OA18480A - Qualitative predictive method for differential diagnosis of pneumococcic, meningococcic and viral meningitis, differential meningitis diagnostic method and kit. - Google Patents

Abstract

The present invention relates to a qualitative predictive method, a method, use and kit for the early differential diagnosis of prevalent forms of bacterial and viral meningitis, which allow the various forms of meningitis to be detected and differentiated. The invention uses a qualitative predictive method based on the combined detection and sequential analysis of the presence or absence of at least three among four specific biomarkers.

Description

QUALITATIVE PREDICTIVE METHOD FOR DIFFERENTIAL

DIAGNOSIS OF PNEUMOCOCCIC, MENINGOCOCC1C AND VIRAL

MENIGITIS, DIFFERENTIAL MENINGITIS DIAGNOSTIC METHOD

AND KIT

The instant invention relates to a qualitative prédictive method, to a method and kit applied to the early differential diagnosis of the most prévalent forms of bacterial and viral meningitis, enabling to detect and distinguish the different forms of meningitis. The invention further relates to the use of biomarker proteins for meningitis prédiction.

The instant invention uses a qualitative prédictive method based on combined détection and sequential analysis of the presence/absence of at least three of four spécifie biomarkers. Biomarkers are proteins of the inflammatory response of the human host présent in patients' cerebrospïnal fluid. The application of the qualitative prédictive method enables to differentiate patients affected by viral meningitis from the ones affected by bacterial meningitis, also determining the etiology of bacterial meningitis, whether pneumococcal or meningococcal. The instant invention further provides a meningitis differential diagnostic method based on the application of the prédictive method and also provides the possibility of incorporating the method of the instant invention into a diagnostic kit contaîning ligands to biomarker proteins such as antibodies or aptamers, adapted to a laboratory évaluation method that can include ELISA (Enzyme Linked Immunosorbent Assay), chromatography, turbidimetry, capillary electrophoresis, inter alia.

BACKGROUND OF THE INVENTION

Many disease conditions are characterized by différences in levels of gene expression in the presence or concentration of spécifie proteins and other biomarkers. Within this context, there is the possibility of using prédictive models (methods) to aid disease diagnosis.

Meningitis is the inflammation of the méningés in response to infection or exposure to chemical agents. According to the etiology, meningitis are classified as aseptie (AM), with no evidence of causative bacterial infection, or bacterial (BM). While AMs, are mostly are benign and of self-limited course, BMs are associated with high mortality and morbidity that hâve remained unchanged in the last décades despîte the advances in antimicrobial therapy and intensive care aîmed at maintaining patients’ vital Systems (Scheld W, Koedel U, Nathan B, Pfister H: Pathophysiology of bacterial meningitis: mechanism(s) of neuronal injury. J Infect Dis 2002, 186 Suppl 2:S225-233).

Bacterial meningitis (BM) is one of the top ten causes of death related to infections worldwide (Fauci A. Infectious diseases: considérations for the 21 st century, Clin Infect Dis 2001, 32(5):675-685), with an estimated incidence of 2-5/100 thousand cases per year in deveioped countries, reaching values up to ten times higher in developing countries (Murray J, Lopez A: Global burden of disease and Injuries sériés. Geneva; 1996.). MB is associated with a mortality rate up to 30%. Furthermore, from 30 to 50 % of patients who survive the Infection deveiop permanent neurological sequelae, including sensorineural deafness, intellectuai disablllty, ieaming disabllities, sensory and physical disabillties and cérébral palsy (Merkelbach S, Sittinger H, Schweizer I, Muller M: Cognitive outcome after bacterial meningltis. Acta Neurol Scand 2000,102(2):118-123).

The most common étiologie agents of MB are Streptococcus pneumonlae (pneumococci), Neisseria men/ngit/dfs (meningococd), and type B Haemophilus influenzae (Hib). Since the création and inclusion of anti-Hlb vaccine ln the basic vaccination schedule, at the end of the 90s, pneumococci hâve become the most frequent causative agent of non-epidemic MB acquired in the communlty among children over one year old (Schuchat A, Robinson K, Wenger J, Harrison L, Fariey M, Relngold A, Lefkowitz L, Perkins B. Bacterial meningitls in the United States in 1995. Active Surveillance Team. N Engi J Med 1997, 337(14):970-976). Among BMs, pneumococcal meningitls Is the one associated with the highest mortality and morbldity rates. Meningococcal meningitls is malnly an épidémie disease and affects malnly children and young adults.

Aseptie meningltis (AM) is defined as an inflammation of the subarachnoid space, characterized by mononuclear cells pleocytosis and by stérile CSF (cerebrospinai fluid or cerebrospinal fluid) culture. Although the primary cause of AMs are virai infections, AM differential diagnosls Includes also tuberculous or fungal meningitis, Inflammation caused by parameningeai infection, collagen vascular diseases and menlngeai inflammations caused by drug (Ravei R: Clinicai Laboratory Mediclne: Clinicai Application of Laboratory Data: Elsevier Health Sciences; 1994). Viral meningitis are common and often not reported. Non-poliovirus enteroviruses (Coxsackievirus and Echovirus) are responsible for 80 to 90% of the cases of virai meningitis with determined etiology (Atkinson P, Shariand M, Maguire H: Prédominant enterovirai serotypes causing meningitls. Archives of Disease In Childhood 1998, 78:373-374).

BMs are characterized by an intense granulocytic Inflammation in the subarachnoid and ventricular spaces, which extends to the perilymphatic space of the inner ear and causes neuronal death, malnly ln the cérébral cortex (CX) and hippocampus (HC) and ln the cochlear spiral ganglion. In pneumococcal meningitis, cortical areas with morphologie evidence of acute neural necrosis are observed, apoptosls being the prédominant form of neuronal damage in the HC (Meli D, Christen S, Leib S, Tauber M: Current concepts in the pathogenesis of meningitis caused by Streptococcus pneumoniae. Curr Opin Infect Dis 2002, 15(3):253-257). The inflammatory response to Infection détermines the BM clinicai resuit. The cascade of inflammatory evens that drives BM pathogenesis Is Inltiated by the presence of the bacteria in the CSF. Bacterial components stimuiate the production and release by endothélial cells, astrocytes and mlcroglia, of inflammatory mediators that can directly injure the brain tissue, interact with other modulators of the inflammatory response, or also induce secondary mechanisms capable of causing brain damage (Koedel U, Pfister H: Oxidatlve stress ln bacterial meningitis. Brain Pathol 1999, 9(1):5767). Another remarkable characteristic of BM Is the Increased permeability of the bloodbrain barrier, which affects homeostasls In the neuronal microenvironment

Most data on the pathophysiology of MB were obtained from studies using experimental models of pneumococcal meningitis. Llttle is known about the pathophysiology of meningococcal meningitis. If, on one hand, It Is reasonable to formulate some extrapolations from the results of studies focused on the pneumococcal disease, the différences In mortaiity and morbidity rates between pneumococcal meningitis and meningococcal hâve remained unexpialned.

The Inflammatory process observed in patients affected by enterovirus AM Is much less weil-known than in the case of BM. It Is known that the Inflammatory response Is triggered by the pénétration of the virus ln the central nervous System (CNS), mainly by hematogenic dissémination from primary Infections sites (Tunkel A, Wispelwey B, Scheld W: Pathogenesis and pathophysiology of meningitis. Infect Dis Clin North Am 1990, 4(4):555-581). The presence of the virus ln the CNS induces the production/release of proinflammatory cytokines that promote Infiltration of leukocytes in the infected area (Sato M, Hosoya M, Honzuml K, Watanabe M, nlnomiya N, Shigeta S, Suzuki H: Cytokine and cellular Inflammatory response In enteroviral meningitis. Pediatrics 2003, 112(5):11031107).

The management of AMs caused by enterovirus Is condueted with support thérapies for symptom control since there is no drug licensed for clinical use that Is effective against these pathogens. In contrast, BM treatment is condueted with antibiotics combined or not with anti-inflammatory drugs. Delays ln the administration of antibiotics and antiInflammatory drugs can hâve devastating conséquences for the patient affected by BM (development of neurosensory sequelae and even death). ln case of any suspicion of BM, empiric antibiotic therapy is Initiated ln order to obtain the results of CSF culture tests. Thus, the fast differential diagnosls of AM and BM could reduce the hospltalization time of patients affected by AM, treatment costs, patients’ exposure to the risk of nosocomial infections, and side effects of antibiotics and anti-inflammatories. Thus, accurate and rapid diagnosls of meningitis is crucial for decision making related to the appropriate therapeutic approach and should be timely for each form of meningitis.

MENINGITIS DIAGNOSIS

Currently, AM and BM are diagnosed based on clinical and laboratory findings, wherein the CSF examination is essential to confirm the meningitis diagnosis based on clinical sIg ns. The main cytochemlcal parameters of cerebrospinal fluid currently used In the differentlal diagnosls of these two types of menlngitis are the overall count and differentlal count of leukocytes, and the levels of total protein and glucose.

These parameters guide the physlcian’s decision to Inltlate empiric treatment until It has a final diagnosls based on the results of the bacterial culture or of the analysis of pathogen antigens. However, différentiation between BM and AM may be hampered by the variability of the parameters currently used for the diagnosls of these diseases (Negrini B, Kelleher K, Wald E. Cerebrospinal fluid flndlngs In aseptie versus bacterial menlngitis. Pediatrics 2000,105(2):316-319).

Currently, multl-parameter models based on combinations of clinical and laboratory parameters for definlng clinical diagnosls of menlngitis hâve been proposed ((Nigrovlc LE, Kuppermann N, Maliey R. Development and validation of a multivariable prédictive model to distingulsh bacterial from aseptie menlngitis in children in the post-Haemophilus Influenzae era. Pediatrics. 2002;110(4):712-9; Jaeger F, Leroy J, Duchêne F, Baty V, Baillet S, Estavoyer JM, et al. Validation of a diagnosls model for differentlating bacterial from viral menlngitis In Infants and children under 3.5 years of âge. Eur J Clin Mlcroblol Infect Dis. 2000; 19(6):418-21). No clinical or laboratory criteria consldered alone can differentlate bacterial menlngitis from viral menlngitis with high sensitivity and good speciflcity. Thus, several teams hâve proposed scoring système that consider combinations of clinical and laboratory parameters to define rules of clinical decision. However, these scoring Systems are difficult to transpose between different hospitals and the distribution of the obtained scores appears to be dispersed due to the variability of menlngitis clinical présentation.

The identification of menlngitis causatîve agent Is fondamental to aid In the cholce of the appropriate therapy and In régional epldemlologlcal tracking of the disease. CSF culture Is consldered the gold standard for the differentlal diagnosls of menlngitis. However, the resuit of the CSF culture Is time-consumlng, requiring the use of empiric therapy with broad-spectrum antibiotics, and in some cases, the administration of antibiotics to patients whose etiology proves to be virai shows In the confirmatory diagnosis. Smears of CSF stalned by Gram staining and the latex agglutination test are also used as auxiliary methods.

Desplte the high specificity, methods for detecting pathogens by molecular biology hâve their sensitivity affected by antibiotic therapy, can produce false positive results due to remnants of nucleic acids of pathogens présent In the cerebrospinal fluid after patient’s healing, and hardly apply to hospital laboratories lacking trained staff and adéquate Infrastructure. In Brazii, such methods hâve been used only for confirmatory diagnosls and epldemlologlcal studies.

Due to the difficulty ln distinguishing patients affected by BM from the one affected by AM during the emergency care, most authors recommend the initiation of antimicrobiai therapy ln ail patients affected by acute menlngitis as long as there 1s any doubt regarding the etiology of the disease until results of CSF bacterial cultures or of antigen identification confirming the pathogen presence are obtained (Tunkel A Hartman B, Kaplan S, Kaufman B, Roos K, Scheld W, Whitley R: Practice guidelines for the management of bacterial meningitis. Curr Opin infect Dis 2004, 39(3):1267-1284). If, on one hand, this recommendation allows to treat, ln a timely fashlon, most cases of BM, it ieads to the hospitalization and the use of empiric antibiotics almost systematically, but useless a posteriori, on patients affected by AM (Swingier G, Delport S, Hussey G: An audit of the use of antibiotics ln presumed virai meningitis in chlldren. Pediatr infect Dis J 1994, 13:1107-1110), which increases the risk of nosocomial Infections and undesirable effects associated with antibiotic use, and Increases conslderably the treatment cost (Parasuraman T, Frenla K, Romero J: Enterovlrai meningitis. Cost of lllness and considérations for the économie évaluation of potential thérapies. 2001;19(1):3-12). Furthermore, the adjunctive therapy with anti-inflammatory (dexamethasone), as recommended in some BM cases, can aggravate the situation of patients affected by viral meningitis.

The described scénario makes it clear that the fast and accurate difierential dlagnosis of meningitis Is currently a technical demand unmet by the available technological approaches, motivated both by the need to improve the effectlveness of patients' management and by the possibility of reducing the costs of the treatment.

The sclentiflc community has conducted research for identifying new blomarkers for the diagnosls of meningitis, for example, assessing the potential of cytokines and chemokines as markers for différentiel dlagnosis between bacterial and viral meningitis and disease prognosis. However, the évaluation of these blomarkers in their potential diagnosis potential différa between the studies, generating conflicting results. ln most studies carried out so far, the concentration of some pro-lnflammatory cytokines is conslderably increased in the CSF or the sérum of patients affected by bacterial meningitis when compared with the ones affected by viral meningitis. However, the évaluation as a diagnostic marker of each cytokine différa between the studies. For example, Vâzquez et al. (Vâzquez JA, Adducci MeC, Coll C, Godoy Monzôn D, Iseraon KV. Acute meningitis prognosis using cerebrospinal fluid interieukln-6 ievels. J Emerg Med. 2012;43(2):322-7.) demonstrated that IL-6 Is a good marker for the diagnosis and prognosis of bacterial meningitis, wherein concentrations of this Interieukin above 35-40 pg/mL would be able to differentlate this disease from virai meningitis or from heaithy subjects with 100% sensitivity and 95% specificlty. However, other authors concluded that iL-6 should not be used as a marker differential of meningitis due to overtaps of this interieukin concentration ranges In the groups of patients affected by bacterial meningitis, by viral meningitis or without It (Pinto Junior VL, Rebelo MC, Gomes RN, Assis EF, Castro-Faria-Neto HC, Bôia MN. IL-6 and IL-8 In cerebrospinal fluid from patients with aseptie meningitis and bacterial meningitis: their potential rôle as a marker for differential diagnosis. Braz J Infect Dis. 2011 ;15(2):156-8., e Mukal AO, Krebs VL, Bertoli CJ, Okay TS. TNF-alpha and IL-6 In the diagnosis of bacterial and aseptie meningitis in children. Pediatr Neurol. 2006;34(1 ):25-9). For examples, some patients affected by menlngococcal meningitis exhibit IL-6 concentrations below 20 pg/mL, while some patients affected by viral meningitis exhibit IL6 concentrations above 50 pg/mL (Mukal AO et al.). These results contradict the cutoff point proposed by Vâzquez and colleagues for the différentiation from cases of bacterial meningitis based on IL-6 dosage.

The diagnostic potential of some blomarkers comprising the qualitative prédictive method proposed by the instant invention has been pointed out by the prior art. High concentrations of the complément C3 fraction In the CSF of patients affected by bacterial meningitis were reported when compared with patients affected by viral meningitis and controls without infection in the CNS (Stahel P, Nadal D, Pfister H, Paradisls p, Bamum S. Complément C3 and factor B cerebrospinal fluid concentrations in bacterial and aseptie meningitis. Ther., 89:1231997, 349.1886-1887; Goonetilieke U, Scarborough M, Ward S, Hussain S, Kadioglu A, Gordon S. Death Is associated with complément C3 déplétion In cerebrospinal fluid of patients with pneumococcal meningitis. mBio 2012, 3(2): e00272-

11). Moreover, patent N°. US 5.778.895 provides a method for the differential diagnosis of bacterial meningitis based on the measure of the levels of C3 and B fractions of the complément in patients* cerebrospinal fluid.

Several studies further demonstrate the existence of increased leveis of C-reactive protein In the CSF and sérum of patients affected by bacterial meningitis when compared with patients affected by viral meningitis or control subjects (Nathan BR, Scheld WM. The potential rôles of C-reactive protein and procalcîtonin concentrations in the sérum and cerebrospinal fluid In the diagnosis of bacterial meningitis. Curr Clin Top Infect Dis. 2002;22:155-65; Donald PR, Strachan AF, Schoeman JF, De Beer FC. Cerebrospinal fluid C-reactive protein In Infective meningitis in childhood. J Lab Clin Med. 1985;106(4):424-7; BenGershôm E, Briggeman-Mol GJ, de Zegher F. Cerebrospinal fluid C-reactive protein In meningitis: diagnostic value and pathophysiology. Eur J Pediatr. 1986;145(4):246-9: Pemde HK, Harish K, Thawranl YP, Shrivastava S, Belapurkar KM. Creactîve protein in childhood menlngitides. Indian J Pediatr. 1996;63(1 ):73-7).

Furthermore, sclentific literature discloses that the concentration of Apolipoprotein A-l in the cerebrospinal fluid of patients affected by meningitis was found to be increased in the acute phase of the disease, retuming to basal concentrations in the convalescent phase, while the level of protein in other neurologlcal disorders remained unchanged when compared with the group of control patients (Apo A-l and apo E concentrations In cerebrospinal fluids of patients with acute meningitis. Ann Clin Biochem. 1998 May;35 (Pt

3):408-14).

However, as observed in the prior art known by the Inventors, there Is no indication of the use of protein marfcers to differentiate between forms of the disease. Therefore, the prior art iacks accurate and efficient means for the differential diagnosls of viral, pneumococcal and menlngococcal meningitis.

The Invention, which will be described ln details below, provides a complote and systematic solution for the diagnosls of meningitis and détermination of its etiology, in a single test More specifically, the Invention herein discloses a qualitative prédictive method based on the combined détection and sequence analysis of the presence/absence of at least three out of four spécifie biomarkers, capable of differentiating between patients affected by viral meningitis from the ones affected by bacterial meningitis, further determining the etiology of the meningitis, whether it Is bacterial, menlngococcal or pneumococcal.

SUMMARY OF THE INVENTION

The instant Invention relates to a qualitative prédictive method , to a method and kit applied to the eariy differential diagnosls of the most prévalent forms of bacterial and viral meningitis, enabling to detect and dlstinguish the different forms of meningitis, which is a prerequisite for the timely application of the appropriate therapeutic approach capable of reducing the mortality and morbidity associated with malignant forms of this disease.

ln general terms, the methodology of the instant Invention employs a qualitative prédictive method based on the combined détection and sequence analysis of spécifie biomarker protelns, represented by the inflammatory response proteins of the human host présent in cerebrospinal fluid. The application of the prédictive method enables to differentiate patients affected by viral meningitis from the ones affected by bacterial meningitis, also determining the etiology of bacterial meningitis, whether pneumococcal or menlngococcal. The qualitative prédictive method was developed on the basis of proteomic studies - by two-dimensional electrophoresis (2D-PAGE) and mass spectrometry (MS) - of the CSF of patients affected by meningitis. The comparison of CSF protein profiles ln the groups ‘viral meningitis, menlngococcal meningitis, pneumococcal meningitis’ and “control was carried out by analyzing the qualitative différences between these profiles. For each etiology of meningitis and for the group of control subjects the respective intersection subsets -consisting of spots generated by two-dimensional electrophoresis présent ln ail samples of a given group, and union sets -consisting of spots observed In at least one sample of a given group, were formed. The comparison between the Intersection subset of the group of patients affected by each studled menlngitis etiology with the union set of the group of patients affected by other menlngitis etiology or with control subjects resulted ln distinctive spots that were found only ln the Intersection subset of each meningitis etiology. The comparative study was made possible by the composition of a matrix of presence (1) or absence (0) of ail the proteins Identified by mass spectrometry ln each menlngitis or control group. n this matrix, proteins were classified as présent or absent for each Intersection subset and union set of menlngitis étiologies or controls. This matrix has ultimately enabled the sélection of distinctive proteins that were selected for the composition of the biomarker panel of the qualitative prédictive method, namely apoiipoprotein A-l, C3 fraction of the complément, C-reactive protein and klninogen.

The qualitative prédictive method Is based on the combined détection and sequence analysis of the presence/absence of at least three out of the four selected spécifie biomarkers, capable of differentiating between patients affected by viral menlngitis from the ones affected by bacterial meningitis, further determining the etiology of the menlngitis, whether It Is bacterial, menlngococcai or pneumococcal.

The qualitative prédictive method consists of three nodes, each one of the nodes being related to the test for detecting at least one out of the four spécifie protein biomarkers. The first node, represented by the test for the presence of apoiipoprotein A-l, allows the distinction between group of patients affected by meningitis from patients without Infection ln the central nervous system or affected by viral meningitis. The second node of the qualitative prédictive method, represented by the test for the presence of C-reactive protein and/or complément C-3 fraction, allows the distinction between the group of patients affected by bacterial meningitis from patients affected by viral menlngitis. The third node of the prédictive method, represented by the test for the presence of Klninogen, allows the distinction between the group of patients affected by menlngococcai menlngitis from patients affected by pneumococcal meningitis.

The Instant Invention further describes a method for the differential diagnosis of menlngitis that comprises the détection, by means of Immunoassay of biomarkers Apoiipoprotein A-l; C-reactive protein and/or complément C-3 fraction in the CSF of patients suspected of having meningitis and sequence analysis of the results, according to the first, second and third nodes of the qualitative prédictive method. After the analysis of the third node, the lab technician will be able to détermine the diagnosis of menlngitis presence and etiology, according to the most prévalent causes: viral menlngitis, menlngococcai meningitis and pneumococcal meningitis.

The method of the Instant invention, based on the combined détection and sequential analysis of the presence/absence of spécifie biomarkers, can be also incorporated Into a diagnostic immonoassay kit containing ligands to biomarker proteins, such as antibodies or aptamers, adapted to a iab évaluation method that can include ELISA (Enzyme Llnked

Immunosorbent Assay), chromatography, turbidimetry, capillary electrophoresis, inter alia.

Said biomarker protein ligand can be represented by antibodies exhibiting the following characteristics:

- primary antibodies of the IgG or IgM types with spécifie affinity against epîtopes of each of the four blomarkers and produced In rabbits, goats, among other animais; and

- secondary antibodies of the IgG type with spécifie affinity against epitopes of the heavy chains of IgG or IgM from rabbits, goats or other animais, according to the source and type of the primary antibody used.

The primary and secondary antibodies are conjugated to the enzyme, or biotin or fluorophore. As for signal détection, in the case of enzyme-llnked antibody, a substrate that changes color after being modified by the enzyme Is used; in the case of biotin-linked antibodies, streptavidin conjugated to the enzyme and the substrate for color production is used; In the case of fluorophore-linked antibodies, détection occurs by light emitted in response to excitation at the appropriate waveiength.

Primary or secondary antibodies coupled to gold or latex particles can also be used for the Visual détection of the resuit.

Altematively, said ligands to biomarkers proteins may be represented by aptamers, for example, of single stranded DNA oligonucleotides which, In physlologlcal conditions of pH, salinity and température, curi assumlng three-dimensional conformations presenting a complementary binding sites with tertiary structure régions of each of the four biomarkers. The détection of aptamers bound to biomarkers can be performed on the basis of their capillary electrophoresis migration properties, or biotin or a fluorochrome can be coupled to the aptamers for détection as described above for the antibodies.

Since the Instant Invention is based on the identification of host Immune response proteins rather than pathogen proteins, even in the case of pathogen éradication, the host inflammatory reaction is maintained In the course of the disease, Increasing the time window for the differential diagnosis of menlngitis through the détection of biomarkers of immune response, which is another advantage associated with the technology. Furthermore, any remnants of nucleic acids of pathogens présent in the cerebrosplnal fluid after patient's healing, which can cause false positive results in molecular biology methods, do not affect the results of the présent invention.

The use of the prédictive method, of the method and kit described herein may decisively contribute to the fast diagnosis of the menlngitis presence and etiology early in the course of the disease in a patient, allowing to make a conscious decision of the appropriate treatment, avoiding indiscriminate hospitalization and antibiotic therapy, besldes reducing ίο treatment costs.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Venn diagram for the distribution of spots in the interception subset of pneumococcal meningitis. The figure discloses the only two spots of the interception subset of the group of patients affected by pneumococcal meningitis after the comparative analysis between the intersection subset of pneumococcal meningitis and the union sets with other étiologies of meningitis and the control group.

Figure 2: Venn diagram for the distribution of spots In the interception subset of meningococcal meningitis. The figure discloses the only five spots of the interception subset of the group of patients affected by meningococcal meningitis after the comparative analysis between the intersection subset of meningococcal meningitis and the union sets with other étiologies of meningitis and the control group.

Figure 3: Venn diagram for the distribution of spots in the interception subset of viral meningitis. The figure discloses the only four spots of the Interception subset of the group of patients affected by virai meningitis after the comparative analysis between the Intersection subset of viral meningitis and the union sets with other etioiogies of meningitis and the control group.

Figure 4: Qualitative prédictive model of meningitis protein biomarkers. The figure depicts the structure of the qualitative prédictive model, Identifying the employed biomarkers and their three analysis nodes, which represent the sequential form for biomarker détection readtng.

Figure 5: Prévision of subcellular localization of Identified proteins that constitute union sets and Intersection subsets of meningitis. A) prédiction of the cellular localization of proteins constituting the union sets of meningitis (n = 131); B) prédiction of the cellular location of proteins constituting the Intersection subsets of meningitis (n = 37).

DETAILED DESCRIPTION OFTHE INVENTION

The instant invention relates to a qualitative prédictive method, to a method and kit applied to the early differential diagnosis of the most prévalent forms of bacterlal and viral meningitis, enabling to detect and distingulsh the different forms of meningitis, In the early phase of the disease. In general terms, the methodology of the Invention employs a prédictive method based on the combined détection and sequence analysis of at least three out of four spécifie biomarker proteins, represented by the inflammatory response proteins of the human host présent In cerebrospinal fluid. The application of the prédictive method enables to differentiate patients affected by viral meningitis from the ones affected by bacterlal meningitis, also determining the etiology of bacterlal meningitis, whether pneumococcal or meningococcal.

The four biomarkers used ln the qualitative prédictive method include: Apolipoprotein A-l,

C-reactlve protein, Complément C-3 fraction and Kininogen.

The qualitative prédictive method consists of three nodes, each one of the nodes being related to the test of at least one out of the four spécifie protein biomarkers. The first node, represented by the test for the presence of apolipoprotein A-l, allows the distinction between group of patients affected by meningitis from patients without Infection in the central nervous system or affected by viral meningitis. The second node of the qualitative prédictive method, represented by the test for the presence of C-reactive protein and/or complément C-3 fraction, allows the distinction between the group of patients affected by bacterial meningitis from patients affected by viral meningitis. The third node of the prédictive method, represented by the test for the presence of Kininogen, allows the distinction between the group of patients affected by meningococcai meningitis from patients affected by pneumococcal meningitis.

More specificaîly, positivity for Apolipoprotein A-l indicates the condition Meningitis, while the négative resuit in the first node of the qualitative prédictive method indicates absence of Infection into the centrai nervous system or viral meningitis. As for the second qualitative prédictive method, positivity for protein of complément C-3 fraction and/or C-reactive protein, combined with the positive resuit in the first node, indicates the condition bacterial meningitis; while negativity for complément C-3 fraction and/or Creactive protein, combined with positive resuit ln the first node, indicates the presence of viral meningitis. As for the third node of the qualitative prédictive method, positivity of kininogen protein, combined with positivity in the first and second nodes, Indicates the condition meningococcai meningitis; while a négative resuit in the analysis of the third node, combined with positivity in the first and second nodes, indicates the condition “pneumococcal meningitis.

Therefore, the issuance of the diagnosis about the presence and etiology of meningitis must be based on the following parameters:

(1) Viral meningitis: négative resuit ln the first node of the prédictive method, combined with a négative resuit in the second node of the prédictive method; or, positive resuit in the first node of the prédictive method, combined with a négative resuit ln the second node of the prédictive method.

(2) Bacterial meningitis, without etiology détermination: positive resuit ln the second node of the prédictive method, combined with a positive resuit ln the first node of the prédictive method.

(3) Meningococcai meningitis: positive resuit ln the third node of the prédictive method, combined with a positive resuit ln the first and second nodes of the prédictive method.

(4) Pneumococcal meningitis: négative resuit in the third node of the prédictive method, combined with a positive resuit In the first and second nodes of the prédictive method.

The method for the differential diagnosls of meningitis comprises the following steps:

(a) Incubation of the patients CSF with spécifie ligands for apolipoprotein A-l; C-reactive protein and/or complément C-3 fraction; kininogen;

(b) Combined détection of apolipoprotein A-i; C-reactive protein and/or complément C-3 fraction; kininogen in the patients CSF by immunoassay, (c) Détermination of the results using the qualitative prédictive method, by means of sequential analysis of the first, second and third nodes, wherein:

(i) a négative resuit In the first node of the prédictive method combined with a négative resuit In the second node of the prédictive method; or, positive resuit ln the first node of the prédictive method combined with a négative resuit ln the second node of the prédictive method indicates the condition “viral meningitis;

(ii) a positive resuit ln the second node of the prédictive method combined with a positive resuit in the first node of the prédictive method Indicates the condition of bacterial meningitis;

(iii) a positive resuit in the third node of the prédictive method combined with a positive resuit ln the first and second nodes of the prédictive method Indicates the condition of “meningococcai meningitis.

(iv) a négative resuit ln the third node of the prédictive method combined with a positive resuit ln the first and second nodes of the prédictive method Indicates the condition of pneumococcal meningitis.

The method of the Instant Invention, based on the combined détection and sequential analysis of the presence/absence of spécifie blomarkers, can be incorporated into a diagnostic immonoassay kït containing ligands to biomarker proteins, such as antibodies or aptamers, adapted to an lab évaluation method that can Include ELISA (Enzyme Llnked Immunosorbent Assay), chromatography, turbidimetry, capillary electrophoresls, Inter alia. ln order to achieve and understand In details the subject matter In which the aforementioned characteristics, advantages and objectives of the Invention, as well as other ones that will become clear, more particular descriptions of the Invention are illustrated ln the following Examples. However, the Examples illustrate prefeaed embodiments of the Invention and, thus, should not be considered limiting in their scope.

EXAMPLE 1 CASUISTRY

For the comparative analysis of the CSF proteome through two-dimensional gels, 18 patients affected by pneumococcal meningitis, meningococcal meningitis or viral 5 meningitis and six control Individuals were selected (Table 1), ali undergolng CSF lumbar puncture. Immediately after CSF collection, the samples were centrifuged for cell séparation and supematants were frozen at -20 ^eC, and subsequently at -80 ’C until analyzed.

_____________TABLE1: CHARACTER1ZATION OFTHE POPULATION UNDER STUDY

SOCIO-DEMOGRAPHICAL LABORATORY CLINICAL DATA

Age

Gen der

H. IJPII

H.GT

Clinical slgn

Fever

Meningeal signs

Leukocyte s

% PMN

Proteins

Glucos e

Culture

Gram

Latex

Patients

PM

1

9y

M

x

vomit

(+)

(+)

5,600

80

441

71

(+)

N/A

N/A

2

22y

M

x

N/A

N/A

N/A

1,438

90

185

26

N/A

(+)

N/A

3

13y

M

x

N/A

N/A

N/A

4,400

93

186

20

(+)

(+)

(+)

4

23y

M

x

N/A

N/A

N/A

645

95

335

2.5

(+)

(+)

N/A

5

36y

M

x

N/A

N/A

N/A

960

87

474

1.5

(+)

(+)

N/A

6

2y

F

x

vomit; headac he

(+)

(+)

240

92

110

1

(+)

(+)

(+)

MM

1

13y

F

x

vomit

(+)

(+)

72

5

66

41

N/A

(+)

N/A

2

5m

F

x

vomit

(+)

(+)

2,560

44

187

3

(+)

(+)

(+)

3

12y

F

x

vomit

(+)

(+)

19,500

96

461

0

(+)

N/A

N/A

4

5y

M

x

vomit

(+)

(+)

6,800

95

202

53

N/A

N/A

(+)

5

8y

F

X

N/A

N/A

N/A

13,500

94

80

2

(-)

(+)

(-)

6

ny

F

x

vomit

(+)

(+)

120

94

281

48

(+)

(+)

N/A

SOCIO-DEMOGRAPHICAL

DATA

LABORATORY CLINICAL DATA

Age

Gen der

H. IJPII

H. GT

Clinical slgn

Fever

Menlngeal signs

Leukocyte s

% PMN

Proteins

GIucos e

Culture

Gram

Latex

Patients

VM

1

4y

F

X

vomit

(+)

(+)

500

30

31

49

N/A

(-)

(-)

2

4y

M

x

(-)

(+)

(-)

2

8

18

51

N/A

(-)

(-)

3

6y

F

x

vomit; headac he

(+)

(+)

44

56

36

43

(-)

(-)

(-)

Age

Gen der

H. IJPII

H. GT

Clinical slgn

Fever

Meningeal signs

Leukocyte s

% PMN

Proteins

GIucos e

Culture

Gram

Latex

4

8y

M

x

vomit; headac he

(+)

(+)

33

73

38

50

N/A

(-)

(-)

5

3y

M

x

malaise

(-)

(+)

53

10

29

47

N/A

N/A

(-)

6

11m

F

x

vomit

(+)

N/A

21

60

32

57

(-)

(-)

N/A

CTRL

1

7y

M

x

vomit

(+)

(+)

6

54

35

72

N/A

N/A

N/A

2

3y

M

x

malaise

(+)

(-)

9

94

20

50

N/A

N/A

N/A

3

33y

M

x

N/A

N/A

N/A

2

80

30

40

(-)

N/A

N/A

SOCIO-DEMOGRAPHICAL LABORATORY CUNICAL DATA

	Age	Gen der	H. IJPII	H.GT	Cllnical slgn	Fever	Menlngeal slgns	Leukocyte s	% PMN	Proteins	Glucos e	Culture	Gram	Latex
Patients
4	2y	M	x		(-)	(-)	N/A	2	13	21	62	N/A	N/A	N/A
5	11m	F	x		vomit	(+)	(-)	1	7	26	58	(-)	(-)	N/A
6	5m	F	X		(-)	(+)	N/A	2	0	24	51	(-)	(-)	N/A

Where: PM Pneumococcal meningitis; MM: Meningococcal meningitis; VM: Viral meningitis; CTRL Control; H. IJPII: Hospital Infantil Joâo Paulo 11; H.GT: Hospital Giselda Trigueiro; y: years; m: months; M: male; F: female; (+): positive; (-): négative; N/A: ignored; Gram: Gram bacterioscopy; Leukocytes (cells/mm3); Proteins and Glucose: mg/dL; culture data, Gram and latex: in the CSF.

Moreover, the results of the CSF cytochemicai parameter tests were analyzed, namely: total and differential leukocyte count, total proteins and glucose of the group of patients constituting this study for comparison with data pre-established ln the scientific literature. The results of these tests were collected from the revlew of the patients* medical records. The confirmatory diagnosis of bacterial meningitis is made by detecting pathogens in CSF, or blood, through one or more of the following tests: culture, Gram bacterioscopy or latex agglutination. The diagnosis of viral meningitis was conducted by exclusion of the bacterial forms of meningitis when there was a clinical condition of meningitis with négative results for the culture, Gram bacterioscopy and latex agglutination, but with a leukocyte count slightly higher (> 50 cells), wherein protein levels may be slightly higher or normal (normal: 15 to 45mg/dL) and glucose levels may be normal or slightly lower (normal: 60mg/dL). The control group consisted of subjects without CNS Infection, systemic Infections, psychiatrie or neurodegenerative diseases checked for suspected meningitis, but whose disease was discarded by confirmatory diagnosis and by normal cytochemicai parameter values.

The évaluation of the total leukocyte count parameter showed significant différences between patients affected by bacterial meningitis compared with those affected by viral meningitis or controls and between the latter two. Médian values of leukocyte count found ln the CSF ln patients affected by pneumococcal meningitis (PM) and meningococcal meningitis (MM) (PM: 1,199 cells/mm3 and MM: 4,680 cells/mm3) were significantly higher than the ones found in patients affected by viral meningitis (VM) and controls (CTRL) (VM: 44.5 cells/mm3 and CTRL: 2 cells/mm3). However, overlaps of the leukocyte values were observed, for example, among patients affected by meningococcal meningitis and viral meningitis, which makes It Impossible to discriminate between these patients.

Protein concentration in the CSF is considered one of the most sensitive Indicators of pathology in the CNS. The results demonstrate significant différences between patients affected by bacterial meningitis (PM: 260.5mg/dL and MM: 194.5mg/dL) when compared with the ones affected by viral meningitis (VM: 31.5mg/dL) or controls (CTRL: 25mg/dL) (p < 0.01), but not between the two latter, given that both show protein concentration values within the normal values (18 to 58mg/dL). Although the total protein levels in CSF of normal subjects are slightly different from the ones observed In patients affected by viral meningitis, the médian value of the latter is within the range considered normal. Moreover, overlaps of total protein values were observed among patients affected by pneumococcal meningitis and meningococcal meningitis, which makes It Impossible to discriminate between these patients.

The analyses conducted for the differential count parameters of polymorphonuclear leukocytes (PMN) and total glucose in the CSF revealed no statistically significant différence between the groups under study. Médian values found for leukocyte differential count of PMN leukocytes In patients affected by bacterial meningitis (PM: 91% and MM: 94%) were higher than the ones found In patients affected by viral meningitis (VM: 43%) and controls (CTRL: 54%), but the différences were not statisticaliy significant (p > 0.05). Médian values for CSF glucose found for patients affected by pneumococcal meningitis (22.5 mg/dL) were lower than the ones found for patients affected by menlngococcal meningitis (41 mg/dL). Ail the patients affected by virai meningitis and the controls exhibit glucose concentrations within normal values (> 40 mg/dL).

Thus, ln this smaii sample none of the classic cytochemicai parameters, when analyzed separately, proved to be sufficiently sensitive and spécifie for the differential diagnosis of pneumococcal, menlngococcal or viral meningitis.

EXAMPLE 2

SAMPLE PROCESSING

CSF contains small molécules, salts, peptides, proteins and enzymes that play critical rôles ln many physiological processes, ln order to improve the resolution of twodimensional electrophoresis most of the sait content of the CSF must be removed to elimlnate possible Interférences.

CSF samples in raw state from patients affected by viral meningitis and controls exhibited a much lower protein concentration when compared with the protein concentrations of CSF samples from patients affected by bacterial meningitis. It Is important to stress that more than 15% of the total protein content of the CSF corresponds to albumin and 15% or more, to immunoglobulins. Therefore, déplétion of abundant proteins is essential to detect less représentative proteins in the CSF. ln order to obtain a sufficient amount of protein for the subséquent steps of the method of the instant invention, twlce the volume of CSF ln the raw state was used for samples from patients affected by viral meningitis and controls compared with patients affected by bacterial meningitis.

Six sampies of each etiology (pneumococcal, meningococcal or viral meningitis) and six samples of control subjects were concentrated from 30 to 35 times for bacterial samples, from 40 to 45 times for viral sampies and controls by acetone précipitation. Albumin and Immunoglobulins were depleted using columns prepared with a mixture of antl-HSA Sepharose and protein G Sepharose of high performance. Then, the proteins were precipitated again with acetone for concentration and desalting. ln this step, ali the sampies were concentrated of approximately 20 times. Précipitâtes were resuspended in a rehydration buffer (IEF) and protein concentrations were determined by Bradford method in a raw state and, after depietion, using standard with bovine sérum albumin. ln order to verify the quality of the samples ln raw state and verify the efficiency of aibumln and IgG depietion, the samples were separated by unidimenslonal electrophoresis ln 12% polyacrylamide denaturing gel (SDS-PAGE) and dyed with sllver nitrate.

EXAMPLE 3

PROTEIN SEPARATION BY TWO-DIMENSIONAL ELECTROPHORESIS

Twelve two-dimensional geis containing 0.5 pg of CSF proteins (representing samples from six subjects and their technical duplicates) and control group were manufactured for each of menlngitis etiology under study, for a total of 48 geis. The fixed amount of 0.5 pg protein was prepared for Isoelectric focusing using 7 cm-IPG strips, pH 3-10NL In PROTEAN IEF Ceii equlpment (BioRad, PA, USA) at 20*C, 50mA/gel under the conditions described below: passive rehydration for 4 hours, 20’C; Step 1:50 V, 12 hours; Step 2: 500 V, 30 minutes; Step 3:1,000 V, 30 minutes; Step 4:4,000 V, 1 hour; and Step 5: 4,000 V, 16,000 V-hr. After conducting the équilibration steps (réduction and alkylation of proteins), the strips were subjected to the process of protein séparation by the second dimension, conducted through PAGE in a 12% polyacrylamide gel, and the gels were dyed with sllver nitrate.

2DE gel images of patients’ samples constituting the group of pneumococcal, meningococcal and viral menlngitis and contrats were analyzed by the software PDQuest 7.3.0 (Bio-Rad, PA, EUA). The scanned images of the two-dimensional gels were used for qualitative analysis of the CSF proteome, that Is, the comparison between CSF protein profites from patients with pneumococcal menlngitis, meningococcal menlngitis and viral menlngitis, and the comparison between these and the protein profiles obtained from CSF of control subjects.

For each menlngitis etiology and for the group of control subjects the respective intersection subsets -consisting of spots présent in ail the 12 two-dimensional gels containing the samples from the six subjects of a given group, and union sets -consisting of the spots observed In at least one out of the 12 two-dimensional gels with samples from the six subjects of each group, as shown In Table 2.

TABLE 2: NUMBER OF SPOTS THAT FORM INTERCEPTION SUBSETS AND UNION

SETS OF THE CSF OF PATIENTS AFFECTED BY MENINGITIS AND CONTROLS
	PM	MM	VM	CTRL
Union (U)	170	105	75	121
Interception (Π)	23	33	27	17

Where: PM: pneumococcal menlngitis; MM: meningococcal menlngitis;

VM: viral menlngitis; CTRL: contrat.

The comparison between the intersection subset of the group of patients affected by each studied menlngitis etiology with the union set of the group of patients affected by other menlngitis etiology or with control subjects resulted in distinctive spots that were found only ln the Intersection subset of each meningitis etiology (Table 3).

TABLE 3: SPOTS DEFINED IN THE COMPARATIVE ANALYSIS BETWEEN THE

DIFFERENT MENINGITIS ETIOLOGIES AND THE CONTROL GROUP

UNION SETS

CTRL (121 spots)

PM (170 spots) MM (105 spots) VM (75 spots)

INTERCEPTI ON SUBSETS	Comm on	Distinct! ve	Comm on	Dlstlnctl ve	Comm on	Dlstlnctl ve	Comm on	Dlstlnctl ve
PM (23 spots)	20	3	—	—	19	4	15	8
MM (33 spots)	12	21	24	9	—	—	19	14
VM (27 spots)	20	7	16	11	16	11

Where: PM: pneumococcal meningitis; MM: menlngococcai meningitis; VM: virai meningitis; CTRL: control.

The results of the comparative analyses between the spots of the Interception subset of patients affected by pneumococcal menlngitis and the spots of the union sets of the control groups and of other meningitis etiology resulted ln single spot of intersection subset of patients affected by pneumococcal menlngitis, as shown ln Figure 1. Only two spots of the Interception subset of the group of patients affected by pneumococcal meningitis were found after the comparative analysis between the Intersection subset of pneumococcal meningitis and the union sets with other étiologies of meningitis and the control group.

The results of the comparative analyses between the spots of the Interception subset of patients affected by menlngococcai meningitis and the spots of the union sets of the control groups and of other menlngitis etiology resulted ln single spot of intersection subset of patients affected by menlngococcai meningitis, as shown ln Figure 2. Five single spots of the Interception subset of the group of patients affected by menlngococcai menlngitis were found after the comparative analysis between the intersection subset of menlngococcai meningitis and the union sets with other étiologies of meningitis and the control group.

The results of the comparative analyses between the spots of the interception subset of patients affected by viral meningitis and the spots of the union sets of the control groups and of other meningitis etiology resulted ln single spot of intersection subset of patients affected by viral meningitis, as shown in Figure 3. Four single spots of the interception subset of the group of patients affected by virai meningitis were found after the comparative analysis between the Intersection subset of virai meningitis and the union sets with other étiologies of meningitis and the control group.

Thus, the single spots found ln the Intersection subsets of each meningitis etiology were prioritized for mass spectrometry Identification for subséquent qualitative composition of the meningitis prédictive method.

EXAMPLE 4

IDENTIFICATION OF DIFFERENTIALLY EXPRESSED PROTEINS BY MASS SPECTROMETRY

The comparative analysis of the two-dimensional gels with CSF proteins of patients affected by meningitis and controls allowed the sélection of the protein spots corresponding to potential biomarkers for each meningitis etiology.

Two-dimensional gels were prepared using pools of three samples from patients affected by pneumococcal meningitis and meningococcai meningitis, or viral meningitis, respectively. For each meningitis etiology, three two-dimensional gels of samples containing 10, 40 and 100pg of the protein were prepared In order to Increase the probability of success in the Identification of distinct proteins with possible overlaps ln the proteomlc map. Ail the proteins Identified ln each produced gel were consîdered in the Instant Invention, ln this step, gels were dyed by colloïdal Coomassie compatible with mass spectrometry.

Out of the 695 protein spots submitted to mass spectrometry, 553 (80%) were Identified and correspond to 131 different protein. Of these, 37 proteins correspond to spots belonglng to the Interception subset of pneumococcal, meningococcai or viral meningitis. ln addition to the spots belonglng to the Intersection subsets of the group of patients affected by meningitis and absent In the union set of other etloiogles of the disease or control subjects, ail spots of the union sets of each meningitis etiology were sought to be identified.

Most of single spots of the interception subsets of each meningitis etiology was discharged as potential biomarker, after Identification by mass spectrometry, for being spots whose identified protein was also found ln other spots of the two-dimensional gels. The same protein may be présent In more than one spot, possibly due to the occurrence of post-translatlonal modifications, such as carbonylation, phosphorylation and méthylation, and in vivo proteolysls, via protéasome and lysosome.

A matrix of presence (1) or absence (0) consisting of the list of ail the proteins corresponding to the Intersection spots belonglng to the Interception subsets Identified by mass spectrometry was built (Table 4, Table 5 and Table 6). ln this matrix, proteins were classified as présent or absent for each Intersection subset and union set of meningitis etiologles or controls. Therefore, this matrix allowed the sélection of the five spots, that, together with the only two spots belonglng to the subset Intersection of menlngococcal menlngitis, correspond to the four proteins used to build the qualitative prédictive method for menlngitis diagnosls.

TABLE 4: MATRIX OF THE SPOTS OF THE INTERCEPTION SUBSETS OF THE GROUP OF PATIENTS AFFECTED BY PNEUMOCOCCAL

MENINGITIS

Spots Revlewed after protein identification

Spot	Proteins	H MM	dvm	U MM	UVM	UCtri	g/	ΠΜΜ	îivm	U MM	UVM	UCtri
p.1	α-2-HS-glycoprotein	0	0	0	0	1	112910	0	0	1	1	1
p.2		0	0	0	0	1	6137432	1	1		1	1
	α-1-antitrypsîn									1
p.3	α-1-antitrypsin	0	0	1	1	1	6137432	1	1	1	1	1
p.4	α-1-antitrypsin	0	0	1	1	1	6137432	1	1	1	1	1
p.5	α-1-antitrypsin	0	0	0	0	0	6137432	1	1	1	1	1
p.6	a-1 -HS-glycoprotein	1	0	1	1	1	223069	1	0	1	0	1
p.7	α-1-antitrypsin	0	0	1	1	1	6137432	1	1	1	1	1
p.8		0	0	1	1	1	386789	1	1	1	1	1
Hemopexin
p.9	Hemopexin	0	0	1	1	1	386789	1	1	1	1	1
p.10	Hemopexin	0	0	1	1	1	386789	1	1	1	1	1
p.11	Albumîn	0	0	1	1	1	28592	1	0	1	1	1
p.12	not identified	0	0	1	1	1	-	0	0	1	1	1
p.13	Transfemn	0	0	1	1	1	115394517	1	1	1	1	1
p.14	Transferrin	0	0	1	1	1	115394517	1	1	1	1	1
p.15	Transfemn	0	0	1	1	1	115394517	1	1	1	1	1
p.16	C3 Component	1	0	1	0	1	179665	1	0	1	0	1
p.17	Haptoglobin	0	0	1	1	1	306882	1	0	1	1	1
p.18	Haptoglobin	0	0	1	1	1	306882	1	0	1	1	1

Spots Reviewed after protein Identification

Spot	Proteins	AMM	avm	U MM	UVM	U Ctrl	gi	AMM	AVM	U MM	UVM	U Ctrl
p.19	Haptoglobin	0	0	1	1	1	306882	1	0	1	1	1
p.20	C-reactive	1	0	1	0	1	1942435	1	0	1	0	1
p.21	Apolipoprotein Al	1	0	1	1	0	90108664	1	0	1	1	0
p.22	Transthyretin	1	0	1	0	1	17942890	1	1	1	1	1
p.23	Transthyretin	0	0	0	0	0	17942890	1	1	1	1	1

Where: Π: interception subset; U: union set; MM: meningococcal meningitis; VM: viral meningitis; Ctrl: control.

TABLE 5: MATRIX OF THE SPOTS OF THE INTERCEPTION SUBSETS OF THE GROUP OF PATIENTS AFFECTED BY MENINGOCOCCAL

MENINGITIS ______________________________________________

Spots Reviewed after protein identification

Spot	Proteins	A PM	AVM	U PM	UVM	U Ctrl	gi	n pm	avm	U PM	UVM	U Ctrl
m.1	Ceruloplasmin	0	0	1	0	1	1620909	0	0	0	1	1
m.2		0	0	1	0	0	69990	0	0	1	1	1
α-1-B-Glycoprotein			1	1	1	69990			1	1
m.3	α-1-B-Glycoprotein	0	0	0	0	1
m.4	chain β T cell receptor	0	0	1	0	0	78101492	0	0	1	1	0
m.5	Hemopexin	0	0	1	0	1	386789	1	1	1	1	1
m.6	Hemopexin	0	0	1	1	1	386789	1	1	1	1	1
m.7	Hemopexin	0	0	0	1	1	386789	1	1	1	1	1
m.8	Hemopexin	0	0	1	1	1	386789	1	1	1	1	1

Spots Reviewed after protein identification

Spot	Proteins	nPM	hvm	U PM	UVM	UCtrl	fff	HPM	hvm	U PM	UVM	UCtrl
m.9	Chain β T cell receptor	1	1	1	1	0	78101492	1	1	1	1	0
m.10	Transfemn	1	1	1	1	1	115394517	1	1	1	1	1
m.11	Transferrin	1	1	1	1	0	115394517	1	1	1	1	1
m.12	Transfemn	1	1	1	1	1	115394517	1	1	1	1	1
m.13	Transferrin	1	1	1	1	1	115394517	1	1	1	1	1
m.14	Transfemn	1	0	1	0	0	115394517	1	1	1	1	1
m.15	a-1 -antichymotrypsin	1	0	1	0	1	177933	1	0	1	0	1
m.16	α-1-antitrypsin	1	1	1	1	0	6137432	1	1	1	1	1
m.17	Kininogen	0	0	0	0	0	4504893	0	0	0	0	0
m.18	Vitamin D binding protein	1	1	1	1	0	181482	1	1	1	1	1
m.19	Vitamin D binding protein	1	0	1	0	0	181482	1	1	1	1	1
m.20	α-1-antitrypsln	0	0	0	0	0	6137432	1	1	1	1	1
m.21	α-1-antitrypsin	1	1	1	1	0	6137432	1	1	1	1	1
m.22	Acid α-1-Glycoprotein	0	0	0	0	0	112877	1	1	1	1	1
m.23	Acid α-1-Glycoprotein	1	0	1	0	0	112877	1	1	1	1	1
m.24	Haptoglobin	0	0	0	1	0	306882	1	0	1	1	1
m.25	C3 Component	0	0	0	0	0	179665	1	0	1	0	1
m.26	Ζη-α-2-glycoprotein	0	0	1	1	1	38026	0	0	1	1	1
m.27	Haptoglobin	1	0	1	1	0	306882	1	0	1	1	1
m.28	Haptoglobin	1	0	1	1	1	306882	1	0	1	1	1
m.29	C-reactive	0	0	1	0	0	1942435	0	0	1	0	1

Spots Reviewed after protein Identification

Spot	Proteins	APM	avm	U PM	UVM	UCtrl	gf	apm	avm	U PM	UVM	UCtrl
m.30	Apolipoprotein A-l	0	0	0	0	0	90108664	1	0	1	1	0
m.31	Apolipoprotein A-l	1	0	1	1	0	90108664	1	0	1	1	0
m.32	Transthyretin	0	1	0	1	0	17942890	1	1	1	1	1
m.33	Transthyretin	0	1	0	1	0	17942890	1	1	1	1	1

Where: A: interception subset; U: union set; PM: pneumococcal meningitis; VM: viral meningitis; Ctrl: control.

TABLE 6: MATRIX OF THE SPOTS OF THE INTERCEPTION SET OF THE GROUP OF PATIENTS AFFECTED BY VIRAL MENINGITIS

		Spots						Reviewed after protein identification
Spot	Proteins	APM	avm	U PM	UVM	UCtrl	g!	APM	AVM	U PM	UVM	UCtrl
v.1	not identifîed	0	0	0	0	0	-	0	0	0	0	0
v.2		0	0	0	1	0		0	0		1	0
not identifîed									0
v.3	not identifîed	0	0	0	0	0	-	0	0	0	0	0
v.4	not identifîed	0	0	1	1	1	*	0	0	1	1	1
not named product			1	1	1	22761380			1	1	1
v.5	0	0	0	0
v.6	Hemopexin	0	0	1	1	1	386789	1	1	1	1	1
v.7	not identifîed	0	0	1	1	1	-	0	0	1	1	1
v.8	Hemopexin	0	0	1	1	0	386789	1	1	1	1	1
v.9	not named product	0	0	1	1	1	22761380	0	0	1	1	1
v.10	Transferrin	0	0	0	1	1	110590597	1	1	1	1	1
v.11	Transferrin	0	0	1	1	1	110590597	1	1	1	1	1

Spots Revlewed after protein Identification

Spot	Proteins	nPM	hvm	U PM	UVM	U Ctri	gi	ΠΡΜ	hvm	U PM	UVM	U Ctri
v.12	Transferrin	0	0	1	1	1	110590597	1	1	1	1	1
v.13	Transferrin	0	0	1	1	1	110590597	1	1	1	1	1
v.14	not identified	0	0	1	1	1	-	0	0	1	1	1
v.15	Transferrin	0	0	1	1	1	110590597	1	1	1	1	1
v.16	Transferrin	0	0	1	0	1	110590597	1	1	1	1	1
v.17	α-1-antitrypsin	0	0	0	1	1	177831	1	1	1	1	1
v.18	not Identified	0	0	1	0	1	-	0	0	0	0	1
v.19	Vitamin D binding protein	0	0	1	0	1	181482	1	1	1	1	1
v.20	Vitamin D binding protein	0	0	1	0	1	181482	1	1	1	1	1
v.21	Acid α-1-Glycoprotein	0	0	0	0	1	112877	1	1	1	1	1
v.22	Transthyretin	0	0	1	0	1	17942890	1	1	1	1	1
v.23	Transthyretin	0	0	0	1	1	17942890	1	1	1	1	1
v.24	Prostaglandin D synthase	0	0	0	0	1	283806778	0	0	0	1	1
v.25	Prostaglandin D synthase	0	0	0	0	0	283806778	0	0	0	1	1
v.26	not identified	0	0	0	1	0	-	0	0	0	1	0
v.27	Transthyretin	0	0	0	0	0	17942890	1	1	1	1	1

Where: Π: interception subset; U: union set; PM: pneumococcal meningitis; VM: viral meningitis; Ctri: control.

EXAMPLE 5

SELECTION OF CANDIDATE BIOMARKERS FOR MENINGITIS DIFFERENTIAL DIAGNOSIS

Based on the analysis of the matrix (Example 4) of presence versus absence, the four selected proteins were combined to préparé a qualitative prédictive method capable of differentiating pneumococcal meningitis from meningococcal meningitis between each other and from viral meningitis or controls (Table 7 and Figure 4).

TABLE 7: MATRIX OF PRESENCE/ABSENCE OF THE PROTEINS SELECTED FOR

THE COMPOSITION OFTHE QUALITATIVE PREDICTIVE METHOD

DESCRIPTION	fll	n pm	H MM	hvm	U PM	U MM	UVM	UCtrl
Apoiipoproteln Al	90108664	1	1	0	1	1	1	0
C-reactive	1942435	1	1	0	1	1	0	1
C3 Component	179665	1	1	0	1	1	0	0
Kininogen	4504893	0	1	0	0	1	0	0

Where: Π: interception subset; U: union set; PM: pneumococcal meningitis; MM: meningococcal meningitis; VM: viral meningitis; CTRL: control.

The absence of apoiipoproteln A-l is associated with the absence of infection in the CNS (control group) or viral meningitis. The spots corresponding to apoiipoproteln A-l occur in the union set of the group of viral meningitis, but not in the union set of the control group, so that the absence of this protein can indicate any one of these two conditions, and its presence Implies the conditions of bacteria! or viral meningitis. The spot corresponding to C-reactive protein was Identified In the union set of the control group, rather than in the union set of the viral meningitis group. Although alone, the presence of C-reactive protein can Indicate the control condition, this can be excluded by the presence of apoiipoproteln A-l. Therefore, the C3 component of the complément system or the c-reactive was used, given that the absence of these proteins defines the condition of virai meningitis. Component C3 of the complément system characterizes the condition of bacterial meningitis without defining the etiological agent, wherein Its corresponding spot was Identified In the Interception subsets of the group affected by bacterial meningitis and not in the union set of the group affected by viral meningitis. For the définition of the etiological agent of bacterial meningitis the kininogen protein Is proposed . The presence of the kininogen protein is associated with meningococcal meningitis, while its absence Indicates pneumococcal meningitis (Figure 4). Kininogen protein was only found in the Interception subset of meningococcal meningitis and its corresponding spot does not belong to the union sets of nelther the other meningitis etiologles nor the control group.

Based on these results, the prédictive method described In Figure 4 was prepared, ln which the presence or absence of the proteins described above must be analyzes sequentlally.

EXAMPLE 6

PREDICTION OFTHE CELLULAR LOCALIZATION OF IDENTIFIED PROTEINS

Cellular locaiization of proteins identified by mass spectrometry was predicted by using their respective amino acid sequences obtained In the NCBI database, in the Fasta format These sequences were inputted ln the web version of the software SherLoc 2 (http://abi.inf.uni-tuebingen.de/Services/SherLoc2).

The results show that most of the proteins identified ln the union sets of meningitis were predicted as cytopiasmic (46%), or extracellular (45%), although they may be also nuclear (3%), of plasmic membrane (4%) or mitochondrial (2%). Furthermore, the identified proteins belonging to the Interception subset for meningitis were predicted only as extracelluiar (71%), or cytopiasmic (29%) (Figure 5).

One of the most distinct features of eukaryotic cells is the compartmentaiization of proteins. The locaiization of a protein is often an essential step to détermine Its function.

The occurrence of nuclear and mitochondrial proteins only in the meningitis union sets suggests that it is the case of proteins occasionally released into the extracellular environment due to the death of neurons, glia, or inflammatory ceils during the course of meningitis.

The fact that the intersection subsets mainly contain extracellular (and some cytopiasmic) protein reinforces the notion that the proteins evaluated for the sélection of meningitis biomarkers particlpate in the physiological processes of host response to bacterial infection.

Moreover, patients included in this study are from different âge groups (infants, children and adults) and their CSF samples were collected along the course of the disease. Another relevant factor of the instant Invention Is that samples from these patients corne from two hospitals of different réglons of the country (Southeast and Northeast) and, possibly represent different genetic backgrounds of the host Thus, every step of the aforementioned experimental design contribute to the reliabllity of the results found in the Instant Invention.

The Examples above and on the results generated from the comparative analysis of the CSF proteome of patients affected by pneumococcal, menlngococcal, viral meningitis and controls allowed identifying proteins spécifie of the host response to Infection by these pathogens. Specifically, among the Identified proteins, four out of these proteins are the subject of the instant invention as biomarkers for the differential diagnosis of malignant and benign meningitis. These biomarker proteins combined ln a prédictive method were capable of distinguishing patients affected by pneumococcal, meningococcal and viral meningitis from the subjects without Infection in the centrai nervous System. Sensltivity, specificlty and accuracy were 100% involving 24 patients (six in each group), tested ln du pi ica te technique.

According to the Example and discussion above, apolipoproteln A-l, C-reactive protein, complément C3 fraction and kininogen were the proteins selected as biomarkers for the differential dlagnosis of meningitis, since they were capable of distinguishing patients affected by pneumococcal, meningococcal and viral meningitis from control subjects 10 (subjects without Infection or psychiatrie or neurodegenerative disease ln the central nervous system).

Kits for the differential diagnosis of meningitis ln the form of EUSA tests, latex agglutination, Westem-biot, side chromatography, capiliary electrophoresis, Inter alia, are produced from antibodies or aptamers capable of recognlzing the biomarkers Identified ln 15 the Instant Invention.

Claims (12)

1. A prédictive method for the differential diagnosis of meningitis characterized by comprising the following steps:

(a) incubation of a patient’s CSF with spécifie ligands for the biomarker proteins apolipoprotein A-l; C-reactive protein and/or complément C-3 fraction; kininogen;

(b) combined détection of apolipoprotein A-l; C-reactive protein and/or complément C-3 fraction; kininogen in the patient’s CSF by immunoassay, (c) détermination of the results by means of sequential analysis of the first, second and third nodes, wherein:

(I) a négative resuit in the first node of the prédictive method combined with a négative resuit in the second node of the prédictive method; or, positive resuit in the first node of the prédictive method combined with a négative resuit in the second node of the prédictive method indicates the condition “viral meningitis;

(ii) a positive resuit in the second node of the prédictive method combined with a positive resuit in the first node of the prédictive method indicates the condition of “bacterial meningitis;

(iii) a positive resuit in the third node of the prédictive method combined with a positive resuit in the first and second nodes of the prédictive method indicates the condition of meningococcal meningitis;

(iv) a négative resuit in the third node of the prédictive method combined with a positive resuit in the first and second nodes of the prédictive method indicates the condition of “pneumococcal meningitis.

2. The method according to ciaim 1 characterized ln that the spécifie ligands are selected among antibodies or aptamers.

3. The method according to claim 2 characterized in that the antibodies binding to biomarker proteins are selected among:

(i) primary antibodies of the IgG or IgM types with spécifie affinity against epitopes of each of the four biomarker proteins and produced ln rabbits, goats, among other animais; and (ii) secondary antibodies of the IgG type with spécifie affinity against epitopes of the heavy chains of IgG or IgM from rabbits, goats or other animais, according to the source and type of the primary antibody used.

4. The method according to claim 1 characterized in that the aptamers binding to biomarker proteins are selected from the group consisting of single-stranded DNA oligonucleotides.

5. ln vitro method for the differential diagnosis of meningitis using the prédictive method as defined in any one of claims 1-3 characterized ln that the détermination of the presence and the absence of biomarker proteins selected among apoiipoprotein A-l, C-reactive protein, complément C3 fraction and kininogen is indicative of disease condition, where (i) a négative resuit in the first node of the prédictive model combined with a négative resuit in the second node of the prédictive model; or, positive resuit in the first node of the prédictive model combined with a négative resuit in the second node of the prédictive model indicates the condition viral meningitis;

(ii) a positive resuit in the second node of the prédictive model combined with a positive resuit in the first node of the prédictive mode! indicates the condition of •bacterial meningitis;

(iii) a positive resuit in the third node of the prédictive model combined with a positive resuit in the first and second nodes of the prédictive model indicates the condition of menlngococcai meningitis;

(iv) a négative resuit in the third node of the prédictive model combined with a positive resuit in the first and second nodes of the prédictive model indicates the condition of pneumococcal meningitis.

6. Biomarker proteins for use in a method of differential détermination of viral meningitis and bacterial meningitis characterized in that the proteins are selected among the group of apoiipoprotein A-l, C-reactive protein, complément C3 fraction and kininogen.

7. Biomarker proteins for use in a method of differential détermination of meningococcal meningitis and pneumococcal meningitis characterized ln that the proteins are selected among the group of apoiipoprotein A-l, C-reactive protein, complément C3 fraction and kininogen.

8. A kit for the differential diagnosis of meningitis for determining whether an Individual is affected by bacterial meningitis or viral meningitis, characterized in that the kit comprises biomarkers proteins selected from apoiipoprotein A-l, C-reactive protein, complément C3 fraction and kininogen; by spécifie ligands that bind to proteins; and by an instruction sheet containing the parameters to correiate the results necessary for differential identification of virai meningitis and bacterial meningitis.

9. The kit according to claim 8 characterized in that the differential détermination occurs between meningococcal meningitis and pneumococcal meningitis.

10. The kit according to claim 8 characterized In that the spécifie ligands are seiected from the group consisting of antibodies or aptamers, wherein said spécifie ligands are adapted to a iaboratory évaluation method, including an ELISA method, chromatography, turbidimetry, capiilary electrophoresis.

11. The kit according to claim 10 characterized in that the antibodies binding to biomarker proteins are selected among:

(i) primary antibodies of the IgG or IgM types with spécifie affinity against epitopes of each of the four biomarkers and produced ln rabbits, goats, among other animais; and

5 (ii) secondary antibodies of the IgG type with spécifie affinity against epitopes of the heavy chains of IgG or IgM from rabbits, goats or other animais, according to the source and type of the primary antibody used.

12. The kit according to claim 10 characterized in that the aptamers bind to the proteins are selected from the group consisting of single-stranded DNA 10 oligonucleotides.