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Malaria Journal                                                                                                                                BioMed Central



Methodology                                                                                                                                  Open Access
Serological detection of Plasmodium vivax malaria using
recombinant proteins corresponding to the 19-kDa C-terminal
region of the merozoite surface protein-1
Maria Helena C Rodrigues1, Maristela G Cunha2, Ricardo LD Machado3,
Orlando C Ferreira Jr4, Mauricio M Rodrigues5 and Irene S Soares*1

Address: 1Departamento de Análises Clínicas e Toxicológicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, Av. Prof. Lineu
Prestes, 580, Cidade Universitária, São Paulo, SP, 05508-900, Brazil, 2Departamento de Patologia, Centro de Ciências Biológicas, Universidade
Federal do Pará, Av. Augusto Correa s/n, Belém, Pa, 66075-900, Brazil, 3Instituto Evandro Chagas, Secretaria de Vigilância em Saúde, Ministério
da Saúde, Av. Almirante Barroso, 492, Belém, Pa, 66.090-000, Brazil, 4Departamento de Hemoterapia, Hospital Israelita Albert Einstein, Av. Albert
Einstein, 627/701, São Paulo, SP, 05651-901, Brazil and 5Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de
São Paulo, Rua Botucatu 862, São Paulo, SP, 04023-062, Brazil
Email: Maria Helena C Rodrigues - helenacr@usp.br; Maristela G Cunha - mgcunha@ufpa.br;
Ricardo LD Machado - ricardomachado@famerp.br; Orlando C Ferreira - orlando@einstein.br;
Mauricio M Rodrigues - mrodrigues@ecb.epm.br; Irene S Soares* - isoares@usp.br
* Corresponding author




Published: 14 November 2003                                                     Received: 09 September 2003
                                                                                Accepted: 14 November 2003
Malaria Journal 2003, 2:39
This article is available from: http://www.malariajournal.com/content/2/1/39
© 2003 Rodrigues et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all
media for any purpose, provided this notice is preserved along with the article's original URL.




                 Abstract
                 Background: Serological tests to detect antibodies specific to Plasmodium vivax could be a valuable
                 tool for epidemiological studies, for screening blood donors in areas where the malaria is not
                 endemic and for diagnosis of infected individuals. Because P. vivax cannot be easily obtained in vitro,
                 ELISA assays using total or semi-purified antigens are rarely used. Based on this limitation, we
                 tested whether recombinant proteins representing the 19 kDa C-terminal region of the merozoite
                 surface protein-1 of P. vivax (MSP119) could be useful for serological detection of malaria infection.
                 Methods: Three purified recombinant proteins produced in Escherichia coli (GST-MSP119, His6-
                 MSP119 and His6-MSP119-PADRE) and one in Pichia pastoris (yMSP119-PADRE) were compared for
                 their ability to bind to IgG antibodies of individuals with patent P. vivax infection. The method was
                 tested with 200 serum samples collected from individuals living in the north of Brazil in areas
                 endemic for malaria, 53 serum samples from individuals exposed to Plasmodium falciparum infection
                 and 177 serum samples from individuals never exposed to malaria.
                 Results: Overall, the sensitivity of the ELISA assessed with sera from naturally infected individuals
                 was 95%. The proportion of serum samples that reacted with recombinant proteins GST-MSP119,
                 His6-MSP119, His6-MSP119-PADRE and yMSP119-PADRE was 90%, 93.5%, 93.5% and 93.5%,
                 respectively. The specificity values of the ELISA determined with sera from healthy individuals and
                 from individuals with other infectious diseases were 98.3% (GST-MSP119), 97.7% (His6-MSP119 and
                 His6-MSP119-PADRE) or 100% (yMSP119-PADRE).
                 Conclusions: Our study demonstrated that for the Brazilian population, an ELISA using a
                 recombinant protein of the MSP119 can be used as the basis for the development of a valuable
                 serological assay for the detection of P. vivax malaria.




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Background                                                        different regions of the world that does not restrict recog-
Plasmodium vivax accounts worldwide for an estimated              nition by human antibodies [10,11]. Together, these
70–80 million cases of malaria per year [1]. In some coun-        results suggested that it could be possible to develop an
tries such as Brazil, P. vivax was responsible for approxi-       ELISA using a single recombinant protein based on the P.
mately 79% of the 389,736 cases of the disease reported           vivax MSP119. This relatively simple and inexpensive tech-
in 2001 [2].                                                      nique could be of great applicability for epidemiological
                                                                  studies, the screening of blood donors and the serological
Laboratory methods are important tools for the control of         diagnosis of malaria caused by P. vivax.
the disease progress. They can be useful for the individual
diagnosis or for the patients' follow-up after specific anti-     In the present study three purified recombinant proteins
malaria treatment. Conventional light microscopy has              produced in E. coli (GST-MSP119, His6-MSP119 and His6-
been widely used for malaria diagnosis. However, outside          MSP119-PADRE) and one in Pichia pastoris (yMSP119-
areas where malaria is endemic, it is rarely performed.           PADRE) were compared for their ability to be recognized
Also, this method is time consuming and requires trained          by IgG antibodies of individuals with patent P. vivax infec-
personnel.                                                        tion. Serological evaluation was performed with serum
                                                                  samples collected from individuals living in areas
In attempts to overcome these problems several rapid              endemic for malaria in the north of Brazil and compared
diagnostic tests have been developed recently. These tests        to serum samples from individuals never exposed to P.
detect specific proteins such as HRP2, a histidine rich pro-      vivax malaria.
tein 2, or pLDH, lactate dehydrogenase, in unfractionated
blood of patients with malaria. However, these assay do           Materials and methods
not differentiate P. vivax from Plasmodium malariae or Plas-      Study population
modium ovale infections, and vary in their sensitivity and        Sera from 430 individuals were used in this study, of
specificity (reviewed in reference [3]).                          which 200 were from patients with patent P. vivax malaria
                                                                  and 230 from persons not exposed to P. vivax malaria
Molecular diagnosis by PCR is described as the most sen-          (negative controls). The blood samples from P. vivax
sitive and specific method for Plasmodium detection.              patients were collected in the state of Pará, in the north of
Genus- and species-specific primers have been used to             Brazil, in areas endemic for malaria: 103 from Belém, 21
amplify Plasmodium ssrRNA genes of the four human                 from Marabá, 20 from Itaituba, 20 from Tailândia, 36
malaria parasites and to detect mixed infections [4-6].           from Igarapé-Açu. Patent infection was documented by
However, this methodology is costly and requires trained          microscopic analysis of Giemsa-stained blood drops.
personnel for its implementation.                                 These samples were obtained between January 1996 and
                                                                  July 1999 with informed consent of all individuals and
Detection of antibodies by immunofluorescence or ELISA            kept at -20°C.
has been used for seroepidemiology of malaria. However,
in the case of P. vivax, the difficulty of blood stage cultiva-   The 230 individuals non-exposed to P. vivax malaria
tion has been hindering the use of these methodologies.           included: (i) 49 donors to blood banks in the city of São
Production of recombinant proteins through the tech-              Paulo, an area where malaria is not endemic (negative
niques of genetic engineering may provide sufficient P.           controls); (ii) 108 blood bank donors with unrelated
vivax blood stage antigen(s) for the establishment of spe-        infectious diseases, detected serologically; among them
cific serological assays.                                         21 with Chagas Disease, 21 with syphilis, 19 with HBV, 21
                                                                  with HCV, 14 with HTLV and 12 with HIV; (iii) 10 indi-
In the course of immuno-epidemiological studies using             viduals positive for antinuclear antibodies (ANA) and 10
recombinant proteins based on the sequence of the Mero-           for rheumatoid factors (RF); (iv) 53 individuals living in
zoite Surface protein-1 (MSP1) of P. vivax, we found that         distinct areas in West Africa where malaria caused by Plas-
a recombinant protein representing the 19 kDa region of           modium falciparum is endemic: a) 26 adults without clear
MSP1 (MSP119) was highly immunogenic during natural               symptoms of malaria or other infectious disease from Sen-
infection in humans [7,8]. This recombinant protein was           egal (n = 19) and Gambia (n = 7), b) seven children from
recognized by antibodies of a large fraction of Brazilian         Gambia during patent infection by P. falciparum, c) 20
individuals recently exposed to P. vivax [7]. This observa-       malaria immune adults from Ghana. Aliquots of these
tion was subsequently confirmed in studies performed in           samples were kindly supplied by Dr. Marcelo Urbano Fer-
Korea, where more than 90% of P. vivax-infected individ-          reira (Universidade de São Paulo, São Paulo) and Dr. Sil-
uals displayed specific antibodies to P. vivax MSP119 [9].        via Di Santi (Superitendência de Controle de Endemias,
Also important was the observation that the P. vivax              SUCEN, São Paulo). Clinical and laboratory data were
MSP119 gene displays very limited allele polymorphism in          reported elsewhere [12-14].


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Table 1: Recombinant proteins based on the MSP119 of P. vivax.

  Recombinant protein              Vector                 Microorganism          Molecular Weight (kDa)                  Ref.

       GST-MSP119                  pGEX-3X                     E. coli                      36                            15
        His6-MSP119                 pET-14b                    E. coli                      18                            15
   His6-MSP119-PADRE*               pET-14b                    E. coli                      18                            15
    yMSP119-PADRE*                  pPIC-9K                Pichia pastoris                  18                            16

 * PADRE epitope is composed of amino acids AKFVAAWTLKAAA.




Recombinant proteins                                                 sera from 49 blood donors plus five standard deviations
The recombinant proteins used in the present study repre-            (SD). The values for sensitivity and specificity were esti-
sent amino acids 1616–1704 of the MSP-1 (Belem strain)               mated as described [18] with microscopy used as the gold
of P. vivax. These proteins were expressed in E. coli (GST-          standard. The Kruskal-Wallis test was used to test the sig-
MSP119, His6-MSP119 and His6-MSP119-PADRE, ref. 15)                  nificance of differences between the group values. Differ-
or Pichia pastoris (yMSP119-PADRE, ref. 16). The genera-             ences between proportions were analyzed by a Chi-square
tion of the plasmids and expression/purification of the              test.
recombinant proteins produced in E. coli or Pichia pas-
toris were performed as described in references 15 and 16,           Results
respectively.                                                        Reactivity of recombinant proteins expressed in distinct
                                                                     vectors with serum samples from individuals with patent P.
ELISA for detection of human antibodies                              vivax
Human IgG antibodies against MSP119 were detected by                 Four recombinant proteins obtained as earlier described
ELISA as described [7]. ELISA plates were coated with 200            in references 15 and 16 (Table 1) were used. SDS-PAGE
ng/well of each recombinant protein. The amount of                   analysis of each recombinant protein revealed a single
recombinant protein gave the same OD492 when we used                 band of the expected molecular weight after Coomassie
an anti-MSP119 MAb [17]. Fifty µl of each solution were              blue [15,16] or Silver staining (data not shown).
added to each of 96 well plates (High binding, Costar).
After overnight incubation at room temperature (rt), the             An IgG survey of the serum samples of individuals with
plates were washed with PBS-Tween (0.05%, v/v) and                   patent P. vivax infection patients showed that the values of
blocked with PBS-milk (5% w/v) for two hours at 37°C.                positivity were quite similar with the four recombinant
Serum samples were diluted 1:100 in this same solution               antigens employed. Ninety to 93.5% of the 200 serum
and 50 µl of each sample was added to each well in dupli-            samples tested were positive for IgG with the recombinant
cate. After incubation for two hours at rt and washes with           proteins based on the MSP119.
PBS-Tween, 50 µl a solution containing peroxidase-conju-
gated goat anti-human IgG (Fc specific) diluted 1:10,000             When we compared the OD492 values from serum samples
(Sigma) were added to each well. The enzymatic reaction              of P. vivax infected individuals, we observed that values
was developed by the addition of 1 mg/ml of o-phenylen-              obtained for the recombinant proteins His6-MSP119 and
ediamine (Sigma) diluted in phosphate-citrate buffer, pH             His6-MSP119-PADRE were significantly higher than those
5.0, containing 0.03% (v/v) hydrogen peroxide, and was               obtained for GST-MSP119 (Fig. 1, P < 0.01, Kruskal-Wallis
stopped by the addition of 50 µl of 4 N H2SO4. Plates were           test). On the other hand, no statistically significant differ-
read at 492 nm (OD492) with an ELISA reader (SLT SPEC-               ence was observed among values obtained by the compar-
TRA, SLT Labinstruments, Austria). The individual values             ison of the recombinant proteins His6-MSP119, His6-
of OD492 obtained for the recombinant proteins of                    MSP119-PADRE and yMSP119-PADRE, or GST-MSP119
MSP119 were corrected by subtraction of the individual               and yMSP119-PADRE (P > 0.05, Kruskal-Wallis test).
values of OD492 obtained against glutathione S-transferase
(GST).                                                               Sera from the few individuals who tested negative towards
                                                                     all four recombinant proteins were also tested for the rec-
Statistical analysis                                                 ognition of recombinant proteins representing the N-ter-
OD492 from different samples were plotted using compu-               minal region of P. vivax MSP1, MSP3α (C-terminal) or
ter graphics software (GraphPad Prism, Version 3.0, San              MSP3β(N and C-terminal and entire protein, references
Diego, California). Cutoff values of each recombinant                19 and 20). All of them failed to recognize any of the
protein in ELISA were calculated as the mean OD492 of                recombinant protein tested (data not shown). However,


                                                                                                                           Page 3 of 7
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                                                                                           GST-MSP119

                          3.5
                                                                                           His6 -MSP119

                          3.0
                                                                                           His6-MSP119-PADRE

                          2.5
                                                                                           yMSP119-PADRE
                  OD492




                          2.0

                          1.5

                          1.0

                          0.5

                          0.0


                                            Recombinant protein




Distribution of OD492 data for 200 sera from individuals with patent malaria infection caused by P.vivax
Figure 1
Distribution of OD492 data for 200 sera from individuals with patent malaria infection caused by P. vivax. The symbols represent
the reactivity of each serum sample tested in duplicate at 1:100 dilution against the indicated recombinant proteins. The hori-
zontal line inside the drops for each recombinant protein represents the cut-off values (0.127, 0.170, 0.198 and 0.500 for GST-
MSP119, His6-MSP119, His6-MSP119-PADRE and yMSP119-PADRE, respectively).




we detected antibodies IgM anti-MSP119 in 90% (9/10) of           individuals with unrelated diseases or healthy individu-
these IgG negative individuals. If we consider the results        als. Due to limitations on the volume of each sample
of detection of antibodies IgG and IgM for MSP119, the            available, sera from African individuals exposed to P. fal-
sensitivity of the assay was of 99.5%. These 10 individuals       ciparum were tested only against recombinant protein
were primo-infected and therefore the presence of IgM but         His6-MSP119. For the calculation of the specificity of the
not IgG may reflect a delay in the immunoglobulin class           assay using the recombinant protein His6-MSP119 the
switch.                                                           results that was obtained did not include sera from Afri-
                                                                  can individuals. The specificity values determined with
From the 200 individuals that we evaluated, 111 were              sera from healthy individuals and sera from individuals
primo-infected. One hundred and one of them had spe-              with other infectious diseases, were 98.3% (GST-MSP119),
cific IgG for the recombinant proteins that we tested. Only       97.7% (His6-MSP119and His6-MSP119-PADRE) and 100%
10 were negative for IgG and, as mentioned above, 9 of            (yMSP119-PADRE). In figure 2, we compared the OD492
them had specific IgM.                                            values from serum samples of P. vivax infected individu-
                                                                  als, individuals exposed to P. falciparum and individuals
Evaluation of the specificity of the recombinant proteins         with unrelated diseases.
tested with serum samples from individuals exposed to P.
falciparum, from unrelated diseases, or healthy donors            Discussion
The specificity of the assay using recombinant P. vivax           Due to the difficulties in cultivating blood stages of P.
antigens was examined with 230 serum samples from                 vivax, serological diagnosis of patent P. vivax malaria can
individuals without previous history of P. vivax malaria,         best be accomplished with the use of recombinant pro-
including individuals exposed to P. falciparum malaria,           teins. In the present study, we compared for purified



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Malaria Journal 2003, 2                                                                              http://www.malariajournal.com/content/2/1/39




                                                    GST-MSP1 19                                    His 6-MSP1 19
                                     3.0                                        3.5

                                     2.5                                        3.0

                                                                                2.5
                                     2.0




                                                                        OD492
                             OD492
                                                                                2.0
                                     1.5
                                                                                1.5
                                     1.0
                                                                                1.0
                                     0.5                                        0.5

                                     0.0                                        0.0




                                                                                      Pv




                                                                                           H RF

                                                                                                   y
                                                                                                ha

                                                                                                  p
                                                                                               BV

                                                                                                  V
                                                                                                  V
                                                                                                 IV

                                                                                                  A
                                                                                                 Pf
                                                      ha


                                                     BV

                                                       V
                                                       V
                                                      IV

                                                       A
                                           Pv

                                                      Pf




                                                H RF

                                                        y
                                                       p




                                                                                              lt h
                                                                                              Sy



                                                                                               C




                                                                                             AN
                                                                                             TL
                                                    lth
                                                   Sy




                                                   TL
                                                     C




                                                  AN




                                                                                              H
                                                                                             C
                                                   H
                                                   C




                                                                                             H
                                                                                             H
                                                  H




                                                                                            ea
                                                  H




                                                 ea




                                                                                            H
                                                 H  Study groups                                   Study groups


                                                 His 6-MSP1 19 -PADRE                             yMSP1 19 -PADRE
                                     3.0                                        3.0

                                     2.5                                        2.5

                                     2.0                                        2.0
                             OD492




                                                                        OD492
                                     1.5                                        1.5

                                     1.0                                        1.0

                                     0.5                                        0.5

                                     0.0                                        0.0
                                           Pv




                                                H RF




                                                                                      Pv




                                                                                                  H RF
                                                     ha

                                                       p
                                                    BV

                                                       V
                                                       V
                                                      IV

                                                       A




                                                                                            ha

                                                                                                         p
                                                                                                      BV

                                                                                                         V
                                                                                                         V
                                                                                                        IV

                                                                                                         A
                                                      Pf




                                                       y




                                                                                             Pf




                                                                                                         y
                                                   lt h




                                                                                                     lt h
                                                   Sy




                                                                                                     Sy
                                                    C




                                                  AN




                                                                                                      C




                                                                                                    AN
                                                  TL




                                                                                                    TL
                                                   H




                                                                                                     H
                                                  C




                                                                                           C
                                                  H




                                                                                                    H
                                                  H




                                                                                                    H
                                                 ea




                                                                                                   ea
                                                 H




                                                                                                   H
                                                    Study groups                                   Study groups




Figure 2 of the OD492 data for sera from individuals with patent P. vivax malaria, from individuals exposed to P. falciparum,
from individuals with unrelated diseases or healthy controls
Distribution
Distribution of the OD492 data for sera from individuals with patent P. vivax malaria, from individuals exposed to P. falciparum,
from individuals with unrelated diseases or healthy controls. The symbols represent the reactivity of each serum sample tested
in duplicate at 1:100 dilution against the indicated recombinant proteins. The abbreviations are as follow: A) Pv= individuals
with P. vivax malaria (n = 200), B) Pf= individuals from areas where P. falciparum malaria is endemic (n = 53), C) Cha = individu-
als with Chagas Disease (n = 21), D) Syp = individuals with syphilis (n = 21), E) HBV = individuals with hepatitis B (n = 19), F)
HCV= individuals with hepatitis C (n = 21), G) HTLV = individuals with HTLV (n = 14), H) HIV= individuals with HIV (n = 12),
I) ANA = individuals positive for antinuclear antibodies (n = 10), J) RF = individuals positive for rheumatoid factors (n = 10), L)
Healthy = Healthy individuals (n = 49). Sera from African individuals exposed to P. falciparum were tested only against recom-
binant protein His6-MSP119. The horizontal line inside the drops for each recombinant protein represents the cut-off values
(0.127, 0.170, 0.198 and 0.500 for GST-MSP119, His6-MSP119, His6-MSP119-PADRE and yMSP119-PADRE, respectively).




recombinant proteins produced in E. coli or in Pichia pas-              during experimental infection in primates. Nevertheless,
toris in their ability to be recognized by IgG antibodies of            we were able to determine that from the 111 primo-
Brazilian individuals with patent P. vivax infection. Our               infected individuals that we evaluated, 101 (90.9%) had
study demonstrated that, for the Brazilian population, an               specific IgG to MSP119. This information is important and
ELISA using a single recombinant protein based on the P.                suggests that IgG antibodies specific for MSP119 are suita-
vivax MSP119 kDa can serve as the basis for the develop-                ble for testing individuals who are traveling or have
ment of a sensitive serological test that can be used for epi-          traveled for the first time through malaria endemic areas.
demiological studies, screening blood donors and                        On the other hand, the persistence of the IgG antibodies
diagnosis of P. vivax malaria.                                          specific for MSP119 is still a matter that has to be further
                                                                        evaluated. In previous studies we determined that the
So far, we do not have an estimate of the timing required               antibody titers decreased relatively rapidly after treatment
after mosquito bite for the appearance of specific antibod-             [8]. However, studies are underway using these recently
ies. To be accurate, this information should be obtained                generated recombinant proteins.


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An important observation from our study was the fact that         Authors' contributions
sera from African individuals naturally exposed to P. falci-      MHCR carried out all the serological assays. MGC gener-
parum failed to cross-react with the recombinant protein          ated and purified the recombinant proteins His6-MSP119
His6-MSP119 (Figure 2). As a control, 39 of these sera were       and His6-MSP119-PADRE. RLDM collected part of the
also tested against a recombinant protein derived of the          serum samples from P. vivax malaria individuals. OCF
MSP-2 of P. falciparum. The percentage of responders was          participated in the design of the study and the supply of
69.2% (data not shown). The lack of cross-reactivity may          the serum samples from blood bank donors and drafted
have several implications and should be further evaluated         the manuscript. MMR participated in the design of the
in the light of the current knowledge that both MSP119            study and drafted the manuscript. ISS conceived of the
share similarities in their predicted tertiary structures [21].   study and participated in all aspects of its design, execu-
For epidemiological studies, for the screening of blood           tion, coordination and manuscript preparation. All
donors and the serological diagnosis of P. vivax malaria,         authors read and approved the final manuscript.
the lack of cross reactivity can be a major advantage. Nev-
ertheless, the absence of cross-recognition of P. vivax           Acknowledgments
MSP119 by antibodies from P. falciparum-exposed individ-          This work was supported by a grant from the Fundação de Amparo a
uals has may also have immunological consequences at              Pesquisa do Estado de São Paulo (FAPESP). MHCR, MMR and ISS are sup-
the level of acquired immunity and vaccine development            ported by fellowships from Conselho Nacional de Desenvolvimento Cientí-
                                                                  fico e Tecnológico (CNPq). Also, the Ethics Committee of the University
in areas where both malarias are prevalent. Detailed stud-
                                                                  of São Paulo approved it. The authors would like to thank Dr. Marcelo U.
ies will be required to determine whether sera from P.            Ferreira and Dr. Silvia di Santi for kindly providing the sera from individuals
vivax infected individuals also fail to recognize recom-          from West Africa, Dr. MUF for providing recombinant protein MSP-2 of P.
binant proteins representing the P. falciparum MSP119.            falciparum, and Drs. Michel Rabinovitch and MUF for critical reading of the
                                                                  manuscript.
Recently, a direct sandwich ELISA to detect antibodies
against the C-terminal region of MSP-1 was proposed as a          References
potential diagnostic method for Plasmodium vivax exposed          1.    Mendis K, Sina BJ, Marchesini P, Carter R: The neglected burden
                                                                        of Plasmodium vivax malaria. Am J Trop Med Hyg 2001, 64:97-106.
individuals from Korea [22]. This assay showed a high             2.    Fundação Nacional de Saúde-FUNASA, Ministério da Saúde.
sensitivity (99.5%) indicating that recombinant proteins                Guia de Doenças: Malária Dados da doença – relatórios gerenciais
containing the C-terminal region of P. vivax MSP-1 may be               2003 [http://www.funasa.gov.br].
                                                                  3.    Moody A: Rapid diagnostic tests for malaria parasites. Clin
used in individuals from different parts of the world.                  Microbiol Rev 2002, 15:66-78.
                                                                  4.    Snounou G, Viriyakosol S, Zhu XP, Jarra W, Pinheiro L, do Rosário
                                                                        VE, Thaithong S, Brown KN: High sensitivity of detection of
It is also important to mention that it is very likely that             human malaria parasites by the use of nested polymerase
serological detection of P. vivax malaria can be further                chain reaction. Mol Biochem Parasitol 1993, 61:315-320.
improved by several distinct strategies. We are currently         5.    Kimura M, Kaneko O, Qing L, Mian Z, Kawamoto F, Wataya Y, Otani
                                                                        S, Yamaguchi Y, Tanabe K: Identification of the four species of
trying to improve the detection level by the combined use               human malaria parasites by nested PCR that targets variant
of other recombinant proteins based on the sequence of                  sequences in the small subunit rRNA gene. Parasitol Int 1997,
                                                                        46:91-95.
other blood stage antigens of P. vivax such as the Plasmo-        6.    Rubio JM, Benito A, Roche J, Berzosa PJ, Garcia ML, Mico M, Edu M,
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                                                                        modium vivax infection in Equatorial Guinea. Am J Trop Med Hyg
[19,20,23,24]. Also, we are developing a chemilumines-                  1999, 60:183-187.
cent enzyme-linked immunosorbent assay that may                   7.    Soares IS, Levitus G, Souza JM, del Portillo HA, Rodrigues MM:
greatly improve the sensitivity, eliminating the few false              Acquired immune responses to the N- and C-terminal
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negative and false positive samples we had [25,26].                     individuals exposed to malaria. Infect Immun 1997, 65:1606-1614.
                                                                  8.    Soares IS, Cunha MG, Silva MN, Souza JM, del Portillo HA, Rodrigues
                                                                        MM: Longevity of the naturally acquired antibody responses
Finally, our results support the notion that recombinant                to the N- and C-terminal regions of Plasmodium vivax MSP1.
proteins based on the P. vivax MSP119 kDa can be useful                 Am J Trop Med Hyg 1999, 60:357-363.
for the development of a rapid immunochromatographic              9.    Park JW, Moon SH, Yeom JS, Lim KJ, Sohn MJ, Jung WC, Cho YJ, Jeon
                                                                        KW, Ju W, Ki CS, Oh MD, Choe K: Naturally acquired antibody
assay for field studies, small laboratories and blood banks             responses to the C-terminal region of merozoite surface
(reviewed in references 3 and 27).                                      protein 1 of Plasmodium vivax in Korea. Clin Diagn Lab Immunol
                                                                        2001, 8:14-20.
                                                                  10.   Pasay MC, Cheng Q, Rzepczyk C, Saul A: Dimorphism of the C
Conclusions                                                             terminus of the Plasmodium vivax merozoite surface protein
Our study demonstrated that for the Brazilian population,               1. Mol Biochem Parasitol 1995, 70:217-219.
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P. vivax MSP119 can serve as the basis for the development              plays limited allele polymorphism, which does not restrict
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                                                                  12.   Ferreira MU, Kimura EAS, Souza JM, Katzin AM: The isotype com-
malaria.                                                                position and avidity of naturally acquired anti-Plasmodium



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Malaria Journal 2003, 2                                                                         http://www.malariajournal.com/content/2/1/39



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      ides brasiliensis. J Clin Lab Anal 1994, 8:424-431.                           Publish with Bio Med Central and every
26.   Almeida IC, Covas DT, Soussumi LM, Travassos LR: A highly sensi-
      tive and specific chemiluminescent enzyme-linked immuno-                     scientist can read your work free of charge
      sorbent assay for diagnosis of active Trypanosoma cruzi                  "BioMed Central will be the most significant development for
      infection. Transfusion 1997, 37:850-857.                                 disseminating the results of biomedical researc h in our lifetime."
27.   Hanscheid T: Current strategies to avoid misdiagnosis of
      malaria. Clin Microbiol Infect 2003, 9:497-504.                               Sir Paul Nurse, Cancer Research UK

                                                                                  Your research papers will be:
                                                                                    available free of charge to the entire biomedical community
                                                                                    peer reviewed and published immediately upon acceptance
                                                                                    cited in PubMed and archived on PubMed Central
                                                                                    yours — you keep the copyright

                                                                           Submit your manuscript here:                                BioMedcentral
                                                                           http://www.biomedcentral.com/info/publishing_adv.asp




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2003 malária e diagnóstico sorológico msp119

  • 1. Malaria Journal BioMed Central Methodology Open Access Serological detection of Plasmodium vivax malaria using recombinant proteins corresponding to the 19-kDa C-terminal region of the merozoite surface protein-1 Maria Helena C Rodrigues1, Maristela G Cunha2, Ricardo LD Machado3, Orlando C Ferreira Jr4, Mauricio M Rodrigues5 and Irene S Soares*1 Address: 1Departamento de Análises Clínicas e Toxicológicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, Av. Prof. Lineu Prestes, 580, Cidade Universitária, São Paulo, SP, 05508-900, Brazil, 2Departamento de Patologia, Centro de Ciências Biológicas, Universidade Federal do Pará, Av. Augusto Correa s/n, Belém, Pa, 66075-900, Brazil, 3Instituto Evandro Chagas, Secretaria de Vigilância em Saúde, Ministério da Saúde, Av. Almirante Barroso, 492, Belém, Pa, 66.090-000, Brazil, 4Departamento de Hemoterapia, Hospital Israelita Albert Einstein, Av. Albert Einstein, 627/701, São Paulo, SP, 05651-901, Brazil and 5Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, Rua Botucatu 862, São Paulo, SP, 04023-062, Brazil Email: Maria Helena C Rodrigues - helenacr@usp.br; Maristela G Cunha - mgcunha@ufpa.br; Ricardo LD Machado - ricardomachado@famerp.br; Orlando C Ferreira - orlando@einstein.br; Mauricio M Rodrigues - mrodrigues@ecb.epm.br; Irene S Soares* - isoares@usp.br * Corresponding author Published: 14 November 2003 Received: 09 September 2003 Accepted: 14 November 2003 Malaria Journal 2003, 2:39 This article is available from: http://www.malariajournal.com/content/2/1/39 © 2003 Rodrigues et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL. Abstract Background: Serological tests to detect antibodies specific to Plasmodium vivax could be a valuable tool for epidemiological studies, for screening blood donors in areas where the malaria is not endemic and for diagnosis of infected individuals. Because P. vivax cannot be easily obtained in vitro, ELISA assays using total or semi-purified antigens are rarely used. Based on this limitation, we tested whether recombinant proteins representing the 19 kDa C-terminal region of the merozoite surface protein-1 of P. vivax (MSP119) could be useful for serological detection of malaria infection. Methods: Three purified recombinant proteins produced in Escherichia coli (GST-MSP119, His6- MSP119 and His6-MSP119-PADRE) and one in Pichia pastoris (yMSP119-PADRE) were compared for their ability to bind to IgG antibodies of individuals with patent P. vivax infection. The method was tested with 200 serum samples collected from individuals living in the north of Brazil in areas endemic for malaria, 53 serum samples from individuals exposed to Plasmodium falciparum infection and 177 serum samples from individuals never exposed to malaria. Results: Overall, the sensitivity of the ELISA assessed with sera from naturally infected individuals was 95%. The proportion of serum samples that reacted with recombinant proteins GST-MSP119, His6-MSP119, His6-MSP119-PADRE and yMSP119-PADRE was 90%, 93.5%, 93.5% and 93.5%, respectively. The specificity values of the ELISA determined with sera from healthy individuals and from individuals with other infectious diseases were 98.3% (GST-MSP119), 97.7% (His6-MSP119 and His6-MSP119-PADRE) or 100% (yMSP119-PADRE). Conclusions: Our study demonstrated that for the Brazilian population, an ELISA using a recombinant protein of the MSP119 can be used as the basis for the development of a valuable serological assay for the detection of P. vivax malaria. Page 1 of 7 (page number not for citation purposes)
  • 2. Malaria Journal 2003, 2 http://www.malariajournal.com/content/2/1/39 Background different regions of the world that does not restrict recog- Plasmodium vivax accounts worldwide for an estimated nition by human antibodies [10,11]. Together, these 70–80 million cases of malaria per year [1]. In some coun- results suggested that it could be possible to develop an tries such as Brazil, P. vivax was responsible for approxi- ELISA using a single recombinant protein based on the P. mately 79% of the 389,736 cases of the disease reported vivax MSP119. This relatively simple and inexpensive tech- in 2001 [2]. nique could be of great applicability for epidemiological studies, the screening of blood donors and the serological Laboratory methods are important tools for the control of diagnosis of malaria caused by P. vivax. the disease progress. They can be useful for the individual diagnosis or for the patients' follow-up after specific anti- In the present study three purified recombinant proteins malaria treatment. Conventional light microscopy has produced in E. coli (GST-MSP119, His6-MSP119 and His6- been widely used for malaria diagnosis. However, outside MSP119-PADRE) and one in Pichia pastoris (yMSP119- areas where malaria is endemic, it is rarely performed. PADRE) were compared for their ability to be recognized Also, this method is time consuming and requires trained by IgG antibodies of individuals with patent P. vivax infec- personnel. tion. Serological evaluation was performed with serum samples collected from individuals living in areas In attempts to overcome these problems several rapid endemic for malaria in the north of Brazil and compared diagnostic tests have been developed recently. These tests to serum samples from individuals never exposed to P. detect specific proteins such as HRP2, a histidine rich pro- vivax malaria. tein 2, or pLDH, lactate dehydrogenase, in unfractionated blood of patients with malaria. However, these assay do Materials and methods not differentiate P. vivax from Plasmodium malariae or Plas- Study population modium ovale infections, and vary in their sensitivity and Sera from 430 individuals were used in this study, of specificity (reviewed in reference [3]). which 200 were from patients with patent P. vivax malaria and 230 from persons not exposed to P. vivax malaria Molecular diagnosis by PCR is described as the most sen- (negative controls). The blood samples from P. vivax sitive and specific method for Plasmodium detection. patients were collected in the state of Pará, in the north of Genus- and species-specific primers have been used to Brazil, in areas endemic for malaria: 103 from Belém, 21 amplify Plasmodium ssrRNA genes of the four human from Marabá, 20 from Itaituba, 20 from Tailândia, 36 malaria parasites and to detect mixed infections [4-6]. from Igarapé-Açu. Patent infection was documented by However, this methodology is costly and requires trained microscopic analysis of Giemsa-stained blood drops. personnel for its implementation. These samples were obtained between January 1996 and July 1999 with informed consent of all individuals and Detection of antibodies by immunofluorescence or ELISA kept at -20°C. has been used for seroepidemiology of malaria. However, in the case of P. vivax, the difficulty of blood stage cultiva- The 230 individuals non-exposed to P. vivax malaria tion has been hindering the use of these methodologies. included: (i) 49 donors to blood banks in the city of São Production of recombinant proteins through the tech- Paulo, an area where malaria is not endemic (negative niques of genetic engineering may provide sufficient P. controls); (ii) 108 blood bank donors with unrelated vivax blood stage antigen(s) for the establishment of spe- infectious diseases, detected serologically; among them cific serological assays. 21 with Chagas Disease, 21 with syphilis, 19 with HBV, 21 with HCV, 14 with HTLV and 12 with HIV; (iii) 10 indi- In the course of immuno-epidemiological studies using viduals positive for antinuclear antibodies (ANA) and 10 recombinant proteins based on the sequence of the Mero- for rheumatoid factors (RF); (iv) 53 individuals living in zoite Surface protein-1 (MSP1) of P. vivax, we found that distinct areas in West Africa where malaria caused by Plas- a recombinant protein representing the 19 kDa region of modium falciparum is endemic: a) 26 adults without clear MSP1 (MSP119) was highly immunogenic during natural symptoms of malaria or other infectious disease from Sen- infection in humans [7,8]. This recombinant protein was egal (n = 19) and Gambia (n = 7), b) seven children from recognized by antibodies of a large fraction of Brazilian Gambia during patent infection by P. falciparum, c) 20 individuals recently exposed to P. vivax [7]. This observa- malaria immune adults from Ghana. Aliquots of these tion was subsequently confirmed in studies performed in samples were kindly supplied by Dr. Marcelo Urbano Fer- Korea, where more than 90% of P. vivax-infected individ- reira (Universidade de São Paulo, São Paulo) and Dr. Sil- uals displayed specific antibodies to P. vivax MSP119 [9]. via Di Santi (Superitendência de Controle de Endemias, Also important was the observation that the P. vivax SUCEN, São Paulo). Clinical and laboratory data were MSP119 gene displays very limited allele polymorphism in reported elsewhere [12-14]. Page 2 of 7 (page number not for citation purposes)
  • 3. Malaria Journal 2003, 2 http://www.malariajournal.com/content/2/1/39 Table 1: Recombinant proteins based on the MSP119 of P. vivax. Recombinant protein Vector Microorganism Molecular Weight (kDa) Ref. GST-MSP119 pGEX-3X E. coli 36 15 His6-MSP119 pET-14b E. coli 18 15 His6-MSP119-PADRE* pET-14b E. coli 18 15 yMSP119-PADRE* pPIC-9K Pichia pastoris 18 16 * PADRE epitope is composed of amino acids AKFVAAWTLKAAA. Recombinant proteins sera from 49 blood donors plus five standard deviations The recombinant proteins used in the present study repre- (SD). The values for sensitivity and specificity were esti- sent amino acids 1616–1704 of the MSP-1 (Belem strain) mated as described [18] with microscopy used as the gold of P. vivax. These proteins were expressed in E. coli (GST- standard. The Kruskal-Wallis test was used to test the sig- MSP119, His6-MSP119 and His6-MSP119-PADRE, ref. 15) nificance of differences between the group values. Differ- or Pichia pastoris (yMSP119-PADRE, ref. 16). The genera- ences between proportions were analyzed by a Chi-square tion of the plasmids and expression/purification of the test. recombinant proteins produced in E. coli or Pichia pas- toris were performed as described in references 15 and 16, Results respectively. Reactivity of recombinant proteins expressed in distinct vectors with serum samples from individuals with patent P. ELISA for detection of human antibodies vivax Human IgG antibodies against MSP119 were detected by Four recombinant proteins obtained as earlier described ELISA as described [7]. ELISA plates were coated with 200 in references 15 and 16 (Table 1) were used. SDS-PAGE ng/well of each recombinant protein. The amount of analysis of each recombinant protein revealed a single recombinant protein gave the same OD492 when we used band of the expected molecular weight after Coomassie an anti-MSP119 MAb [17]. Fifty µl of each solution were blue [15,16] or Silver staining (data not shown). added to each of 96 well plates (High binding, Costar). After overnight incubation at room temperature (rt), the An IgG survey of the serum samples of individuals with plates were washed with PBS-Tween (0.05%, v/v) and patent P. vivax infection patients showed that the values of blocked with PBS-milk (5% w/v) for two hours at 37°C. positivity were quite similar with the four recombinant Serum samples were diluted 1:100 in this same solution antigens employed. Ninety to 93.5% of the 200 serum and 50 µl of each sample was added to each well in dupli- samples tested were positive for IgG with the recombinant cate. After incubation for two hours at rt and washes with proteins based on the MSP119. PBS-Tween, 50 µl a solution containing peroxidase-conju- gated goat anti-human IgG (Fc specific) diluted 1:10,000 When we compared the OD492 values from serum samples (Sigma) were added to each well. The enzymatic reaction of P. vivax infected individuals, we observed that values was developed by the addition of 1 mg/ml of o-phenylen- obtained for the recombinant proteins His6-MSP119 and ediamine (Sigma) diluted in phosphate-citrate buffer, pH His6-MSP119-PADRE were significantly higher than those 5.0, containing 0.03% (v/v) hydrogen peroxide, and was obtained for GST-MSP119 (Fig. 1, P < 0.01, Kruskal-Wallis stopped by the addition of 50 µl of 4 N H2SO4. Plates were test). On the other hand, no statistically significant differ- read at 492 nm (OD492) with an ELISA reader (SLT SPEC- ence was observed among values obtained by the compar- TRA, SLT Labinstruments, Austria). The individual values ison of the recombinant proteins His6-MSP119, His6- of OD492 obtained for the recombinant proteins of MSP119-PADRE and yMSP119-PADRE, or GST-MSP119 MSP119 were corrected by subtraction of the individual and yMSP119-PADRE (P > 0.05, Kruskal-Wallis test). values of OD492 obtained against glutathione S-transferase (GST). Sera from the few individuals who tested negative towards all four recombinant proteins were also tested for the rec- Statistical analysis ognition of recombinant proteins representing the N-ter- OD492 from different samples were plotted using compu- minal region of P. vivax MSP1, MSP3α (C-terminal) or ter graphics software (GraphPad Prism, Version 3.0, San MSP3β(N and C-terminal and entire protein, references Diego, California). Cutoff values of each recombinant 19 and 20). All of them failed to recognize any of the protein in ELISA were calculated as the mean OD492 of recombinant protein tested (data not shown). However, Page 3 of 7 (page number not for citation purposes)
  • 4. Malaria Journal 2003, 2 http://www.malariajournal.com/content/2/1/39 GST-MSP119 3.5 His6 -MSP119 3.0 His6-MSP119-PADRE 2.5 yMSP119-PADRE OD492 2.0 1.5 1.0 0.5 0.0 Recombinant protein Distribution of OD492 data for 200 sera from individuals with patent malaria infection caused by P.vivax Figure 1 Distribution of OD492 data for 200 sera from individuals with patent malaria infection caused by P. vivax. The symbols represent the reactivity of each serum sample tested in duplicate at 1:100 dilution against the indicated recombinant proteins. The hori- zontal line inside the drops for each recombinant protein represents the cut-off values (0.127, 0.170, 0.198 and 0.500 for GST- MSP119, His6-MSP119, His6-MSP119-PADRE and yMSP119-PADRE, respectively). we detected antibodies IgM anti-MSP119 in 90% (9/10) of individuals with unrelated diseases or healthy individu- these IgG negative individuals. If we consider the results als. Due to limitations on the volume of each sample of detection of antibodies IgG and IgM for MSP119, the available, sera from African individuals exposed to P. fal- sensitivity of the assay was of 99.5%. These 10 individuals ciparum were tested only against recombinant protein were primo-infected and therefore the presence of IgM but His6-MSP119. For the calculation of the specificity of the not IgG may reflect a delay in the immunoglobulin class assay using the recombinant protein His6-MSP119 the switch. results that was obtained did not include sera from Afri- can individuals. The specificity values determined with From the 200 individuals that we evaluated, 111 were sera from healthy individuals and sera from individuals primo-infected. One hundred and one of them had spe- with other infectious diseases, were 98.3% (GST-MSP119), cific IgG for the recombinant proteins that we tested. Only 97.7% (His6-MSP119and His6-MSP119-PADRE) and 100% 10 were negative for IgG and, as mentioned above, 9 of (yMSP119-PADRE). In figure 2, we compared the OD492 them had specific IgM. values from serum samples of P. vivax infected individu- als, individuals exposed to P. falciparum and individuals Evaluation of the specificity of the recombinant proteins with unrelated diseases. tested with serum samples from individuals exposed to P. falciparum, from unrelated diseases, or healthy donors Discussion The specificity of the assay using recombinant P. vivax Due to the difficulties in cultivating blood stages of P. antigens was examined with 230 serum samples from vivax, serological diagnosis of patent P. vivax malaria can individuals without previous history of P. vivax malaria, best be accomplished with the use of recombinant pro- including individuals exposed to P. falciparum malaria, teins. In the present study, we compared for purified Page 4 of 7 (page number not for citation purposes)
  • 5. Malaria Journal 2003, 2 http://www.malariajournal.com/content/2/1/39 GST-MSP1 19 His 6-MSP1 19 3.0 3.5 2.5 3.0 2.5 2.0 OD492 OD492 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 Pv H RF y ha p BV V V IV A Pf ha BV V V IV A Pv Pf H RF y p lt h Sy C AN TL lth Sy TL C AN H C H C H H H ea H ea H H Study groups Study groups His 6-MSP1 19 -PADRE yMSP1 19 -PADRE 3.0 3.0 2.5 2.5 2.0 2.0 OD492 OD492 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 Pv H RF Pv H RF ha p BV V V IV A ha p BV V V IV A Pf y Pf y lt h lt h Sy Sy C AN C AN TL TL H H C C H H H H ea ea H H Study groups Study groups Figure 2 of the OD492 data for sera from individuals with patent P. vivax malaria, from individuals exposed to P. falciparum, from individuals with unrelated diseases or healthy controls Distribution Distribution of the OD492 data for sera from individuals with patent P. vivax malaria, from individuals exposed to P. falciparum, from individuals with unrelated diseases or healthy controls. The symbols represent the reactivity of each serum sample tested in duplicate at 1:100 dilution against the indicated recombinant proteins. The abbreviations are as follow: A) Pv= individuals with P. vivax malaria (n = 200), B) Pf= individuals from areas where P. falciparum malaria is endemic (n = 53), C) Cha = individu- als with Chagas Disease (n = 21), D) Syp = individuals with syphilis (n = 21), E) HBV = individuals with hepatitis B (n = 19), F) HCV= individuals with hepatitis C (n = 21), G) HTLV = individuals with HTLV (n = 14), H) HIV= individuals with HIV (n = 12), I) ANA = individuals positive for antinuclear antibodies (n = 10), J) RF = individuals positive for rheumatoid factors (n = 10), L) Healthy = Healthy individuals (n = 49). Sera from African individuals exposed to P. falciparum were tested only against recom- binant protein His6-MSP119. The horizontal line inside the drops for each recombinant protein represents the cut-off values (0.127, 0.170, 0.198 and 0.500 for GST-MSP119, His6-MSP119, His6-MSP119-PADRE and yMSP119-PADRE, respectively). recombinant proteins produced in E. coli or in Pichia pas- during experimental infection in primates. Nevertheless, toris in their ability to be recognized by IgG antibodies of we were able to determine that from the 111 primo- Brazilian individuals with patent P. vivax infection. Our infected individuals that we evaluated, 101 (90.9%) had study demonstrated that, for the Brazilian population, an specific IgG to MSP119. This information is important and ELISA using a single recombinant protein based on the P. suggests that IgG antibodies specific for MSP119 are suita- vivax MSP119 kDa can serve as the basis for the develop- ble for testing individuals who are traveling or have ment of a sensitive serological test that can be used for epi- traveled for the first time through malaria endemic areas. demiological studies, screening blood donors and On the other hand, the persistence of the IgG antibodies diagnosis of P. vivax malaria. specific for MSP119 is still a matter that has to be further evaluated. In previous studies we determined that the So far, we do not have an estimate of the timing required antibody titers decreased relatively rapidly after treatment after mosquito bite for the appearance of specific antibod- [8]. However, studies are underway using these recently ies. To be accurate, this information should be obtained generated recombinant proteins. Page 5 of 7 (page number not for citation purposes)
  • 6. Malaria Journal 2003, 2 http://www.malariajournal.com/content/2/1/39 An important observation from our study was the fact that Authors' contributions sera from African individuals naturally exposed to P. falci- MHCR carried out all the serological assays. MGC gener- parum failed to cross-react with the recombinant protein ated and purified the recombinant proteins His6-MSP119 His6-MSP119 (Figure 2). As a control, 39 of these sera were and His6-MSP119-PADRE. RLDM collected part of the also tested against a recombinant protein derived of the serum samples from P. vivax malaria individuals. OCF MSP-2 of P. falciparum. The percentage of responders was participated in the design of the study and the supply of 69.2% (data not shown). The lack of cross-reactivity may the serum samples from blood bank donors and drafted have several implications and should be further evaluated the manuscript. MMR participated in the design of the in the light of the current knowledge that both MSP119 study and drafted the manuscript. ISS conceived of the share similarities in their predicted tertiary structures [21]. study and participated in all aspects of its design, execu- For epidemiological studies, for the screening of blood tion, coordination and manuscript preparation. All donors and the serological diagnosis of P. vivax malaria, authors read and approved the final manuscript. the lack of cross reactivity can be a major advantage. Nev- ertheless, the absence of cross-recognition of P. vivax Acknowledgments MSP119 by antibodies from P. falciparum-exposed individ- This work was supported by a grant from the Fundação de Amparo a uals has may also have immunological consequences at Pesquisa do Estado de São Paulo (FAPESP). MHCR, MMR and ISS are sup- the level of acquired immunity and vaccine development ported by fellowships from Conselho Nacional de Desenvolvimento Cientí- fico e Tecnológico (CNPq). Also, the Ethics Committee of the University in areas where both malarias are prevalent. Detailed stud- of São Paulo approved it. The authors would like to thank Dr. Marcelo U. ies will be required to determine whether sera from P. Ferreira and Dr. Silvia di Santi for kindly providing the sera from individuals vivax infected individuals also fail to recognize recom- from West Africa, Dr. MUF for providing recombinant protein MSP-2 of P. binant proteins representing the P. falciparum MSP119. falciparum, and Drs. Michel Rabinovitch and MUF for critical reading of the manuscript. Recently, a direct sandwich ELISA to detect antibodies against the C-terminal region of MSP-1 was proposed as a References potential diagnostic method for Plasmodium vivax exposed 1. Mendis K, Sina BJ, Marchesini P, Carter R: The neglected burden of Plasmodium vivax malaria. Am J Trop Med Hyg 2001, 64:97-106. individuals from Korea [22]. This assay showed a high 2. Fundação Nacional de Saúde-FUNASA, Ministério da Saúde. sensitivity (99.5%) indicating that recombinant proteins Guia de Doenças: Malária Dados da doença – relatórios gerenciais containing the C-terminal region of P. vivax MSP-1 may be 2003 [http://www.funasa.gov.br]. 3. Moody A: Rapid diagnostic tests for malaria parasites. Clin used in individuals from different parts of the world. Microbiol Rev 2002, 15:66-78. 4. Snounou G, Viriyakosol S, Zhu XP, Jarra W, Pinheiro L, do Rosário VE, Thaithong S, Brown KN: High sensitivity of detection of It is also important to mention that it is very likely that human malaria parasites by the use of nested polymerase serological detection of P. vivax malaria can be further chain reaction. Mol Biochem Parasitol 1993, 61:315-320. improved by several distinct strategies. We are currently 5. Kimura M, Kaneko O, Qing L, Mian Z, Kawamoto F, Wataya Y, Otani S, Yamaguchi Y, Tanabe K: Identification of the four species of trying to improve the detection level by the combined use human malaria parasites by nested PCR that targets variant of other recombinant proteins based on the sequence of sequences in the small subunit rRNA gene. Parasitol Int 1997, 46:91-95. other blood stage antigens of P. vivax such as the Plasmo- 6. Rubio JM, Benito A, Roche J, Berzosa PJ, Garcia ML, Mico M, Edu M, dium vivax Merozoite Surface Proteins-3α and β, Apical Alvar J: Semi-nested, multiplex polymerase chain reaction for Membrane Antigen-1 and the Duffy Binding Protein detection of human malaria parasites and evidence of Plas- modium vivax infection in Equatorial Guinea. Am J Trop Med Hyg [19,20,23,24]. Also, we are developing a chemilumines- 1999, 60:183-187. cent enzyme-linked immunosorbent assay that may 7. Soares IS, Levitus G, Souza JM, del Portillo HA, Rodrigues MM: greatly improve the sensitivity, eliminating the few false Acquired immune responses to the N- and C-terminal regions of Plasmodium vivax merozoite surface protein 1 in negative and false positive samples we had [25,26]. individuals exposed to malaria. Infect Immun 1997, 65:1606-1614. 8. Soares IS, Cunha MG, Silva MN, Souza JM, del Portillo HA, Rodrigues MM: Longevity of the naturally acquired antibody responses Finally, our results support the notion that recombinant to the N- and C-terminal regions of Plasmodium vivax MSP1. proteins based on the P. vivax MSP119 kDa can be useful Am J Trop Med Hyg 1999, 60:357-363. for the development of a rapid immunochromatographic 9. 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