Parasitol Res (2014) 113:2569–2575 DOI 10.1007/s00436-014-3908-7
ORIGINAL PAPER
Diethylaminoethyl (DEAE) binding fraction from Taenia solium metacestode improves the neurocysticercosis serodiagnosis Vanessa da S. Ribeiro & Daniela da S. Nunes & Henrique T. Gonzaga & Jair P. da Cunha-Junior & Julia M. Costa-Cruz
Received: 16 January 2014 / Accepted: 9 April 2014 / Published online: 29 April 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Neurocysticercosis (NC) is one of the most important diseases caused by parasites affecting the central nervous system. We fractionated by ion-exchange chromatography using diethylaminoethyl (DEAE)-sepharose resin the total saline extract (S) from Taenia solium metacestodes and evaluated obtained fractions (DEAE S1 and DEAE S2) by enzyme-linked immunosorbent assay (ELISA, n=123) and immunoblotting (IB, n=22) to detect human NC in serum. Diagnostic parameters were established by ROC and TG ROC curves for ELISA tests. IB was qualitatively analyzed. S and DEAE S1 presented sensitivity of 87. 5 % and DEAE S2 90 %. The best specificity was observed for DEAE S2 (90.4 %). In IB, using DEAE S2 samples from NC patients presented bands of 20–25, 43–45, 55–50, 60–66, 82, 89, and 140 kDa. The great diagnostic parameters reached by DEAE S2 suggest the potential applicability of this fraction in NC immunodiagnosis. Keywords Taenia solium . Ion-exchange chromatography . Neurocysticercosis . Diagnosis
Introduction First recognized as a disease of pork in the ancient Greece, neurocysticercosis (NC) is now considered the most common helminthic disease of the central nervous system in humans (Del Brutto 2012). V. da S. Ribeiro : D. da S. Nunes : H. T. Gonzaga : J. P. da Cunha-Junior : J. M. Costa-Cruz (*) Departamento de Imunologia, Microbiologia e Parasitologia Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, Avenida Pará 1720, CEP 38400-902 Uberlândia, Minas Gerais, Brazil e-mail: [email protected]
The imaging techniques are useful for diagnosis but may overlook the infection when the number of parasites is low and/or the figures are not clear or are not typical. Moreover, these techniques are really not suitable for the diagnosis of NC in endemic areas because of the high cost, being available at major urban centers, whereas the principal population at risk is mostly rural (Rajkotia et al. 2007; Sako et al. 2013). Despite the advances in neuroimaging and immune diagnostic tests, the diagnosis of neurocysticercosis is a challenge in many patients because clinical manifestations are nonspecific; neuroimaging findings are most often not pathognomonic; and serological tests are faced with problems related to relatively poor specificity and sensitivity (Del Brutto 2012; Garcia et al 2012). Therefore, the development of an immunodiagnostic test that detects specific antibodies in either sera or cerebrospinal fluid (CSF) is necessary due to its simplicity and reliability especially when examining serum. Efforts have been directed towards identification and characterization of specific antigens (Sako et al. 2013). Although the immunological tests used for the diagnosis of cysticercosis have been considerably refined, attempts have been made to obtain more sensitive and specific antigens, since difficulties in the immunodiagnosis of NC have been attributed to the cross-reactivity between antigens from cysticerci and antibodies present in sera from patients with other parasitic diseases. Previous studies have shown that purified fractions had better diagnostic performance than total extracts (Silva et al. 2000; Machado et al. 2007; Ribeiro et al. 2010; Nunes et al. 2010; Nunes et al. 2013). The development of an inexpensive accurate serological assay could be of importance as a surveillance tool for diagnosis and seroepidemiology of NC. Considering this, in this study, we used ion-exchange chromatography to fractionate the total saline extract (S) from Taenia solium metacestodes producing potential useful fractions to be used in the immunodiagnosis of human neurocysticercosis.
2570
Material and methods Ethics statement This study was conducted according to the ethical guidelines of the Brazilian Health Ministry after being approved by the Research Ethics Committee of Universidade Federal de Uberlândia (UFU), state of Minas Gerais, Brazil. Antigen preparation Fresh T. solium metacestodes were collected from massively infected pigs. Metacestodes were carefully dissected from the host tissues, washed repeatedly, and stored at −20 °C. Total saline extract (S) from T. solium metacestodes was prepared as previously described (Costa et al. 1982). Serum samples Serum samples were collected from 123 subjects attended at the Laboratory of Clinical Analysis at the Clinical Hospital (groups 1 and 2) and from the Laboratory of Parasitology (group 3 (G3)) of the Federal University of Uberlândia (UFU) in the state of Minas Gerais, Brazil, maintained in the Biological Samples Bank from the Laboratório de Diagnóstico de Parasitoses. Group 1 (G1) consisted of 40 patients with definitive diagnosis of NC, as follows: (a) all patients presented at least one type of clinical manifestation suggestive of NC such as: epilepsy, cephalea, dizziness, dementia, faintness, hydrocephalus, and no signs or symptoms of cysticercosis in other organs; (b) all patients came from or lived in an area where cysticercosis is endemic; and (c) they presented evidence of parasite in neuroimaging (Del Brutto, 2012). Group 2 (G2) consisted of 43 patients who harbored other parasites: Ascaris lumbricoides (n=3), Echinococcus granulosus (n=3), Enterobius vermicularis (n=3), hookworm (n = 6), Hymenolepis nana (n = 4), Schistosoma mansoni (n=7), Strongyloides stercoralis (n=7), Taenia sp. (n=6), and Trichuris trichiura (n=4). Group 3 consisted of 40 individuals from an endemic area for cysticercosis, as patients from G2. All volunteers from group 3 did not present evidence of household contact with T. solium infection or a history of taeniasis or cysticercosis; they had three fecal samples tested negative by conventional methods of Baermann (1917) and Lutz (1919). Ion-exchange chromatography Fractions from total saline extract (S) were obtained by ionexchange chromatography developed in microtubes according to Gonzaga et al. (2013) as follows. Briefly, 1,200 μg of S was loaded onto a 200 μl diethylaminoethyl (DEAE)-sepharose resin (GE Healthcare Life Sciences), previously equilibrated
Parasitol Res (2014) 113:2569–2575
with 10 volumes of phosphate-buffered saline (PBS) by three cycles of centrifugation (2,000×g, 2 min). The suspension S/DEAE-resin was maintained under gently mixing for 20 min at 4 °C. After, the suspension was centrifuged as described and the supernatant was recovered and considered as nonbinding 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 analyzed for protein content according to Lowry et al. (1951) and by 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions, as described by Laemmli (1970). The proteins were visualized in SDS-PAGE by a silver staining (Friedman 1982). IgG detection ELISA using total S and DEAE S1 and DEAE S2 fractions from T. solium metacestodes was carried out according to Ribeiro et al. (2010), with modifications. Preliminary experiments were done out to determine the optimal conditions for ELISA through block titrations of reagents (antigens, sera, and conjugate). Briefly, polystyrene microplates (Interlab, São Paulo, Brazil) were used for a final assay volume of 50 μL/ well. Between each step of the reaction, microplates were washed three times, for 5 min each time, with PBS containing 0.05 % Tween 20 (PBS-T). For coating, S, DEAE S1, and DEAE S2 were diluted 10 μg/mL in 0.06 mol/L carbonate– bicarbonate buffer, pH 9.6, and incubated overnight at 4 °C. Serum samples diluted 1:200 were incubated for 45 min at 37 °C. The immunoenzymatic conjugate (peroxidase-goat antihuman IgG, Fc specific; Sigma) was diluted 1:2,000 in PBS-T and incubated for 45 min at 37 °C. The assay was developed by adding the enzymatic substrate consisting of H2O2 and o-phenylenediamine in 0.1 mol/L citrate phosphate buffer, pH 5.5, for 15 min followed by 25 μL/well of 2 N H2SO4 to stop the reaction. Optical densities (OD) were determined at 492 nm in an ELISA reader (TitertekPlus; Flow Laboratories, McLeanVA). ELISA reactivity index (RI) was obtained by the ratio between OD and cutoff. Values of RI greater than the optimum point of reaction for each extract were considered positive (RI>1). Immunoblot assay The immunoblot assay (IB) was performed according to Nunes et al. (2010). Antigen preparations were subjected to electrophoresis as previously and transferred to nitrocellulose membranes (0.20 μm; Bio-Rad) using a Trans-Blot SD semidry electrophoretic transfer cell (Bio-Rad, Hercules, California, USA). Nitrocellulose strips containing antigen preparations were blocked with 5 % nonfat milk in PBS-T (PBS-TM) for 2 h at room temperature and incubated overnight with
Parasitol Res (2014) 113:2569–2575
serum samples diluted 1:50 in 1 % PBS-TM at 4 °C. After six washes with 1 % PBS-TM, the strips were incubated for 2 h at room temperature with peroxidase-labeled goat IgG antihuman IgG conjugate, whole molecule (Sigma), diluted 1:1,500 in 1 % PBS-TM. The strips were washed in PBS and developed for 3 min in substrate solution containing hydrogen peroxide and 3,3-diaminobenzidine tetrahydrochloride (DAB-Sigma). The reaction was stopped by washing the strips with distilled water, and positive reactions were determined by the appearance of clearly defined bands. The relative molecular weight of the recognized bands were determined by comparison with a protein standard marker (Real Biotech, RECOMTM Blue Wide Range Prestain Marker, Banqiao, Taiwan). Analysis was performed using a graphical method to generate protein lane profile plot of each antigenic fraction, by Image J version 1.44 software (National Institutes of Health, Bethesda, USA). Statistical analyses of ELISA data Analyses were performed using the GraphPad software package 5.0 (GraphPad Software Inc., San Diego, USA). Optimum point for each condition of ELISA reaction and cutoff points were established using a two-graph receiver operating characteristic curve (TG-ROC) (Greiner et al. 1995) based on ELISA results from patients positive for NC (positive controls) and individuals from the other two groups (other parasites and healthy). Sensitivity (Se) and specificity (Sp) were calculated accordingly to the formulas: Se=a/(a+b) and Sp=d/(c+d), where a=true positive, b=false positive, c=false negative, and d=true negative (Youden 1950). Receiver operating characteristic curves (ROC) were built to describe the tests (Martinez et al. 2003). The area under ROC curve (AUC) Fig. 1 Detection of immunoglobulin G antibodies anti-T. solium metacestodes in serum samples from patients with a definitive diagnosis of neurocysticercosis (NC) (G1; n= 40), other parasites (G2; n=43), and apparently healthy individuals (G3; n=40) by enzyme-linked immunosorbent assay using the total saline extract (S), DEAE S1, and DEAE S2 fractions from T. solium metacestodes. Dotted line indicates the cutoff (reactivity index, RI=1); horizontal bars indicates median
2571
was calculated, and values close to 1 indicate an informative test and close to 0.5 indicate an uninformative test (Hanley and McNeil, 1982). AUC comparison between antigenic preparations was made using the method described previously (Hanley and McNeil; 1983). Likelihood ratio (LR), a parameter independent of prevalence (Greiner et al. 1995), was calculated, as follows: LR=Se/(1−Sp). Probability (P) values of
ORIGINAL PAPER
Diethylaminoethyl (DEAE) binding fraction from Taenia solium metacestode improves the neurocysticercosis serodiagnosis Vanessa da S. Ribeiro & Daniela da S. Nunes & Henrique T. Gonzaga & Jair P. da Cunha-Junior & Julia M. Costa-Cruz
Received: 16 January 2014 / Accepted: 9 April 2014 / Published online: 29 April 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Neurocysticercosis (NC) is one of the most important diseases caused by parasites affecting the central nervous system. We fractionated by ion-exchange chromatography using diethylaminoethyl (DEAE)-sepharose resin the total saline extract (S) from Taenia solium metacestodes and evaluated obtained fractions (DEAE S1 and DEAE S2) by enzyme-linked immunosorbent assay (ELISA, n=123) and immunoblotting (IB, n=22) to detect human NC in serum. Diagnostic parameters were established by ROC and TG ROC curves for ELISA tests. IB was qualitatively analyzed. S and DEAE S1 presented sensitivity of 87. 5 % and DEAE S2 90 %. The best specificity was observed for DEAE S2 (90.4 %). In IB, using DEAE S2 samples from NC patients presented bands of 20–25, 43–45, 55–50, 60–66, 82, 89, and 140 kDa. The great diagnostic parameters reached by DEAE S2 suggest the potential applicability of this fraction in NC immunodiagnosis. Keywords Taenia solium . Ion-exchange chromatography . Neurocysticercosis . Diagnosis
Introduction First recognized as a disease of pork in the ancient Greece, neurocysticercosis (NC) is now considered the most common helminthic disease of the central nervous system in humans (Del Brutto 2012). V. da S. Ribeiro : D. da S. Nunes : H. T. Gonzaga : J. P. da Cunha-Junior : J. M. Costa-Cruz (*) Departamento de Imunologia, Microbiologia e Parasitologia Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, Avenida Pará 1720, CEP 38400-902 Uberlândia, Minas Gerais, Brazil e-mail: [email protected]
The imaging techniques are useful for diagnosis but may overlook the infection when the number of parasites is low and/or the figures are not clear or are not typical. Moreover, these techniques are really not suitable for the diagnosis of NC in endemic areas because of the high cost, being available at major urban centers, whereas the principal population at risk is mostly rural (Rajkotia et al. 2007; Sako et al. 2013). Despite the advances in neuroimaging and immune diagnostic tests, the diagnosis of neurocysticercosis is a challenge in many patients because clinical manifestations are nonspecific; neuroimaging findings are most often not pathognomonic; and serological tests are faced with problems related to relatively poor specificity and sensitivity (Del Brutto 2012; Garcia et al 2012). Therefore, the development of an immunodiagnostic test that detects specific antibodies in either sera or cerebrospinal fluid (CSF) is necessary due to its simplicity and reliability especially when examining serum. Efforts have been directed towards identification and characterization of specific antigens (Sako et al. 2013). Although the immunological tests used for the diagnosis of cysticercosis have been considerably refined, attempts have been made to obtain more sensitive and specific antigens, since difficulties in the immunodiagnosis of NC have been attributed to the cross-reactivity between antigens from cysticerci and antibodies present in sera from patients with other parasitic diseases. Previous studies have shown that purified fractions had better diagnostic performance than total extracts (Silva et al. 2000; Machado et al. 2007; Ribeiro et al. 2010; Nunes et al. 2010; Nunes et al. 2013). The development of an inexpensive accurate serological assay could be of importance as a surveillance tool for diagnosis and seroepidemiology of NC. Considering this, in this study, we used ion-exchange chromatography to fractionate the total saline extract (S) from Taenia solium metacestodes producing potential useful fractions to be used in the immunodiagnosis of human neurocysticercosis.
2570
Material and methods Ethics statement This study was conducted according to the ethical guidelines of the Brazilian Health Ministry after being approved by the Research Ethics Committee of Universidade Federal de Uberlândia (UFU), state of Minas Gerais, Brazil. Antigen preparation Fresh T. solium metacestodes were collected from massively infected pigs. Metacestodes were carefully dissected from the host tissues, washed repeatedly, and stored at −20 °C. Total saline extract (S) from T. solium metacestodes was prepared as previously described (Costa et al. 1982). Serum samples Serum samples were collected from 123 subjects attended at the Laboratory of Clinical Analysis at the Clinical Hospital (groups 1 and 2) and from the Laboratory of Parasitology (group 3 (G3)) of the Federal University of Uberlândia (UFU) in the state of Minas Gerais, Brazil, maintained in the Biological Samples Bank from the Laboratório de Diagnóstico de Parasitoses. Group 1 (G1) consisted of 40 patients with definitive diagnosis of NC, as follows: (a) all patients presented at least one type of clinical manifestation suggestive of NC such as: epilepsy, cephalea, dizziness, dementia, faintness, hydrocephalus, and no signs or symptoms of cysticercosis in other organs; (b) all patients came from or lived in an area where cysticercosis is endemic; and (c) they presented evidence of parasite in neuroimaging (Del Brutto, 2012). Group 2 (G2) consisted of 43 patients who harbored other parasites: Ascaris lumbricoides (n=3), Echinococcus granulosus (n=3), Enterobius vermicularis (n=3), hookworm (n = 6), Hymenolepis nana (n = 4), Schistosoma mansoni (n=7), Strongyloides stercoralis (n=7), Taenia sp. (n=6), and Trichuris trichiura (n=4). Group 3 consisted of 40 individuals from an endemic area for cysticercosis, as patients from G2. All volunteers from group 3 did not present evidence of household contact with T. solium infection or a history of taeniasis or cysticercosis; they had three fecal samples tested negative by conventional methods of Baermann (1917) and Lutz (1919). Ion-exchange chromatography Fractions from total saline extract (S) were obtained by ionexchange chromatography developed in microtubes according to Gonzaga et al. (2013) as follows. Briefly, 1,200 μg of S was loaded onto a 200 μl diethylaminoethyl (DEAE)-sepharose resin (GE Healthcare Life Sciences), previously equilibrated
Parasitol Res (2014) 113:2569–2575
with 10 volumes of phosphate-buffered saline (PBS) by three cycles of centrifugation (2,000×g, 2 min). The suspension S/DEAE-resin was maintained under gently mixing for 20 min at 4 °C. After, the suspension was centrifuged as described and the supernatant was recovered and considered as nonbinding 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 analyzed for protein content according to Lowry et al. (1951) and by 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions, as described by Laemmli (1970). The proteins were visualized in SDS-PAGE by a silver staining (Friedman 1982). IgG detection ELISA using total S and DEAE S1 and DEAE S2 fractions from T. solium metacestodes was carried out according to Ribeiro et al. (2010), with modifications. Preliminary experiments were done out to determine the optimal conditions for ELISA through block titrations of reagents (antigens, sera, and conjugate). Briefly, polystyrene microplates (Interlab, São Paulo, Brazil) were used for a final assay volume of 50 μL/ well. Between each step of the reaction, microplates were washed three times, for 5 min each time, with PBS containing 0.05 % Tween 20 (PBS-T). For coating, S, DEAE S1, and DEAE S2 were diluted 10 μg/mL in 0.06 mol/L carbonate– bicarbonate buffer, pH 9.6, and incubated overnight at 4 °C. Serum samples diluted 1:200 were incubated for 45 min at 37 °C. The immunoenzymatic conjugate (peroxidase-goat antihuman IgG, Fc specific; Sigma) was diluted 1:2,000 in PBS-T and incubated for 45 min at 37 °C. The assay was developed by adding the enzymatic substrate consisting of H2O2 and o-phenylenediamine in 0.1 mol/L citrate phosphate buffer, pH 5.5, for 15 min followed by 25 μL/well of 2 N H2SO4 to stop the reaction. Optical densities (OD) were determined at 492 nm in an ELISA reader (TitertekPlus; Flow Laboratories, McLeanVA). ELISA reactivity index (RI) was obtained by the ratio between OD and cutoff. Values of RI greater than the optimum point of reaction for each extract were considered positive (RI>1). Immunoblot assay The immunoblot assay (IB) was performed according to Nunes et al. (2010). Antigen preparations were subjected to electrophoresis as previously and transferred to nitrocellulose membranes (0.20 μm; Bio-Rad) using a Trans-Blot SD semidry electrophoretic transfer cell (Bio-Rad, Hercules, California, USA). Nitrocellulose strips containing antigen preparations were blocked with 5 % nonfat milk in PBS-T (PBS-TM) for 2 h at room temperature and incubated overnight with
Parasitol Res (2014) 113:2569–2575
serum samples diluted 1:50 in 1 % PBS-TM at 4 °C. After six washes with 1 % PBS-TM, the strips were incubated for 2 h at room temperature with peroxidase-labeled goat IgG antihuman IgG conjugate, whole molecule (Sigma), diluted 1:1,500 in 1 % PBS-TM. The strips were washed in PBS and developed for 3 min in substrate solution containing hydrogen peroxide and 3,3-diaminobenzidine tetrahydrochloride (DAB-Sigma). The reaction was stopped by washing the strips with distilled water, and positive reactions were determined by the appearance of clearly defined bands. The relative molecular weight of the recognized bands were determined by comparison with a protein standard marker (Real Biotech, RECOMTM Blue Wide Range Prestain Marker, Banqiao, Taiwan). Analysis was performed using a graphical method to generate protein lane profile plot of each antigenic fraction, by Image J version 1.44 software (National Institutes of Health, Bethesda, USA). Statistical analyses of ELISA data Analyses were performed using the GraphPad software package 5.0 (GraphPad Software Inc., San Diego, USA). Optimum point for each condition of ELISA reaction and cutoff points were established using a two-graph receiver operating characteristic curve (TG-ROC) (Greiner et al. 1995) based on ELISA results from patients positive for NC (positive controls) and individuals from the other two groups (other parasites and healthy). Sensitivity (Se) and specificity (Sp) were calculated accordingly to the formulas: Se=a/(a+b) and Sp=d/(c+d), where a=true positive, b=false positive, c=false negative, and d=true negative (Youden 1950). Receiver operating characteristic curves (ROC) were built to describe the tests (Martinez et al. 2003). The area under ROC curve (AUC) Fig. 1 Detection of immunoglobulin G antibodies anti-T. solium metacestodes in serum samples from patients with a definitive diagnosis of neurocysticercosis (NC) (G1; n= 40), other parasites (G2; n=43), and apparently healthy individuals (G3; n=40) by enzyme-linked immunosorbent assay using the total saline extract (S), DEAE S1, and DEAE S2 fractions from T. solium metacestodes. Dotted line indicates the cutoff (reactivity index, RI=1); horizontal bars indicates median
2571
was calculated, and values close to 1 indicate an informative test and close to 0.5 indicate an uninformative test (Hanley and McNeil, 1982). AUC comparison between antigenic preparations was made using the method described previously (Hanley and McNeil; 1983). Likelihood ratio (LR), a parameter independent of prevalence (Greiner et al. 1995), was calculated, as follows: LR=Se/(1−Sp). Probability (P) values of
