WO2015056244A1 - Kit and immunodiagnostic method for detecting anaemia caused by vivax malaria, synthetic peptides and uses - Google Patents

Kit and immunodiagnostic method for detecting anaemia caused by vivax malaria, synthetic peptides and uses Download PDF

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WO2015056244A1
WO2015056244A1 PCT/IB2014/065429 IB2014065429W WO2015056244A1 WO 2015056244 A1 WO2015056244 A1 WO 2015056244A1 IB 2014065429 W IB2014065429 W IB 2014065429W WO 2015056244 A1 WO2015056244 A1 WO 2015056244A1
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malaria
vivax
immunodiagnostic
detecting
peptide
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PCT/IB2014/065429
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French (fr)
Portuguese (pt)
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Carlos DELFIN CHÁVEZ OLÓRTEGUI
Ricardo ANDREZ MACHADO DE ÁVILA
Erika MARTINS BRAGA
Ingrid Carla DE OLIVEIRA
Luiza CARVALHO MOURÃO
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Universidade Federal De Minas Gerais - Ufmg
Fundação De Amparo À Pesquisa Do Estado De Minas Gerais- Fapemig
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Priority claimed from BR102013026897A external-priority patent/BR102013026897A2/en
Application filed by Universidade Federal De Minas Gerais - Ufmg, Fundação De Amparo À Pesquisa Do Estado De Minas Gerais- Fapemig filed Critical Universidade Federal De Minas Gerais - Ufmg
Priority claimed from BR102014025967-8A external-priority patent/BR102014025967B1/en
Publication of WO2015056244A1 publication Critical patent/WO2015056244A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56905—Protozoa
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/445—Plasmodium
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/44—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from protozoa
    • G01N2333/445—Plasmodium
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present technology describes a kit and an immunodiagnostic method for the detection of Plasmodium vivax malaria using as a marker antigen a peptide (SEQ ID NQ 1) that composes Plasmodium vivax merozoite surface protein 1 (PvMSP). -1), or even this polymerized peptide.
  • SEQ ID NQ 1 a marker antigen a peptide that composes Plasmodium vivax merozoite surface protein 1 (PvMSP). -1), or even this polymerized peptide.
  • PvMSP Plasmodium vivax merozoite surface protein 1
  • Malaria is caused by parasites of the genus Plasmodium, belonging to the phylum Apicomplexa, family Plasmodidae. Five species can infect humans - Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and currently Plasmodium knowlesy, which, although described as a simian parasite, have now been reported in human infections (COX-SINGH, J .; DAVIS, TME; LEE, KS; SHASUL, SSG; MATUSOP, A.; RATNAM, S.; RAHMAN, HA; CONWAY, DJ; SINGH, B.
  • Plasmodium knowlesi malaria in Humans is widely distributed and potentially life threatening. , v. 46, no. 2, pp. 165-171, 2008; TILLEY, L.; DIXON, MWA; KIRK, K. The International Journal of Biochemistry & Cell Biology, v. 43, 839-842, 2011).
  • falciparum accounts for about 20% and P. malariae for less than 1% of occurrences (OLIVEIRA- FERREIRA, J .; LACERDA, MVG; BRAZIL, P.; LADISLAU, JLB; TAUIL, PL; DANIEL-RIBEIRO, CT Malaria in Brazil: in the overview. Malaria Journal, v. 9, No. 1 15, 2010).
  • P. falciparum is the species responsible for the highest mortality - about 95% of all reported deaths (WORLD HEALTH ORGANIZATION (WHO). World Malaria Report, 201 1)
  • P. vivax is the most common (KASLIWAL RAO, MS; KUJUR, R. Plasmodium vivax malaria: an unusual presentation Indian Journal of Critical Care Medicine, v.13, p.103-105, 2009) and has a wide geographic distribution, with about 2.6 billion people in South and Southeast Asia, Central America and South America at risk of contracting the disease (HULDEN, L; HULDEN, L. Activation of hipnozoyte: part of Plasmodium Vivax Wie cycle and survive Malaria Journal, v.
  • the Plasmodium vivax is able to complete its cycle esporogônico at relatively low temperatures (around 16 Q C), and this fact has contributed substantially to its success in establishing stable foci transmission, not only in tropical areas, but also in temperate zone locations (CUI, L; ESCALANTE, AA; IMWONG, M .; SNOUNOU, G.
  • C temperate zone locations
  • Symptoms of vivax malaria are similar to those caused by other plasmodia and include fever with chills, headache, weakness, diarrhea, vomiting, pallor, splenomegaly and hepatomegaly (KASLIWAL, P .; RAO, MS; KUJUR, R. Plasmodium vivax malaria: an unusual presentation Indian Journal of Critical Care Medicine, v. 13, pp. 103-105, 2009; ECHEVERRI, M .; TOB ⁇ N, A.; ALVAREZ, G .; CARMONA, J .; BLAIR S. Clinical and laboratory findings of Plasmodium vivax malaria in Colombia, 2001. Journal of the Institute of Tropical Medicine, v. 45, no. 1, pp. 29-34, 2003).
  • Hematological changes such as anemia, leukopenia and thrombocytopenia are also reported (ECHEVERRI et al., 2003; SONG, HH; O, SO; KIM, SH; MOON, SH; KIM, JB; YOON, JW; KOO, JR; HONG, KS; LEE, MG; KIM, DJ; SHIN, DH; KANG, SH; CHOI, MG; LEE, KH Clinical features of Plasmodium vivax malaria The Korean Journal of International Medicine, v.18, n.4, p.220 -224, 2003).
  • Vivax malaria is no longer a benign disease and in some cases has clinical profiles similar to those seen in severe P. falciparum malaria. Signs and symptoms observed include renal failure, respiratory syndrome, and especially severe anemia (KOCHAR, DK; SAXENA, V.; SINGH, N .; KOCHAR, SK; KUMAR, SV; DAS, A. Plasmodium vivax malaria. Emerging Infectious Diseases , v. 11, pp. 132-134, 2005; ANSTEY, NM; RUSSELL, B.; YEO, TW; PRICE, RN The pathophysiology of vivax malaria Trends in Parasitology, v. 25, no.
  • BRAGA EM
  • SOURCES CJF Plasmodium - Malaria.
  • NEVES NEVES
  • MELO A.L
  • LINARDI PM
  • VITOR RWA Human Parasitology. 12th ed. S ⁇ o Paulo: Publisher Atheneu, 201 1
  • BRAZIL Ministry of Health. Health Surveillance Department of Epidemiological Surveillance Guide for Epidemiological Surveillance 7. ed. Brasilia: Ministry of Health, 2009. 816 P. (Series A. Standards and Technical Manuals)).
  • Immunochromatographic tests represent novel methods of rapid diagnosis of malaria by detecting antigenic components of plasmodium. These tests are useful for screening or even diagnostic confirmation, especially in remote areas or areas that are difficult to access to health services, because they are quick and easy to perform (BRAZIL. Ministry of Health. Health Surveillance Secretariat. Surveillance Department Epidemiological Guide to Epidemiological Surveillance 7th ed Brasilia: Ministry of Health, 2009. 816 pp. (Series A. Technical Standards and Manuals)). These methods utilize monoclonal and polyclonal antibodies directed against P. falciparum histidine-rich protein 2 (PfHRP-2) and against lactate dehydrogenase enzyme (pDHL) of the four plasmid species.
  • PfHRP-2 P. falciparum histidine-rich protein 2
  • pDHL lactate dehydrogenase enzyme
  • pDHL is an intracellular enzyme produced in abundance by living parasites, which allows differentiating between acute and convalescent phase of infection.
  • a disadvantage is that it does not allow the diagnosis of a mixed infection (BRAGA, EM; SOURCES, CJF Plasmodium - Malaria. In: NEVES, DP; MELO, A.; LINARDI, PM; VICTOR, RWA. Human Parasitology. 12 Ed S ⁇ o Paulo: Publisher Atheneu, 201 1).
  • a more specific method is required to diagnose cases of malaria in anasic patients infected with Plasmodium vivax, as the decision on how to treat the patient should be preceded by information regarding disease severity, plasmodium species, age. history of exposure prior to infection, as well as susceptibility of parasites to conventional antimalarial drugs (BRAZIL. Ministry of Health. Department of Health Care. Department of Primary Care. Health Surveillance: Dengue, Schistosomiasis, Leprosy, Malaria, Trachoma and Tuberculosis. 2nd ed. rev., Brasilia: Ministry of Health, 2008. 197 p .: il. (Series A. Standards and Technical Manuals) (Primary Care Notebooks, # 21)).
  • biomarkers can provide important information about the patient's biological state, degree of morbidity and disease prognosis. Biomarkers capable of differentiating Plasmodium species or effective in determining malaria prognosis could expedite treatment and, if employed in rapid diagnostic tests, facilitate early detection of disease in difficult to reach endemic regions. In addition, they could have a positive impact on control and prevention measures.
  • Biomarkers may be derived from the parasite or host, or a combination thereof, including any measurable and evaluated substance (CONROY et al., 2012; LUCCHI, NW; JAIN, V .; WILSON, NO; SINGH, N UDHAYAKUMAR, V; STILES, JK Potential serological biomarkers of cerebral malaria. Disease Markers, v. 31, no. 6, pp. 327-335, 2011).
  • Plasmodium Genome Sequencing vivax has enabled many advances in research, including strain differentiation and characterization. Unlike P. falciparum, which has hundreds of sequenced isolates, only the P. vivax Salvador I and IQ07 (Peruvian Isolate) strains are complete (CARLTON, JM; ADAMS, JH; SILVA, J. C; BIDWELL, S.
  • PAULSEN I.; WHITE, O .; RALPH, SA; REN, Q .; SARGEANT, TJ; SALZBERG, S.; STOECKERT, C; SULLIVAN, SA; YAMAMOTO, MM; HOFFMAN, S.; WORTMAN, JR; GARDNER, MJ; GALINSKI, MR; BARNWELL, JW; FRASER-LIGGETT, CM Comparative genomics of the neglected human malaria parasite Plasmodium vivax Nature, No.
  • MSP merozoite surface proteins
  • MSP-1 merozoite surface protein 1
  • EGAN AF; BURGHAUS, P.; DRUILHE, P.; HOLDER, AA; RILEY, E Human antibodies to the 19 kDa C-terminal fragment of Plasmodium falciparum merozoite surface protein 1 inhibit parasite growth in vitro Parasite Immunology, v. 21, pp. 133-139, 1999; STOWERS, AW; CIOCE, R. L; LAWSON , M .; HUI, G .; MURATOVA, O .; KASLOW, D. C.
  • MSP-1 Sal-I comprises 1751 amino acids and is frequently tested for vaccine candidate (GIBSON, H.L; TUCKER, JE; KASLOW, D.C; KRETTLI, AU; COLLINS, WE; KIEFER, M.C; BATHURST, I.C.
  • peptides are synthesized, simultaneously on a cellulose membrane using special amino acids protected by the Fmoc group (Fluorenyl Methyl Oxicarbonyl) which are deposited on the automatic synthesizer (LAUNE, D .; MOLINA, F .; FERRI ⁇ RIS, G .; VILLARD, S .; B ⁇ S, C; RIEUNIER, F .; CHARD ⁇ S, T .; GRANIER C.
  • LAUNE D .
  • MOLINA F .
  • F FERRI ⁇ RIS G .
  • VILLARD S .
  • B ⁇ S C
  • RIEUNIER F .
  • CHARD ⁇ S T .
  • GRANIER C Application of the Spot method to the Identification of peptides and amino acids from the paratope antibody that contributes to antigen binding.Journal Immunol Methods, v. 267, p 53-70, 2002).
  • This mapping allows the identification of immunogenic protein epitopes, as well as the characterization of the reactivity profile of different epitopes against sera from different groups of infected or uninfected patients. Given the impossibility of in vivo cultivation of P. vivax, chemical synthesis of peptides is an important tool for the development of molecular technologies that enable the diagnosis of malaria caused by this parasite.
  • SEQ ID NQ 1 a 15 amino acid peptide (SEQ ID NQ 1) has been identified, the sequence of which is derived from the surface protein of Plasmodium vivax merozoite 1, which is selectively reactive only to sera from patients with anemia due to P. vivax infection, showing no reactivity to sera from non-anemic patients infected with this parasite, as well as to sera from patients infected with P. P. falciparum, whether anemic or not, and patients not infected with anemia or not.
  • the breakthroughs of the present technology are based on spot-synthesis identification of a peptide originating from the protein PvMSP-1 (SEQ ID NQ 1) which is useful as a biomarker in a kit.
  • immunodiagnosis for the detection of anemia caused by vivax malaria This type of diagnosis is of paramount importance for the prognosis of the disease as well as to guide the choice of the appropriate treatment to be employed, considering that anemia is one of the most important, if not the most important, clinical manifestations observed in the severe forms of the disease.
  • vivax malaria (CHANG, KH; STEVENSON, MM Malaria anemia: mechanisms and implications of insufficient erythropoiesis during blood-stage malaria. International Journal for Parasitology, v.
  • Patent and patent documents dealing with the use of proteins and peptides in malaria immunodiagnosis have been found in the prior art.
  • WO0068270 entitled "Antibodies and peptides for detection of Plasmodum vivax” describes new peptides whose basic amino acid sequence corresponds to a 12-amino acid sequence of P. vivax ESP-1 which repeats 3 times in the protein, as well as antibodies produced by mammals in response to this antigen.
  • the application further encompasses immunodiagnostic assays and kits employing such antibodies.
  • the present technology differs from that presented in the cited document, since the protein that originated the marker peptide for vivax malaria is PvMSP-1 and not PvESP-1.
  • US8030471 entitled “Plasmodium malar ⁇ ae and Plasmodium ovale genes and uses thereof", describes nucleotide and amino acid sequences of MSP-1 from Plasmodium species, namely P. malar ⁇ ae and P. ovale, as well as methods for detection of antibodies to such plasmid species by using the proteins protected by the patent. Greater than 90% identity of the peptide of this patent application has been observed with two protein sequences protected by the aforementioned document, however these sequences refer to polypeptides with approximate amino acid number of 1,700, ie it does not deal with small peptides such as This is the case with this application.
  • US7931908 entitled “Chimeric MSP-based malaria vaccine” describes nucleotide and amino acid sequences of P. vivax and P. falciparum as well as the fusion thereof to form a chimeric protein for the purpose of obtaining an antimalarial vaccine. Greater than 90% identity of the peptide of the present application has been observed with a protein sequence protected by the above document. However, in addition to the protected sequence referring to a polypeptide with approximately amino acid number of 1,700 and not small peptides, as in the present application, there is also the fact that it is a chimeric protein for vaccination purpose. that is not claimed in the present technology.
  • US20041321 17 entitled “Immunoassay and diagnostic reagent for malaria” deals with an immunoassay for the diagnosis of malaria which detects specific malaria antibodies in the blood using Plasmodium vivax Merozoite Surface Protein.
  • This patent application further describes obtaining such protein by means of transformed yeast or Escherichia coli. Unlike the present technology, this document deals with the complete sequence of PvMSP and not specific small peptides extracted from this sequence.
  • EP035461 1 entitled "Synthtic antigens useful in the diagnosis of malaria induced by Plasmodium vivax” describes the use of new synthetic antigens for the detection of P. vivax anti-sporozoite antibodies. in human blood samples by an enzyme immunoassay employing such antigens. These antigens consist of peptides originating from the circumsporozoite protein of P. vivax, and are useful in immunodiagnostic methods and immunodiagnostic kit for detection of antibodies with high specificity, sensitivity and rapidity. Because it deals with peptides originating from the circumsporozoite protein of P. vivax and not from the surface protein of merozoite (MSP), this document is not similar to the present technology.
  • MSP surface protein of merozoite
  • US2007036818 entitled "Recombinant protein containing a C-terminal fragment of Plasmodium MSP-1", which is a continuation of US6958235, deals with a recombinant protein that includes the 19 kDa C-terminal protein fragment. of the merozoite of an infectious Plasmodium parasite in humans other than P. vivax.
  • MSP-1 19 was confirmed, however the results showed no statistically significant difference between reactivity indices obtained for sera from infected patients and individuals never exposed to malaria, as will be described in the examples below. This demonstrates that this fraction of MSP-1 is not the most satisfactory for developing a malaria biomarker.
  • US2010166794 entitled “Plasmodium liver stage antigens”, covers peptides, immunogenic compositions, sequences of nucleotides as well as the use of the compositions for the diagnosis of malaria in the liver state and induction of the immune response.
  • This document lists peptides derived from proteins specifically expressed in the liver phase of Plasmodium infection. Among these proteins is not included MSP-1, which differs from this document of this patent application.
  • EP1526178 entitled “MSP-3-like family of genes” describes the use of Plasmodium merozoite-3 (MSP-3) surface protein-derived peptides in protection against malaria. This document differs from the present technology in that the peptide sequences originate from distinct proteins and therefore do not coincide.
  • the present technology differs from the others found in the prior art for dealing specifically with a peptide (SEQ ID NO: Q 1) and / or its polymer originated from the sequence of MSP-1 of P. vivax and not the complete sequence of this protein, or any other P. vivax protein. It is also noteworthy that the peptides protected by the above patents or patent applications do not correspond to the peptide sequence of the present application. The technology is also distinguished by addressing the differential diagnosis of anemia due to P. vivax.
  • Figure 1 represents membrane I, with the result of immunodetection.
  • A Using pool of sera from anemic individuals - infected by P. vivax (IAN); infected with P. falciparum (PfAN); not infected with microcytic and hypochromic anemia (NIM) and infected with normocytic and normochromic anemia (NIN).
  • B Using pool of sera from non-anemic individuals infected with P. vivax (INA); infected with P. falciparum (PfNA); and uninfected (NINA).
  • Figure 2 Represents the Spot Reactivity Index (RI). Red dashes represent the medians. *** indicates P ⁇ 0.0001 compared to other groups (Kruskal-Wallis one way ANOVA).
  • IAN P. vivax-infected anemic patients;
  • INA Non-anemic patients infected with P. vivax;
  • PFAN Anemic patients infected with P. falciparum;
  • NAFL Non-anemic patients infected with P. falciparum;
  • NIM Uninfected individuals with microcytic and hypochromic anemia;
  • NIN Uninfected individuals with normocytic and nomochromic anemia.
  • IAN P. vivax-infected anemic patients;
  • INA Non-anemic patients infected with P. vivax;
  • PFAN Anemic patients infected with P. falciparum;
  • NAFL Non-anemic patients infected with P.
  • Figure 4 Represent the reactions of different sera against peptide 70 (SEQ ID NO: 1) (arrow). All sera from P.vivax (IAN) infected anemic patients were seropositive to this peptide, unlike the other populations.
  • IAN P. vivax-infected anemic patients
  • INA Non-anemic patients infected with P. vivax
  • PFAN Anemic patients infected with P. falciparum
  • NAFL Non-anemic patients infected with P. falciparum
  • NIM Uninfected individuals with microcytic and hypochromic anemia
  • NIN Uninfected individuals with normocytic and nomochromic anemia
  • NINA Uninfected non-anemic individuals.
  • Figure 5 represents the association between MSP1 peptide 70 absorbance (SEQ ID NO: 1) and P. vivax anemia.
  • IAN Anemic individuals infected with P. vivax.
  • INA Non-anemic individuals infected with P. vivax.
  • NINA uninfected and non anemic individuals.
  • an antigenic peptide originated from Plasmodium vivax protein PvMSP-1, as well as its use in the immunodiagnosis of anemic patients due to P. vivax infection, is comprised.
  • Immunodiagnostics may be performed by a serological test, which may be ELISA, Western-Blot, dot-blot, immunodiffusion or immunochromatographic.
  • This invention further describes the polymeric form of peptide 70 (SEQ ID NO: 1) and the use of this polymeric peptide in the immunodiagnosis of anemic patients due to P. vivax infection. Polymerization of this peptide may be obtained, without limitation, by the use of glutaraldehyde. Immunodiagnosis may be performed by a serological test, which may be ELISA, Western-Blot, dot-blot, immunodiffusion or immunochromatographic.
  • kits for immunodiagnosis of vivax anemia are also understood, whose operation is based on the identification of the sera of anemic patients infected with Plasmodium.
  • vivax by use of the antigenic peptide (SEQ ID NQ 1) originating from the PvMSP-1 protein, in its simple or polymerized form.
  • the immunodiagnostic kits described in the present technology comprise: a) Peptide SEQ ID NO: 1 in its simple or polymerized form; (b) an enzyme or marker-conjugated secondary antibody specific for recognizing the primary antibody contained in samples from anemic individuals with vivax malaria; c) Reagent for detecting the enzyme or label mentioned in step "b".
  • the "a” step peptide should be attached to a solid support or carrier.
  • the peptides may or may not be subjected to a solid or carrier support fixation step using methanol.
  • Detection of primary antibodies in the sera of anemic individuals with vivax malaria will be done by using secondary antibodies (step "b") of the IgG, IgM, IgA or IgE type and their subclasses.
  • an enzyme or a marker conjugated to an enzyme or a marker, the enzyme being selected from the group comprising alkaline phosphatase, peroxidase, ⁇ -galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase.
  • Markers may be selected from the group consisting of enzymes, radioisotopes, biotin, chromophores, fluorophores and chemiluminescent. Secondary antibodies may be detected by fluorescence, immunoluminescence, absorbance or radioisotope techniques.
  • the method for immunodiagnosis of anemia due to vivax malaria may be performed by serological tests, which may be ELISA, Western-blot, dot-blot, immunodiffusion and / or immunochromatography, which comprise the following steps: a) expose the primary antibodies of a sample to the peptide having amino acid sequence SEQ ID NQ 1, in simple or polymerized form; b) contacting the antibodies of step "a" in contact with a secondary antibody, conjugated to an enzyme or label, which binds to the antibodies of step "a”; c) detecting anti-malaria antibodies unique to anemic individuals due to P. vivax infection in the above sample by detecting the secondary antibody specifically bound to said anti-malaria antibody.
  • serological tests which may be ELISA, Western-blot, dot-blot, immunodiffusion and / or immunochromatography, which comprise the following steps: a) expose the primary antibodies of a sample to the peptide having amino acid sequence SEQ ID NQ 1, in simple or polymerized
  • the peptide of step "a” must be attached to a solid support or a carrier.
  • the peptides may or may not be subjected to a solid or carrier support fixation step using methanol.
  • Detection of primary antibodies in the sera of anemic individuals with vivax malaria will be done by using secondary antibodies (step "b") of the IgG, IgM, IgA or IgE type and their subclasses. These are conjugated to an enzyme or a marker, the enzyme being selected from the group comprising alkaline phosphatase, peroxidase, ⁇ -galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase.
  • Markers may be selected from the group consisting of enzymes, radioisotopes, biotin, chromophores, fluorophores and chemiluminescent. Secondary antibodies may be detected by fluorescence, immunoluminescence, absorbance or radioisotope techniques.
  • Peptide identification was done by spot-synthesis technique, which allowed epitope mapping of the complete PvMSP-1 sequence comprising 1751 amino acids.
  • the spot synthesis technique allowed the multiple synthesis of peptides in cellulose membrane, covering the entire length of the protein. The synthesis of these peptides was made according to the methodology described by Laune et al., 2002 (LAUNE, D., MOLINA, F., FERRI ⁇ RIS, G., VILLARD, S., B ⁇ S, C, RIEUNIER, F., CHARD ⁇ S, T., GRANIER.
  • C Application of the Spot method to the identification of peptides and amino acids from the antibody paratope that contributes to antigen binding.
  • each spot consisted of an array of fifteen amino acid residues overlapping 12 residues totaling 580 spots. This membrane was used to screen differently for reactive sera groups against each peptide, and these sera groups consisted of:
  • Membrane I containing the synthetic peptides was washed with ethanol PA, then Phosphate Buffer Solution 1X (PBS) for ten minutes, and then a blocking solution ⁇ Bovine Serum Albumine 3% and 5% sucrose. in PBS-Tween 0.1%) was added and maintained overnight. The following day, the blocking solution was removed and the membrane was washed with 0.1% PBS-Tween for ten minutes. The membrane was then incubated with the sera (antibody) pool diluted in blocking solution (1: 500) for 2 hours at room temperature and under constant agitation. After incubation, the membrane was again washed with 0.1% PBS-Tween for ten minutes three times.
  • PBS Phosphate Buffer Solution 1X
  • the alkaline phosphatase-conjugated secondary antibody was diluted in the blocking solution (1: 2000) and incubated with the membrane for 1 hour under shaking at room temperature. Then, two washes of ten minutes each, with PBS-Tween 0.1%, and two washes, also ten minutes each, with Coating Buffer Solution (CBS, pH 7.0).
  • CBS Coating Buffer Solution
  • a solution composed of CBS pH 7.0, Thiazolyl Blue Tetrazolium Bromide (MTT), 5-bromo-4-chloro-3-indolyl phosphate disodium (BCIP) and Magnesium Chloride (MgCl2) was added and stirred under stirring. 15 to 30 minutes. The development reaction was stopped with distilled water and the membrane was digitized for further analysis.
  • a regeneration step was performed in which the membrane was subjected to three consecutive washes of ten minutes each with DMF (dimethylformamide) plus three washes with Reagent A (8M Urea + 2.5% Sodium Dodecil Sulfate + 0.1% 2-mercaptoethanol) and three washes with Reagent B (50ml_ ethanol + 40ml_ water + 10ml_ acetic acid), also for ten minutes each.
  • DMF dimethylformamide
  • Reagent A 8M Urea + 2.5% Sodium Dodecil Sulfate + 0.1% 2-mercaptoethanol
  • Reagent B 50ml_ ethanol + 40ml_ water + 10ml_ acetic acid
  • FIG. 1 shows the image of Membrane I after the tests.
  • Sera from individuals with anemia concomitant with P. vivax infection recognized ten peptides that were unreactive to the other pools used, including the peptide inserted at position 70 (SEQ ID NO: 1). Reactivity values for each of the peptides are shown in Table 1, which shows that peptide 70 (SEQ ID No. 1) stands out as a possible biomarker for anemia due to vivax malaria.
  • peptide 70 (SEQ ID NO: 1) was polymerized by glutaraldehyde.
  • a peptide solution previously synthesized manually by FMOC-Synthesis Method and lyophilized, was prepared in PBS (pH 7.4).
  • 1% v / v solution of glutaraldehyde in water was prepared, which was added dropwise to the above solution, constantly stirred by magnetic stirrer, also in a cold chamber. After one hour, the reaction was stopped by adding enough sodium borohydrate (NaBH 4 ) to bring it to a final concentration of 10 mg / mL in the solution.
  • NaBH 4 sodium borohydrate
  • 96-well microplates (Costar, Cambridge, MA) were sensitized with 100 ⁇ of antigens (peptide consisting of polymerized sequence SEQ ID No. 1) at a concentration of 2.5 mg / mL, diluted in carbonate-bicarbonate buffer (pH 9.6; 0.1M) for 18 hours (overnight) at 4 Q C. After incubation, was added 50 uL / well of methanol to the peptide fixation plate and expected 1 Q hour at 37 C until complete evaporation of the liquid.
  • antigens peptide consisting of polymerized sequence SEQ ID No. 1
  • carbonate-bicarbonate buffer pH 9.6; 0.1M
  • the washing process was performed five consecutive times and 100 ⁇ of 1: 100 diluted serum samples in PBST containing 1% BSA (PBST + BSA) were added to each well in duplicates. After 1 hour incubation at 37 Q C, the plates were subjected to five washes with PBST and peroxidase conjugated antibody, anti-human IgG diluted 1: 2000 in PBST + BSA (100 ⁇ / ⁇ ) was added, followed once again by incubating at 37 C for Q
  • the sera samples evaluated consisted of 15 sera from anemic infected individuals, 129 non-anemic infected and 22 non-anemic uninfected individuals.
  • Peptide concentration 1 ug / well; serum dilution: 1: 100; anemic infected (IAN), infected nonanemic (INA) and uninfected (NI) sera.
  • IAN anemic infected
  • INA infected nonanemic
  • NI uninfected
  • Table 3 Average absorbance values for polymerized or unpolymerized peptide 70 with or without methanol for fixation to individual sera from P. vivax parasitized individuals or not and healthy controls.
  • Peptide concentration 1 ug / well; serum dilution: 1: 100; anemic infected (IAN), infected nonanemic (INA) and uninfected (NI) sera.
  • IAN anemic infected
  • INA infected nonanemic
  • NI uninfected

Abstract

The present technology describes a kit and an immunodiagnostic method for detecting malaria caused by Plasmodium vivax, which uses a peptide (SEQ ID NO. 1) comprising Plasmodium vivax merozoite surface protein 1 (PvMSP-1) as the antigenic marker, or the polymerized form thereof. Said peptides have proven to be highly specific in the identification of serum from anaemic individuals infected with Plasmodium vivax.

Description

KIT E MÉTODO IMUNODIAGNÓSTICO PARA DETECÇÃO DE ANEMIA EM DECORRÊNCIA DE MALÁRIA VIVAX, PEPTÍDEOS SINTÉTICOS E USOS  IMMUNODIAGNOSTIC KIT AND METHOD FOR DETECTION OF ANEMIA DUE TO VIVAX MALARIA, SYNTHETIC PEPTIDES AND USES
[001 ] A presente tecnologia descreve um kit e um método imunodiagnóstico para a detecção de malária causada por Plasmodium vivax, utilizando como antígeno marcador um peptídeo (SEQ ID NQ1 ) que compõe a proteína 1 da superfície de merozoíto de Plasmodium vivax (PvMSP-1 ), ou ainda este peptídeo polimerizado. Estes peptídeos se apresentaram altamente específicos na identificação de soros de indivíduos anêmicos com infecção patente por Plasmodium vivax. [001] The present technology describes a kit and an immunodiagnostic method for the detection of Plasmodium vivax malaria using as a marker antigen a peptide (SEQ ID NQ 1) that composes Plasmodium vivax merozoite surface protein 1 (PvMSP). -1), or even this polymerized peptide. These peptides were highly specific in the identification of sera from anemic individuals with Plasmodium vivax infection.
[002] A malária é causada por parasitos do género Plasmodium, pertencentes ao filo Apicomplexa, família Plasmodidae. Cinco espécies podem infectar os humanos - Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale e, atualmente, Plasmodium knowlesy, que, embora descrito como um parasito de símios, agora tem sido relatado em infecções humanas (COX-SINGH, J.; DAVIS, T. M. E.; LEE, K. S.; SHASUL, S. S. G.; MATUSOP, A.; RATNAM, S.; RAHMAN, H. A.; CONWAY, D. J.; SINGH, B. Plasmodium knowlesi malária in Humans is widely distributed and potentially life threatening. Clinicai Infectious Disease, v. 46, n. 2, p. 165-171 , 2008; TILLEY, L; DIXON, M. W. A.; KIRK, K. The Plasmodium falciparum infected red blood cell. The International Journal of Biochemistry & Cell Biology, v. 43, p. 839-842, 201 1 ).  Malaria is caused by parasites of the genus Plasmodium, belonging to the phylum Apicomplexa, family Plasmodidae. Five species can infect humans - Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and currently Plasmodium knowlesy, which, although described as a simian parasite, have now been reported in human infections (COX-SINGH, J .; DAVIS, TME; LEE, KS; SHASUL, SSG; MATUSOP, A.; RATNAM, S.; RAHMAN, HA; CONWAY, DJ; SINGH, B. Plasmodium knowlesi malaria in Humans is widely distributed and potentially life threatening. , v. 46, no. 2, pp. 165-171, 2008; TILLEY, L.; DIXON, MWA; KIRK, K. The International Journal of Biochemistry & Cell Biology, v. 43, 839-842, 2011).
[003] Atualmente, são registrados no mundo cerca de 200-300 milhões de casos de malária a cada ano. Deste número alarmante, 90% se concentram na África Tropical, com aproximadamente 0,9 milhão de mortes, principalmente entre crianças menores de cinco anos de idade (85%). O restante está distribuído nas Américas Central e do Sul, Sudeste Asiático e Ilhas da Oceania (BRAGA, E. M.; FONTES, C. J. F. Plasmodium - Malária. In: NEVES, D. P.; MELO, A. L; LINARDI, P. M.; VÍTOR, R. W. A. Parasitologia Humana. 12° ed. São Paulo: Editora Atheneu, 201 1 ). [003] Currently, around 200-300 million cases of malaria are reported worldwide each year. Of this alarming figure, 90% is concentrated in Tropical Africa, with approximately 0.9 million deaths, mostly among children under five (85%). The remainder is distributed in Central and South America, Southeast Asia and the Oceania Islands (BRAGA, EM; SOURCES, CJF Plasmodium - Malaria. In: NEVES, DP; MELO, A.; LINARDI, PM; VICTOR, RWA Human Parasitology 12th ed São Paulo: Publisher Atheneu, 201 1).
[004] No Brasil, a malária permanece como grande causa de morbidade, apesar de anos de tentativas de controle da doença (SILVA-NUNES, M.; CODEÇO, C. T.; MALAFRONTE, R. S.; SILVA, N. S.; JUNCANSEN, C; MUNIZ, P. T.; FERREIRA, M. U. Malária on the amazonian frontier: transmission dynamics, risk factors, spatial distribution, and prospects for control. American Journal of Tropical Medicine and Hygiene, v. 79, n. 4, p. 624- 635, 2008). Atualmente, os registros brasileiros de malária restringem-se à Amazónia Legal - região que compreende nove estados: Amazónia, Acre, Amapá, Maranhão, Mato Grosso, Pará, Rondônia, Roraima e Tocantins - onde as condições ambientais são altamente favoráveis ao desenvolvimento de Anopheles darlingi, principal vetor da doença no território (GIL, L. H. S. G.; TADA, M. S.; KATSURAGAWA, T. H.; RIBOLLA, P. E. M.; SILVA, L. H. P. Urban and suburban malária in Rondônia (Brazilian Western Amazon) II. Perennial transmission with high anopheline densities are associated with human environmental changes. Memórias do Instituto Oswaldo Cruz, v. 102, n. 3, p. 271 -276, 2007). Nesta região, as medidas de controle estabelecidas durante a criação da Campanha de Erradicação da Malária (CEM), em 1965, não obtiveram o êxito esperado, e este fato foi associado à presença da floresta tropical úmida que contribui para a proliferação dos vetores, à presença de indivíduos expostos, como é o caso dos garimpeiros, madeireiros e agricultores, e à inexistência de serviços permanentes de saúde e de infraestrutura social (LOIOLA, C. C. P.; SILVA, C. J. M.; TAUIL, P. L. Controle da malária no Brasil: 1965 a 2001 . Revista Panamericana de Saúde Pública, v. 1 1 , n. 4, p. 235-244, 2002). Desta forma, até os dias atuais, a transmissão da doença não foi interrompida nestes estados. Três espécies de Plasmodium estão envolvidas nas infecções maláricas no país, sendo que P. vivax é responsável por aproximadamente 80% dos casos, enquanto P. falciparum responde por cerca de 20% e P. malariae, menos que 1 % das ocorrências (OLIVEIRA-FERREIRA, J.; LACERDA, M. V. G.; BRASIL, P.; LADISLAU, J. L. B.; TAUIL, P. L.; DANIEL-RIBEIRO, C. T. Malária in Brazil: na overview. Malária Journal, v. 9, n. 1 15, 2010). [004] In Brazil, malaria remains a major cause of morbidity, despite years of attempts to control the disease (SILVA-NUNES, M .; CODEÇO, CT; MALAFRONTE, RS; SILVA, NS; JUNCANSEN, C; MUNIZ, PT; FERREIRA, MU Malaria on the amazonian frontier: transmission dynamics, risk factors, spatial distribution, and prospects for control. American Journal of Tropical Medicine and Hygiene, v. 79, no. 4, p. 624-635, 2008). Currently, Brazilian records of malaria are restricted to the Legal Amazon - a region comprising nine states: Amazonia, Acre, Amapá, Maranhão, Mato Grosso, Pará, Rondonia, Roraima and Tocantins - where environmental conditions are highly favorable to Anopheles development. darlingi, the main disease vector in the territory (GIL, LHSG; TADA, MS; KATSURAGAWA, TH; RIBOLLA, PEM; SILVA, LHP Urban and suburban malaria in Rondonia (Brazilian Western Amazon) II. Perennial transmission with high anopheline densities are associated with human environmental changes Memories of the Oswaldo Cruz Institute, v. 102, no. 3, pp. 271-276, 2007). In this region, the control measures established during the creation of the Malaria Eradication Campaign (CEM) in 1965 did not achieve the expected success, and this fact was associated with the presence of tropical rainforest that contributes to the proliferation of vectors, presence of exposed individuals, such as prospectors, loggers and farmers, and the lack of permanent health services and social infrastructure (LOIOLA, CCP; SILVA, CJM; TAUIL, PL. Malaria control in Brazil: 1965 to 2001. Pan American Journal of Public Health, v. 11, No. 4, pp. 235-244, 2002). Thus, until the present day, the transmission of the disease has not been interrupted in these states. Three species of Plasmodium are involved in malaria infections in the country, with P. vivax accounting for approximately 80% of cases, while P. falciparum accounts for about 20% and P. malariae for less than 1% of occurrences (OLIVEIRA- FERREIRA, J .; LACERDA, MVG; BRAZIL, P.; LADISLAU, JLB; TAUIL, PL; DANIEL-RIBEIRO, CT Malaria in Brazil: in the overview. Malaria Journal, v. 9, No. 1 15, 2010).
[005] Apesar de P. falciparum ser a espécie responsável por maior mortalidade - cerca de 95% de todos os óbitos registrados (WORLD HEALTH ORGANIZATION (WHO). World Malária Report, 201 1 ), P. vivax é a mais comum (KASLIWAL, P.; RAO, M. S.; KUJUR, R. Plasmodium vivax malária: an unusual presentation. Indian Journal of Criticai Care Medicine, v.13, p.103-105, 2009) e apresenta ampla distribuição geográfica, com cerca de 2,6 bilhões de pessoas no sul e sudeste da Ásia, América Central e América do Sul sob o risco de contrair a doença (HULDEN, L; HULDEN, L. Activation of hipnozoyte: a part of Plasmodium Vivax Wíe cycle and survive. Malária Journal, v. 10, n. 90, 201 1 ). O plasmódio P. vivax é capaz de completar o seu ciclo esporogônico em temperaturas relativamente baixas (em torno de 16Q C), e este fato tem contribuído substancialmente para o seu sucesso em estabelecer focos estáveis de transmissão, não só em zonas tropicais, mas também em localidades de zonas temperadas (CUI, L; ESCALANTE, A. A.; IMWONG, M.; SNOUNOU, G. The genetic diversity of Plasmodium vivax populations. Trends in Parasitology, v. 19, n. 5, p. 220-226, 2003). Although P. falciparum is the species responsible for the highest mortality - about 95% of all reported deaths (WORLD HEALTH ORGANIZATION (WHO). World Malaria Report, 201 1), P. vivax is the most common (KASLIWAL RAO, MS; KUJUR, R. Plasmodium vivax malaria: an unusual presentation Indian Journal of Critical Care Medicine, v.13, p.103-105, 2009) and has a wide geographic distribution, with about 2.6 billion people in South and Southeast Asia, Central America and South America at risk of contracting the disease (HULDEN, L; HULDEN, L. Activation of hipnozoyte: part of Plasmodium Vivax Wie cycle and survive Malaria Journal, v. 10, no 90, 201 1). The Plasmodium vivax is able to complete its cycle esporogônico at relatively low temperatures (around 16 Q C), and this fact has contributed substantially to its success in establishing stable foci transmission, not only in tropical areas, but also in temperate zone locations (CUI, L; ESCALANTE, AA; IMWONG, M .; SNOUNOU, G. The genetic diversity of Plasmodium vivax populations. Trends in Parasitology, v. 19, n. 5, p. 220-226, 2003).
[006] Os sintomas da malária vivax são semelhantes àqueles causados pelos demais plasmódios e incluem febre com calafrios, dores de cabeça, fraqueza, diarréia, vómitos, palidez, esplenomegalia e hepatomegalia (KASLIWAL, P.; RAO, M. S.; KUJUR, R. Plasmodium vivax malária: an unusual presentation. Indian Journal of Criticai Care Medicine, v. 13, p. 103-105, 2009; ECHEVERRI, M.; TOBÓN, A.; ALVAREZ, G.; CARMONA, J.; BLAIR S. Clinicai and laboratory findings of Plasmodium vivax malária in Colômbia, 2001 . Revista do Instituto de Medicina Tropical, v. 45, n. 1 , p. 29-34, 2003). Alterações hematológicas como anemia, leucopenia e trombocitopenia, são também relatadas (ECHEVERRI et al., 2003; SONG, H. H.; O, S. O.; KIM, S. H.; MOON, S. H.; KIM, J. B.; YOON, J. W.; KOO, J. R.; HONG, K. S.; LEE, M. G.; KIM, D. J.; SHIN, D. H.; KANG, S. H.; CHOI, M. G.; LEE, K. H. Clinicai features of Plasmodium vivax malária. The Korean Journal of Internai Medicine, v.18, n.4, p.220-224, 2003).  Symptoms of vivax malaria are similar to those caused by other plasmodia and include fever with chills, headache, weakness, diarrhea, vomiting, pallor, splenomegaly and hepatomegaly (KASLIWAL, P .; RAO, MS; KUJUR, R. Plasmodium vivax malaria: an unusual presentation Indian Journal of Critical Care Medicine, v. 13, pp. 103-105, 2009; ECHEVERRI, M .; TOBÓN, A.; ALVAREZ, G .; CARMONA, J .; BLAIR S. Clinical and laboratory findings of Plasmodium vivax malaria in Colombia, 2001. Journal of the Institute of Tropical Medicine, v. 45, no. 1, pp. 29-34, 2003). Hematological changes such as anemia, leukopenia and thrombocytopenia are also reported (ECHEVERRI et al., 2003; SONG, HH; O, SO; KIM, SH; MOON, SH; KIM, JB; YOON, JW; KOO, JR; HONG, KS; LEE, MG; KIM, DJ; SHIN, DH; KANG, SH; CHOI, MG; LEE, KH Clinical features of Plasmodium vivax malaria The Korean Journal of International Medicine, v.18, n.4, p.220 -224, 2003).
[007] A malária vivax deixou de ser uma doença benigna para, em alguns casos, apresentar perfis clínicos semelhantes aos observados na malária grave causada por P. falciparum. Os sinais e sintomas observados incluem falência renal, síndrome respiratória e, principalmente anemia grave (KOCHAR, D. K.; SAXENA, V.; SINGH, N.; KOCHAR, S. K.; KUMAR, S. V.; DAS, A. Plasmodium vivax malária. Emerging Infectious Diseases, v. 1 1 , p. 132-134, 2005; ANSTEY, N. M.; RUSSELL, B.; YEO, T. W.; PRICE, R. N. The pathophysiology of vivax malária. Trends in Parasitology, v. 25, n. 5, p. 220-227, 2009; KASLIWAL, P.; RAO, M. S.; KUJUR, R. Plasmodium vivax malária: an unusual presentation. Indian Journal of Criticai Care Medicine, v. 13, n. 2, p. 103-105, 2009). Vivax malaria is no longer a benign disease and in some cases has clinical profiles similar to those seen in severe P. falciparum malaria. Signs and symptoms observed include renal failure, respiratory syndrome, and especially severe anemia (KOCHAR, DK; SAXENA, V.; SINGH, N .; KOCHAR, SK; KUMAR, SV; DAS, A. Plasmodium vivax malaria. Emerging Infectious Diseases , v. 11, pp. 132-134, 2005; ANSTEY, NM; RUSSELL, B.; YEO, TW; PRICE, RN The pathophysiology of vivax malaria Trends in Parasitology, v. 25, no. 220-227, 2009; KASLIWAL, P .; RAO, MS; KUJUR, R. Plasmodium vivax malaria: an unusual presentation. Indian Journal of Critical Care Medicine, v. 13, no. 2, p. 103-105, 2009).
[008] A ocorrência da anemia tem sido relatada em diversos estudos e desperta grande interesse no entendimento de sua etiologia, dada sua importância nos índices de mortalidade associados à malária, principalmente a infantil (GENTON, B.; D'ACREMONT, V.; RARE, L; BAEA, K.; REEDER, J. C; ALPERS, M. P.; MLILLER, I. Plasmodium vivax and mixed infections are associated with severe malária in children: A Prospective cohort study from Papua New Guinea. Pios Medicine, v. 5, n. 6, 2008). [008] The occurrence of anemia has been reported in several studies and arouses great interest in understanding its etiology, given its importance in mortality rates associated with malaria, especially childhood (GENTON, B .; D'ACREMONT, V .; RARE, L; BAEA, K.; REEDER, J. C. ALPERS, MP; MLILLER, I. Plasmodium vivax and mixed infections are associated with severe malaria in children: A Prospective cohort study from Papua New Guinea. 5, no. 6, 2008).
[009] A cada ano, entre 72 e 80 milhões de novos casos de malária vivax são diagnosticados mundialmente. Com milhares de pessoas sob o risco de contrair a doença, o desenvolvimento de ferramentas diagnosticas se faz necessário tanto para estimar a carga parasitária, quanto para manter os órgãos de saúde pública atentos aos novos casos (CONROY, A. L; MCDONALD, C. R.; KAIN, K. C. Malária in pregnancy: diagnosing infection and identifying fetal risk. Expert Review Anti Infective Therapy, v. 10, n.1 1 , p. 1331 - 1342, 2012).  [009] Each year, between 72 and 80 million new cases of vivax malaria are diagnosed worldwide. With thousands of people at risk of contracting the disease, the development of diagnostic tools is needed both to estimate parasite burden and to keep public health agencies alert to new cases (CONROY, A. L; MCDONALD, CR; KAIN, KC Malaria in pregnancy: diagnosing infection and identifying fetal risk Expert Review Anti Infective Therapy, v. 10, n.1 1, pp. 1331 - 1342, 2012).
[010] Tendo em vista que as manifestações graves apresentadas por P. vivax têm aumentado o número de internações hospitalares (SANTOS-CIMINERA, P. D.; ROBERTS, D. R.; ALECRIM, M. G.; COSTA, M. R. F.; JR, G. V. Q. Malária diagnosis and hospitalization trends, Brazil. Emerging Infectious Diseases, v. 13, p. 1597-1599, 2007; ALEXANDRE, M. A.; FERREIRA, C. O.; SIQUEIRA, A. M.; MAGALHÃES, B. L; MOURÃO, M. P. G.; LACERDA, M. V.; ALECRIM, M. G. C. Severe Plasmodium vivax malária, Brazilian Amazon. Emerging Infectious Diseases, v. 16, p. 161 1 -1614, 2010), e que o diagnóstico é fundamental para a terapêutica apropriada, a busca por novos biomarcadores de infecção e/ou morbidade se faz necessária. A despeito do grande avanço nas técnicas imunológicas de diagnóstico ocorrido nas últimas décadas, o diagnóstico da malária continua sendo feito pela tradicional pesquisa do parasito no sangue periférico, seja pelo método da gota espessa, ou pelo esfregaço sanguíneo. Estas técnicas baseiam-se na visualização do parasito através de microscopia ótica, após coloração com corante vital (azul-de-metileno e Giemsa). Estes são os únicos métodos que permitem a diferenciação específica dos parasitos a partir da análise da sua morfologia e das alterações provocadas no eritrócito infectado. Em função de sua simplicidade de realização, seu baixo custo e sua eficiência diagnostica, o exame da gota espessa tem sido utilizado em todo o mundo para o diagnóstico específico da malária (BRAGA, E. M.; FONTES, C. J. F. Plasmodium - Malária. In: NEVES, D. P.; MELO, A. L; LINARDI, P. M.; VÍTOR, R. W. A. Parasitologia Humana. 12° ed. São Paulo: Editora Atheneu, 201 1 ) e, no Brasil, é o método oficialmente adotado (BRASIL. Ministério da Saúde. Secretaria de Vigilância em Saúde. Departamento de Vigilância Epidemiológica. Guia de vigilância epidemiológica. 7. ed. Brasília : Ministério da Saúde, 2009. 816 p. (Série A. Normas e Manuais Técnicos)). [010] Given that the severe manifestations presented by P. vivax have increased the number of hospital admissions (SANTOS-CIMINERA, PD; ROBERTS, DR; ALECRIM, MG; COSTA, MRF; JR, GVQ. Malaria diagnosis and hospitalization trends, Emerging Infectious Diseases, v. 13, pp. 1597-1599, 2007; ALEXANDRE, MA; FERREIRA, CO; SIQUEIRA, AM; MAGALHÃES, L; MOURÃO, MPG; LACERDA, MV; ALECRIM, MGC Severe Plasmodium vivax malaria, Brazilian Amazon, Emerging Infectious Diseases, v. 16, pp. 161 1 -1614, 2010), and since diagnosis is essential for appropriate therapy, the search for new biomarkers of infection and / or morbidity is necessary. Despite the great advance in the diagnostic immunological techniques that occurred in the last decades, the diagnosis of malaria continues to be made by the traditional research of the parasite in the peripheral blood, either by the thick drop method or by the blood smear. These techniques are based on the visualization of the parasite by optical microscopy after staining with vital dye (methylene blue and Giemsa). These are the only methods that allow the specific differentiation of parasites from the analysis of their morphology and the alterations caused in the infected erythrocyte. Due to its simplicity of performance, its low cost and its diagnostic efficiency, thick gout examination has been used worldwide for the specific diagnosis of malaria (BRAGA, EM; SOURCES, CJF Plasmodium - Malaria. In: NEVES, DP; MELO, A.L; LINARDI, PM; VITOR, RWA Human Parasitology. 12th ed. São Paulo: Publisher Atheneu, 201 1) and, in Brazil, is the officially adopted method (BRAZIL. Ministry of Health. Health Surveillance Department of Epidemiological Surveillance Guide for Epidemiological Surveillance 7. ed. Brasilia: Ministry of Health, 2009. 816 P. (Series A. Standards and Technical Manuals)).
[01 1 ] Apesar de sua inquestionável vantagem, o diagnóstico parasitologico da malária pela gota espessa é dependente de fatores como a habilidade técnica no preparo da lâmina, seu manuseio e coloração, a qualidade ótica e iluminação do microscópio, a competência e o cuidado por parte do microscopista, entre outros. Realizar o diagnóstico específico de malária atendendo a todos esses quesitos é impraticável em muitos locais onde a malária ocorre, seja pela precariedade dos serviços de saúde, seja pela dificuldade de acesso da população aos centros de diagnósticos (BRAGA, E. M.; FONTES, C. J. F. Plasmodium - Malária. In: NEVES, D. P.; MELO, A. L; LINARDI, P. M.; VÍTOR, R. W. A. Parasitologia Humana. 12° ed. São Paulo: Editora Atheneu, 201 1 ). Além disso, a baixa sensibilidade da técnica de microscopia é destacada por vários autores (OKELL, L. C; GHANI, A. C; LYONS, E.; DRAKELEY, C. J. Submicroscopic Infection in Plasmodium falciparum endemic populations: a systematic review and meta-analysis. The Journal of Infectious Diseases, v. 200, p. 1509-1517, 2009; HARRIS, I.; SHARROCK, W. W.; BAIN, L. M.; GRAY, K. A.; BOBOGARE, A.; BOAZ, L; LILLEY, K.; KRAUSE, D.; VALLELY, A.; JOHNSON, M. L; GATTON, M. L; SHANKS, G. D.; CHENG, Q. A large proportion of asymptomatic Plasmodium infections with low and sub-microscopic parasite densities in the low transmission setting of Temotu Province, Solomon Islands: challenges for malária diagnostics in an elimination setting. Malária Journal, v. 9, 2010; OKELL, L. C; BOUSEMA, T.; GRIFFIN, J. T.; OUEDRAOGO, A. L; GHANI, A. C; DRAKELEY, C. J. Factors determining the occurrence of submicroscopic malária infections and their relevance for control. Nature Communications, v. 3, 2012). Por estas razões, nos últimos dez anos, métodos rápidos, práticos e sensíveis vêm sendo desenvolvidos. [01 1] Despite its unquestionable advantage, parasitological diagnosis of malaria by thick drop is dependent on factors such as the technical ability to prepare the slide, its handling and staining, the optical quality and illumination of the microscope, the competence and care of it. part of the microscopist, among others. Performing a specific malaria diagnosis meeting all of these requirements is impractical in many places where malaria occurs, either because of poor health services or the difficulty of population access to diagnostic centers (BRAGA, EM; SOURCES, CJF Plasmodium - In: NEVES, DP; MELO, A.L; LINARDI, PM; VICTOR, RWA Human Parasitology. 12th ed. São Paulo: Publisher Atheneu, 201 1). In addition, the low sensitivity of the microscopy technique is highlighted by several authors (OKELL, L.C; GHANI, A.C; LYONS, E.; DRAKELEY, CJ; Submicroscopic Infection in Plasmodium falciparum endemic populations: a systematic review and meta- The Journal of Infectious Diseases, v. 200, pp. 1509-1517, 2009; HARRIS, I.; SHARROCK, WW; BAIN, LM; GRAY, KA; BOBOGARE, A .; BOAZ, L; LILLEY, K. ; KRAUSE, D .; VALLELY, A.; JOHNSON, M. L.; GATTON, M. L.; SHANKS, GD; CHENG, Q. A large proportion of asymptomatic Plasmodium infections with low and sub-microscopic parasite densities in the low transmission setting of Temotu Province, Solomon Islands: challenges for malaria diagnostics in an elimination setting Malaria Journal, v. 9, 2010; OKELL, L. C; BOUSEMA, T .; GRIFFIN, JT; OUEDRAOGO, A. L; GHANI, A. C; DRAKELEY, CJ Factors determining the occurrence of submicroscopic malaria infections and their relevance for control. Nature Communications, v. 3, 2012). For these reasons, in the last ten years, fast, practical and sensitive methods have been developed.
[012] Testes imunocromatográficos representam novos métodos de diagnóstico rápido de malária, por meio da detecção de componentes antigênicos de plasmódio. Estes testes são úteis para triagem ou mesmo confirmação diagnostica, especialmente em áreas longínquas ou de difícil acesso aos serviços de saúde, devido ao fato de serem rápidos e de fácil realização (BRASIL. Ministério da Saúde. Secretaria de Vigilância em Saúde. Departamento de Vigilância Epidemiológica. Guia de vigilância epidemiológica. 7° ed. Brasília: Ministério da Saúde, 2009. 816 p. - (Série A. Normas e Manuais Técnicos)). Estes métodos utilizam anticorpos monoclonais e policlonais dirigidos contra a proteína 2 rica em histidina do P. falciparum (PfHRP-2) e contra a enzima desidrogenase do lactato (pDHL) das quatro espécies de plasmódio. Tem a vantagem de diferenciar o P. falciparum das demais espécies, as quais são identificadas como não-P. falciparum pelo teste. Além disso, a pDHL é uma enzima intracelular produzida em abundância pelos parasitos vivos, o que permite diferenciar entre fase aguda e convalescença da infecção. Uma desvantagem, no entanto, é não permitir o diagnóstico de uma infecção mista (BRAGA, E. M.; FONTES, C. J. F. Plasmodium - Malária. In: NEVES, D. P.; MELO, A. L; LINARDI, P. M.; VÍTOR, R. W. A. Parasitologia Humana. 12° ed. São Paulo: Editora Atheneu, 201 1 ).  [012] Immunochromatographic tests represent novel methods of rapid diagnosis of malaria by detecting antigenic components of plasmodium. These tests are useful for screening or even diagnostic confirmation, especially in remote areas or areas that are difficult to access to health services, because they are quick and easy to perform (BRAZIL. Ministry of Health. Health Surveillance Secretariat. Surveillance Department Epidemiological Guide to Epidemiological Surveillance 7th ed Brasilia: Ministry of Health, 2009. 816 pp. (Series A. Technical Standards and Manuals)). These methods utilize monoclonal and polyclonal antibodies directed against P. falciparum histidine-rich protein 2 (PfHRP-2) and against lactate dehydrogenase enzyme (pDHL) of the four plasmid species. It has the advantage of differentiating P. falciparum from other species, which are identified as non-P. falciparum by the test. In addition, pDHL is an intracellular enzyme produced in abundance by living parasites, which allows differentiating between acute and convalescent phase of infection. A disadvantage, however, is that it does not allow the diagnosis of a mixed infection (BRAGA, EM; SOURCES, CJF Plasmodium - Malaria. In: NEVES, DP; MELO, A.; LINARDI, PM; VICTOR, RWA. Human Parasitology. 12 Ed São Paulo: Publisher Atheneu, 201 1).
[013] É necessário um método mais específico para diagnosticar os casos de malária em pacientes anêmicos infectados por Plasmodium vivax, visto que a decisão sobre a forma de tratamento do paciente deve ser precedida de informações referentes à gravidade da doença, espécie de plasmódio, idade do paciente, histórico de exposição anterior à infecção, bem como susceptibilidade dos parasitos aos antimaláricos convencionais (BRASIL. Ministério da Saúde. Secretaria de Atenção à Saúde. Departamento de Atenção Básica. Vigilância em Saúde: Dengue, Esquistossomose, Hanseníase, Malária, Tracoma e Tuberculose. 2° ed. rev., Brasília: Ministério da Saúde, 2008. 197 p.: il. (Série A. Normas e Manuais Técnicos) (Cadernos de Atenção Básica, n. 21 )). Estes aspectos são de fundamental importância, pois a base para o controle da malária atualmente consiste no tratamento adequado e oportuno da doença e, ademais, no caso de P. vivax, são observadas formas hipnozoítas que permanecem em estado de latência no tecido hepático e são responsáveis pelas recidivas da doença (HULDEN, L; HULDEN, L. Activation of hipnozoyte: a part of Plasmodium Vivax Wíe cycle and survive. Malária Journal, v. 10, n. 90, 201 1 ). A eliminação destes hipnozoítos em P. vivax requer administração da droga específica, sendo a cloroquina a mais utilizada atualmente (BAIRD, J. K. Chloroquine resistance in Plasmodium. Antimicrobial Agents Chemotherapics, v. 48, n. 1 1 , 2004), cuja resistência em P. falciparum é relatada desde os anos 50 (WELLEMS, T. E.; PLOWE, C. V. Chloroquine-resistant malária. The Journal of lnfectious Diseases, v. 184, p. 770-776, 2001 ). [013] A more specific method is required to diagnose cases of malaria in anasic patients infected with Plasmodium vivax, as the decision on how to treat the patient should be preceded by information regarding disease severity, plasmodium species, age. history of exposure prior to infection, as well as susceptibility of parasites to conventional antimalarial drugs (BRAZIL. Ministry of Health. Department of Health Care. Department of Primary Care. Health Surveillance: Dengue, Schistosomiasis, Leprosy, Malaria, Trachoma and Tuberculosis. 2nd ed. rev., Brasilia: Ministry of Health, 2008. 197 p .: il. (Series A. Standards and Technical Manuals) (Primary Care Notebooks, # 21)). These aspects are of fundamental importance, as the basis for malaria control nowadays is the adequate and timely treatment of the disease. Moreover, in the case of P. vivax, hypnozoite forms are observed that remain latent in the liver tissue and are responsible for disease relapses (HULDEN, L; HULDEN, L. Activation of hypnozoyte: part of Plasmodium Vivax cycle and survive. Malaria Journal, v. 10, n. 90, 201 1). Elimination of these hypnozoites in P. vivax requires specific drug administration, with chloroquine being the most widely used today (BAIRD, JK Chloroquine resistance in Plasmodium. Antimicrobial Agents Chemotherapics, v. 48, No. 11, 2004), whose resistance in P Falciparum has been reported since the 1950s (WELLEMS, TE; PLOWE, CV Chloroquine-resistant malaria. The Journal of Infective Diseases, v. 184, p. 770-776, 2001).
[014] As limitações dos métodos diagnósticos atuais associadas às dificuldades de acesso aos serviços de saúde implicam em tratamento tardio e aumentam os riscos de desenvolvimento de malária grave. Neste contexto, os biomarcadores podem fornecer importantes informações sobre o estado biológico do paciente, grau de morbidade e prognóstico da doença. Biomarcadores capazes de diferenciar as espécies de Plasmodium ou eficazes na determinação do prognóstico da malária poderiam agilizar o tratamento e ainda, se empregados em testes rápidos de diagnóstico, facilitar a detecção precoce da doença em regiões endémicas de difícil acesso. Além disso, poderiam ter um impacto positivo sobre as medidas de controle e prevenção. Os biomarcadores podem ser derivados do parasito ou do hospedeiro, ou ainda uma associação destes, incluindo qualquer substância passível de ser medida e avaliada (CONROY et al., 2012; LUCCHI, N. W.; JAIN, V.; WILSON, N. O.; SINGH, N.; UDHAYAKUMAR, V.; STILES, J. K. Potential serological biomarkers of cerebral malária. Disease Markers, v. 31 , n. 6, p. 327-335, 201 1 ).  [014] The limitations of current diagnostic methods associated with difficulties in accessing health services imply late treatment and increase the risks of developing severe malaria. In this context, biomarkers can provide important information about the patient's biological state, degree of morbidity and disease prognosis. Biomarkers capable of differentiating Plasmodium species or effective in determining malaria prognosis could expedite treatment and, if employed in rapid diagnostic tests, facilitate early detection of disease in difficult to reach endemic regions. In addition, they could have a positive impact on control and prevention measures. Biomarkers may be derived from the parasite or host, or a combination thereof, including any measurable and evaluated substance (CONROY et al., 2012; LUCCHI, NW; JAIN, V .; WILSON, NO; SINGH, N UDHAYAKUMAR, V; STILES, JK Potential serological biomarkers of cerebral malaria. Disease Markers, v. 31, no. 6, pp. 327-335, 2011).
[015] Apenas recentemente as pesquisas em malária vêm avançando na identificação destas moléculas, contudo, a sua utilização em doenças parasitárias é ainda limitada. O sequenciamento do genoma do Plasmodium vivax possibilitou muitos avanços na pesquisa, incluindo a diferenciação e caracterização de cepas. Ao contrário de P. falciparum, que tem centenas de isolados sequenciados, apenas as cepas Salvador I e IQ07 (Isolado peruano) de P. vivax estão completas (CARLTON, J. M.; ADAMS, J. H.; SILVA, J. C; BIDWELL, S. L; LORENZI, H.; CALER, E.; CRABTREE, J.; ANGIUOLI, S. V.; MERINO, E. F.; AMEDEO, P.; CHENG, Q.; COULSON, R. M. R.; CRABB, B. S.; DEL PORTILLO, H. A.; ESSIEN, K.; FELDBLYUM.T. V.; FERNANDEZ- BECERRA, C; GILSON, P. R.; GUEYE, A. H.; GUO, X.; KANG'A, S.; KOOIJ, T. W. A.; KORSINCZKY, M.; MEYER, E. V. S.; NENE, V.; PAULSEN, I.; WHITE, O.; RALPH, S. A.; REN, Q.; SARGEANT, T. J.; SALZBERG, S. L; STOECKERT, C. J.; SULLIVAN, S. A.; YAMAMOTO, M. M.; HOFFMAN, S. L; WORTMAN, J.R.; GARDNER, M. J.; GALINSKI, M. R.; BARNWELL, J. W.; FRASER-LIGGETT, C. M. Comparative genomics of the neglected human malária parasite Plasmodium vivax. Nature, n. 455, p. 757-763, 2008; DHARIA, N. V.; BRIGHT, A. T.; WESTENBERGER, S. J.; BARNES, S. W.; BATA LO V, S.; KUHEN, K.; BORBOA, R.; FEDERE, G. C; MCCLEAN, C. M.; VINETZ, J. M.; NEYRA, V.; LLANOS-CUENTAS, A.; BARNWELL, J. W.; WALKER, J. R.; WINZELER, E. A. Whole-genome sequencing and microarray analysis of ex vivo Plasmodium vivax reveal selective pressure on putative drug resistance genes. Proceedings of the National Academy of Science, v. 107, n. 46, 2010DHARIA et al., 2010). Desde seu isolamento em 1969, a partir de um paciente proveniente de Congrejera, La Paz, El Salvador, a cepa Salvador I (Sal-I) tem sido amplamente utilizada em trabalhos experimentais (COLLINS, W. E.; SULLIVAN, J. S.; STROBERT, E.; GALLAND, G. G.; WILLIAMS, A.; NACE, D.; WILLIAMS, T.; BARNWELL, J. W. Studies on the Salvador I Strain of Plasmodium vivax in Non-human Primates and Anopheline Mosquitoes. American Journal of Tropical Medicine and Hygien, v. 80, n. 2, 2009). [015] Only recently has malaria research been advancing in the identification of these molecules, however, their use in parasitic diseases is still limited. Plasmodium Genome Sequencing vivax has enabled many advances in research, including strain differentiation and characterization. Unlike P. falciparum, which has hundreds of sequenced isolates, only the P. vivax Salvador I and IQ07 (Peruvian Isolate) strains are complete (CARLTON, JM; ADAMS, JH; SILVA, J. C; BIDWELL, S. L; LORENZI, H.; CALER, E.; CRABTREE, J .; ANGIUOLI, SV; MERINO, EF; AMEDEO, P.; CHENG, Q .; COULSON, RMR; CRABB, BS; DEL PORTILLO, HA; ESSIEN, KEL; FELDBLYUM.TV; FERNANDEZ-BECERRA, C.GILSON, PR; GUEYE, AH; GUO, X .; KANG'A, S.; KOOIJ, TWA; KORSINCZKY, M .; MEYER, EVS; NENE, V. PAULSEN, I.; WHITE, O .; RALPH, SA; REN, Q .; SARGEANT, TJ; SALZBERG, S.; STOECKERT, C; SULLIVAN, SA; YAMAMOTO, MM; HOFFMAN, S.; WORTMAN, JR; GARDNER, MJ; GALINSKI, MR; BARNWELL, JW; FRASER-LIGGETT, CM Comparative genomics of the neglected human malaria parasite Plasmodium vivax Nature, No. 455, pp 757-763, 2008; DHARIA, NV; AT; WESTENBERGER, SJ; BARNES, SW; BATA LO V, S.; KUHEN, K.; BORBOA, R.; FEDERE, G. C. MCCLEAN, CM; VINETZ, J ;; NEYRA, V .; LLANOS-CUENTAS, A. BARNWELL, JW; WALKER, JR; WINZELER, E Whole-genome sequencing and microarray analysis of ex vivo Plasmodium vivax reveal selective pressure on putative drug resistance genes. Proceedings of the National Academy of Science, v. 107, no. 46, 2010DHARIA et al., 2010). Since its isolation in 1969 from a patient from Congrejera, La Paz, El Salvador, the Salvador I (Sal-I) strain has been widely used in experimental work (COLLINS, WE; SULLIVAN, JS; STROBERT, E. ; GALLAND, GG; WILLIAMS, A.; NACE, D.; WILLIAMS, T .; BARNWELL, JW Studies on the Salvador I Strain of Plasmodium vivax in Non-human Primates and Anopheline Mosquitoes. American Journal of Tropical Medicine and Hygien, v 80, No 2, 2009).
[016] Na busca por possíveis biomarcadores para diagnóstico de infecção por malária, as proteínas de superfície dos merozoítos (MSP) estão entre os candidatos de primeira escolha, por se exporem a diversos mecanismos da resposta imune efetora do hospedeiro. Dez membros da família MSP já foram descritos em P. falciparum, e oito destes foram detectados em P. vivax (PvMSP-1 , PvMSP-185, PvMSP-3a, PvMSP-3b, PvMSP3c, PvMSP-4, PvMSP- 5, PvMSP-8, PvMSP-9 e PVMSP-10). E, neste contexto, a proteína 1 de superfície de merozoíto (MSP-1 ) se destaca por estar presente em todas as espécies de Plasmodium (EGAN, A. F.; BURGHAUS, P.; DRUILHE, P.; HOLDER, A. A.; RILEY, E. Human antibodies to the 19 kDa C-terminal fragment of Plasmodium falciparum merozoite surface protein 1 inhibit parasite growth in vitro. Parasite Immunology, v. 21 , p. 133-139, 1999; STOWERS, A. W.; CIOCE, R. L; LAWSON, M.; HUI, G.; MURATOVA, O.; KASLOW, D. C; ROBINSON, R.; LONG, C. A.; MILLER, A. H. Efficacy of two alternate vaccines based on Plasmodium falciparum merozoite surface protein 1 in an Aotus challenge trial. Infection and Immunity, v. 3, p. 1536-1546, 2001 ). A MSP-1 Sal- I compreende 1751 aminoácidos e é frequentemente testada a candidato vacinai (GIBSON, H. L; TUCKER, J. E.; KASLOW, D. C; KRETTLI, A. U.; COLLINS, W. E.; KIEFER, M. C; BATHURST, I. C; BARR, P. J. Structure and expression of the gene for Pv200, a major blood-stage surface antigen of Plasmodium vivax. Molecular and Biochemical Parasitology, v. 50, p. 325-334, 1992; COLLINS, W. E.; KASLOW, D. C; SULLIVAN, J. S.; MORRIS, C. L; GALLAND, G. G.; YANG, C; SAEKHOU, A. M.; XIAO, L; LAL, A. A. Testing the efficacy of a recombinant merozoite surface protein (MSP-1 19) of Plasmodium vivax in Saimiri boliviensis monkeys. American Journal Tropical Medicine and Hygiene, n. 60, p. 350-356, 1999; YANG, C; COLLINS, W. E.; SULLIVAN, J. S.; KASLOW, D. C; XIAO, L; LAL, A. Partial protection against Plasmodium vivax blood-stage infectionin Saimiri monkeys by immunization with a recombinant C-terminal fragment of merozoite surface protein 1 in block copolymer adjuvant. Infection and Immunity, v. 67, p. 342-349, 1999). [016] In the search for possible biomarkers for diagnosis of malaria infection, merozoite surface proteins (MSP) are among the first choice candidates because they expose themselves to various mechanisms of the host effector immune response. Ten members of the MSP family have already been described in P. falciparum, and eight of these have been detected in P. vivax. (PvMSP-1, PvMSP-185, PvMSP-3a, PvMSP-3b, PvMSP3c, PvMSP-4, PvMSP-5, PvMSP-8, PvMSP-9, and PVMSP-10). And in this context, the merozoite surface protein 1 (MSP-1) stands out for being present in all Plasmodium species (EGAN, AF; BURGHAUS, P.; DRUILHE, P.; HOLDER, AA; RILEY, E Human antibodies to the 19 kDa C-terminal fragment of Plasmodium falciparum merozoite surface protein 1 inhibit parasite growth in vitro Parasite Immunology, v. 21, pp. 133-139, 1999; STOWERS, AW; CIOCE, R. L; LAWSON , M .; HUI, G .; MURATOVA, O .; KASLOW, D. C. ROBINSON, R.; LONG, CA; MILLER, AH Efficacy of two alternate vaccines based on Plasmodium falciparum merozoite surface protein 1 in An Aotus challenge trial Infection and Immunity, v. 3, pp. 1536-1546, 2001). MSP-1 Sal-I comprises 1751 amino acids and is frequently tested for vaccine candidate (GIBSON, H.L; TUCKER, JE; KASLOW, D.C; KRETTLI, AU; COLLINS, WE; KIEFER, M.C; BATHURST, I.C. BARR, PJ Structure and expression of the gene for Pv200, the major blood-stage surface antigen of Plasmodium vivax.Molecular and Biochemical Parasitology, v. 50, pp 325-334, 1992; COLLINS, WE; KASLOW, D. C. SULLIVAN, JS; MORRIS, C. L; GALLAND, GG; YANG, C.; SAEKHOU, AM; XIAO, L; LAL, AA Testing the efficacy of a recombinant merozoite surface protein (MSP-1 19) of Plasmodium vivax in Saimiri boliviensis monkeys, American Journal Tropical Medicine and Hygiene, 60, pp 350-356, 1999; Yang, C., Collins, We; Suviniv, JS; Kaslow, D. C.; Xiao, L., Lal, A; Partial protection against Plasmodium vivax blood-stage infectionin Saimiri monkeys by immunization with a C-terminal recombinant fragment of merozoite surface protein 1 in block copolymer adjuvant Infection and Immunity, v. 67, pp. 342-349, 1999).
[017] Entre os avanços na pesquisa possibilitados pelos dados de sequenciamento de P. vivax, inclui-se a busca por peptídeos que possam ser úteis como biomarcadores para a malária por meio de diversas metodologias. Uma delas é a técnica de Spot-Syntesis, a qual consiste na síntese automática, em membrana de celulose, de uma série de peptídeos que podem ser utilizados de forma a mapear uma proteína e verificar quais deles são reativos a determinados anticorpos. Esses peptídeos são sintetizados, simultaneamente, em uma membrana de celulose, utilizando-se aminoácidos especiais, protegidos pelo grupamento Fmoc (Fluorenil Metil Oxicarbonila) que são depositados no sintetizador automático (LAUNE, D.; MOLINA, F.; FERRIÈRIS, G.; VILLARD, S.; BÈS, C; RIEUNIER, F.; CHARDÈS, T.; GRANIER. C. Application of the Spot method to the Identification of peptides and amino acids from the antibody paratope that contribute to antigen binding. Journal Immunol Methods, v. 267, p. 53-70, 2002). Este mapeamento permite a identificação de epitopos imunogênicos da proteína, bem como a caracterização do perfil de reatividade de diferentes epitopos frente a soros de grupos distintos de pacientes infectados ou não. Tendo em vista a impossibilidade de realizar o cultivo in vivo de P. vivax, a síntese química de peptídeos é uma importante ferramenta para o desenvolvimento de tecnologias moleculares que possibilitem o diagnóstico da malária causada por este parasito. [017] Advances in research made possible by P. vivax sequencing data include the search for peptides that may be useful as biomarkers for malaria through various methodologies. One of them is the Spot-Syntesis technique, which consists in the automatic cellulose membrane synthesis of a series of peptides that can be used to map a protein and verify which ones are reactive to certain antibodies. These peptides are synthesized, simultaneously on a cellulose membrane using special amino acids protected by the Fmoc group (Fluorenyl Methyl Oxicarbonyl) which are deposited on the automatic synthesizer (LAUNE, D .; MOLINA, F .; FERRIÈRIS, G .; VILLARD, S .; BÈS, C; RIEUNIER, F .; CHARDÈS, T .; GRANIER C. Application of the Spot method to the Identification of peptides and amino acids from the paratope antibody that contributes to antigen binding.Journal Immunol Methods, v. 267, p 53-70, 2002). This mapping allows the identification of immunogenic protein epitopes, as well as the characterization of the reactivity profile of different epitopes against sera from different groups of infected or uninfected patients. Given the impossibility of in vivo cultivation of P. vivax, chemical synthesis of peptides is an important tool for the development of molecular technologies that enable the diagnosis of malaria caused by this parasite.
[018] Lima-Junior e colaboradores relataram, pela primeira vez, o uso da técnica de spot-synthesis no mapeamento completo de epitopos antigênicos em uma proteína de P. vivax (PvMSP-3a). Por meio desta técnica, os autores caracterizaram os anticorpos anti-PvMSP-3a de P. Vivax naturalmente induzidos em indivíduos expostos a infecções por malária (LIMA-JUNIOR JC, JIANG J, RODRIGUES-DA-SILVA RN, BANIC DM, TRAN TM. B cell epitope mapping and characterization of naturally acquired antibodies to the Plasmodium vivax merozoite surface protein-3a (PvMSP-3a) in malária exposed individuais from Brazilian Amazon. Vaccine, v. 29, p. 1801 -181 1 , 201 1 ). Contudo, diferentemente da presente tecnologia, o objetivo destes autores foi apenas caracterizar a resposta imune de indivíduos expostos à malária avaliando a reação de pools de soros frente à proteína PvMSP-3a íntegra e dos peptídeos originados da mesma (os quais foram sintetizados por spot-synthesis), sem identificação de marcadores para a infecção. Como será descrito a seguir, nesta tecnologia foi selecionado um biomarcador originado de PvMSP-1 para anemia por malária vivax.  [018] Lima-Junior and colleagues first reported the use of spot-synthesis in the complete mapping of antigenic epitopes on a P. vivax protein (PvMSP-3a). By this technique, the authors characterized P. Vivax anti-PvMSP-3a antibodies naturally induced in individuals exposed to malaria infections (LIMA-JUNIOR JC, JIANG J, RODRIGUES-DA-SILVA RN, BANIC DM, TRANTM). B cell epitope mapping and characterization of naturally acquired antibodies to the Plasmodium vivax merozoite surface protein-3a (PvMSP-3a) in exposed individual malaria from Brazilian Amazon. Vaccine, v. 29, pp. 1801-181 1, 201 1). However, unlike the present technology, the aim of these authors was only to characterize the immune response of individuals exposed to malaria by evaluating the reaction of serum pools against intact PvMSP-3a protein and peptides originating from it (which were synthesized by spot- synthesis), without identifying markers for the infection. As will be described below, a biomarker originating from PvMSP-1 for vivax malaria anemia was selected in this technology.
[019] Na presente tecnologia, foi identificado um peptídeo de 15 aminoácidos (SEQ ID NQ1 ), cuja sequencia é derivada da proteína de superfície de merozoíto 1 de Plasmodium vivax, o qual é seletivamente reativo apenas a soros de pacientes com anemia em decorrência da infecção por P. vivax, não apresentando reatividade frente a soros de pacientes não anêmicos infectados por tal parasito, bem como a soros de pacientes infectados por P. falciparum, anêmicos ou não, e de pacientes não infectados por malária anêmicos ou não. In the present technology, a 15 amino acid peptide (SEQ ID NQ 1) has been identified, the sequence of which is derived from the surface protein of Plasmodium vivax merozoite 1, which is selectively reactive only to sera from patients with anemia due to P. vivax infection, showing no reactivity to sera from non-anemic patients infected with this parasite, as well as to sera from patients infected with P. P. falciparum, whether anemic or not, and patients not infected with anemia or not.
[020] Os pontos inovadores da presente tecnologia baseiam-se na identificação, por meio da técnica de spot-synthesis, de um peptídeo originado da proteína PvMSP-1 (SEQ ID NQ1 ), o qual seja útil como biomarcador em um kit imunodiagnóstico para a detecção de anemia causada por malária vivax. Este tipo de diagnóstico é de primordial importância para o prognóstico da doença bem como para nortear a escolha do tratamento adequado a ser empregado, tendo em vista que a anemia é uma das mais importantes manifestações clínicas, senão a mais importante, observada nas formas graves da malária vivax (CHANG, K. H.; STEVENSON, M. M. Malarial anaemia: mechanisms and implications of insufficient erythropoiesis during blood-stage malária. International Journal for Parasitology, v. 34, p. 1501 -1516, 2004; QUINTERO, J. P.; SIQUEIRA, A. M.; TOBÓN, A.; BLAIR, S.; MORENO, A.; ARÉVALO-HERRERA, M.; LACERDA, M. V. G.; VALENCIA, S. H. Malaria- related anaemia: a Latin American perspective. Memórias Do Instituto Oswaldo Cruz, v. 106, 201 1 ). [020] The breakthroughs of the present technology are based on spot-synthesis identification of a peptide originating from the protein PvMSP-1 (SEQ ID NQ 1) which is useful as a biomarker in a kit. immunodiagnosis for the detection of anemia caused by vivax malaria. This type of diagnosis is of paramount importance for the prognosis of the disease as well as to guide the choice of the appropriate treatment to be employed, considering that anemia is one of the most important, if not the most important, clinical manifestations observed in the severe forms of the disease. vivax malaria (CHANG, KH; STEVENSON, MM Malaria anemia: mechanisms and implications of insufficient erythropoiesis during blood-stage malaria. International Journal for Parasitology, v. 34, pp. 1501-1516, 2004; QUINTERO, JP; SIQUEIRA, AM; TOBÓN, A .; BLAIR, S .; MORENO, A .; ARÉVALO-HERRERA, M .; LACERDA, MVG; VALENCIA, SH Malaria-related anemia: a Latin American perspective. Memories of the Oswaldo Cruz Institute, v. 106, 201 1 ).
[021 ] Foram encontrados, no estado da técnica, documentos de patente e de pedidos de patente que tratam do uso de proteínas e peptídeos no imunodiagnóstico de malária.O documento WO0068270, intitulado "Antibodies and peptides for detection of Plasmodum vivax", descreve novos peptídeos cuja sequencia básica de aminoácidos corresponde a uma sequencia de 12 aminoácidos de ESP-1 de P. vivax a qual se repete por 3 vezes na proteína, bem como anticorpos produzidos por mamíferos em resposta a este antígeno. O pedido abrange ainda ensaios e kits de imunodiagnóstico empregando tais anticorpos. A presente tecnologia se difere da apresentada no documento citado, pois a proteína que deu origem ao peptídeo marcador para anemia por malária vivax trata-se da PvMSP-1 e não da PvESP-1 . [022] A patente US8030471 , intitulada "Plasmodium malaríae and Plasmodium ovale genes and uses thereof", descreve sequências nucleotídicas e aminoacídicas de MSP-1 de espécies de Plasmodium, sendo elas P. malaríae e P. ovale, bem como métodos de detecção de anticorpos anti tais espécies de plasmódio, por meio da utilização das proteínas protegidas pela patente. Foi observada identidade superior a 90% do peptídeo deste pedido de patente com duas sequencias proteicas protegidas pelo documento supracitado, entretanto estas sequências referem-se a polipeptídeos com número de aminoácidos aproximado de 1 .700, ou seja, não trata de pequenos peptídeos, como é o caso do presente pedido. Patent and patent documents dealing with the use of proteins and peptides in malaria immunodiagnosis have been found in the prior art. WO0068270 entitled "Antibodies and peptides for detection of Plasmodum vivax" describes new peptides whose basic amino acid sequence corresponds to a 12-amino acid sequence of P. vivax ESP-1 which repeats 3 times in the protein, as well as antibodies produced by mammals in response to this antigen. The application further encompasses immunodiagnostic assays and kits employing such antibodies. The present technology differs from that presented in the cited document, since the protein that originated the marker peptide for vivax malaria is PvMSP-1 and not PvESP-1. [022] US8030471, entitled "Plasmodium malaríae and Plasmodium ovale genes and uses thereof", describes nucleotide and amino acid sequences of MSP-1 from Plasmodium species, namely P. malaríae and P. ovale, as well as methods for detection of antibodies to such plasmid species by using the proteins protected by the patent. Greater than 90% identity of the peptide of this patent application has been observed with two protein sequences protected by the aforementioned document, however these sequences refer to polypeptides with approximate amino acid number of 1,700, ie it does not deal with small peptides such as This is the case with this application.
[023] O documento US7931908, intitulado "Chimeric MSP-based malária vaccine", descreve sequencias nucleotídicas e aminoacídicas de MSP-8 e MSP-1 19 de P. vivax e P. falciparum, bem como a fusão das mesmas, de maneira a formar uma proteína quimérica com o objetivo de se obter uma vacina antimalárica. Foi observada identidade superior a 90% do peptídeo do presente pedido de patente com uma sequência proteica protegida pelo documento supracitado. Entretanto além de a sequência protegida referir-se a um polipeptídeo com número de aminoácidos aproximado de 1 .700 e não de pequenos peptídeos, como no presente pedido, há ainda o fato de que trata-se de uma proteína quimérica com finalidade vacinai, o que não é pleiteado na presente tecnologia.  [023] US7931908, entitled "Chimeric MSP-based malaria vaccine", describes nucleotide and amino acid sequences of P. vivax and P. falciparum as well as the fusion thereof to form a chimeric protein for the purpose of obtaining an antimalarial vaccine. Greater than 90% identity of the peptide of the present application has been observed with a protein sequence protected by the above document. However, in addition to the protected sequence referring to a polypeptide with approximately amino acid number of 1,700 and not small peptides, as in the present application, there is also the fact that it is a chimeric protein for vaccination purpose. that is not claimed in the present technology.
[024] O documento US20041321 17, intitulado "Immunoassay and diagnostic reagent for malária", trata de um ensaio imunológico para diagnóstico de malária o qual detecta anticorpos específicos para malária no sangue por meio do uso da Proteína de Superfície de Merozoíto de Plasmodium vivax. Este pedido de patente descreve ainda a obtenção de tal proteína por meio de leveduras ou Escherichia coli transformadas. Diferentemente da presente tecnologia, este documento trata da sequência completa da PvMSP e não de pequenos peptídeos específicos extraídos desta sequência.  [024] US20041321 17, entitled "Immunoassay and diagnostic reagent for malaria", deals with an immunoassay for the diagnosis of malaria which detects specific malaria antibodies in the blood using Plasmodium vivax Merozoite Surface Protein. This patent application further describes obtaining such protein by means of transformed yeast or Escherichia coli. Unlike the present technology, this document deals with the complete sequence of PvMSP and not specific small peptides extracted from this sequence.
[025] O documento EP035461 1 , intitulado "Synthtic antigens useful in the diagnosis of malária induced by Plasmodium vivax", descreve o uso de novos antígenos sintéticos para a detecção de anticorpos anti-esporozoíto de P. vivax em amostras de sangue humano, por meio de um ensaio imunoenzimático empregando tais antígenos. Estes antígenos consistem de peptídeos originados na proteína circunsporozoitica de P. vivax, e são úteis em métodos de imunodiagnóstico e kit imunodiagnóstico para detecção de anticorpos com alta especificidade, sensibilidade e rapidez. Por tratar de peptídeos originados da proteína circunsporozoitica de P. vivax e não da proteína de superfície de merozoíto (MSP), este documento não é similar à presente tecnologia. [025] EP035461 1 entitled "Synthtic antigens useful in the diagnosis of malaria induced by Plasmodium vivax" describes the use of new synthetic antigens for the detection of P. vivax anti-sporozoite antibodies. in human blood samples by an enzyme immunoassay employing such antigens. These antigens consist of peptides originating from the circumsporozoite protein of P. vivax, and are useful in immunodiagnostic methods and immunodiagnostic kit for detection of antibodies with high specificity, sensitivity and rapidity. Because it deals with peptides originating from the circumsporozoite protein of P. vivax and not from the surface protein of merozoite (MSP), this document is not similar to the present technology.
[026] O documento US2007036818, intitulado "Recombinant protein containing a C-terminal fragment of Plasmodium MSP-1 ", o qual é uma continuação da patente US6958235, trata de uma proteína recombinante que inclui o fragmento C-terminal de 19 kDa da proteína de superfície do merozoíto de um parasita do tipo Plasmodium infeccioso em humanos, que não seja o P. vivax. AA maioria dos estudos apontam a porção C-terminal da PvMSP-1 como a mais imunogênica, principalmente, a extremidade de 19 kDa (RODRIGUES, M. H. C; CUNHA, M. G.; MACHADO, R. L. D.; FERREIRA, O. C; RODRIGUES, M. M.; SOARES, I. S. Serological detection of Plasmodium vivax malária using recombinant proteins corresponding to the 19-kDa C-terminal region of the merozoite surface protein-1 . Malária Journal, v. 2, n. 1 , 2003; PARK, J. W.; MOON, S. H.; YEOM, J. S.; LIM, K. J.; SOHN, M. J.; JUNG, W. C; CHO, Y. J.; JEON, K. W.; JU, W.; Kl, C. S.; OH, M. D.; CHOE, K. Naturally acquired antibody responses to the C-terminal region of merozoite surface protein 1 of Plasmodium vivax in Korea. Clinicai and Vaccine Immunology, v. 8, p. 14-20, 2001 ). A tecnologia ora pleiteada avaliou se os peptídeos que correspondem a esta extremidade de 19 kDa apresentariam elevado reconhecimento. E, realmente, a elevada imunogenicidade da MSP-1 19 foi confirmada, entretanto os resultados não apontaram diferença estatística significativa entre os índices de reatividade obtidos para soros de pacientes infectados e de indivíduos nunca expostos à malária, como será descrito nos exemplos adiante. Isto demonstra que esta fração da MSP-1 não é a mais satisfatória para o desenvolvimento de um biomarcador para malária. US2007036818, entitled "Recombinant protein containing a C-terminal fragment of Plasmodium MSP-1", which is a continuation of US6958235, deals with a recombinant protein that includes the 19 kDa C-terminal protein fragment. of the merozoite of an infectious Plasmodium parasite in humans other than P. vivax. Most studies point to the C-terminal portion of PvMSP-1 as the most immunogenic, especially the 19 kDa end (RODRIGUES, MH C; CUNHA, MG; MACHADO, RLD; FERREIRA, O; C; RODRIGUES, MM; SOARES, IS Serological detection of Plasmodium vivax malaria using recombinant proteins corresponding to the 19-kDa C-terminal region of the merozoite surface protein 1. Malaria Journal, v. 2, no. 1, 2003; PARK, JW; MOON, SH ; YEOM, JS; LIM, KJ; SOHN, MJ; JUNG, W.C; CHO, YJ; JEON, KW; JU, W .; Kl, CS; OH, MD; CHOE, K. Naturally acquired antibody responses to the C-terminal region of merozoite surface protein 1 of Plasmodium vivax in Korea Clinical and Vaccine Immunology, v. 8, pp 14-20, 2001). The technology claimed now evaluated whether the peptides corresponding to this 19 kDa end would be highly recognized. And indeed, the high immunogenicity of MSP-1 19 was confirmed, however the results showed no statistically significant difference between reactivity indices obtained for sera from infected patients and individuals never exposed to malaria, as will be described in the examples below. This demonstrates that this fraction of MSP-1 is not the most satisfactory for developing a malaria biomarker.
[027] O documento US2010166794, intitulado "Plasmodium liver stage antigens", abrange peptídeos, composições imunogênicas, sequências de nucleotídeos bem como o uso das composições para o diagnóstico de malária no estado hepático e indução da resposta imune. Neste documento são relacionados peptídeos derivados de proteínas especificamente expressas na fase hepática da infecção por Plasmodium. Dentre estas proteínas não está incluída a MSP-1 , o que difere este documento do presente pedido de patente. [027] US2010166794, entitled "Plasmodium liver stage antigens", covers peptides, immunogenic compositions, sequences of nucleotides as well as the use of the compositions for the diagnosis of malaria in the liver state and induction of the immune response. This document lists peptides derived from proteins specifically expressed in the liver phase of Plasmodium infection. Among these proteins is not included MSP-1, which differs from this document of this patent application.
[028] O documento US2007141075, intitulado "Plasmodium vivax blood stage antigens, PvESP-1 , antibodies and diagnostic assays" descreve a utilização de polipetídeos de P. vivax secretados no plasma de indivíduos infectados e anticorpos anti tais peptídeos, para o diagnóstico diferencial de P. vivax. Considerando que a presente tecnologia trata do diagnóstico diferencial de pacientes anêmicos em decorrência de malária vivax por meio da utilização de um peptídeo originado de PvMSP-1 , nota-se que não aborda o assunto do documento acima mencionado. [028] US2007141075 entitled "Plasmodium vivax blood stage antigens, PvESP-1, antibodies and diagnostic assays" describes the use of P. vivax polypeptides secreted in the plasma of infected individuals and antibodies against such peptides for the differential diagnosis of P. vivax. Considering that the present technology deals with the differential diagnosis of anemic patients due to vivax malaria through the use of a peptide originated from PvMSP-1, it is noteworthy that it does not address the subject of the above mentioned document.
[029] O documento EP1526178, intitulado "MSP-3-like family of genes", descreve a utilização de peptídeos derivados da proteína de superfície de merozoíto-3 (MSP-3) de Plasmodium na proteção contra malária. Este documento difere da presente tecnologia pelo fato de as sequencias dos peptídeos serem originadas de proteínas distintas e, portanto, não coincidirem.  EP1526178, entitled "MSP-3-like family of genes", describes the use of Plasmodium merozoite-3 (MSP-3) surface protein-derived peptides in protection against malaria. This document differs from the present technology in that the peptide sequences originate from distinct proteins and therefore do not coincide.
[030] Como pode ser constatado, a presente tecnologia se diferencia das demais encontradas no estado da técnica por tratar especificamente de um peptídeo (SEQ ID NQ1 ) e/ou seu polímero originado da sequencia da MSP-1 de P. vivax, e não da sequência completa desta proteína, ou de qualquer outra proteína de P. vivax. É digno de nota, ainda, que os peptídeos protegidos pelas patentes ou pedidos de patentes supracitados não correspondem à sequência do peptídeo do presente pedido. A tecnologia se distingue ainda por tratar do diagnóstico diferencial de anemia em decorrência de P. vivax. [030] As can be seen, the present technology differs from the others found in the prior art for dealing specifically with a peptide (SEQ ID NO: Q 1) and / or its polymer originated from the sequence of MSP-1 of P. vivax and not the complete sequence of this protein, or any other P. vivax protein. It is also noteworthy that the peptides protected by the above patents or patent applications do not correspond to the peptide sequence of the present application. The technology is also distinguished by addressing the differential diagnosis of anemia due to P. vivax.
[031 ] Na presente tecnologia, foi identificado, pela técnica spot-synthesis, um novo antígeno capaz de identificar soros de pacientes anêmicos infectados por Plasmodium vivax, o qual se mostrou bastante específico, não apresentando reação frente a soros de indivíduos infectados por P. falciparum ou indivíduos anêmicos por outras razões que não sejam a infecção por P. vivax. O kit imunodiagnóstico contendo este antígeno possibilitará a identificação imunológica e laboratorial específica de indivíduos anêmicos portadores de malária vivax, o que o torna uma importante ferramenta no prognóstico da doença e na escolha do tratamento. [031] In the present technology, a new antigen capable of identifying serum from Plasmodium vivax-infected anemic patients was identified by the spot-synthesis technique, which was quite specific and did not react to sera from P. infected individuals. falciparum or anemic individuals for reasons other than P. vivax infection. The immunodiagnostic kit containing this antigen will enable the identification of specific immunological and laboratory tests of anemic individuals with vivax malaria, which makes it an important tool in disease prognosis and treatment choice.
DESCRIÇÃO DAS FIGURAS DESCRIPTION OF THE FIGURES
[032] A Figura 1 Representa a membrana I, com o resultado das imunodetecções. (A) Utilizando pool de soros de indivíduos anêmicos - infectados por P. vivax (IAN); infectados por P. falciparum (PfAN); não infectados com anemia microcítica e hipocrômica (NIM) e infectados com anemia normocítica e normocrômica (NIN). (B) Utilizando pool de soros de indivíduos não anêmicos - infectados por P. vivax (INA); infectados por P. falciparum (PfNA); e não infectados (NINA). [032] Figure 1 represents membrane I, with the result of immunodetection. (A) Using pool of sera from anemic individuals - infected by P. vivax (IAN); infected with P. falciparum (PfAN); not infected with microcytic and hypochromic anemia (NIM) and infected with normocytic and normochromic anemia (NIN). (B) Using pool of sera from non-anemic individuals infected with P. vivax (INA); infected with P. falciparum (PfNA); and uninfected (NINA).
[033] A Figura 2 Representa o índice de Reatividade dos spots (IR). Traços vermelhos representam as medianas. *** indica P<0,0001 comparado com outros grupos (Kruskal-Wallis one way ANOVA). IAN = Pacientes anêmicos infectados por P. vivax; INA = Pacientes não anêmicos infectados por P. vivax; PFAN = Pacientes anêmicos infectados por P. falciparum; PFNA = Pacientes não anêmicos infectados por P. falciparum; NIM = Indivíduos não infectados com anemia microcítica e hipocrômica; NIN = Indivíduos não infectados com anemia normocítica e nomocrômica. [033] Figure 2 Represents the Spot Reactivity Index (RI). Red dashes represent the medians. *** indicates P <0.0001 compared to other groups (Kruskal-Wallis one way ANOVA). IAN = P. vivax-infected anemic patients; INA = Non-anemic patients infected with P. vivax; PFAN = Anemic patients infected with P. falciparum; NAFL = Non-anemic patients infected with P. falciparum; NIM = Uninfected individuals with microcytic and hypochromic anemia; NIN = Uninfected individuals with normocytic and nomochromic anemia.
[034] A Figura 3 representa (A) Prevalência (Teste exato de Fisher) e (B) magnitude da resposta (Kruskal-Wallis one way ANOVA acompanhado pelo teste pos hoc de Dunns) contra a região C-terminal MSP-119, Sal-I. IR = índice de reatividade. Traços vermelhos indicam as medianas. Gráficos não apresentaram diferença estatística significativa. IAN = Pacientes anêmicos infectados por P. vivax; INA = Pacientes não anêmicos infectados por P. vivax; PFAN = Pacientes anêmicos infectados por P. falciparum; PFNA = Pacientes não anêmicos infectados por P. falciparum; NIM = Indivíduos não infectados com anemia microcítica e hipocrômica; NIN = Indivíduos não infectados com anemia normocítica e nomocrômica. [035] A Figura 4 Representa as reações de diferentes soros frente ao peptídeo 70 (SEQ ID N°1 ) (seta). Todos os soros de pacientes anêmicos infectados por P.vivax (IAN), apresentaram-se soropositivos a este peptídeo, ao contrário das demais populações. IAN = Pacientes anêmicos infectados por P. vivax; INA = Pacientes não anêmicos infectados por P. vivax; PFAN = Pacientes anêmicos infectados por P. falciparum; PFNA = Pacientes não anêmicos infectados por P. falciparum; NIM = Indivíduos não infectados com anemia microcítica e hipocrômica; NIN = Indivíduos não infectados com anemia normocítica e nomocrômica; NINA = Indivíduos não infectados não anêmicos. [034] Figure 3 represents (A) Prevalence (Fisher's exact test) and (B) Response magnitude (Kruskal-Wallis one way ANOVA accompanied by Dunns pos hoc test) against C-terminal region MSP-119, Sal -I. IR = reactivity index. Red dashes indicate the medians. Graphs showed no statistically significant difference. IAN = P. vivax-infected anemic patients; INA = Non-anemic patients infected with P. vivax; PFAN = Anemic patients infected with P. falciparum; NAFL = Non-anemic patients infected with P. falciparum; NIM = Uninfected individuals with microcytic and hypochromic anemia; NIN = Uninfected individuals with normocytic and nomochromic anemia. [035] Figure 4 Represent the reactions of different sera against peptide 70 (SEQ ID NO: 1) (arrow). All sera from P.vivax (IAN) infected anemic patients were seropositive to this peptide, unlike the other populations. IAN = P. vivax-infected anemic patients; INA = Non-anemic patients infected with P. vivax; PFAN = Anemic patients infected with P. falciparum; NAFL = Non-anemic patients infected with P. falciparum; NIM = Uninfected individuals with microcytic and hypochromic anemia; NIN = Uninfected individuals with normocytic and nomochromic anemia; NINA = Uninfected non-anemic individuals.
[036] A Figura 5 representa a associação entre a absorbância ao peptídeo 70 (SEQ ID N°1 ) da MSP1 e a anemia por P. vivax. IAN = Indivíduos anêmicos infectados por P. vivax. INA = Indivíduos não anêmicos infectados por P. vivax. NINA = indivíduos não infectados e não anêmicos. Figure 5 represents the association between MSP1 peptide 70 absorbance (SEQ ID NO: 1) and P. vivax anemia. IAN = Anemic individuals infected with P. vivax. INA = Non-anemic individuals infected with P. vivax. NINA = uninfected and non anemic individuals.
DESCRIÇÃO DETALHADA DA TECNOLOGIA DETAILED DESCRIPTION OF TECHNOLOGY
[037] Na presente tecnologia, está compreendido um peptídeo antigênico (SEQ ID N°1 ) originado da proteína PvMSP-1 de Plasmodium vivax, bem como seu uso no imunodiagnostico de pacientes anêmicos em decorrência de infecção por P. vivax. O imunodiagnostico pode ser realizado através de um teste sorológico, podendo ser ensaio tipo ELISA, Western-Blot, dot-blot, imunodifusão ou imunocromatografia. [037] In the present technology, an antigenic peptide (SEQ ID NO: 1) originated from Plasmodium vivax protein PvMSP-1, as well as its use in the immunodiagnosis of anemic patients due to P. vivax infection, is comprised. Immunodiagnostics may be performed by a serological test, which may be ELISA, Western-Blot, dot-blot, immunodiffusion or immunochromatographic.
[038] Esta invenção descreve, ainda, a forma polimérica do peptídeo 70 (SEQ ID N°1 ) e a utilização deste peptídeo polimérico no imunodiagnóstico de pacientes anêmicos em decorrência de infecção por P. vivax. A polimerização deste peptídeo pode ser obtida, de forma não limitante, por meio da utilização de glutaraldeído. O imunodiagnóstico pode ser realizado através de um teste sorológico, podendo ser ensaio tipo ELISA, Western-Blot, dot-blot, imunodifusão ou imunocromatografia. [038] This invention further describes the polymeric form of peptide 70 (SEQ ID NO: 1) and the use of this polymeric peptide in the immunodiagnosis of anemic patients due to P. vivax infection. Polymerization of this peptide may be obtained, without limitation, by the use of glutaraldehyde. Immunodiagnosis may be performed by a serological test, which may be ELISA, Western-Blot, dot-blot, immunodiffusion or immunochromatographic.
[039] Na presente tecnologia, estão compreendidos, ainda, kits para imunodiagnóstico de anemia vivax, cujo funcionamento é baseado na identificação dos soros de pacientes anêmicos infectados por Plasmodium vivax por meio da utilização do peptídeo antigênico (SEQ ID NQ1 ) originado da proteína PvMSP-1 , na sua forma simples ou polimerizado. [039] In the present technology, kits for immunodiagnosis of vivax anemia are also understood, whose operation is based on the identification of the sera of anemic patients infected with Plasmodium. vivax by use of the antigenic peptide (SEQ ID NQ 1) originating from the PvMSP-1 protein, in its simple or polymerized form.
[040] Os kits para imunodiagnóstico descritos na presente tecnologia compreendem: a) Peptídeo SEQ ID N° 1 , na sua forma simples ou polimerizado; b) Anticorpos secundários conjugados a uma enzima ou a um marcador, específicos para reconhecerem o anticorpo primário contido nas amostras de indivíduos anêmicos com malária vivax; c) Reagente para detectar a enzima ou o marcador mencionado na etapa "b".  The immunodiagnostic kits described in the present technology comprise: a) Peptide SEQ ID NO: 1 in its simple or polymerized form; (b) an enzyme or marker-conjugated secondary antibody specific for recognizing the primary antibody contained in samples from anemic individuals with vivax malaria; c) Reagent for detecting the enzyme or label mentioned in step "b".
[041 ] Considerando os diferentes testes imunodiagnósticos, o peptídeo da etapa "a" deve estar ligado a um suporte sólido ou a um carreador. Os peptídeos podem ou não ser submetidos a uma etapa de fixação ao suporte sólido ou carreador por meio da utilização de metanol. A detecção dos anticorpos primários nos soros de indivíduos anêmicos com malária vivax será feita através do uso de anticorpos secundários (etapa "b") do tipo IgG, IgM, IgA ou IgE e subclasses deles. Esses são conjugados a uma enzima ou um marcador, sendo a enzima selecionada do grupo compreendendo fosfatase alcalina, peroxidase, β-galactosidase, urease, xantina oxidase, glicose oxidase e penicilinase. Os marcadores poderão ser selecionados do grupo consistindo de enzimas, radioisótopos, biotina, cromóforos, fluoróforos e quimioluminescentes. Os anticorpos secundários poderão ser detectados por técnicas de fluorescência, de imunoluminescência, de absorbância ou de radioisótopos.  [041] Considering the different immunodiagnostic tests, the "a" step peptide should be attached to a solid support or carrier. The peptides may or may not be subjected to a solid or carrier support fixation step using methanol. Detection of primary antibodies in the sera of anemic individuals with vivax malaria will be done by using secondary antibodies (step "b") of the IgG, IgM, IgA or IgE type and their subclasses. These are conjugated to an enzyme or a marker, the enzyme being selected from the group comprising alkaline phosphatase, peroxidase, β-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase. Markers may be selected from the group consisting of enzymes, radioisotopes, biotin, chromophores, fluorophores and chemiluminescent. Secondary antibodies may be detected by fluorescence, immunoluminescence, absorbance or radioisotope techniques.
[042] O método para imunodiagnóstico de anemia em decorrência de malária vivax pode ser realizado através de testes sorológicos, podendo ser ELISA, Western-Blot, dot-blot, imunodifusão e/ou imunocromatografia, os quais compreendem as seguintes etapas: a) expor os anticorpos primários de uma amostra ao peptídeo que apresenta sequência de aminoácidos SEQ ID NQ 1 , em sua forma simples ou polimerizado; b) colocar os anticorpos da etapa "a" em contato com um anticorpo secundário, conjugado a uma enzima ou a um marcador, que se liga aos anticorpos da etapa "a"; c) detectar os anticorpos anti-malária exclusivos dos indivíduos anêmicos em decorrência de infecção por P. vivax na amostra supracitada pela detecção do anticorpo secundário especificamente ligado ao dito anticorpo anti-malária. [042] The method for immunodiagnosis of anemia due to vivax malaria may be performed by serological tests, which may be ELISA, Western-blot, dot-blot, immunodiffusion and / or immunochromatography, which comprise the following steps: a) expose the primary antibodies of a sample to the peptide having amino acid sequence SEQ ID NQ 1, in simple or polymerized form; b) contacting the antibodies of step "a" in contact with a secondary antibody, conjugated to an enzyme or label, which binds to the antibodies of step "a"; c) detecting anti-malaria antibodies unique to anemic individuals due to P. vivax infection in the above sample by detecting the secondary antibody specifically bound to said anti-malaria antibody.
[043] O peptídeo da etapa "a" deve estar ligado a um suporte sólido ou a um carreador. Os peptídeos podem ou não ser submetidos a uma etapa de fixação ao suporte sólido ou carreador por meio da utilização de metanol. A detecção dos anticorpos primários nos soros de indivíduos anêmicos com malária vivax será feita através do uso de anticorpos secundários (etapa "b") do tipo IgG, IgM, IgA ou IgE e subclasses deles. Esses são conjugados a uma enzima ou um marcador, sendo a enzima selecionada do grupo compreendendo fosfatase alcalina, peroxidase, β-galactosidase, urease, xantina oxidase, glicose oxidase e penicilinase. Os marcadores poderão ser selecionados do grupo consistindo de enzimas, radioisótopos, biotina, cromóforos, fluoróforos e quimioluminescentes. Os anticorpos secundários poderão ser detectados por técnicas de fluorescência, de imunoluminescência, de absorbância ou de radioisótopos.  [043] The peptide of step "a" must be attached to a solid support or a carrier. The peptides may or may not be subjected to a solid or carrier support fixation step using methanol. Detection of primary antibodies in the sera of anemic individuals with vivax malaria will be done by using secondary antibodies (step "b") of the IgG, IgM, IgA or IgE type and their subclasses. These are conjugated to an enzyme or a marker, the enzyme being selected from the group comprising alkaline phosphatase, peroxidase, β-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase. Markers may be selected from the group consisting of enzymes, radioisotopes, biotin, chromophores, fluorophores and chemiluminescent. Secondary antibodies may be detected by fluorescence, immunoluminescence, absorbance or radioisotope techniques.
[044] A tecnologia descrita pode ser mais bem compreendida, de forma não limitante, por meio dos exemplos seguintes.  The technology described can be better understood, but not limited to, by the following examples.
Exemplo 1 - Síntese do peptídeo 70 (SEQ ID N°1) em membrana de celulose e avaliação da reatividade de pools de soros de pacientes com malária vivax frente a este peptídeo Example 1 - Synthesis of peptide 70 (SEQ ID NO: 1) on cellulose membrane and evaluation of serum pools reactivity of patients with vivax malaria against this peptide
[045] A identificação do peptídeo (SEQ ID N°1 ) foi feita através da técnica de spot-synthesis, a qual permitiu o mapeamento de epitopos da sequência completa de PvMSP-1 , que compreende 1751 aminoácidos. A técnica de spot- synthesis permitiu a síntese múltipla de peptídeos em membrana de celulose, cobrindo toda a extensão da proteína. A síntese destes peptídeos foi feita conforme metodologia descrita por Laune et al., 2002 (LAUNE, D., MOLINA, F., FERRIÈRIS, G., VILLARD, S., BÈS, C, RIEUNIER, F., CHARDÈS, T., GRANIER. C. Application of the Spot method to the identification of peptides and amino acids from the antibody paratope that contribute to antigen binding. Journal Immunol Methods, v. 267, p. 53-70, 2002). Na Membrana I, cada spot consistiu em um arranjo de peptídeos de quinze resíduos de aminoácidos, com sobreposição de 12 resíduos, totalizando 580 spots. Esta membrana foi utilizada para realização de um screening de grupos de soros reativos diferencialmente frente a cada peptídeo, sendo que estes grupos de soros consistiam de: [045] Peptide identification (SEQ ID NO: 1) was done by spot-synthesis technique, which allowed epitope mapping of the complete PvMSP-1 sequence comprising 1751 amino acids. The spot synthesis technique allowed the multiple synthesis of peptides in cellulose membrane, covering the entire length of the protein. The synthesis of these peptides was made according to the methodology described by Laune et al., 2002 (LAUNE, D., MOLINA, F., FERRIÈRIS, G., VILLARD, S., BÈS, C, RIEUNIER, F., CHARDÈS, T., GRANIER. C. Application of the Spot method to the identification of peptides and amino acids from the antibody paratope that contributes to antigen binding. Journal Immunol Methods, v. 267, p. 53-70, 2002). In Membrane I, each spot consisted of an array of fifteen amino acid residues overlapping 12 residues totaling 580 spots. This membrane was used to screen differently for reactive sera groups against each peptide, and these sera groups consisted of:
1 ) IAN - pacientes com infecção por P. wVax anêmicos (n=10);  1) IAN - patients with anemic P. wVax infection (n = 10);
2) INA - pacientes com infecção por P. wVax não-anêmicos (n=10);  2) INA - patients with non-anemic P. wVax infection (n = 10);
3) PfAN - pacientes com infecção por P. falciparum anêmicos (n=6);  3) PfAN - patients with anemic P. falciparum infection (n = 6);
4) PfNA - pacientes com infecção por P. falciparum não-anêmicos (n=6);  4) PfNA - patients with nonanemic P. falciparum infection (n = 6);
5) NINA - pacientes não-infectados não-anêmicos (n=10);  5) NINA - noninfected nonanemic patients (n = 10);
6) NIM - pacientes não-infectados com anemia microcítica e hipocrômica, neste caso, anemia decorrente de outras etiologias (n=10);  6) NIM - uninfected patients with microcytic and hypochromic anemia, in this case anemia due to other etiologies (n = 10);
7) NIN - pacientes não-infectados com anemia normocítica e normocrômica, neste caso, anemia decorrente de outras etiologias (n=10).  7) NIN - uninfected patients with normocytic and normochromic anemia, in this case anemia due to other etiologies (n = 10).
[046] A Membrana I, contendo os peptídeos sintéticos, foi lavada com etanol P.A., em seguida, com Phosphate Buffer Solution 1 X (PBS) durante dez minutos, e então uma solução de bloqueio {Bovine Serum Albumine 3% e sacarose 5% em PBS-Tween 0,1 %) foi adicionada e mantida overnight. No dia seguinte, a solução de bloqueio foi retirada e a membrana foi lavada com PBS- Tween 0,1 % durante dez minutos. A membrana foi então incubada com o pool de soros (anticorpo) diluído em solução de bloqueio (1 :500), por 2 horas à temperatura ambiente e sob agitação constante. Após a incubação, a membrana foi novamente lavada com PBS-Tween 0,1 % durante dez minutos, por três vezes. Para a revelação, o anticorpo secundário conjugado com fosfatase alcalina foi diluído na solução de bloqueio (1 :2000) e incubado com a membrana por 1 hora, sob agitação à temperatura ambiente. Em seguida, foram realizadas duas lavagens de dez minutos cada, com PBS-Tween 0,1 %, e mais duas lavagens, também de dez minutos cada, com Coating Buffer Solution (CBS, pH 7.0). Como substrato, uma solução composta por CBS pH 7.0, Thiazolyl Blue Tetrazolium Bromide (MTT), 5-bromo-4-chloro-3-indolyl phosphate disodium (BCIP) e Cloreto de Magnésio (MgCl2), foi adicionada e mantida sob agitação por 15 a 30 minutos. A reação de revelação foi interrompida com água destilada e a membrana foi digitalizada para posterior análise. Membrane I containing the synthetic peptides was washed with ethanol PA, then Phosphate Buffer Solution 1X (PBS) for ten minutes, and then a blocking solution {Bovine Serum Albumine 3% and 5% sucrose. in PBS-Tween 0.1%) was added and maintained overnight. The following day, the blocking solution was removed and the membrane was washed with 0.1% PBS-Tween for ten minutes. The membrane was then incubated with the sera (antibody) pool diluted in blocking solution (1: 500) for 2 hours at room temperature and under constant agitation. After incubation, the membrane was again washed with 0.1% PBS-Tween for ten minutes three times. For the development, the alkaline phosphatase-conjugated secondary antibody was diluted in the blocking solution (1: 2000) and incubated with the membrane for 1 hour under shaking at room temperature. Then, two washes of ten minutes each, with PBS-Tween 0.1%, and two washes, also ten minutes each, with Coating Buffer Solution (CBS, pH 7.0). As a substrate, a solution composed of CBS pH 7.0, Thiazolyl Blue Tetrazolium Bromide (MTT), 5-bromo-4-chloro-3-indolyl phosphate disodium (BCIP) and Magnesium Chloride (MgCl2) was added and stirred under stirring. 15 to 30 minutes. The development reaction was stopped with distilled water and the membrane was digitized for further analysis.
[047] A fim de reutilizar-se a membrana para imunodetecção com os diferentes pools de soros, foi realizada uma etapa de regeneração, na qual a membrana foi submetida a três lavagens consecutivas, de dez minutos cada, com DMF (dimetilformamida), mais três lavagens com o Reagente A (ureia 8M + Sodium Dodecil Sulfate a 2,5% + 2-mercaptoetanol 0,1 %) e três lavagens com o Reagente B (50ml_ de etanol + 40ml_ de água + 10ml_ de ácido acético), também durante dez minutos cada.  In order to reuse the membrane for immunodetection with the different serum pools, a regeneration step was performed in which the membrane was subjected to three consecutive washes of ten minutes each with DMF (dimethylformamide) plus three washes with Reagent A (8M Urea + 2.5% Sodium Dodecil Sulfate + 0.1% 2-mercaptoethanol) and three washes with Reagent B (50ml_ ethanol + 40ml_ water + 10ml_ acetic acid), also for ten minutes each.
[048] Ressalta-se que a primeira imunodetecção foi realizada apenas com o anticorpo conjugado com fosfatase alcalina, ou seja, sem adição do anticorpo primário, seguindo-se das demais etapas (revelação e regeneração), para controle da membrana e verificação de reações cruzadas. Nesse sentido, dez peptídeos foram descartados por assumirem reatividade frente ao teste controle. As imunodetecçoes seguintes foram realizadas utilizando-se os pools de soros dos sete grupos de indivíduos analisados (diluição 1 :500): infectados por P. wVax anêmicos (IAN) e não anêmicos (INA); infectados por P. falciparum anêmicos (PfAN) e não anêmicos (PfNA); e não infectados controles anêmicos (NIM e NIN) e não anêmicos (NINA). Após cada detecção, a membrana foi regenerada para utilização com o pool àe soros seguinte. A Figura 1 apresenta a imagem da Membrana I, após os testes.  It is noteworthy that the first immunodetection was performed only with the alkaline phosphatase conjugated antibody, ie without the addition of the primary antibody, followed by the other steps (development and regeneration), for membrane control and reaction verification. crusades. In this sense, ten peptides were discarded for assuming reactivity to the control test. The following immunodetections were performed using the sera pools of the seven groups of individuals analyzed (1: 500 dilution): anemic (IAN) and non-anemic (INA) P. wVax infected; P. falciparum infected anemic (PfAN) and non anemic (PfNA); and uninfected anemic (NIM and NIN) and non-anemic (NINA) controls. After each detection, the membrane was regenerated for use with the next pool of sera. Figure 1 shows the image of Membrane I after the tests.
[049] Inicialmente, foi realizada uma análise visual da Membrana I para identificação dos pontos reativos e não reativos. A quantificação dos spots foi realizada utilizando-se um software para edição de imagens. As membranas digitalizadas foram adicionadas ao programa e cada spot foi marcado, manualmente, para quantificação, permitindo assim a comparação entre as reatividades obtidas em cada ponto. Após conversão para preto e branco, as regiões não reativas (negativas) da membrana foram quantificadas e a média dos valores obtidos nesta quantificação foi subtraída do valor de cada spot reativo, de modo a diminuir as interferências do background. Dessa forma, a comparação dos spots entre as diferentes membranas tornou-se mais fidedigna. Valores negativos obtidos após este cálculo foram considerados nulos. [049] Initially, a visual analysis of Membrane I was performed to identify reactive and nonreactive points. Spot quantification was performed using image editing software. The digitized membranes were added to the program and each spot was manually marked for quantification, thus allowing the comparison between the reactivities obtained at each point. After conversion to black and white, non-reactive (negative) membrane regions were quantified and the mean The values obtained in this quantification were subtracted from the value of each reactive spot, in order to reduce background interference. Thus, the comparison of the spots between the different membranes became more reliable. Negative values obtained after this calculation were considered null.
[050] Soros de indivíduos que apresentaram anemia concomitante à infecção por P. vivax reconheceram dez peptídeos que não foram reativos aos demais pools utilizados, incluindo o peptídeo inserido na posição 70 (SEQ ID N°1 ). Os valores da reatividade para cada um dos peptídeos encontram-se na Tabela 1 , na qual se observa que o peptídeo 70 (SEQ ID N°1 ) se destaca como possível biomarcador para anemia em decorrência de malária vivax.  [050] Sera from individuals with anemia concomitant with P. vivax infection recognized ten peptides that were unreactive to the other pools used, including the peptide inserted at position 70 (SEQ ID NO: 1). Reactivity values for each of the peptides are shown in Table 1, which shows that peptide 70 (SEQ ID No. 1) stands out as a possible biomarker for anemia due to vivax malaria.
Tabela 1 - Valores atribuídos aos peptídeos considerados como possíveis biomarcadores Table 1 - Values attributed to peptides considered as possible biomarkers
QPOT índice de Reatividade  QPOT Reactivity Index
IAN INA NINA NIM NIN PFAN PFNA  IAN INA NINA NIM NIN PFAN
69 12.171 2.929 0 0 0 0 0 69 12.171 2.929 0 0 0 0 0
314 57.314 3.695 0 0 0 0 0314 57,314 3,695 0 0 0 0 0
50 14.006 0 0 0 0 0 050 14,006 0 0 0 0 0 0
51 8.014 0 0 0 0 0 051 8.014 0 0 0 0 0 0
70 30.888 0 0 0 0 0 070 30,888 0 0 0 0 0 0
1 1 1 9.034 0 0 0 0 0 01 1 1 9,034 0 0 0 0 0 0
181 6.624 0 0 0 0 0 0181 6,624 0 0 0 0 0 0
184 10.868 0 0 0 0 0 0184 10,868 0 0 0 0 0 0
272 16.955 0 0 0 0 0 0272 16,955 0 0 0 0 0 0
275 6.410 0 0 0 0 0 0275 6,410 0 0 0 0 0 0
506 1.832 0 0 0 0 0 0506 1,832 0 0 0 0 0 0
507 3.511 0 0 0 0 0 0507 3,511 0 0 0 0 0 0
Obs.: índices de ι reatividade obtidos através do software para edição de imagens. Note: reactivity indexes obtained through the image editing software.
Exemplo 2 - Reconhecimento da porção correspondente à PvMSP-1 19 pelos diferentes pools de soros Example 2 - Recognition of PvMSP-1 19 portion by different serum pools
[051 ] Grande parte dos estudos para identificação de imunógenos de PvMSP- 1 apontam a porção C-terminal desta proteína como a mais imunogênica, principalmente, a extremidade de 19kDa (RODRIGUES et al., 2003; PARK et al., 2001 ). Desta forma, durante o desenvolvimento desta tecnologia foi avaliado se os peptídeos que correspondem a esta região apresentariam elevado reconhecimento. E, realmente, a elevada imunogenicidade da MSP-1 19 foi confirmada, entretanto não foi possível observar diferença entre o número de spots reconhecidos pelos diferentes grupos de soros (Figura 3A), e entre as magnitudes das respostas obtidas (Figura 3B), ou seja, os resultados não apontaram diferença estatística significativa entre os índices de reatividade obtidos para soros de pacientes infectados e de indivíduos nunca expostos à malária. Isto demonstra que esta fração da MSP-1 não é a mais adequada para utilização como biomarcador para malária. [051] Most studies for identification of PvMSP-1 immunogens point to the C-terminal portion of this protein as the most immunogenic, especially the 19kDa end (RODRIGUES et al., 2003; PARK et al., 2001). This way, during the development of this technology it was whether peptides corresponding to this region would be highly recognized. And indeed, the high immunogenicity of MSP-1 19 was confirmed, however, no difference could be observed between the number of spots recognized by the different groups of sera (Figure 3A), and between the magnitudes of the responses obtained (Figure 3B), or That is, the results showed no statistically significant difference between the reactivity indices obtained for sera from infected patients and individuals never exposed to malaria. This shows that this fraction of MSP-1 is not best suited for use as a biomarker for malaria.
Exemplo 3 - Imunodetecção do peptídeo biomarcador para anemia por Plasmodium vivax (SEQ ID N°1) Example 3 - Immunodetection of Plasmodium vivax Anemia Biomarker Peptide (SEQ ID NO: 1)
[052] Após a identificação dos possíveis biomarcadores listados na Tabela 1 , estes peptídeos, incluindo o peptídeo 70 (SEQ ID N°1 ), foram incluídos na segunda etapa de testes, a síntese da Membrana II. Desta forma, a membrana II foi feita com peptídeos selecionados com base na reatividade dos pools de soros observada na Membrana I.  [052] Following identification of the possible biomarkers listed in Table 1, these peptides, including peptide 70 (SEQ ID NO: 1), were included in the second testing step, Membrane II synthesis. Thus, membrane II was made with peptides selected based on the reactivity of the sera pools observed in Membrane I.
[053] No intuito de identificar um biomarcador para a infecção e para a anemia por P. vivax, as imunodetecções da Membrana II foram realizadas com a incubação, individual, de cada soro que compôs os pools IAN (n=10) e INA (n=10), utilizados nos testes com a Membrana I. Também se testou, individualmente, os soros dos pools PfAN (n=3) e PfNA (n=3). Os controles (NINA (n=10), NIM (n=10) e NIN (n=10)), por sua vez, foram testados em pool, já que não se pretendia obter reatividade frente aos soros destes grupos.  [053] In order to identify a biomarker for P. vivax infection and anemia, Membrane II immunodetections were performed by individually incubating each serum that comprised the IAN (n = 10) and INA pools ( n = 10), used for Membrane I tests. Sera from the PfAN (n = 3) and PfNA (n = 3) pools were also tested individually. The controls (NINA (n = 10), NIM (n = 10) and NIN (n = 10)), in turn, were pool tested, as reactivity to the sera from these groups was not intended.
[054] Inicialmente, realizou-se a imunodetecção na Membrana II sem a adição do anticorpo primário para a determinação de reações cruzadas entre o conjugado e os peptídeos sintéticos, e não foi observada reatividade cruzada frente ao anticorpo secundário. Com relação à reatividade dos soros de indivíduos com malária, observou-se que todos os soros de pacientes anêmicos infectados por P. vivax (IAN), apresentaram-se soropositivos ao peptídeo 70 (SEQ ID NQ1 ), ao contrário das demais populações. Sendo assim, o peptídeo 70 foi considerado um possível biomarcador para a anemia durante a infecção por este plasmódio (Figura 4). Exemplo 4 - Avaliação da reatividade do peptídeo 70 (SEQ ID N°1) em sua forma polimerizada frente a soros de pacientes anêmicos infectados por malária vivax Initially, Membrane II immunodetection was performed without the addition of the primary antibody for the determination of cross-reactions between the conjugate and the synthetic peptides, and no cross-reactivity with the secondary antibody was observed. Regarding the reactivity of sera from individuals with malaria, it was observed that all sera from P. vivax (IAN) infected anemic patients were seropositive to peptide 70 (SEQ ID N Q 1), unlike the other populations. . Thus, peptide 70 was considered a possible biomarker for anemia during infection with this plasmid (Figure 4). Example 4 - Evaluation of Peptide 70 Reactivity (SEQ ID NO: 1) in its polymerized form against sera from vivax malaria infected anemic patients
[055] Primeiramente, foi realizada a polimerização do peptídeo 70 (SEQ ID N°1 ) por glutaraldeído. Foi preparada uma solução de peptídeo, previamente sintetizado manualmente por FMOC-Synthesis Method e liofilizado, em PBS (pH 7,4). Posteriormente, foi preparada solução 1 % v/v de glutaraldeído em água, sendo esta adicionada gota a gota à solução anterior, constantemente agitada por agitador magnético, também em câmara fria. Após uma hora, a reação foi parada pela adição de borohidrato de sódio (NaBH4) em quantidade suficiente para que esse ficasse em concentração final de 10 mg/mL na solução. Foi feita diálise da solução final e obtida solução de peptídeo polimerizado a 2,5 mg/mL. First, peptide 70 (SEQ ID NO: 1) was polymerized by glutaraldehyde. A peptide solution, previously synthesized manually by FMOC-Synthesis Method and lyophilized, was prepared in PBS (pH 7.4). Subsequently, 1% v / v solution of glutaraldehyde in water was prepared, which was added dropwise to the above solution, constantly stirred by magnetic stirrer, also in a cold chamber. After one hour, the reaction was stopped by adding enough sodium borohydrate (NaBH 4 ) to bring it to a final concentration of 10 mg / mL in the solution. The final solution was dialysed and 2.5 mg / mL polymerized peptide solution was obtained.
[056] Para o teste de ELISA, microplacas de 96 poços (Costar, Cambridge, MA) foram sensibilizadas com 100 μΐ dos antígenos (peptídeo consistindo da sequência SEQ ID N°1 polimerizado), em concentração de 2,5 mg/mL, diluídos em tampão carbonato-bicarbonato (pH 9,6; 0,1 M), por 18 horas (overnight) a 4QC. Após a incubação, foram adicionados 50 uL/well de metanol para fixação do peptídeo à placa e esperada 1 hora a 37QC até a evaporação completa do líquido. As microplacas foram lavadas cinco vezes com PBS contendo 0,05% de Tween 20 (PBST) e, em seguida, bloqueadas com PBST contendo 5% de leite desnatado (Moliço Nestlé®), por 1 hora a 37QC. Posteriormente, repetiu-se[056] For the ELISA assay, 96-well microplates (Costar, Cambridge, MA) were sensitized with 100 μΐ of antigens (peptide consisting of polymerized sequence SEQ ID No. 1) at a concentration of 2.5 mg / mL, diluted in carbonate-bicarbonate buffer (pH 9.6; 0.1M) for 18 hours (overnight) at 4 Q C. After incubation, was added 50 uL / well of methanol to the peptide fixation plate and expected 1 Q hour at 37 C until complete evaporation of the liquid. The plates were washed five times with PBS containing 0.05% Tween 20 (PBST) and then blocked with PBST containing 5% skim milk (Moliço NESTLÉ®) for 1 hour at 37 Q C. Subsequently, repeated up
0 processo de lavagem por cinco vezes consecutivas e adicionou-se a cada orifício, em duplicatas, 100 μΐ das amostras de soros diluídas 1 :100 em PBST contendo 1 % de BSA (PBST+BSA). Após incubação de 1 hora a 37QC, as placas foram novamente submetidas a cinco lavagens com PBST e o anticorpo conjugado a peroxidase, anti-lgG humano diluído 1 :2000 em PBST+BSA (100 μί/ροςο) foi adicionado, seguindo-se mais uma vez por incubação a 37 QC porThe washing process was performed five consecutive times and 100 μΐ of 1: 100 diluted serum samples in PBST containing 1% BSA (PBST + BSA) were added to each well in duplicates. After 1 hour incubation at 37 Q C, the plates were subjected to five washes with PBST and peroxidase conjugated antibody, anti-human IgG diluted 1: 2000 in PBST + BSA (100 μί / ροςο) was added, followed once again by incubating at 37 C for Q
1 hora. Transcorrido esse período, as placas foram lavadas por cinco vezes. Pelo uso do anticorpo conjugado a peroxidase, faz-se necessário o uso da Streptoavidina diluída em PBS em concentração determinada pelo fabricante (100μί/ροςο). A revelação foi realizada por meio da adição de OPD (o- phenylenediamine dihydrochloride substrate - Sigma, EUA) diluído em tampão fosfato-citrato, pH 5,0 contendo peróxido de hidrogénio (H2O2) (100μΙ_/ρος:ο). Em seguida, as placas foram incubadas protegidas da luz por 1 hora a 37QC, até a reação ser interrompida pela adição de 50μΙ_ de uma solução de H2SO4 4 N. 1 hour. After this time, the plates were washed five times. The use of peroxidase-conjugated antibody requires the use of Streptoavidin diluted in PBS at a concentration determined by the manufacturer (100μί / ροςο). The revelation was accomplished by the addition of OPD phenylenediamine dihydrochloride substrate - Sigma, USA) diluted in phosphate-citrate buffer, pH 5.0 containing hydrogen peroxide (H2O2) (100μΙ_ / ρος: ο). Then the plates were incubated protected from light for 1 hour at 37 Q C until the reaction is stopped by adding 50μΙ_ a 4 N solution of H2SO4
[057] As amostras de soros avaliadas consistiram de 15 soros de indivíduos infectados anêmicos, 129 infectados não anêmicos e 22 não infectados não anêmicos.  [057] The sera samples evaluated consisted of 15 sera from anemic infected individuals, 129 non-anemic infected and 22 non-anemic uninfected individuals.
[058] Os valores de absorbância foram, então, medidos a 492 nm utilizando-se um leitor de microplacas automático (SpectraMax 240 PC, Molecular Devices). Como não há absorbância determinada (padrão) do peptídeo 70 (SEQ ID N°1 ), as análises foram feitas com base nas absorbâncias de cada amostra.  Absorbance values were then measured at 492 nm using an automated microplate reader (SpectraMax 240 PC, Molecular Devices). Since there is no determined (standard) absorbance of peptide 70 (SEQ ID No. 1), analyzes were performed based on the absorbances of each sample.
[059] No ELISA, foi analisado um total de 166 soros (IAN - n=15, INA - n=129, NINA - n=22) e foi observado que soros de indivíduos anêmicos infectados por P. vivax (IAN) apresentaram maiores absorbâncias frente o peptídeo 70 (SEQ ID N°1 ), comparado ao grupo dos indivíduos infectados não anêmicos (Figura 5). Entretanto, foi possível verificar que pacientes infectados por P. vivax, mas sem anemia e também poucos indivíduos não infectados apresentavam valores altos de absorbância frente ao peptídeo 70. In the ELISA, a total of 166 sera were analyzed (IAN - n = 15, INA - n = 129, NINA - n = 22) and it was observed that sera from P. vivax infected anemic individuals (IAN) had higher absorbances against peptide 70 (SEQ ID NO: 1), compared to the group of non-anemic infected individuals (Figure 5). However, it was possible to verify that patients infected by P. vivax, but without anemia and also few uninfected individuals had high absorbance values against the peptide 70.
Exemplo 5 - Avaliação comparativa da reatividade do peptídeo 70 (SEQ ID N°1) em sua forma simples ou polimerizado e avaliação da etapa de fixação do peptídeo à placa de ELISA com metanol Example 5 - Comparative Evaluation of Peptide 70 Reactivity (SEQ ID NO: 1) in its Simple or Polymerized Form and Evaluation of the Peptide Fixation Step to the Methanol ELISA Plate
[060] A fim de se determinar as melhores condições para realização dos testes imunoenzimáticos (ELISA) procederam-se análises comparativas das reatividades obtidas a partir do uso do peptídeo 70 da MSP1 (SEQ ID N°1 ) na sua forma simples ou polimerizada. Nesta análise também foi avaliado o efeito da fixação dessas duas formulações à placa de ELISA a partir da adição de metanol. A Tabela 2 representa os valores médios de absorbância quando se utilizou pools de soros de indivíduos e pacientes dos diferentes grupos e a Tabela 3 representa a média de absorbância dos soros dos diferentes grupos, testados individualmente. Tabela 2 - Valores médios de absorbância para o peptídeo 70 polimerizado ou não polimerizado e com uso de metanol ou não para fixação frente a diferentes pools de soros. In order to determine the best conditions for performing enzyme-linked immunosorbent assays (ELISA), comparative analyzes of reactivity obtained from the use of MSP1 peptide 70 (SEQ ID NO: 1) in its simple or polymerized form were performed. In this analysis we also evaluated the effect of fixing these two formulations to the ELISA plate by adding methanol. Table 2 represents the mean absorbance values when serum pools of individuals and patients from different groups were used and Table 3 represents the mean absorbance of serum from different groups, tested individually. Table 2 - Average absorbance values for polymerized or unpolymerized peptide 70 with or without methanol for fixation in front of different serum pools.
Figure imgf000027_0001
Figure imgf000027_0001
Concentração dos peptídeos: 1 ug/well; diluição dos soros: 1 :100; soros infectados anêmicos (IAN), infectados não anêmicos (INA) e não infectados (NI).  Peptide concentration: 1 ug / well; serum dilution: 1: 100; anemic infected (IAN), infected nonanemic (INA) and uninfected (NI) sera.
Tabela 3 - Valores médios de absorbância para o peptídeo 70 polimerizado ou não polimerizado e com uso de metanol ou não para fixação frente a soros individuais de indivíduos parasitados por P. vivax anêmicos ou não e controles saudáveis. Table 3 - Average absorbance values for polymerized or unpolymerized peptide 70 with or without methanol for fixation to individual sera from P. vivax parasitized individuals or not and healthy controls.
Figure imgf000027_0002
Figure imgf000027_0002
Concentração dos peptídeos: 1 ug/well; diluição dos soros: 1 :100; soros infectados anêmicos (IAN), infectados não anêmicos (INA) e não infectados (NI).  Peptide concentration: 1 ug / well; serum dilution: 1: 100; anemic infected (IAN), infected nonanemic (INA) and uninfected (NI) sera.
[061 ] Foi constatado que o uso do peptídeo polimerizado por glutaradeído aliado à sua prévia fixação com metanol à placa de ELISA fornece maiores diferenças entre as absorbâncias detectadas para os diferentes grupos clínicos, em ELISA. [061] It has been found that the use of the glutaradeid-polymerized peptide coupled with its prior fixation with methanol to the ELISA plate provides greater differences between the absorbances detected for the different clinical groups in ELISA.

Claims

REIVINDICAÇÕES
1 . Kit imunodiagnóstico para detecção de malária caracterizado por detectar anemia em decorrência de malária vivax e compreender: a) Peptídeo SEQ ID N° 1 , na sua forma simples ou polimerizado; b) Anticorpos secundários conjugados a uma enzima ou a um marcador, específicos para reconhecerem o anticorpo primário contido nas amostras de indivíduos anêmicos com malária vivax; c) Reagente para detectar a enzima ou o marcador mencionado na etapa" b". 1 . Malaria immunodiagnostic kit for detecting malaria due to vivax malaria and comprising: (a) peptide SEQ ID NO: 1, in its simple or polymerized form; (b) an enzyme or marker-conjugated secondary antibody specific for recognizing the primary antibody contained in samples from anemic individuals with vivax malaria; c) Reagent for detecting the enzyme or label mentioned in step "b".
2. Kit imunodiagnóstico para detecção de malária, de acordo com a reivindicação 1 , etapa "a", caracterizado pelo peptídeo estar ligado a um suporte sólido ou carreador podendo opcionalmente ser fixado a este suporte sólido ou carreador com metanol. Immunodiagnostic malaria detection kit according to claim 1, step "a", characterized in that the peptide is attached to a solid support or carrier and may optionally be attached to this solid support or carrier with methanol.
3. Kit imunodiagnóstico para detecção de malária, de acordo com a reivindicação 1 , etapa "b", caracterizado pelo anticorpo secundário ser selecionado do grupo consistindo de IgG, IgM, IgA, IgE e subclasses deles. Immunodiagnostic malaria detection kit according to claim 1, step "b", characterized in that the secondary antibody is selected from the group consisting of IgG, IgM, IgA, IgE and their subclasses.
4. Kit imunodiagnóstico para detecção de malária, de acordo com a reivindicação 1 , etapa "b", caracterizado pela enzima ser selecionada do grupo compreendendo fosfatase alcalina, peroxidase, β- galactosidase, urease, xantina oxidase, glicose oxidase e penicilinase. Malaria detection immunodiagnostic kit according to claim 1, step "b", characterized in that the enzyme is selected from the group comprising alkaline phosphatase, peroxidase, β-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase.
5. Kit imunodiagnóstico para detecção de malária, de acordo com a reivindicação 1 , etapa "b", caracterizado pelo marcador ser selecionado do grupo consistindo de enzimas, radioisótopos, biotina, cromóforos, fluoróforos e quimioluminescentes. Malaria detection immunodiagnostic kit according to claim 1, step "b", characterized in that the marker is selected from the group consisting of enzymes, radioisotopes, biotin, chromophores, fluorophores and chemiluminescent.
6. Uso do kit imunodiagnóstico para detecção de malária descrito nas reivindicações 1 a 5 caracterizado por ser para imunodiagnóstico de pacientes anêmicos em decorrência de infecção por Plasmodium vivax através da utilização de testes sorologicos que podem ser do tipo ELISA, Western-Blot, dot-blot, imunodifusão ou imunocromatografia. Use of the malaria immunodiagnostic kit described in claims 1 to 5 for immunodiagnosis of anemic patients due to Plasmodium vivax infection. by the use of serological tests which may be ELISA, Western-Blot, dot-blot, immunodiffusion or immunochromatographic.
7. Método de imunodiagnóstico para detecção de malária caracterizado por detectar anemia em decorrência de malária vivax e compreender as seguintes etapas: a) expor os anticorpos primários de uma amostra ao peptídeo que apresenta sequência de aminoácidos SEQ ID NQ 1 , em sua forma simples ou polimerizado; b) colocar os anticorpos da etapa "a" em contato com um anticorpo secundário, conjugado a uma enzima ou a um marcador que se liga aos anticorpos da etapa "a"; c) detectar os anticorpos anti-malária exclusivos dos indivíduos anêmicos em decorrência de infecção por P. vivax na amostra supracitada pela detecção do anticorpo secundário especificamente ligado ao dito anticorpo anti-malária. 7. An immunodiagnostic method for detecting malaria, characterized by detecting anemia due to vivax malaria and comprising the following steps: (a) exposing the primary antibodies of a sample to the peptide having the amino acid sequence SEQ ID NQ 1 in simple form or polymerized; b) contacting the antibodies of step "a" in contact with a secondary antibody, conjugated to an enzyme or a marker that binds to the antibodies of step "a"; c) detecting anti-malaria antibodies unique to anemic individuals due to P. vivax infection in the above sample by detecting the secondary antibody specifically bound to said anti-malaria antibody.
8. Método de imunodiagnóstico para detecção de malária, de acordo com a reivindicação 7, caracterizado por ser realizado por meio de testes sorologicos que podem ser do tipo ELISA, Western-Blot, dot-blot, imunodifusão e/ou imunocromatografia. An immunodiagnostic method for detecting malaria according to claim 7, characterized in that it is performed by serological tests which may be ELISA, Western-Blot, dot-blot, immunodiffusion and / or immunochromatographic.
9. Método de imunodiagnóstico para detecção de malária, de acordo com a reivindicação 7, etapa "b", caracterizado pelo anticorpo secundário ser selecionado do grupo consistindo de IgG, IgM, IgA, IgE e subclasses deles. An immunodiagnostic method for detecting malaria according to claim 7, step "b", characterized in that the secondary antibody is selected from the group consisting of IgG, IgM, IgA, IgE and their subclasses.
10. Método de imunodiagnóstico para detecção de malária, de acordo com a reivindicação 7, etapa "b", caracterizado pela enzima ser selecionada do grupo compreendendo fosfatase alcalina, peroxidase, β- galactosidase, urease, xantina oxidase, glicose oxidase e penicilinase. An immunodiagnostic method for detecting malaria according to claim 7, step "b", characterized in that the enzyme is selected from the group comprising alkaline phosphatase, peroxidase, β-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase.
1 1 . Método de imunodiagnóstico para detecção de malária, de acordo com a reivindicação 7, etapa "b", caracterizado pelo marcador ser selecionado do grupo consistindo de enzimas, radioisótopos, biotina, cromóforos, fluoróforos e quimioluminescentes. 1 1. Immunodiagnostic method for malaria detection according to claim 7, step "b", characterized in that the marker is selected from the group consisting of enzymes, radioisotopes, biotin, chromophores, fluorophores and chemiluminescent.
12. Uso do método de imunodiagnóstico para detecção de malária descrito nas reivindicações 7 a 11 caracterizado por ser para imunodiagnóstico de pacientes anêmicos em decorrência de infecção por Plasmodium vivax. Use of the malaria detection immunodiagnostic method described in claims 7 to 11 for immunodiagnosis of anemic patients due to Plasmodium vivax infection.
13. Peptídeo sintético caracterizado por ser a SEQ ID N° 1 . Synthetic peptide characterized in that it is SEQ ID NO: 1.
14. Peptídeo sintético, de acordo com a reivindicação 13, caracterizado por ser a SEQ ID N°1 em sua forma simples ou polimerizado. Synthetic peptide according to claim 13, characterized in that it is SEQ ID NO: 1 in its simple or polymerized form.
15. Uso dos peptídeos sintéticos descritos nas reivindicações 13 e 14 caracterizado por ser no imunodiagnóstico de pacientes anêmicos em decorrência de infecção por Plasmodium vivax, através de um ensaio sorológico que pode ser do tipo ELISA, Western-Blot, dot-blot, imunodifusão ou imunocromatografia. Use of the synthetic peptides described in claims 13 and 14 for the immunodiagnosis of anemic patients due to Plasmodium vivax infection by a serological assay which may be ELISA, Western-Blot, dot-blot, immunodiffusion or immunochromatography.
PCT/IB2014/065429 2013-10-18 2014-10-17 Kit and immunodiagnostic method for detecting anaemia caused by vivax malaria, synthetic peptides and uses WO2015056244A1 (en)

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BR102014025967-8A BR102014025967B1 (en) 2014-10-17 2014-10-17 IMMUNODIAGNOSIS KIT AND METHOD FOR DETECTION OF ANEMIA RESULTING FROM VIVAX MALARIA, SYNTHETIC PEPTIDES AND USES
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CA2271451A1 (en) * 1999-04-20 2000-10-20 Medical Research Council Variant c-terminal fragment of plasmodium falciparum msp-1
WO2000063245A2 (en) * 1999-04-20 2000-10-26 Medical Research Council Variant of the plasmodium merozoite surface protein (msp-1) and vaccine comprising said variant
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