INTRODUCTION
Strongyloidiasis is caused by Strongyloides stercoralis, an intestinal nematode which is mainly endemic in tropical and subtropical regions (Grove, Reference Grove1996; Paula and Costa-Cruz, Reference Paula and Costa-Cruz2011) and affects 30–100 million people in the world (Siddiqui and Berk, Reference Siddiqui and Berk2001). The parasite causes chronic asymptomatic infections in immunocompetent human hosts and systemic invasion in immunocompromised patients, developing into a fatal hyperinfection syndrome (Marcos et al. Reference Marcos, Terashima, Canales and Gotuzzo2011).
Clinical diagnosis is uncertain because most cases are oligo- or asymptomatic or present as pulmonary and intestinal symptoms common to other parasitic diseases (Agrawal et al. Reference Agrawal, Agarwal and Ghoshal2009). Parasitological diagnosis presents low sensitivity, due to an intermittent larval shedding (Uparanukraw et al. Reference Uparanukraw, Phongsri and Morakote1999; Blatt and Cantos, 2003; Carvalho et al. Reference Carvalho, Moreira, Pena, Marinho, Bahia and Machado-Coelho2012).
Immunological methods, such as enzyme-linked immunosorbent assay (ELISA), have been widely used in the diagnosis of human strongyloidiasis due to its applicability, safety, availability of reagents, and high diagnostic parameters, being considered the best test for serological screening (Mangali et al. Reference Mangali, Chaicumpa, Nontasut, Chantavanij, Tapchaisri and Viravan1991; Atkins et al. Reference Atkins, Conway, Lindo, Bailey and Bundy1999; Schaffel et al. Reference Schaffel, Nucci, Carvalho, Braga, Almeida, Portugal and Pulcheri2001; Siddiqui and Berk, Reference Siddiqui and Berk2001; Koosha et al. Reference Koosha, Fesharaki and Rokni2004; Feliciano et al. Reference Feliciano, Gonzaga, Gonçalves-Pires, Gonçalves, Rodrigues, Ueta and Costa-Cruz2010; Gonzaga et al. Reference Gonzaga, Ribeiro, Feliciano, Manhani, Silva, Ueta and Costa-Cruz2011b).
As strongyloidiasis immunodiagnosis continues to be a challenge because of the increasing difficulty in obtaining sufficient quantities of S. stercoralis larvae (Rossi et al. Reference Rossi, Partel, Teixeira, Takahashi, De Barros Mazon and da Silva1993; Costa-Cruz et al. Reference Costa-Cruz, Machado and Campos1998) alternative antigens, including heterologous ones from Strongyloides venezuelensis have also been used, with satisfactory results, to diagnose human strongyloidiasis (Sato et al. Reference Sato, Kobayashi, Toma and Shiroma1995; Machado et al. Reference Machado, Faccioli, Costa-Cruz, Lourenço, Roque-Barreira, Gonçalves-Pires and Ueta2008; Ribeiro et al. Reference Ribeiro, Feliciano, Gonzaga, Costa, Gonçalves-Pires, Ueta and Costa-Cruz2010; Gonçalves et al. Reference Gonçalves, Rocha, Gonzaga, Gonçalves-Pires, Ueta and Costa-Cruz2012).
Recent research studies have shown that fractions of total saline extract of S. venezuelensis filariform larvae, obtained by different separation principles, can be used to detect IgG in S. stercoralis infection (Rigo et al. Reference Rigo, Lescano, de Marchi and Amato Neto2008; Feliciano et al. Reference Feliciano, Gonzaga, Gonçalves-Pires, Gonçalves, Rodrigues, Ueta and Costa-Cruz2010; Gonzaga et al. Reference Gonzaga, Ribeiro, Cunha-Júnior, Ueta and Costa-Cruz2011a).
The aim of this study was to fractionate, for the first time, the antigenic extract from filariform larvae of S. venezuelensis in ion-exchange resin diethylaminoethyl sepharose (DEAE), to obtain antigenic fractions potentially applicable in immunoassays.
MATERIALS AND METHODS
Serum samples
Serum samples were collected from 155 subjects attending the Laboratory of Clinical Analysis at the Clinical Hospital (Groups 1 and 2) and from the Laboratory of Parasitology (Group 3) of the Federal University of Uberlândia in the State of Minas Gerais, Brazil. Group 1 (G1) consisted of 50 patients living in an endemic area and with confirmed parasitological diagnosis of strongyloidiasis using the Baermann-Moraes (Baermann, Reference Baermann1917; Moraes, Reference Moraes1948) method, based on positive larval thermo-hydrotropism, and the method of Lutz (Reference Lutz1919), a gravity sedimentation technique.
Group 2 (G2) consisted of 55 patients who harboured other parasites: hookworm (Necator americanus or Ancylostoma spp.): (n = 16; hookworm only (n = 12); co-infected patients (n = 4): (hookworm + A. lumbricoides + T. trichiura (n = 2); hookworm + A. lumbricoides (n = 1); and hookworm + Hymenolepis nana (n = 1)), Ascaris lumbricoides (n = 6), Trichuris trichiura (n = 2), A. lumbricoides + T. trichiura (n = 1), Entamoeba histolytica (n = 1), Enterobius vermicularis (n = 5), Giardia lamblia (n = 6), Hymenolepis nana (n = 5), Schistosoma mansoni (n = 5), Taenia sp. (n = 8; taeniasis (n = 5) and cysticercosis (n = 3)).
Group 3 (G3) consisted of 50 healthy volunteers based on their clinical profile. Although they live in an endemic area for strongyloidiasis, none had had household contact with S. stercoralis infection or any previous clinical related history of strongyloidiasis. Group 3 individuals underwent 3 faecal samples which were negative by Lutz (Reference Lutz1919) and Baermann-Moraes (Baermann, Reference Baermann1917; Moraes, Reference Moraes1948). This study was approved by the Research Ethics Committee of the Federal University of Uberlândia.
Larvae obtention
S. venezuelensis third-stage larvae (L3) were obtained from charcoal cultures of feces from experimentally infected rats (Rattus norvegicus; L-2 strain; 1500 L3 larvae; abdominal subcutaneous inoculation). Fecal samples were mixed with equal parts of charcoal, moistened with water, spread in uniform layers on Petri dishes, and incubated at 28 °C for 72 h. Infective larvae were collected and concentrated using a Rugai apparatus (Rugai et al. Reference Rugai, Mattos and Brisola1954). L3 were washed 5 times in phosphate-buffered saline (PBS), 0·01 M, pH 7·2, and stored at −20 °C in PBS until use in antigen preparation.
Somatic antigen (SA) of Strongyloides venezuelensis filariform larvae
Somatic antigen was prepared according to the method of Gonzaga et al. (Reference Gonzaga, Ribeiro, Cunha-Júnior, Ueta and Costa-Cruz2011a) with minor modifications. Briefly, S. venezuelensis filariform larvae (300 000) were re-suspended in PBS containing protease inhibitors (ethylenediaminetetraacetic acid 1 mmol/L, benzamidine 1 mmol/L, phenyl methyl sulfonyl fluoride 1 mmol/L, aprotinin 1 μg/mL, and leupeptin 2 μg/mL) and disrupted by 10 cycles of freezing (1 min, −196 °C) and thawing/sonication (5 min, 40 kHz, 37 °C) (Thornton, Inspec Eletrônica, São Paulo, Brazil). After a 2-h incubation period at 4 °C under gentle shaking, the suspension was centrifuged (12400 g, 30 min, 4 °C) and the supernatant was analysed for protein content according to the method of Lowry et al. (Reference Lowry, Rosebrough, Farr and Randall1951), and then stored at −20 °C until use.
Ion-exchange chromatography
Fractions of SA were obtained by ion-exchange chromatography developed in microtubes as follows. Briefly, 1200 μg of SA was loaded onto a 200 μl diethylaminoethyl Sepharose-DEAE resin (GE Healthcare Life Sciences), previously equilibrated with 10 volumes of PBS by 3 cycles of centrifugation (2000 g, 2 min). The suspension SA/resin was maintained under gentle mixing for 20 min at 4 °C. After, the suspension had been centrifuged as described, the supernatant was recovered and considered as the non-binding resin fraction (DEAE S1). Resin was washed by centrifugation with 10 volumes of PBS, and the retained proteins (DEAE S2) were eluted using PBS supplemented with 0·5 M NaCl. The obtained fractions were analysed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions, as described by Laemmli (Reference Laemmli1970). The proteins were visualized in SDS-PAGE by silver staining (Friedman, Reference Friedman1982). Gel analysis was performed using a graphical method to generate a protein lane profile plot of each antigenic fraction, by Image J version 1.44 software (National Institutes of Health, Bethesda, EUA); and then peaks referring to bands were compared with a protein standard marker (Real Biotech, RECOM™ Blue Wide Range Prestain Marker, Banqiao City, Taiwan) to estimate their relative molecular weights.
ELISA to detect IgG anti-Strongyloides
Preliminary experiments were carried out to determine the optimal conditions for ELISA, through block titration of reagents (antigens, sera and conjugate). ELISA was carried out as described by Gonzaga et al. (Reference Gonzaga, Ribeiro, Feliciano, Manhani, Silva, Ueta and Costa-Cruz2011b) with modifications. Polystyrene microplates (Interlab, Brazil) were coated with each antigen (SA, DEAE S1 and DEAE S2) at a concentration of 5 μg/ml in carbonate-bicarbonate buffer, pH 9·6, and incubated overnight at 4 °C. Microplates were washed 3 times with PBS containing 0·05% Tween 20 (PBS-T). Then serum samples were diluted 1:160 in PBST containing 3% skimmed milk (PBST-M) and incubated for 60 min at 37 °C. After another step of washing, the immunoenzymatic conjugate (peroxidase-goat anti-human IgG, Fc specific; Sigma) was added diluted 1:2000 in PBS-T and incubated for 60 min at 37 °C. The assay was developed after an additional washing procedure by adding the enzyme substrate consisting of hydrogen peroxide and orthophenylenediamine (OPD) in 0·1 M citrate phosphate Na2HPO4 buffer, pH 5·5, for 15 min. The reaction was interrupted by addition of 25 μl per well of 2M H2SO4. Optical densities (OD) were determined at 492 nm in an ELISA reader (Titertek Plus, Flow Laboratories, USA).
Statistical analyses
Analyses were performed using GraphPad software package 5.0 (GraphPad Software Inc., San Diego, USA). Cut-off points were established using a two-graph receiver operating characteristic curve (TG-ROC) (Greiner et al. Reference Greiner, Sohr and Göbel1995) based on ELISA results from patients positive for S. stercoralis in stool samples (positive controls) and individuals from the other 2 groups (strongyloidiasis negative controls). The ELISA reactivity index (RI) was obtained by the ratio between OD and cut-off. Values of RI greater than the optimum point of reaction for each extract were considered positive (RI >1). Sensitivity (Se) and specificity (Sp) were calculated according to the formulae: Se=a/(a + b) and Sp=d/(c + d) where a = is true positive, b=false positive, c=false negative and d=true negative (Youden, Reference Youden1950).
Friedman (F) and post-hoc Wilcoxon Signed-Rank tests (W), with Bonferroni-Holm (Holm, Reference Holm1979) correction for a multiple comparison situation, were conducted to evaluate statistical differences of IgG levels using the 3 antigenic fractions among studied groups.
Receiver operating characteristic curves (ROC) were built to describe the tests (Martinez et al. Reference Martinez, Louzada-Neto and Pereira2003). The area under the ROC curve (AUC), an overall index of diagnostic accuracy, was calculated, values close to 1 indicate an informative test; and close to 0·5 indicate an uninformative test (Hanley and McNeil, Reference Hanley and Macneil1982). An AUC comparison between antigenic preparations, which were derived from the same sera panel, was made using the method described by Hanley and McNeil (Reference Hanley and Mcneil1983). The likelihood ratio (LR), an efficiency diagnostic parameter independent of prevalence (Greiner et al. Reference Greiner, Sohr and Göbel1995) was calculated, as follows: LR + =Se/(1 − Sp), which indicates how likely patients with strongyloidiasis are to have a positive test result compared with other patients; and LR − =(1 − Se)/Sp, defined as the probability of having a negative test result for strongyloidiasis patients. Probability (P) values of <0·05 were regarded as significant and 95% confidence intervals (CI) were provided to Se, Sp and AUC statistical calculations.
RESULTS
Eletrophoretical characterization
Figure 1 shows the eletrophoretical profile of each antigenic fraction after 12% SDS-PAGE. SA showed several proteic compounds with molecular weights ranging from <15 to 240 kDa. In the DEAE S1 fraction an enriched fraction of proteins with molecular weight from <15 to 21 kDa was observed. Otherwise, the analysis of DEAE S2 revealed a wider range of protein profile throughout the major antigens of 21 to 50 kDa.
Fig. 1. Electrophoretic profiles of somatic antigen (SA) of Strongyloides venezuelensis filariform larvae and fractions obtained in ion-exchange diethylaminoethyl Sepharose resin – DEAE (DEAE S1 and a DEAE S2, that interacted with the resin) and Image J software analysis, in 12% SDS-PAGE, silver stained are shown. MW, molecular weight standard in kilodaltons (kDA).
IgG detection by ELISA using SA, DEAE S1 and DEAE S2
All serum samples were tested by ELISA using SA and its fractions obtained after ion-exchange chromatography (DEAE S1 and DEAE S2). At G1, 88% (44/50) of positivity for SA, 80% (40/50) and 92% (46/50) for DEAE S1 and DEAE S2 was observed, respectively. By analysis of detection patterns in G2, positivity rates of 9% (5/55) were observed when using SA and DEAE S1, and 5% (3/55) to DEAE S2. At G3 positivity was 20% (10/50), 26% (13/50) and 12% (6/50) for SA, DEAE S1 and DEAE S2, respectively. Differences in positivity were accompanied by a statistically significant difference in IgG antibody levels (F > 21·71; P < 0·0001) according to each antigenic fraction used (Fig. 2); notably the DEAE S2 fraction presented the highest RI median in G1 (1·924; SA x DEAE S2, W = − 769·0, P=0·0002; DEAE S1 x DEAE S2, W = − 973·0, P < 0·0001) and the lowest in G2 (0·512; SA x DEAE S2, W = 1134·0, P < 0·0001; DEAE S1 x DEAE S2, W = 950·0, P < 0·0001) and G3 (0·687; SA x DEAE S2, W = 845·0, P < 0·0001; DEAE S1 x DEAE S2, W = − 1099·0, P < 0·0001).
Fig. 2. Detection of IgG anti-Strongyloides in serum samples from patients with strongyloidiasis (G1, n = 50), other parasitic infections (G2, n = 55) and healthy individuals (G3, n = 50) analysed by ELISA (enzyme-linked immunosorbent assay) using somatic antigen (SA) of S. venezuelensis, and DEAE fractions DEAE S1 (unbound fraction) and DEAE S2 (bound fraction). Data by groups represented in box and whisker plot, with box indicating median and 25–75% percentiles, and whiskers indicating data range. Statistical significance was calculated by the Friedman test and post-hoc Wilcoxon Signed-Rank test, with Bonferroni-Holm correction (*P<0·017).
Cross-reactivity at G2 (RI >1) was observed when testing serum samples from patients infected with hookworm only (3/12 SA, DEAE S1 and DEAE S2), E. vermicularis (1/5 SA and DEAE S1), G. lamblia (1/6 SA) and A. lumbricoides (1/6 DEAE S1).
According to the cut-off point established by TG-ROC, sensitivity and specificity were 88% and 85·7% for SA; 80% and 82·9%, respectively for DEAE S1. When DEAE S2 was used an antigenic fraction having the best diagnostic performance with the highest values for sensitivity (92%) and specificity (91·4%) (Fig. 3) for IgG detection was observed.
Fig. 3. Receiver operating characteristic curves (ROC) indicating the optimum point of reaction (cut-off), sensitivity (Se), specificity (Sp), area under curve (AUC) and likelihood ratios (LR+ and LR−) for IgG detection in serum samples using somatic antigen (SA) of Strongyloides venezuelensis and DEAE fractions (DEAE S1 and DEAE S2).
Analysis of the ROC curve (Fig. 3), demonstrated that the DEAE S2 fraction efficiently distinguished patients with strongyloidiasis from control groups (G2 and G3). Test performance, indicated by AUC, showed that IgG detection using the DEAE S2 fraction (0·981) had the closest to the maximum value (1·00) of efficiency. Otherwise, when considering AUC values from SA (0·957) and DEAE S1 (0·915), efficiency was lower. Comparing AUC by the method of Hanley and McNeil (Reference Hanley and Mcneil1983) it was observed that the DEAE S2 fraction achieved a value statistically better than SA (z = −3·464; P=0·0008) and DEAE S1 (z = −3·509; P=0·0005).
LR+ to SA was 6·17 and for its fraction DEAE S1 was 4·67, indicating a moderate and small probability of a true positive strongyloidiasis, respectively. However, for the DEAE S2 fraction, a value of LR+ (10·73) points to an efficient test. The LR− values for SA (LR − =0·14) and DEAE S1 (LR − =0·14) indicate that both tests had a moderate effect on diminishing the probability of disease, while a low LR− (<0·1) for DEAE S2 (LR − =0·09) virtually excludes the chance that a patient is infected.
DISCUSSION
Considering the difficulties regarding to S. stercoralis immunodiagnosis, mainly in the cross-reactivity with other parasitic infections and normal endemic sera, efforts to achieve a reliable diagnostic test are needed. The present study was the first attempt using ion-exchange chromatography to obtain fractions potentially applicable in human strongyloidiasis diagnosis. Several studies have demonstrated the partial fractionation of Strongyloides antigenic extracts using other chromatographic approaches, including: gel filtration by S-200 (Mangali et al. Reference Mangali, Chaicumpa, Nontasut, Chantavanij, Tapchaisri and Viravan1991), hydrophobic interaction by octyl-Sepharose® (Rigo et al. Reference Rigo, Lescano, de Marchi and Amato Neto2008) and carbohydrate-affinity interaction by concanavalin-A-agarose resin (Gonzaga et al. Reference Gonzaga, Ribeiro, Cunha-Júnior, Ueta and Costa-Cruz2011a). In the present study, electrophoretical analysis of DEAE S1 and S2 fractions showed a complementary profile when compared with SA. In the DEAE S1 fraction, an enrichment of proteins with low molecular weight was detected, including polypeptides from 21 to <15 kDa, indicating that these molecules do not interact with the DEAE resin. On the other hand, the DEAE S2 fraction is mainly enriched with proteins from 21 to 50 kDa that interact tightly with cationic diethylaminoethyl group. These findings suggest that proteins belonging to DEAE S2 display a negative liquid charge under pH physiological conditions.
DEAE resin was applied as a first fractionation step of Strongyloides larval extract to evaluate ELISA protocols. Despite having lower ELISA diagnostic parameters, the DEAE S1 fraction included regions of immunodominant proteins recently described: a 10–30 kDa region (Rigo et al. Reference Rigo, Lescano, de Marchi and Amato Neto2008), and a 28 kDa band (Feliciano et al. Reference Feliciano, Gonzaga, Gonçalves-Pires, Gonçalves, Rodrigues, Ueta and Costa-Cruz2010) of S. venezuelensis larval preparations were detected by IgG from infected patients, even after different detergent fractionations. Other studies have demonstrated reactivity with 10, 17, 19 kDa proteic bands in the sera of S. stercoralis patients (Atkins et al. Reference Atkins, Conway, Lindo, Bailey and Bundy1999; Silva et al. Reference Silva, Barcelos, Passos-Lima, Espindola, Campos and Costa-Cruz2003).
Previous studies that employed homologous and/or heterologous Strongyloides antigens showed serum reactivity in a comparable proteic region to that obtained in this study, especially in DEAE S2. Machado et al. (Reference Machado, Faccioli, Costa-Cruz, Lourenço, Roque-Barreira, Gonçalves-Pires and Ueta2008) identified a 45 kDa immunodominant antigenic fraction, by immunoblot, which was recognized by IgG antibodies using S. venezuelensis antigenic extract. Using homologous antigens, other authors have shown IgG reactivity with 21, 26, 31, 32 and 33 kDa bands from S. stercoralis antigens (Sato et al. Reference Sato, Inoue, Matsuyama and Shiroma1990; Atkins et al. Reference Atkins, Conway, Lindo, Bailey and Bundy1999; Sudré et al. Reference Sudré, Siqueira, Barreto, Peralta, Macedo and Peralta2007). Although similar electrophoretic motility does not characterize an absolute measure for polypeptide identity (Gomez-Munoz et al. Reference Gomez-Muñoz, Cuquerella and Alunda1996) it is suggested that the bands described are equivalent to the ones found herein, and the partially purified DEAE S2 fraction should be relevant in human strongyloidiasis immunodiagnosis.
Analysis of the DEAE S2 fraction performance within groups, patients with strongyloidiasis had 4 sera with false-negative results, possibly because these patients did not reach detectable levels of antibodies. Other parasitic infections group (G2) is representative of the general population, especially in developing countries where parasitic diseases are highly prevalent (Grove, Reference Grove1996; CDC, 2012). Since serum samples from Toxocara-infected patients were not available, especially due to the low occurrence, it was not possible to determine cross-reactivity indices for this parasite. In G2 cases of cross-reactivity occurred in patients infected with hookworm, who also were false-positive in the SA and DEAE S1 fraction. In addition, hookworm is one of the main contributors to cross-reactivity in strongyloidiasis serology. There were endemic normal sera (G3) with false-positive results, and it is suggested that these patients only had detectable IgG anti-Strongyloides titres due to constant antigenic exposure. These false-positive results in G2 and G3 may also possibly be the result of individuals who previously had an asymptomatic and undetected strongyloidiasis infection and therefore may have had antibodies against Strongyloides. In this way, it could be hypothesized that the difference at the composition of the DEAE S1 and DEAE S2 fractions actually affected test performance. The use of DEAE S2 contributed to an increase in G1 IgG detection with possible selection of more sensible epitopes and with a decrease in antibody median of other groups with less cross-reactivity.
ROC and TG-ROC curve analysis provided an optimal cut-off point for each antigenic fraction studied. Information obtained from ELISA showed a different IgG detection profile for each fraction tested. DEAE S1 diagnostic parameters were inferior if compared with those from DEAE S2, which exhibited an increased sensitivity, specificity and a higher AUC value. Therefore, it is suggested that different epitopes could be recognized (Atkins et al. Reference Atkins, Conway, Lindo, Bailey and Bundy1999; Gonzaga et al. Reference Gonzaga, Ribeiro, Cunha-Júnior, Ueta and Costa-Cruz2011a); however, IgG possibly has a higher affinity for proteic epitopes with selected anionic residues, once the DEAE resin is positively charged. According to Jaeschke et al. (Reference Jaeschke, Guyatt and Sacket1994), values of likelihood ratio LR+ greater than 10, practically confirm the diagnosis, and LR− below 0·1 exclude diagnosis. Therefore, the LRs showed that the DEAE S2 fraction had a remarkable diagnostic performance to detect IgG in patients with strongyloidiasis, reaching values of LR + =10·73 and LR − =0·09.
A simple step to partial purification, as accomplished with DEAE anion-exchange chromatography, already showed appreciable results in immunoassay tests in other parasitic species studies to detect serum antibodies in Wuchereria bancrofti filariasis (Chenthamarakshan et al. Reference Chenthamarakshan, Reddy and Harinath1996) or for localization of fractionated antigen in Paragonimus infection (Lee and Chung, Reference Lee and Chung2001); and also for Trichinela spiralis tubulin purification (Martínez-González et al. Reference Martínez-González, Jiménez-González and Rodríguez-Caabeiro1998). Regarding diagnosis, the unbound fraction obtained in DEAE from heterologous filarial antigen provided better results in IgG detection for bancroftian filariasis (Chenthamarakshan et al. Reference Chenthamarakshan, Reddy and Harinath1996), in contrast with our results.
A detailed immunochemical characterization of the DEAE S2 fraction, would be considerable using other approaches, for example, sequential gel filtration chromatography as previously used for diagnostic purposes in other parasite infections (de Graaf et al. 1993; Camargo et al. Reference Camargo, Uzcanga and Bubis2004; Revilla-Nuín et al. Reference Revilla-Nuín, Manga-González, Miñambres and González-Lanza2005; Kaushal et al. Reference Kaushal, Srivastava, Mustafa, Tandon, Singh and Kaushal2009). However, it should be considered that a high-level purification of parasitic antigens may be related to an increase in specificity accompanied by a reduction in sensitivity indices (Chenthamarakshan et al. Reference Chenthamarakshan, Vadivelu and Puthucheary2001). Thus, a defined antigenic fraction, such as DEAE S2, that contains more antigenic polypeptides could ensure an appropriate ELISA system.
In conclusion, the DEAE S2 fraction, obtained from Strongyloides venezuelensis somatic antigen fractionated on DEAE ion-exchange resin, showed high diagnostic parameters thereby suggesting its potential for strongyloidiasis diagnosis and as a source of immunodominant polypeptides for IgG detection.
FINANCIAL SUPPORT
This research was supported by the Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and by the Federal University of Uberlândia (UFU), Brazil.
CONFLICT OF INTEREST
The authors declare that there are no conflicts of interest.
