Kit and method for sorodiagnosis of chagas disease using synthetic peptides, and uses

Description

"KIT AND METHOD FOR SERODIAGNOSIS OF CHAGAS DISEASE USING SYNTHETIC PEPTIDES, AND USES"

[01] The present technology relates to a kit and a method for the serodiagnosis of Chagas disease using four synthetic peptides derived from conserved motifs present in polymorphic antigenic proteins expressed by the parasite. The peptides were selected from the analysis of a large number of isolates from different endemic areas of the American continent and exhibit high sensitivity and specificity in samples from humans infected with T. cruzi. Furthermore, the technology is inexpensive, since the peptides are small in size and do not necessarily need to be recombinantly expressed by modified bacteria, as they can be chemically produced in an automated synthesizer.

[02] Trypanosoma cruzi is a flagellated protozoan that is exclusively parasitic, belonging to the Discoba Group, Kinetoplastea Class, and Trypanosoma Genus, causing American trypanosomiasis or Chagas disease (CD), an anthropozoonosis whose different clinical manifestations, etiological agent, transmission cycle, and reservoirs were initially described in 1909 by Carlos Chagas. It is classified among the 17 neglected tropical diseases according to the World Health Organization. It is estimated that approximately eight million individuals are infected with T. cruzi and 70 million are at risk of acquiring the infection. In addition, more than 10,000 deaths per year can be attributed to CD, making it a serious public health problem. Due to migration, recent cases of CD have also been reported in other countries, such as Europe, North America, Japan, and Australia.

[03] According to data from the Ministry of Health, it is estimated that there are currently at least one million people infected with T. Petition 870250007999, dated 01 / 30 / 2025, page 20 / 46 2 / 21 cruzi. Despite the elimination of T. infestans, the risk of vector-borne transmission of the parasite continues, due to the existence of other autochthonous triatomine species with a high colonization potential, as well as the presence of T. cruzi reservoirs and the increasingly frequent proximity of human populations to these environments. Other factors have also contributed to the maintenance of Chagas disease transmission in Brazil, such as the occurrence of cases and outbreaks through oral transmission via ingestion of food (sugarcane juice, açaí, bacaba, among others) contaminated with infected vector insects, through domestic vector transmission without colonization, and through extradomiciliary vector transmission, mainly in the Legal Amazon.

[04] DC effectively has two clinical phases. The acute phase is characterized by intense parasitemia and intense tissue parasitism involving the infection of various nucleated host cells, including cardiac, skeletal and smooth muscle cells, liver cells, adipose and neural cells. The host's immune response in the acute phase is characterized by polyclonal activation of B and T lymphocytes and hypergammaglobulinemia, as in many diseases. Symptoms in the acute phase, when apparent, can persist for approximately two months. In this phase, the mortality rate varies from 2 to 8%, occurring more frequently in children. Blood parasitism is high and the symptomatology is quite variable, ranging from asymptomatic to mild to severe. The clinical diagnosis of acute DC is a challenge due to the infrequent occurrence of specific infection symptoms. Among the symptoms that can aid diagnosis in this phase is painless, unilateral, bipalpebral ocular edema.General symptoms and systemic changes include fever, malaise, headache, asthenia, hyporexia, enlarged lymph nodes, hepatomegaly and splenomegaly, and acute myocarditis. Subsequently, infected individuals progress to... Petition 870250007999, dated 01 / 30 / 2025, page 21 / 46 3 / 21 The chronic phase of the disease can be divided into two stages: the asymptomatic (or indeterminate) chronic phase, which can last for decades after infection, in which patients test positive in serological tests but do not present evident clinical symptoms. In this phase, the infection is controlled, but the immune system may not prevent the progression of the disease. Thus, the portion of asymptomatic individuals who become symptomatic, generally decades after infection, corresponds to 10-40% of infected patients, with cardiomyopathies and pathologies of the digestive tract.

[05] Only two trypanocidal drugs are available for the etiological treatment of Chagas disease: benznidazole and nifurtimox. Both are nitroheterocyclic, oral compounds that require prolonged administration, can have serious adverse effects, cannot be used to treat pregnant women due to their uncertain teratogenic risks (in the case of nifurtimox), and, most importantly, are highly effective only if administered early in the infection, being able to cure about 70% of acute cases. From a serological and parasitological point of view, there is evidence that cure rates after specific treatment are variable in the chronic phase of the disease and depend on the duration of treatment. Data have been reported that after etiological treatment there is a gradual decrease in serological titers of anti-T. cruzi antibodies, and that treatment failure can sometimes manifest itself, even decades after persistently negative xenodiagnosis and blood culture.Therefore, diagnosing acute and recent chronic cases of Crohn's disease is extremely important, increasing the chances of cure through early chemotherapy treatment.

[06] The diagnosis of individuals infected with T. cruzi can be carried out through parasitological or serological methods. In parasitological methods, techniques are used to detect forms Petition 870250007999, dated 01 / 30 / 2025, page 22 / 46 4 / 21 circulating parasite samples, such as stained smears or microhematocrit, or even parasite DNA, as in conventional or real-time PCR. However, it is important to emphasize that these techniques often do not perform satisfactorily, especially in the chronic phase of infection, where parasitemia is lower, in addition to requiring highly trained professionals and / or involving high costs for an endemic area.

[07] An alternative is often serological diagnosis, which can efficiently and rapidly confirm the etiology of Chagas disease, especially during the chronic phase. Antibody detection can be based on various immunological techniques such as Enzyme-Linked Immunosorbent Assay (ELISA), Indirect Hemagglutination Assay (HAI), Indirect Immunofluorescence Antibody Test (IFAT), and Western Blotting (WB), for example. Most techniques are based on the detection of IgG, which can be considered conventional tests because they use the whole parasite or purified extracts as antigens, or non-conventional tests that use recombinant antigens or synthetic peptides. Conventional tests often cause problems due to cross-reactivity, mainly with T. rangeli and parasites of the genus Leishmania. Therefore, non-conventional tests are recommended to increase the specificity of the diagnosis in endemic areas where, in addition to T.cruzi, these parasites are also endemic.

[08] However, even after successive improvements, none of the available tests has emerged as a “gold standard,” that is, with approximately 100% specificity and sensitivity. In this context, current guidelines developed by the WHO, the Pan American Health Organization (PAHO), and the Brazilian Consensus on Chagas disease from the Brazilian Ministry of Health recommend the use of at least two serological tests based on different principles to achieve a Petition 870250007999, dated 01 / 30 / 2025, page 23 / 46 5 / 21 “conclusive” diagnosis. In the case of ambiguous or discordant results, a third technique should be performed. These guidelines increase the cost of diagnosis and the risk of patient “loss,” especially in endemic areas. Furthermore, the known variability of antigens expressed by the trypomastigote forms of the parasite, particularly those derived from multigene families, can lead to variations in the antigenic constitution of different isolates and DTUs circulating in different endemic areas. Thus, antigens expressed in various cellular compartments of the parasite, such as cytoplasm, cytoskeleton, cell surface, flagellum, ribosomes, among others, have been identified as potential marker targets for serological tests.In the acute phase of infection, for example, antigenic proteins such as transialidases (TS), mucin-associated surface proteins (MASPs), and mucins have been described as antigenic proteins in the acute phase of T. cruzi infection. However, the use of these antigens for immunodiagnosis is a challenge due to their high polymorphism in different isolates of the parasite.

[09] To solve the problem of surface protein polymorphism, genomic approaches can be used. However, the exclusive use of assembled genomes can limit the scope of the great variability found in isolates from endemic areas. This scenario can be attributed to the fact that, due to the repetitive nature of certain genomic regions, multigene families tend to collapse during the assembly process, resulting in an underrepresentation of these regions. Additionally, in genomic assemblies, only one chromosome copy is represented, which can suppress relevant information, such as the presence of heterozygosity and aneuploidies. Petition 870250007999, dated 01 / 30 / 2025, page 24 / 46 6 / 21

[10] In this context, a promising alternative is the use of unassembled reads, which can circumvent these difficulties. In addition, such reads have the advantage of being widely available in public databases, containing a vast number of isolates. By exploring these unassembled reads, it is possible to capture genetic diversity in a complete and representative manner, contributing to a better understanding of T. cruzi protein polymorphism, and potentially encompassing a wider range of targets.

[11] The state of the art presents some documents that describe the use of peptides for the diagnosis of Chagas disease. Patent document BR1020180691074, whose priority date is 20 / 09 / 2018, entitled “Synthetic peptides, method and kit for diagnosis of the chronic phase of Chagas disease, and use”, describes three sequences conserved in several strains of the parasite Trypanosoma cruzi, and their use as highly specific and reactive antigens in sera from individuals in the chronic phase of Chagas disease, presenting the clinical forms Indeterminate or Chagasic Cardiomyopathy. Patent document WO2023114960, whose priority date is 16 / 12 / 2022, entitled “Antigenic peptides and methods of use for diagnosis of Chagas disease”, describes peptides and peptide mixtures for serological diagnosis of Chagas disease. These technologies differ because they use different antigens than those described in the present technology.

[12] In the state of the art, no technology has been found that uses the synthetic peptides described in the present technology for the diagnosis of Chagas disease.

[13] The technology developed and proposed here has advantages over other similar technologies by identifying peptides originating from conserved repetitive regions in more than 40 T. cruzi isolates. Petition 870250007999, dated 01 / 30 / 2025, page 25 / 46 7 / 21 peptides originating from various areas of the American continent showed sensitivity above 77% and specificity above 89% in assays performed with humans infected with T. cruzi, carriers of visceral leishmaniasis, and uninfected donors. In diagnostic validation assays, all peptides showed a large area under the curve (AUC) with values ​​above 0.9. Furthermore, the technology is inexpensive, since the peptides are small in size and do not necessarily need to be recombinantly expressed by modified bacteria; they can be chemically produced in an automated synthesizer. BRIEF DESCRIPTION OF THE FIGURES

[14] Figure 1 shows the antigenicity of peptides derived from conserved 80-mer consensuses using different human samples. On the left are individuals in the chronic phase of T. cruzi infection, in the middle individuals with visceral leishmaniasis, and on the right uninfected donors. Each point corresponds to a peptide synthesized in the nitrocellulose membrane. The reactivity of each is measured on a scale ranging from black (low), orange (medium), and white (high). Green circles indicate peptides whose densitometric value is above the cut-off (average of negatives + 2 * standard deviation of negatives) for Chagas disease patients and below for the other sample groups.

[15] Figure 2 demonstrates the recognition of soluble peptides by sera from humans with chronic Chagas disease (CD) and uninfected individuals. To the left of each graph are individuals with CD, in the center those with visceral leishmaniasis, and to the right those uninfected. The dashed line indicates the cutoff based on the ROC curve and Youden index, which had values ​​of 0.34; 0.79; 0.36; and 1.16 for NK4 (SEQ ID No1), NK6 (SEQ ID No2), NK8 (SEQ ID No3), and NK9 (SEQ ID No4), respectively. Petition 870250007999, dated 01 / 30 / 2025, p. 26 / 46 8 / 21

[16] Figure 3 represents the ROC curve of NK4 (SEQ ID No1), NK6 (SEQ ID No2), NK8 (SEQ ID No3) and NK9 (SEQ ID No4) peptides for the ELISA assay with patients in the chronic phase of T. cruzi infection and uninfected donors. The x-axis represents the false negative rate (100 - specificity) and the y-axis the true positive rate (sensitivity). The red line indicates the threshold for a sample to be randomly classified as positive or negative. DETAILED DESCRIPTION OF THE TECHNOLOGY

[17] The present technology deals with a kit and method for the serodiagnosis of Chagas disease using four synthetic peptides derived from conserved motifs present in polymorphic antigenic proteins expressed by the parasite from the analysis of a large number of isolates from different endemic areas of the American continent and show high sensitivity and specificity in samples from humans infected with T. cruzi. In addition, the technology is inexpensive, since the peptides are small in size, and it is not necessary for them to be recombinantly expressed by modified bacteria, and they can be chemically produced in an automated synthesizer.

[18] More specifically, the Chagas disease serodiagnosis kit is characterized by comprising: a) At least one of the synthetic peptides defined by SEQ ID No. 1 to 4; (b) A secondary antibody or a protein, where the secondary antibody (IgG, IgM, IgA, IgE and / or their respective subclasses), or the protein (protein A and / or protein G), is conjugated to an enzyme or a marker; c) A reagent to detect the enzyme or marker. Petition 870250007999, dated 01 / 30 / 2025, p. 27 / 46 9 / 21

[19] In item “a”, the peptides can be linked to a solid support, selected from the group of materials comprising nitrocellulose, nylon, latex, polypropylene and / or polystyrene; or to a carrier, preferably gold particles.

[20] In item “b”, the marker may be selected from the group comprising enzymes, radioisotopes, biotin, chromophores, fluorophores and chemiluminescents. The enzyme may be selected from the group comprising peroxidase, alkaline phosphatase, beta-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase.

[21] The method for serodiagnosis of Chagas disease comprises the following steps: a) Exposure of a sample to at least one of the synthetic peptides defined by the SEQ ID sequences No. 1 to 4, with such antigens bound to a solid support or carrier; b) Addition of an antibody or a protein, which are conjugated to an enzyme or a marker and which bind to the antibodies in the sample from step (a); c) Detection of specific antibodies for Chagas disease in the sample mentioned in step (a), using reagents capable of detecting the enzyme or marker mentioned in step (b).

[22] In step “a”, the sample may be selected from the group comprising blood, serum, plasma or other body fluid, without the need for large quantities thereof, and the solid support may comprise materials selected from the group comprising nitrocellulose, nylon, latex, polypropylene, polystyrene.

[23] In step “b”, the secondary antibody may comprise IgG, IgM, IgA, IgE and their respective subclasses; the protein may be Protein A and / or Protein G; the enzyme may be selected from the group comprising peroxidase, alkaline phosphatase, beta-galactosidase, urease, xanthine Petition 870250007999, dated 01 / 30 / 2025, page 28 / 46 10 / 21 oxidase, glucose oxidase, and penicillinase; and the marker may be selected from the group comprising enzymes, radioisotopes, biotin, chromophores, fluorophores, and chemiluminescent agents.

[24] In step “c”, the detection antibody can be selected from the group comprising fluorescence, immunofluorescence, immunoluminescence, absorbance or radioisotope detection.

[25] The synthetic peptides defined by SEQ ID No. 1 to 4 and the kit can be used for serodiagnosis of Chagas disease.

[26] The present invention can be better understood through the following examples, which are not limiting. EXAMPLE 1 - MAPPING, SELECTION AND SYNTHESIS OF SYNTHETIC PEPTIDES

[27] The present technology consists of obtaining serological markers capable of detecting IgG subclass antibodies in individuals chronically infected with T. cruzi, differentiating them from others affected by visceral leishmaniasis and from uninfected individuals. For this, 42 libraries of T. cruzi genomic DNA reads present in the Sequence Read Archive (SRA) of the National Center for Biotechnology Information (NCBI) were used, being 25 TcI, 8 TcII, 1 TcIII, 1 TcIV, 1 TcV and 6 TcVI. The reads were filtered using the Trimmomatic software (Anthony M. Bolger, Marc Lohse, Bjoern Usadel, Trimmomatic: a flexible trimmer for Illumina sequence data, Bioinformatics, Volume 30, Issue 15, August 2014, Pages 2114-2120, https: / / doi.org / 10.1093 / bioinformatics / btu170) using an average Phred quality of 25, a sliding window of 5 nucleotides, and a minimum size of 50 bases.

[28] Adapters, when present, were also removed using the same program. Libraries that had more than 50% of their reads that did not meet the criteria described above were Petition 870250007999, dated 01 / 30 / 2025, page 29 / 46 11 / 21 excluded. In addition, all had more than 70% of the trimmed reads mapped to reference genomes using BWA-mem v0.7.12r1039 software (LI, 2013). For this, assembled genomes of the DTUs TcI (clone SylvioX10 / 1), TcII (clone Y C6), TcIII (strain 231), TcIV (clone SylvioX10 / 1), TcV (clone CL Brener Esmeraldo-like haplotype) and TcVI (CL Brener Esmeraldo-like haplotype), obtained from version 62 of TriTrypDB (http: / / tritrypdb.org / ), were used as references.

[29] The number of reads mapped in the reference was verified using the flagstat command of the SAMtools v1.3.1 software (Heng Li, Bob Handsaker, Alec Wysoker, Tim Fennell, Jue Ruan, Nils Homer, Gabor Marth, Goncalo Abecasis, Richard Durbin, 1000 Genome Project Data Processing Subgroup, The Sequence Alignment / Map format and SAMtools, Bioinformatics, Volume 25, Issue 16, August 2009, pages 2078-2079, https: / / doi.org / 10.1093 / bioinformatics / btp352). Unmapped reads were also used in subsequent analyses to avoid the loss of repetitive sequences that may not be present in the assembled reference and, consequently, not be mapped.

[30] All libraries also had genomic coverage equal to or greater than 20 times. This value was estimated using CADIn software. In this process, only one assembled DTU genome corresponding to the reads in question was used as a reference. Since files in General Feature Format (GFF), necessary for program execution, were only available for TcI, TcII, and TcVI, the TcIII and TcIV libraries were mapped to the SylvioX10 / 1 clone (TcI). The TcV and TcVI samples were mapped to the Emerald-like haplotype of the CL Brener clone.

[31] The 80-nucleotide k-mers (80-mers) corresponding to the chosen reads were generated by counting using the count command of the Jellyfish v2.2.4 software (https: / / doi.org / 10.1093 / bioinformatics / btr011). Petition 870250007999, dated 01 / 30 / 2025, page 30 / 46 12 / 21 To avoid incorporating spurious 80-mers generated by sequencing errors, only those with a minimum occurrence equal to 30% of the genomic coverage value of their original sample were included in the analyses. Furthermore, to normalize the frequency of k-mers across samples, these values ​​were divided by the average genomic coverage of the corresponding libraries. In all rounds, the hash table considered was 275M (-s 275M) and 10 threads were used (-t 10). Additionally, since the direction of the reads was unknown, as they were joined and treated with single-end data, the -C subcommand was used as suggested by the program's manual. Thus, for any 80-mer m, its canonical representation is m itself or its reverse complement, whichever came first lexicographically. With the -C option, only the canonical representation of the 80-mers is stored in the hash, and the count value is the number of occurrences of the 80-mer and its reverse complement.The dump command was then used to convert the sequences to FASTA format, and their headers were modified using the replace command of the SeqKit v0.12.0 program (Shen W, Le S, Li Y, Hu F (2016) SeqKit: A CrossPlatform and Ultrafast Toolkit for FASTA / Q File Manipulation. PLoS ONE 11(10): e0163962. https: / / doi.org / 10.1371 / journal.pone.0163962) in order to point to the DTU and source library of each 80-mer.

[32] Next, the 80-mers from all samples were joined into a single FASTA file, redundancy between them was removed, and then they were clustered with CD-HIT-EST v4.8.1 (Limin Fu, Beifang Niu, Zhengwei Zhu, Sitao Wu, Weizhong Li, CD-HIT: accelerated for clustering the next-generation sequencing data, Bioinformatics, Volume 28, Issue 23, December 2012, Pages 3150-3152, https: / / doi.org / 10.1093 / bioinformatics / bts565; Weizhong Li, Adam Godzik, Cd-hit: a fast program for clustering and comparing large sets of protein Petition 870250007999, dated 01 / 30 / 2025, p. 31 / 46 13 / 21 or nucleotide sequences, Bioinformatics, Volume 22, Issue 13, July 2006, Pages 1658-1659, https: / / doi.org / 10.1093 / bioinformatics / btl158). A 95% cutoff was employed to evaluate sequences common to all DTUs that could be used as potential diagnostic targets. Only clusters with 80-mers from all evaluated isolates were considered conserved and included in the next steps. In all clustering, sequences were grouped to the first cluster that reached the cutoff (-d 0), with the alignment needing to cover at least 97% of the sequence size (-aL 0.97 and -aS 0.97), using 16 CPU threads (-T 16) and 75000 GB of RAM (-M 75000).

[33] The MAFFT v7.427 software (Kazutaka Katoh, Daron M. Standley, MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability, Molecular Biology and Evolution, Volume 30, Issue 4, April 2013, Pages 772-780, https: / / doi.org / 10.1093 / molbev / mst010), with the adjustdirectionaccurately subcommand enabled, was used to perform a multiple alignment with the sequences of each cluster and a consensus was generated with the consensus function of the seqinr v4 package. 2.16 (Charif, D., Lobry, JR (2007). SeqinR 1.0-2: A package contributed to the R project for Statistical Computing Devoted to Biological Sequences Retrieval and Analysis. https: / / doi.org / 10.1007 / 978-3-540-35306-5_10) of R.A minimum relative frequency (threshold) of 90% was defined to indicate a nucleotide as consensus at a given position. If this was not the case, an "N" was returned. Due to alignment, "N"s and "-"s were frequently returned at the ends. When this... Petition 870250007999, dated 01 / 30 / 2025, pp. 32 / 46 If the 14 / 21 rule was met, these were removed, reducing the sequence size. Therefore, those with a size smaller than 70 nucleotides were excluded. Consensus sequences with "N"s in internal positions were also not considered.

[34] To reduce the probability of cross-reactivity in diagnostic tests, a Nucleotide BLAST (blastn) (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) was performed against the NCBI Nucleotide collection (nr / nt) non-redundant sequence database, considering the following algorithm parameters: short queries selected, expected threshold word size of 0.05, max matches in a query range of 0, matches / mismatch scores of 1,-2, gap costs linear, filter selecting the low complexity regions option and mask selecting mask for lookup table only.

[35] The first 1000 matches were returned for each evaluated sequence. Consensus clusters with any matches with organisms other than Trypanosoma cruzi (taxid: 5693), Trypanosoma cruzi marinkellei (taxid: 85056), Trypanosoma cruzi subsp.marinkellei (taxid: 85056), Trypanosoma cruzi dionisii (taxid: 78083) and Trypanosoma cruzi cruzi (taxid: 85057) had their headers identified with an “others” tag, and those that did not have matches only for these were marked with “tcruzi”. Subsequently, a CD-HIT was performed with 50% identity and other parameters as described above, and clusters with sequences marked with “others” were excluded, while those formed only by sequences with “tcruzi” were retained.

[36] The consensuses that passed this stage were submitted to a blastx v2.2.30 locally against the proteomes of SylvioX10 / 1, Y C6, CL Brener Emerald-like and Non Emerald-like. Only matches with identity and coverage above 90% were considered. A bed file was generated from the blastx output table, containing information from Petition 870250007999, dated 01 / 30 / 2025, pp. 33 / 46 15 / 21 sequence name, start and end match coordinates, and reference protein. The bedtools getfasta command was used to retrieve amino acid sequences from the reference proteomes.

[37] A blastp was performed locally using the nr / nt database as a reference to once again try to reduce cross-reactivity with other organisms. The parameters used were based on the online version of the NCBI: - task blastp-short -gapopen 9 -max_target_seqs 1000 -word_size 2 -matrix PAM30 -threshold 16 -comp_based_stats 0 evalue 1000 -window_size 15 -num_threads 20. The process of marking the headers and running CD-HIT was also performed after this step. Finally, the selected peptides were fragmented into 15-amino acid portions with a 2-residue sliding window using the sliding command of SeqKit v0.12.0 (Shen W, Le S, Li Y, Hu F (2016) SeqKit: A Cross-Platform and Ultrafast Toolkit for FASTA / Q File Manipulation. PLoS ONE 11(10): e0163962. (https: / / doi.org / 10.1371 / journal.pone.0163962). Sequences shorter than 15 amino acids were not fragmented and were kept in their original form.

[38] The peptides were then synthesized using a ResPep SL automated synthesizer (Intavis) and the MultiPep program (Intavis), according to the SPOT technique (Frank, R. (1992) Tetrahedron 42, 92179232). Briefly, the peptides were synthesized in spots on a nitrocellulose membrane, starting from the C-terminal of the last amino acid. The Fmoc group was removed with 25% 4-methylpiperidine in dimethylformamide (DMF). The amino acids were activated using 1.1 M Oxyma Pure DIC (diisopropylcarbodiimide) and deposited onto the membrane in two coupling cycles without a further deprotection step between them. The free amine residues were acetylated with acetic anhydride in 20% DMF. The new Fmoc protecting group of Petition 870250007999, dated 01 / 30 / 2025, pp. 34 / 46 The 16 / 21 linked amino acid was then removed with 25% 4-methylpiperidine in DMF. These steps were repeated until all amino acids were added. At the end of the synthesis, the membrane was submerged for one hour in a cleavage solution composed of 95% (v / v) trifluoroacetic acid (TFA) associated with 2.5% (v / v) water and 2.5% (v / v) triisopropylsilane (TIPS) to remove the protecting groups from the amino acid side chains. Afterwards, the membrane was washed 4 times with dichloromethane (DCM), 4 times with DMF, and 2 times with ethanol. The membrane was dried and the spots checked under ultraviolet light. Known functional peptides, obtained from the work of Ricci et al. 2023 (Ricci, AD, Bracco, L., Salas-Sarduy, E. et al. The Trypanosoma cruzi Antigen and Epitope Atlas: antibody specificities in Chagas disease patients across the Americas. Nat Commun 14, 1850 (2023). (https: / / doi.org / 10.1038 / s41467-023-37522-9), were used as reactivity controls, located in rows 1 to 18 of the first column of each membrane.

[39] The antigenicity of the membrane peptides was evaluated separately with three groups of serum pools from individuals in the state of Minas Gerais - Brazil: individuals in the chronic phase of DC without parasite genotyping, individuals with visceral leishmaniasis and uninfected donors.

[40] In summary, the membrane was initially blocked with a solution of PBS + 5% BSA and 4% sucrose for 16 h, washed with PBS + 0.1% Tween 20 and incubated with the primary antibody (serum pools) diluted at a ratio of 1:1,000 in washing solution for two hours at 37 °C. It was then washed again and incubated with anti-human IgG conjugated to peroxidase (Sigma-Aldrich) diluted 1:10,000 in washing solution for one hour at 37 °C. The membrane was washed again and visualized by chemiluminescence using the developer. Petition 870250007999, dated 01 / 30 / 2025, pages 35 / 46 17 / 21 Immobilon Forte Western HRP (Merck) was exposed for 30 seconds on the ImageQuant LAS 4000 digital imaging system (GE Healthcare). After development, the membrane was regenerated for testing with a new group of samples. Three 10-minute washes with N,N-dimethylformamide were performed, followed by incubation with a denaturing solution (8 M urea + 1% sodium dodecyl sulfate) for 16 hours. Then, two half-hour washes with denaturing solution, one two-minute wash with deionized water, and three 10-minute washes with a solution of 55% ethanol, 35% deionized water, and 10% acetic acid were performed. Finally, one more wash with deionized water and two with absolute ethanol were performed, and the membrane was then allowed to air dry at room temperature for reuse.

[41] The densitometric value of each spot was calculated using ImageJ software (Schneider, C., Rasband, W. & Eliceiri, K. NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9, 671-675 (2012). https: / / doi.org / 10.1038 / nmeth.2089) with the Protein Array Analyser plug-in (http: / / image.bio.methods.free.fr / ImageJ / ?Protein-Array-Analyzer-forImageJ.html). The minimum reactivity cut-off was 17,672.72, based on the mean plus twice the standard deviation of the value of all spots on the membrane evaluated with uninfected individuals. Peptides that had a value above the cut-off in the assay with sera from patients infected with T. cruzi and below with negative and LV samples were considered.

[42] Table 1 shows the position, sequence, densitometry values ​​for each sample group as well as the ratio between them for the 12 peptides, named NK1 to NK12, selected and highlighted in green circles in Figure 1. They were synthesized on a solid-phase resin at a scale of 25 μmol also on the ResPep SL synthesizer. In short, the Fmoc-amino acid conjugate was activated with a Petition 870250007999, dated 01 / 30 / 2025, pages 36 / 46 18 / 21 pure oxime solution (Merk) and DIC (Sigma-Aldrich). The activated amino acids were incorporated into an H-Rink Amide ChemMatrix resin (Sigma-Aldrich). Deprotection of the Fmoc group was performed using 25% 4-methylpiperidine (25% v / v in DMF). The steps were repeated until the end of the peptide sequence. The peptides were released from the resin by treatment with a solution of 92.5% trifluoroacetic acid, 2.5% water, 2.5% triisopropyl silane, and 2.5% beta-mercaptoethanol under stirring for 3 h. Then the peptides were precipitated with methyl tert-butyl ether and lyophilized. The synthesized peptides were characterized using an Autoflex III Mass Spectrometer (Bruker Daltonics™). For the analysis, 0.5 μL of the concentrated sample was mixed with 0.5 μL of a saturated matrix solution of 10 mg / mL α-cyano-4-hydroxycinnamic acid (Sigma-Aldrich™) in 50% acetonitrile / 0.1% trifluoroacetic acid.The samples were applied to an MTP AnchorChip™ 600 / 384 plate (Bruker Daltonics™) and dried at room temperature. Each spectrum was produced by accumulating data from 200 consecutive laser shots using a positive / reflector mode controlled by FlexControl™ 3.3 software. Instrument calibration was performed using reference peptides (Peptide Standard, Bruker Daltonics). Table 1. Densitometry and molecular weight of the 12 best peptides in the different sample groups evaluated in immunoblotting. Peptide Membrane Position Densitometry Chagas Negative ss Ratio Chagas / Negative Chagas / LV Molecular Weight NK1 1 L20_C7 28241 6675 8955 3.15 4.23 1551 NK2 1 L19_C8 21844 5202 6997 3.12 4.2 1625 NK3 1 L9_C10 21018 9842 8424 2.5 2.14 1567 NK4 (SEQ ID No1) 1 L3_C19 23287 9323 123 189 2.5 1680 Petition 870250007999, dated 01 / 30 / 2025, pp. 37 / 46 19 / 21 NK5 L18_C1 9 30029 7701 584 51.4 3.9 1592 NK6 (SEQ ID No2) L13_C2 0 21831 1236 215 102 17.7 1632 NK7 L18_C2 0 24000 3926 523 45.9 6.11 1539 NK8 (SEQ ID No3) 1 L9_C21 19220 1346 7 2705 7.11 1.43 1666 NK9 (SEQ ID No4) L16_C2 2 24809 6645 9581 2.59 3.73 1655 NK10 L16_C2 3 20629 8582 12903 1.6 2.4 1717 NK11 L19_C2 5 20798 9022 15917 1.31 2.31 1655 NK12 L20_C2 6 19143 9438 16666 1.15 2.03 1629 EXAMPLE 2 - DIAGNOSTIC VALIDATION TESTS OF SYNTHETIC PEPTIDES

[43] The antigens were evaluated individually in triplicates of sera from individuals infected with T. cruzi without parasite genotyping, with visceral leishmaniasis and uninfected donors. Each well of a Costar Polystyrene Half-Area high-binding ELISA microplate was sensitized with 0.2 pg / well diluted in 100 pL of sodium carbonate buffer pH 9.6 and incubated for 16 h at 37°C. The plates were manually washed four times with PBS + 0.05% Tween 20 and then blocked with PBS + 5% BSA for 1 h at 37°C. After that, the individual serum samples diluted 1:100 in PBS + 2.5% BSA, final volume of 25 pL, were added and incubated for one hour at 37°C. Next, the plates were washed as previously described, incubated with 25 pL of peroxidase-conjugated anti-human IgG antibody (Sigma-Aldrich), diluted at a ratio of 1:10,000 in PBS + 2.5% BSA for 1 hour at 37°C.Next, the plates were washed, incubated with 25 pL of developing solution (0.1M citrate buffer and 0.2M phosphate buffer) pH 5.0 for 15 min, and then 12.5 pL of... Petition 870250007999, dated 01 / 30 / 2025, pp. 38 / 46 20 / 21 H2SO4 4N was added to stop the reaction. Absorbance readings were taken at 492 nm using a Thermo Scientific™ Multiskan™ FC Microplate Photometer.

[44] For ELISA, duplicates had their mean and standard deviation calculated, with those with a standard deviation less than 0.2 being retained. The cut-off was calculated based on the Receiver Operating Characteristic (ROC) curve, with a 95% confidence interval to estimate the area under the curve, and using the Youden index (IY) (sensitivity (%) + specificity (%) - 100) as a metric (Lai CY, Tian L, Schisterman EF. Exact confidence interval estimation for the Youden index and its corresponding optimal cut-point. Comput Stat Data Anal 2012;56(5):1103e14) with the cutpointr function from the cutpointr v1.1.2 package (Thiele, C., & Hirschfeld, G. (2021). cutpointr: Improved Estimation and Validation of Optimal Cutpoints in R. Journal of Statistical Software, 98(11), 1-27. https: / / doi.org / 10.18637 / jss.v098.i11) in R. This tool only accepts two sample groups for its analysis.Thus, individuals with visceral leishmaniasis were included in the negative group, since they are not the primary target for antigen detection. Positive predictive values ​​(PPV), negative predictive values ​​(NPV), and accuracy were also calculated.

[45] In Figure 2, it can be observed that for the four peptides evaluated, most samples from positive individuals are above the cutoff line, while most of those with visceral leishmaniasis and uninfected donors are below this line. In addition, all presented a large area under the curve (AUC) with values ​​above 0.9 (Figure 3 and Table 2). Positive and negative predictive values, as well as accuracy, were considered satisfactory. Petition 870250007999, dated 01 / 30 / 2025, pp. 39 / 46 21 / 21 Table 2. Diagnostic parameters of the ELISA for the four peptides Soluble derivatives of 80-mers preserved with improved parameters. Sequence VPP VPN Sensitivity AUC Specificity Accuracy Youden Index NK4 (SEQ ID No1) LREIDDSLSSSAW VC 0.878 8 0.897 4 0.8788 0.8974 0.938 0 0.8889 0.7762 NK6 (SEQ ID No2) LREIDGSLSSPAW VC 0.803 0 0.974 4 0.8030 0.9744 0.934 5 0.8958 0.7774 NK8 (SEQ ID No3) LREIDGSLSSSAW VF 0.863 6 0.948 7 0.8636 0.9487 0.947 6 0.9097 0.8124 NK9 (SEQ ID No4) LRKIDGSFSSSAW VC 0.772 7 1 0.7727 1 0.925 9 0.8958 0.7727 PPV: positive predictive value; NPV: negative predictive value; AUC: area under the curve.

Claims

1. A kit for serodiagnosis of Chagas disease, characterized by comprising: a) At least one of the synthetic peptides defined by SEQ ID No. 1 to 4; b) A secondary antibody or a protein, wherein the secondary antibody (IgG, IgM, IgA, IgE and / or their respective subclasses), or the protein (protein A and / or protein G), conjugated to an enzyme or a marker; c) A reagent for detecting the enzyme or marker.

2. A KIT, according to claim 1, characterized in that, in item “a”, the peptides are linked to a solid support, selected from the group of materials comprising nitrocellulose, nylon, latex, polypropylene and / or polystyrene; or to a carrier, preferably gold particles.

3. A KIT, according to claim 1, characterized in that, in item “b”, the marker is selected from the group comprising enzymes, radioisotopes, biotin, chromophores, fluorophores and chemiluminescents; and the enzyme is selected from the group comprising peroxidase, alkaline phosphatase, beta-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase.

4. METHOD FOR SERODIAGNOSIS OF CHAGAS DISEASE using the kit defined in claim 1, characterized by comprising the following steps: a) Exposure of a sample to at least one of the synthetic peptides defined by the SEQ ID sequences No. 1 to 4, such antigens being linked to a solid support or carrier; Petition 870250007999, dated 01 / 30 / 2025, p. 41 / 46 2 / 2 b) Addition of an antibody or a protein, which are conjugated to an enzyme or a marker and which bind to the antibodies in the sample from step (a); c) Detection of specific antibodies for Chagas disease in the sample mentioned in step (a), using reagents capable of detecting the enzyme or marker mentioned in step (b).

5. METHOD, according to claim 4, characterized in that, in step “a”, the sample is selected from the group comprising blood, serum, plasma or other body fluid, without the need for large quantities thereof, and the solid support comprises materials selected from the group comprising nitrocellulose, nylon, latex, polypropylene, polystyrene.

6. METHOD, according to claim 4, characterized in that, in step “b”, the secondary antibody comprises IgG, IgM, IgA, IgE and their respective subclasses; the protein is Protein A and / or Protein G; the enzyme is selected from the group comprising peroxidase, alkaline phosphatase, beta-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase; and the marker is selected from the group comprising enzymes, radioisotopes, biotin, chromophores, fluorophores and chemiluminescents.

7. METHOD, according to claim 4, characterized in that, in step “c”, the detection antibody is selected from the group comprising fluorescence, immunofluorescence, immunoluminescence, absorbance or radioisotope detection.

8. USE of synthetic peptides defined by SEQ ID No. 1 to 4, characterized by being in the serodiagnosis of Chagas disease.

9. USE of the kit defined in claim 1, characterized by being in the serodiagnosis of Chagas disease.

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