Carole Eldin

Philippe Parola

Didier Raoult
email: didier.raoult@gmail.com

Limitations of diagnostic tests for bacterial infections

Keywords: sérologie, PCR, réactions croisées, maladie de Lyme serology, PCR, cross-reactions, Lyme disease

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Spirochetes and the example of syphilis

Syphilis, caused by Treponema pallidum (a spirochete similar to Borrelia), is an example of infection that has been accused of causing a variety of unexplained syndromes because of the use of poorly reliable serological tests. Serological testing for syphilis, known as "Wasserman test", used to be based on the detection of human cardiolipin [START_REF] Cirera | Rapid technic for the Bordet-Wasserman sero-reaction with hemolytic complex[END_REF]. The serology was positive in many situations other than the syphilis infection, and was possibly temporarily positive, such as during pregnancy, viral infections, parasitic infections, or autoimmune diseases [START_REF] Berntsson | The Wasserman, Kline and VDRL reactions in routine syphilis serodiagnosis[END_REF][START_REF] Null | Pneumonia with a transitory positive Bordet-Wasserman in the blood, and a constant serologic reaction in the cerebrospinal fluid[END_REF][START_REF] Carpio | Non-specific Wassermann reaction in cerebrospinal fluid in a case of cerebral cysticercosis[END_REF]. As a consequence, between the first and the second world war, physicians used to believe that Rh incompatibility between mother and child was due to seropositive or seronegative congenital syphilis. However, nontreponemal serological tests used in the diagnosis of syphilis have always lacked specificity.

As early as the 1980s, our team and other authors demonstrated the existence of crossreactions between all spirochetes and mainly between the causative agents of syphilis, leptospirosis, and Borrelia infections [START_REF] Hechemy | Cross-reaction of immune sera from patients with rickettsial diseases[END_REF]. It was demonstrated that the cross-reaction between Borrelia and Treponema pallidum could partly be inhibited by prior absorption by Reiter's treponema (treponema strain that can be cultured in vitro, non-pathogenic species) [START_REF] Hunter | Evaluation of sera from patients with Lyme disease in the fluorescent treponemal antibody-absorption test for syphilis[END_REF]. We therefore used to systematically perform this prior absorption at the laboratory to avoid crossreactions with other spirochetes before performing the Lyme serology [START_REF] Raoult | Cross-reaction with Borrelia burgdorferi antigen of sera from patients with human immunodeficiency virus infection, syphilis, and leptospirosis[END_REF]. The development of confirmatory Western Blot technology was associated with better specificity [START_REF] Raoult | Crossed reactions in Lyme disease. Value of the Western blot[END_REF][START_REF] Arzouni | Comparison of western blot and microimmunofluorescence as tools for Lyme disease seroepidemiology[END_REF].

However, the Western Blot technique still does not distinguish Borrelia species responsible for recurrent fever from Borrelia species responsible for Lyme disease [START_REF] Krause | Human Borrelia miyamotoi infection in California: Serodiagnosis is complicated by multiple endemic Borrelia species[END_REF]. The serological diagnosis of borrelioses, including Lyme disease, is imperfect and will always be imperfect because of consubstantial limitations associated with bacterial serology.

Other historical examples of cross-reactions in bacterial serology

In 1916, as major typhus epidemics sparked during the first world war, Edmund Weil and Arthur Felix developed a serological test based on the finding that the serum of patients presenting with typhus cross-reacted with bacteria of the Proteus genus [START_REF] Wilson | The Wilson-Weil-Felix Reaction in Typhus Fever1[END_REF]. It is only in the 1940s that serological tests based on Rickettsia spp. antigens (true causative agents of typhus)

were developed. For many years -and still today in countries such as India -Weil-Felix serological test was used with OX19 (Proteus vulgaris) yielding cross-reactions with typhusgroup Rickettsia spp. [START_REF] Eremeeva | Serological response of patients suffering from primary and recrudescent typhus: comparison of complement fixation reaction, Weil-Felix test, microimmunofluorescence, and immunoblotting[END_REF], with OX2 (Proteus vulgaris) yielding cross-reactions with spottedgroup Rickettsia spp. [START_REF] Raoult | Immunoblot cross-reactions among Rickettsia, Proteus spp. and Legionella spp. in patients with Mediterranean spotted fever[END_REF], and with OXK (Proteus mirabilis) yielding cross-reactions with

Orientia tsutsugamushi [START_REF] Swierzko | Structural and serological studies of the O-antigen of the bacterium Proteus mirabilis OXK (serogroup O3) used in the Weil-Felix test[END_REF]. Lipopolysaccharides of Proteus are responsible for crossreactions, not only with Rickettsia spp. but also with Legionella spp. [START_REF] Raoult | Immunoblot cross-reactions among Rickettsia, Proteus spp. and Legionella spp. in patients with Mediterranean spotted fever[END_REF][START_REF] Swierzko | Structural and serological studies of the O-antigen of the bacterium Proteus mirabilis OXK (serogroup O3) used in the Weil-Felix test[END_REF][START_REF] Amano | Serological studies of the antigenic similarity between typhus group rickettsiae and Weil-Felix test antigens[END_REF].

Another example taken from the research field of Rickettsia spp. led to incorrect assumptions: the microagglutination test, developed by Giroud in the 1940s [START_REF] Giroud | Rickettsia-Agglutina-tion Test ; Serum-Protection Test ; and Reaction of Cutaneous Hyper-sensitivity[END_REF]. This method had very poor specificity and yielded many false positive results (25%-30%), as reported by Edlinger and Raoult [START_REF] Edlinger | Les séquelles neuro-vasculaires des Rickettsioses: Une appréciation critique du diagnostic[END_REF][START_REF] Raoult | The rickettsial origin of multiple sclerosis: the end of a myth[END_REF]. This technique thus led several authors to believing that diseases without any proven etiology such as multiple sclerosis, vasculitis including Buerger's disease, schizophrenia or "tetany", were caused by Rickettsia spp. [START_REF] Gac | Tetany and rickettsiosis. Rickettsia mooserii in the etiology of tetany[END_REF][START_REF] Jadin | Maladies rickettsiennes et sclérose en plaques[END_REF][START_REF] Gac | On a possible rickettsial and neo-rickettsial etiology of multiple sclerosis][END_REF][START_REF] Gac | The treatment of multiple sclerosis of rickettsial or neo-rickettsial origin][END_REF][START_REF] Legac | APROPOS OF NEGATIVE MICROAGGLUTINATION REACTIONS AGAINST RICKETTSIA AND NEORICKETTSIA IN MULTIPLE SCLEROSIS[END_REF]. This technique was then no longer used in France because of its lack of specificity. It is, however, still being used in some regions of the world such as South Africa, leading some to say that Rickettsia spp. are responsible for syndromes such as chronic fatigue and fibromyalgia [25].

This type of cross-reactions may be observed with other bacteria. Musso et al.

demonstrated that up to 35% of patients presenting with Coxiella burnetii infection had a cross-reaction with Legionella micdadei, that could also lead to pulmonary involvement [START_REF] Musso | Serological cross-reactions between Coxiella burnetii and Legionella micdadei[END_REF].

These cross-reactions were once again mainly due to proteins and lipopolysaccharide antigens. Another study reported that the serum of patients presenting with a persisting C.

burnetii infection was positive for Bartonella henselae in 50% of cases, as detected by serology [START_REF] Scola | Serological cross-reactions between Bartonella quintana, Bartonella henselae, and Coxiella burnetii[END_REF].

PCR detection techniques

Bacterial DNA amplification by PCR contributed to major advances in human microbiology. However, just like any other diagnostic tests, it is associated with a risk of false positive and false negative results. False positive results may be explained by an aspecific amplification of environmental contaminants or, within laboratories performing routine PCR tests, of previously amplified products. This is why laboratories performing these tests should meet specific certification requirements. An external validation, performed by the reference centers for infectious diseases, is required for all commercialized tests [START_REF]Liste et coordonnées des CNR / Centres nationaux de référence / Espace professionnels / Accueil[END_REF]. These reference centers have many samples available and perfectly documented, i.e. corresponding to various stages of the disease for positive specimens or collected from "healthy" patients or from patients not carrying the disease for negative specimens. Laboratories performing medical diagnoses should also acquire the COFRAC certification, as this agency has the monopoly on evaluating procedures in French medical laboratories. This certification is now mandatory in the French private and public sectors, and each step of the procedure, each reagent, and each technique has to be validated. Positive and negative controls should be performed.

Some patients practicing doctor-hopping to obtain a diagnosis have their samples tested -sometimes with a prescription delivered by a physician -in veterinary laboratories which do not have to comply with the above-mentioned obligations and required certifications for the diagnosis in humans. Results from such laboratories are therefore not reliable. These biological results usually do not inform on the technique used, the positive and negative controls, nor do they specify the method used. Such practices cannot be accepted in the diagnosis of human infectious diseases.

Besides the issue of false positive results yielded by PCR tests and of the quality of the technique used, the PCR diagnosis also raises the question of the viability of microorganisms, which DNA is detected inside tissues, and of their pathogenicity. Our team has, for instance, reported a case of Streptococcus pneumoniae endocarditis with a PCR result positive for S.

pneumoniae on heart valve tissue seven years after the endocarditis episode, while the patient was clinically cured for the endocarditis [START_REF] Branger | Streptococcus pneumoniae endocarditis: persistence of DNA on heart valve material 7 years after infectious episode[END_REF]. A similar case was reported with Streptococcus mutans endocarditis and a positive PCR on heart valve tissue 31 months following the episode [START_REF] Gauduchon | Molecular diagnosis of recurrent Streptococcus mutans endocarditis by PCR amplification and sequencing[END_REF]. Both these cases raise the question of the residual presence of bacterial DNA without any particular meaning. Several in vitro studies also demonstrated that the DNA of bacteria killed via exposure to ultraviolet rays, boiling water, acid, or autoclave could still be detected by PCR, and the same probably holds in the tissues of living organisms [START_REF] Branger | Streptococcus pneumoniae endocarditis: persistence of DNA on heart valve material 7 years after infectious episode[END_REF]. Studies trying to demonstrate the persistence of Borrelia infection on the sole basis of PCR detection, without any specific clinical sign, cannot prove the active infection.

Benefits and limitations of biological tests

How can the "reliability" of a test be assessed? We usually talk about the "performance" of a test, which can be characterized by its sensitivity and specificity. Lyme disease diagnosis does not solely rely on the result of a serological test or of a PCR test.

Clinical and epidemiological characteristics should be taken into consideration in the test interpretation. This holds for Lyme disease, but also for any other disease. The predictive value of the serological test depends on the intrinsic characteristics of the test (sensitivity and specificity) and on the likelihood of having the disease (prevalence), which in turn depends on the tested sample. Let's take an example: a positive serology in a patient who do not present with any objective lesions suggestive of Lyme disease and who do not live nor has traveled to an endemic area, has a predictive value close to zero. Let's take another example: when performing seroprevalence studies in individuals excessively exposed to the disease (for instance forest rangers living in Alsace, France), up to 14% of them can have a positive serology without presenting any sign of the disease [START_REF] Rigaud | Seroprevalence of seven pathogens transmitted by the Ixodes ricinus tick in forestry workers in France[END_REF]. The proportion of people who contract Lyme disease following contact with Borrelia burgdorferi is variable, but the bacterium presence is not necessarily associated with disease onset in 100% of individuals following inoculation by a tick. This is why biological results cannot be interpreted on their own. Physicians' knowledge is required to interpret biological results in light of the patient's clinical features.

Conclusion

Borrelia burgdoferi sensu lato early acute infections have well-defined clinical characteristics and should thus be treated without biological diagnosis. The issue of subjective manifestations in people with tick bites and/or in people who never had confirmed Lyme disease, will not be solved based on current medical and scientific knowledge. However, no literature data currently suggests that species of the Borrelia burgdoferi sensu lato complex play a role in the genesis of chronic subjective symptoms, as suggested in the history of microbiology for other intracellular bacteria for which the diagnosis relied on serological testing.

1

  RésuméLe diagnostic de la maladie de Lyme est actuellement basé sur la sérologie, méthode de diagnostic indirect, du fait d'une culture en laboratoire fastidieuse. Le seul moyen de diagnostic direct qui peut être utile dans certains prélèvements (biopsies cutanées ou liquides de ponction) est la PCR. Nous détaillons dans cet article les principales limites de ces deux techniques pour le diagnostic des infections bactériennes, qui sont illustrées par des exemples tirés de l'histoire récente de la microbiologie. La principale limite de la sérologie bactérienne est la présence de nombreuses réactions croisées du fait de nombreux gènes communs entre espèces bactériennes. Certaines techniques sérologiques, pour le diagnostic des rickettsioses Current controversies on the diagnosis of Lyme disease contribute to putting into perspective the issue of the relevance of currently available diagnostic tools for bacterial infections. Species of the Borrelia burgdorferi sensu lato complex, i.e. the causative agents of Lyme disease, are associated with fastidious laboratory cultures and with a highly transitory bacteremia only occurring at the initial stage of the infection [1]. The main diagnostic method for this infection is serology, i.e. an indirect diagnostic method. Another method relies on DNA detection by PCR when tissue biopsies or synovial fluid or cerebrospinal fluid aspiration are performed. In the present article we detail the main limitations of both of these diagnostic tests, i.e. serology and PCR testing, drawing on examples from the recent history of microbiology.

	Bacterial serology
	Bacterial serology differs from viral serology. Viruses have great genetic variability
	and as a consequence, they have specific and rare proteins associated with an extremely low
	risk of cross-reactions. However, bacteria have a pool of common genes -especially genes
	encoding for the ribosome and genes encoding for heat-shock proteins -to which human
	serum is reactive. Bacterial peptidoglycan and lipopolysaccharide are also associated with
	numerous cross-reactions. Diagnostic interpretation difficulties thus remain for bacterial
	infections that can only be diagnosed by serology. The history of microbiology offers several
	examples of bacterial serologies based on cross-reactions. Serological testing may still
	contribute to the diagnosis when interpreted in light of the clinical context.

notamment, ont même été basées sur l'existence de réactions croisées. La principale limite de la PCR est la présence de contaminations de laboratoire possibles, ce qui nécessite une qualification des laboratoires qui pratiquent cette technique. De plus, la PCR ne renseigne pas sur la viabilité des bactéries détectées et doit donc être interprétée en fonction de la présentation clinique. Ceci illustre le fait que tout test de diagnostic microbiologique ne doit pas être interprété isolément mais doit toujours prendre en compte le contexte clinique et épidémiologique dans lequel il est effectué.


Carole Eldin

Benoit Jaulhac

Oleg Mediannikov

Jean-Pierre Arzouni

Didier Raoult

Performances des tests diagnostiques pour les différentes espèces de spirochètes Values of diagnostic tests for the various species of spirochetes

Keywords: spirochètes, Lyme, Borrelia, Leptospira, Treponema pallidum spirochetes, Lyme, Borrelia, Leptospira, Treponema pallidum

Les bactéries du complexe B. burgdorferi sensu lato, qui causent la maladie de Lyme, appartiennent au phylum des spirochètes. Les difficultés diagnostiques rencontrées dans la maladie de Lyme sont en partie dues aux caractéristiques des spirochètes qui sont des bactéries dont la culture est fastidieuse, voire impossible pour certaines d'entre elles. Nous avons réalisé une revue de la littérature concernant les performances des différents tests diagnostiques dans les spirochétoses d'intérêt médical que sont la borréliose de Lyme, les borrélioses récurrentes, la syphilis et la leptospirose. Cette revue permet de dégager un certain nombre de points communs pour ces quatre infections. La PCR en temps réel a pris une place importante ces dernières années dans le diagnostic direct de ces infections. Cependant, le diagnostic direct reste difficile du fait d'un manque de sensibilité persistant et les sérologies gardent donc un rôle central dans le raisonnement diagnostique. Tous les outils diagnostiques actuels présentent des imperfections avec un risque éventuel de faux positifs et de faux négatifs en fonction du contexte clinique. Ceci doit inciter les cliniciens à une interprétation des tests diagnostiques en cas de suspicion de maladie de Lyme, Borréliose récurrente, syphilis ou leptospirose, toujours en relation avec le contexte clinique et épidémiologique dans lequel ils se trouvent.

Introduction

Bacteria of the Borrelia burgdorferi sensu lato group (responsible for Lyme borreliosis) are spirochetes just like Borrelia spp. responsible for relapsing fever, Leptospira spp. responsible for leptospirosis (worldwide zoonosis transmitted through contact with urines of rodents), and Treponema spp. including the causative agents of syphilis (a sexuallytransmitted disease). Bacteria belonging to this phylum have specific characteristics, such as a helical form and motility due to pseudoflagella which help them move in viscous media [START_REF] Wolgemuth | The flagellar cytoskeleton of the spirochetes[END_REF]. Some spirochetes are commensal bacteria of humans, mainly of the oral flora, and others play a specific role in human pathology. Some Borrelia spp. transmitted by ticks or lice, are responsible for infections very different from Lyme disease, and are called relapsing fever borreliae, caused by ticks or lice. These infections all have very different clinical symptoms, and their diagnosis is based on the physical examination and physician's judgment as well as on confirmatory microbiological tests. Other specificities of bacteria belonging to this phylum are their non-Gram staining characteristics and their inability to be cultured using standard culture media. Laboratory diagnosis is therefore tedious.

Current debates are mainly sparked by patients' associations and some physicians who are questioning the reliability of Lyme borreliosis diagnostic tests. There is no such debate for other spirochetes. We assessed literature data on the value of diagnostic tests for Borrelia spp. responsible for Lyme disease and for other spirochetes of medical interest.

Current quality standards and French regulations for diagnostic tests in humans

Before analyzing scientific literature data, we should note that biomedical laboratories commercializing and performing diagnostic tests in humans have to comply with various regulations and standards in Europe and France: CE marking, inspections performed by the French Agency for the Safety of Health Products (French acronym ANSM), and COFRAC accreditation for laboratories performing biological tests. Manufacturers apply the CE marking on their tests, thus guarantying the conformity of their products with legal European requirements. The ANSM (https://ansm.sante.fr/) also performs a retrospective inspection to ensure conformity of the various biological tests marketed in France with health and safety requirements. The French accreditation committee, named COFRAC (https://www.cofrac.fr/), is the only national agency responsible for the accreditation of both public and private biology laboratories. For molecular biology techniques such as PCR detection of Borrelia, Treponema, and Leptospira, laboratories must therefore have their methods validated.

Additional tests must therefore be performed to ensure the absence of laboratory contaminations for instance. Quality of the tests performed by laboratories not complying with these requirements is not ensured. It should be noted that veterinary laboratories do not have to obtain the COFRAC accreditation, and that they are not allowed to perform human biological tests since May 30, 2013.

Lyme disease diagnosis

Except in case of typical erythema migrans, a positive biological test is required to confirm Lyme borreliosis diagnosis.

Direct diagnosis

Just like other spirochetes of medical interest, Borrelia spp. responsible for Lyme borreliosis cannot be detected by standard optical microscopy nor by Gram staining. They can, however, be detected by dark-field microscopy or phase-contrast microscopy [START_REF] Cutler | Diagnosing Borreliosis[END_REF]. However, bacteremia observed during Lyme borreliosis, especially with European clinical presentations, is moderate, of a short duration, and only occurs at the beginning of dissemination [START_REF] Cutler | Diagnosing Borreliosis[END_REF]. Looking for the bacterium in blood samples when patients do not have fever is therefore useless in case of Lyme borreliosis. The few pieces of data available after so-called positive microscopy have actually been demonstrated to be artifacts and not Borrelia [START_REF] Aase | Validate or falsify: Lessons learned from a microscopy method claimed to be useful for detecting Borrelia and Babesia organisms in human blood[END_REF]. The microscopic examination does not perform well enough to be a useful tool for the biological diagnosis of Lyme borreliosis.

The in vitro culture of Borrelia from tissue biopsy (skin, synovial membrane) or from biological fluid (CSF, synovial fluid) is performed on specific liquid media: BSK-II, MKP, or BSK-H (commercialized), incubated at 32-34°C. As the mean time to Borrelia growth is 7-20 hours, the time to positive culture is usually more than 15 days, or even more than eight weeks [START_REF] Schramm | La borréliose de Lyme[END_REF]. Cultures should therefore be checked once a week with a dark-field microscope for at least eight weeks before being able to conclude to a negative result, because Borrelia growth does not cloud the culture medium [START_REF] Schramm | La borréliose de Lyme[END_REF]. As Borrelia are highly fragile bacteria, specimens should be inoculated directly at the patient's bedside. Considering these technical requirements, Borrelia culture from human specimens is only performed by a few specialized laboratories in Europe. Although cultures perform well with erythema migrans specimens, this method lacks sensitivity when it is used with other biological specimens (CSF, skin biopsy of acrodermatitis chronica atrophicans, and synovial fluid where only a few isolates are observed) [START_REF] Cutler | Diagnosing Borreliosis[END_REF]. Moreover, no strain has so far been isolated from blood specimens of patients presenting with chronic disorders several years after a tick bite; culture from blood specimens is therefore currently not recommended.

The PCR detection of Borrelia is not associated with the constraints of culture.

Several PCR techniques are available and their value varies in terms of sensitivity, specificity, and detection spectrum [START_REF] Faller | EU-wide external quality assessement study on the sensitivity and specificity of different amplication protocol for détection of Borrelia burgdorferi sensu lato. 14th[END_REF]. Several PCR kits are currently commercialized, but their performance is poorly known. Direct detection of Borrelia by PCR test should thus only be performed by official and accredited specialized laboratories. However, sensitivity of the PCR test is much higher than that of culture in disseminated skin and articular presentations of the disease [START_REF] Aguero-Rosenfeld | Diagnosis of lyme borreliosis[END_REF]. Nonetheless, a negative PCR test result cannot rule out the Lyme borreliosis diagnosis. Physicians should always keep in mind that prescribing Borrelia PCR testing in case of a negative serology is not recommended, except for patients presenting with early atypical skin lesions of erythema migrans or very early Lyme neuroborreliosis [START_REF] Dessau | To test or not to test? Laboratory support for the diagnosis of Lyme borreliosis: a position paper of ESGBOR, the ESCMID study group for Lyme borreliosis[END_REF]. Among all direct diagnostic techniques, only PCR testing and culture -despite their limitations -can be used and have been validated for the detection of Borrelia. Microscopy techniques are indeed too often associated with artifacts and should be avoided.

Indirect diagnosis

Because of the above-mentioned limitations of direct techniques, the biological diagnosis of Lyme borreliosis is currently mainly based on serological testing. Results should always be interpreted in light of the clinical context and the incidence of the disease as they impact pre-test probability and consequently the positive and negative predictive values of the tests. Lyme borreliosis diagnosis in France is based on two-tiered serological testing in France, just like in most European countries and in the United States. This process improves the specificity of the final result [START_REF] Dessau | To test or not to test? Laboratory support for the diagnosis of Lyme borreliosis: a position paper of ESGBOR, the ESCMID study group for Lyme borreliosis[END_REF]. The first test is usually an ELISA assay; in case of a positive or dubious result, a second confirmatory test is performed by immunoblot technique (or Western blot) to confirm the specificity of anti-Borrelia antibodies [START_REF] Dessau | To test or not to test? Laboratory support for the diagnosis of Lyme borreliosis: a position paper of ESGBOR, the ESCMID study group for Lyme borreliosis[END_REF].

A European meta-analysis recently demonstrated that immunoblot tests used on their own were not associated with better sensitivity or specificity than ELISA tests for the diagnosis of Lyme borreliosis in Europe, whether it be for Lyme arthritis or Lyme neuroborreliosis [START_REF] Leeflang | The diagnostic accuracy of serological tests for Lyme borreliosis in Europe: a systematic review and meta-analysis[END_REF]. The overall sensitivity on serum in cases of Lyme neuroborreliosis was 81% (CI: 70-89%) for ELISA tests and 81% (CI: 57-96%) for immunoblot tests, with 92% specificity (CI: 88-89%) for ELISA tests and 94% (CI: 91-96%) for immunoblot tests. For Lyme arthritis, the overall sensitivity of ELISA tests was 94% (CI: 86-98%) and 95% (CI: 84-98%) for immunoblot tests, with 97% specificity (CI: 94-98%) for ELISA tests and 92% (CI: 84-96%) for immunoblot tests. This study also highlighted the lower accuracy of ELISA tests compared with immunoblot tests [START_REF] Leeflang | The diagnostic accuracy of serological tests for Lyme borreliosis in Europe: a systematic review and meta-analysis[END_REF]. The authors of this meta-analysis also reported a slight overall improvement of these tests, although statistically non-significant, when using purified antigens and/or recombinant antigens, mainly in patients presenting with Lyme neuroborreliosis [START_REF] Leeflang | The diagnostic accuracy of serological tests for Lyme borreliosis in Europe: a systematic review and meta-analysis[END_REF]. This two-tiered serological testing process improves the positive predictive value of the final result compared with a single ELISA or immunoblot test (Western blot) as they may yield up to 27% of false IgM positive results [START_REF] Seriburi | High frequency of false positive IgM immunoblots for Borrelia burgdorferi in clinical practice[END_REF]. As both methods have some degree of specificity, their successive use contributes to increasing the specificity of the final result [START_REF] Leeflang | The diagnostic accuracy of serological tests for Lyme borreliosis in Europe: a systematic review and meta-analysis[END_REF].

However, these specificity and sensitivity values vary depending on the infection stage. No biological test is required for erythema migrans as the serological test is negative in more than 50% of cases in Europe [START_REF] Stanek | Lyme borreliosis: clinical case definitions for diagnosis and management in Europe[END_REF]. Erythema migrans is the most frequent clinical presentation of Lyme borreliosis. It is a localized infection, associated with a low immune system response. A negative serological test result is therefore associated with a probability of wrongly ruling out the diagnosis. The biological diagnosis of Lyme neuroborreliosis is based on the joint analysis of blood and CSF [START_REF] Blanc | Relevance of the antibody index to diagnose Lyme neuroborreliosis among seropositive patients[END_REF]. CSF analysis reveals early lymphocytic cellular reaction in more than 93% of patients presenting with meningoradiculitis [START_REF] Ogrinc | Course and Outcome of Early European Lyme Neuroborreliosis (Bannwarth Syndrome): Clinical and Laboratory Findings[END_REF]. Lyme serology is positive in serum specimens at the acute phase of Lyme neuroborreliosis in 70-89% of cases, and sensitivity in CSF specimens at the acute phase is >90% [START_REF] Ogrinc | Course and Outcome of Early European Lyme Neuroborreliosis (Bannwarth Syndrome): Clinical and Laboratory Findings[END_REF]. At the stage of Lyme arthritis or acrodermatitis chronica atrophicans, the sensitivity of Lyme serology is 95% and 98%, respectively [START_REF] Leeflang | The diagnostic accuracy of serological tests for Lyme borreliosis in Europe: a systematic review and meta-analysis[END_REF]. The very high negative predictive value of serology should lead to question the suggested diagnosis in case of a negative result in patients presenting with clinical signs and symptoms. One should also bear in mind that, following effective treatment, anti-Borrelia antibodies (including IgM) may persist for months or even years after clinical cure [START_REF] Dessau | To test or not to test? Laboratory support for the diagnosis of Lyme borreliosis: a position paper of ESGBOR, the ESCMID study group for Lyme borreliosis[END_REF]. Serological testing is therefore not useful for the follow-up of treated patients and the presence of IgM is not indicative of a persistent Borrelia infection.

Various species of the B. burgdorferi sensu lato complex

Various species have been defined as belonging to the B. burgdorferi sensu lato complex. Their geographical distribution is varied and they are responsible for various clinical signs and symptoms [START_REF] Stanek | The expanding Lyme Borrelia complex--clinical significance of genomic species?[END_REF]. Predominant species in Europe are B. garinii and B. afzelii, while B. burgdorferi sensu stricto is most frequently observed in the United States [START_REF] Stanek | The expanding Lyme Borrelia complex--clinical significance of genomic species?[END_REF].

Partial differentiation of species among the B. burgdorferi sensu lato complex by serological testing is possible because of molecular and antigen changes of surface proteins. garinii, and B. burgdorferi sensu stricto [START_REF] Norman | Serodiagnosis of Lyme borreliosis by Borrelia burgdorferi sensu stricto, B. garinii, and B. afzelii western blots (immunoblots)[END_REF]. The authors revealed that European sera were more reactive to Western blot prepared with strains of B. garinii and B. afzelii, while sera collected from Northern American patients were more reactive to B. burgdorferi sensu stricto antigens [START_REF] Norman | Serodiagnosis of Lyme borreliosis by Borrelia burgdorferi sensu stricto, B. garinii, and B. afzelii western blots (immunoblots)[END_REF]. Sera of patients presenting with neurological signs were also more reactive to B. garinii antigens and those of patients with dermatological clinical presentations were rather reactive to B. afzelii antigens [START_REF] Norman | Serodiagnosis of Lyme borreliosis by Borrelia burgdorferi sensu stricto, B. garinii, and B. afzelii western blots (immunoblots)[END_REF]. One should note that, in this study, the interpretation of the Western blot result (positive or negative) could vary depending on the strain used for 8% of tested specimens. Other European studies using the Western blot technique reported similar results, with a preferential association of B. garinii and neurological disorders, B. afzelii and late cutaneous signs, and a slight predominance of B. burgdorferi sensu stricto in joint manifestations [START_REF] Ryffel | Scored antibody reactivity determined by immunoblotting shows an association between clinical manifestations and presence of Borrelia burgdorferi sensu stricto, B. garinii, B. afzelii, and B. Valaisiana in humans[END_REF][START_REF] Péter | Association of distinct species of Borrelia burgdorferi sensu lato with neuroborreliosis in Switzerland[END_REF][START_REF] Grygorczuk | Assessment of the frequency of different Borrelia burgdorferi sensu lato species in patients with Lyme borreliosis from north-east Poland by studying preferential serologic response and DNA isolates[END_REF]. Consequently, as early as 1999, standardization of Western blot techniques prepared with B. garinii and B. afzelii strains was suggested in Europe [START_REF] Hauser | Validity of interpretation criteria for standardized Western blots (immunoblots) for serodiagnosis of Lyme borreliosis based on sera collected throughout Europe[END_REF]. To do so, the authors used a panel of sera from patients coming from various European regions, that had been provided by members of the European Union Concerted Action on Lyme Borreliosis (EUCALB), and compared them with those of healthy blood donors [START_REF] Dickeson | Concordance of four commercial enzyme immunoassay and three immunoblot formats for the detection of Lyme borreliosis antibodies in human serum: the two-tier approach remains[END_REF]. However, composition and sometimes performance differences are observed between ELISA and Western blot techniques commercialized in the various European markets [START_REF] Leeflang | The diagnostic accuracy of serological tests for Lyme borreliosis in Europe: a systematic review and meta-analysis[END_REF][START_REF] Dickeson | Concordance of four commercial enzyme immunoassay and three immunoblot formats for the detection of Lyme borreliosis antibodies in human serum: the two-tier approach remains[END_REF]. These tests are usually prepared with a mixture of recombinant antigens from the three main pathogenic species for humans (B. burgdorferi sensu stricto, B. garinii, and B. afzelii) [START_REF]dediagnostic-in-vitro-Operations-d-evaluations-et-de-controle-du-marche/Dispositifs-medicauxde-diagnostic-in-vitro-Operations-d-evaluations-et-de-controle-du-marche/Reactifs-deserologie[END_REF]. Including antigens from these various species mainly aims to improve the sensitivity of serological tests.

However, accurate differentiation of Borrelia species can only be performed with molecular techniques; reactivity differences observed with serological tests in antigens of various species do not always result from culture or PCR, and may lead to interpretation errors.

Other diagnostic tests for Lyme borreliosis

Some tests are sometimes suggested for the diagnosis of Lyme borreliosis, although their bioclinical value has not been extensively assessed. Other tests are not validated using the methodology approved in medical biology [START_REF] Dessau | To test or not to test? Laboratory support for the diagnosis of Lyme borreliosis: a position paper of ESGBOR, the ESCMID study group for Lyme borreliosis[END_REF]. These various techniques include lymphocyte transformation tests (LTT). LTTs currently lack validation and published studies have substantial methodological biases [START_REF] Dessau | The lymphocyte transformation test for the diagnosis of Lyme borreliosis has currently not been shown to be clinically useful[END_REF]. For instance, the diagnostic value of LTTs in Lyme neuroborreliosis is low (36% sensitivity and 82% specificity) [START_REF] Nordberg | Can ELISPOT Be Applied to A Clinical Setting as A Diagnostic Utility for Neuroborreliosis?[END_REF]. Another test relies on CD57 marker detection. Little data is available and the only case-control study performed (NIH study) reported the complete lack of specificity of this test [START_REF] Marques | Natural killer cell counts are not different between patients with post-Lyme disease syndrome and controls[END_REF]. As for rapid diagnostic tests for self-testing, sensitivity and specificity data is currently very limited [START_REF]dediagnostic-in-vitro-Operations-d-evaluations-et-de-controle-du-marche/Dispositifs-medicauxde-diagnostic-in-vitro-Operations-d-evaluations-et-de-controle-du-marche/Reactifs-deserologie[END_REF].

Relapsing fever borreliae

Relapsing fever borreliae are mainly observed in tropical and subtropical regions, and mainly in Africa. The causative agents are usually B. duttonii, B. crocidurae, B. recurrentis, B. persica, and B. hispanica [START_REF] Talagrand-Reboul | Relapsing Fevers: Neglected Tick-Borne Diseases[END_REF]. They are transmitted by soft ticks (Argasidae) and for some of them by body lice (B. recurrentis) [START_REF] Talagrand-Reboul | Relapsing Fevers: Neglected Tick-Borne Diseases[END_REF]. An emerging species, B. miyamotoi, is transmitted by hard ticks [START_REF] Talagrand-Reboul | Relapsing Fevers: Neglected Tick-Borne Diseases[END_REF].

Direct diagnosis

Relapsing fever borreliae, unlike Lyme borreliosis, can lead to severe bacteremia during febrile episodes. This is why the usual diagnostic method is optical microscopy after Giemsa staining of blood smear or thick blood drop. This technique may detect concentrations of 10 3 to 10 5 microorganisms/mL [START_REF] Parola | Ticks and tickborne bacterial diseases in humans: an emerging infectious threat[END_REF][START_REF] Trape | Tick-borne borreliosis in west Africa[END_REF][START_REF] Vial | Incidence of tick-borne relapsing fever in west Africa: longitudinal study[END_REF]. Comparative studies reported that additional methods could increase the sensitivity of optical microscopy. Differential centrifugation, acridine orange, quantitative buffy coat (QBC), and fluorescence microscopy could contribute to increasing microscopy sensitivity [START_REF] Larsson | A novel and simple method for laboratory diagnosis of relapsing Fever borreliosis[END_REF][START_REF] Cobey | Short report: Detection of borrelia (relapsing fever) in rural Ethiopia by means of the quantitative buffy coat technique[END_REF][START_REF] Van Dam | Tick-borne relapsing fever imported from West Africa: diagnosis by quantitative buffy coat analysis and in vitro culture of Borrelia crocidurae[END_REF]. Quantitative buffy coat is supposed to be 100 times more sensitive than thick blood drop for the diagnosis of relapsing fever borreliae [START_REF] Van Dam | Tick-borne relapsing fever imported from West Africa: diagnosis by quantitative buffy coat analysis and in vitro culture of Borrelia crocidurae[END_REF].

However, such technique requires specific laboratory equipment that is often not available in endemic regions.

Molecular biology techniques such as standard PCR and quantitative PCR have been developed. Several comparative studies reported the better sensitivity of these techniques compared with usual microscopy techniques. These findings are detailed in Table 1 [START_REF] Elbir | Multiplex Real-Time PCR Diagnostic of Relapsing Fevers in Africa[END_REF][START_REF] Karan | Dynamics of Spirochetemia and Early PCR Detection of Borrelia miyamotoi[END_REF][START_REF] Antinori | Diagnosis of Louse-Borne Relapsing Fever despite Negative Microscopy in Two Asylum Seekers from Eastern Africa[END_REF].

Culture may be performed but it requires expert skills, thus limiting its routine use. It is thus very rarely used with clinical samples. BSK-H culture medium is the same as the one for Borrelia spp. responsible for Lyme disease. All Borrelia species responsible for relapsing fever do not grow on this medium though. Growth is detected by dark-field microscopy.

Antigen detection in blood or other tissue specimens is being developed, but it is currently not available in clinical practice. Specific monoclonal antibodies have for instance been developed to detect B. crocidurae and B. hermsii [START_REF] Fotso | Monoclonal Antibodies for the Diagnosis of Borrelia crocidurae[END_REF][START_REF] Schwan | Identification of the tick-borne relapsing fever spirochete Borrelia hermsii by using a speciesspecific monoclonal antibody[END_REF]. Another innovative technique is the MALDI-TOF test. It has been used in the detection of Borrelia crocidurae in Ornithodoros sonrai ticks [START_REF] Fotso | MALDI-TOF mass spectrometry detection of pathogens in vectors: the Borrelia crocidurae/Ornithodoros sonrai paradigm[END_REF].

Indirect diagnosis

Specific serological tests for the causative agents of relapsing fever borreliae have been developed. They are based on the use of GlpQ antigen or BipA antigen, that are supposed to be absent from species of the burgdorferi group [START_REF] Cutler | Diagnosing Borreliosis[END_REF][START_REF] Fritz | Serologic evidence for Borrelia hermsii infection in rodents on federally owned recreational areas in California[END_REF][START_REF] Lopez | A novel surface antigen of relapsing fever spirochetes can discriminate between relapsing fever and Lyme borreliosis[END_REF]. However, several studies reported cross reactions with bacteria of the burgdorferi sensu lato complex responsible for Lyme disease. This has for instance been observed between B. miyamotoi, B. burgdorferi, and B. hermsii in the United States [START_REF] Krause | Human Borrelia miyamotoi infection in California: Serodiagnosis is complicated by multiple endemic Borrelia species[END_REF]. The serological test is also often negative during the first fever episodes of relapsing fever and should therefore rather be used as a retrospective diagnostic tool.

Syphilis: Treponema pallidum

Syphilis is a sexually-transmitted infection caused by T. pallidum. It is a public health problem as its incidence has been on the rise in France since the years 2000 [START_REF] Morshed | Recent trends in the serologic diagnosis of syphilis[END_REF]. The main obstacle to diagnosis and to the study of T. pallidum is the inability to culture the bacterium in an axenic medium.

Direct diagnosis

Direct examination by dark-field microscopy of chancre or skin lesion samples is highly contributory and immediate. Performances vary according to laboratories and staff. Specific material and qualified staff are required. False positive results (commensal spirochetes, especially on the mouth or anus) and false negative results (a negative direct examination should not rule out syphilis) may be observed [START_REF] Wheeler | Dark ground microscopy and treponemal serological tests in the diagnosis of early syphilis[END_REF][START_REF] Organization | Guidelines for the Management of Sexually Transmitted Infections[END_REF]. The sensitivity of the examination is higher at the primary and secondary phases and in early congenital syphilis (chancres, condyloma latum, mucous patches, adenopathy) [START_REF] Larsen | Laboratory diagnosis and interpretation of tests for syphilis[END_REF].

Direct immunofluorescence for the detection of Treponema has been associated with satisfactory results at the mucous, skin lesion, or tissue levels. The sensitivity of these techniques is close to 80% [START_REF] Daniels | Specific direct fluorescent antibody detection of Treponema pallidum[END_REF][START_REF] Romanowski | Detection of Treponema pallidum by a fluorescent monoclonal antibody test[END_REF][START_REF] Ito | Specific immunofluorescent staining of pathogenic treponemes with a monoclonal antibody[END_REF]. However, these techniques have become obsolete since the commercialization of PCR tests. Various methods such as standard PCR, nested PCR, RT-PCR, or quantitative PCR, have been used by targeting several genes such as bmp, tpp47, tmpA encoding for surface lipoproteins, or pol A involved in genome replication [START_REF] Grange | Evaluation of a PCR test for detection of treponema pallidum in swabs and blood[END_REF]. Recent studies reported no performance differences between PCR testing targeting the gene encoding for the membrane protein of 47 Kd and that targeting the gene encoding for the "pol A" polymerase DNA gene [START_REF] Gayet-Ageron | Comparison of Diagnostic Accuracy of PCR Targeting the 47-Kilodalton Protein Membrane Gene of Treponema pallidum and PCR Targeting the DNA Polymerase I Gene: Systematic Review and Meta-analysis[END_REF]. Sensitivity is approximately 80% and specificity 95%; and 95% and 89% negative and positive predictive values have been reported, respectively [START_REF] Gayet-Ageron | Comparison of Diagnostic Accuracy of PCR Targeting the 47-Kilodalton Protein Membrane Gene of Treponema pallidum and PCR Targeting the DNA Polymerase I Gene: Systematic Review and Meta-analysis[END_REF][START_REF] Heymans | Clinical value of Treponema pallidum real-time PCR for diagnosis of syphilis[END_REF]. Just like for other spirochetes of medical interest, the values of PCR tests depend on biological samples tested and infection stages. For illustrative purposes, patients presenting with secondary syphilis or patients co-infected with HIV with a low level of CD4 lymphocytes, have higher rates of spirochetemia than patients presenting with primary syphilis or patients at the early latency phase. Blood PCR testing is therefore associated with better sensitivity in these patients [START_REF] Wu | Spirochetemia due to Treponema pallidum using polymerase-chain-reaction assays in patients with early syphilis: prevalence, associated factors and treatment response[END_REF]. PCR techniques have been assessed in the detection of T. pallidum DNA in swabs of chancre, cutaneous biopsy of secondary syphilis, bones, various biological fluids such as serum, CSF, urines, placenta, gastric lesions [START_REF] Sánchez | Evaluation of molecular methodologies and rabbit infectivity testing for the diagnosis of congenital syphilis and neonatal central nervous system invasion by Treponema pallidum[END_REF][START_REF] Buffet | Diagnosing Treponema pallidum in secondary syphilis by PCR and immunohistochemistry[END_REF][START_REF] Burstain | Sensitive detection of Treponema pallidum by using the polymerase chain reaction[END_REF][START_REF] Centurion-Lara | Detection of Treponema pallidum by a sensitive reverse transcriptase PCR[END_REF][START_REF] Gayet-Ageron | Performance of the 47-kilodalton membrane protein versus DNA polymerase I genes for detection of Treponema pallidum by PCR in ulcers[END_REF][START_REF] Gordon | The response of symptomatic neurosyphilis to high-dose intravenous penicillin G in patients with human immunodeficiency virus infection[END_REF][START_REF] Hay | Use of the polymerase chain reaction to detect DNA sequences specific to pathogenic treponemes in cerebrospinal fluid[END_REF][START_REF] Kouznetsov | Detection of the 47-kilodalton membrane immunogen gene of Treponema pallidum in various tissue sources of patients with syphilis[END_REF][START_REF] Wicher | Detection of Treponema pallidum in early syphilis by DNA amplification[END_REF]. PCR testing is thus recommended as a diagnostic tool for primary and secondary syphilis. However, because of an insufficient sensitivity, its negativity should not rule out the diagnosis and an indirect diagnostic test should also be performed.

Indirect diagnosis

No serological test for syphilis is able to differentiate venereal syphilis caused by Treponema pallidum pallidum, and non-venereal Treponema infections caused by T. pallidum subsp. endemicum (responsible for Bejel), T. pallidum subsp. pertenue (responsible for Yaws), and T. pallidum carateum (responsible for Pinta). Two types of tests are available: nontreponemal tests (NTT) and treponemal tests (TT).

Nontreponemal tests (NTT)

These tests are based on the use of a complex antigen, made of cardiolipin, lecithin, and cholesterol [START_REF] Larsen | Laboratory diagnosis and interpretation of tests for syphilis[END_REF]. The venereal disease research laboratory test (VDRL) and rapid plasma reagin test (RPR) are the most frequently used. The RPR test is made of charcoal particles coated with a mixture of lipid antigens [START_REF] Larsen | Laboratory diagnosis and interpretation of tests for syphilis[END_REF]. Consequently, anti-lipid antibodies detected by these tests are not only produced during Treponema infection, but also in case of any other disease responsible for tissue lesions. As many cross reactions are observed, these tests alone cannot confirm the syphilis diagnosis. In case of infection, these tests are positive 10 to 15 days after primary chancre, i.e. six weeks following contact. In the absence of treatment, the highest level is reached between one and two years and remains positive at lower levels during late phases of syphilis [START_REF] Larsen | Laboratory diagnosis and interpretation of tests for syphilis[END_REF]. Complete seronegativation during tertiary syphilis is extremely rare. NTT titers are also correlated with the infection activity, and are thus used to control treatment effectiveness. Sensitivity values of these tests thus vary depending on the disease stage [START_REF] Larsen | Laboratory diagnosis and interpretation of tests for syphilis[END_REF]. During the primary phase (at the chancre stage), sensitivity of the RPR and VDRL tests is low, respectively 86% and 78% [START_REF] Larsen | Laboratory diagnosis and interpretation of tests for syphilis[END_REF][START_REF] Cantor | Screening for Syphilis: Updated Evidence Report and Systematic Review for the US Preventive Services Task Force[END_REF]. Sensitivity is 100% for both NTTs at the secondary phase, and 71% for VDRL and 71% for RPR at the late phase [START_REF] Larsen | Laboratory diagnosis and interpretation of tests for syphilis[END_REF][START_REF] Cantor | Screening for Syphilis: Updated Evidence Report and Systematic Review for the US Preventive Services Task Force[END_REF].

Specificity is 98% at all infection stages [START_REF] Larsen | Laboratory diagnosis and interpretation of tests for syphilis[END_REF]. The risk of prozone reaction triggering false negative results is another problem observed with NTTs. This reaction is observed in up to 2% of patients presenting with secondary syphilis, in serum specimens with a high number of antibodies. The normal antigen-antibody reaction is therefore inhibited [START_REF] Larsen | Laboratory diagnosis and interpretation of tests for syphilis[END_REF]. Weakly positive results, dubious results, or even negative results are in that case observed. It is thus recommended to dilute samples to first obtain increased titers and then a progressive decrease [START_REF] Larsen | Laboratory diagnosis and interpretation of tests for syphilis[END_REF].

by Western blot technique in children enables the distinction between passive transmission of IgG mother antibodies and active synthesis of IgM by the newborn.

Strategy for serological diagnosis

The French National Authority for Health (French acronym HAS) suggested a new screening strategy in 2015. This strategy is based on two-tiered serological testing: an automated TT (ELISA, EIA, or CMIA) to detect IgG and IgM [START_REF]HAS 2015 syphilis -Recherche Google[END_REF], followed by confirmatory quantitative NTT if the first test result is positive. A recent study performed by the centers for disease control and prevention (CDC) evaluated five laboratories using this strategy in the United States. The authors revealed that up to 56% of patients with a positive automated TT result had a negative NTT result (RPR) and that 36% of them also had a negative TPPA or FTA result [START_REF] Centers For | Discordant results from reverse sequence syphilis screening--five laboratories, United States, 2006-2010[END_REF]. This proportion of false positive results was higher among populations known to be associated with a low prevalence of syphilis [START_REF] Centers For | Discordant results from reverse sequence syphilis screening--five laboratories, United States, 2006-2010[END_REF]. Such evaluation has so far not been conducted in France, where the use of this strategy is more recent.

Leptospirosis

Leptospirosis is a zoonosis caused by pathogenic spirochetes of the Leptospira genus.

It is transmitted to humans by direct or indirect contact (through contaminated water mainly) with the urine of infected animals [START_REF] Levett | Leptospirosis[END_REF]. The clinical presentation of the disease is divided into two phases: a febrile, bacteremia phase of variable duration and an immune phase characterized by antibody production and excretion of Leptospira in urines, that may be associated with fever [START_REF] Levett | Leptospirosis[END_REF]. Most symptoms associated with disease severity (jaundice, lung involvement, renal involvement, meningitis) are observed during the immune phase. Just like other spirochetes mentioned in the present article, the value and interpretation of biological tests depend on the infection stage.

Direct diagnosis

The detection of Leptospira requires a dark-field microscope or a phase-contrast microscope [START_REF] Levett | Leptospirosis[END_REF]. Leptospira are spiral-shaped bacteria (6 to 12 µm long and 0.1 µm in diameter), with mobile hooked ends [START_REF] Levett | Leptospirosis[END_REF]. The direct examination with a dark-field microscope is weakly sensitive and poorly specific, with a detection threshold of 10 4 Leptospira/mL [START_REF] Levett | Leptospirosis[END_REF]. This method only yields positive results during the bacteremia phase of the infection, but has to be performed by a laboratory technician. The risk of false positive results is also high due to the presence of fibrin filaments or protein fragments which are moved by Brownian motion process [START_REF] Levett | Leptospirosis[END_REF]. Leptospira may be cultured from biological fluids such as blood, CSF, or urine. Blood samples should be collected in heparin tubes during the febrile period and inoculated on the specific EMJH culture medium [START_REF] Toyokawa | Diagnosis of acute leptospirosis[END_REF]. Samples should be incubated in a dark room at 28-30°C for at least eight weeks, and analyzed weekly with a dark-field microscope [START_REF] Levett | Leptospirosis[END_REF]. CSF may be cultured in case of meningitis signs and symptoms, with the same modalities. Urines are alkalized and inoculated within four hours (the acidity of urines lyses Leptospira) with serial dilutions at 1/10 and 1/100 [START_REF] Levett | Leptospirosis[END_REF]. Isolated strains are identified by agglutination tests on specific antiserum. Genotypic identification techniques are also more frequently used [START_REF] Ahmed | Multilocus sequence typing (MLST): markers for the traceability of pathogenic Leptospira strains[END_REF][START_REF] Boonsilp | A single multilocus sequence typing (MLST) scheme for seven pathogenic Leptospira species[END_REF].

PCR is currently the most frequently used direct diagnostic method and allows for the early diagnosis of the infection before antibody detection. secY (flagellin), rrs, flaB, and rrl genes and the LA 3521 locus are used in the various methods currently available [START_REF] Toyokawa | Diagnosis of acute leptospirosis[END_REF]. PCR detection has been used on blood, urine, CSF, and aqueous humor samples with different performances [START_REF] Toyokawa | Diagnosis of acute leptospirosis[END_REF]. The sensitivity and specificity values on blood samples are unfortunately highly variable and are even lower with late PCR testing than early PCR testing at the start of the infection (from 9% to 73% and 52% to 100%, respectively) [START_REF] Smythe | A quantitative PCR (TaqMan) assay for pathogenic Leptospira spp[END_REF][START_REF] Slack | Identification of pathogenic Leptospira species by conventional or real-time PCR and sequencing of the DNA gyrase subunit B encoding gene[END_REF]. The detection threshold is set at 10 to 50 Leptospira/mL of blood. PCR testing on urine samples is associated with better performance and specificity and sensitivity values of 100%, although based on only one study [START_REF] Toyokawa | Diagnosis of acute leptospirosis[END_REF][START_REF] Slack | Identification of pathogenic Leptospira species by conventional or real-time PCR and sequencing of the DNA gyrase subunit B encoding gene[END_REF]. Quantitative PCR has now replaced standard PCR, with increasing sensitivity and specificity values (96% and 100%, respectively) [START_REF] Espy | Real-time PCR in clinical microbiology: applications for routine laboratory testing[END_REF].

Indirect diagnosis

The historical reference serological technique is the microscopic agglutination test (MAT) [START_REF] Levett | Leptospirosis[END_REF]. Developed in 1918 by Martin and Pettit, this method relies on serum incubation with Leptospira antigen suspension of various serovars and on looking for agglutination with a dark-field microscope. Serological titer can thus be determined, corresponding to the highest dilution at which a 50% agglutination is observed [START_REF] Levett | Leptospirosis[END_REF]. This method requires particular expertise to maintain representative strains of each serovar in culture. It is thus only performed by reference centers. It also helps identify the causative serogroup, which is useful for epidemiological purposes. Although considered a reference method, sensitivity values are not optimal at all stages of the infection (82% at Week 2 of the infection, 96% after Week 4) [START_REF] Levett | Leptospirosis[END_REF]. Significant titers are 100 in endemic regions and 400 in non-endemic regions [START_REF] Wuthiekanun | Clinical diagnosis and geographic distribution of leptospirosis, Thailand[END_REF].

Various ELISA serological tests enabling the detection of anti-Leptospira IgM, have been commercialized over the past few years. These tests are more easily used and are better standardized. The value of these commercialized tests is highly variable depending on studies (sensitivity ranging from 4% to 97%), mainly because some studies performed the serological test during the first week of the infection. Nonetheless, studies performing the serological test after Day 7 reported sensitivity and specificity values between 75% and 100% and 78% and 97%, respectively [START_REF] Yersin | Field evaluation of a one-step dipstick assay for the diagnosis of human leptospirosis in the Seychelles[END_REF][START_REF] Bajani | Evaluation of four commercially available rapid serologic tests for diagnosis of leptospirosis[END_REF][START_REF] Vijayachari | Evaluation of Lepto Dri Dot as a rapid test for the diagnosis of leptospirosis[END_REF][START_REF] Levett | Evaluation of two enzyme-linked immunosorbent assay methods for detection of immunoglobulin M antibodies in acute leptospirosis[END_REF][START_REF]Haute Autorité de Santé -Diagnostic biologique de la leptospirose[END_REF]. These tests are therefore currently recommended as first-line strategies during the immune phase of the infection, with secondary confirmation by MAT [START_REF]Haute Autorité de Santé -Diagnostic biologique de la leptospirose[END_REF].

Another type of technique based on unit tests with visual interpretation on strips has been developed [START_REF]Haute Autorité de Santé -Diagnostic biologique de la leptospirose[END_REF]. It is based on the same principle as the ELISA assay, but the antigen is fixed on a strip. The added value of this test relies on its ease of use, although limited by its expensive price. This method has never been fully assessed in metropolitan France, but its sensitivity is believed to be lower than the usual ELISA techniques. Its use is therefore not currently recommended, except in isolated regions of French overseas territories [START_REF]Haute Autorité de Santé -Diagnostic biologique de la leptospirose[END_REF]. The slide microagglutination test with a thermoresistant antigen has long been used as a screening technique before MAT confirmation. Total antibodies can thus be detected during the second week of the infection [START_REF]Haute Autorité de Santé -Diagnostic biologique de la leptospirose[END_REF]. Tests used in France are associated with sensitivity values ranging from 45% to 63% and specificity values from 84% to 88%. The HAS thus decided in 2011 to no longer use this test as part of the diagnostic strategy of leptospirosis [START_REF]Haute Autorité de Santé -Diagnostic biologique de la leptospirose[END_REF].

Diagnostic scores

Considering the limitations of the various diagnostic tests of leptospirosis and their poor availability in resource-limited countries where the infection prevalence is high, the WHO developed specific diagnostic criteria in 1982: Faine criteria (Table 3) [START_REF] Faine | Guidelines for the control of leptospirosis[END_REF]. These criteria are divided into a clinical category, an epidemiological category, and a biological diagnosis category (Table 3). The latter category was modified in 2012 to include more recent diagnostic methods than MAT, as well as the risk factor of "rainfall before symptom onset" [START_REF] Shivakumar | Diagnosis of leptospirosis utilizing modified Faine's criteria[END_REF]. The leptospirosis diagnosis is highly suspected when the score is >20, with high probability when >24 (Table 3) [START_REF] Doudier | Prognostic factors associated with severe leptospirosis[END_REF]. This modified score has newly been evaluated in a recent study conducted in Sri Lanka, and sensitivity and specificity values of 95% and 56%, respectively, have been reported [START_REF] Bandara | Utility of modified Faine's criteria in diagnosis of leptospirosis[END_REF]. Several predictive diagnostic scores have since been developed. A Sri Lankan team thus suggested using a score based on a derivation cohort of 450 patients suspected of having leptospirosis and on a validation cohort of 142 patients with a confirmed diagnosis of leptospirosis [START_REF] Rajapakse | A Diagnostic Scoring Model for Leptospirosis in Resource Limited Settings[END_REF]. This score includes five items, each with values ranging from 4 to 6: bilirubinemia >30 mmol/L, >80% of neutrophils, exposure to contaminated water or soil, creatininemia >150 μmol/L, platelet count <85 Giga/L [START_REF] Rajapakse | A Diagnostic Scoring Model for Leptospirosis in Resource Limited Settings[END_REF]. A threshold score of 14 was associated with an 80% sensitivity, a 62% specificity, a positive predictive value of 54%, and a negative predictive value of 84% [START_REF] Rajapakse | A Diagnostic Scoring Model for Leptospirosis in Resource Limited Settings[END_REF].

Conclusion

This literature review on biological diagnostic tests for spirochetes responsible for infections in humans, highlighted several similarities between these bacteria. For all studied bacteria, the direct diagnostic methods are not optimal. Dark-field microscopy has to be performed by a laboratory technician. Culture is always tedious or even impossible on an axenic medium such as for syphilis. The commercialization of real-time PCR techniques represented major progress for the four infections studied in the present article, as they are more sensitive than culture and as they can be more easily standardized. However, the likelihood of a positive PCR result depends on the infection stage (bacteremia stage for Leptospira or relapsing fever borreliae) and on the type of sample collected (erythema migrans biopsy for Lyme disease, genital chancre for syphilis).

As for indirect diagnostic methods, false negative results are mainly observed at the early stage of the four infections when no or not enough specific antibodies are produced.

Serological tests are also all associated with cross reactions, with a risk of false positive results, and consequently with a risk of excessive diagnoses and treatments. This risk of false positive results is known and can be measured thanks to Bayes' theorem, developed in the 18 th century. False positive results are indeed an inherent difficulty to all medical diagnostic tests as no test is perfect. This risk is higher if the probability of having the disease in the studied population is low. Thus, to reduce the risk of false positive results with a given test, one should select a population associated with an "a priori" higher disease prevalence than in the general population. Several strategies have been developed.

The first strategy consists in performing the test in a population that has been selected for its risk factors for the infection. For instance, when performing an HIV serological test in a group of intravenous drug users sharing syringes, the infection prevalence will be much higher than if we were testing random people among the general population. Such strategy thus leads to decreasing the number of false positive results. Diagnostic scores such as those developed for leptospirosis are also based on that same principle. Another strategy consists in performing two tests: a screening test, more sensitive than specific, which will yield many false positive results although enabling the selection of a population subgroup where the disease prevalence is a priori higher. The first test should then be confirmed with a more specific test to improve the overall relevance of the test result. For Lyme borreliosis, this strategy is applied with a sensitive but weakly specific ELISA serological test -and thus associated with false positive results -followed by a more specific confirmatory Western blot test.

Disclosure of interests

Didier Raoult and Oleg Mediannikov applied for a patent for a monoclonal antibody enabling the detection of Borrelia crocidurae. Thick blood drop 10 4 -10 5 [START_REF] Van Dam | Tick-borne relapsing fever imported from West Africa: diagnosis by quantitative buffy coat analysis and in vitro culture of Borrelia crocidurae[END_REF] Quantitative buffy coat 10 3 [START_REF] Van Dam | Tick-borne relapsing fever imported from West Africa: diagnosis by quantitative buffy coat analysis and in vitro culture of Borrelia crocidurae[END_REF] Inoculation to mice Borrelia vivantes in culture [START_REF] Diatta | A comparative study of three methods of detection of Borrelia crocidurae in wild rodents in Senegal[END_REF] qPCR: quantitative polymerase chain reaction Tableau 2. Résumé des valeurs de sensibilité et spécificité des différents tests tréponémiques

  In 1994 Wilske et al. developed an immunoblot technique based on the use of five recombinant antigens. This technique helped detect seroreactivity differences between patients infected with B. afzelii, B. garinii, or B. burgdorferi sensu stricto [18]. Two years later, Norman et al. developed an immunoblot technique using several strains of B. afzelii, B.

Tableau 1 .

 1 Sensibilité des différentes méthodes de diagnostic direct des borrélioses récurrentes

Table 1 .

 1 Sensitivity of the various direct diagnostic methods for relapsing fever borreliae

	Method	Characteristics	Reference
	Multiplex qPCR for	Cut-off 36 Ct=100 copies	[32]
	B.crocidurae,	of plasmids 5 µl
	B.duttoni/recurrentis, and
	B. hispanica
	B. recurrentis qPCR	3 copies 40.94	[87]
		32 copies 36.92

Table 2 .

 2 Summary of sensitivity and specificity values of the various treponemal testsPrimary phase Secondary phase Latency phase Late phase Specificity

	FTA-ABS 84 (70-100) 100	100	96	97 (94-100)
	TPHA	76 (69-90)	100	97 (97-100) 94	99 (98-100)
	TPPA		84-100		99.5
	CMIA		99.38			99
	ELISA		98.12

These tests are also associated with a risk of false positive results, mainly because of the type of lipid antigens used: they yield a positive result in cases such as acute hepatitis, infectious mononucleosis, pneumonia, chickenpox, measles, pregnancy, or malaria [START_REF] Larsen | Laboratory diagnosis and interpretation of tests for syphilis[END_REF].

Chronic diseases such as autoimmune diseases (mainly lupus), cancers, leprosy, or intravenous drug use may lead to positive results with NTTs.

Treponemal tests (TT)

TTs are based on the detection of antibodies targeted against antigens that are part of Treponema. All TTs use T. pallidum as antigen. Their specificity is better than that of NTTs, but they always yield positive results after treatment. Active syphilis thus cannot be distinguished from a serological scar [START_REF] Larsen | Laboratory diagnosis and interpretation of tests for syphilis[END_REF]. These tests are T. pallidum hemagglutination assay (TPHA), T. pallidum particle agglutination assay (TPPA), and fluorescent treponemal antibody absorbed test (FTA) [START_REF] Marangoni | Evaluation of the BioPlex 2200 syphilis system as a first-line method of reverse-sequence screening for syphilis diagnosis[END_REF]. More recent immunoenzyme techniques are available, such as the ELISA assay or CMIA (chemiluminescent microparticles immunoassay).

Agglutination and fluorescence tests are usually manually performed (TPHA, TPPA, FTA), whereas immunoenzyme tests are automated and multiplex tests have even been recently developed [START_REF] Marangoni | Evaluation of the BioPlex 2200 syphilis system as a first-line method of reverse-sequence screening for syphilis diagnosis[END_REF]. Sensitivity and specificity values of the various TTs are reported in Table 2.

Automated techniques (ELISA, CMIA BIOPLEX) are usually more sensitive and specific than manually performed tests (FTA, TPPA, and TPHA) [START_REF] Marangoni | Evaluation of the BioPlex 2200 syphilis system as a first-line method of reverse-sequence screening for syphilis diagnosis[END_REF][START_REF] Fakile | Correlation of Treponemal Immunoassay Signal Strength Values with Reactivity of Confirmatory Treponemal Testing[END_REF].

Western blot tests may also be used to confirm results, with up to 99.9% sensitivity and specificity [START_REF] Morshed | Recent trends in the serologic diagnosis of syphilis[END_REF][START_REF] Janier | European guideline on the management of syphilis[END_REF][START_REF] Novikov | Western blot as a confirming test in the laboratory diagnosis of syphilis][END_REF][START_REF] Ebel | Validation of the INNO-LIA syphilis kit as a confirmatory assay for Treponema pallidum antibodies[END_REF]. These tests are able to detect antibodies targeted against antigen proteins such as TpN15, TpN17, TpN45, or TmpA. They are considered positive with at least two strips of IgG or one strip of IgM. They are particularly useful when other serological tests yield discordant results, or for congenital syphilis diagnosis because pregnancy is a cause of false positive results with NTTs and possibly with the more usual TTs [START_REF] Morshed | Recent trends in the serologic diagnosis of syphilis[END_REF]. Looking for IgM Tableau 3. Score diagnostique de la leptospirose