relevant for host defence in infections. DDX58 corresponds to an antiviral innate immune response receptor retinoic acid-induced gene-I (RIG-I), that senses viral nucleic acids in the cytoplasm and further activates a signalling cascade that leads to the production of pro-inflammatory cytokines and type I interferons, which also may play important roles in bacterial infections [21]. Finally, ITGB6, integrin subunit beta 6, constitutes a part of the heterodimeric integrin αvβ6 transmembrane receptor exclusively expressed in epithelial cells. Integrin αvβ6 may serve as a receptor for fibronectin, but one of its major functions is to activate transforming growth factor-β1, a key regulator of the innate anti-inflammatory surveillance of the body [22]. Taken together, the combined profile suggests that these six proteins may represent relevant reactions caused by the pathogen in combination with the host immune defence thus including cellular signal transduction, regulation of oxidative stress and protein synthesis, and regulation of cellular immunity as well as inflammatory and anti-inflammatory reactions necessary for clearance of the pathogen. Interestingly, the markers may be both up- and downregulated, and in ongoing LB both PRDX5 and DDX58 were upregulated while the remaining markers were downregulated, suggesting importance of this specific pattern in the present ongoing LB infection phase. To our knowledge, these six markers have not previously been investigated and shown relevant in the perspective of LB. Thus, the significance of them in the discrimination of active from previous infection needs further confirmation in other clinical LB materials as well as in differential conditions to LB including other infectious and inflammatory conditions. Furthermore, longitudinal studies of confirmed LB patients with follow-up sampling after treatment and resolution of symptoms would be of great value as well as patients classified after additional and other LB serological assays.

The strengths of this study include the well-characterised and relevant clinical LB patients together with subjects representing both healthy controls (blood donors) with and without anti-Borrelia antibodies. Moreover, the included LB patients represent the major and most important of the various clinical manifestations of LB in Europe. Additionally, the proximity extension assay methodology applied to the serum material is sensitive and specific [14]. Regarding limitations, although a reasonable number of LB patients and blood donors were investigated, the material is still limited in number, and follow-up samples were not available for the patients which would have been a valuable source of confirmation. Also, the number of studied parameters in relation to the number of available samples needs to be handled with caution in order to avoid random statistical effects.

To conclude, we here show a possibility to discriminate ongoing LB from previous exposure to Borrelia using a powerful multiplex serum protein approach. This new strategy is interesting and opens for improvements in the diagnostics of LB in principle although not adapted for clinical use at this point. Further studies on well-characterised clinical materials including follow-up samples and relevant control conditions are warranted. Additional screening using multiplex technology may confirm our data and possibly also detect new and fewer biomarkers with the same diagnostic power suitable for routine use.

 Supporting information <LOD; Frequency of samples with results below the lowest concentration possible to detect.

*Highest p-value below 0.1 of two randomly selected halves of the study population calculated using Mann-Whitney’s U-test (for details see the original publication).

(DOCX)

The authors would like to acknowledge support of the Clinical biomarker facility at SciLifeLab Sweden for providing assistance in protein analyses. We would also like to thank the patients and blood donors for participating in this study as well as all involved health care professionals and departments for valuable assistance.

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Epub 20180418. doi: 10.1016/j.biocel.2018.04.013 29678785. 10.1371/journal.pone.0287586.r001 Decision Letter 0 Chegou Novel Njweipi Academic Editor 2023 Novel Njweipi Chegou This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 0

30 Mar 2023

PONE-D-23-05893A novel laboratory approach to discriminate active Lyme borreliosis from previous Borrelia exposurePLOS ONE

Dear Dr. Tjernberg,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Additional Editor Comments (if provided): Major Revision

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Reviewers' comments:

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Comments to the Author

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: No

Reviewer #3: No

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

Reviewer #1: I Don't Know

Reviewer #2: No

Reviewer #3: No

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Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In the manuscript by Dr. Tjernberg et al, the authors evaluated if detemining of selected immune response-related human protein biomarkers in serum using a proximity extension assay could be used to differentiate between patients with active Lyme borreliosis and healthy controls. The study seaks answers to an important clinical question i.e. how one could exclude with high probabilty the possibility of an active borrelial infection (and defer antibiotics) in patients with clinicaly suspected Lyme borreliosis, but not fulfiling criteria for definite LB. A new diagnostic approach beside serology would be of great value particularly in LB endemic areas with high background Borrelia seropositivity.

I have listed some concerns regarding the study design in the attached manuscript. Furthermore, I think the rational/reasons for selecting the particular set of immune response-related human protein biomarkers should be provided in the Introduction part. The comparative group of healthy blood donors could be described in more detail or defined more specificaly e.g. did seropositive asymptomatic controls had documented LB in the past? As I understand, the approach described would be a valuable addition to Borrelia serologic tests therfore, I would propose to make also a comparison between patients with active LB and those who are Borrelia seropositive, but do not have LB.

Reviewer #2: The manuscript by Tjernberg et al. addressed an important problem, which is that of discrimination between active Lyme borreliosis from resolved Borrelia infection after exposure. The paper however is poorly writing and needs major revisions before being considered for publication. There are major concerns associated with the study design and the proper discrimination of false positive.

Major concerns

Study design

There is logical and/or methodological problem with the study design, as the category ‘previous exposure’ can be included under ‘ongoing LB infection’. This is; patients with ‘previous exposure’ could have an ‘ongoing LB infection’. How the authors rule out that ‘blood donors with’ ‘anti-Borrelia antibodies’ did not had an ‘ongoing LB infection’? Are the authors referring to ‘acute’ vs. ‘chronic’ LB? In either case, the patients included in the study do not seem to be properly classified and/or the classification does not separate individuals beyond ‘seropositive’ or ‘seronegative’ to LB. Therefore, the question set by the authors need revision.

Methodology

It is known that several Borrelia burgdorferi proteins cross-react with that from other bacteria (see e.g., Eur J Clin Microbiol Infect Dis. 1992;11(3):224-32). Particularly, excessively high rates of false positive results of ELISA tests for Lyme Borreliosis (> 90% of positives were found to be false positives) were significantly reduced after adsorption of patient sera with Escherichia coli antigens (see e.g., J Rheumatol. 1995;22(4):684-8). Not clear how the authors dealt here with ‘false positive’ samples in the present study. For example, will sera with anti-Borrelia antibodies be still positive if adsorption with Escherichia coli antigens is performed?

In general, the methodology is poorly described. Scientific papers must include details so that others can repeat the experiments and/or analysis published.

Minor comments

Abstract

Line 58: It is confusing that the authors set to ‘discriminate an ongoing Lyme borreliosis (LB) infection from a previous exposure.’ (Lines 53-54), however, the cohort of LB patients (as mentioned in line 58) does not include patients with ‘ongoing LB infection’ and other with ‘previous exposure’. If these two groups of patients were included, this has to be mentioned here.

Line 65: Not clear what ‘immune-related proteins’ mean.

Line 68: These abbreviations are not very helpful, even less in an abstract.

Methods

Line 112: Based on what criteria was the ‘subset of serum samples from a previous prospective study’ selected? What the authors mean by ‘well characterised adult LB’? Provide further details.

Lines 116-117: Please, include general information on the location of origin of the ‘patients’ and ‘blood donors’.

Lines 118-120: ‘Classification and confirmation of LB diagnosis’ is poorly described. Improve.

Lines 120-122: Provide details about this kit. What antigen(s) the kit uses? What dilution of reagents was used?

Line 125: What ‘symptoms or signs of disease’ were considered?

Lines 130-141: This section has to be separated into different subsections and each one of these detail the methodologies used. For example, what ‘multiple analytes are detected in parallel’? How the ‘unique strands of DNA oligonucleotides’ and ‘individually paired matched antibodies’ were generated? How were these specific to LB? How the Borrelia proteins tested in the assay were obtained? How the ‘quantitative PCR’ was performed? What adaptors were used?

Reviewer #3: 1. According to the objectives, the authors aimed to investigate immunological protein biomarkers to search for specific patterns for active Lyme borreliosis. They conclude that levels of a number of them differed significantly from healthy blood donors, whether or not previously exposed to Borrelia.

My major comment is that it is not surprising that patients with an active infectious disease differ from healthy controls with regard to some immunological protein biomarkers. This is also the case for CXCL13 in CSF for active neuroborreliosis. However, in that entity patients with neuroborreliosis have also extensively compared with patients with other infectious and non-infectious diseases and high levels of this specific biomarker are rather specific for neuroborreliosis.

In the present paper, any comparison of the relevance of these immunological protein biomarkers in Lyme borreliosis and in other infectious and non-infectious diseases is lacking. Whereas I agree that in a pilot study like this one it may not yet be relevant to test such patient groups, I suppose that these biomarkers have also been studied in other diseases. Therefore I think it is necessary to include a section about what is known of the supposedly relevant biomarkers in other diseases. Are they non-specific and increased with inflammation, like CRP, or are their levels increased –or decreased- especially in Lyme borreliosis?

2. Some of the blood donors were seropositive for LB, others were not. This is extensively mentioned in the abstract. However, it is not at all stated whether seropositive donors differed from seronegative donors with regard to any marker. Probably, this is not the case, but the authors should clarify this. If any difference would exist between these populations, follow-up procedures comparing all blood donors and LB patients would not be valid.

3. The title suggests that the paper was aimed at comparing active Lyme borreliosis with previous Borrelia exposure. However, as stated in 2., patients with active LB are not compared to patients with previous Borrelia exposure – and follow-up samples of treated LB patients would be more relevant to provide insight in differences between active and past LB. The title should rather be “A novel laboratory approach to discriminate active Lyme borreliosis form healthy controls” or more appropriate, “Identification of potential biomarkers in active Lyme borreliosis”

4. Another limitation of the study is that most patients (41/52) were patients with neuroborreliosis. The number of patients with arthritis, acrodermatitis and erythema migrans was small and results might not be applicable to those patients. The authors might also consider to calculate the AUC for the comparison blood donors – neuroborreliosis patients, since this is a more clearly described clinical entity and this comparison could be more relevant for clinical practice.

5. It is not clear what is represented in table S1, probably same as in table 2?

Minor comment:

L.172: sentence is incomplete.

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Reviewer #1: Yes: Daša Stupica

Reviewer #2: No

Reviewer #3: Yes: Alje P. van Dam

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Submitted filename: PONE-D-23-05893_reviewer.pdf

 10.1371/journal.pone.0287586.r002 Author response to Decision Letter 0 Submission Version 1

30 May 2023

We thank the reviewers for valuable comments and suggestions in order to improve the quality of the manuscript and refer to the document responses to reviewers for point-by-point responses.

Best regards,

Ivar Tjernberg, MD PhD Associate professor

Submitted filename: Responses to reviewers.docx

 10.1371/journal.pone.0287586.r003 Decision Letter 1 Chegou Novel Njweipi Academic Editor 2023 Novel Njweipi Chegou This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Submission Version 1

8 Jun 2023

Identification of potential biomarkers in active Lyme borreliosis

PONE-D-23-05893R1

Dear Dr. Tjernberg

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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Kind regards,

Novel N. Chegou, Ph.D

Academic Editor

PLOS ONE

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Reviewers' comments:

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Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: I Don't Know

Reviewer #2: Yes

**********

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

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: Thank you to the authors for addressing the previous concerns positively............................

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Reviewer #1: No

Reviewer #2: No

**********

 10.1371/journal.pone.0287586.r004 Acceptance letter Chegou Novel Njweipi Academic Editor 2023 Novel Njweipi Chegou This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

19 Jun 2023

PONE-D-23-05893R1

Identification of potential biomarkers in active Lyme borreliosis

Dear Dr. Tjernberg:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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