# Lyme borreliosis diagnosis: state of the art of improvements and innovations

Mickaël Guérin, M. Shawky, Ahed Zedan, Stéphane Octave, Bérangère Avalle, Irene Maffucci, Séverine Padiolleau-Lefèvre

<figure><img src="image_1.png" /></figure>

## To cite this version:

Mickaël Guérin, M. Shawky, Ahed Zedan, Stéphane Octave, Bérangère Avalle, et al., Lyme borreliosis diagnosis: state of the art of improvements and innovations. BMC Microbiology, 2023, 23 (1), pp.204. 10.1186/s12866-023-02935-5. http://dx.doi.org/10.1186/s12866-023-02935-5

HAL Id: hal-04192113

https://hal.science/hal-04192113v1

Submitted on 31 Aug 2023

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
REVIEW

Open Access

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

# Lyme borreliosis diagnosis: state of the art of improvements and innovations

Mickaël Guérin¹, Marc Shawky², Ahed Zedan³, Stéphane Octave¹, Bérangère Avalle¹, Irene Maffucci¹ and Séverine Padiolleau-Lefèvre¹*

**Abstract**

With almost 700 000 estimated cases each year in the United States and Europe, Lyme borreliosis (LB), also called Lyme disease, is the most common tick-borne illness in the world. Transmitted by ticks of the genus *Ixodes* and caused by bacteria *Borrelia burgdorferi* sensu lato, LB occurs with various symptoms, such as erythema migrans, which is characteristic, whereas others involve blurred clinical features such as fatigue, headaches, arthralgia, and myalgia. The diagnosis of Lyme borreliosis, based on a standard two-tiered serology, is the subject of many debates and controversies, since it relies on an indirect approach which suffers from a low sensitivity depending on the stage of the disease. Above all, early detection of the disease raises some issues. Inappropriate diagnosis of Lyme borreliosis leads to therapeutic wandering, inducing potential chronic infection with a strong antibody response that fails to clear the infection. Early and proper detection of Lyme disease is essential to propose an adequate treatment to patients and avoid the persistence of the pathogen. This review presents the available tests, with an emphasis on the improvements of the current diagnosis, the innovative methods and ideas which, ultimately, will allow more precise detection of LB.

**Keywords** Lyme disease, Tick, Diagnostic, Culture, PCR, Serology detection, Direct and indirect methods

**Introduction**

Lyme borreliosis (LB), discovered in 1975 and commonly known as Lyme disease, is transmitted by ticks of the genus *Ixodes*. Etiological agents are the spirochete bacteria from the *Borrelia burgdorferi* sensu lato complex (*Bbsl*). The distribution of the various genospecies is detailed in Fig. 1 [1, 2]. Recent studies aim to update the genospecies distribution worldwide for better diagnosis of LB [3–6].

The genospecies, reported inside the *Borreliae* Lyme Group (*BLG*), are different in terms of clinical manifestation, dissemination insides the human body but also in terms of genomes/antigens expression. Regarding the symptoms, it has been described for example that, *Bbss* has shown to be arthritogenic, whereas *B. afzelii* causes skin infections and *B. garinii* is especially neurotropic. Recently, *B. mayonni*, member of the *BLG*, has been associated with unusually high spirochaetemia, like Tick Borrelia Relapsing Fever (TBRF) genospecies, while the others genospecies causing LB are characterized by being organotrophic with a very low level of spirochaetemia [2, 7, 8].

The heterogeneity of both the clinical manifestation and the species distribution worldwide leads to regional differences in terms of diagnosis [9]. Indeed, when focusing on the regional genospecies differences and concerning the genome of the complex *Bbsl*, it has been shown that even if the linear chromosome of 910 kilobases is

<footnote>*Correspondence:
Séverine Padiolleau-Lefèvre
severine.padiolleau@utc.fr

¹ Unité de Génie Enzymatique Et Cellulaire (GEC), CNRS UMR 7025,
Université de Technologie de Compiègne, 60203 Compiègne, France

² Connaissance Organisation Et Systèmes TECHniques (COSTECH), EA
2223, Université de Technologie de Compiègne, 60203 Compiègne,
France

³ Polyclinique Saint Côme, 7 Rue Jean Jacques Bernard,
60204 Compiègne, France</footnote>
<table><caption>**Fig. 1** Geographic distribution of *Borrelia* species</caption><thead><tr><th>Localization</th><th>Borrelia genospecies</th></tr></thead><tbody><tr><td rowspan="3">NORTH AMERICA</td><td>Bbss</td></tr><tr><td>B. mayonii</td></tr><tr><td>B. bissettii</td></tr><tr><td rowspan="10">EUROPE</td><td>Bbss</td></tr><tr><td>B. afzelii</td></tr><tr><td>B. garinii</td></tr><tr><td>B. spielmanii</td></tr><tr><td>B. bavariensis</td></tr><tr><td>B. bissettii</td></tr><tr><td>B. lusitaniae</td></tr><tr><td>B. mayonii</td></tr><tr><td>B. valaisiana</td></tr><tr><td rowspan="7">ASIA</td><td>Bbss</td></tr><tr><td>B. afzelii</td></tr><tr><td>B. garinii</td></tr><tr><td>B. spielmanii</td></tr><tr><td>B. bavariensis</td></tr><tr><td>B. bissettii</td></tr><tr><td>B. valaisiana</td></tr></tbody></table>

highly conserved among the BLG, the plasmids (9 linear plasmids and 12 circular plasmids) [10] show a high degree of variation [9, 11, 12]. Some researchers have shown, for example by studying OspC (Outer Surface Proteins) typing or by multilocus sequence analysis (MLSA), many differences between genospecies in Europe and the United States [13–15]. Intraspecies diversity has also been noted, in the US, where patients from New York and Wisconsin were infected by two distinct populations of *Bbss* but genetically and phylogenetically closely related [16, 17]. The intricate nature of diverse Borrelia genospecies within Europe and in the US, added to the antigenic variation abilities of the genospecies, underscores the need for diagnostic assays capable of detecting all pathogenic strains.

LB is the most common tick-borne illness and every year affects for 476 000 estimated cases in the United States and more than 200 000 cases in Europe [18–20]. LB is underdiagnosed and/or misdiagnosed, making the estimated incidence controversial with probably a considerably higher number of cases [21].

Patients suffering from this disease have various symptoms depending on the stage of its evolution. Commonly divided into three stages—early localized, early disseminated, and late disseminated—the only pathognomonic symptom, erythema migrans (EM), is a cutaneous manifestation, appearing during the first stage, at the site of the tick bite. However, EM is not systematically detected and only non-characteristic symptoms linked to stages 2 and 3 (Lyme neuroborreliosis, carditis, or arthritis) can

be expressed [1, 22]. It is important to emphasize that the risk of the appearance of other symptoms from LB (especially neurologic and articular manifestations) is significantly reduced after the recognition of EM and the rapid initiation of antibiotic treatment [23].

Indeed, if LB is detected correctly, timely and early enough, its treatment is simple and involves an antibiotic therapy, which allows solving 80 to 90% of *Borrelia* infection cases [24]. However, the Lyme disease diagnosis is not straightforward, because of the above-mentioned blurred symptoms and the limits of the approved tests. Indeed, although other tests exist, the diagnosis of LB is currently based on serology using two tests: an enzyme-linked immunosorbent assay (ELISA) and a Western Blot (WB). These serological tests are indirect diagnostic methods measuring the presence of anti-*Bb* antibodies (Abs) [25]. Unfortunately, these techniques have many limitations [26]. *Bbsl* is able to escape the immune system according to a large variety of mechanisms, inducing a reduced immune protection despite the activation of innate and adaptive immunity [27]. Nevertheless, even if secreted antibodies fail to efficiently protect individuals, they should be exploitable for serodetection and indirect diagnosis. But, in practice, serodetection lacks of sensitivity in the early stage of the disease for many reasons, including, possibly, an insufficient time interval between the infection and the detection test, also called “window period”, or the use of antibiotic treatment limiting the development of a strong antibody response (see Sect. 1.1). Added to
the possible cross-reactivity of antibodies, due to common antigens with other diseases, or to the co-infection issues suggesting that targeting a specific antibody is not sufficient [28], indirect measurement causes false positive or false negative results. Finally, the indirect approach doesn't allow to distinguish between a past but cleared infection, and an active one [29]. Besides hampering the LB diagnosis and healing, the question of long or chronic form of the disease frequently raises [30]. Although still unclear, some hypotheses on the etiology of the persistent symptoms have been done, involving autoimmunity, cross-reactivity, molecular mimicry, co-infections/co-transmission, or borrelial tolerance to antibiotics [24, 31–33]. Detecting *Bbsl* bacteria despite treatment could help to decide if the antibiotics-based therapy has to be prolonged or not.

In addition to previously published reports [18, 34–37], this review aims to update and wholly collect the knowledge and data available on the Lyme diagnosis issue, to summarize the limitations of direct and indirect LB detection methods, but also to present new strategies required to avoid therapeutic wandering (Fig. 2).

## Indirect diagnosis

### Standard two-tiered testing (STTT) and modified two-tiered testing (MTTT)

Currently, serology testing, i.e. indirect diagnosis, is the official recommendation for the diagnosis of LB [38]. Serology is based on Abs detection following the immune response of the host after infection. However, serology encounters limitations to detect early localized Lyme disease, as well as early disseminated or late-stage LB. Indeed, due to the latency time of the immune response after bacterial or viral infection, detecting early Lyme disease, especially during its acute phase, using serology may be useless, since the diagnosis rarely exceeds a sensitivity beyond 50% [39]. Moreover, after treatment of an active infection, Abs can still be detected in serum from months to years after the infection [40]. This makes the serology unsuitable for monitoring response to treatment or identifying new infections.

Since 1994, the Centers for Disease Control and Prevention (CDC) guidelines and recommendations consist of a standard two-tiered serologic testing approach, called STTT, to maintain a high sensitivity and optimize the specificity. Firstly, an ELISA is performed. If the

<figure><img src="image_4.png" /><figcaption>**Fig. 2** Holistic view of diagnostic approaches performed from patient fluids. Boxes in pink are direct detection methods whereas boxes in blue are indirect ones, potentially applied in the absence of Erythema migrans (represented by concentric circles). Others red spots represent some possible localizations of symptoms</figcaption></figure>
result is borderline or positive, a confirmatory test by WB
is required [25].

The ELISA method is a very well-known technique
used in diagnosis. It allows the detection of immuno-
globulins (Ig) G and/or IgM. Briefly, antigens specific to
*Bb* are coated on a plate and the patient's sera are chal-
lenged for these antigens' recognition. A secondary Ab
labelled with an enzyme or a tag will bind to the crys-
tallizable fragment (Fc) of the patient's Ab (Fig. 3A).
Adding the substrate induces a color change or fluo-
rescence reaction that can be measured with a suit-
able detection system. The reliability of ELISA depends
on the antigen's identity and its preparation. The first
generation of ELISA for LB diagnosis was based on spi-
rochete lysates obtained by sonication. However, this
generation lacked sensitivity. Indeed, a large diversity of
the antigen is thus coated on a plate, among which only
a few are immunodominant. Thus, the ability to cap-
ture predominant antibodies is reduced. Furthermore,
the expression of multiple antigens of *Bb* changes,
depending of the bacteria's localization (*in-vitro* cul-
ture, midgut of unfed ticks, when the tick starts feeding
on mammals, or at different stages of human infection).

The first generation of ELISA does not contain multi-
ple antigens expressed later during the human infec-
tion such as VlsE (Variable major protein Like sequence
Expressed) or OspC [41, 42]. This generation based on
the Whole-Cell Sonicate (WCS) also lacked of specific-
ity due to antibody cross-reactivity with proteins con-
served between *Bb* and other commonly encountered
bacteria (heat shock and flagellar proteins) [43]. Then,
a second generation has been developed using purified,
synthetic or recombinant antigens, such as the surface
lipoproteins OspC, OspA, or VlsE [44]. Thus, the use
of a synthetic peptide derived from the VlsE sequence
(C6 peptide), which is highly immunogenic and well
conserved [45, 46], or C10 peptide derived from the
conserved amino-terminal portion of OspC (namely
pepC10), is also used [47].

After a positive or borderline result, a WB is usually
performed. The principle of WB is the same as ELISA.
Abs produced consecutively to a *Bb* infection are even-
tually detected. The separation of *Bb* antigens by SDS-
PAGE according to a characteristic migration profile
preceding the detection confirms the data by increas-
ing the specificity and by reinforcing the diagnosis. WB

<figure><img src="image_5.png" /><figcaption>**Fig. 3** Indirect detection tests</figcaption></figure>
usually relies on *Bb* cell lysates and/or recombinant antigens [48, 49].

Sensitivity and specificity remain major issues for these techniques (Table 1). Besides the risk of false negative results due to poor sensitivity for early Lyme disease, a recent study showed that current tests generate also many false positives, i.e. showed poor specificity, leading to incorrect treatment of the patient [50]. In this work, the authors evaluated the reactivity of sera from patients with viral infections (Epstein-Barr virus (EBV) or cyto-megalovirus (CMV)) with the *Borrelia* antigens used in the serological tests. Many false positives have been observed and are probably related to the cross-reaction of Abs produced during the lymphocyte response activated by the presence of viral superantigens. False positive enzyme immunoassay (EIA) is also reported due to the presence of cross-reactive Abs, due to common antigens with other diseases, including, but not only, tick-borne relapsing fever, anaplasmosis, and diseases related to the presence of *Helicobacter pylori* [51], *Treponema denticola* [38] and *T. pallidum*, the etiological agent of syphilis [52]. Much effort is made to overcome this problem. Recently, Arnaboldi et al. generated peptides from linear B cell epitope mapping. The purpose is dual: i) solve the specificity issues thanks to peptides—rather than complex protein mixes—able to specifically capture serum Abs, and ii) solve the sensitivity issues thanks to the use of a rationalized variety of such peptides, each representing an immunodominant epitope from a bacterial mix. The final aim is to ensure the development of a multi-peptide-based assay [53].

Many reasons can also be singled out for influencing the STTT sensitivity and specificity, and should be nuanced. The first one is related to the studies themselves. Tests on patients with early LB have a lower sensitivity in the STTT and MTTT than patients with late

LB. The main reason is the early LB patients do not have enough time to develop an effective antibody response. The second reason is depending on the patient's treatment. Indeed, an effective antibiotic treatment during the early LB diminishes the antibody response from developing, meaning that patients are less likely to be tested positive on serological tests [62].

In contrast, patients with late LB such as Lyme arthritis, Lyme neuroborreliosis, or acrodermatitis chronica atrophicans (ACA) often have strong antibody responses to serological tests with high sensitivity and high specificity. Moreover, serological tests specificity is very dependent to the number of samples, especially between (i) healthy controls, (ii) potentially-cross reactive patients with others diseases, and (iii) LB patients. Finally, the patients' localization used for the studies also greatly influences the sensitivity and the specificity. The main reasons are (i) the species involved in the disease (*Bbss* in North America and *B. garinii*, *B. afzelii*, *B. spielmanii*, *B. bavariensis*, in Europe) and (ii) the criteria applied for considering the positivity of the diagnosis are different [58].

Besides the specificity and sensibility issues, STTT is time-consuming, requires skilled operators, suffers from subjective interpretation and from its inability to distinguish between an active and past infection [63]. These drawbacks highlight the need for improvements.

Since the introduction of the STTT at the Dearborn conference in 1994, variations in the combination of these tests have been proposed to improve the sensitivity and specificity [64, 65]. In July 2019, the Food and Drug Administration (FDA) cleared some EIAs to be used in a MTTT that might help to resolve the previously mentioned issues [66]. Replacing the WB, which can be time consuming and whose interpretation can be subjective, with another serological test, and the use of recombinant

<table><caption>**Table 1** Sensitivity and specificity for STTT in America and in Europe</caption><thead><tr><th rowspan="3">Symptoms / Stage of the disease</th><th colspan="3">Patient's localization</th><th colspan="3">Europe</th></tr><tr><th colspan="3">America</th><th colspan="3">Europe</th></tr><tr><th>Sensitivity<sup>a</sup></th><th>Specificity<sup>b</sup></th><th>References</th><th>Sensitivity</th><th>Specificity</th><th>References</th></tr></thead><tbody><tr><td>Erythema migrans or early Lyme borreliosis<br><i>Acute phase<sup>c</sup> (AP)</i></td><td>11–50%</td><td>99%</td><td>[29, 34, 54–57]</td><td>23–55%</td><td>75–99%</td><td>[34, 58–61]</td></tr><tr><td>Erythema migrans or early Lyme borreliosis<br><i>Convalescent phase<sup>d</sup> (CP)</i></td><td>18–78%</td><td>99%</td><td></td><td>30–67%</td><td>95%</td><td>[61]</td></tr><tr><td>Acute-phase neurologic or cardiac involvement</td><td>60–100%</td><td>99%</td><td></td><td>69–97%</td><td>98–99%</td><td>[58, 61]</td></tr><tr><td>Arthritis or late neurologic involvement</td><td>97–100%</td><td>99%</td><td></td><td>78–100%</td><td>98–99%</td><td></td></tr></tbody></table>

<footnote><sup>a</sup> Ability of the test to identify the presence of a disease correctly (CDC)</footnote>

<footnote><sup>b</sup> Ability of the test to identify the absence of a disease correctly (CDC)</footnote>

<footnote><sup>c</sup> Phase during an active infection, characterized by active multiplication of the pathogens and pronounced symptoms</footnote>

<footnote><sup>d</sup> Phase after the antibiotic treatment</footnote>
or synthetic antigens increased the specificity and sensitivity (at an early stage) of the two-tiered testing [34, 66]. A non-exhaustive list of the different used techniques is reported in Table 2. In this context, Waddell and collaborators reviewed the sensitivity and specificity of these tests according to the stage of the disease and the combination of the approaches [67]. The two-tiered testing is nowadays still necessary because unless single test have some close sensitivity or specificity, none attained or surpassed the traditional two-tiered testing [59].

## Antibodies-based approaches

The development of a screening test which would not require specific materials, nor laboratory equipment, but available for a self-test, appears to be an appealing

option. One of the main methods with these characteristics, which was widely used for the COVID-19 crisis, is called Lateral Flow ImmunoAssay (LFIA) technology. It has many advantages: it is fast and simple to perform, easy to interpret, and can be performed outside the laboratory. This technology is simple and derived from the ELISA principle. A previously treated and dried sample paper receives the biological sample and the migration buffer. This sample, named analyte, migrates by capillary action on the paper containing a colored particle (latex, colloidal gold...) coupled to Abs against human IgG or IgM, considered as antigen. The analyte-colored particle complex then migrates through a nitrocellulose membrane coated with a bacterial protein able to capture the complex. The formation of the complete complex will

<table><caption>**Table 2** Examples of sensitivity and specificity for MTTT in America and in Europe</caption><thead><tr><th colspan="2">Methods used</th><th rowspan="2">Symptoms / Stage of the disease</th><th colspan="4">Patient's localization</th><th colspan="3">Europe</th></tr><tr><th>First test</th><th>Second test</th><th colspan="3">America</th><th colspan="3">Europe</th></tr><tr><th></th><th></th><th></th><th>Sensitivity</th><th>Specificity</th><th>References</th><th>Sensitivity</th><th>Specificity</th><th>References</th></tr></thead><tbody><tr><td rowspan="4">VISE C6-ELISA</td><td rowspan="4">na</td><td>Erythema migrans or early Lyme borreliosis AP</td><td>29%</td><td>96%</td><td>[68]</td><td>65-68%</td><td>91-92%</td><td>[34, 61]</td></tr><tr><td>Erythema migrans or early Lyme borreliosis CP</td><td>56%</td><td>96%</td><td></td><td>80-82%</td><td></td><td></td></tr><tr><td>Acute-phase neurologic or cardiac involvement</td><td>98-100%</td><td>96%</td><td></td><td>94-100%</td><td></td><td></td></tr><tr><td>Arthritis or late neurologic involvement</td><td>98-100%</td><td>96%</td><td></td><td>94-100%</td><td></td><td></td></tr><tr><td>Enzygnost-2®</td><td>C6-ELISA</td><td>Erythema migrans or early Lyme borreliosis</td><td>na</td><td></td><td></td><td>78%</td><td>96%</td><td>[59]</td></tr><tr><td rowspan="2">Whole-Cell Sonicate ELISA</td><td rowspan="2">C6-ELISA</td><td>Erythema migrans or early Lyme borreliosis AP</td><td>38-58%</td><td>98-100%</td><td>[20, 34]</td><td>na</td><td></td><td></td></tr><tr><td>Erythema migrans or early Lyme borreliosis CP</td><td>76-79%</td><td>98-100%</td><td></td><td></td><td></td><td></td></tr><tr><td>Borrelia DotBlot G®</td><td>na</td><td>nd</td><td>93%</td><td>35%</td><td>[69]</td><td></td><td></td><td></td></tr><tr><td>MarDX® EIA</td><td>na</td><td></td><td>100%</td><td>92%</td><td></td><td></td><td></td><td></td></tr><tr><td>VIDAS®</td><td>na</td><td></td><td>100%</td><td>90%</td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="4">VISE1-IgG</td><td rowspan="4">pepC10-IgM</td><td>Erythema migrans or early Lyme borreliosis AP</td><td>46%</td><td>96%</td><td>[54]</td><td></td><td></td><td></td></tr><tr><td>Erythema migrans or early Lyme borreliosis CP</td><td>89%</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Acute-phase neurologic or cardiac involvement</td><td>100%</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Arthritis or late neurologic involvement</td><td>100%</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td rowspan="4">VISE1-pepC10</td><td rowspan="4">C6-ELISA</td><td>Erythema migrans or early Lyme borreliosis AP</td><td>na</td><td></td><td></td><td>51%</td><td>94-95%</td><td>[61]</td></tr><tr><td>Erythema migrans or early Lyme borreliosis CP</td><td></td><td></td><td></td><td>67%</td><td></td><td></td></tr><tr><td>Acute-phase neurologic or cardiac involvement</td><td></td><td></td><td></td><td>88%</td><td></td><td></td></tr><tr><td>Arthritis or late neurologic involvement</td><td></td><td></td><td></td><td>90%</td><td></td><td></td></tr></tbody></table>

AP Acute phase, CP Convalescent phase, na Not applicable, nd not determined
provide a colored line according to a sandwich model. A line test containing Ig against conjugated Abs is used for control (Fig. 3B). Few commercialized tests exist, based on the antibody response of the host, such as the Lyme IgM or IgG (VEDA.LAB, Alençon, France) and the Keulo-test *Borreliose Complete IgM and IgG test* (Borreliose Complete; BioGenTechnologies, Steinfurt, Germany). However, they suffer from low sensitivity (30% on average) despite a quite good specificity: 85% to 88% [70]. The inclusion of a reader, as in the ReaScan + C6 LYME IgG test (index test; Reagena, Toivala, Finland), can help in the analysis of the results to avoid subjective interpretations by the user, which is a major drawback of these assays. For this device, a recent study determined sensitivity and specificity between 83 and 91% respectively [71]. However, these products are currently not recommended for diagnostic use by either the USA or the European institutions, since the sensitivity and specificity are lower than the traditional two-step testing process [72].

Considering the coinfection notion appears highly appealing since ticks transmit several pathogens. In this context, a multiplexed vertical flow assay (xVFA) is under development. It consists of different layers of paper driving a uniform vertical flow of buffer and serum through a detection membrane, allowing the detection of antibodies directed against numerous Bb-specific antigens on the sensing membrane such as OspC, BmpA, P41, decorin

binding protein B (DbpB), Crasp1, P35, or Erpd/Arp37, as well as the C6-like peptide. A colorimetric signal is observed after 15 min. The specificity and sensitivity of the test are 96.3% and 85.7%, respectively [73].

A new paramagnetic bead-based multiplex assay using Luminex™ xMAP™ INTELLIFLEX System for the simultaneous detection of Borrelia specific IgG/IgM class antibodies allowed to reduce experiment time and biosample material requirements (sensitivity and specificity are reported Table 3) [74].

Diminishing volumes and reagents is one of the ways to improve the LB diagnosis since the sample collection can be invasive. By combining these characteristics, microfluidics seems the most suitable method. Recently, Nayak et al. developed a point of care test based on this principle. It allows dealing with very small volumes of fluids, down to femtoliters. This rapid 15-min multiplexed laboratory test consists of a chip called mChip-Ld that detects anti-*B. burgdorferi* antibodies using the OspC-K antigen [75]. This test, very similar to LFIA, also uses a signal detection device. Thus, specific antibodies from the patient blood sample will bind to antigens immobilized on the surface of the microfluidic cassette. The peculiarity of this test is the silver amplification protocol to enhance the signal thanks to the silver ion reduction on gold nanoparticles attached to the cassette surface [88]. With a sensitivity and specificity of 84% and 92%,

<table><caption>**Table 3** Sensitivity and specificity for emerging but not yet accredited laboratory tests</caption><thead><tr><th>Classification</th><th>Tests</th><th>Sensitivity</th><th>Specificity</th><th>References</th></tr></thead><tbody><tr><td rowspan="5">Microfluidics, lateral and vertical flow immunoassay</td><td>mChip-Ld</td><td>84%</td><td>92%</td><td>[75]</td></tr><tr><td>Lyme IgM and IgG</td><td>26%</td><td>85%</td><td>[70]</td></tr><tr><td>Keul-o-test Borreliose Complete IgM and IgG test</td><td>32%</td><td>88%</td><td></td></tr><tr><td>ReaScan + C6 LYME IgG</td><td>83%</td><td>91%</td><td>[71]</td></tr><tr><td>Multiplexed vertical flow assay (xVFA)</td><td>86%</td><td>96%</td><td>[73]</td></tr><tr><td>Luminex</td><td>Paramagnetic bead-based multiplex assay and INTELLIFLEX System</td><td>94%</td><td>97%</td><td>[74]</td></tr><tr><td rowspan="5">ELISpot and cells-based approaches</td><td>iSpot LymeTM</td><td>54-84%</td><td>54-94%</td><td>[76, 77]</td></tr><tr><td>QuantiFERON-Lyme</td><td>70%</td><td>na</td><td>[78]</td></tr><tr><td>Spirofind</td><td>43%</td><td>82%</td><td>[77]</td></tr><tr><td>LTT-MELISA</td><td>30%</td><td>53%</td><td></td></tr><tr><td>TCR sequencing</td><td>56%</td><td>99%</td><td>[79]</td></tr><tr><td rowspan="4">Biomarkers detection</td><td>ReaScan CXCL13 assay</td><td>78%</td><td>95%</td><td>[80, 81]</td></tr><tr><td>RecomBead CXCL13 assay</td><td>86-100%</td><td>91-97%</td><td></td></tr><tr><td>Quantikine CXCL13 ELISA</td><td>88-100%</td><td>89-99%</td><td>[81, 82]</td></tr><tr><td>Metabolomics techniques</td><td>88%</td><td>95%</td><td>[83]</td></tr><tr><td rowspan="4">Raman Spectroscopy (RS)</td><td>SERS and DNA aptamers</td><td>91%</td><td>96%</td><td>[84]</td></tr><tr><td>Raman Spectroscopy</td><td>85%</td><td>90%</td><td>[85]</td></tr><tr><td>Raman Spectroscopy & Chemometrics</td><td>83%</td><td>91%</td><td>[86]</td></tr><tr><td>Immuno-PCR</td><td>69%</td><td>98%</td><td>[87]</td></tr></tbody></table>

Na not applicable
respectively for early diagnosis of the disease [75], this technique is promising, as confirmed by other studies, which showed that it provides an increase in sensitivity compared to the STTT approach [89].

## ELISpot and derived approaches

Many studies show that *Borrelia burgdorferi* can enter endothelial cells and macrophages [90–92]. This ability is one of the main components of the humoral immune escape mechanism of *Bb* [27]. Similarly to viral infections, intracellular activation of type I interferons (IFN) plays an important role in *B. burgdorferi* infection [93]. One of the immune responses of the host in LB infection is characterized by a cytokine response and the secretion of IFN-γ followed by a high expression of IL-4, by T helper 1 lymphocytes, which are associated with non-chronic manifestations of LB. However, persistent IFN-γ expression could lead to chronicity of the LB [94, 95].

In light of this, the secretion of IFN-γ can be exploited to detect the presence of Ag from *Bb* using ELISpot methods [96]. It measures the antigen-specific cellular responses by quantifying the number of IFN-γ-producing T cells [97]. The sensitivity of this kind of method is known to be about 20 to 200 times greater than ELISA or flow cytometry, and it is based on conditions (antigens and cellular medium) used for LT activation [98]. The iSpot Lyme™ is an ELISpot method (Autoimmun Diagnostika / Genome Identification Diagnostics) that uses recombinant *Borrelia* antigens (recombinant DbpA, OspC, p100, and VlsE) to stimulate specific effector/ memory T cells combined to a signal enhancer medium CTL Test Plus. This assay showed significantly higher sensitivity (84%) as compared to the WB (30%) [76]. However, as for conventional serology, traditional or improved ELISPOT assays cannot differentiate active from past LB [99].

The ELISpot principle has been combined with the QuantiFERON technology (QIAGEN Sciences) for the detection and measurement of IFN-γ in the case of *Mycobacterium tuberculosis* infection [100]. Taking inspiration from this technology, Callister's team developed the QuantiFERON-Lyme assay, which consists in the detection of IFN-γ in whole blood after incubation with synthetic *Borrelia* antigens (p66, DbpB, OspC, and flagellin) [78]. This test showed a sensitivity of approximately 70% in patients with EM versus 17% for standard serology. In addition, this test allows the distinction between an active or past infection because of the significant decrease of the immune response in patients who have been treated. However, this technique seems to be suitable only in the United States where *Bbss*, one of the species belonging to the *B. burgdorferi* sensu lato complex, dominates. In Europe, where *B. afzelii* and *B. garinii* are

the dominant species, induced responses are low and a more sensitive test is therefore required [101]. The use of IFN-γ for diagnosis could be challenging, and dependent on the cell type used for the assay. Indeed, a recent study confirmed that *Borrelia burgdorferi* is a poor inducer of IFN-γ production by peripheral blood mononuclear cells (PBMCs) [102] but a strong inducer by human primary NK cells [103]. More studies have to be done regarding specificity and reliability.

Many other methods are based on the ELISpot principle, such as the Spirofind Revised (Oxford Immunotec), which quantifies the IL-1β produced by primary PBMC cells after contact with *Borrelia* mix, using bead ELISA assay [104].

Also based on T cells, some authors exploited the massive sequencing of T-cell receptor repertoire to highlight the specificity of T-cell responses and probe pathogen exposure. Indeed, it is known that the serological approach based on specific antibody-response suffers from a seronegative window period of 2 to 4 weeks [105], whereas T-cell response is detectable before the humoral response [79]. To circumvent both the co-infection issue and the difference between the kinetics of T-cell response and the humoral response, Greissl and collaborators recently proposed an aid for diagnosis based on a tool allowing to analyze and classify TCR sequencing in order to ensure higher sensitivity testing compared with STTT, in particular concerning early LB (Table 3).

Other tests also based on cellular proliferation are available. A few years ago, a Lymphocyte Transformation Test-Memory Lymphocyte Immunostimulation Assay (LTT-MELISA®, InVitaLab) was developed. This assay is based on two steps: uptake of radioisotope by dividing lymphocytes, followed by their detection [106]. This method evaluates the lymphoproliferative response of PBMCs to *B. burgdorferi* antigens. iSpotLyme™, Spirofind™, and LTT-MELISA® assays have been recently studied and have been compared to classical serology testing [104]. According to the sensitivity and specificity reported in Table 3, it has been confirmed that these cellular tests lead to a high number of false-positive results and are unfit for clinical use at this stage [77].

## Biomarkers-based approaches

The use of the omics (transcriptomics, metabolomics, inflammatomics) approach could identify biomarkers or biosignature of a LB [107]. Several studies have shown that some molecules, related to the immune response of the host, can be used as biomarkers for the diagnosis of LB.

Chemokines and cytokines are key signaling molecules for inflammation and modulation of the immune cells. For Lyme neuroborreliosis (LNB) diagnosis,
inflammatory cerebrospinal fluid (CSF) changes (pleocytosis, blood-CSF barrier dysfunction, and intrathecal Ig synthesis) can be expected. Laboratory diagnosis is made by calculating an antibody index based on intrathecal production of antibodies against *Bb* by comparing CSF and serum antibody levels [22, 108, 109]. To improve LNB diagnosis, many studies and tests are based on the quantification of the C-X-C motif chemokine ligand 13 (CXCL13). This chemokine is produced by antigen-presenting cells such as dendritic cells and macrophages. Via its receptor, namely CXCR5, this chemokine is used to guide B cells towards secondary lymphoid organs [110, 111]. Thus, this chemokine has many advantages as compared to the antibody index method, for example (i) its CSF level is high [112, 113], (ii) this chemokine is detectable before antibodies whose levels can be very low at early stage neuroborreliosis [114], and (iii) its level rapidly declines after antibiotics treatment, while CSF pleocytosis remains elevated and antibody level remains positive for years after treatment [115]. Thus, this marker could be an important element for the diagnosis of Lyme neuroborreliosis. Many methods allow its detection and quantification. In a recent study, Haglund and collaborators compared 2 commercial assays: the ReaScan CXCL13 (Reagena Ltd, Toivala, Finland) and the recomBead CXCL13 (Mikrogen Diagnostik, GmbH, Neuried, Germany) assays. The ReaScan CXCL13 assay is a rapid cassette-based immunochromatographic system, similar to LFIA approach. The reader values are translated to semi-quantitative CXCL13 concentrations interpreted as <250 pg/mL (negative), 250–500 pg/mL (grey zone), and >500 pg/mL (positive/suspected LNB). As in all LFIA assays, CXCL13 in the sample interacts with an antibody conjugated to colloidal gold, and the complex is then captured onto the test line by anti-CXCL13 antibody. The recomBead CXCL13 (Mikrogen Diagnostik, GmbH, Neuried, Germany) assay, based on the Luminex xMAP® technology, is interpreted as CXCL13 < 190 pg/mL (negative), 190–300 pg/mL (grey zone), and > 300 pg/mL (positive/suspected LNB). In this type of technology, beads coated with anti-CXCL13 antibodies are incubated with a sample containing unbound biotinylated antibodies against CXCL13. After washing, incubation with a reporter streptavidin-R-Phycoerythrin is made and a sandwich is generated. By using a dual laser system, the presence and intensity of the reporter associated with the bead are detected providing information about CXCL13 concentration in the sample [80]. While the sensitivity of recomBead is higher than that of ReaScan (see Table 3), the recomBead is less specific. ELISA methods can also be used for measuring CXCL13 levels with a sensitivity of 88–100% and a specificity of 89–99% [81, 82, 116]. However, determining levels of CXCL13 as a marker for LNB

can aid in the diagnosis but should be interpreted with care since CXCL13 has been reported to be nonspecific to Lyme neuroborreliosis. Increased CSF values have also been found in patients affected by neurosyphilis [117], CNS lymphomas [118] or also in immunocompromised patients and patients with an autoimmune disorder [82]. To overcome this limit, a recent study proposed to titrate the interleukin-6 (IL-6) in addition to the CXCL13 chemokine. High concentrations of IL-6 have been found in CSF samples from patients suffering from neuroinfекtions due to bacterial or viral etiology, while lower levels have been detected in CSF specimens from cases of LNB. However, the use of CXCL13 and IL-6 needs to be evaluated further in future studies [119].

CXCL9, CXCL10, and C–C Motif Chemokine Ligand 19 (CCL19) levels are significantly elevated in the serum during acute infection depending on the severity of the disease, but they usually decrease after the proper treatment and the resolution of EM [120]. Elevated levels of CXCL9 and CXCL10 are linked to the Th1 immune response due to bacterial and virus infection [121]. The exploitation of these chemokines, knowing that the list isn’t exhaustive and will be enriched in the future, with studies under consideration (such as CCL20, IL-17A...) [122], will therefore give important information for the diagnosis and the understanding of the disease, as well as for the optimization of the treatment, if needed.

Nowadays, a controversy exists when patients, after treatment, develop persistent symptoms, qualified either as chronic LB or post-treatment Lyme disease syndrome (PTLDS). A study showed that CCL19 levels are elevated in patients with the development of these persistent symptoms [123]. The identification of this biomarker, by using a method similar to ELISA and based on the Luminex-xMAP® technology described before, called Bio-Plex bead array system, will offer the opportunity to better understand this phenomenon, its diagnosis, and treatment. Within the related study, 14.5% of treated patients have developed persistent symptoms, and, notably, after 1-year, high CCL-19 levels were only observed in patients with PTLDS, although these observations need to be confirmed by further studies. Other molecules such as autoantigens, specifically apolipoprotein B, present in large quantities in patients with Lyme arthritis, could serve as diagnostic elements [124].

Besides measuring the levels of the above-mentioned chemokines, the analysis of metabolomics can be used to determine molecules exploitable as biomarkers or biosignatures of specific disease states [125]. Indeed, since metabolic activity strongly depends on environmental factors, including infections [126], the liquid chromatography-mass spectrometry approach could be potentially exploited for diseases diagnosis, as it has
been shown for the diagnosis of schistosomiasis [127]. This approach seems to be promising for the LB diagnosis as demonstrated by Molins and collaborators who established a metabolic signature with a sensitivity of 88% and a specificity of 95% for the early detection of LB [83]. Using a similar method, some isoprostenes and neuroprostane, such as malondialdehyde and 4-hydroxy-2-nonenal, can serve as biological markers [128]. Indeed, Ligor et al. showed that the identification by spectrometry and liquid and gas chromatography of these compounds in urine, blood, or CSF can help to diagnose the early stage of Lyme disease. Another recent and interesting research article from Magni et al. aims to present a new experimental procedure for characterizing the urinary pathogen-derived proteome of patients from LB. This innovative method, combining the mass spectrometry technique with a sample concentration methodology (affinity hydrogel particles) and a bioinformatic authentication method, allows to better identify patients with the presence or persistence of the BLG, even after antibiotic treatment [129].

Also based on biosignatures, many interesting research articles investigate the potential Raman spectroscopy (RS) diagnostic capacity for LB. This method does not detect *Bb* spirochetes in blood, but rather biochemical changes associated with *Bb* infection. Goff et al. demonstrated that blood samples from mice but also from humans were analysed with quite high sensitivity and specificity [85, 130]. In a similar approach, combining RS with chemometrics, Senger et al. developed an interesting test allowing to distinguish an LB molecular signature from healthy volunteers, end-stage kidney disease patients, and patients with active or remissive bladder cancer (Table 3) [86].

Altered metabolic biomarkers profiles can reflect a disease state and be exploited for LB diagnosis. In this context, and by analyzing the metabolic pathways of *Borrelia* bacteria, it has been shown an evolutionarily reduced genome because of its close association with its vertebrate and tick hosts [131]. For this reason, many metabolic pathways lack making the bacteria incorporating host lipids for growth. By including host's lipoproteins and lipids, such as phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylserine (PS) and cholesterol, the host immune system develops autoantibodies to those lipids. The detection of the elevation of antiphospholipid antibodies against PC, PG, or PS, but not against cardiolipin, may first aid in the diagnosis of Lyme disease, but also distinguished LB from syphilis and some other diseases [132, 133]. Interestingly, Molins et al. managed, by analyzing a metabolomic signature, to differentiate early LB from southern tick-associated rash illness (STARI) with an accuracy of 85 to 98%. Indeed,

early LB can be clinically diagnosed by EM, but can be confused with other illnesses like cutaneous manifestation from STARI [134].

In combination with AI approach, some studies linked to biomarkers are reported in the Sect. 4 [135–137]. These innovations pave the way for an approved diagnostic test.

## Direct diagnosis

Direct diagnosis, mainly based on the detection of the pathogen, is for many infectious diseases the golden standard for proving an active infection. Currently, a direct diagnosis of a disease can rely on many methods: culture of the pathogen, microscopic observation, xeno-diagnosis, detection and amplification of the pathogen DNA/RNA, and proteins/antigens (Ag) detection [36].

## Culture of *Borrelia burgdorferi*

The culture of *Bb* represents a difficult task at multiple levels. First of all, obtaining samples can be extremely invasive. Indeed, although blood or urine samples can sometimes be used to collect the etiological pathogen of LB, in some cases skin biopsy from EM or ACA, or even synovial, cerebrospinal fluid (CSF) or myocardium need to be used [138, 139]. Therefore, the patient's pain, the associated risks, the available technical skills, and the relative costs need to be considered when culturing *Bb*.

In addition, the *Borrelia* culture is a fastidious and long process, making this approach unsuitable for rapid detection of the pathogen. Indeed, *Bb* grows on a very specific and rich medium, i.e. Barbour-Stoënner-Kelly (BSK) - II or BSK-H supplemented with rabbit serum or modified Kelly-Pettenkofer (MKP) [140]. Many comparisons have been done regarding the interest of each medium. For example, according to a study from Ružić-Sabljić et al., BSK-H medium supports the growth of borrelial strains but MKP is superior with regard to the isolation rate, morphology and motility of strains. However, even if BSK-H medium supports fast initial growth of *borreliae*, this is followed by rapid deformation and death of the spirochaetes [141]. Regarding the different genospecies insides BLG, the comparison of MKP and BSK-H medium for *Borrelia* culturing from skin specimens of European patients with EM revealed the advantage of MKP over BSK-H [142]. Indeed, when using *Borrelia* isolates from tick- or host-derived samples, MKP medium should be preferred [143].

The growth rate ranges between 8 and 12 h at the appropriate temperature (between 30 and 35 °C) [144], which justifies that pathogen detection takes around 24 days for blood culture and skin biopsy samples [145]. Furthermore, the use of a rich medium makes the culture more sensitive to contamination by fast-growing
bacteria. Thus, to prevent bacterial contamination, an antibiotic should be used, such as rifampicin or phosphomycin, since *Borrelia* is intrinsically resistant to these antibiotics. Amphotericin B, in combination with rifampicin and phosphomycin, can also be used to suppress fungal growth [146]. Therefore, the culture should be kept for at least 8 to 12 weeks to consider a negative result [29]. As previously mentioned, despite *Bb* culture being considered as the gold standard with a specificity close to 100%, the sensitivity of this approach remains very low, depending on the clinical stage of the disease, the sample origin, and the genospecies responsible for LB [147]. Sensitivities as a function of the type of clinical sample are reported in Table 4. Moreover, the clinical samples often contain a small number of living bacteria, sometimes lower than the measurable level, and the authors underlined the extreme variability of borrelia quantity collection in samples [36, 148, 149]. Finally, previous works suggest that *Bbsl* could adopt a persistence state including a viable-but-nonculturable (VBNC) state [24, 150]. This, together with the above-mentioned sample harvesting and bacterial growth problems, puts bacterial culture beyond the capabilities of most clinical laboratories and therefore not suitable for diagnosis.

### Microscopic observation and xenodiagnosis

The clinical utility of direct microscopic observation of *Bb* is limited due to the low concentration of bacteria in body fluids. Indeed, Wormser et al. considered the number of 0.1 cultivable cell/mL of whole blood [149], even

if such quantification can vary according to the authors [148, 157].

This low load means that the detection by light microscopy is not feasible in clinical practice, ruling out such an approach for routine diagnosis. Furthermore, the specificity of this approach is moderate, as artifacts can indeed be responsible for false positive interpretations, even by a trained biologist. For example, recently, Laane et al. carried out a study based on a modified dark-field microscopy technique, allowing the detection of *Borrelia* structure [158]. However, using the same method, Aase et al. showed that 85% of blood samples from the healthy control group were detected positive when only 66% of those with LB were detected, making this approach unsuitable for Lyme disease diagnosis [159].

Alternatively, the presence of an infection can be determined by xenodiagnosis. This method is based on the biting and feeding of an uninfected tick on the suspected infected patient. Then, the tick is used to check the presence of *Bb* by Polymerase Chain Reaction (PCR) assay. Trials have been performed in murine models or in Rhesus Macaques [160, 161]. In this last-mentioned trial, small numbers of intact spirochetes were recovered by xenodiagnosis from treated monkeys, also demonstrating that *B. burgdorferi* can withstand antibiotic treatment. Even if xenodiagnosis appears safe and generally well tolerated in humans [162], Bockenstedt and Radolf underlined a flawed result since xenodiagnosis works in inbred mice because spirochetes persist in the distal skin, but not in humans [163].

<table><caption>**Table 4** Sensitivity of culture detection and PCR assays depending on the origin of clinical sample</caption><thead><tr><th>Methods</th><th>Origin of clinical sample</th><th>Sensitivity range</th><th>References</th></tr></thead><tbody><tr><td rowspan="10">Culture of <i>B. burgdorferi</i></td><td>Skin biopsies from EM</td><td>40 – 90%</td><td>[36, 149, 151]</td></tr><tr><td>Skin biopsies from ACA</td><td>20 – 60%</td><td>[36]</td></tr><tr><td>Skin biopsies from Borrelial lymphocytoma</td><td>24 – 32%</td><td>[147]</td></tr><tr><td>Synovial fluid/biopsy</td><td>&lt; 1%</td><td>[36]</td></tr><tr><td>Cerebrospinal fluid</td><td>10 – 26%</td><td>[29, 36]</td></tr><tr><td>Blood</td><td>5 – 40%</td><td>[29, 36, 149]</td></tr><tr><td>Urine</td><td>nd</td><td>[152]</td></tr><tr><td>Skin biopsies from EM</td><td>30 – 89%</td><td>[29, 153]</td></tr><tr><td>Skin biopsies from ACA</td><td>20 – 100%</td><td></td></tr><tr><td>Skin biopsies from Borrelial lymphocytoma</td><td>67.5%</td><td>[154]</td></tr><tr><td rowspan="4">Traditional PCR assays</td><td>Synovial fluid/biopsy</td><td>40 – 96%</td><td>[36, 155, 156]</td></tr><tr><td>Cerebrospinal fluid</td><td>5 – 30%</td><td>[36, 147, 155]</td></tr><tr><td>Blood</td><td>10 – 20%</td><td></td></tr><tr><td>Urine</td><td>nd</td><td>[152]</td></tr></tbody></table>

Nd not detectable
Antigens detection

Antigen detection assays suffer from the same limitations
as microscopic detection. Few diagnostic tests captur-
ing antigen from Bb exist [164–166] and the reliability of
these methods for clinical practice is poor [36]. Due to
low level of bacteria in body fluids, which depends mainly
on the origin of the sample or the LB stages, many meth-
ods have been developped to improve antigen/bacteria
detection in a clinical sample. They consist in, but are
not restricted to, the concentration of antigens or bacte-
ria, increasing of the initial clinical sample volume, and
improvement of antibodies affinity, peptides, or aptamers
for one or several specific targets.

With this aim, centrifugation helps to concentrate
membrane proteins (OspA, flab, p66, OspC and BmpA)
after bacterial lysis. This method allows the detection of
*B. burgdorferi* membrane OspA proteins at low concen-
trations of about 4 fmol OspA/mg serum protein [167].
Other concentration methods such as the Nanotrap
[168] or the Hydrogel Microparticles technology [169,
170] are also available. In both approaches, nanoparti-
cles will bind to the OspA antigen in urine or any other
body fluid. An elution can then be performed in a smaller
volume allowing its concentration before detection using
any immunoassay technique. Magnetic particles as simi-
lar diagnostic tools can also offer a large signal amplifi-
cation by concentration of Lyme antigens or by bonding
many labels such as enzymes (like horseradish peroxidase
(HRP)) [171].

Regarding the affinity of molecules to their target, a
recent study presents an improved direct method, where
the protein OspA is detected in clinical serum sam-
ples, combining enhanced Raman scattering (SERS) and
aptamers. A DNA aptamer, exhibiting a high specificity
and a dissociation constant (K_D) of 2.18 nM for OspA,
shows a sensitivity of 91%, using serum samples from
Lyme patients. In addition, the OspA limit-of-detection
was determined to be 1×10⁻⁴ ng/mL, four orders of
magnitude lower than that found in serum samples from
early Lyme disease patients. This promising application
may help Lyme diagnosis [84].

Flynn et al. utilized the inherent binding affinity of the
BBK32 protein for human fibronectin to propose a highly
promising electrochemical sensing methodology. As a
proof of concept, they employed a biomimetic electrode
to measure impedance changes induced by protein inter-
actions, thereby detecting the presence of bacteria [172].

Traditional PCR assays and PCR improvements

PCR is a widely known in vitro method for the amplifica-
tion and detection of DNA. Briefly, copies of a targeted
DNA fragments will be recovered, if present, in the sample.
*B. burgdorferi*'s genome which is very complex and consists

of a ~950-kb linear chromosome and many additional cir-
cular plasmids and linear plasmids that range in size from
9 to 62 kb [173]. Because of this complexity, many genes
can be chosen as the target DNA fragment, like a unique
rRNA gene (16S rDNA, 23S rDNA, 23S-5S rDNA intergenic
spacer), or a single gene in the Bb chromosome (flagellin
(fla), hbb, rrf-rrl, polC, SrRNK, p66, recA, bmpA, rpoB, rpoC,
or gyr). Regarding the plasmid targets, many genes can be
used such as dbpA, vlsE, and outer surface protein genes
(ospA, ospB, ospC) [155]. The sensitivity varies depending
on the sample. Interestingly, authors recently demonstrated
significance to perform an early PCR detection of Borrelia,
directly on the bite site, using a new non-invasive sampling
device, based on a microneedles patch [174].

Table 4 reports the sensitivity of PCR assays accord-
ing to the origin of the clinical sample. As before men-
tioned, besides the very low level of bacteria according to
the sample collections, the main limitation of PCR assay
is its inability to distinguish between active and past
infection, since *B. burgdorferi* DNA remains present for
weeks, or even months, after antibiotic treatment [11,
175]. Additional reasons justify this limitation: Firstly,
nondividing *Borrelia* can remain in body fluids and can
be detected, leading to positive results despite past infec-
tion [176, 177]. Moreover, it has also been shown that *Bb*
produces outer membrane vesicles containing virulence
factors, such as Outer surface proteins (OspA, B, and C),
as well as DNA [178]. Finally, even if antibiotic treatment
has been successful, the residual DNA or antigenic pro-
teins included in such blebs could induce positive results
despite a past infection.

Thus, no conclusion can be made by PCR on the dis-
crimination between residual DNA and viable organisms,
or the success or lack of, a therapy.

As previously mentioned, the improvement of tradi-
tional PCR assays is one of the top research subjects for
LB diagnosis. Many studies showed that a simple way of
improving the sensibility of PCR assays can rely on the
preparation and enrichment of bacteria/DNA in the
clinical sample. For example, the application of a cen-
trifugation step to concentrate the bacteria in the sam-
ple, the use of cDNA as a template, and the removal of
erythrocytes improve the sensitivity of PCR assays [179].
Alternatively, a lysis step of PBMCs containing masked
intracellular bacteria can be added, increasing the
amount of *Bb* DNA copies in the initial sample. For this
purpose, different lysis techniques can be used such as
hypotonic water shock, solutions containing ammonium
chloride (NH₄Cl), or the use of chaotropic and/or deter-
gent agents (e.g. SDS, triton, etc.) [180].

An innovative method based on PCR called immuno-
PCR (iPCR) allows to increase the detection limit, for some
cases, of a conventional ELISA about 100- to 10,000-fold
[181, 182]. Applied to Lyme borreliosis diagnosis, the immuno-PCR uses a recombinant *B. burgdorferi* protein antigen coupled to magnetic beads to capture *B. burgdorferi*-specific host-generated antibodies. A biotinylated DNA oligonucleotide reporter molecule coupled to a streptavidin-conjugated reporter antibody is then amplified by qPCR for detection and quantification (Fig. 4). This method, described by Halpern et al., shows better sensitivity (reported Table 3) than the STTT method. [87, 183].

Besides these considerations, the PCR approach should also consider potential co-infections. Indeed, cases of co-infections comprising two or more species belonging to the genus *Borrelia*, but also cases comprising the genus *Borrelia* and other pathogens such as *Rickettsia* spp or *Babesia* spp are reported: According to Raileanu et al., 64.5% of infected *I. ricinus* were positive for more than one pathogen. In Romania for example, the most common co-infection was between *B. garinii* and *B. afzelii* (4.3%), followed by *B. garinii* and *B. lusitaniae* (3.0%). Co-infections between *Borrelia* spp. and *Rickettsia* spp. represented 1.3% of the investigated cases [31]. In addition, cases of human co-transmissions and co-infections have been listed [33, 184–187]. Using traditional PCR assays, the identification of the different species within the complex sensu lato can be based on a multi-locus detection [188, 189] ensuring the differentiation of *Bb* strains. At this scope, multi-locus sequence typing (MLST) and variable number tandem repeat (VNTR) analysis are commonly used and are based on housekeeping genes such as the intergenic spacer rrs-rrlA (IGS) and the ospC gene [190, 191]. Indeed, these sequences allow the distinction between strains because they represent different alleles.

These co-infections change the severity of the symptoms and the effectiveness of the treatments. This shows the need for a multiplexed test on several strains of the *Borrelia* genus, but ultimately for a multiplexed test against all tick-borne diseases. In this context, much effort still must be made to develop a multiplexed PCR to increase the attractiveness of this method for clinical use and to assure proper treatment. Another type of PCR, called quantitative PCR (real-time PCR or qPCR) has shown to be able not only to quantify the DNA and to determine the amplification purity by analysis of the melting curve, but also in an original manner, it has been used to detect different species namely *B. burgdorferi*, *B. afzelii*, and *B. garinii* by using a post-PCR denaturation profile analysis and a single molecular beacon probe. This latter is a hairpin-shaped oligonucleotide probe, highly specific for its target sequences, and usually labelled with distinguishably colored fluorophores [28]. Another qPCR method is based on FRET (Fluorescence Resonance Energy Transfert) and allows to improve the detection limit, with only 10 copies of *Borrelia* per PCR reaction [192]. Thus, the development of these types of multiplexed PCR methods allows for the identification of multiple tick-borne pathogens such as *Bbsl*, *Anaplasma/Ehrlichia* spp., and *Babesia* spp. [28, 193, 194] helping the future diagnosis and choosing the needed treatment.

As previously mentioned, one of the major limits of classical PCR assays is the difficulty in distinguishing between living and dead organisms, and therefore between an active or past infection [175]. A new method exploiting bacteriophages has been recently developed to face this issue. Bacteriophages, viruses which infect

<figure><img src="image_6.png" /><figcaption>**Fig. 4** iPCR principle</figcaption></figure>
bacteria, are only present in active bacterial infections since they require living bacteria for their replication. Therefore, a phage-based test is considered as direct evidence of an active infection. An important characteristic of bacteriophages is their ability to shift from a state of lysogenic activity to a lytic one following unfavorable physicochemical and/or biological conditions. Thus, the lytic stage will lead to the release of phages that had proliferated -and therefore of viral DNA- using the host's cellular machinery, and finally lead to the lysis of bacteria [195] (Fig. 5). The high number of viral DNA copies determines and increases the test sensitivity since the presence of one copy of the bacterial DNA implies the presence of many viral DNA copies. Moreover, bacteriophages have a specific tropism towards certain bacteria. In other words, phages infect a specific bacterial species and their genetic material is specific to the bacteria they infect, resulting in a high specificity of the test exploiting this approach, such as the Phelix Phage test patented by Dr. Louis Teulières [196].

The applied principle is the following: after the extraction of the pathogenic DNA from blood samples, a PCR for the detection of phages is performed using primers known to specifically target distinct phages [157]. The identification of the primers panel allowing amplification leads to the identification of the bacteria panel having infected the patient. Finally, the amplified fragments are sequenced to confirm the positivity of the sample and exclude false positives. Based on this principle, a recent study managed to reach a sensitivity of 3.3 *Borrelia* cells per ml of blood, which corresponds to the bacteria level in LB in clinical blood samples, by

targeting the prophage *terL* gene (NC_000948.1) [157]. This gene encodes for the *terL*, a protein responsible for packing phage genomes that were found in three linear plasmids and seven of the circular plasmids of the cp32 series. These latter have been determined to be *Bb* prophages within the B31 genome [197, 198]. Even if this technique is promising, an analysis of this study shows inaccuracies in terms of statistical analysis or cohort composition and comparison with healthy controls, thus requiring validation [199].

## Artificial Intelligence (AI) advent in diagnosis

When it comes to disease diagnosis, accuracy is critical to avoid therapeutic wandering and to plan and prescribe an effective treatment, ensuring the well-being of patients. Artificial Intelligence (AI) can be a strong ally in this scope, as shown by [200]. It could be exploited for LB diagnosis, for example to identify new biomarkers of the pathology. However, it must be recognized that AI studies are still in progress. Their validation must be realized and confirmed. In this context, a machine-learning algorithm was developed and it allowed identification of 20 genes that discriminated LB from other bacterial and viral infections [135]. Interestingly, these novel LB biomarkers not only identified subjects with acute disseminated LB, but also distinguished between subjects with an acute and resolved disease with 97% accuracy. Alternatively, the machine learning-based analyses of RNA-Seq data are promising and allow Servellita et al. to define 31 differentially expressed genes in PBMC cells between healthy and LB patients (Table 5) [136]. Similarly, an up-to-date research article demonstrates that

<figure><img src="image_7.png" /><figcaption>**Fig. 5** Phage PCR principle</figcaption></figure>
<table><caption>**Table 5** Sensitivity and specificity of tests relied on AI</caption><thead><tr><th>Classification</th><th>Tests</th><th>Sensitivity</th><th>Specificity</th><th>References</th></tr></thead><tbody><tr><td rowspan="2">Bioinformatics approaches</td><td>Transcriptomic: 20 classifier genes as biomarkers</td><td>100%</td><td>90%</td><td>[135]</td></tr><tr><td>Transcriptomic: 31 differentially expressed genes in PBMC cells</td><td>90%</td><td>100%</td><td>[136]</td></tr></tbody></table>

machine learning has managed to identify 35 genes as biomarkers for post-treatment LB. A RNA sequencing of PBMCs patients with post-treatment Lyme disease showed a differential expression with acute LB patients and uninfected people [137].

In a near future, AI tools will reach new heights, and increase the accuracy and velocity of LB clinical diagnosis. For example, studies showed that deep learning, based on a dataset of medical images, which allows the discrimination of EM from ambiguous skin manifestations such as cellulitis or herpes zoster (which can be simulated by LD [201]), can help clinicians with early diagnosis and reduce further complications [202, 203].

## Conclusion

Lyme disease is the most frequent tick-borne disease in the world. Even if EM is the traditional clinical manifestation, the pathogen can spread to other tissues and organs, leading to blurred symptoms. Many direct and indirect tests for Lyme diagnosis are available. Despite the innovation of new or improved assays, the diagnosis of LB remains a challenge. Immune escape mechanisms, the difference in protein and antigen expression, and the low level of bacteria in clinical samples are some of the factors that explain the difficulties encountered. Due to the heterogeneous distribution of genospecies in the world, unique recommendation, unique target and unique technology do not appear to be appropriate for the diagnosis of LB. Not only sensitive and specific assays but also multiplexed tests are required, in order to identify the co-infectious profile of patients frequently mentioned in such pathologies so as to adapt the appropriate treatment. Therefore, it appears that more work needs to be done to develop reliable and unambiguous diagnostic test for LB. Moreover, clinicians need assistance with diagnostic evaluation since symptoms are diverse and blurred. In such a context, the development and application of AI techniques could improve detection and diagnosis of LB.

## Acknowledgements

The authors thank Anne-Louise Lamontagne for correcting the manuscript, and the Institut des Sciences du Calcul et des Données (ISCD) of Sorbonne University. Parts of the figure were drawn by using pictures from Servier Medical Art. Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/).

## Authors' contributions

MG and SP conceptualized the idea for manuscript. MG performed literature review and drafted the manuscript. MG and SP made the figures. MG, AZ, SO, BA, IM and SP evaluated the manuscript and improved the content. All authors contributed to the article and approved the submitted version.

## Funding

This work is supported by the French ministry of higher education, research and innovation (MESRI). The funding body played no role in the design of the study and collection, analysis, interpretation of data, and in writing the manuscript.

## Availability of data and materials

Not applicable.

## Declarations

### Ethics approval and consent to participate

Not applicable.

### Consent for publication

Not applicable.

### Competing interests

The authors declare no competing interests.

Received: 20 March 2023 Accepted: 4 July 2023

Published online: 01 August 2023

## References

1. Stanek G, Wormser GP, Gray J, Strle F. Lyme borreliosis. The Lancet. 2012;379:461–73.

2. Trevisan G, Cinco M, Trevisini S, di Meo N, Chersi K, Ruscio M, et al. Borreliae Part 1: Borrelia Lyme Group and Echidna-Reptile Group. Biology. 2021;10:1036.

3. Wodecka B, Kolomiiets V. Genetic Diversity of Borreliaceae Species Detected in Natural Populations of Ixodes ricinus Ticks in Northern Poland. Life. 2023;13:972.

4. Karageorgou I, Koutantou M, Papadogiannaki I, Voulgari-Kokota A, Makka S, Angelakis E. Serological evidence of possible Borrelia afzelii lyme disease in Greece. New Microbes New Infect. 2022;46:100978.

5. Mancilla-Agrono LY, Banguero-Micolta LF, Ossa-López PA, Ramírez-Chaves HE, Castaño-Villa GJ, Rivera-Páez FA. Is Borrelia burgdorferi Sensu Stricto in South America? First Molecular Evidence of Its Presence in Colombia. Trop Med Infect Dis. 2022;7:428.

6. Rudenko N, Golovchenko M, Horak A, Grubhoffer L, Mongodin EF, Fraser CM, et al. Genomic Confirmation of Borrelia garinii. United States Emerg Infect Dis. 2023;29:64–9.

7. Trevisan G, Cinco M, Trevisini S, di Meo N, Ruscio M, Forgione P, et al. Borreliae Part 2: Borrelia Relapsing Fever Group and Unclassified Borrelia. Biology. 2021;10:1117.

8. Trevisan G, Cinco M, Ruscio M, Forgione P, Bonoldi VLN. Borrelia Lyme Group. J Dermatol Res Rev Rep. 2022;3:1–12.

9. Steere AC, Strle F, Wormser GP, Hu LT, Branda JA, Hovius JWR, et al. Lyme borreliosis. Nat Rev Dis Primer. 2016;2:16090.

10. Casjens S, Palmer N, Van Vugt R, Mun Huang W, Stevenson B, Rosa P, et al. A bacterial genome in flux: the twelve linear and nine circular
extrachromosomal DNAs in an infectious isolate of the Lyme disease spirochete <i>Borrelia burgdorferi</i>. *Mol Microbiol.* 2000;35:490–516.

11. Iyer R, Mukherjee P, Wang K, Simons J, Wormser GP, Schwartz I. Detection of Borrelia burgdorferi Nucleic Acids after Antibiotic Treatment Does Not Confirm Viability. J Clin Microbiol. 2013;51:857–62.

12. Schüler W, Bunikis I, Weber-Lehman J, Comstedt P, Kutschan-Bunikis S, Stanek G, et al. Complete Genome Sequence of Borrelia afzelii K78 and Comparative Genome Analysis. PLoS One. 2015;10:e0120548.

13. Chiappa G, Perini M, Cafiso A, Nodari R, Wilhelmsson P, Lindgren P-E, et al. A Novel High Discriminatory Protocol for the Detection of Borrelia afzelii, Borrelia burgdorferi Sensu Stricto and Borrelia garinii in Ticks. Pathogens. 2022;11:1234.

14. Pearson P, Skaltsos O, Luo C-Y, Xu G, Oppler Z, Brisson D, et al. A Borrelia burgdorferi outer surface protein C (OspC) genotyping method using Luminex technology. PLoS One. 2022;17:e0269266.

15. Golidonova KA, Андреевна ГК, Korenberg ЕІ, Исаевич КЭ, Gintsburg AL, Леонидович ГА. Optimized multilocus sequence analysis for laboratory identification of pathogens of ixodid tick-borne borreliosis. J Microbiol Epidemiol Immunobiol. 2022;99:514–24.

16. Hanincova K, Mukherjee P, Ogden NH, Margos G, Wormser GP, Reed KD, et al. Multilocus sequence typing of Borrelia burgdorferi suggests existence of lineages with differential pathogenic properties in humans. PLoS ONE. 2013;8:e73066.

17. Cerar T, Strle F, Stupica D, Ruzic-Sabljic E, McHugh G, Steere AC, et al. Differences in Genotype, Clinical Features, and Inflammatory Potential of Borrelia burgdorferi sensu stricto Strains from Europe and the United States. Emerg Infect Dis. 2016;22:818–27.

18. Bobe JR, Jutras BL, Horn EJ, Embers ME, Bailey A, Moritz RL, et al. Recent Progress in Lyme Disease and Remaining Challenges. Front Med. 2021;8:666554.

19. Sykes RA, Makiello P. An estimate of Lyme borreliosis incidence in Western Europe+. J Public Health Oxf Engl. 2017;39:74–81.

20. Marques AR, Strle F, Wormser GP. Comparison of Lyme Disease in the United States and Europe. Emerg Infect Dis. 2021;27:2017–24.

21. Cook MJ, Puri BK. Estimates for Lyme borreliosis infections based on models using sentinel canine and human seroprevalence data. Infect Dis Model. 2020;5:871–88.

22. Stanek G, Strle F. Lyme borreliosis—from tick bite to diagnosis and treatment. FEMS Microbiol Rev. 2018;42:233–58.

23. Trevisan G, Nan K, di Meo N, Bonin S. The Impact of Telemedicine in the Diagnosis of Erythema Migrans during the COVID Pandemic: A Comparison with In-Person Diagnosis in the Pre-COVID Era. Pathogens. 2022;11:1122.

24. Cabello FC, Embers ME, Newman SA, Godfrey HP. Borreliella burgdorferi Antimicrobial-Tolerant Persistence in Lyme Disease and Posttreatment Lyme Disease Syndromes. mBio. 2022;13:e0344021.

25. Centers for Disease Control and Prevention (CDC). Recommendations for test performance and interpretation from the Second National Conference on Serologic Diagnosis of Lyme Disease. Morb Mortal Wkly Rep. 1995;44:590–1.

26. Aslam B, Nisar MA, Khurshid M, Farooq Salamat MK. Immune escape strategies of Borrelia burgdorferi. Future Microbiol. 2017;12:1219–37.

27. Anderson C, Brissette CA. The Brilliance of Borrelia: Mechanisms of Host Immune Evasion by Lyme Disease-Causing Spirochetes. Pathogens. 2021;10:281.

28. Chan K, Marras SA, Parveen N. Sensitive multiplex PCR assay to differentiate Lyme spirochetes and emerging pathogens Anaplasma phagocytophilum and Babesia microti. BMC Microbiol. 2013;13:295.

29. Aguero-Rosenfeld ME, Wang G, Schwartz I, Wormser GP. Diagnosis of Lyme Borreliosis. Clin Microbiol Rev. 2005;18:484–509.

30. Dumes AA. Lyme Disease and the Epistemic Tensions of “Medically Unexplained Illnesses.” Med Anthropol. 2020;39:441–56.

31. Raileanu C, Moutailler S, Pavel I, Porea D, Mihalca AD, Savuta G, et al. Borrelia Diversity and Co-infection with Other Tick Borne Pathogens in Ticks. Front Cell Infect Microbiol. 2017;7:36.

32. Moutailler S, Moro CV, Vaumourin E, Michelet L, Tran FH, Devillers E, et al. Co-infection of Ticks: The Rule Rather Than the Exception. PLoS Negl Trop Dis. 2016;10:e0004539.

33. Moniuszko A, Dunaj J, Święcicka I, Zambrowski G, Chmielewska-Badora J, Żukiewicz-Sobczak W, et al. Co-infections with Borrelia species,

Anaplasma phagocytophilum and Babesia spp. in patients with tick-borne encephalitis. Eur J Clin Microbiol Infect Dis. 2014;33:1835–41.

34. Branda JA, Body BA, Boyle J, Branson BM, Dattwyler RJ, Fikrig E, et al. Advances in Serodiagnostic Testing for Lyme Disease Are at Hand. Clin Infect Dis. 2018;66:1133–9.

35. Chou E, Lin Y-P, Cady NC. Recent Strategies for the Diagnosis of Early Lyme Disease. Sci Prog. 2018;101:311–31.

36. Lohr B, Fingerle V, Norris DE, Hunfeld K-P. Laboratory diagnosis of Lyme borreliosis: Current state of the art and future perspectives. Crit Rev Clin Lab Sci. 2018;55:219–45.

37. Kobayashi T, Auwaerter PG. Diagnostic Testing for Lyme Disease. Infect Dis Clin North Am. 2022;36:605–20.

38. Miraglia CM. A Review of the Centers for Disease Control and Prevention's Guidelines for the Clinical Laboratory Diagnosis of Lyme Disease. J Chiropr Med. 2016;15:272–80.

39. Marques A. Laboratory Diagnosis of Lyme Disease - Advances and Challenges. Infect Dis Clin North Am. 2015;29:295–307.

40. Feder HM Jr, Gerber MA, Luger SW, Ryan RW. Persistence of Serum Antibodies to Borrelia burgdorferi in Patients Treated for Lyme Disease. Clin Infect Dis. 1992;15:788–93.

41. Cinco M, Murgia R. Evaluation of the C6 enzyme-linked immunosorbent assay for the serodiagnosis of Lyme borreliosis in north-eastern Italy. New Microbiol. 2006;29:139–41.

42. Kumaran D, Eswaramoorthy S, Luft BJ, Koide S, Dunn JJ, Lawson CL, et al. Crystal structure of outer surface protein C (OspC) from the Lyme disease spirochete. Borrelia burgdorferi EMBO J. 2001;20:971–8.

43. Theel ES. The Past, Present, and (Possible) Future of Serologic Testing for Lyme Disease. J Clin Microbiol. 2016;54:1191–6.

44. Kaiser R, Rauer S. Advantage of recombinant borreliol proteins for serodiagnosis of neuroborreliosis. J Med Microbiol. 1999;48:5–10.

45. Nyman D, Willén L, Jansson C, Carlsson S-A, Granlund H, Wahlberg P. VlsE C6 peptide and IgG ELISA antibody analysis for clinical diagnosis of Lyme borreliosis in an endemic area. Clin Microbiol Infect. 2006;12:496–7.

46. van Burgel ND, Brandenburg A, Gerritsen HJ, Kroes ACM, van Dam AP. High sensitivity and specificity of the C6-peptide ELISA on cerebrospinal fluid in Lyme neuroborreliosis patients. Clin Microbiol Infect. 2011;17:1495–500.

47. Davis IRC, McNeil SA, Allen W, MacKinnon-Cameron D, Lindsay LR, Bernat K, et al. Performance of a Modified Two-Tiered Testing Enzyme Immunoassay Algorithm for Serologic Diagnosis of Lyme Disease in a Nova Scotia. J Clin Microbiol. 2020;58:e01841–e1919.

48. Mavin S, Milner RM, Evans R, Chatterton JMW, Joss AWL, Ho-Yen DO. The use of local isolates in Western blots improves serological diagnosis of Lyme disease in Scotland. J Med Microbiol. 2007;56:47–51.

49. Mavin S, Evans R, Milner RM, Chatterton JMW, Ho-Yen DO. Local Borrelia burgdorferi sensu stricto and Borrelia afzelii strains in a single mixed antigen improves western blot sensitivity. J Clin Pathol. 2009;62:552–4.

50. Wojciechowska-Koszko I, Kwiatkowski P, Sienkiewicz M, Kowalczyk M, Kowalczyk E, Dołęgowska B. Cross-Reactive Results in Serological Tests for Borreliosis in Patients with Active Viral Infections. Pathogens. 2022;11:203.

51. Zóka A, Gönçzi M, Kádár B, Steinhauser R, Baráth A, Bekő G. The potential impact of Helicobacter pylori seropositivity on recombinant antigen-based Lyme serology. J Microbiol Methods. 2021;188:106298.

52. Naesens R, Vermeiren S, Van Schaeren J, Jeurissen A. False Positive Lyme Serology Due to Syphilis: Report of 6 Cases and Review of the Literature. Acta Clin Belg. 2011;66:58–9.

53. Arnaboldi PM, Katseff AS, Sambir M, Dattwyler RJ. Linear Peptide Epitopes Derived from ErpP, p35, and FlaB in the Serodiagnosis of Lyme Disease. Pathogens. 2022;11:944.

54. Porwancher RB, Hagerty CG, Fan J, Landsberg L, Johnson BJB, Kopnitsky M, et al. Multiplex Immunoassay for Lyme Disease Using VlsE1-IgG and pepC10-IgM Antibodies: Improving Test Performance through Bioinformatics. Clin Vaccine Immunol CVI. 2011;18:851–9.

55. Steere AC, McHugh G, Damle N, Sikand VK. Prospective Study of Serologic Tests for Lyme Disease. Clin Infect Dis Off Publ Infect Dis Soc Am. 2008;47:188–95.

56. Branda JA, Steere AC. Laboratory Diagnosis of Lyme Borreliosis. Clin Microbiol Rev. 2021;34:e00018-19.
57. Marques AR. Revisiting the Lyme Disease Serodiagnostic Algorithm: the Momentum Gathers. J Clin Microbiol. 2018;56:e00749-e818.

58. Branda JA, Strle F, Strle K, Sikand N, Ferraro MJ, Steere AC. Performance of United States Serologic Assays in the Diagnosis of Lyme Borreliosis Acquired in Europe. Clin Infect Dis Off Publ Infect Dis Soc Am. 2013;57:333-40.

59. Baarsma M, Schellekens J, Meijer B, Brandenburg A, Souilljee T, Hofhuis A, et al. Diagnostic parameters of modified two-tier testing in European patients with early Lyme disease. Eur J Clin Microbiol Infect Dis. 2020;39:2143-52.

60. Leeflang MMG, Ang CW, Berkhout J, Bijlmer HA, Van Bortel W, Brandenburg AH, et al. The diagnostic accuracy of serological tests for Lyme borreliosis in Europe: a systematic review and meta-analysis. BMC Infect Dis. 2016;16:140.

61. Baarsma ME, Vrijlandt A, Ursinus J, Zaaijer HL, Jurriaans S, van Dam AP, et al. Diagnostic performance of the ZEUS Borrelia VlsE1/pepC10 assay in European LB patients: a case-control study. Eur J Clin Microbiol Infect Dis. 2022;41:387-93.

62. Quintero JA, Attah R, Khianey R, Capitle E, Schutzer SE. Arthritis and Diagnostics in Lyme Disease. Trop Med Infect Dis. 2021;6:18.

63. Kenyon SM, Chan SL. A focused review on Lyme disease diagnostic testing: An update on serology algorithms, current ordering practices, and practical considerations for laboratory implementation of a new testing algorithm. Clin Biochem. 2021;S0009-9120(21)00318.

64. Branda JA, Linskey K, Kim YA, Steere AC, Ferraro MJ. Two-Tiered Antibody Testing for Lyme Disease With Use of 2 Enzyme Immunoassays, a Whole-Cell Sonicate Enzyme Immunoassay Followed by a VlsE C6 Peptide Enzyme Immunoassay. Clin Infect Dis. 2011;53:541-7.

65. Molins CR, Delorey MJ, Sexton C, Schriefer ME. Lyme Borreliosis Serology: Performance of Several Commonly Used Laboratory Diagnostic Tests and a Large Resource Panel of Well-Characterized Patient Samples. J Clin Microbiol. 2016;54:2726-34.

66. Miraglia CM. An Update to a Review of Guidelines for the Clinical Laboratory Diagnosis of Lyme Disease. J Chiropr Med. 2020;19:201-2.

67. Waddell LA, Greig J, Mascarenhas M, Harding S, Lindsay R, Ogden N. The Accuracy of Diagnostic Tests for Lyme Disease in Humans, A Systematic Review and Meta-Analysis of North American Research. PLoS One. 2016;11:e0168613.

68. Branda JA, Strle K, Nigrovic LE, Lantos PM, Lepore TJ, Damle NS, et al. Evaluation of Modified 2-Tiered Serodiagnostic Testing Algorithms for Early Lyme Disease. Clin Infect Dis. 2017;64:1074-80.

69. Jespersen DJ, Smith TF, Rosenblatt JE, Cockerill FR. Comparison of the Borrelia DotBlot G, MarDx, and VIDAS Enzyme Immunoassays for Detecting Immunoglobulin G Antibodies to Borrelia burgdorferi in Human Serum. J Clin Microbiol. 2002;40:4782-4.

70. Smit PW, Kurkela S, Kuusi M, Vapalhti O. Evaluation of two commercially available rapid diagnostic tests for Lyme borreliosis. Eur J Clin Microbiol Infect Dis. 2015;34:109-13.

71. Pietikäinen A, Backman I, Henningsson AJ, Hytönen J. Clinical performance and analytical accuracy of a C6 peptide-based point-of-care lateral flow immunoassay in Lyme borreliosis serology. Diagn Microbiol Infect Dis. 2022;103:115657.

72. Eldin C, Raffetin A, Bouiller K, Hansmann Y, Roblot F, Raoult D, et al. Review of European and American guidelines for the diagnosis of Lyme borreliosis. Médecine Mal Infect. 2019;49:121-32.

73. Joung H-A, Ballard ZS, Wu J, Tseng DK, Teshome H, Zhang L, et al. Point-of-Care Serodiagnostic Test for Early-Stage Lyme Disease Using a Multiplexed Paper-Based Immunoassay and Machine Learning. ACS Nano. 2020;14:229-40.

74. Häring J, Hassenstein MJ, Becker M, Ortmann J, Junker D, Karch A, et al. Borrelia multiplex: a bead-based multiplex assay for the simultaneous detection of Borrelia specific IgG/IgM class antibodies. BMC Infect Dis. 2022;22:859.

75. Nayak S, Sridhara A, Melo R, Richer L, Chee NH, Kim J, et al. Microfluidics-based point-of-care test for serodiagnosis of Lyme Disease. Sci Rep. 2016;6:35069.

76. Jin C, Roen DR, Lehmann PV, Kellermann GH. An Enhanced ELISPOT Assay for Sensitive Detection of Antigen-Specific T Cell Responses to Borrelia burgdorferi. Cells. 2013;2:607-20.

77. Baarsma ME, van de Schoor FR, Gauw SA, Vrijmoeth HD, Ursinus J, Goudriaan N, et al. Diagnostic parameters of cellular tests for Lyme

borreliosis in Europe (VICTORY study): a case-control study. Lancet Infect Dis. 2022;22:1388-96.

78. Callister SM, Jobe DA, Stuparic-Stancic A, Miyamasu M, Boyle J, Datwyler RJ, et al. Detection of IFN-γ Secretion by T Cells Collected Before and After Successful Treatment of Early Lyme Disease. Clin Infect Dis. 2016;62:1235-41.

79. Greissl J, Pesesky M, Dalai SC, Rebman AW, Soloski MJ, Horn EJ, et al. Immunosequencing of the T-Cell Receptor Repertoire Reveals Signatures Specific for Identification and Characterization of Early Lyme Disease. 2022;2021.07.30.21261353.

80. Haglund S, Lager M, Gyllemark P, Andersson G, Ekelund O, Sundqvist M, et al. CXCL13 in laboratory diagnosis of Lyme neuroborreliosis— the performance of the recomBead and ReaScan CXCL13 assays in human cerebrospinal fluid samples. Eur J Clin Microbiol Infect Dis. 2022;41:175-9.

81. van Gorkom T, van Arkel GHJ, Heron M, Voet W, Thijns SFT, Kremer K. The Usefulness of Two CXCL13 Assays on Cerebrospinal Fluid for the Diagnosis of Lyme Neuroborreliosis: a Retrospective Study in a Routine Clinical Setting. J Clin Microbiol. 2021;59:e0025521.

82. van Burgel ND, Bakels F, Kroes ACM, van Dam AP. Discriminating Lyme neuroborreliosis from other neuroinflammatory diseases by levels of CXCL13 in cerebrospinal fluid. J Clin Microbiol. 2011;49:2027-30.

83. Molins CR, Ashton LV, Wormser GP, Hess AM, Delorey MJ, Mahapatra S, et al. Development of a Metabolic Biosignature for Detection of Early Lyme Disease. Clin Infect Dis. 2015;60:1767-75.

84. Tabb J, Rapoport E, Han I, Lombardi J, Green O. An antigen-targeting assay for Lyme disease: Combining aptamers and SERS to detect the OspA protein. Nanomedicine Nanotechnol Biol Med. 2022;41:102528.

85. Goff NK, Dou T, Higgins S, Horn EJ, Morey R, McClellan K, et al. Testing Raman spectroscopy as a diagnostic approach for Lyme disease patients. Front Cell Infect Microbiol. 2022;12:1006134.

86. Senger RS, Sayed Issa A, Agnor B, Talty J, Hollis A, Robertson JL. Disease-Associated Multimolecular Signature in the Urine of Patients with Lyme Disease Detected Using Raman Spectroscopy and Chemometrics. Appl Spectrosc. 2022;76:284-99.

87. Halpern MD, Molins CR, Schriefer M, Jewett MW. Simple Objective Detection of Human Lyme Disease Infection Using Immuno-PCR and a Single Recombinant Hybrid Antigen. Clin Vaccine Immunol. 2014;21:1094-105.

88. Rodríguez MO, Covián LB, García AC, Blanco-López MC. Silver and gold enhancement methods for lateral flow immunoassays. Talanta. 2016;148:272-8.

89. Arumugam S, Nayak S, Williams T, di Santa Maria FS, Guedes MS, Chaves RC, et al. A Multiplexed Serologic Test for Diagnosis of Lyme Disease for Point-of-Care Use. J Clin Microbiol. 2019;57:e01142-e1219.

90. Livengood JA, Gilmore RD. Invasion of human neuronal and glial cells by an infectious strain of Borrelia burgdorferi. Microbes Infect. 2006;8:2832-40.

91. Ma Y, Sturrock A, Weis JJ. Intracellular localization of Borrelia burgdorferi within human endothelial cells. Infect Immun. 1991;59:671-8.

92. Woitzik P, Linder S. Molecular Mechanisms of Borrelia burgdorferi Phagocytosis and Intracellular Processing by Human Macrophages. Biology. 2021;10:567.

93. Petnicki-Ocwieja T, Kern A. Mechanisms of Borrelia burgdorferi internalization and intracellular innate immune signaling. Front Cell Infect Microbiol. 2014;4:175.

94. Oksi J, Savolainen J, Pêne J, Bôusquet J, Laippala P, Viljanen MK. Decreased interleukin-4 and increased gamma interferon production by peripheral blood mononuclear cells of patients with Lyme borreliosis. Infect Immun. 1996;64:3620-3.

95. Widhe M, Jarefors S, Ekerfelt C, Vrethem M, Bergstrom S, Forsberg P, et al. Borrelia-specific interferon-gamma and interleukin-4 secretion in cerebrospinal fluid and blood during Lyme borreliosis in humans: association with clinical outcome. J Infect Dis. 2004;189:1881-91.

96. Forsberg P, Ernerudh J, Ekerfelt C, Roberg M, Vrethem M, Bergström S. The outer surface proteins of Lyme disease borrelia spirochetes stimulate T cells to secrete interferon-gamma (IFN-gamma): diagnostic and pathogenic implications. Clin Exp Immunol. 1995;101:453-60.

97. Lehmann PV, Zhang W. Unique strengths of ELISPOT for T cell diagnostics. Methods Mol Biol Clifton NJ. 2012;792:3-23.
98. Prabhakar U, Kelley M. Validation of cell-based assays in the GLP setting: a practical guide. Pennsylvania: Wiley; 2008.

99. van Gorkom T, Sankatsing SUC, Voet W, Ismail DM, Muilwijk RH, Salomons M, et al. An Enzyme-Linked Immunosorbent Spot Assay Measuring Borrelia burgdorferi B31-Specific Interferon Gamma-Secreting T Cells Cannot Discriminate Active Lyme Neuroborreliosis from Past Lyme Borreliosis: a Prospective Study in the Netherlands. J Clin Microbiol. 2018;56:e01695-e1717.

100. Syed Ahamed Kabeer B, Raman B, Thomas A, Perumal V, Raja A. Role of QuantiFERON-TB Gold, Interferon Gamma Inducible Protein-10 and Tuberculin Skin Test in Active Tuberculosis Diagnosis. PLoS One. 2010;5:e9051.

101. Baarsma ME, van de Schoor FR, Van den Wijngaard CC, Joosten LAB, Kullberg BJ, Hovius JW. The Initial QuantiFERON-Lyme Prototype is Unsuitable for European Patients. Clin Infect Dis. 2021;73:1125-6.

102. van de Schoor FR, Vijrijoeth HD, Brouwer MAE, ter Hofstede HJM, Lemmers HLM, Dijkstra H, et al. Borrelia burgdorferi Is a Poor Inducer of Gamma Interferon: Amplification Induced by Interleukin-12. Infect Immun. 2022;90:e0055821.

103. Oosting M, Brouwer M, Vijrijoeth HD, Pascual Domingo R, Greco A, ter Hofstede H, et al. Borrelia burgdorferi is strong inducer of IFN-γ production by human primary NK cells. Cytokine. 2022;155:155895.

104. van de Schoor FR, Baarsma ME, Gauw SA, Joosten LAB, Kullberg BJ, van den Wijngaard CC, et al. Validation of cellular tests for Lyme borreliosis (VICTORY) study. BMC Infect Dis. 2019;19:732.

105. Talagrand-Reboul E, Raffetin A, Zachary P, Jaulhac B, Eldin C. Immunoserological Diagnosis of Human Borrelioses: Current Knowledge and Perspectives. Front Cell Infect Microbiol. 2020;10:241.

106. Valentine-Thon E, Ilsemann K, Sandkamp M. A novel lymphocyte transformation test (LTT-MELISA) for Lyme borreliosis. Diagn Microbiol Infect Dis. 2007;57:27-34.

107. Badawi A. The Potential of Omics Technologies in Lyme Disease Biomarker Discovery and Early Detection. Infect Dis Ther. 2017;6:85-102.

108. Rauer S, Kastenbauer S, Hofmann H, Fingerle V, Huppertz H-I, Hunfeld K-P, et al. Guidelines for diagnosis and treatment in neurology - Lyme neuroborreliosis. GMS Ger Med Sci. 2020;18:Doc03.

109. Strle F, Stanek G. Clinical Manifestations and Diagnosis of Lyme Borreliosis. Lyme Borreliosis. 2009;37:51-110.

110. Ansel KM, Harris RBS, Cyster JG. CXCL13 Is Required for B1 Cell Homing, Natural Antibody Production, and Body Cavity Immunity. Immunity. 2002;16:67-76.

111. Legler DF, Loetscher M, Roos RS, Clark-Lewis I, Baggiolini M, Moser B. B Cell-attracting Chemokine 1, a Human CXC Chemokine Expressed in Lymphoid Tissues, Selectively Attracts B Lymphocytes via BLR1/CXCR5. J Exp Med. 1998;187:655-60.

112. Rupprecht TA, Kirschning JC, Popp B, Kastenbauer S, Fingerle V, Pfister H-W, et al. Borrelia garinii Induces CXCL13 Production in Human Monocytes through Toll-Like Receptor 2. Infect Immun. 2007;75:4351-6.

113. Yang J, Han X, Liu A, Bao F, Peng Y, Tao L, et al. Chemokine CXC Ligand 13 in Cerebrospinal Fluid Can Be Used as an Early Diagnostic Biomarker for Lyme Neuroborreliosis: A Meta-Analysis. J Interferon Cytokine Res. 2017;37:433-9.

114. Rupprecht TA, Koedel U, Angele B, Fingerle V, Pfister H-W. Zyktokin CXCL13. Nervenarzt. 2006;77:470-3.

115. Senel M, Rupprecht TA, Tumani H, Pfister HW, Ludolph AC, Brettschneider J. The chemokine CXCL13 in acute neuroborreliosis. J Neurol Neurosurg Psychiatry. 2010;81:929-33.

116. Wagner JN, Weis S, Kubasta C, Panholzer J, von Oertzen TJ. CXCL13 as a diagnostic marker of neuroborreliosis and other neuroinflammatory disorders in an unselected group of patients. J Neurol. 2018;265:74-81.

117. Dersch R, Hottenrott T, Senel M, Lehmensiek V, Tumani H, Rauer S, et al. The chemokine CXCL13 is elevated in the cerebrospinal fluid of patients with neurosyphilis. Fluids Barriers CNS. 2015;12:12.

118. Rubenstein JL, Wong VS, Kadoch C, Gao H-X, Barajas R, Chen L, et al. CXCL13 plus interleukin 10 is highly specific for the diagnosis of CNS lymphoma. Blood. 2013;121:4740-8.

119. Leth TA, Dessau RB, Møller JK. Discriminating between Lyme neuroborreliosis and other central nervous system infections by use of biomarkers CXCL13 and IL-6: Supplemental laboratory analysis in diagnostics of Lyme neuroborreliosis. Ticks Tick-Borne Dis. 2022;13:101984.

120. Soloski MJ, Crowder LA, Lahey LJ, Wagner CA, Robinson WH, Aucott JN. Serum Inflammatory Mediators as Markers of Human Lyme Disease Activity. PLoS One. 2014;9:e93243.

121. Liu M, Guo S, Hibbert JM, Jain V, Singh N, Wilson NO, et al. CXCL10/IP-10 in infectious diseases pathogenesis and potential therapeutic implications. Cytokine Growth Factor Rev. 2011;22:121-30.

122. Gyllemark P, Sjöwall J, Forsberg P, Ernerudh J, Henningsson A. Intrathecal Th17-driven inflammation is associated with prolonged post-treatment convalescence for patients with Lyme neuroborreliosis. 2023.

123. Aucott JN, Soloski MJ, Rebman AW, Crowder LA, Lahey LJ, Wagner CA, et al. CCL19 as a Chemokine Risk Factor for Posttreatment Lyme Disease Syndrome: a Prospective Clinical Cohort Study. Clin Vaccine Immunol. 2016;23:757-66.

124. Crowley JT, Drouin EE, Pianta A, Strle K, Wang Q, Costello CE, et al. A Highly Expressed Human Protein, Apolipoprotein B-100, Serves as an Autoantigen in a Subgroup of Patients With Lyme Disease. J Infect Dis. 2015;212:1841-50.

125. Swan AL, Mobasheri A, Allaway D, Liddell S, Bacardit J. Application of Machine Learning to Proteomics Data: Classification and Biomarker Identification in Postgenomics Biology. OMICS J Integr Biol. 2013;17:595-610.

126. Vinayavekhin N, Homan EA, Saghatelat A. Exploring Disease through Metabolomics. ACS Chem Biol. 2010;5:91-103.

127. Balog CIA, Meissner A, Göraler S, Bladergroen MR, Vennervald BJ, Mayboroda OA, et al. Metabonomic investigation of human Schistosoma mansoni infection. Mol Biosyst. 2011;7:1473-80.

128. Ligor M, Olszowy P, Buszewski B. Application of medical and analytical methods in Lyme borreliosis monitoring. Anal Bioanal Chem. 2012;402:2233-48.

129. Magni R, Almofee F, Yusuf S, Mueller C, Vuong N, Almosuli M, et al. Evaluation of pathogen specific urinary peptides in tick-borne illnesses. Sci Rep. 2020;10:19340.

130. Farber C, Morey R, Krimmer M, Kurouski D, Rogovskyy AS. Exploring a possibility of using Raman spectroscopy for detection of Lyme disease. J Biophotonics. 2021;14:e202000477.

131. Crowley JT, Toledo AM, LaRocca TJ, Coleman JL, London E, Benach JL. Lipid Exchange between Borrelia burgdorferi and Host Cells. PLOS Pathog. 2013;9:e1003109.

132. Arora G, Hart T, Fikrig E. Use of host lipids by the Lyme disease spirochete may lead to biomarkers. J Clin Invest. 2022;132:e158254.

133. Gwynne PJ, Clendenen LH, Turk S-P, Marques AR, Hu LT. Antiphospholipid autoantibodies in Lyme disease arise after scavenging of host phospholipids by Borrelia burgdorferi. J Clin Invest. 2022;132:e152506.

134. Molins CR, Ashton LV, Wormser GP, Andre BG, Hess AM, Delorey MJ, et al. Metabolic differentiation of early Lyme disease from southern tick-associated rash illness (STARI). Sci Transl Med. 2017;9:eaa12717.

135. Petzke MM, Volyanskyy K, Mao Y, Arevalo B, Zohn R, Quituisaca J, et al. Global Transcriptome Analysis Identifies a Diagnostic Signature for Early Disseminated Lyme Disease and Its Resolution. mBio. 2020;11:e00047-20.

136. Servellita V, Bouquet J, Rebman A, Yang T, Samayoa E, Miller S, et al. A diagnostic classifier for gene expression-based identification of early Lyme disease. Commun Med. 2022;2:92.

137. Clarke DJB, Rebman AW, Fan J, Soloski MJ, Aucott JN, Ma'ayan A. Gene set predictor for post-treatment Lyme disease. Cell Rep Med. 2022;3:100816.

138. Trevisan G, Bonin S, Ruscio M. A Practical Approach to the Diagnosis of Lyme Borreliosis: From Clinical Heterogeneity to Laboratory Methods. Front Med. 2020;7:265.

139. Lardieri G, Salvi A, Camerini F, Ciñco M, Trevisan G. Isolation of Borrelia burgdorferi from myocardium. Lancet. 1993;342:490.

140. Ružić-Sabljić E, Maraspin V, Cimperman J, Strle F, Lotrič-Furlan S, Stupica D, et al. Comparison of isolation rate of Borrelia burgdorferi sensu lato in two different culture media. MKP and BSK-H Clin Microbiol Infect. 2014;20:636-41.

141. Ružić-Sabljić E, Maraspin V, Stupica D, Rojko T, Bogović P, Strle F, et al. Comparison of MKP and BSK-H media for the cultivation and isolation of Borrelia burgdorferi sensu lato. PLoS One. 2017;12:e0171622.

142. Ružić-Sabljić E, Maraspin V, Cimperman J, Lotrič-Furlan S, Stupica D, Cerar T. Comparison of isolation rate of Borrelia burgdorferi sensu lato
in two different culture media. MKP and BSK-H Clin Microbiol Infect. 2014;20:636-41.

143. Berthold A, Faucillion M-L, Nilsson I, Golovchenko M, Lloyd V, Bergström S, et al. Cultivation Methods of Spirochetes from Borrelia burgdorferi Sensu Lato Complex and Relapsing Fever Borrelia. J Vis Exp JoVE. 2022. https://doi.org/10.3791/64431.

144. Jutras BL, Chenail AM, Stevenson B. Changes in Bacterial Growth Rate Govern Expression of the Borrelia burgdorferi OspC and Erp Infection-Associated Surface Proteins. J Bacteriol. 2013;195:757-64.

145. Coulter P, Lema C, Flayhart D, Linhardt AS, Aucott JN, Auwaerter PG, et al. Two-Year Evaluation of Borrelia burgdorferi Culture and Supplemental Tests for Definitive Diagnosis of Lyme Disease. J Clin Microbiol. 2005;43:5080-4.

146. Zückert WR. Laboratory Maintenance of Borrelia burgdorferi. Curr Protoc Microbiol. 2007;4:12C.1.1-12C.1.10.

147. Kullberg BJ, Vrijmoeth HD, van de Schoor F, Hovius JW. Lyme borreliosis: diagnosis and management. BMJ. 2020;369:m1041.

148. Goodman JL, Bradley JF, Ross AE, Goellner P, Lagus A, Vitale B, et al. Bloodstream invasion in early Lyme disease: Results from a prospective, controlled, blinded study using the polymerase chain reaction. Am J Med. 1995;99:6-12.

149. Wormser GP, Bittker S, Cooper D, Nowakowski J, Nadelman RB, Pavia C. Yield of Large-Volume Blood Cultures in Patients with Early Lyme Disease. J Infect Dis. 2001;184:1070-2.

150. Mali S, Mitchell M, Havis S, Bodunrin A, Rangel J, Olson G, et al. A Proteomic Signature of Dormancy in the Actinobacterium Micrococcus luteus. J Bacteriol. 2017;199:e00206-e217.

151. Stupica D, Lusa L, Maraspin V, Bogović P, Vidmar D, O'Rourke M, et al. Correlation of Culture Positivity, PCR Positivity, and Burden of Borrelia burgdorferi Sensu Lato in Skin Samples of Erythema Migrans Patients with Clinical Findings. PLoS One. 2015;10:e0136600.

152. Rauter C, Mueller M, Diterich I, Zeller S, Hassler D, Meergans T, et al. Critical Evaluation of Urine-Based PCR Assay for Diagnosis of Lyme Borreliosis. Clin Vaccine Immunol. 2005;12:910-7.

153. Ruzić-Sabljić E, Cerar T. Progress in the molecular diagnosis of Lyme disease. Expert Rev Mol Diagn. 2017;17:19-30.

154. Colli C,leinweber B, Müllegger R, Chott A, Kerl H, Cerroni L. Borrelia burgdorferi-associated lymphocytoma cutis: clinicopathologic, immunophenotypic, and molecular study of 106 cases. J Cutan Pathol. 2004;31:232-40.

155. Lager M, Faller M, Wilhelmsson P, Kjelland V, Andreassen Å, Dargis R, et al. Molecular detection of Borrelia burgdorferi sensu lato – An analytical comparison of real-time PCR protocols from five different Scandinavian laboratories. PLoS ONE. 2017;12:e0185434.

156. Arvikar SL, Steere AC. Diagnosis and treatment of Lyme arthritis. Infect Dis Clin North Am. 2015;29:269-80.

157. Shan J, Jia Y, Teulières L, Patel F, Clokie MRJ. Targeting Multicopy Prophage Genes for the Increased Detection of Borrelia burgdorferi Sensu Lato (s.l.), the Causative Agents of Lyme Disease, in Blood. Front Microbiol. 2021;12:e651217.

158. Laane M, Mysterud I. A simple method for the detection of live Borrelia spirochaetes in human blood using classical microscopy techniques. Biol Biomed Rep. 2013;3:15-28.

159. Aase A, Hajdusek O, Øines Ø, Quarsten H, Wilhelmsson P, Herstad TK, et al. Validate or falsify: Lessons learned from a microscopy method claimed to be useful for detecting Borrelia and Babesia organisms in human blood. Infect Dis Lond Engl. 2016;48:411-9.

160. Bockenstedt LK, Mao J, Hodzic E, Barthold SW, Fish D. Detection of attenuated, noninfectious spirochetes in Borrelia burgdorferi-infected mice after antibiotic treatment. J Infect Dis. 2002;186:1430-7.

161. Embers ME, Barthold SW, Borda JT, Bowers L, Doyle L, Hodzic E, et al. Persistence of Borrelia burgdorferi in Rhesus Macaques following Antibiotic Treatment of Disseminated Infection. PLoS ONE. 2012;7:e29914.

162. Marques A, Telford SR III, Turk S-P, Chung E, Williams C, Dardick K, et al. Xenodiagnosis to Detect Borrelia burgdorferi Infection: A First-in-Human Study. Clin Infect Dis. 2014;58:937-45.

163. Bockenstedt LK, Radolf JD. Editorial Commentary: Xenodiagnosis for Posttreatment Lyme Disease Syndrome: Resolving the Conundrum or Adding to It? Clin Infect Dis. 2014;58:946-8.

164. Coyle PK, Deng Z, Schutzer SE, Belman AL, Benach J, Krupp LB, et al. Detection of Borrelia burgdorferi antigens in cerebrospinal fluid. Neurology. 1993;43:1093-1093.

165. Dorward DW, Schwan TG, Garon CF. Immune capture and detection of Borrelia burgdorferi antigens in urine, blood, or tissues from infected ticks, mice, dogs, and humans. J Clin Microbiol. 1991;29:1162-70.

166. Hyde FW, Johnson RC, White TJ, Shelburne CE. Detection of antigens in urine of mice and humans infected with Borrelia burgdorferi, etiologic agent of Lyme disease. J Clin Microbiol. 1989;27:58-61.

167. Cheung CSF, Anderson KW, Benitez KYV, Soloski MJ, Aucott JN, Phinney KW, et al. Quantification of Borrelia burgdorferi Membrane Proteins in Human Serum: A New Concept for Detection of Bacterial Infection. Anal Chem. 2015;87:11383-8.

168. Magni R, Espina BH, Shah K, Lepene B, Mayuga C, Douglas TA, et al. Application of Nanotrap technology for high sensitivity measurement of urinary outer surface protein A carboxyl-terminus domain in early stage Lyme borreliosis. J Transl Med. 2015;13:346.

169. Douglas T, Tamburro D, Fredolini C, Espina B, Lepene BS, Ilag L, et al. The Use of Hydrogel Microparticles to Sequester and Concentrate Bacterial Antigens in a Urine Test for Lyme Disease. Biomaterials. 2011;32:1157-66.

170. Luchini A, Geho DH, Bishop B, Tran D, Xia C, Dufour R, et al. Smart Hydrogel Particles: Biomarker Harvesting: One-step affinity purification, size exclusion, and protection against degradation. Nano Lett. 2008;8:350-61.

171. Otieno BA, Krause CE, Rusling JF. Chapter Seven - Bioconjugation of Antibodies and Enzyme Labels onto Magnetic Beads. In: Kumar CV, editor. Methods in Enzymology. Academic Press; 2016. p. 135-50.

172. Flynn CD, Sandomierski M, Kim K, Lewis J, Lloyd V, Ignaszak A. Electrochemical Detection of Borrelia burgdorferi Using a Biomimetic Flow Cell System. ACS Meas Sci Au. 2023. https://doi.org/10.1021/acsmesasuresciau.3c00004.

173. Brisson D, Drecktrah D, Eggers CH, Samuels DS. Genetics of Borrelia burgdorferi. Annu Rev Genet. 2012;46:515-36.

174. Kight E, Alfaro R, Gadila SKG, Chang S, Evans D, Embers M, et al. Direct Capture and Early Detection of Lyme Disease Spirochete in Skin with a Microneedle Patch. Biosensors. 2022;12:819.

175. Buniksen J, Barbour AG. Laboratory testing for suspected lyme disease. Med Clin North Am. 2002;86:311-40.

176. Barthold SW, Hodzic E, Imai DM, Feng S, Yang X, Luft BJ. Ineffective-ness of Tigecycline against Persistent Borrelia burgdorferi. Antimicrob Agents Chemother. 2010;54:643-51.

177. Hunfeld K-P, Ruzic-Sabljić E, Norris DE, Kraiczy P, Strle F. In Vitro Susceptibility Testing of Borrelia burgdorferi Sensu Lato Isolates Cultured from Patients with Erythema Migrans before and after Antimicrobial Chemotherapy. Antimicrob Agents Chemother. 2005;49:1294-301.

178. Karvonen K, Tammisto H, Nykkjy J, Gilbert L. Borrelia burgdorferi Outer Membrane Vesicles Contain Antigenic Proteins, but Do Not Induce Cell Death in Human Cells. Microorganisms. 2022;10:212.

179. Lager M, Wilhelmsson P, Matussek A, Lindgren P-E, Hennningsson AJ. Molecular Detection of Borrelia Bacteria in Cerebrospinal Fluid-Optimisation of Pre-Analytical Sample Handling for Increased Analytical Sensitivity. Diagnostics. 2021;11:2088.

180. Lacout A, Thariat J, Hajjam ME, Marcy PY, Perronne C. Lyme disease & co-infections: role of adaptive immune system. Future Microbiol. 2018;13:613-5.

181. Adler M, Wacker R, Niemeyer CM. A real-time immuno-PCR assay for routine ultrasensitive quantification of proteins. Biochem Biophys Res Commun. 2003;308:240-50.

182. Niemeyer CM, Adler M, Wacker R. Immuno-PCR: high sensitivity detection of proteins by nucleic acid amplification. Trends Biotechnol. 2005;23:208-16.

183. Halpern MD, Jain S, Jewett MW. Enhanced Detection of Host Response Antibodies to Borrelia burgdorferi Using Immuno-PCR. Clin Vaccine Immunol. 2013;20:350-7.

184. Hoversten K, Bartlett MA. Diagnosis of a tick-borne coinfection in a patient with persistent symptoms following treatment for Lyme disease. Case Rep. 2018;2018:bcr2018225342.

185. Krause PJ, McKay K, Thompson CA, Sikand VK, Lentz R, Lepore T, et al. Disease-Specific Diagnosis of Coinfecting Tickborne Zoonoses:
Babesiosis, Human Granulocytic Ehrlichiosis, and Lyme Disease. Clin Infect Dis. 2002;34:1184-91.

186. Scott JD, Pesapane RR. Detection of Anaplasma phagocytophilum, Babesia odocoilei, Babesia sp., Borrelia burgdorferi Sensu Lato, and Hepatozoon canis in Ixodes scapularis Ticks Collected in Eastern Canada. Pathogens. 2021;10:1265.

187. Tijsse-Klasen E, Sprong H, Pandak N. Co-infection of Borrelia burgdorferi sensu lato and Rickettsia species in ticks and in an erythema migrans patient. Parasit Vectors. 2013;6:347.

188. Crowder CD, Matthews HE, Schutzer S, Rounds MA, Luft BJ, Nolte O, et al. Genotypic Variation and Mixtures of Lyme Borrelia in Ixodes Ticks from North America and Europe. PLoS One. 2010;5:e10650.

189. Eshoo MW, Crowder CC, Rebman AW, Rounds MA, Matthews HE, Picuri JM, et al. Direct Molecular Detection and Genotyping of Borrelia burgdorferi from Whole Blood of Patients with Early Lyme Disease. PLoS One. 2012;7:e36825.

190. Farlow J, Postic D, Smith KL, Jay Z, Baranton G, Keim P. Strain Typing of Borrelia burgdorferi, Borrelia afzelii, and Borrelia garinii by Using Multiple-Locus Variable-Number Tandem Repeat Analysis. J Clin Microbiol. 2002;40:4612-8.

191. Margos G, Gatewood AG, Aanensen DM, Hanincová K, Terekhova D, Vollmer SA, et al. MLST of housekeeping genes captures geographic population structure and suggests a European origin of Borrelia burgdorferi. Proc Natl Acad Sci. 2008;105:8730-5.

192. Hanschmann H, Rödiger S, Kramer T, Hanschmann K, Steidle M, Fingerle V, et al. LoopTag FRET Probe System for Multiplex qPCR Detection of Borrelia Species. Life. 2021;11:1163.

193. Maggi R, Breitschwerdt EB, Qurollo B, Miller JC. Development of a Multi-plex Droplet Digital PCR Assay for the Detection of Babesia, Bartonella, and Borrelia Species. Pathogens. 2021;10:1462.

194. Rodríguez I, Burri C, Noda AA, Douet V, Gern L. Multiplex PCR for molecular screening of Borrelia burgdorferi sensu lato, Anaplasma spp. and Babesia spp. Ann Agric Environ Med AAEM. 2015;22:642-6.

195. Argov T, Sapir SR, Pasechnek A, Azulay G, Stadnyuk O, Rabinovich L, et al. Coordination of cohabiting phage elements supports bacteriophage cooperation. Nat Commun. 2019;10:5288.

196. Teulières LC. Test Phelix Phage Borrelia. Dr. Louis Teulières. 2021. https://louisteulières.com/test-phage/. Accessed 23 Nov 2021.

197. Eggers CH, Samuels DS. Molecular evidence for a new bacteriophage of Borrelia burgdorferi. J Bacteriol. 1999;181:7308-13.

198. Sun S, Gao S, Kondabagil K, Xiang Y, Rossmann MG, Rao VB. Structure and function of the small terminase component of the DNA packaging machine in T4-like bacteriophages. Proc Natl Acad Sci U S A. 2012;109:817-22.

199. van de Schoor FR, Baarsma ME, Leeflang MMG, Fingerle V, Margos G, Hovius JW, et al. Opinion: Methodological Shortcomings in the Study on a Prophage-based PCR Test for Lyme Borreliosis. Front Microbiol. 2021;12:802131.

200. Kumar Y, Koul A, Singla R, Ijaz MF. Artificial intelligence in disease diagnosis: a systematic literature review, synthesizing framework and future research agenda. J Ambient Intell Humaniz Comput. 2023;14:8459-86.

201. Trevisan G, Stinco G, Cattonar P, Nobile C. Unusual onset of Lyme borreliosis simulating herpes zoster. J Eur Acad Dermatol Venereol. 1995;4:299-300.

202. AlZubi AA, Tiwari S, Walia K, Alanazi JM, AlZobi FI, Verma R. An Efficient Stacked Deep Transfer Learning Model for Automated Diagnosis of Lyme Disease. Comput Intell Neurosci. 2022;2022:e2933015.

203. Burlina PM, Joshi NJ, Mathew PA, Paul W, Rebman AW, Aucott JN. Al-based detection of erythema migrans and disambiguation against other skin lesions. Comput Biol Med. 2020;125:103977.

**Publisher's Note**

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Ready to submit your research? Choose BMC and benefit from:

* fast, convenient online submission

* thorough peer review by experienced researchers in your field

* rapid publication on acceptance

* support for research data, including large and complex data types

* gold Open Access which fosters wider collaboration and increased citations

* maximum visibility for your research: over 100M website views per year

At BMC, research is always in progress.

Learn more biomedcentral.com/submissions

<figure><img src="image_8.png" /></figure>