AJTMH ASTMH Job Mart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am. J. Trop. Med. Hyg., 73(3), 2005, pp. 510-516
Copyright © 2005 by The American Society of Tropical Medicine and Hygiene

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via ISI Web of Science (6)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by VILLAGRÁN, M. E.
Right arrow Articles by SÁNCHEZ-MORENO, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by VILLAGRÁN, M. E.
Right arrow Articles by SÁNCHEZ-MORENO, M.
Related Collections
Right arrow Trypanosomiasis
Right arrow Diagnosis
Right arrow Chagas Disease

USE OF AN IRON SUPEROXIDE DISMUTASE EXCRETED BY TRYPANOSOMA CRUZI IN THE DIAGNOSIS OF CHAGAS DISEASE: SEROPREVALENCE IN RURAL ZONES OF THE STATE OF QUERETARO, MEXICO

MARÍA E. VILLAGRÁN, CLOTILDE MARÍN, ISABEL RODRÍGUEZ-GONZALEZ, JOSE A. DE DIEGO, AND MANUEL SÁNCHEZ-MORENO*
Laboratorio de Tripanosomiasis Americana, Facultad de Química, Universidad Autónoma de Querétaro, Querétaro, Mexico; Departamento de Parasitología, Facultad de Ciencias, Instituto de Biotecnología, Universidad de Granada, Granada, Spain; Unidad de Parasitología y Medicina Tropical, Departamento de Medicina Preventiva y Salud Pública, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Four iron superoxide dismutase (SODI, SODII, SODIII, and SODIV) activities with pI values of 6.9, 6.8, 5.25, and 3.8, respectively, were isolated from epimastigote forms of the Maracay strain of Trypanosoma cruzi cultivated at 28°C in Grace’s medium supplemented with 10% heat-inactivated fetal bovine serum. The activity of SODe (pI 3.8), which coincides with that of SODIV, was detected in Grace’s medium without serum in which T. cruzi epimastigotes were cultivated for 24 hours at 28°C. SODe, which was excreted into the medium by the parasite, was immunogenic and antibodies to SODe were detected in serum to a dilution of 1:2,500 by Western blot. The role of SODe is related to the establishment of the parasite within the host, and its high immunogenicity and specificity make it a useful molecular marker in diagnosing infection with this parasite. To validate a Western blot result using SODe as a antigen fraction, 1,029 sera of individuals from 11 municipalities in the state of Queretaro, Mexico were analyzed. Sampling was done randomly and results were compared with those for the same sera with three conventional serologic methods: an enzyme-linked immunosorbent assay (ELISA), indirect hemagglutination (IHA), and an indirect immunofluorescence assay (IFA) to detect antibodies to T. cruzi SODe. Samples that were positive by these three techniques were also positive by the Western blot method. The seroprevalence values for SODe (8.16% by ELISA and Western blot) in Queretaro were considerably higher than those reported in regions of Mexico considered to be endemic for Chagas disease. These results support the use of SODe in the serodiagnosis of Chagas disease.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chagas disease, which is caused by the protozoan parasite Trypanosoma cruzi, is now ranked as the most serious parasite disease in Latin America, and has a heavy economic impact.1 The disease is transmitted in two ways: by vectors (triatomes) and by transfusions with infected blood.2 In recent years, because of migration from rural to urban areas, Chagas disease has become a serious health problem in cities in Latin America, where it can be transmitted vertically as well as by blood transfusions3 or organ transplants.4

Infection with this parasite is characterized by an acute parasitemic phase of 1–2 months, followed by a life-long chronic phase in which bloodstream parasites are detectable only by indirect methods such as xenodiagnosis5 or hemoculture.6 Most patients are diagnosed during the chronic phase by conventional serologic tests, such as an indirect inmunofluorescence assay (IFA), an enzyme-linked immunosorbent assay (ELISA), a complement fixation test, or indirect hem-agglutination (IHA).

Although these assays when used with crude parasite extracts are very sensitive in detecting T. cruzi infections, a problem with these assays is that they yield a small but significant number of false-positive results, especially when sera from patients with leishmaniasis7 are tested. This can make a correct diagnosis difficult, especially in areas in which both diseases are endemic, resulting in the rejection of a large number of blood donations. Another problem of the serologic methods concerns their sensitivity, which is not always adequate, leading to increased transmission of the disease.

This situation has resulted in the search for antigens from T. cruzi that could be used for the detection of this parasite. Two molecules have been detected: 1) a cysteine proteinase (GP57/51) that is released during metacyclogenesis,8 and 2) a 70-kD heat-shock protein9 that may be useful in diagnosing infection with this parasite.

For many intracellular parasites and bacterial pathogens, superoxide dismutase (SOD), a metalloenzyme present in all oxygen-breathing organisms, constitutes the first line of defense against oxidative damage caused by the formation of superoxide radicals. This enzyme catalyzes the reduction of superoxide radicals to molecular oxygen and hydrogen peroxide.10 Three classes of SODs, each with different metal cofactors (iron, manganese, and copper-zinc), have been identified. They also differ in their location in the cell (cytosol, other cell organelles, and cell secretions).

All parasitic protozoa studied to date have SODs with only iron cofactors.11,12 Ever since it was discovered that SODs from parasitic protozoa had iron cofactors and that they differed from those of the vertebrate host not only structurally but also terms of specific inhibitors, they have been proposed as potential chemotherapeutic targets.11 In some parasitic protozoa, it has been demonstrated that some SODs involved in oxidative attack are highly immunogenic, raising the possibility of using them in the diagnosis of Chagas disease.13

We recently characterized two iron superoxide dismutase activities (SODI and SODII) from a plant trypanosomatid isolated from Euphorbia characias.14 The isoenzyme SODII is immunogenic and is excreted into the culture medium by this plant trypanosomatid (Villagrán ME and others, unpublished data). In T. cruzi, SODs have been detected in the epimastigote stage; however, their presence and role in other developmental stages of the parasite is unknown.15

The purpose of this study was to determine whether T. cruzi excretes an SOD (SODe) similar to that excreted by plant trypanosomatids and to analyze its potential immunologic properties and usefulness in diagnosing infection with this parasite. To demonstrate the diagnostic usefulness of SODe, we tested 1,029 sera from individuals in the state of Queretaro, Mexico by an IFA, IHA, and an ELISA, as well as by a Western blot, against the antigenic SODe fraction obtained from cultures of the Maracay strain of T. cruzi. This enables us to obtain new data on the prevalence of Chagas disease in this area. In addition, assays were conducted with sera from patients with Chagas disease, as well as with sera from patients in Peru with leishmaniasis and from patients in Spain infected with Toxoplasma gondii and Mycobacterium leprae.


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Parasites and culture. The Maracay strain of T. cruzi was isolated from a clinical specimen at the Institute of Malariology and Environmental Health in Maracay, Venezuela. Epimastigotes were grown in axenic Grace’s medium (Gibco-BRL, Gaithersburg, MD) supplemented with 10% heat-inactivated fetal bovine serum at 28°C. Epimastigote cultures (in the exponential growth phase) were concentrated by centrifugation at 600 x g for 10 minutes, and cells was washed twice and resuspended in ice-cold STE buffer (0.25 M sucrose, 25 mM Tris-HCl, 1 mM EDTA, pH 7.8) (buffer 1).

Extraction and purification of SOD isoenzymes. Cells were suspended (0.5–0.6 grams wet weight/mL) in 3 mL of buffer 1 and disrupted by three cycles of sonic disintegration (each for 30 seconds at 60 volts). The sonicated homogenate was centrifuged at 1,500 x g for 10 minutes at 4°C, and the pellet was washed three times with buffer 1 and resuspended in this buffer to obtain a total supernatant fraction of 9 mL. This fraction was centrifuged (2,500 x g for 10 min at 4°C), the supernatant was collected, and solid ammonium sulfate was added. The protein fraction, which precipitated at salt concentrations of 30–85%, was centrifuged (9,000 x g for 20 minutes at 4°C), redissolved in 2.5 mL of 20 mM potassium phosphate buffer, pH 7.8, containing 1 mM EDTA (buffer 2), and purified on a Sephadex G-25 column (PD10; Pharmacia, Piscataway, NJ) that was equilibrated with buffer 2. This resulted in a final volume of 3.5 mL (fraction P85).16

Enzyme assay. The SOD activity was determined by NAD(P)H oxidation according to Paoletti and Mocali.17 One unit was the amount of enzyme required to inhibit the rate of NAD(P)H reduction by 50%. The protein content was determined in all fractions by the Bio-Rad (Hercules, CA) test, which was based on the Bradford method (Sigma, St. Louis, MO), with bovine serum albumin as a standard.

Gel electrophoresis. Isoelectric focusing in a polyacrylamide Phast (Pharmacia) gel pI 3–9 was performed as previously described.18 The gels were stained according to a protocol reported elsewhere19 and stained for protein with silver nitrate as described by the manufacturer.

Determination of the isoelectric point of purified enzymes. Isoelectric points were determined in the Phast System using trypsinogen, lentillectin acidic band, horse heart myoglobin, carbonic anhydrase II, ß-lactoglobulin A, soybean trypsin inhibitor, and amyloglucosidase from Aspergillus niger (pI values of 9.3, 8.15, 6.8, 5.9, 5.1, 4.6, and 3.6, respectively) as standards. The gels were stained for protein with silver nitrate and for SOD activity as described elsewhere.19

Antigen preparations. Epimastigote forms in the exponential growth phase were concentrated by centrifugation at 600 x g for 10 minutes. The cell pellet was washed twice in Grace’s medium without serum, and the number of cells was counted in a hemocytometric chamber and distributed into aliquots of 5 x 109 parasites/mL. These cells were then grown in Grace’s medium without serum for 24 hours. The supernatant was collected by centrifugation at 600 x g for 10 minutes, passed through a 200-µm pore size filter, and solid ammonium sulfate was added until the desired antigen fraction (SODe) was obtained. This fraction was concentrated by ultrafiltration in Centriprep-10 tubes (Amicon, Danvers, MA) by centrifugation at 3,000 x g to give a final concentration of 2 mg/mL.

Polyclonal serum. To obtain specific antibodies against SODe, we immunized two female four-week-old BALB/c mice (protein concentration = 2 mg /mL) and performed the separation by electrophoresis of IEF 3–9 in polyacrylamide gels. Afterwards, the lane corresponding to the first well was removed from the gel and the SOD activity was analyzed.19 From the remaining gel the zones corresponding to the activity bands of SODe (pI 3.8) were cut, ground, and homogenized with sterile buffer phosphate (PBS). The mice were then each injected intraperitoneally in four immunizations, the first with Complete Freund’s Adjuvant (CFA) and the following three (booster immunizations) with Incomplete Freund’s Adjuvant (IFA) at 10-day intervals. The sera were collected 15 days after the last booster immunization by cardiac puncture. In addition, serum from the mouse that had not been immunized with the antigen fraction (control serum) was collected.

Western blot analysis. The antigen fraction (SODe) from T. cruzi was run on IEF 3–9 gels (protein concentration = 2 mg/mL), and transferred to a nitrocellulose membrane for 30 minutes, as described the manufacturer. The membrane was blocked for two hours at room temperature with 0.4% gelatin and 0.2% Tween 20 in PBS, washed three times with 0.1% Tween 20 in PBS, and incubated for two hours at room temperature with either negative serum at a dilution of 1:100 or anti-SODe serum at a dilution of 1:500. The membrane was then incubated for two hours at room temperature with an anti-human IgG (Fc specific) peroxidase conjugate (Sigma) diluted 1:1,000. After washing as above, substrate (diamino-benzidine, 0.5 mg/mL in 0.1 M Tris-HCl, pH 7.4, containing a 1:5,000 dilution of H2O2) was added. The reaction was stopped by washing in distilled water.

Human sera. A total of 1,029 sera were obtained from individuals in Queretaro, Mexico and screened for Chagas disease by conventional serologic tests (ELISA, IHA, and IFA). All localities were randomly sampled, taking into account that houses where the sampled persons lived were within high-risk areas of colonization by triatomines. Many of these dwellings had patios that increased the risk. In addition, most humans cohabitated with both domestic and synanthropic animals. Five sera from patients in Peru with Chagas disease that were reactive against the Maracay strain of T. cruzi at a titer of 1:80 measured by IHA and an IFA were used as positive controls. Sera from healthy persons in Spain that were not reactive by IHA or the IFA (titers < 1:8) were used as negative controls. Similarly, sera from patients with other parasitic infections (five with leishmaniasis, four with toxoplasmosis, and three with Mycobacterium leprae) were also assayed. The use of serum samples in the study was reviewed and approved by the Health Secretary of Queretaro State (Mexico) and the Scientific Ethics Committee of the University of Granada (Spain).

Serologic assays. Epimastigotes of the Maracay strain of T. cruzi were used as the antigenic fraction in these assays. For the ELISA, optimal concentrations of antigen, serum, and conjugate were determined by checkerboard titration, and epimastigote homogenates were used at a concentration of 1.25 µg/mL following the procedure of Krautz and others.9 Samples showing reactivity at dilutions > 1:32 were considered positive. The IFA was carried out following the method of Camargo,20 and samples with titers > 1:16 were considered positive. Indirect hemagglutination was performed according to the method of Camargo and others.21 All samples were analyzed in triplicate in polystyrene microtiter plates, and samples with titers > 1:32 were considered positive. In all cases, positive and negative controls were also analyzed simultaneously.


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
When a partially purified fraction of the epimastigote forms of the Maracay strain of T. cruzi was used for isoelectric focusing polyacrylamide gel electrophoresis (pI 3–9), followed by staining for SOD, four different SOD bands (SODI, SODII, SODIII, and SODIV) were detected (Figure 1Go, lane A). The SODI and SODII bands had similar isoelectric points (6.8–6.9) and were considered a double-banded isoenzyme. The other two bands (SODIII and SODIV) were single bands with isoelectric points of 5.25 and 3.8, respectively.



View larger version (77K):
[in this window]
[in a new window]
  		    FIGURE 1. Determination of the isoelectric points by isoelectric focusing (pI 3–9) of isoenzymes of superoxide dismutase (SOD) of the Maracay strain of Trypanosoma cruzi. Lane M, marker proteins with various pI values stained with Coomassie Blue; lane A, purified fraction of T. cruzi; lane B, SOD fraction excreted by T. cruzi. The SOD activity was determined according to the technique of Beyer and Fridovic.19

 
To identify the metal cofactors of the SODs, we tested inhibitors that have known effects on various cofactors (Cu/Zn, Mn, or Fe) of these enzymes. The four SODs were inhibited by H2O2, but not by cyanide, indicating that all four enzymes had an iron cofactor.

When epimastigote forms of T. cruzi (5 x 1010 parasites) were cultured for 24 hours in Grace’s medium without serum and the cell-free supernatant was collected, concentrated, and subjected to isoelectric focusing (pI 3–9), followed by staining for SOD activity, only a single SOD band was detected (Figure 1Go, lane B). The isoelectric point of this SODe was consistent with the isoelectric point of the SODIV band detected in the partially purified fraction of T. cruzi epimastigotes cultured under normal conditions.

We identified specific antibodies against SODe obtained from immunized BALB/c mice and identified by Western blot using SODe as the antigen fraction (Figure 2Go). With the control serum (Figure 2Go, lane 4), the reaction was negative at a dilution of 1:100. Sera containing antibodies to SODe (Figure 2Go, lanes 1–3) showed reactivity at dilutions of 1:100, 1:500, and 1:2,500.



View larger version (74K):
[in this window]
[in a new window]
  		    FIGURE 2. Detection of the excreted superoxide dismutase (SODe) isoenzyme of Trypanosoma cruzi by Western blot with various dilutions of polyclonal antibodies to SODe. Lane 1, 1:100; lane 2, 1:500; lane 3, 1:2,500; lane 4, control serum diluted 1:100.

 
To confirm the usefulness of SODe in diagnosing Chagas disease, we tested 1,029 blood samples from 11 municipalities in Queretaro, Mexico by Western blot, using the purified SODe extract as the antigen fraction (Table 1Go). We used the results of three conventional serologic methods (ELISA, IHA, and IFA) as standards. Eighty-five samples at a dilution of 1:100 were positive by Western blot against the SODe antigen fraction (Figure 3Go, lanes 1–6), giving a seroprevalence of 8.26% (Table 1Go). The seroprevalences of the different Mexican municipalities ranged from 22.44% in San Juan del Rio to 2.75% in Toliman (Figure 4Go). Serum at a dilution of 1:100 from a patient in Peru with Chagas disease was also positive (Figure 3Go, lane 10), while sera from a patient in Peru with leishmaniasis (Figure 3Go, lane 7) and from patients in Spain with leprosy (Figure 3Go, lane 8) and toxoplasmosis (Figure 3Go, lane 9) were negative.


View this table:
[in this window]
[in a new window]
 
TABLE 1
Seroprevalence of antibodies against Trypanosoma cruzi in individuals in different manicipalities of the State of Queretaro, Mexico by conventional serologic tests and Western blot against the SODe antigen fraction of T. cruzi*
 


View larger version (58K):
[in this window]
[in a new window]
  		    FIGURE 3. Immunoblot of sera from patients with various diseases against the superoxide dismutase excreted by the Maracay strain of Trypanosoma cruzi. Lanes 1–6, samples (diluted 1:100) from patients with Chagas disease in the state of Queretaro, Mexico; lane 7, sample from a patient with leishmaniasis in Peru; lane 8, sample from a patient with leprosy in Spain; lane 9, sample (diluted 1:50) from a patient with toxoplasmosis in Spain; lane 10, sample (diluted 1:100) from a patient with Chagas disease in Peru.

 


View larger version (30K):
[in this window]
[in a new window]
  		    FIGURE 4. Distribution of antibodies to Trypanosoma cruzi detected by an enzyme-linked immunosorbent assay and Western blot in individuals from different municipalities in the State of Queretaro, Mexico. Numbers indicate the percentages of seropositive individuals.

 
Of the 1,029 samples assayed, 54 were positive by least two of the conventional serologic tests used, giving a seroprevalence of 5.25%. However, when the ELISA results were compared with those of the Western blot, the seroprevalence increased to 8.16% (Figure 5Go). The concordance between various combinations of the tests used is shown in Figure 6Go.



View larger version (10K):
[in this window]
[in a new window]
  		    FIGURE 5. Comparative results of an enzyme-linked immunosorbent assay (ELISA), an indirect immunofluorescence assay (IFA), an indirect hemagglutination (IHA) test, and Western blot (SODe) in determining the seroprevalence of antibodies to the excreted super-oxide dismutase (SODe) of Trypanosoma cruzi in 1,029 individuals in of the State of Queretaro, Mexico.

 


View larger version (13K):
[in this window]
[in a new window]
  		    FIGURE 6. Concordance of results of an enzyme-linked immunosorbent assay (ELISA), an indirect hemagglutination (IHA) test, an indirect immunofluorescence assay (IFA), and Western blot (SODe) in the diagnosis of infection with Trypanosoma cruzi in 1,029 individuals in the State of Queretaro, Mexico. SODe = excreted super-oxide dismutase.

 

DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We recently observed that a trypanosomatid isolated from a plant (Phytomonas spp. isolated from Euphorbia characias) has two SODs isoenzymes with iron cofactors.16 On studying its subcellular location, we found that 20% of the activity was not solubilized even at high concentrations of digitonin, indicating that some of the SOD activity could be associated with membranes or even be excreted to the exterior by the parasite.14 This has been confirmed in species of the genus Phytomonas in our laboratory (Villagrán ME and others, unpublished data), and it has also been demonstrated that this excreted SOD is highly immunogenic. Phytomonas shows metabolic similarities to other trypanosomatids of animals and humans, such as T. cruzi.22 For these reasons, we investigated whether T. cruzi, in which two SOD activities had been demonstrated,15 excreted an SOD that serves the parasite as a defense mechanism against toxic radicals generated by the host.11

In the present study, we have demonstrated that T. cruzi excreted an SOD with an iron cofactor and a pI of 3.8. SODe was obtained from epimastigote forms cultured for 24 hours in Grace’s medium without serum. Marker enzymes (pyruvate kinase and hexokinase) confirmed that there was no lysis of the parasite during this culture period, and that the presence of the SOD was due its excretion by the parasite. In addition to this SODe, three other isoenzymes with different pIs were also detected. The presence of several isoenzymes can be explained by the fact that each has a different location in the parasite and carries out different functions in various developmental stages.14,15,23

It is known that in other parasitic protozoa, SOD is an immunogenic protein.13 One aim of the present study was to study the immunogenic properties of SODe. To achieve this, we isolated polyclonal antibodies against SODe. Control serum showed no reactivity against SODe by Western blot, while serum containing antibodies to SODe showed reactivity up a dilution of 1:2,500. This result indicates that SODe is a highly immunogenic protein, raising the possibility of its use in the diagnosis of Chagas disease.

To determine whether SODe could be used in the diagnosis of Chagas disease, we conducted serologic studies in Queretaro, Mexico. We investigated the use of SODe as the antigenic fraction in a diagnostic Western blot, and determined the seroprevalence of this disease in an area of Mexico in which few data are available, but which is close to other states (Hidalgo, Guanajuato, and San Luis de Potosí) that are endemic for this disease.24,25 This is important because Chagas disease is a serious health problem in Mexico26 (e.g., in the Yucatan, its has a prevalence of 11.20%27).

The Pan American Health Organization recommends that when performing an epidemiologic study of Chagas disease, more than one serologic procedure should be used to reduce diagnostic errors.28,29 Confirmation of undetermined cases of American trypanosomiasis by serologic diagnosis is made when there are positive results in at least two serologic tests.30 For this reason, in the present study samples were analyzed by three conventional serologic tests, in addition to the Western blot with SODe as antigen. In the ELISA, IHA, IFA test, and Western blot, the Maracay strain of T. cruzi was used as antigen because other investigators have reported that the strain of T. cruzi used does not appear to influence the results of serologic testing.29

When we compared values obtained with two of the conventional serologic tests (ELISA and IFA), we found seroprevalence values of 5.64%. However, when we compared the results of the ELISA with those of the Western blot, the seroprevalence increased to 8.16% and the concordance between the two techniques was nearly 100%. Seroprevalence in a given municipality was random, fluctuating between 22.45% for San de los Ríos and 2.75% for Toliman. When we analyzed seroprevalence values independently from the different tests used, we concluded that the Western blot with the SOD antigen excreted by the parasite was more specific and sensitive than the ELISA (seroprevalence of the Western blot = 8.26%).

It is known that at least 10% of patients with Chagas disease have serum antibodies that cross-react with some species of the genus Leishmania.31 Therefore, we also tested sera from patients with leishmaniasis, toxoplasmosis, and leprosy. None of these sera showed a positive reaction. However, the dilution titers used may have been too high to detect cross-reactivity.31

The results of the present study confirm that T. cruzi excretes an SOD (with a pI of 3) that has a role in the establishment of the parasite in the host. This SOD is highly immunogenic and specific, and therefore should be a useful tool in the diagnosis of Chagas disease. The seroprevalence values found among the rural population of the state of Queretaro, Mexico are considerably higher than those published by the health authorities. It would be advisable to establish obligatory diagnosis of Chagas disease by means of serologic tests (including the Western blot with SODe) for all blood donors. This is necessary because as a consequence of demographic shifts, the parasite has migrated beyond the disease-endemic area due to transmission by blood transfusions.31

Received September 8, 2004. Accepted for publication December 2, 2004.

Financial support: This work was supported by the ATP 2002/03: Circulation of Trypanosomatidae Project

* Address correspondence to Manuel Sánchez-Moreno, Departamento de Parasitología, Facultad de Ciencias, Instituto de Biotecnología, Universidad de Granda, Spain. E-mail: msanchem{at}ugr.es Back

Authors’ addresses: María E. Villagrán, Laboratorio de Tripanosomiasis Americana, Facultad de Química, Universidad Autónoma de Querétaro, Querétaro, Mexico. Clotilde Marín, Isabel Rodríguez-Gonzalez, and Manuel Sánchez-Moreno, Departamento de Parasitología, Facultad de Ciencias, Instituto de Biotecnología, Universidad de Granada, Granada, Spain. Jose A. De Diego, Unidad de Parasitología y Medicina Tropical, Departamento de Medicina Preventiva y Salud Pública, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain, E-mail: msanchem{at}ugr.es.


REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Dias JC, Schofield CJ, 1999. The evolution of Chagas disease (American trypanosomiasis). Control after 90 years since Carlos Chagas discovery. Mem Inst Oswaldo Cruz 94: 103–121.
  2. Dodd RY, 1998. Transmission of parasites by blood transfusion. Vox Sang 74: 161–163.[Medline]
  3. Schumunis GA, 1999. Prevention of transfusional Trypanosoma cruzi infection in Latin America. Mem Inst Oswaldo Cruz 94: 93–101.
  4. Carvalho MF, de Franco MF, Sooares VA, 1997. Amastigotes forms of Trypanosoma cruzi detected in a renal allograft. Rev Inst Med Trop Sao Paulo 39: 223–226.[Medline]
  5. Castro CN, Alves MT, Macedo VO, 1983. Importancia da repeticao do xenodiagnistico para avaliacao da parasitemia na fase cronica da doenca de Chagas. Rev Soc Bras Med Trop 22:19–23.
  6. Luz ZM, Coutinho MG, Cancado JR, Krettli AU, 1994. Hemoculture sensitive technique in the detection of Trypanosoma cruzi in chagasic patients in the chronic phase of Chagas disease. Rev Soc Bras Med Trop 27: 143–148.[Medline]
  7. Schechter M, Flint JE, Voller A, Guhi E, Marinkelle CJ, Miles MA, 1983. Purified T. cruzi specific glycoprotein for discriminative serological diagnosis of South American trypanosomiasis. Lancet 2: 939–941.[Medline]
  8. Ronaldo MC, D’Escoffier LN, Salles JM, Goldenberg S, 1991. Characterization and expression of proteases during T. cruzi metaciclogenesis. Exp Parasitol 73: 44–51.[Medline]
  9. Krautz GM, Peterson JD, Godsel LM, Krettli AU, Engman DM, 1998. Human antibody responses of Trypanosoma cruzi 70-kD heat-shock proteins. Am J Trop Med Hyg 58: 137–143.[Abstract]
  10. Ahmed H, Schott EJ, Gauthier JD, Vasta GR, 2003. Superoxide dismutase from the oyster parasite Perkinsus marinus: purification, biochemical characterization, and development of a plate microassay for activity. Anal Biochem 318: 132–141.[Medline]
  11. Kabiri M, Steverding D, 2001. Identification of a developmentally regulated iron superoxide dismutase of Trypanosome brucei. J Biochem 360: 173–177.
  12. Quesada JM, Entrala E, Fernández-Ramos C, Marín C, Sánchez-Moreno M, 2001. Phytomonas spp.: superoxide dismutase in plant trypanosomes. Mol Biochem Parasitol 115: 123–127.[Medline]
  13. Pérez-Fuentes R, Guégan JF, Barnabé C, López-Colombo A, Salgado-Rosas H, Torres-Rasgado E, Briones B, Romero-Diaz M, Ramos-Jiménez J, Sánchez-Guillén MC, 2003. Severity of chronic Chagas disease is associated with cytokine/antioxidant imbalance in chronically infected individuals. Int J Parasitol 33: 293–299.[Medline]
  14. Marín C, Hitos AB, Rodríguez-Gonzalez I, Dollet M, Sánchez-Moreno M, 2004. Phytomonas iron superoxide dismutase: a possible molecular marker. FEMS Microbiol Lett 234: 69–74.[Medline]
  15. Ismail SA, Paranchuk W, Skeiky YA, Reed SG, Bhatia A, Gedamu L, 1997. Molecular cloning and characterization of two iron superoxide dismutase cDNAs from Trypanosoma cruzi. Mol Biochem Parasitol 86: 187–197.[Medline]
  16. Marín C, Rodriguez-Gonzalez I, Hitos AB, Rosales MJ, Dollet M, Sánchez-Moreno M, 2004. Purification and characterization of two iron superoxide dismutase of Phytomonas sp. isolated from Euphorbia characias (plant trypanosomatids). Parasitology 129: 79–86.[Medline]
  17. Paoletti F, Mocali A, 1990. Determination of superoxide dismutase activity by purely chemical system based on NAD(P)H oxidation. Methods Enzymol 186: 209–265.[Medline]
  18. Bécuwe P, Gratepanche S, Fourmaux MN, van Beeumen J, Samyn B, Mercereau-Puijalon O, Touzel JP, Slomianny C, Camus D, Dive D, 1996. Characterization of iron-dependent endogenous superoxide dismutase of Plasmodium falciparum. Mol Biochem Parasitol 76: 125–134.[Medline]
  19. Beyer WF, Fridovich I, 1987. Assaying for superoxide dismutase activity: some large consequences of minor changes in conditions. Anal Biochem 161: 559–566.[ISI][Medline]
  20. Camargo ME, 1966. Fluorescent antibody test for serodiagnosis technical modification employing preserved cultured forms of T. cruzi in slide test. Rev Inst Med Trop Sao Paulo 8: 227–234.[Medline]
  21. Camargo ME, Hshino S, Sequeira GRV, 1973. Hemagglutination, with preserved, sensitized cells, a practical test for routine serological diagnosis of American trypanosomiasis. Rev Inst Med Trop Sao Paulo 15: 81–85.[Medline]
  22. Uttaro AD, Mirkin N, Rider MH, Michels PAM, Opperdoes FR, 1999. Phytomonas sp. as a model of trypanosomatid metabolism and drug. Mem Inst Oswaldo Cruz 94 (Suppl II): C19.
  23. Bannister JV, Bannister WH, Rottilio G, 1987. Aspects of the structure, function and application of superoxide dismutases. Crit Rev Biochem 22: 111–180.[ISI][Medline]
  24. Velasco-Castrejón O, Valdespino JL, Tapia CR, Salvatierra B, Guzmán-Bracho C, Magos C, 1992. Seroepidemiología de la enfermedad de Chagas en México. Salud Publica Mex 34: 186–196.[Medline]
  25. Guzmán-Bracho C, García L, Verdugo JF, Guerrero S, Torres M, Ramírez C, Veslcao-Castrejón O, 1998. Riesgo de transmisión de Tripanosoma cruzi por transfusión de sangre en México. Rev Panam Salud Publica 4: 94–99.[Medline]
  26. UNDP/World Bank/WHO, 1997. Special Programme for Research and Training in Tropical Diseases. TDR News 52: 2.
  27. Zavala-Velázquez JE, 2003. La enfermdad de Chagas en el Estado de Yucatán,México. Rev Biomed 14: 35–43.
  28. Camargo ME, Segura EL, Kagan IG, Pacheco-Souza JM, Carvalheiro JR, Yanovsky JF, 1987. Normalización del diagnóstico sexológico de la enfermedad de Chagas en Las Américas: evaluación de tres años de colaboración. Bol Oficina Sanit Panam 102: 449–463.
  29. Sousa-Jurado F, Zumaquero JL, Reyes PA, Cruz-García A, Guzmán-Bracho C, Monteón VM, 2004. Factores bióticos y abióticos que determinan la seroprevalencia de anticuerpos contra Trypanosoma cruzi en el municipio de Bravo, Puebla, México. Salud Publica Mex 46: 39–48.[ISI][Medline]
  30. Secretaria de Salud, PROY-NOM-032-SSA2, 2000. Para la Vigilancia, Prevención y Control de Enfermedades Transmitidas por Vector. Mexico City, Mexico: Diario Oficial de la Federación, November 21, 2000. Second sección.
  31. Ramos-Echeverría AA, Monteon-Padilla VM, Reyes PA, 1993. Detección de anticuerpos contra Tripanosoma cruzi en donadores de sangre. Salud Publica Mex 35: 1–9.




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via ISI Web of Science (6)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by VILLAGRÁN, M. E.
Right arrow Articles by SÁNCHEZ-MORENO, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by VILLAGRÁN, M. E.
Right arrow Articles by SÁNCHEZ-MORENO, M.
Related Collections
Right arrow Trypanosomiasis
Right arrow Diagnosis
Right arrow Chagas Disease

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS