Ptx3 biomarker for reducing antibiotic treatment in newborns

Abstract

The invention relates to an in vitro method for determining whether a newborn patient has late-onset neonatal sepsis, which comprises determining the PTX-3 protein concentration in a biological sample previously obtained from said patient.

Description

Description Title: PTX3 BIOMARKER FOR REDUCING ANTIBIOTIC THERAPY IN NEWBORN INFANTS Technical field

[0001] The invention relates to an in vitro method for determining whether a newborn patient has late neonatal sepsis comprising determining the protein concentration of PTX-3 in a biological sample previously obtained from said patient. Prior Art

[0002] Sepsis is a major cause of morbidity and mortality in newborns. Neonatal sepsis is an invasive infection, usually bacterial, that occurs during the neonatal period. There are two categories of neonatal sepsis: early neonatal sepsis, which generally appears before the first three days of life, or late neonatal sepsis, which appears on day four or later. Neonatal sepsis is called late in nature in contrast to early infections, which are mostly transmitted from mother to newborn during pregnancy or delivery. Symptoms are multiple, nonspecific, and complex, and diagnosis is based on laboratory blood culture results. Diagnosis by blood culture carries a risk of false negatives, and the volume of blood required for an optimal culture may sometimes not be collected in very low-birth-weight newborns.Additionally, blood culture results are typically obtained within 48 hours.

[0003] Given the rapid progression of sepsis and its high mortality rate, prompt empiric antibiotic therapy is usually administered to newborns upon arrival in the neonatal unit in cases of suspected sepsis and while awaiting blood culture results. Thus, a certain proportion of patients receive antibiotic treatment when it is ultimately not necessary.

[0004] However, the systematic use of antibiotic therapy in these newborns, in addition to contributing to the emergence of multi-resistant bacteria, can also have deleterious effects, including mortality during hospitalization (Ting et al. 2016. JAMA pediatrics, 170(12):1181-1187) or in the medium / long term, a disruption of the microbiota which can be involved in the occurrence of subsequent pathologies such as respiratory difficulties (Kummeling I. et al. Pediatrics, 2007.119(1):e225-231).

[0005] It is therefore imperative to develop rapid and reliable diagnostic tests to determine early the absence of late neonatal sepsis in newborns in order to minimize the use of antibiotic therapy, particularly prolonged use in newborns who ultimately do not have an infection. Rapid diagnosis would thus make it possible to determine whether antibiotic treatment should be initiated or continued when it has been prescribed and administered in cases of suspected late neonatal sepsis and thus maintain antibiotic therapy only for newborns who actually have late neonatal sepsis.

[0006] A number of biomarkers have been studied for the diagnosis of sepsis in neonates. Procalcitonin (PCT), an early inflammatory factor used as a biomarker for bacterial infections in adults, has been frequently used as a diagnostic marker for neonatal sepsis. However, its clinical use for diagnosing neonatal sepsis is debated (Pontrelli G. et al. BMC Infect Dis. 2017;17:302; Eschborn S and Weitkamp JH, Journal of Perinatology, 2019, ;39(7):893-903) and this marker should be used in combination with other clinical markers and laboratory data to assess the need for continued antibiotic therapy.

[0007] Thus, there is a need to identify a biomarker allowing the rapid and reliable diagnosis of late neonatal sepsis to precisely exclude cases of neonatal sepsis but also to avoid false positives and avoid or reduce exposure to antibiotics in newborns.

[0008] Pentraxin 3 (PTX-3) is a glycoprotein expressed by endothelial cells and mononuclear phagocytes. Elevated PTX-3 levels have been shown to be an indicator of meningococcal shock in children. In contrast, The role of PTX3 in the diagnosis of neonatal sepsis has been poorly studied (Sharma et al. 2018, J Matern Fetal Neonatal Med. 2018 Jun;31(12):1646-1659 ; Baumert M. et al. Biomed Res Int, 2021 Jun 30;2021:6638622 ). Thus, larger, rigorous studies are needed to accurately assess the efficacy of this biomarker for its clinical use in reducing antibiotic therapy in newborns and in the diagnosis of neonatal sepsis. Abstract

[0009] The present disclosure improves the situation. The inventors in the present application conducted a prospective, multicenter study in two large-scale neonatal intensive care units to evaluate the reliability of different biomarkers as tools for reducing antibiotic therapy in newborns, for example in stopping antibiotic treatments initially administered following a suspicion of infection, or to diagnose the absence of late neonatal sepsis. The inventors surprisingly showed that among these biomarkers and unlike IL-6, the determination of the protein concentration of PTX3 makes it possible to exclude the presence of late neonatal sepsis.Furthermore, the determination of PTX3 protein concentration, advantageously combined with the determination of NGAL and possibly Gelsolin protein concentration, makes it possible to avoid or reduce antibiotic therapy early in more than half of patients diagnosed as not having late neonatal sepsis, for example following blood culture results or expert opinion. This performance is maintained when this biomarker is combined for example with other biomarkers such as IL-10, IL-6, Calprotectin, LBP, PCT, sCD14 or IP10.

[0010] The PTX3 biomarker, used alone or in combination, thus finds a very particular application in patients hospitalized in the neonatology department as a tool in the early cessation of antibiotic treatments initially prescribed following a suspicion of late neonatal sepsis.

[0011] The present invention relates to an in vitro method for determining whether treatment with an antibiotic is likely to be stopped in a newborn patient susceptible to late neonatal sepsis and having previously received an antibiotic treatment, said method comprising determining the protein concentration of PTX-3 in a biological sample previously obtained from said patient and comparing the concentration thus determined to a reference threshold value, characterized in that a protein concentration of PTX-3 lower than said reference threshold value indicates that the antibiotic treatment is likely to be stopped and that the patient does not have late neonatal sepsis.

[0012] According to a preferred embodiment, the method also comprises determining the protein concentration of NGAL in a biological sample previously obtained from said patient and comparing the concentration thus determined to a reference threshold value. According to this embodiment, protein concentrations of PTX-3 and NGAL lower than the reference threshold values ​​indicate that the antibiotic treatment is likely to be stopped.

[0013] According to another preferred embodiment, the method also comprises determining the protein concentrations of NGAL and Gelsolin in a biological sample previously obtained from said patient and comparing the concentrations thus determined to reference threshold values. According to this embodiment, protein concentrations of PTX-3, NGAL and Gelsolin lower than the reference threshold values ​​indicate that the antibiotic treatment is likely to be stopped.

[0014] Preferably, the protein concentration of PTX-3 is determined by an enzyme-linked immunosorbent assay (ELISA), preferably a microfluidic ELISA method. In a particular embodiment, said biological sample is a biological sample of blood, plasma or serum, preferably serum, and preferably the volume of the biological sample previously obtained from the patient is less than 50 µL, preferably less than 20 µL, and more preferably less than 5 µL.

[0015] In a particular embodiment, the reference threshold value of the protein concentration of PTX-3 is less than 3000 pg / mL, preferably less than 2000 pg / mL, preferably less than 1300 pg / mL, and more preferably, between 1000 and 1300 pg / mL, for example 1266 pg / mL.

[0016] In another particular embodiment, the method according to the present invention may comprise determining the concentration of at least one other protein selected from: IL-6, IL-10, PCT, IP10, sCD14, LBP, NGAL, Calprotectin and Gelsolin, said method being characterized in that lower concentrations of the PTX-3 protein and at least one of said other proteins at corresponding reference threshold values ​​indicate that the patient does not have late neonatal sepsis and / or that the antibiotic treatment is likely to be stopped.Preferably, according to this embodiment, said method comprises determining the concentration of the NGAL protein or the concentrations of the NGAL and Gelsolin proteins, said method being characterized in that concentrations of the PTX3, NGAL, and optionally Gelsolin proteins, lower than corresponding reference threshold values ​​indicate that the patient does not have late neonatal sepsis and / or that the antibiotic treatment is likely to be stopped.

[0017] According to a variant of this embodiment, the method according to the present invention may comprise the determination of the concentration of at least two other proteins chosen from: IL-6, IL-10, PCT, IP10, sCD14, LBP, NGAL, Calprotectin and Gelsolin, and more preferably, chosen from IL-10, NGAL and Gelsolin.

[0018] In another particular embodiment, the method according to the present invention is characterized in that only the concentration of PTX-3 is determined.

[0019] In a preferred embodiment, the biological sample is previously obtained from a patient presenting at least one clinical sign associated with a late neonatal sepsis syndrome, preferably chosen from: fever ≥ 38°C, tachycardia ≥ 160 / min, skin recoloration time (SRT) ≥ 3 seconds, gray and / or pale complexion, apnea and bradycardia syndrome, increased ventilatory assistance and / or increased FiO2, digestive bloating, rectal bleeding, hypotonia, lethargy, convulsions without other obvious cause, skin rash or inflammation at the puncture site of the central venous catheter, or a combination of these signs.

[0020] In another aspect, the present invention relates to an in vitro method for determining whether a newborn patient has late neonatal sepsis, said method comprising determining the protein concentration of PTX-3 in a biological sample of said patient, and comparing the concentration thus determined to a reference threshold value, characterized in that a higher concentration of PTX-3 than the reference threshold value indicates that the patient has late neonatal sepsis.

[0021] In a final aspect, the present invention relates to an antibiotic for use in the treatment of late neonatal sepsis in a newborn patient, characterized in that said antibiotic is administered to said patient previously assessed as having late neonatal sepsis by the method as described above, preferably said antibiotic being selected from amoxicillin, gentamicin and cefotaxime. Brief description of the drawings Fig.1

[0022] [Fig.1] shows a diagram of the organization of the study follow-up. Fig.2

[0023] [Fig.2] shows a ROC and AUC curve of clinical signs. Fig.3

[0024] [Fig. 3] shows the ROC curves and corresponding AUCs for the PTX3 biomarkers, as well as for the combination of PTX3 / NGAL and PTX3 / NGAL / Gelsolin biomarkers. Description of the embodiments

[0025] In order to determine whether a patient has late neonatal sepsis and to assess whether antibiotic treatment should be administered or stopped in the patient, the inventors have shown by carrying out a large-scale study on a large cohort of patients, that the determination of the protein concentration of PTX3 in a biological sample from the patient in comparison with a reference threshold value allows us to assess whether the patient has late neonatal sepsis. This makes it possible to limit antibiotic therapy in a patient who does not need it.

[0026] The present application relates to an in vitro method for determining whether a newborn patient has late neonatal sepsis, said method comprising determining the protein concentration of PTX3 in a biological sample previously obtained from said patient and comparing the concentration thus determined to a reference threshold value, characterized in that a protein concentration of PTX3 lower than the reference threshold value indicates that said patient does not have late neonatal sepsis.

[0027] The present application also relates to an in vitro method for determining whether antibiotic treatment is likely to be stopped in a newborn patient likely to have late neonatal sepsis and having previously received antibiotic treatment, said method comprising determining the protein concentration of PTX-3 in a biological sample previously obtained from said patient and comparing the concentration thus determined to a reference threshold value, characterized in that a protein concentration of PTX-3 lower than said reference threshold value indicates that the antibiotic treatment is likely to be stopped and that the patient does not have late neonatal sepsis.

[0028] Neonatal sepsis is an invasive infection, usually bacterial, occurring during the neonatal period. Symptoms are multiple, nonspecific, and include decreased spontaneous activity, less vigorous sucking, apnea, bradycardia, temperature instability, respiratory distress, vomiting, diarrhea, abdominal distension, nervousness, seizures, jaundice, or other abnormal symptoms. Neonatal sepsis can be categorized into early neonatal sepsis, which usually appears before the first 3 days of life, or late neonatal sepsis, which appears on day 4 or later (Pontrelli G. et al. BMC Infect Dis. 2017; 17: 302; Sharma et al. 2018, J Matern Fetal Neonatal Med. 2018 Jun; 31(12): 1646-1659).

[0029] As explained previously, late neonatal sepsis is usually acquired from the environment (neonatal nosocomial infections) and occurs after 3 days of life, as opposed to early neonatal sepsis which is an infection generally transmitted from mother to newborn during pregnancy or delivery (Sharma et al. 2018, J Matern Fetal Neonatal Med. 2018 Jun;31(12):1646-1659). Staphylococci are responsible for 30-60% of late infections and are most often due to intravascular devices (especially central vascular catheters). E. coli bacteria are also increasingly responsible for late sepsis especially in very low birth weight infants. Organisms that can cause neonatal sepsis include, but are not limited to, coagulase-negative staphylococci, including S. epidermidis, S. haemolyticus, S. hominis, S. warneri, S. saprophyticus, S. cohnii, and S.capitis, group B streptococci, Staphylococcus aureus, Enterococcus fecalis and E. faecium, Listeria monocytogenes, Escherichia coli, P. aeruginosa, Haemophilus influenzae, Streptococcus bovis, α-hemolytic streptococci, Streptococcus pneumoniae, Neisseria meningitides and N. gonorrhoeae. .

[0030] Newborns suspected of having late-onset neonatal sepsis, particularly those presenting with clinical signs suggestive of late-onset neonatal sepsis, are treated with antibiotics. Indeed, as previously discussed, in the neonatology department, while the blood culture results are available to confirm the presence or absence of late-onset neonatal sepsis, which in practice represents 48 hours, newborns are treated with antibiotics.The antibiotic that may be administered to the newborn patient may include one of the following antibiotics as examples: amoxicillin, amikacin, aztreonam, chloramphenicol, ceftazidime, clindamycin, cefalotin, ciprofloxacin, colistin, cefotetan, cefotaxime, erythromycin, fusidic acid, fosfomycin, cefoxitin, furans, gentamicin, imipenem, kanamycin, lincomycin, cefamandole, minocycline, latamoxef, metronidazole, nalidixic acid, netilmicin, oxacillin, benzylpenicillin, pefloxacin, piperacillin, pristinamycin, rifampicin, spiramycin, sulfonamides, streptomycin, trimethoprim, sulfametoxazole, tetracycline, teicoplanin, ticarcillin, tobramycin, trimethoprim, vancomycin, preferably amoxicillin. gentamicin or cefotaxime. Antibiotics can be adapted according to the antibiogram and the location of the infection.

[0031] The inventors have shown that pentraxin 3 (PTX3) is a biomarker that allows for the reliable and rapid assessment of the absence of late neonatal sepsis in the newborn and / or whether antibiotic treatment is likely to be initiated or stopped in said newborn.

[0032] In humans, the PTX3 gene, also known as TSG-14 or TNFAIP5 (Gene ID: 5806 updated on March 20, 2022) encodes a member of the pentraxin protein family, PTX3 (UniProtKB – P26022, modified on February 23, 2022) comprising a 17-amino acid signal peptide. The protein without a signal peptide comprises the sequence SEQ ID NO: 1. PTX3 is a protein involved in immunity and inflammation by recognizing pathogenic molecules. PTX3 expression is induced by inflammatory cytosines in response to inflammatory stimuli in several mesenchymal and epithelial cell types, including endothelial cells and mononuclear phagocytes. The protein promotes fibrocyte differentiation and is involved in the regulation of inflammation and complement activation. It also plays a role in angiogenesis and tissue remodeling.

[0033] Determining the protein concentration of PTX3 in a biological sample previously taken from a newborn and comparing it to a reference threshold value makes it possible to determine whether the newborn has late neonatal sepsis and / or whether antibiotic treatment is likely to be initiated or stopped in said newborn, and preferably stopped.

[0034] The protein concentration of the biomarker in a biological sample according to the present disclosure, in particular of PTX3, can be determined by any suitable method known to those skilled in the art. The protein concentration can be measured, for example, by semi-quantitative Western blotting, enzyme-labeled immunoassays, such as enzyme-linked immunosorbent assay (ELISA), biotin / avidin assays, radioimmunoassays, immunoelectrophoresis, mass spectrometry, or immunoprecipitation or by protein or antibody arrays.

[0035] In a preferred embodiment, the protein concentration is determined by an ELISA method.

[0036] The ELISA method is a solid enzyme immunoassay. This biochemical analysis technique falls within the more general framework of enzyme immunoassays (EIA), in which the recognition of a studied antigen by a specific antibody is monitored through an enzyme-catalyzed reaction and can generate the emission of a signal by a chromogenic or fluorogenic substrate. To do this, the antigen is recognized by an antibody covalently coupled to an enzyme. The binding of the labeled molecule will result, after using a chromogenic or fluorogenic substrate, in the enzyme bound to the antibody, in the emission of a colored or fluorescent signal. The technique uses one or two antibodies.To facilitate the implementation of the technique, particularly in the case of a large number of samples to be analyzed, the antibody specific to the antigen sought or the antigen recognized by the antibody sought is bound to the support used which is covered with it, hence the name immunoabsorption technique. Several variations of operating protocols exist to increase the specificity or sensitivity of antigen recognition (indirect, sandwich or competitive ELISA).

[0037] The antigen-binding antibodies or fragments thereof used for protein detection and protein concentration measurement are the antigen-binding antibodies or fragments specifically binding to the proteins of the present application (eg PTX3). The antibodies suitable for the present method may be selected from all the antibodies known in the prior art.

[0038] The term "antibody" as used herein includes monoclonal antibodies, polyclonal antibodies, and chimeric antibodies. The antibody may be derived from recombinant sources and / or produced by transgenic animals. The term "antibody fragment" as used herein is intended to include Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, and multimers thereof and Bispecific antibody fragments. Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bonds to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab', and F(ab')2 fragments, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments, and other fragments can also be synthesized by recombinant techniques.

[0039] Antibodies having specificity for one of the proteins described herein such as PTX3, NGAL, IL-10, IL-6, Calprotectin, LBP, PCT, sCD14, IP10 or Gelsolin may be prepared by conventional methods. A mammal (e.g., a mouse, hamster or rabbit) may be immunized with an immunogenic form of the peptide that elicits an antibody response in the mammal. Techniques for conferring immunogenicity to a peptide include conjugation to carriers or other techniques well known in the art. For example, the peptide may be administered in the presence of an adjuvant. The progress of immunization may be monitored by detecting antibody titers in plasma or serum. Standard ELISA or other immunoassay procedures may be used with the immunogen as the antigen to assess antibody levels.After immunization, antisera can be obtained and, if desired, polyclonal antibodies can be isolated from the sera.

[0040] To produce monoclonal antibodies, antibody-producing cells (lymphocytes) can be taken from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures, thereby immortalizing these cells and producing hybridoma cells. Such techniques are well known in the art (e.g., the hybridoma technique originally developed by Kohler and Milstein (Nature 256:495-497 (1975)) as well as other techniques such as the human B-cell hybridoma technique (Kozbor et al., Immunol. Today 4:72 (1983)), the EBV hybridoma technique for producing human monoclonal antibodies (Cole et al., Methods Enzymol, 121:140-67 (1986)), and screening of combinatorial antibody libraries (Huse et al., Science 246:1275 (1989)). Hybridoma cells can be immunochemically screened for the production of antibodies reacting specifically with the peptide, and monoclonal antibodies can be isolated.

[0041] Alternatively, antibodies can be commercial antibodies, for example, referenced in antibody databases such as the online database Antibodypedia, (Kiermer V. (2008) “Antibodypedia - A web portal to share antibody validation data” Nature Methods.5: 860).

[0042] In one embodiment of the present application, the antibodies or antibody fragments used in the ELISA methods, which bind to proteins (eg PTX3), are labeled with a detectable label. The label is preferably capable of producing, directly or indirectly, a detectable signal. For example, the label may be radiopaque or a radioisotope, such as 3H, 14C, 32P, 35S, 123I, 125I or 131I; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such as alkaline phosphatase, beta-galactosidase or peroxidase; an imaging agent; or a metal ion. In another embodiment, the detectable signal is indirectly detectable. For example, a labeled secondary antibody can be used to detect the protein of interest.

[0043] In newborns, it is difficult to obtain a large volume of biological sample due to technical and legal constraints (Jardé Law). Thus, in a preferred embodiment in order to limit the volume of sample collected, the protein concentration of PTX3 is determined by a microfluidic ELISA method (Lee et al., "Microfluidic enzyme-linked immunosorbent assay technology," Adv. CHn. Chem. 42: 255-259 (2006)). An example of an automatic microfluidic platform that can be used in the context of the present disclosure is described in Aldo P. et al. American Journal of Reproductive Immunology 75 (2016): 678-693.

[0044] The biological sample previously collected from a patient may be a sample of blood, plasma, serum or urine, preferably a sample of blood, plasma or serum, and more preferably a sample of serum.

[0045] In a preferred embodiment, the volume of the biological sample previously collected from the patient is less than 50 µL, preferably less than 20 µL, more preferably less than 5 µL, such as for example 3 µL.

[0046] The patient from whom the sample is previously taken is a newborn.

[0047] The term "newborn" refers to an infant within the first 28 days of birth. In some embodiments, the newborn may be within the first 14 days of birth. Preferably, the patient is a newborn over 3 days old, preferably over 7 days old. A newborn evaluated in the present application may be premature or full-term, for example, more particularly, a premature newborn weighing less than 1500g.

[0048] According to the method of the present application, said patient is a newborn suspected of having late neonatal sepsis. As mentioned previously, it is commonly accepted that late neonatal sepsis is sepsis occurring after 72 hours following birth.

[0049] According to a particular embodiment, the patient is a newborn suspected of having late neonatal sepsis and who is treated with antibiotics following said suspicion.

[0050] Clinical signs that may suggest late neonatal sepsis are a deterioration of general signs such as fever with a temperature above 38°C, hypothermia with a temperature below 36°C, a deterioration of respiratory signs such as respiratory distress, for example, whining, nasal flaring or signs of retraction, tachypnea with a respiratory rate above 60 / min and apnea, a deterioration of hemodynamic signs such as tachycardia above 160 bpm or bradycardia below 80 bpm, signs of shock such as an increase in skin recoloration time, pallor, arterial hypotension, oliguria, worsening of neurological signs such as drowsiness, irritability, hypotonia or convulsions, or worsening of digestive signs such as refusal to drink or vomiting.

[0051] In particular, said patient presents at least one of the clinical signs of late neonatal sepsis, preferably chosen from: fever ≥ 38°C, tachycardia ≥ 160 / min, skin recoloration time (SRT) ≥ 3 seconds, gray and / or pale complexion, apnea and bradycardia syndrome, increased ventilatory assistance and / or increased FiO2, digestive bloating, rectal bleeding, hypotonia, lethargy, convulsions without other obvious cause, skin rash or inflammation at the puncture site of the central venous catheter, or a combination of these signs.

[0052] According to the present disclosure, the protein concentration of PTX3 in a biological sample previously collected from said patient is then compared to a reference threshold value. Comparing the protein concentration of PTX3 to a reference threshold value quickly and reliably indicates whether or not a patient has late neonatal sepsis. It can thus make it possible to determine whether treatment with an antibiotic should be initiated or stopped in the case where the latter has been administered preventively to the patient when said sepsis is suspected.

[0053] According to the present disclosure, a protein concentration of PTX3 in a patient biological sample below a reference threshold value indicates that the patient does not have late neonatal sepsis and preferably that antibiotic treatment is likely to be stopped. In contrast, a protein concentration of PTX3 in a patient biological sample above a reference threshold value indicates that the patient has late neonatal sepsis and preferably indicates that antibiotic treatment should be continued.

[0054] The term "reference threshold value" as used herein refers to a reference value that may be predetermined, specifying, for example, a confidence interval or a threshold value for evaluating data obtained from a biological sample previously collected from a newborn patient and for diagnosing neonatal sepsis. The reference threshold value may also be derived from the protein concentration of one or several biomarkers, preferably of the protein concentration of the corresponding PTX3 in a "control" biological sample, for example a negative control (uninfected) or a positive control (infected). The reference threshold value may be based on a large number of biological samples, such as from a population of subjects in the chronological age-matched group, or based on a pool of samples including or excluding the sample to be tested. In a particular embodiment, the reference threshold value according to the method of the present application may be obtained from one or more subjects who do not suffer from sepsis (i.e., negative control subjects). A subject is considered not to suffer from sepsis if he or she has not been diagnosed as having sepsis, typically performed by blood culture or by expert opinion.

[0055] The reference threshold value can also be obtained by determining the optimal sensitivity and specificity using a ROC (Receiver Operating Characteristic) curve based on experimental data. For example, as illustrated in the examples of the present application, after determining the protein concentration of the biomarker (e.g. PTX3) in a reference group, an algorithmic analysis can be used for the statistical processing of the values ​​measured in the samples to be tested, and thus obtain a classification standard having significance for the classification of the samples. In a particular embodiment, an ROC analysis with the protein concentration of the biomarker (e.g. PTX3) providing the highest specificity for a sensitivity of at least 0.895 was chosen as the reference threshold value, and as described in the examples of the application.This algorithmic method is preferably carried out using a computer. Software or systems existing in the art can be used for plotting the ROC curve, such as: MedCalc 9.2.0.1, medical statistics software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER.SAS, CREATE-ROC.SAS, GB STAT VI0.0 (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.

[0056] In a preferred embodiment, according to the method of the present disclosure, the reference threshold value of the protein concentration of PTX3 is less than 3000 pg / mL, preferably less than 2000 pg / mL, preferably less than 1300 pg / mL and more preferably between 1000 and 1300 pg / mL, such as for example 1266 pg / mL.

[0057] In a preferred embodiment, according to the method of the present disclosure, the reference threshold value of the protein concentration of PTX3 is from 1000 to 3000 pg / mL, preferably from 1000 to 2000 pg / mL, and more preferably from 1000 to 1300 pg / mL.

[0058] A PTX3 protein concentration in a biological sample previously obtained from said patient that is higher than a reference threshold value as described above indicates that the patient has late neonatal sepsis. In contrast, a PTX3 protein concentration in a biological sample previously obtained from said patient that is lower than said reference threshold value indicates that the patient does not have late neonatal sepsis and preferably indicates that antibiotic treatment is likely not to be initiated or to be stopped if prescribed.

[0059] The inventors in the present application have shown that it is possible to identify a threshold value of PTX3 protein concentration making it possible to avoid or stop antibiotic therapy in patients who are ultimately not infected. Thus, in a particular embodiment, only the PTX3 protein concentration is determined.

[0060] In another particular embodiment, the method according to the present disclosure may further comprise determining the protein concentration of at least one other protein selected from: IL-6, IL-10, procalcitonin (PCT), IP10, sCD14, LBP, NGAL, Calprotectin and Gelsolin, preferably NGAL and optionally Gelsolin, and comparing the concentrations thus determined to corresponding reference threshold values. According to this embodiment, concentrations of said proteins lower than corresponding reference threshold values ​​indicate that the patient does not have late neonatal sepsis. The reference threshold values ​​are as defined below.

[0061] According to a variant of this embodiment, the method according to the present disclosure may comprise determining the protein concentration of at least two other proteins chosen from: IL-6, IL-10, PCT, IP10, sCD14, LBP, NGAL, Calprotectin and Gelsolin, and more preferably, chosen from IL-10, NGAL and Gelsolin, and comparing the concentrations thus determined with corresponding reference threshold values.

[0062] According to a preferred embodiment, the method according to the present application comprises determining the protein concentration of PTX3 and NGAL and comparing the concentrations thus determined to corresponding reference threshold values. According to this embodiment, concentrations of said proteins lower than corresponding reference threshold values ​​indicate that the patient does not have late neonatal sepsis and / or that the antibiotic treatment is likely to be stopped.

[0063] According to another preferred embodiment, the method according to the present application comprises determining the protein concentration of PTX3, NGAL and Gelsolin, and comparing the concentrations thus determined to corresponding reference threshold values. According to this embodiment, concentrations of said proteins below corresponding reference threshold values ​​indicate that the patient does not have late neonatal sepsis and / or that the antibiotic treatment is likely to be stopped.

[0064] In humans, interleukin 6 (IL6), also known as IFNB2, BSF-2, CDF, BSF2, HGF, or HSF, is a 212-amino acid cytokine (UniProKB – P05231, updated February 23, 2022) comprising a 29-amino acid signal peptide encoded by the IL-6 gene (Gene ID: 3569, updated March 27, 2022). IL-6 is produced by B and T lymphocytes as well as monocytes, endothelial cells, and fibroblasts and has a wide variety of biological functions in immunity, tissue regeneration, and metabolism. IL6 binds to the IL6R, and the complex then associates with the IL6ST / gp130 signaling subunit to trigger the intracellular IL6 signaling pathway. Advantageously, the reference threshold value of the protein concentration of IL6 is 15 pg / mL, preferably 10 pg / mL, preferably 7 pg / mL, and very particularly between 4 and 5 pg / mL, such as for example 4.8 pg / mL.

[0065] In humans, the interleukin-10 (IL-10) gene (Gene ID: 3586, updated on March 27, 2022) encodes a 178-amino acid protein (UniProtKB – P22301, amended on February 23, 2022) comprising an 18-amino acid signal peptide. Interleukin 10, sometimes also called CSIF (human cytokine synthesis inhibitory factor), is a protein produced by monocytes and to a lesser extent by lymphocytes and having numerous effects on immunoregulation and inflammation. IL-10 binds to its heterotetrameric receptor comprising IL10RA and IL10RB leading to the phosphorylation of STAT3 via JAK1 and STAT-2. STAT3 is then translocated into the cell nucleus to regulate the expression of anti-inflammatory mediators. Advantageously, the reference threshold value of the protein concentration of IL10 is 45 pg / mL, preferably 20 pg / mL, preferably 15 pg / mL and more preferably between 5 and 10 pg / mL, such as for example 7 pg / mL.

[0066] CD14 is a surface antigen primarily expressed by macrophages. It cooperates with other proteins to mediate the innate immune response to bacterial lipopolysaccharide (LPS) and viruses. In humans, CD14 (UniprotKB – P08571, updated on February 23, 2022) is encoded by the CD14 gene (Gene ID: 929, updated on March 27, 2022). CD14 is a 375-amino acid protein comprising a signal peptide (the first 19 amino acids of the sequence) and a 30-amino acid propeptide (sequence between positions 346 and 375). There is a soluble form (amino acids at positions 20 to 367) and a membrane-bound form (amino acids at positions 20 to 345). The soluble form is cleaved by proteases and the cleavage product is a 13 kDa N-terminal fragment called presepsin or sCD14. Presepsin loses its ability to bind LPS.Presepsin exhibits a different immunogenicity from the soluble form of CD14 and can be distinguished using specific antibodies (WO2004 / 044005). Many techniques, including ELISA methods using specific antibodies recognizing presepsin, for determining the protein concentration of presepsin are known to those skilled in the art (For review, Memar MY et al. Biomed Pharmacother, 2019 Mar;111:649-656). Advantageously, the reference threshold value for the protein concentration of CD14 is 2000 ng / mL, preferably 1500 ng / mL, preferably 1000 ng / mL, more preferably 800 ng / mL, and most preferably between 700 and 750 ng / mL, such as for example 712 ng / mL.

[0067] Interferon gamma-induced protein 10 (IP10), also known as CXC motif chemokine ligand 10 (CXCL10), or small inducible cytokine B10 (UniProKB – P02778, updated on February 23, 2022) is an 8.7 kDa protein that in humans is encoded by the CXCL10 gene (Gene ID: 3627, updated on March 27, 2022). IP10 is a pro-inflammatory cytokine that is involved in a wide variety of processes such as chemotaxis, differentiation and activation of peripheral immune cells, regulation of cell growth, apoptosis, and modulation of angiostatic effects. It thus plays an important role during viral infections by stimulating the activation and migration of immune cells to infected sites.Mechanistically, binding of CXCL10 to the CXCR3 receptor activates G-protein mediated signaling and results in downstream activation of the phospholipase C-dependent pathway, increased intracellular calcium production, and actin reorganization. Advantageously, the reference threshold value of the protein concentration of IP10 is 200 ng / mL, preferably 150 ng / mL, preferably 100 ng / mL, more preferably 80 ng / mL, and most preferably between 75 and 80 ng / mL, such as 78 pg / mL.

[0068] Neutrophil gelatinase-associated lipocalin (NGAL / Lipocalin-2 Lcn-2) is a 21 kDa human protein of the lipocalin superfamily (UniProtKB-P80188, updated on February 23, 2022) comprising a signal peptide consisting of the first 20 amino acids of the sequence. The protein is encoded in humans by the LCN2 gene (Gene ID: 3934, updated on March 20, 2022). Lipocalins are transporters of hydrophobic molecules, such as lipids, steroid hormones, and retinoids. NGAL is a neutrophil gelatinase-associated lipocalin that plays a role in innate immunity by limiting bacterial growth through the sequestration of iron-containing sidephores.Advantageously, the reference threshold value of the NGAL protein concentration is 300 ng / mL, preferably 200 ng / mL, preferably 150 ng / mL, more preferably 100 ng / mL, and very particularly between 90 and 100 ng / mL, such as for example 96 ng / mL.

[0069] Lipopolysaccharide binding protein (LBP) (UniProtKB – P18428, updated on February 23, 2022) is encoded in humans by the LBP gene also called BPIFD2 (Gene ID: 3929, updated on January 25, 2022). LBP is a soluble acute phase protein that binds to bacterial lipopolysaccharide (or LPS) to induce immune responses by presenting LPS to important cell surface pattern recognition receptors called CD14 and TLR42. Advantageously, the reference threshold value of LPB protein concentration is 8000 ng / mL, preferably 7000 ng / mL, preferably 6500 ng / mL, more preferably 6200 ng / mL, and most preferably between 6100 and 6200 ng / mL, such as 6172 ng / mL.

[0070] Calprotectin is a heterocomplex of two calcium-binding proteins, the S100-A8 protein also called calgranulin A or MRP8 (UniProtKB – P05109, updated on February 23, 2022) encoded in humans by the S100A8 gene (Gene ID: 6279, updated on March 7, 2022) and the S100-A9 protein also called MRP8 / 14 (UniProtKB – P06702, updated on February 23, 2022) encoded in humans by the S100A9 gene (Gene ID: 6280, updated on March 27, 2022). Other names for calprotectin include MRP8-MRP14, calgranulin A and B, cystic fibrosis antigen, L1, 60BB antigen, and 27E10 antigen. In the presence of calcium, calprotectin is able to sequester the transition metals iron, manganese, and zinc by chelation. This sequestration of metals gives the complex antimicrobial properties.Advantageously, the reference threshold value of the calprotextin protein concentration is 2000 ng / mL, preferably 1500 ng / mL, preferably 1000 ng / mL, more preferably 800 ng / mL, and very particularly between 740 and 760 ng / mL, such as for example 748 ng / mL.

[0071] Gelsolin is a cytosolic protein (82kDa) (UniProtKB – P06396, updated on February 23, 2022) encoded by the GSN gene also called ADF or AGEL (Gene ID: 2394, updated on March 13, 2022). Gelsolin, activated by the presence of calcium ions in high concentration, is able to bind to actin filaments and create a local dislocation of these. In addition, it remains fixed at the end (+) of the microfilament (polymerization end) and thus prevents rapid re-polymerization of actin. Advantageously, the reference threshold value for the protein concentration of Gelsolin is 300 µg / mL, preferably 200 µg / mL, more preferably 150 µg / mL, and particularly between 130 and 140 µg / mL, such as 133 µg / mL.

[0072] Procalcitonin is a 116-amino acid (12.6 kDa) polypeptide encoded in humans by the calcitonin gene (Gene ID: 796, updated March 13, 2022) which is the peptide precursor of calcitonin. It corresponds to the prohormone calcitonin (CT) (NCBI Sequence Reference: NP_001029124.1, updated February 27, 2022) comprising a 25-amino acid signal peptide. PCT is primarily synthesized by thyroid C cells and to a lesser extent in neuroendocrine tissue of other organs such as the lungs and intestines. PCT production can be stimulated in almost all organs by inflammatory cytokines and more particularly bacterial endotoxins present during sepsis.Advantageously, the reference threshold value of the PCT protein concentration is 1500 pg / mL, preferably 1000 pg / mL, and more preferably 750 pg / mL, and very particularly between 450 and 550 pg / mL, such as for example 510 pg / mL.

[0073] The protein concentration of at least one other protein selected from: IL-6, IL-10, procalcitonin (PCT), IP10, CD14, LBP, NGAL, Calprotectin and Gelsolin, preferably NGAL and Gelsolin can be measured by any suitable method known to those skilled in the art as described above. In particular, the protein concentration is determined by an ELISA method, preferably a microfluidic ELISA method. The antibodies used in the ELISA methods for these biomarkers are known to those skilled in the art and are for example referenced in antibody databases such as the online database Antibodypedia, (Kiermer V. (2008) “Antibodypedia - A web portal to share antibody validation data” Nature Methods.5: 860).

[0074] The protein concentration of at least one other protein selected from: IL-6, IL-10, procalcitonin (PCT), IP10, CD14, LBP, NGAL, Calprotectin and Gelsolin, preferably NGAL and Gelsolin, is compared to a corresponding reference threshold value. Concentrations of the PTX3 protein and at least one of said other proteins above corresponding reference threshold values ​​indicate that the patient has late neonatal sepsis, and preferably that antibiotic treatment is likely to be continued. Conversely, concentrations of the PTX3 protein and at least one of said other proteins below corresponding reference threshold values ​​indicate that the patient does not have late neonatal sepsis and preferably that antibiotic treatment is likely to be stopped.

[0075] The present disclosure relates to an in vitro method for determining whether antibiotic treatment is likely to be discontinued in a newborn patient susceptible to late neonatal sepsis and having previously received antibiotic treatment, said method comprising determining the protein concentration of PTX-3 in a biological sample previously obtained from said patient and comparing the concentration thus determined to a reference threshold value, characterized in that a protein concentration of PTX-3 lower than said threshold value indicates that the antibiotic treatment is likely to be discontinued and that the patient does not have late neonatal sepsis.

[0076] In another aspect, the present application relates to an antibiotic for use in the treatment of late neonatal sepsis syndrome in a newborn patient, characterized in that said antibiotic is administered to said patient previously assessed as having late neonatal sepsis by a method as previously described.

[0077] The present application also relates to a method for treating late neonatal sepsis in a newborn patient comprising: a) determining the protein concentration of PTX3 and preferably at least one other protein chosen from: IL-6, IL-10, procalcitonin (PCT), IP10, CD14, LBP, NGAL, Calprotectin and Gelsolin in a biological sample previously obtained from said patient and comparing the concentration(s) thus determined with one or more reference threshold values, characterized in that a protein concentration of PTX3 and preferably at least one of said other proteins above said reference threshold value(s) indicate that the patient has late neonatal sepsis, b) administering to said patient assessed as having late neonatal sepsis a therapeutically effective amount of an antibiotic.

[0078] The present application also relates to the use of an antibiotic in the preparation of a medicament for the treatment of late neonatal sepsis in a newborn patient characterized in that said antibiotic is administered to said patient after having been assessed as having late neonatal sepsis by the method described above.

[0079] In another particular embodiment, the present application also relates to a method for treating late neonatal sepsis in a newborn patient comprising: a) administering to said patient an effective therapeutic amount of an antibiotic, b) determining the protein concentration of PTX-3 and preferably at least one other protein chosen from: IL-6, IL-10, procalcitonin (PCT), IP10, CD14, LBP, NGAL, Calprotectin and Gelsolin in a biological sample from said patient and comparing the concentration(s) thus determined with one or more reference threshold values, characterized in that a protein concentration of PTX3 and preferably at least one other protein chosen from: IL-6, IL-10, procalcitonin (PCT), IP10, CD14, LBP, NGAL,Calprotectin and Gelsolin lower than said reference threshold value(s) indicates that antibiotic treatment is likely to be stopped in said patient and that said patient does not have late neonatal sepsis, c) stopping the administration of an antibiotic to said patient, for whom antibiotic treatment is assessed as likely to be stopped.

[0080] Preferably, the antibiotic that can be administered to the newborn patient may include one of the following antibiotics as examples: amoxicillin, amikacin, aztreonam, chloramphenicol, ceftazidime, clindamycin, cefalotin, ciprofloxacin, colistin, cefotetan, cefotaxime, erythromycin, fusidic acid, fosfomycin, cefoxitin, furans, gentamicin, imipenem, kanamycin, lincomycin, cefamandole, minocycline, latamoxef, metronidazole, nalidixic acid, netilmicin, oxacillin, benzylpenicillin, pefloxacin, piperacillin, pristinamycin, rifampicin, spiramycin, sulfonamides, streptomycin, trimethoprim, sulfametoxazole, tetracycline, teicoplanin, ticarcillin, tobramycin, trimethoprim, vancomycin, preferably amoxicillin, gentamicin or cefotaxime.

[0081] In the context of this disclosure, the term "treat" or "treatment," as used herein, means to reverse, alleviate, inhibit the progression of, or prevent the disorder or condition to which such term applies, or to reverse, alleviate, inhibit the progression of, or prevent one or more symptoms of the disorder or condition to which such term applies.

[0082] As used herein, a "therapeutically effective amount" or "effective amount" means the amount of a composition that, when administered to a subject to treat a condition, disorder, or state, is sufficient to effect treatment. The therapeutically effective amount varies depending on the compound, formulation, or composition, the disease and its severity, and the age, weight, physical condition, and responsiveness of the subject to be treated.

[0083] The antibiotic described herein may be administered by any means known to those skilled in the art, including, without limitation, intravenously, orally, intraperitoneally, intramuscularly, parenterally, subcutaneously, and topically, preferably intravenously and orally. Thus, the compositions may be formulated as an injectable, topical, or ingestible formulation. Administration of the compounds or therapeutic agents to a subject in accordance with the present disclosure may exhibit beneficial effects in a dose-dependent manner. Thus, within broad limits, administering larger amounts of the compositions is expected to achieve greater beneficial biological effects than administering a smaller amount. In addition, efficacy is also contemplated at doses below the level at which toxicity is observed.

[0084] It will be appreciated that the specific dosage of the antibiotic in a given case will be adjusted depending on the composition administered, the volume of the composition that can be effectively delivered to the site of administration, the disease to be treated or to inhibit, the condition of the subject, and other relevant medical factors which may alter the activity of the compositions or the response of the subject, as is well known to those skilled in the art.

[0085] In another aspect, the present disclosure relates to a kit comprising a set of reagents which makes it possible to determine the protein concentration of PTX3, and preferably of at least one of the proteins selected from: NGAL, IL-10, IL-6, Calprotectin, LBP, PCT, sCD14, IP10 or Gelsolin, preferably of PTX3, NGAL and Gelsolin.

[0086] Preferably, the reagents comprise the antibodies or antibody fragments as previously defined for the method.

[0087] According to a particular embodiment, the kit comprises a set of reagents which makes it possible to determine the protein concentration of PTX3 and at least two other proteins chosen from: NGAL, IL-10, IL-6, Calprotectin, LBP, PCT, sCD14, IP10 and Gelsolin, preferably from NGAL, IL-10 and Gelsolin.

[0088] Preferably, the kit comprises a control sample calibrated to contain the threshold value of the PTX3 concentration as defined above, and optionally one or more other control samples calibrated to each contain the threshold value of the concentration of at least one of the proteins chosen from: NGAL, IL-10, IL-6, Calprotectin, LBP, PCT, sCD14, IP10 or Gelsolin, preferably NGAL and / or Gelsolin and / or IL-10.

[0089] More preferably, the kit comprises containers each comprising one or more compounds at a concentration or in an amount which facilitates the reconstitution and / or use of a set of reagents, preferably antibodies, preferentially coupled to a fluorophore or a chromogen which allows the specific determination of the protein concentration of PTX3, and preferably of at least one of the proteins chosen from: NGAL, IL-10, IL-6, Calprotectin, LBP, PCT, sCD14, IP10 or Gelsolin, preferably of PTX3, NGAL and Gelsolin and the implementation of the method according to the disclosure.

[0090] The kit may also include instructions indicating methods of preparing and / or using reagents to determine the level of expression of said genes according to the methods of the disclosure.

[0091] The kit according to the present disclosure is therefore particularly suitable for use in determining whether antibiotic treatment is likely to be stopped in a newborn patient likely to have late neonatal sepsis and who has previously received antibiotic treatment. Examples Material and method 1. Study design

[0092] A prospective, multicenter cohort study was conducted in two neonatal intensive care units (Hôpital Femme Mère Enfant, Hospices Civils de Lyon, Bron; Centre Hospitalier Universitaire de Nantes, Nantes) between November 19, 2017, and November 20, 2020 (clinicaltrials.gov ID: NCT03299751). Hospitalized newborns over 7 days old, covered by health insurance, with signs suggestive of late onset sepsis were consecutively included.

[0093] Suspicion of late neonatal sepsis was defined by at least one of the following clinical criteria: Fever > 38°C - tachycardia > 160 bpm, - skin recoloration time (SRT) > 3 seconds, - gray and / or pale complexion, - apnea / bradycardia syndrome, - digestive bloating, - rectal bleeding, - hypotonia, - lethargy, - convulsions without other obvious cause, - increased ventilatory support and / or increased FiO2, - skin rash or inflammation at the central venous catheter puncture site.

[0094] Exclusion criteria in the study included newborns treated with antibiotics for a bacteriologically documented infection at the time of blood sampling or within 48 hours prior to blood sampling, newborns who had undergone surgery within the previous 7 days, newborns vaccinated within the previous 7 days, newborns who had received systemic corticosteroid therapy within 48 hours prior to sampling, and newborns with severe combined immunodeficiency.

[0095] At the time of inclusion, investigators recorded demographic data, medical history, disease history, neonatal data, presence of neonatal transfusion within 7 days prior to inclusion, and physical examination data.

[0096] The results of additional tests performed (chest X-ray, bacteriological samples, C-reactive protein (CRP), white blood cell count, absolute neutrophil count) were also recorded. Standard care, according to the investigators' assessment, included blood culture and in some patients the performance of other tests (e.g., CRP, urinalysis, lumbar puncture, stool culture, and chest X-ray). The decision to treat the newborn with antibiotics was left to the physician's discretion. At 48 hours post-inclusion, a clinical reassessment was performed, including clinical data as well as intubation data, CRP and bacteriology laboratory assays, and antibiotic therapy data. 2. Adjudication procedure

[0097] In the absence of a gold standard for diagnosing infection in newborns, a reference standard was established by a panel of experts, following existing medical recommendations.

[0098] Thus, an independent adjudication committee was formed with three pediatric neonatal resuscitation experts, with expertise in diseases neonatal infectious diseases and independent of the team carrying out the inclusion. The experts assigned each included newborn to one of the following 3 diagnostic categories: (i) confirmed infection, (ii) unconfirmed infection, (iii) indeterminate infection. The classification by the experts of the adjudication committee was based on the clinical data collected at inclusion and at 48 hours blinded to the results of the biomarker measurement and the decision of their peers. The final diagnosis was determined by the majority agreement of the committee (at least two concordant classifications out of three). In case of non-majority, the three experts made a consensus decision on the diagnosis. 3. Sample collection and biomarker measurements

[0099] At inclusion, 0.4 mL of blood was collected at the time of venipuncture prescribed as part of standard care, for the measurement of the following 11 protein markers: - Interleukin 10 (IL-10), - Procalcitonin (PCT), - Interleukin 6 (IL-6), - Interferon gamma induced protein 10 (IP-10)(also called CXCL10), - Neutrophil gelatinase associated lipocalin (NGAL)(also called lipocalin-2), - Pentraxin 3 (PTX3)(also called TSG-14), - Presepsin (sCD14), - Lipopolysaccharide binding protein (LBP), - Calprotectin, - Gelsolin, and - Interleukin 27 (IL-27).

[0100] The assay of these 11 biomarkers was performed in a central laboratory (HCL-bioMérieux Joint Research Unit, Lyon, France), in serum samples prepared from 0.4 mL of whole blood collected in BD Microtainer™ serum separator tubes (Beckon Dickinson, reference BD365968). After 2 hours of coagulation at room temperature and centrifugation at 2500 g for 10 min, sera were aliquoted and stored at -80°C until biomarker measurements.

[0101] Concentrations of IL-10, PCT, IP-10, IL-6, NGAL, PTX3, Presepsin and LBP were measured in serum samples using the Simple Plex automated immunoassay platform. TM (Simple Protein©, CA, USA) according to the manufacturer's instructions. The Simple Plex TM is an integrated immunoassay system consisting of a disposable microfluidic cartridge and an automated analyzer, the ELLA instrument.

[0102] Quantification of IL10, PCT, IP-10, and IL-6 was performed simultaneously in a multiplex cartridge format using 50 µL of twice-diluted serum. NGAL, PTX3, Presepsin, and LBP concentrations were measured in a multiplex cartridge format using 50 µL of 1:400 diluted serum.

[0103] Gelsolin concentrations were measured using the GS(Gelsolin) Human ELISA kit (Elabsciences®) using serum diluted 1:2000, according to the manufacturer's instructions.

[0104] Calprotectin concentrations were measured using the Human S100A8 / S100A9 Heterodimer Quantikine ELISA kit (R&D Systems, MN, USA) with serum diluted 1:200.

[0105] Finally, IL-27 levels were measured using the DuoSet ELISA kit (R&D Systems, MN, USA) with twofold diluted serum. All measurements were performed in duplicate by manual ELISA. 4. Statistical analyses

[0106] Data are described by median and range (quantitative data) and numbers and percentages for categorical data. Comparison of marker values ​​between the “confirmed infection” and “disproven infection” groups is performed for exploratory purposes, using a Shapiro-Wilk test.

[0107] The assessment of the diagnostic capacity of clinical markers / signs is carried out on the groups of patients with “confirmed infection” or “unconfirmed infection”.

[0108] Univariate logistic regressions were performed to assess the association between symptoms and confirmed infection; the association is quantified by an odds ratio with its 95% confidence interval. Symptoms with a p-value less than 0.2 in univariate analysis, with little collinearity, and clinically relevant, were combined through a multivariate logistic regression model.

[0109] The markers were combined to predict confirmed infection using a logistic regression model assuming an additive effect on the linear predictor scale. Log transformations of the markers were considered to meet the model assumptions. The predictions from the logistic regression models were then used as a new marker summarizing the marker combination. Any combination of 2, 3, or 4 markers was considered.

[0110] The performance of markers, marker combinations, symptoms, and symptom combinations was assessed by constructing ROC curves and calculating areas under the ROC curve and partial areas under the ROC curve (area in the zone where sensitivity is greater than 0.90) with associated confidence intervals. For each marker and marker combination, the threshold associated with the highest specificity and a sensitivity of at least 0.9 was determined. At this threshold, the following were assessed: specificity, positive predictive value (PPV), negative predictive value (NPV), and positive and negative likelihood ratios with their associated 95% confidence intervals.

[0111] The combination of markers is carried out within the framework of the study on a training set, which will require confirmation on a subsequent confirmation set.

[0112] Statistical analyses were performed using R and SAS software. 5. Ethics

[0113] Written informed consent was obtained from at least one of the patients' parents or legal guardians. The study was approved by the Ethics Committee (Comité de Protection des Personnes [CPP]) Sud-Ouest et Outremer III under registration number 2017-A02492-51, and was conducted in accordance with the recommendations for good clinical practice and the Declaration of Helsinki. The study was registered in clinicaltrials.gouv.fr under the number NCT03299751. Results 1. Presentation of the patient cohort and the classification carried out by the adjudication committee

[0114] As shown in [Fig. 1], out of 234 patients included, 230 could be classified by the adjudication committee into the three previously defined diagnostic categories. In particular, 51 patients were classified as having a confirmed infection, 153 patients as having an unconfirmed infection (i.e., infection ruled out), and 26 patients for whom it was not possible to conclude on the presence or absence of an infection (i.e., infection indeterminate). 2. Demographic and clinical characteristics of patients at admission

[0115] All demographic and clinical characteristics of patients at admission are presented in [Table 1] below:

[0116] [Table 1] 3. Clinical condition of patients at the time of sampling

[0117] The clinical status of the patients at the time of sampling is presented in [Table 2] below:

[0118] [Table 2] 4. Additional examinations

[0119] The results of the additional biological analyses are presented in [Table 3] below:

[0120] [Table 3] 5. Results of blood cultures and antibiotic therapy

[0121] The results are presented in [Table 4] below:

[0122] [Table 4]

[0123] Thus, 84% of patients classified as having a confirmed infection by the adjudication committee have a positive blood culture. This percentage does not reach 100% due to the limitations of blood culture, notably the false negative rate as well as the volume of blood required for an optimal culture, which cannot always be collected from very low birth weight newborns with insufficient total blood volume.

[0124] The inventors also note that all patients classified as having a confirmed infection did indeed receive antibiotic treatment, confirming that there is no need for new biomarkers for the positive diagnosis of the infection. However, among patients classified as having a ruled-out infection, 27.5% received antibiotic therapy when it was not necessary, thus confirming the need for new biomarkers to stop antibiotic treatment at an early stage.

[0125] Indeed, as soon as there is a suspicion of late neonatal sepsis, medical recommendations consist of treating the newborn with antibiotics until the blood culture results are available, which in practice represents a delay of 48 hours.

[0126] Therefore, in the event that the blood culture proves sterile and the decision is made by the doctor to stop the antibiotic treatment, the newborn has still received the treatment for approximately 48 hours.

[0127] Given the harmful consequences of antibiotic use on newborns, including mortality during hospitalization (see in particular Ting et al. JAMA Pediatr. 2016 Dec 1;170(12):1181-1187 - doi: 10.1001 / jamapediatrics.2016.2132) or even the modification of the microbiota with long-term effects (see in particular Kummeling et al. https: / / doi.org / 10.1542 / peds.2006-0896), there is a need to be able to stop treatment as early as possible once it has been prescribed.

[0128] As demonstrated in the following section, this need is all the more important since clinical signs alone do not provide sufficient performance to make a decision on stopping antibiotic treatment. 6. Performance of the model based on clinical signs alone

[0129] The clinical model is derived from a multivariate analysis based on clinical signs with a p-value less than 0.2 in univariate analysis. Thus, the clinical signs that were retained are presented in the following [Table 5]:

[0130] [Table 5]

[0131] The ROC curve of clinical signs is presented in [Fig.2] and confirms that clinical signs alone do not have sufficient performance to determine the discontinuation of antibiotic treatment in case of suspected late neonatal sepsis. 7. Analysis of biomarker performance 7.1. Description of biomarkers according to patient status [Table 6]

[0132] [Table 6] 7.2. ROC and AUC curves

[0133] The analyses are carried out considering the outcome "Occurrence of a confirmed infection" (versus infection denied). The performances of the biomarkers taken one by one were notably evaluated by the area under the ROC curve and are presented in part in Figure 3 and repeated in Table 7 below.

[0134] For each marker, the threshold selected is the one with the highest specificity and leading to a sensitivity of at least 0.895. Sensitivity, specificity, positive and negative predictive values, as well as positive and negative likelihood ratios were estimated at the optimal threshold, with their associated 95% confidence intervals. Biomarkers alone [Table 7]

[0135] In view of the performance, and in particular through the identified threshold, the PTX3 biomarker according to the invention can thus be used as a marker of late neonatal sepsis in newborns, thus supplementing the tools available to practitioners for the identification of infections in the neonatal population. Combinations of biomarkers

[0136] The PTX3 biomarker was combined with the other biomarkers in a logistic regression model, assuming an additive effect of the markers after logarithmic transformation and performance results are shown in [Table 8] below:

[0137] [Table 8]

[0138] The performance thus confirms that the PTX3 biomarker can advantageously be combined with other biomarkers in the identification of late neonatal sepsis in newborns. 7.3 Combination of biomarkers to determine discontinuation of antibiotic treatment

[0139] [Table 9]

[0140] (a)Logistic regression-based models; (b)By adjudication committee experts; (c)Denominator < 51 in some cases for logistic regression-based models due to two patients with missing biomarker data. (d)Denominator < 42 in some cases due to one patient with missing biomarker data.

[0141] Thus, among the patients who received antibiotic therapy, five of them identified as having an infection by the adjudication committee were reclassified by the models as not having an infection, but this result was expected because the sensitivity of the model is set at 0.9.

[0142] However, it was found that more than half (respectively 64.3% and 56.1%) of antibiotic treatments could have been avoided by using the combination of PTX3 / NGAL or PTX3 / NGAL / Gelsolin biomarkers.

[0143] Conversely, the IL6 biomarker, which presented the best results among all the biomarkers evaluated in terms of area under the ROC curve (AUC 0.864; Se 0.90; Sp 5.536 [0.799; 0.929]), does not allow a sufficient number of antibiotic therapies to be avoided (only 23.8%) to be implemented and present a benefit for patients, thus confirming the surprising nature obtained with the PTX3 biomarker.

[0144] The PTX3 biomarker according to the invention, used in combination, therefore also finds a very particular application in patients in neonatology units as a tool in the early discontinuation of antibiotic treatments initially prescribed following a suspicion of late neonatal sepsis. Free text of the sequence listing

[0145] In this application, reference is made to the sequence listing(s) whose identifier(s) (or “SEQ ID NO”) are listed below.

[0146] SEQ ID NO: 1 (PTX-3):

[0147] Regardless of the form in which the listings are provided, these listings form part of this application.

Claims

Claims

1. An in vitro method for determining whether antibiotic treatment is likely to be discontinued in a newborn patient at risk of developing late-onset neonatal sepsis who has previously received antibiotic treatment, said method comprising determining the protein concentration of PTX-3 in a biological sample previously obtained from said patient and comparing the concentration thus determined to a reference threshold value, characterized in that a PTX-3 protein concentration below said threshold value indicates that antibiotic treatment is likely to be discontinued and that the patient does not have late-onset neonatal sepsis.

2. The method according to claim 1, characterized in that the protein concentration of PTX-3 is determined by an enzyme-linked immunosorbent assay (ELISA), preferably a microfluidic ELISA.

3. The method according to claim 1 or 2, characterized in that said biological sample is a biological sample of blood, plasma, or serum, preferably serum.

4. The method according to claim 3, characterized in that the volume of the biological sample previously obtained from the patient is less than 50 µL, preferably less than 20 µL, and preferably, even more preferably, less than 5 µL.

5. The method according to any one of claims 1 to 4, characterized in that the reference threshold value of the PTX-3 protein concentration is less than 3000 pg / mL, preferably less than 2000 pg / mL, preferably less than 1300 pg / mL, and preferably, even more preferably, between 1000 and 1300 pg / mL.

6. The method according to any one of claims 1 to 5, comprising the determination of the concentration of at least one other protein selected from: IL-6, IL-10, PCT, IP10, sCD14, LBP, NGAL, Calprotectin and Gelsolin, said method being characterized in that concentrations of the PTX-3 protein and at least one of said other proteins below corresponding reference threshold values ​​indicate that the treatment at. The antibiotic is likely to be discontinued and the patient does not have late neonatal sepsis.

7. The method according to claim 6, characterized in that it comprises the determination of NGAL protein concentrations, said method being characterized in that PTX-3, NGAL protein concentrations below corresponding reference threshold values ​​indicate that antibiotic treatment is likely to be discontinued and that the patient does not have late neonatal sepsis.

8. The method according to claim 7, characterized in that the reference threshold value for the NGAL protein concentration is less than 300 ng / mL, preferably less than 200 ng / mL, more preferably 100 ng / mL, and particularly between 90 and 100 ng / mL.

9. The method according to any one of claims 6 to 8, characterized in that it comprises the determination of the concentrations of NGAL and Gelsolin proteins, said method being characterized in that concentrations of PTX-3 protein, NGAL, and Gelsolin below corresponding reference threshold values ​​indicate that antibiotic treatment can be discontinued and that the patient does not have late neonatal sepsis.

10. The method according to claim 9, characterized in that the reference threshold value for the Gelsolin concentration is less than 300 µg / mL, preferably less than 200 µg / mL, preferably still less than 150 µg / mL, and particularly between 130 and 140 µg / mL.

11. The method according to any one of claims 1 to 5, characterized in that only the concentration of PTX-3 is determined.

12. An in vitro method for determining whether a newborn patient has late neonatal sepsis, said method comprising determining the protein concentration of PTX-3 in a biological sample previously obtained from said patient and preferably the protein concentration of at least one other protein selected from: IL-6, IL-10, PCT, IP10, sCD14, LBP, NGAL, Calprotectin and Gelsolin and the. Comparison of the concentration(s) thus determined to one or more corresponding reference threshold values, characterized in that one or more concentrations of PTX-3 and preferably at least one other protein selected from: IL-6, IL-10, PCT, IP10, sCD14, LBP, NGAL, Calprotectin, and Gelsolin above the reference threshold value indicate that the patient has late-onset neonatal sepsis.

13. An antibiotic for use in the treatment of late-onset neonatal sepsis in a newborn patient, characterized in that said antibiotic is in a form suitable for administration to said patient previously assessed as having late-onset neonatal sepsis by the method according to claim 12, preferably said antibiotic being selected from amoxicillin, gentamicin, and cefotaxime.

14. A kit comprising reagents for determining the protein concentration of PTX3 and optionally at least one other protein selected from NGAL, IL-10, IL-6, Calprotectin, LBP, PCT, sCD14, IP10, and Gelsolin.

15. Use of the kit according to claim 14 to determine whether antibiotic treatment is likely to be discontinued in a newborn patient suspected of having late-onset neonatal sepsis and who has previously received antibiotic treatment.

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