Cysticercus Antigens in Cerebrospinal Fluid

8/13/2019 Cysticercus Antigens in Cerebrospinal Fluid

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10.1128/JCM.39.9.3368-3372.2001.2001, 39(9):3368. DOI:J. Clin. Microbiol.

Machado and Jos Antnio LivramentoAlessandra Xavier Pardini, Adelaide Jos Vaz, Luis Dos Ramos

NeurocysticercosisSamples from Patients withCysticercus Antigens in Cerebrospinal Fluid

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JOURNAL OFCLINICALMICROBIOLOGY,0095-1137/01/$04.000 DOI: 10.1128/JCM.39.9.33683372.2001

Sept. 2001, p. 33683372 Vol. 39, No. 9

Copyright 2001, American Society for Microbiology. All Rights Reserved.

Cysticercus Antigens in Cerebrospinal Fluid Samples fromPatients with Neurocysticercosis

ALESSANDRA XAVIER PARDINI,1 ADELAIDE JOSE VAZ,1* LUIS DOS RAMOS MACHADO,2

ANDJOSE ANTONIO LIVRAMENTO2

Laboratory of Clinical Immunology, Faculty of Pharmaceutical Sciences, 05508-900 Sao Paulo,1 and Center ofNeurological Investigation, Faculty of Medicine, University of Sao Paulo, 01246-903 Sao Paulo,2 SP, Brazil

Received 27 January 2001/Returned for modification 3 April 2001/Accepted 6 July 2001

Antigens were detected in cerebrospinal fluid (CSF) samples from patients with neurocysticercosis (NC) byenzyme-linked immunosorbent assay (ELISA) using polyclonal sera of rabbit anti- Taenia solium cysticerci(anti-Tso) and anti- Taenia crassiceps cysticerci vesicular fluid (anti-Tcra or anti-Tcra


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pH was adjusted to 4.5. Caprylic acid (25 l/ml) was slowlyadded dropwise with thorough mixing, and the solution wascentrifuged at 10,000 g for 30 min. The supernantant wasfiltered and mixed with 1/10 volume of 10-concentratedphosphate-buffered saline (PBS); and the pH was adjusted to7.4. The supernatant was cooled to 4C and fractionated withammonium sulfate (0.277 g/ml), and the sample was stirred for30 min before the precipitated IgG was collected by centrifu-gation at 5,000 gfor 15 min. The IgG pellet was resuspendedin PBS and dialyzed against PBS.

Samples.The protocol was approved by the Ethics Commit-tee for the Analysis of Research Projects of the Clinical Di-rectors Office of the Hospital (approval no. 072/97). All of the

patients in the NC group had a diagnosis of NC on the basis ofthe criteria of the General NC Investigation Protocol of theHospital of the Faculty of Medicine, University of Sao Paulo.A total of 104 CSF samples from patients with a diagnosis ofNC were analyzed. For 40 patients, it was possible to obtainresults of imaging exams, with 27 of them being classified as theactive form (cysts associated with an inflammatory process)and 13 being classified as the inactive form (nodular calcifica-tions). All of these 40 patients had been clinically followed upfor periods of time ranging from 2 to 10 years. Seventy CSFsamples were obtained from patients in the control group witha negative clinical laboratory diagnosis of NC.

ELISA.Plates with 96 wells (Nunc) were sensitized with 50l of CSF plus 50 l of 0.02 M carbonate-bicarbonate buffer,pH 9.6, for 18 h in a humidified chamber at 4C. The plateswere blocked with 5% skim milk (Molico skim milk; Nestle,Aracatuba, Sao Paulo, Brazil) in 0.01 M PBS (0.0075 MNa

2HPO

4, 0.025 M NaH

2PO

4, 0.14 M NaCl, pH 7.2) contain-

ing 0.05% Tween 20 (Merck, Schudart, Munich, Germany)(PBS-T). The ideal immune serum and conjugate concentra-tions were obtained by titration. We diluted control (nonim-mune rabbit) serum to 1:100, anti-Tcra 30 kDa serum to 1:50,anti-Tcra serum to 1:500, and anti-Tso serum to 1:100 andadded peroxidase-labeled rabbit anti-IgG (Sigma ChemicalCo.). The enzymatic reaction was developed with the chromo-

genic substrate tetramethylbenzidine and hydrogen peroxide(Bio-Rad Laboratories, Inc., Hercules, Calif.) for 20 min in thedark and blocked with 4 N sulfuric acid. Labeling intensity wasquantified with a plate reader at 450 nm (Diagnostics Pasteur,Strasburg-Schiltigheim, France). The absorbance (optical den-sity [OD]) obtained for each test was subtracted from thecontrol (nonimmune rabbit) reading. All incubations were car-ried out at 37C for 1 h, except for the blocking step, which wascarried out for 2 h. Between the sample, conjugate, and sub-strate incubation steps, the plates were washed in an automaticwasher with four cycles of saline solution containing 0.05%Tween. All plates contained a control with T-Tso and VF-Tcra

FIG. 1. ELISA results, expressed as ODs, for the detection of antigens in 104 CSF samples from the NC group and 70 from the control groupassayed with anti-T. soliumcysticercus (Tso), anti-T. crassicepscysticercus (Tcra) and anti-T. crassiceps 30 kDa (Tcra30) sera. The cutoff pointsfor the reactions are shown as horizontal lines, and the numbers of samples assayed are shown at the bottom.

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antigens (0.001g). The cutoff was determined based on theanalysis of the results of the control group. Samples presentingan OD equal to or higher than the cutoff OD were consideredto be positive.

Immunoblotting.For the initial characterization of the pep-tides detected in the CSF samples (selected from the availablevolume), the samples were treated with 15 mM sample bufferconcentratedfive fold with dithiothreitol (Amersham Pharma-cia Biotech, Piscataway, N.J.), submitted to sodium dodecyl-sulfate15% polyacrylamide gel electrophoresis (19), and

then transferred to a 0.1-m polyvinylidene difluoride mem-brane (Millipore Corp., Bedford, Mass.) in an electrophoreticcuvette (TE42 Transphor Unit; Amersham Pharmacia Bio-tech) for 16 to 18 h at 4C. The membranes were cut into 3-mmstrips, washed three times with PBS-T for 10 min, and blockedwith 5% skim milk in PBS-T (diluting solution). The controlserum and immune sera were added to the strips and incubatedovernight at 4C under constant shaking. Alkaline phospha-tase-labeled rabbit anti-IgG conjugate (Bio-Rad Laboratories,Inc.) was added, and the strips were incubated for 2 h underconstant shaking. The enzymatic reaction was developed with5-bromo-4-chloro-3-indolylphosphatenitroblue tetrazolium(Sigma Chemical Co.), and the strips were then washed indistilled water. All incubations were carried out for 2 h at 25 C.

Between the sample, conjugate, and substrate incubation steps,the strips were washed three times with PBS-T or with distilledwater (final block) for 10 min.

The ELISA results obtained for the 104 CSF samples fromthe NC group and the 70 samples from the control group areshown in Fig. 1. Fifteen (18.75%) of the samples from the NCgroup did not react with anti-Tso serum, while 9 (18%) of thecontrol samples showed reactivity. Eight (10%) of the samplesfrom the NC group were not reactive with anti-Tcra serum,while one control sample was. One (4.2%) of the samples fromthe NC group did not react with anti-Tcra 30 kDa serum, andno sample from the control group showed reactivity. ELISAs

carried out with the anti-Tso, anti-Tcra, and anti-Tcra 30kDa antigens showed sensitivities of 81.2, 90, and 95.8% andspecificities of 82, 98, and 100%, respectively. No differencebetween anti-Tso and anti-Tcra sera was observed in the 40-sample group with image diagnosis. Antigens were detected in100% of the samples from patients with the active from byusing the two sera; antigens were detected in 76.9% of theinactive-form samples assayed with anti-Tso serum and in92.3% of the samples assayed with anti-Tcra serum.

Two reactive samples from the NC group and two from thecontrol group were assayed by immunoblotting for antigencharacterization. The 14- and 18-kDa peptides were only iden-tified in samples from the NC group, while the 34-kDa proteinwas considered nonspecific since it was also identified in con-trol samples (Fig. 2).

The sera used in the present study for the detection ofantigens in CSF samples from patients with NC were found tobe efficient. The hyperimmune sera obtained from the heter-ologousT. crassicepsantigen showed the highest sensitivity andspecificity in ELISA, reaching sensitivities of 81.2, 90, and

95.8% and specificities of 82, 98, and 100% for the anti-Tso,anti-Tcra, and anti-Tcra 30 kDa sera, respectively, with91.5% concordance. The cutoff points (T-Tso, 0.68; VF-Tcra,0.48; Tcra 30 kDa, 0.81) were chosen in order to obtainhigher specificity than sensitivity. This high background may bedue to nonspecific binding of the conjugate to the microplates(Maxisorp) or to minimal cross-reactivity with adsorbed hu-man IgG from the samples.

Other authors, using anti-T. saginatamonoclonal antibodies,detected antigens in CSF samples (4, 7) and in sera fromhumans and infected cattle (1, 9).

Anti-Tso sera have been used for the detection of antigens inCSF samples from patients with NC. Tellez-Giron et al. (29)using dot-ELISA and ELISA, showed that 59 and 77%, re-

spectively, of 17 CSF samples from patients with NC containedantigens. Velasco-Castrejon et al. (33) detected T. solium an-tigens in 88% of 215 CSF samples from patients with NC byagglutination of latex particles adsorbed to anti-vesicular fluid,anti-excretion-secretion, and anti-total T. solium cysticercusextract immunoglobulins.

In the present study, the 34-kDa peptide was considered tobe nonspecific since it was also identified in control samples(Fig. 2). Low-molecular-mass peptides (20 kDa) have beenidentified by antibodies in samples from patients with cystic-ercosis (15, 17, 26, 27, 30). Our group has recently reportedthat the 14- and 18-kDa peptides are responsible for the cross-reactivity between the T. soliumand T. crassiceps species (10)and are specific for antibody detection in serum and CSF

samples from patients with cysticercosis (2). In the presentstudy, these peptides were strongly recognized in the two CSFsamples using anti-Tso and anti-Tcra sera in immunoblots,suggesting that they may interact more intensely with the host,possibly representing excretion and secretion products re-leased into CSF during the different phases of the parasiticevolution of NC (active and inactive forms).

In contrast to the present results, other investigators de-tected high-molecular-mass peptides by using anti-Tso serum.Tellez-Giron et al. (29) characterized a circulating antigen of66 kDa in CSF samples. Estrada et al. (12) identified twoantigens of 190 and 230 kDa in 14 of 18 CSF samples from

FIG. 2. Immunoblots of T-Tso (A) and VF-Tcra (B) antigens as-sayed with negative control (lanes 1), anti-T. soliumcysticercus(lanes2), and anti-T. crassicepscysticercus (lanes 3) sera. Two CSF samplesfrom the NC group (lanes 4 and 5) and two control CSF samples (lanes6 and 7) were assayed with anti-T. soliumcysticercus (C) and anti-T.crassicepscysticercus (D) sera. Molecular size standards (94, 67, 43, 20,and 14 kDa) are shown, and the arrows indicate the 14- and 18-kDa

peptides.

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patients with suspected NC and in the cysticercus vesicularfluid. Choromanski et al. (5) identified two antigens of 110 and400 kDa in CSF samples by high-performance liquid chro-matography.

Some authors have suggested that the detection of antibod-ies against low-molecular-mass peptides may be associatedwith the developmental phase of the parasite (6, 24, 28, 35),whereas Bueno et al. (2) did not find an association betweenantibody detection and the phase of the disease. The detectionof antigens in patients in different phases of the disease, asanalyzed in the present study, revealed practically the samereactivity with anti-Tso and anti-Tcra sera. It is important tonote that the difficulty in detecting larval antigens in CSF maybe related to their low concentration, antigen degradation, orthe release of a still unidentified antigen. Among the 40 sam-ples from patients with imaging results, antigens were identi-fied in 100% of the cases of the active form with anti-Tso andanti-Tcra sera, whereas 3 (23%) of the 13 cases of the inactiveform showed a negative result with anti-Tso serum and one(8%) of these samples was also negative with anti-Tcra serum.

Anti-Tcra serum was more sensitive for diagnostic purposeseven during the calcification phase (92%), and anti-Tso serumcould be used to distinguish the disease phase in a more ade-quate manner. It should be pointed out that the patients understudy had been followed up for periods of 2 to 10 years andthose in the calcification phase were in the initial part of thisprocess, which may last several years. Verification of the testwith a larger number of samples is required to determinewhether there is a correlation with the developmental phase ofthe parasite or with the hosts immune inflammatory process.

The sera used in the present study proved to be efficient forantigen identification in CSF samples from patients with NC,suggesting that antigen identification may contribute as anadditional marker to the study and understanding of the dis-

ease, in addition to being of help in the diagnosis and prognosisof cysticercosis.In future studies, analysis by immunoblotting using sensitive

systems for protein detection such as enhanced chemilumines-cence (Amersham Pharmacia Biotech) and monoclonal anti-bodies for various antigenic epitopes may define the peptidespresent during different phases of the infection.

This work was supported by FAPESP (grant 98/04473-9) and by aCNPq fellowship to A. X. Pardini.

We are indebted to Paulo Mutuko Nakamura for help in obtainingimmune sera.

REFERENCES

1. Brandt, J. R. A., S. Geerts, R. De Deken, V. Kumar, F. Ceulemans, L. Brijs,and N. Falla. 1992. A monoclonal antibody-based ELISA for the detection

of circulating excretory-secretory antigens in Taenia saginata cysticercosis.Int. J. Parasitol. 22:471477.

2. Bueno, E. C., A. J. Vaz, L. R. Machado, J. A. Livramento, and S. Mielle.2000. Specific Taenia crassiceps and Taenia solium antigenic peptides forneurocysticercosis immunodiagnosis using serum samples. J. Clin. Microbiol.38:146151.

3. Cantu, C., and F. Barinagarrementeria. 1996. Cerebrovascular complica-tions of neurocysticercosis clinical and neuroimaging spectrum. Arch. Neu-rol. 53:233239.

4. Chang-Yuan, W., Z. Hong-Hua, and G. Ling-Yun. 1992. A MAb-basedELISA for detection circulating antigen in CSF of patients with neurocys-ticercosis. Hybridoma 11:825827.

5. Choromanski, L., J. J. Estrada, and R. E. Kuhn. 1990. Detection of antigensof larvalTaenia soliumin the cerebrospinal fluid of patients with the use ofHPLC and ELISA. J. Parasitol. 76:6973.

6. Chung, J. Y., Y. Y. Bahk, S. Huh, S. Y. Kang, Y. Kong, and S. Y. Cho. 1999.

A recombinant 10-kDa protein of Taenia solium metacestodes specific toactive neurocysticercosis. J. Infect. Dis. 180:13071315.

7. Correa, M. D., A. Plancarte, M. A. Sandoval, E. Rodriguez-del-Rosal, A.Meza-Lucas, and A. Flisser. 1989. Immunodiagnosis of human and porcine

cysticercosis detection of antibodies and parasite products. Acta Leidensia57:9399.

8. Diaz, J. F., M. Verastegui, R. H. Gilman, V. C. W. Tsang, J. B. Pilcher, C.Gallo, H. H. Garcia, P. Torres, T. Montenegro, E. Miranda, and The Cys-

ticercosis Working Group in Peru.1992. Immunodiagnosis of human cystic-

ercosis (Taenia solium): a field comparison of antibody-enzyme-linked assay(ELISA) an antigen-ELISA, and enzyme-linked-immunoelectrotransfer blot(EITB) assay in Peru. Am. J. Trop. Med. Hyg. 46:610615.

9. Draelants, E., J. Brandt, V. Kumar, and S. Geerts. 1995. Characterization ofepitopes on excretory-secretory antigens of Taenia saginata metacestodesrecognized by monoclonal antibodies with immunodiagnostic potential. Par-asite Immunol. 17:119126.

10. Espindola, N. M., E. N. De Gaspari, P. M. Nakamura, and A. J. Vaz.2000.Cross-reactivity of anti-Taenia crassiceps cysticerci immune antibodies withTaenia solium antigens. Vet. Parasitol. 89:321326.

11. Espinoza, B., G. Ruiz-Palacios, A. Tovar, M. A. Sandoval, A. Plancarte, andA. Flisser. 1986. Characterization by enzyme-linked immunosorbent assay ofthe humoral immune response in patients with neurocysticercosis and itsapplication in immunodiagnosis. J. Clin. Microbiol. 24:536541.

12. Estrada, J. J., J. A. Estrada, and R. E. Kuhn. 1989. Identification ofTaeniasoliumantigens in cerebrospinalfluid and larval antigens from patients withneurocysticercosis. Am. J. Trop. Med. Hyg. 41:5055.

13. Feldman, M., A. Plancarte, M. Sandoval, M. Wilson, and A Flisser. 1990.Comparison of two assays (EIA and EITB) and two samples (serum and

saliva) for the diagnosis of neurocysticercosis. Trans. R. Soc. Trop. Med.Hyg.84:559562.

14. Freeman, R. S. 1962. Studies on the biology of Taenia crassiceps. Can. J.Zool. 40:969990.

15. Gottstein, B., D. Zini, and P. M. Schantz.1987. Species-specific immunodi-agnosis of Taeniasolium cysticercosisby ELISA and immunoblotting. Trop.Med. Parasitol. 38:299303.

16. Ito, A., A. Plancarte, L. Ma, Y. Kong, A. Flisser, S. Y. Cho, Y. H. Liu, S.Kamhawi, M. W. Lightowlers, and P. M. Schantz. Novel antigens for neu-rocysticercosis: simple method for preparation and evaluation for serodiag-nosis. Am. J. Trop. Med. Hyg. 59:291294.

17. Kaur, M., R. Goyal, N. K. Ganguly, R. C. Mahajan, and N. Malla. 1996.Evaluation and characterization of purified antigenic fraction-II of Cystic-ercus cellulosae by enzyme-linked immunosorbent assay for the diagnosis ofneurocysticercosis before and after treatment. Immunol. Infect. Dis. 6:2529.

18. Kunz, J., B. Kallina, V. Watschke, and E. Geyer. 1989. Taenia crassicepsmetacestode vesicularfluid antigens shared with Taenia solium larval stageand reactive with serum antibodies from patients with neurocysticercosis.Zentbl. Bakteriol. 271:510520.

19. Laemmli, U. K.1970. Cleavage of structural proteins during the assembly ofhead of bacteriophage T4. Nature 227:680685.

20. Lightowlers, and P. M. Schantz.1998. Novel antigens for neurocysticercosis:simple method for preparation and evaluation for serodiagnosis. Am. J.Trop. Med. Hyg. 59:291294.

21. Machado, L. R., J. P. S. Nobrega, N. G. Barros, J. A. Livramento, L. A.Bacheschi, and A. Spina-Franca.1990. Computed tomography in neurocys-ticercosis. A 10-long year evolution analysis of 100 patients with an appraisalof new classification. Arq. Neuro-Psiquiatr. 48:414418.

22. McKinney, M. M., and A. Parkinson.1987. A simple, non-chromatographicprocedure to purify immunoglobulins from serum and ascites fluid. J. Im-munol. Methods 96:271278.

23. McManus, D. P. 1990. Molecular technology: improving strategies for con-trolling hydatid diseases and cysticercosis. Southeast Asian J. Trop. Med.Public Health 21:161173.

24. Michault, A., B. Riviere, P. Fressy, J. P. Laporte, G. Bertil, and C. Mignardo.1990. Apport de lenzyme-linked immunoelectrotransfer blot assay au diag-nostic de la neurocysticercosis humaine. Pathol. Biol. 38:119125.

25. Pammenter, M. D., and E. J. Rossouw. 1987. The value of an antigenic

fraction of Cysticercus cellulosae in the serodiagnosis of cysticercosis. Ann.Trop. Med. Parasitol. 81:117123.

26. Rodriguez-Canul, R., J. C. Allan, C. Fletes, I. P. Sutisna, I. N. Kapti, andP. S. Craig. 1997. Comparative evaluation of purified Taenia solium glyco-proteins and crude metacestode extracts by immunoblotting for the serodi-agnosis of humanT. solium cysticercosis. Clin. Diagn. Lab. Immunol. 4:579582.

27. Rodriguez-Canul, R., J. C. Allan, J. L. Dominguez, S. Villegas, L. Cob, R. I.Rodriguez, A. J. Cook, J. Willians, F. Argaez, and P. S. Craig.1998. Appli-cation of immunoassay to determine risk factors associated with porcinecysticercosis in rural areas of Yucatan, Mexico. Vet. Parasitol. 79:165180.

28. Simac, C., P. Michel, A. Andriantsimahavandy, P. Esterre, and A. Michault.1995. Use of enzyme-linked immunosorbent assay and enzyme-linked im-munoelectrotransfer blot for the diagnosis and monitoring of neurocysticer-cosis. Parasitol. Res. 81:132136.

29. Tellez-Giron, E., M. C. Ramos, P. Alvarez, L. Dufour, and M. Montante.

VOL. 39, 2001 NOTES 3371


6/6

1989. Detection and characterization of antigens from Taenia soliumcystic-ercus in cerebrospinal fluid. Acta Leidensia 57:101105.

30. Tsang, V. G. W., J. A. Brand, and A. E. Boyer. 1989. An enzyme-linkedimmunoelectrotransfer blot assay and glycoprotein antigens for diagnosis ofhuman cysticercosis (Taenia solium). J. Infect. Dis. 159:5059.

31. Valdez, F., M. Hernandez, T. Govezenky, G. Fragoso, and E. Sciutto. 1994.Immunization against Taenia crassiceps cysticercosis: identification of themost promising antigens in the induction of protective immunity. J. Parasitol.80:931936.

32. Vaz, A. J., C. M. Nunes, R. M. Piazza, J. A. Livramento, M. V. Silva, P. M.Nakamura, and A. W. Ferreira. 1997. Immunoblot with cerebrospinalfluid

from patients with neurocysticercosis using antigen from cysticerci ofTaeniasoliumand Taenia crassiceps. Am. J. Trop. Med. Hyg. 57:354357.

33. Velasco-Castrejon, O., M. Gutierrez-Quiroz, V. Romero, and C. Guzman-Bracho. 1989. Detection de antigenos solubles de Cysticercus cellulosae enliquido cefalorraqudeo como medio diagnostico en neurocisticercosis. Rev.Latinoam. Microbiol. 31:235239.

34. White, A. C., Jr. 1997. Neurocysticercosis: a major cause of neurologicaldisease worldwide. Clin. Infect. Dis. 24:101115.

35. Yang, H.-J., J. Y. Chung, D. H. Yun, Y. Kong, A. Ito, L. Ma, Y. H. Liu, S. C.

Lee, and S. Y. Kang. 1998. Immunoblot analysis of a 10kDa antigen in cystfluid ofTaenia soliummetacestodes. Parasite Immunol. 20:483488.

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Cysticercus Antigens in Cerebrospinal Fluid