WO2012007622A1 - Method for the differential diagnosis of chagas disease - Google Patents

Abstract

The present invention relates to a peptide and to the method for producing data that can be used in the differential diagnosis of Chagas disease, which allows differentiation of patients with chronic Chagas from healthy individuals and from those with other, similar infectious diseases, which is based on the detection of antibodies to said peptide. Furthermore, this method allows differentiation of patients with Chagas disease in an indeterminate clinical form from patients with Chagas who are suffering from pathological cardiac and digestive system conditions. The invention likewise allows differentiation of patients with Chagasic cardiopathy from those with non-Chagas cardiac disorders (myocarditis, idiopathic cardiopathies, myocardial infarction, etc). The present invention also allows evaluation of the response to the treatment of said disease in patients suffering from Chagas in chronic phase, especially with cardiac disorders. The present invention also relates to a kit for implementing said differential diagnostic method.

Description

 Differential diagnosis method of Chaqas disease

The present invention is within medicine, molecular biology, immunology and parasitology, and refers to a method of obtaining useful data for the differential diagnosis of Chagas disease, allowing to differentiate patients in an undetermined phase from chronic patients. with cardiac and digestive pathologies, as well as to evaluate the response to the treatment of said disease in Chagas patients in chronic cardiac phase.

STATE OF THE PREVIOUS TECHNIQUE

Chagas disease (Chagas-Mazza disease, Chagas disease or American trypanosomiasis), is a tropical parasitic disease mainly from Central and South America, generally chronic and whose etiologic agent is the Trypanosoma cruzi protozoan (T. cruzi). It is estimated to result in about 21,000 deaths each year (WHO, 2002, 2005), with approximately 50,000-200,000 new cases diagnosed per year (Tarleton RL, 2007. PLoS Med 4 (12): e332). Although the disease has traditionally been confined to Latin America, it is currently expanding to non-endemic areas as a result of migratory processes, so it has been necessary to implement diagnostic tests in blood banks and health centers in those countries with a high rate of immigrant population from endemic areas. Thus, the incidence of the disease in said immigrant population is 16 per 1,000 in Australia, 9 per 1,000 in Canada, 25 per 1,000 in Spain and 8-50 per 1,000 in the US (Schmunís GA et al, 2007. Mem tnst Oswaldo Cruz. 102 Suppl 1: 75-85).

T. cruzi is a flagellated parasitic protozoan that belongs to the Kinetoplastid Order and presents an obligatory phase of intracellular multiplication in the vertebrate host, in which it is capable of infecting the different types of cells. The infective form of the parasite or metacyclic trypomastigote is It is found in the gastrointestinal tract of the invertebrate host or insect that transmits the disease and is transmitted to the vertebrate host after the bite of the insect, which, in addition, defecates at the time of the bite, favoring the entry of the parasite by scratching caused by the character stool irritant The metacyclic trypomastigote is visible in the blood as a fusiform tripomastigote, in the form of "C" or "S", 20 μιη long by 1 μιτι wide and has no replicative capacity. When the parasite infects the cells of different tissues in the vertebrate host, mainly the striated cardiac muscle, skeletal muscle and smooth muscle, the scourge is shortened and transformed into a round amastigote of 2 to 5 μιτι in diameter. The amastigote is multiplied by binary fission forming "clusters" or "nests" that accumulate in the host cell until it breaks. The parasites released from the cell are transformed into the form of blood trypomastigote and are released into the bloodstream. They have a size that varies between 15 and 20 μηι, have free scourge thanks to which they can move, a bulky, terminal or underground cinetoplast, and an oval nucleus. These trypomastigotes can infect other cells to repeat the cycle, but they are not able to multiply in the blood, since the only replicative form in the vertebrate is the intracellular amastigote form.

Invertebrate hosts are hematophagous and acquire the parasite by feeding on man or infected domestic or wild animals. The trypomastigotes migrate to the midgut of the insect, where they become epimmastigotes, wide flagellate, very mobile, with the kinetoplast between the nucleus and the free scourge. There they divide a large number of times, leaving the insect infected for life. The epímastigotes migrate to the posterior intestine where, due to the acidity of the area, they are transformed into metacyclic tripomastigotes being excreted with feces at the time of the bite. In the case of infection caused by T, cruzi in man, the disease presents two severe states. The acute phase takes place after the bite of the insect and, although it usually goes unnoticed, it is associated with it approximately 10% mortality. The chronic phase, which can develop even after more than ten years, is characterized by the appearance of cardiomegaly, electrocardiographic abnormalities, arrhythmias (Chronic Chagas with cardiac involvement), aperistalsis, megaesophagus and megacolon {Chronic Chagas with digestive involvement), being able to reach cause death. The acute phase lasts approximately two to three months, progressing to give rise to an asymptomatic chronic phase, currently called an undetermined phase, which is characterized by the persistence of the infection without presenting apparent clinical problems. About 40% of serologically positive cases are in an undetermined phase, which frequently evolves into a chronic symptomatic phase several years later.

Chagas disease is routinely diagnosed by various commercial serological methods such as ELISA techniques, indirect immunofluorescence (IFI) or hemagglutination, for which complete or semi-purified protein extracts of the epimastigote forms of the T. cruzi parasite are used, mixtures of recombinant proteins or synthetic peptides corresponding to antigens or antigenic fragments of the parasite (Umezawa et al., 1996. J. Clin Microbiot. 34: 2143-2147; da Silveira et al., 2001. Trend Parasite !. 1 7: 286- 291). Another method of serological diagnosis developed is the so-called ID-PaGIA-Chagas (Rabello et al., 1999. Mem Inst Oswaldo Cruz 94 (1): 77-82), based on a gel agglutination reaction using three synthetic peptides derived from two antigens of T. cruzi, or the so-called INNO-LIA (Saez-Alquezar et al., 2000. J. Clin Microbio !. 38: 851-854), based on the reactivity against seven recombinant proteins fixed on a support of nylon. The Stat-Pack immunochromatographic kit uses six recombinant proteins and presents high levels of sensitivity and specificity (Ponce et al., 2005. J Clin Microbio! 43 (10): 5065-5068). These methods have been specially proposed to obtain rapid results of seropositivity in screenings in blood banks, medical emergencies and in organ transplants. However, they only allow you to obtain a qualitative result (positive / negative) and, therefore, are of low utility to evaluate the evolution of the chagasic patient. Recently, the efficacy of nine different serological diagnosis systems of Chagas disease has been evaluated, seven of them commercial (Caballero et al., 2007. Clinical and Máceme Immunology. 14 (8): 1045-1049), showing percentages of sensitivity and specificity ranging from 75% to 100%, using a Western blot (immunoblot) assay with secretory-excretory antigens of trypomastigote forms of T. cruzi. However, none of the methods described allow differential diagnosis between the different clinical forms of the chronic phase of Chagas disease.

In general, serological diagnostic systems allow antibodies to be detected in sera of chronic patients, but they are not very useful for evaluating the evolution of patients under treatment, since antibody levels persist stably for a long time. Probably, the treatment, although decreasing the parasite load can influence the reduction of the level of antibodies, destroying the parasite can lead to exposure to the immune system of new parasitic components, with the consequent possible generation of antibodies with other specificities. These two conjugated effects (increase in the titer of some antibodies and decrease in others) give rise to an apparent stability of the titer measured against total lysate proteins of the parasite or combinations of antigens. Therefore, these commercial kits do not allow to detect the dynamics of response after the treatment nor, therefore, to recognize the cases in which the therapy is not effective.

The only two medications available for the treatment of Chagas disease are Nifurtimox, developed in 1960 by Bayer, and Benznidazol, developed in 1974 by Roche. The therapeutic advantage of these drugs in the chronic phases of the disease remains controversial and, in addition, both have high rates of undesirable side effects. Consequently, it is still essential to develop tools that allow differential diagnosis and thus determine on the one hand, at what stage of the disease the patient is and, on the other, monitor the course of the disease and assess the evolution of the disease and treatment effectiveness

DESCRIPTION OF THE INVENTION

The present invention provides a new marker and a new method of obtaining useful data for the diagnosis of Chagas disease, for the differential diagnosis of the chronic stages (with cardiac or digestive alterations) of the undetermined stage, allowing the establishment of groups of patients according to the degree of the pathology, as well as to monitor and evaluate the response to the treatment and evolution of said disease in patients with chagasic heart disease. Another aspect of the present invention relates to a kit comprising said new marker and the use of said kit for the diagnosis of Chagas disease, for the differential diagnosis of the different clinical forms of said disease and for monitoring the patient and / or evaluation of the state of your disease after the administration of the treatment.

This marker makes it possible to differentiate patients with Chagas disease in any of the forms of chronic disease (indeterminate, cardiac, digestive) from those patients with related infectious diseases (leishmaniasis, malaria, tuberculosis). This marker distinguishes patients with chagasic heart disease from those patients with non-chagasic cardiac disorders (acute myocarditis, acute myocardial infarction, idiopathic cardiomyopathy, heart failure, etc.). Thus, the present invention provides a solution, under a non-invasive serological technique of easy and rapid realization as well as economical, to the need to identify the phase in which the chagasic patients are, since It allows differentiating patients in chronic phase from patients in undetermined phase. Moreover, the present invention makes it possible to differentiate whether the patient in the chronic phase has a cardiac or digestive condition from those who do not.

The present invention is useful for the follow-up of chronic chagasic patients, mainly with cardiac alterations, after the administration of the treatment, since the level of specific antibodies against this marker decreases, which can be considered as an index of treatment efficacy and improvement. of the illness. The non-modification of the level of antibodies against the marker of the present invention could imply therapeutic failure or the abandonment or non-correct monitoring of the treatment by the patient. Therefore, a first aspect of the invention relates to a peptide (hereinafter called the peptide of the invention) of amino acid sequence:

FX QX ₂ -DKP-X ₃ , where Xi represents an amino acid that is selected from G and A; where X ₂ is a group of 4 amino acids that are selected from A, E and G; where X ₃ is a group of 2 amino acids that are selected from S, L, P and A.

Preferably, X ₂ is AAX ₄ , where X ₄ is a group of two amino acids that are selected from A, E and G. Preferably, X ₄ is AA, AG or EG.

Preferably, X ₃ is SL, PP, AP or AA.

In a preferred embodiment of the peptide of the invention, the amino acid sequences are selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO : 6, SEQ ID NO: 7 and SEQ ID NO: 8. In a more preferred embodiment of the peptide of the invention, the amino acid sequence is SEQ ID NO: 1.

A peptide is a molecule formed by the union of several amino acids by peptide bonds. Amino acids are organic molecules that have an amino group and a carboxyl group and are part of the proteins. The amino acids that are part of the proteins are 20, and can be represented by three letters or by a single letter. Thus, F is phenylalanine, Q is Glutamine, D is aspartic acid, K is lysine, P is proline, G is glycine, A is alanine, E is glutamic acid, S is serine and L is leucine.

In another preferred embodiment of the peptide of the invention, said peptide is characterized in that its amino acid sequence comprises the amino acid sequence of the peptide of the invention as described above. The label may be a peptide of the invention flanked by amino acids or other molecules not relevant for its function as a marker, but which may allow anchoring to a solid support so that all relevant antigenic sequence of the peptide is available to be recognized by a antibody.

In another preferred embodiment of the peptide of the invention, said peptide is characterized in that its sequence is repeated at least twice. Thus, the peptide sequence of the invention can be repeated at least twice, each repetition being linked by amino acids or molecules not relevant for the specific recognition of the peptide by the antibodies.

A second aspect of the invention relates to the use of the peptide of the invention as a marker for the differential diagnosis, prognosis or monitoring of the stage of Chagas disease in an isolated biological sample. The terms "diagnosis" and "differential diagnosis", as used in the present invention, refer to the ability to discriminate between individuals infected by the T. cruzi parasite from those not infected by T. cruzi or infected by other agents. Infectious infections (such as the causes of leishmaniosis, malaria, tuberculosis, etc.). It also refers, but not limited to, the ability to discriminate between samples from patients presenting with different clinical forms of Chagas disease: the acute phase, shortly after infection, the undetermined phase and the chronic phase. In turn, other subclassifications could be established within this principal, thus facilitating the choice and establishment of appropriate therapeutic regimens. This discrimination, as understood by an expert in the field, is not intended to be correct in 100% of the samples analyzed. However, it requires that a statistically significant amount of the analyzed samples be classified correctly.

The term "prognosis", as used in the description of the invention, refers to the ability to assign a probability of certain situations occurring in the course of Chagas disease, when a sample classification method is applied. based on the analysis of the amount of antibodies against the peptide of the invention and on the comparison of the amount detected with a reference amount. This assignment, as understood by a person skilled in the art, is not intended to be correct in 100% of the samples analyzed. However, it requires that a statistically significant amount of the analyzed samples be classified correctly. This term refers, for example, but not limited to the probability of developing or suffering from a cardiac pathology or the probability of developing or suffering from a digestive pathology, as well as the prediction of response to a certain treatment of Chagas disease. An "isolated biological sample" refers, but is not limited to, cells, tissues and / or biological fluids of an organism, obtained by any method known to a person skilled in the art. Preferably, the isolated biological sample is a biological fluid, such as, but not limited to, blood, plasma or blood serum. More preferably, the biological fluid is blood serum. The detection of antibodies against the peptide of the invention in an isolated biological sample of an individual is indicative that it has been or continues to be parasitized by T. cruzi. The term "individual", as used in the description, refers to animals, preferably mammals, and more preferably, humans. The term "individual" is not intended to be limiting in any aspect, and may be of any age, sex, physical condition and be originating and / or proceeding from any part of the world.

Organisms of the Trypanosoma cruzi species belong to Superuk Eukaryota, Order Kinetoplastida, Family Trypanosomatidae, Genus Trypanosoma and subgenus Schizotrypanum.

A third aspect of the invention relates to a method of obtaining useful data for the diagnosis and monitoring of Chagas disease, which comprises:

 to. detecting the existence and / or quantifying the amount of antibodies against the peptide of the invention in an isolated biological sample, by contacting said peptide with said sample. In a preferred embodiment of the invention, the method of obtaining useful data further comprising:

b. compare the amount of antibodies quantified in step (a) with a reference amount. In a more preferred embodiment, the method of obtaining useful data for the diagnosis or monitoring of Chagas disease also comprises: C. assign the individual in the sample to the group of individuals with Chagas disease, when they present a quantity of antibodies quantified in step (a), greater and statistically significant compared to a reference amount.

The measurement of the amount or concentration, preferably semi-quantitatively or quantitatively, can be carried out directly or indirectly. Direct measurement refers to the measurement of the amount or concentration of antibodies, based on a signal that is obtained directly from the antibodies, and that is directly correlated with the number of antibody molecules present in the sample. Said signal - which we can also refer to as an intensity signal - can be obtained, for example, by measuring an intensity value of a chemical or physical property of said antibodies. The indirect measurement includes the measurement obtained from a secondary component or a biological measurement system (for example the measurement of cellular responses, ligands, "tags" or enzymatic reaction products). The term "antibody", as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, that is, molecules that contain an antigen binding site to which they specifically bind (immunoreact) which can be a natural or chemically synthesized peptide or a natural or recombinant protein, produced and purified by molecular and chemical techniques. There are five main isotypes or classes of immunoglobulins: immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin A (IgA) and immunoglobulin E (IgE). The term "comparison", as used in the description, refers to, but is not limited to, the comparison of the amount of antibodies against the peptide of the invention in the biological sample to be analyzed, also called the sample. biological problem, with an amount of antibodies against the peptide of the invention of one or more reference samples. The reference sample can be analyzed, for example, simultaneously or consecutively, together with the problem biological sample. The comparison described in section (b) of the method of the present invention can be performed manually or automatically and / or assisted by a computer.

The term "reference amount", as used in the description, refers to the absolute or relative amount of antibodies against the peptide of the invention that allows to discriminate a certain stage of Chagas disease from the other stages of the disease. Suitable reference amounts can be determined by the method of the present invention from a reference sample that can be analyzed, for example, simultaneously or consecutively, together with the problem biological sample. Thus, for example, but not limited to, the reference sample may be the negative controls, that is, the amounts detected by the method of the invention in samples of individuals that have not been parasitized by T. cruzi. Also, for example, in the case of obtaining useful data for the differential diagnosis of chronic Chagas disease, the reference amount would be the amount of antibodies against the peptide of the invention detected in patients with undetermined Chagas disease. In addition, in the case of monitoring the evolution of Chagas disease, the reference amount could be, but not limited to, the amount of these antibodies detected in a biological sample of the same individual obtained previously.

Thus, the sample or reference samples can be, for example, obtained from the serum of a patient with Chagas disease in a certain clinical phase. In another preferred embodiment of this aspect of the present invention, the reference amount is obtained from a reference sample. The reference quantity can also be obtained, for example, from the normal distribution limits of an amount found in samples obtained from a population of patients with Chagas disease in different phases, using well-known statistical techniques.

The amount that is statistically significant can be established by a person skilled in the art by using different statistical tools, for example, but not limited, by determining confidence intervals, determining the p-value, Student's test or discriminant functions of Fisher Preferably, the confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. Preferably, the value of p is less than 0.1, 0.05, 0.01, 0.005 or 0.0001. Preferably, the present invention allows the disease to be correctly detected in at least 60%, in at least 70%, in at least 80%, or in at least 90% of the subjects of a certain group or population analyzed.

Another aspect of the invention is a method for the differential diagnosis of Chagas disease comprising steps (a) and (b) of the method of obtaining useful data of the invention, which further comprises:

 C'. assign the individual of the sample to the group of individuals with chronic Chagas disease with cardiac or digestive pathology when they present a quantity of antibodies quantified in step (a), greater and statistically significant compared to a reference amount, or

c ". assign the individual of the sample to the group of individuals with Chagas disease in an undetermined phase when it presents a quantity of antibodies quantified in step (a), smaller and statistically significant compared to a reference amount. The term" diagnosis differential ", as used in the present invention, refers to discrimination, within Chagas patients, of chronic patients, with cardiac and / or digestive pathologies, of undetermined patients.

Another aspect of the invention is a method of monitoring the evolution of Chagas disease in chronic chagasic individuals with heart disease which comprises performing at least twice the sequence of steps (a) and (b) of the method of obtaining useful data from the invention, in biological samples from the same individual, and isolated at different times. Preferably, the monitoring method is performed post-treatment.

The term "evolution monitoring", as used in the present description, refers to the monitoring of the development of the disease, such as, but not limited to, the evaluation of the response to a particular treatment of the disease. Chagas disease.

Preferably, the method of monitoring the evolution of Chagas disease further comprises comparing the amount of antibodies quantified in step (a) before the start of treatment, with the amount of antibodies quantified in step (a) at different times after the start of treatment

Preferably, the quantification of the antibodies is performed by an immunoassay. Preferably, the immunoassay is an ELISA. Preferably, the ELISA is an indirect ELISA.

Examples of immunoassays known in the state of the art are, for example, but not limited to: immunoblot, enzyme-linked immunoadsorbent assay (ELISA), linear immunoassay (LIA), radioimmunoassay (RIA), immunofluorescence, immunohistochemistry or protein microarray. The ELISA is based on the premise that an immunoreactive can be immobilized on a solid support, then bringing that system into contact with a fluid phase containing the complementary reagent that can bind to a marker compound. There are different types of ELISA: direct ELISA, indirect ELISA or sandwich ELISA. The term "marker compound", as used herein, refers to a compound capable of giving rise to a chromogenic, fluorogenic, radioactive and / or chemiluminescent signal that allows the detection and / or quantification of the amount of antibodies The marker compound is selected from the list comprising radioisotopes, enzymes, fluorophores or any molecule capable of being conjugated with another molecule or detected and / or quantified directly. This marker compound can bind to the antibody directly, or through another compound. Some examples of marker compounds that bind directly are, but are not limited to, enzymes such as alkaline phosphatase or peroxidase, radioactive isotopes such as ³² P or ³⁵ S, fluorochromes such as fluorescein or metal particles, for direct detection by colorimetry, auto-radiography , fluorimetry, or metallography, respectively.

Another aspect of the invention relates to a kit (hereinafter called the kit of the invention) comprising the peptide of the invention for quantifying antibodies against said peptide in an isolated biological sample. Preferably, the peptide is immobilized in a solid phase.

Another aspect of the invention relates to the use of the kit of the invention for the diagnosis of Chagas disease.

Another aspect of the invention relates to the use of the kit of the invention for the differential diagnosis of the stage of Chagas disease.

Another aspect of the invention relates to the use of the kit of the invention for monitoring Chagas disease after the start of treatment. Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For experts in the field, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Figure 1. It shows the reactivity of sera from chagasic patients against peptide 3973. 50 sera from Chagas disease patients and 28 sera from healthy donors (C-, negative control) were used. Serums from Chagas disease patients recognize this peptide significantly higher (p <0.001) in relation to healthy donors. The results shown are those obtained at a dilution of the sera of 1: 800.

Figure 2. Shows the specificity of the serological response against peptide 3973 (related infectious diseases). 50 sera from Chagas disease patients, 5 from patients with leishmaniasis, 5 from patients with tuberculosis, 4 from patients with malaria and 28 sera from healthy donors (C-, negative control) were used. The sera of Chagas patients recognize peptide 3973 significantly higher (p <0.001) than those of patients with other infectious diseases. The results shown are those obtained at a dilution of the sera of 1: 800. Figure 3. It shows the recognition of peptide 3973 depending on the associated chagasic pathology. 50 sera from Chagas disease patients were used, of which 30 were in the undetermined phase (IND), 30 in the chronic phase with cardiac pathology (CARD) and 21 in the chronic phase with digestive pathology (DIG). The sera of patients with chronic chagasic or digestive heart disease recognize peptide 3973 significantly higher (p <0.001 in both cases) than patients in the indeterminate phase. The results shown are those obtained at a dilution of the sera of 1: 1 .600.

Figure 4. Shows the specificity of the serological response against peptide 3973 (other heart diseases). The sera of patients with chronic chagasic or digestive heart disease have a recognition of peptide 3973 significantly higher than those of patients who have other non-chagasic heart disease (p <0.001). The results shown are those obtained at a dilution of the sera of 1: 1 .600.

Figure 5. It shows the reactivity of the sera of 23 chagasic patients to peptide 3973 before and during treatment with benznidazole (at 3, 6, 9, and 12 months of treatment). Only Chagas patients with chronic chagasic heart disease had a significant decrease in recognition of peptide 3973 (p <0.05 at 6 months, p <0.1 at 9 months and p <0.02 at 12 months of treatment) . The results shown are those obtained at a dilution of the sera of 1: 1 .600.

EXAMPLES

Next, the invention will be illustrated by tests carried out by the inventors, which shows the specificity and effectiveness of the methods of the invention for obtaining useful data in the differential diagnosis of Chagas disease and for monitoring said disease.

MATERIALS AND METHODS Subjects of the study The study subjects, coming from different Latin American countries and residing in Spain, were diagnosed as Chagas patients or healthy donors, using two conventional serological techniques. The Chagas patients underwent different medical tests such as electrocardiogram, chest x-ray, echocardiography and digestive radiography with contrast (digestive cases) for detailed clinical assessment. The results of the aforementioned tests allowed patients to be classified clinically according to the stage of the disease as asymptomatic or indeterminate (IND), with cardiac (CARD) or digestive (DIG) pathology. Serum samples were taken before the start of benznidazole treatment and at 3, 6, 9 and 12 months after the start of treatment. Subjects with non-chagasic heart disease are residents in Spain and from non-endemic areas.

Sequencing and peptide synthesis

Peptide 3973 was obtained by multiple solid phase synthesis (SMPS) using p-methylbenzhydrylamide (MBHA) resin (Houghten, Proc Nati Acad Sci U S A. 1985 82 (15): 5131-5). The purity of the peptide was analyzed by high pressure liquid chromatography (HPLC), according to the method previously described (Hunkapiller and Hood, Biochemistry. 1978 30; 17 (1 1): 2124-33). ELISA (Enzyme-Linked ImmunoSorbent Assay)

The levels of specific IgG antibodies against peptide 3973 were determined in chagasic sera and sera of healthy donors, as well as in sera of individuals suffering from infectious diseases that may coexist territorially with that of Chagas. Briefly, the ELISA wells (Nunc) were covered with 10 μg / ml of the 3973 peptide dissolved in PBS ("Phosphate Buffer Satine" or phosphate buffer, at pH = 7.2) and stored at -20 ^to C until its use. Subsequently, the wells were washed twice with 200 μΙ of PBS-0.05% Tween-20 and incubated for 30 minutes with blocking solution (2.5% skimmed milk powder in PBS-0.05% Tween-20 ). Then, sera from patients diluted in blocking solution were added and incubated for 1 hour at 37 ^° C. As a secondary antibody, a secondary antibody was used. peroxidase-conjugated anti-IgG antibody (Biosource) at a 1: 2,000 dilution and incubated for one hour at 37 ^s C. After washing the wells, the reaction was developed with a mixture of equal volumes of tetramethylbenzidine (TMB 0.4 g / l) and hydrogen peroxide (H ₂ 0 ₂ 0.02%) for 10 minutes at room temperature and absorbance at 620 nm was measured. The sera were tested in triplicate and in three dilutions (1: 400, 1: 800 and 1: 1 .600). The corresponding positive controls (fixation of peptide to the plate, serum reactivity against the fixed and revealed peptide of the reaction) and negatives (background of the reaction) were included in each test, and those tests in which the optical densities were discarded (DO) of the controls were more than 20% different than expected values.

Statistical analysis The SPSS 15.0 program was used. Statistically significant differences between different groups of patients were analyzed using the non-parametric Mann-Withney test. The analysis of the post-treatment longitudinal differences was performed with the non-parametric Wilcoxon method. The differences are considered statistically significant if the confidence interval is at least 90% (p≤0.1).

Example 1: Reactivity of sera from chagasic patients against peptide 3973 and other peptides with formula FX Q-X2-DKP-X ₃ The presence of specific antibodies as well as the antibody titer against a synthetic peptide was analyzed by ELISA, smaller in size than 20 amino acids, whose sequence corresponds to a part of the T. cruzi membrane protein (TcMp) (Table 1). It was determined that this was specifically recognized by more than 95% of the sera of chagasic individuals (Tablal). Likewise, we studied the specific sequence against which the detected antibodies are specifically directed, analyzing by ELISA the presence and level of antibodies against 19 different peptides whose sequences correspond to that of peptide 3973 {Table 1) but also carry one or more modifications in the amino acid sequence.

The results obtained showed that 7 of these peptides were recognized in the same way and percentage (95%) by the sera of the chagasic patients and that, in 12 of the peptides, the level of recognition decreased from 50% to less than 5 % (Table 1 ). Therefore, the results obtained and represented in Table 1 showed the existence of a 12 amino acid sequence, which is recognized by ELISA and statistically significant by more than 95% of the sera of Chagas patients and not by sera from healthy donors. In addition, the results obtained and shown in Table 1 show the identity and relative position of 5 amino acids contained in the 3973 peptide that are involved in said recognition and that are responsible for it.

Table 1 . Table of reactivity of peptides analogous to 3973. "N" indicates that the peptide is a natural molecule and "Q" indicates that it is a chimeric molecule.

6174 Q (SEQ ID NO: 12) FGQAAAGDGPSL 1090,3 <5%

6174 Q (SEQ ID NO: 12) FGQAAAGDGPSL 1090.3 <5%

 6173 Q (SEQ ID NO: 13) FGQAAAGGKPSL 1 103.4 <5%

 6172 Q (SEQ ID NO: 14) FGQAAAGERPSL 1203.4 <5%

 6381 Q (SEQ ID NO: 15) AAAAAAGDKPAA 998.3 <5%

 6382 Q (SEQ ID NO: 16) AAQAAAGDKPSL 1056.4 <10%

 6175 Q (SEQ ID NO: 17) AGQAAAGDKPSL 1085.3 50%

 6380 Q (SEQ ID NO: 7) FAQAAAGDKPSL 1 189.4> 95%

 6383 Q (SEQ ID NO: 18) FGAAAAGDKPSL 1 104.5 <10%

 6384 Q (SEQ ID NO: 19) AAGDKPSL FGQA 1 161, 4 <5%

 6385 Q (SEQ ID NO: 20) QAAAGDKPSL FG 1 161, 4 <5%

 6386 Q (SEQ ID NO: 8) FAQAAAADKPAA 329.38> 95

Example 2: The serological response against peptide 3973 is specific to Chagas disease versus other related infectious diseases. The reactivity against peptide 3973 of sera from patients with Chagas disease (50 patients) and 28 healthy donor subjects (called negative controls or C-) was tested, observing that the recognition of peptide 3973, by Chagas patients, It is significantly higher (p <0.001) than that of healthy donors (Fig 1). Likewise, the reactivity against peptide 3973 of sera from patients with Chagas disease (50 patients) and other related and endemic infectious diseases of the regions where Chagas disease is found, such as leishmaniasis (5 patients) ), tuberculosis (5 patients) or malaria (4 patients). The recognition of peptide 3973, by Chagas patients, is significantly higher (p <0.001) than that of patients with other infectious diseases endemic to the affected regions. Likewise, no significant serological recognition of said peptide was observed by sera from patients with related infectious pathologies, which presented levels similar to those of healthy donors (Fig. 2). Example 3: The serological response against peptide 3973 makes it possible to discriminate the stage of Chagas disease.

Serums from patients with Chagas disease were tested against peptide 3973 and D.O. they were represented according to the pathology of each patient (chronic phase with heart disease (CARD), chronic phase with digestive pathology (DIG) and undetermined phase (IND). Significant recognition of the 3973 peptide by Chagas patients regardless of stage of the disease Likewise, it is observed that patients with associated pathology such as chronic chagasic (30 patients) or digestive heart disease (21 patients) recognize this peptide significantly higher (p <0.001) than those of Chagas patients who apparently they do not have an associated pathology (30 patients) (Fig. 3) Example 4: The serological response against peptide 3973 is specific to Chagas disease in chronic stage and with evidenced pathology and not other non-chagasic diseases that have associated heart disease 50 sera from Chagas disease patients were tested against peptide 3973 and OD values were represented. They were grouped according to the absence or presence of chagasic pathology of each patient: IND: patients in an undetermined phase without apparent symptoms; CARD: with chronic chagasic heart disease; DIG: with digestive pathology. Patients with non-chagasic heart disease were also included. The results obtained show that the recognition of peptide 3973 by patients with chronic chagasic or digestive heart disease is significantly higher than that of patients with other non-chagasic heart disease (Fig. 4).

Example 5: Modifications in the recognition of peptide 3973 induced by treatment with benznidazole depend on the associated chagasic pathology. 50 sera from patients with Chagas disease were tested against peptide 3973 before the start of benznidazole (T0) treatment and, for 23 patients, at regular intervals of 3 months after the start of treatment (at 3, 6, 9 and 12 months post-treatment). The patients were grouped according to the associated chagasic pathology: cardiac, digestive pathology or in indeterminate patients without apparent pathology. A significant decrease in the recognition of peptide 3973 was observed at 6 months (p <0.05), 9 months (p <0.1) and 12 months (p <0.02) post-treatment, only in patients with chronic chagasic heart disease (Fig. 5).

Claims

1. A peptide of amino acid sequence: FX QX ₂ -DKP-X ₃ Where: Xi represents an amino acid that is selected from G and A; where X2 is a group of 4 amino acids that are selected from A, E and G; where X ₃ is a group of 2 amino acids that are selected from S, L, P and A. 2. The peptide according to the preceding claim, wherein X is G. 3. The peptide according to any of the two preceding claims, wherein 4. The peptide according to any of the three preceding claims, wherein X2 is AAX ₄ and X ₄ is a group of two amino acids that are selected from A, E and G. 5. The peptide according to the preceding claim, wherein X ₄ is AA, AG or EG. 6. The peptide according to any of the five preceding claims, wherein X ₃ is SL, PP, AP or AA. 7. The peptide according to any of the preceding six claims, wherein the amino acid sequence of the peptide is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8. 8. The peptide according to any of the seven preceding claims, wherein the amino acid sequence of the peptide is SEQ ID NO: 1. 9. A peptide characterized in that its amino acid sequence comprises the amino acid sequence of the peptide according to any of the previous eight claims. 10. The peptide according to the preceding claim characterized in that its sequence is repeated at least twice. eleven . A use of the peptide according to any of the previous ten claims as a marker for the differential diagnosis, prognosis or monitoring of the stage of Chagas disease in an isolated biological sample. 12. A method of obtaining useful data for the diagnosis and monitoring of Chagas disease, which includes:  to. detecting the existence and / or quantifying the amount of antibodies against the peptide according to claims 1 to 10 in an isolated biological sample, by contacting said peptide with said sample. 13. The method according to the preceding claim, further comprising:  b. compare the amount of antibodies quantified in step (a) with a reference amount. 14. The method according to any of the two preceding claims for the diagnosis or monitoring of Chagas disease, which further comprises: C. assign the individual in the sample to the group of individuals with Chagas disease, when they present a quantity of antibodies quantified in step (a), greater and statistically significant compared to a reference amount. 15. A method for the differential diagnosis of Chagas disease comprising steps (a) and (b) according to any of claims 12 and 13, further comprising:  c \ assign the individual of the sample to the group of individuals with chronic Chagas disease with cardiac or digestive pathology when it presents a quantity of antibodies quantified in step (a), greater and statistically significant compared to a reference amount, or  c ". assign the individual of the sample to the group of individuals with Chagas disease in an undetermined phase when it presents a quantity of antibodies quantified in step (a), smaller and statistically significant compared to a reference amount. 16. The method of monitoring the evolution of Chagas disease in chronic chagasic individuals with heart disease comprising performing at least twice the sequence of steps (a) and (b) according to any of claims 12 and 13, in biological samples from the same individual, and isolated at different times. 17. The monitoring method according to the preceding claim, wherein the monitoring is carried out post-treatment. 18. The monitoring method according to the preceding claim, further comprising comparing the amount of antibodies quantified in the step (a) before the start of treatment, with the amount of antibodies quantified in step (a) at different times after the start of treatment. 19. The method according to any of the seven preceding claims, wherein the quantification of the antibodies is performed by an ELISA. 20. The method according to the preceding claim, wherein the ELISA is an indirect ELISA. 21. A kit comprising the peptide according to any one of claims 1 to 10 for quantifying antibodies against said peptide in an isolated biological sample. 22. The kit according to the preceding claim wherein the peptide is immobilized in a solid phase. 23. Use of the kit according to any of the two preceding claims for the diagnosis of Chagas disease. 24. Use of the kit according to any of claims 21 and 22 for the differential diagnosis of the stage of Chagas disease. 25. Use of the kit according to any of claims 21 and 22 for monitoring Chagas disease after the start of treatment.