Antibody specific to 43 kDa excretory–secretory antigenic peptide of Taenia solium metacestode as a potential diagnostic marker in human neurocysticercosis

Acta Tropica 115 (2010) 257–261

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Antibody specific to 43 kDa excretory–secretory antigenic peptide of Taenia solium metacestode as a potential diagnostic marker in human neurocysticercosis Priyadarshi Soumyaranjan Sahu a,c,∗ , Subhash Chandra Parija a , Seetharaman Jayachandran b a

Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry 605006, India Department of Biotechnology, School of Life Sciences, Pondicherry University, Kalapet, Pondicherry 605014, India c South Texas Centre for Emerging Infectious Diseases, University of Texas at San Antonio, San Antonio, TX 78249, USA b

a r t i c l e

i n f o

Article history: Received 4 September 2009 Received in revised form 20 March 2010 Accepted 8 April 2010 Available online 23 April 2010 Keywords: Taenia solium Neurocysticercosis EITB Excretory–secretory Antigen

a b s t r a c t Recent studies suggest excretory–secretory (ES) antigen specific antibody detection tests to be of promising utility in laboratory diagnosis of many parasitic diseases in human including neurocysticercosis (NCC). The objective of the present study was to characterize the ES antigens collected from in vitro culture of Taenia solium metacestode larvae, and to identify specific ES peptides as diagnostic markers. Three ES peptides viz., 67 kDa, 43 kDa and 32 kDa, were found to be diagnostic for NCC based on high sensitivity and specificity of their reactivity to either serum or cerebrospinal fluid (CSF) specimens. More remarkably, the 43 kDa ES peptide was found reactive with CSF and serum specimens from confirmed NCC patients with absolute specificity and a high sensitivity (88.23% in serum and 89.28% in CSF). This peptide was also detected by sera and CSF from clinically suspected NCC patients but with a decreased sensitivity correlating with the decreasing order of the certainty of diagnosis as per a criteria proposed earlier. The 43 kDa ES peptide is suggested to be an important peptide of diagnostic utility in NCC. Published by Elsevier B.V.

1. Introduction Neurocysticercosis (NCC) in human is a major public health problem, and it is identified as the single most common cause of community acquired active epilepsy in the developing world including Indian subcontinent and Latin American countries. In a recent report it is suggested that Taenia solium cysticercosis disease burden in India surpasses many other developing countries (Prasad et al., 2008). The diagnosis of NCC is given by the combined analysis of clinical data, neuro imaging features, immunological test reports and epidemiological information (Garcia et al., 2005). The majority of NCC cases do not always show typical neuro imaging features with invaginated scolex. Moreover, in developing countries especially in epidemiological studies neuro imaging methods are not suitable due to cost and time (Flisser et al., 2003). So, serology using specific antigens of the parasite, either semi-purified native or recombinant antigens, is essential for confirming the diagnosis. The test of choice for serological diagnosis of human cysticercosis is by enzyme-linked immuno electro transfer blot (EITB), a

∗ Corresponding author at: South Texas Centre for Emerging Infectious Diseases, 1.204 MBT Building, University of Texas at San Antonio, San Antonio, TX 78249, USA. Tel.: +1 210 458 7032/7025; fax: +1 210 458 7025. E-mail address: priyadarshi [email protected] (P.S. Sahu). 0001-706X/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.actatropica.2010.04.006

Western blot assay that relies on the use of seven lentil lectin purified glyco proteins derived from T. solium metacestodes and it has been approved by The Centres for Disease Control and Prevention (CDC). This EITB test has a sensitivity of 98% and a specificity of 100% in diagnosis of cysticercosis (Tsang et al., 1989). Several investigators have reported the usefulness of this EITB assay for diagnosis of NCC (Zini et al., 1990; Aguilar-Rebolledo et al., 2002; ProanoNarvaez et al., 2002; Kojic and White, 2003; Villota et al., 2003; Hancock et al., 2004). Yet the high cost with unavailability of the CDC recommended EITB diagnostic material and even the laborintensive process of metacestode glycoprotein purification hinder its practicality to be used in average laboratories in developing countries (Flisser et al., 2003). The excretory–secretory (ES) antigens collected from the in vitro culture of T. solium metacestode larvae are found to be an important homologous source of antigen for demonstration of serum or CSF antibodies in diagnosis of NCC as reported previously (Sahu et al., 2009). There are also a couple of other studies which show the usefulness of the parasite ES antigens in diagnosis of cysticercosis in humans (Molinari et al., 2002; Lopez et al., 2004; Atluri et al., 2009). The aim of the present study was to characterize the ES antigens collected from in vitro culture of T. solium metacestodes and to identify immunologically reactive and specific ES peptides to be used as diagnostic markers in human cysticercosis.

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2. Patients and methods 2.1. Patients and study groups Serum and cerebrospinal fluid (CSF) samples from patients with NCC were examined to determine the frequency of distribution of reactive ES peptides. We also determined whether there were cross-reacting serum antibodies to these ES peptides in case of patients infected with other related helminthic infections (viz., Echinococcus granulosus, and Hymenolepis nana). Study protocol was reviewed and approved by the Institutional Ethical Committee at the Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry (India). Patients provided voluntary consent and signed written consent forms. A presumptive diagnosis of NCC was made based on the clinical presentation, physical examination, history and imaging features. A total of 160 cases were identified on the basis of a diagnostic criteria proposed by Del Brutto et al. (2001). The following groups of patients and controls were included in the study: (I) Cases of NCC with a definitive diagnosis (n = 34): These patients fulfilled the diagnostic criteria to be definitively NCC. CDC-approved immunoblot was performed on sera from this group for further verifying their diagnosis. The test was performed using QualicodeTM Cysticercosis EITB Kit following manufacturer’s instruction (Immunetics Inc., USA). (II) Cases of NCC with a probable diagnosis (n = 67): This group included cases strongly suspected to be NCC again based on Del Brutto’s criterion. (III) Cases of NCC with a possible diagnosis (n = 59): This group included clinically suspected NCC cases having a lower certainty of diagnosis. (IV) Controls with other infectious diseases (n = 98 for serum and n = 34 for CSF): This group included patients with other parasitic diseases or infectious disorders of CNS (tubercular meningitis, 12; toxoplasmosis, 20; cystic echinococcosis, 16; filariasis, 37; cryptococcal meningitis, 10; hymenolepiasis, 3). These control cases were diagnosed based on clinical symptoms, laboratory culture, serology, and imaging techniques. (V) Healthy controls (n = 100 for serum and n = 50 for CSF): This group included healthy adults (blood donors, healthy staff and students) with no clinical history of cysticercosis or any other disease in the recent past for serum samples. For control CSF samples, individuals with non-inflammatory neurological disorders such as benign intracranial hypertension and non-compressive myelopathy (n = 50), were used as negative controls with respect to NCC as accepted in study before (Manoutcharian et al., 1999). 2.2. Serum and CSF specimens Five milliliters of venous blood were collected from all the study subjects including controls under aseptic precautions and was allowed to clot. One to two milliliters of CSF were collected from the NCC patients and controls with other infectious diseases by lumbar puncture under aseptic precautions. The CSF samples were collected from 70 NCC patients (Group-1, 28; Group-2, 22; Group-3, 20); 34 Group-IV controls with other infectious diseases (tubercular meningitis, 12; cryptococcal meningitis, 10; toxoplasmosis, 12), and 50 Group-V controls with non-inflammatory neurological disorders. All the serum and CSF samples were stored at −20 ◦ C until use. 2.3. Preparation of T. solium metacestode total soluble ES antigenic peptides T. solium metacestode whole larval cysts were dissected immediately after postmortem from naturally infected pigs obtained from local slaughter house. The ES antigen was prepared by in vitro culture of the larvae in RPMI-1640 medium (HiMedia, India) following the procedure as described earlier (Sahu et

al., 2009). Briefly, freshly dissected cysts from pig muscle were washed thoroughly with PBS, pH 7.2 containing antibiotics: Penicillin G (100 U/mL) + Streptomycin (100 U/mL) + Amphoterecin B (0.25 ␮g/mL). Twenty intact cysts were kept in tissue culture flasks (NUNC, New Zealand) in 20 mL of RPMI as stated above. The medium was supplemented with calcium carbonate (CaHCO3 ) at the concentration of 2.2 g/L. The flasks were incubated at 37 ◦ C and 3% CO2 . Growth and viability were monitored at regular interval of 12 h. The total observation period was up to 13 days, but the ESantigen was collected up to 6 days. The medium was changed once in every 12 h and the old medium was centrifuged at 5000 RPM for removal of suspended solids followed by concentration by ultra membrane filtration using Centricon tubes of 10 kDa cut off (Amicon, USA). The concentrate was stored at −20 ◦ C in aliquots with 0.006 mM phenylmethylsulfonyl fluoride (AppliChem GmbH, Germany) as the protease inhibitor. The protein content of every batch of ES antigen was determined by standard method using Pierce BCA Protein Assay Kit (Thermo Scientific). 2.4. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electro immuno transfer blotting (EITB) The T. solium metacestode ES protein preparation was analyzed by SDS-PAGE (Bio-Rad, USA) under non-reducing condition. Concentrated ES protein preparation was subjected to SDS-PAGE in a 10% gel for resolving the antigenic peptides and the protein banding pattern was visualized by Coomassie brilliant blue staining. Molecular weights of the antigenic peptides were determined by comparing with standard molecular weight marker (New England Biolab) run along side. The SDS-PAGE resolved ES antigens were subjected to EITB to elucidate the distribution of reactive peptides where sera from NCC cases and control subjects were tested for identifying reactive peptide bands on the blot. Reactive peptides were visualized after incubating with an anti-human immunoglobulin peroxidase labeled antibody (Bangalore Genei, India) and subsequently with 3,3-diaminobenzidine (Sigma, USA) solution. CSF samples from all the study groups were also assayed for detecting reactivity to different ES peptides. 3. Results 3.1. SDS-PAGE profile of the ES protein preparation A total of 12 major peptide bands were detected in the T. solium metacestode ES protein preparation. The major antigenic peptides were found to be of 100 kDa, 75 kDa, 67 kDa, 62 kDa, 51 kDa, 47 kDa, 43 kDa, 40 kDa, 32 kDa, 28 kDa, 21 kDa and 14 kDa. 3.2. EITB for detection of reactive ES peptides Using serum samples, a total of five antigenic ES peptides: the 67 kDa, 51 kDa, 43 kDa, 40 kDa and 32 kDa are demonstrated by EITB. The sera reacting with one or more of these peptides was considered positive by the test. The EITB test for anti-ES antibodies in serum was positive in 94.11% confirmed cases of cysticercosis (Table 1). The test showed a false positive reaction in 9.1% and 2% controls with other parasitic diseases and healthy controls respectively. Three antigenic peptides (67 kDa, 43 kDa and 32 kDa) were found to be more specific. The 43 kDa peptide was found to be reactive predominantly with 88.23% sera of Group I, 67.16% of Group II and 57.62% of Group III cases. This peptide did not react with any of the control sera. The 51 kDa, 40 kDa, 32 kDa peptides were the commonly recognized cross-reactive peptides detected by negative control sera (Fig. 1A).

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Table 1 EITB using T. solium metacestode ES antigen for detection of antibody in sera from cases of NCC and controls. Study groups

No. of subjects

I II III IV V

34 67 59 98 100

Frequency (%) of reactive T. solium metacestode ES antigenic peptides with sera 67 kDa

51 kDa

43 kDa

40 kDa

32 kDa

23 (67.64) 37 (55.22) 30(50.84) 1 (1.02) 0

24 (70.58) 40.(59.7) 32(54.23) 3 (3.03) 0

30 (88.23) 45(67.16) 34(57.62) 0 0

31 (91.17) 47(70.14) 35(59.32) 4 (4.08) 2 (2)

28 (82.35) 36(52.73) 29(49.15) 2 (2.04) 0

Overall 32 (94.11) 52(77.61) 35(59.32) 9 (9.1) 2 (2.0)

Fig. 1. Representative EITB images showing reactive T. solium metacestode ES antigenic peptides with serum (A) and CSF (B) from cases of NCC and controls. M: Molecular weight markers; Lane 1–10: ten Group-1 cases with definitive diagnosis of NCC; Lane 11–16: six cases of NCC with a possible diagnosis (Group-2); Lane 17–22: six cases with a probable diagnosis (Group-3); Lane 23–27: five controls with other diseases (Group-4); Lane 28–32: five healthy controls (Group-5).

The ES antigenic peptides reactive to CSF samples are presented in Table 2. The CSF reacting with one or more of the five antigenic peptides (67 kDa, 51 kDa, 43 kDa, 40 kDa, and 32 kDa peptides) was considered positive by the test. The three antigenic peptides viz., 67 kDa, 51 kDa, and 43 kDa peptides, were found to be predominantly reactive and 100% specific. The EITB test showed a false positive reaction with 8.82% CSF and 2% CSF from controls with other parasitic diseases and healthy controls respectively. The 43 kDa peptide was found to be reactive with 89.28% CSF from Group-1 patients. The 40 kDa and 32 kDa peptides were the commonly recognized cross-reactive peptides that were found reactive with sera from controls with other parasitic diseases (Fig. 1B).

Common antigenic ES peptides reactive to both serum and CSF are 67 kDa, 51 kDa, 43 kDa, 40 kDa and 32 kDa peptides. Among these 43 kDa ES peptide was found to be predominantly reactive and 100% specific when tested either with serum or CSF specimens. Though the specificity of 51 kDa ES peptide was found to be 100% in CSF, it was reactive to 3.03% control sera. Also 67 kDa ES peptide was detected with less sensitivity using either specimen (Table 3). 4. Discussion Serological diagnosis has been always an adjunct to neuro imaging based modalities for diagnosis of NCC in hospitals. In field-based

Table 2 EITB using T. solium metacestode ES antigen for detection of antibodies in CSF from cases of NCC and controls. Study groups

No. of subjects

I II III IV V

28 22 20 34 50

Frequency (%) of reactive T. solium metacestode ES antigenic peptides with CSF 67 kDa

51 kDa

43 kDa

40 kDa

14 (50) 12(54.54) 8(40) 0 0

12(42.85) 10(45.45) 10(50) 0 0

25 (89.28) 18(81.81) 12(60) 0 0

27 (96.42) 20(90.9) 9(45) 2 (5.88) 0

32 kDa 12 (42.85) 17(72.27) 14(70) 2 (5.88) 1 (2)

Overall 28 (100) 20(90.9) 14(70.0) 3 (8.82) 1 (2.0)

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Table 3 Comparison of the sensitivity and specificity of detection of reactive ES antigenic peptides by EITB. ES antigenic peptides

Sensitivity and specificity of ES peptides reactive with

Serum Sensitivity 67 kDa 51 kDa 43 kDa 40 kDa 32 kDa

67.64% 70.58% 88.23% 90.17% 82.35%

CSF Specificity

Sensitivity

Specificity

99.49% 98.48% 100% 96.96% 98.98%

50% 42.85% 89.28% 96.42% 42.85%

100% 100% 100% 97.61% 96.42%

Sensitivity and specificity was calculated based on 34 definitive cases and 198 controls for serum; 28 definitive cases and 84 controls for CSF.

studies or in hospitals with lack of availability of imaging tools in endemic areas, serology becomes the primary method of its diagnosis. However, still there is lack of a suitable and affordable serological test in many developing countries where the disease is actually more prevalent. To the best of our knowledge, the present study is one of the few reports of EITB using ES antigens obtained from cysticerci for demonstration of specific antibodies in serum and CSF for diagnosis of NCC (Espindola et al., 2002; Fleury et al., 2003; Lopez et al., 2004). Also we have reported in our previous study about the utility of an ELISA strategy employing ES antigens for diagnosis of NCC (Sahu et al., 2009). The overall sensitivities of EITB in the present study showed a higher value in CSF than serum, however, the specificities were comparable in both the specimens. In our study, the 43 kDa ES peptide was detected in sera as well as CSF with a specificity of 100%. The 43 kDa ES peptide, which was found predominantly reactive using serum and CSF specimens, is considered as a potent diagnostic marker of NCC. The 43 kDa ES antigen was also reactive to sera and CSF from study group-2 and 3 with a decreased sensitivity; this correlated with the decreasing order of the certainty of the diagnosis as per the criteria used. The 67 kDa and 51 kDa ES peptides were also detected with higher frequency using sera and CSF from Group-I cases but a low level of cross-reactivity was observed when control sera were used. The 43 kDa ES antigenic peptide is thought to be unique to T. solium infection for two reasons. First, this peptide did not react with any sera from patients having other helminthic infections such as E. granulosus, and H. nana. Second, CSF from patients with other infectious diseases in brain also did not react with this particular ES antigenic peptide. CSF from the control group recognized 40 kDa antigen with a frequency of 5.88% cases. There were three ES antigens (95 kDa, 49 kDa and 29 kDa), recognized by 86–100% of the ELISA-positive CSF in a study elsewhere (Lopez et al., 2004). However, the sensitivity in clinically suspected NCC patients is not reported by these authors. In our knowledge our present report is the only report where we have evaluated the EITB test for antibody detection in serum as well CSF employing T. solium metacestode homologous ES antigens and the results are presented against NCC patients of various certainty of diagnosis based on a well defined criteria. Though antibody reactivity to ES antigens (using sera and CSF from NCC patients) is documented in few earlier studies, those have employed ES antigens from non-homologous sources (Espindola et al., 2002; Molinari et al., 2002; Lopez et al., 2004). The 43 kDa, 58 kDa and 66 kDa ES antigens were found to be more specific in diagnosing human cysticercosis in a Korean study (Ko and Ng, 1998a), which correlates with our observations. Our 43 kDa and 67 kDa antigens are comparable with 66 kDa and 43 kDa antigens in the Korean study. EITB showed ES antigens at 22 kDa, 64 kDa, and 70 kDa recognized by sera from patients with NCC in a study

from Mexico (Molinari et al., 1993). Subsequently, 45 kDa ES antigen was predominantly recognized in 6 of 7 cases of NCC patients in a study by the same group (Molinari et al., 2002). ES peptides of 18 kDa and 14 kDa were recognized by polyclonal antibodies produced in rabbits immunized either with the vesicular fluid antigens or with a total antigen preparation of T. solium metacestode larvae in a report from Brazil (Espindola et al., 2002). In a recent report from Texas, the use of purified antigens of T. solium metacestode ES proteins in EITB is suggested three ES peptides (95 kDa, 49 kDa and 29 kDa) to be diagnostic which were recognized by 86–100% of the ELISA-positive CSF from NCC patients. When these three antigens were isolated and tested, as a pool, against all the CSF samples in double-blind ELISA, almost all (96.6%) of the CSF samples from patients with metacestodes at the vesicular stage were recognized (Lopez et al., 2004). The reason for finding different molecular weight reactive peptides from different studies may be due to variation in the preparation protocol of antigens and difference in methodologies for their separation and immunoreactivity detection systems. In an earlier study proteomic analysis of T. solium metacestode cyst fluid, a protein complex of 120 kDa was showed to have 2 major components of 42–46 kDa and 22–28 kDa each sharing 3 subunits of 14 kDa, 16 kDa and 18 kDa. The 42–46 kDa component was determined to contain 3 additional subunits of 22 kDa, 28 kDa and 38 kDa. These 6 subunits were shown to originate from either the 14 kDa or 18 kDa precursor (Lee et al., 2005). This study suggested that the higher molecular weight antigens may be actually the combination of multiple subunits having similar or different antigenic epitopes. So the reactive antigenic peptides of marginally different molecular weights reported from different studies may be due to variation in the degree of glycosylation. There are reports showing usefulness of low molecular weight ES peptides in diagnosis of NCC (Obregón-Henao et al., 2001; Haslam et al., 2003; Villota et al., 2003; Atluri et al., 2009). In our study, though low molecular weight peptides (lower than 32 kDa) were present in the ES antigens and detected by Coomassie brilliant blue staining, those could not be detected by the patient specimens. This may be due to low concentration of these small peptides to be detected by the method used. Otherwise these may be partially degraded products of larger ES peptides with loss of their antibody binding clefts. The cross-reactivity of antigen is closely related to the antigenic homology between the parasites more particularly among closely related species, as observed in earlier studies (Ko and Ng, 1998a,b; Wilkins et al., 1999) and also demonstrated in the present study. The cross-reaction is observed with the sera from 5 hydatid patients and 3 filariasis patients in the present study. The coexistence of NCC and other lesions may be an incidental observation in few patients particularly from areas of high prevalence and endemicity contributing to cross-reaction (Azad et al., 2003). In our study, the 40 kDa ES peptide showed cross-reaction more frequently in CSF than sera. The 32 kDa ES-antigen also showed cross-reaction with control sera and CSF, and it was more frequent in CSF than sera. The general impression is that detection of anti-Cysticercus antibodies by EITB and ELISA may be useful in identifying a population at risk of contact with the parasite, but not necessarily indicating an active infection. However, in a previous study, we demonstrated that antibodies to two unique antigenic preparations from the larval T. solium were associated with biological stage of the parasite in host; so antibody detection test can differentiate an active infection with a live parasite from a case with a degenerated one (Sahu et al., 2009). In the present study, we demonstrated that these antigens are indeed unique to T. solium metacestode infection. We did not observe any definite pattern when the reactive peptide bands were compared between patients with either vesicular or degenerated cysts in brain. This may be due to higher sensitivity of the EITB where even low level of circulating antibodies in serum and CSF

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could bind to the ES peptides concentrated in form of bands on the nitro cellulose membrane blot. Further analysis may be required employing purified ES peptides in ELISA format for their individual diagnostic performance. Cyst fluid or vesicular fluid from T. solium metacestode is an important source of ES antigens and a number of papers have described their serodiagnostic potential. Protein profile of the cyst fluid is also quite similar to that of T. solium metacestode ES substances collected from in vitro culture (data not presented). However, the ease of obtaining vesicular fluid from infected pigs is having its limitation in terms of very low quantity of the fluid and it is practically difficult and time consuming to isolate intact cysts from infected muscle. The in vitro culture of live T. solium metacestode larvae as employed in the present study can yield more quantity of ES antigen. Also there is no alternative method for obtaining ES antigens of more quantity until recombinant antigens are available at affordable costs. 5. Conclusion The 43 kDa ES peptide is suggested to be an important peptide of diagnostic utility in NCC. Further work is to be continued using purified 43 kDa ES peptide to exploit its diagnostic efficacy in formats suitable for rapid field based diagnosis of NCC in human. Acknowledgements We are grateful to Dr. Sunil K. Narayan, D.M. (Professor and Head, Department of Neurology) for valuable suggestions in classifying cases/controls. The first author is personally thankful to Dr. Devendra Kumar, Ph.D. (Professor and Head, Department of Parasitology) for helpful discussions. References Aguilar-Rebolledo, F., Meza-Lucas, A., Torres, J., Cedillo-Rivera, R., Enciso, A., Garcia, R.C., Munoz, O., Correa, D., 2002. Evaluation of the enzyme-linked immuno electro transfer blot assay for diagnosis of neurocysticercosis in children. J. Child Neurol. 17, 416–420. Atluri, S.R., Singhi, P., Khandelwal, N., Malla, N., 2009. Evaluation of excretory secretory and 10–30 kDa antigens of Taenia solium cysticerci by EITB assay for the diagnosis of neurocysticercosis. Para. Immunol. 31, 151–155. Azad, R., Gupta, R.K., Kumar, S., Pandey, C.M., Prasad, K.N., Husain, N., Husain, M., 2003. Is neurocysticercosis a risk factor in coexistent intracranial disease? An MRI based study. J. Neuro. Neurosurg. Psych. 74, 359–361. Del Brutto, O.H., Rajshekhar, V., White Jr., A.C., Tsang, V.C.W., Nash, T.E., Takayanagui, O.M., Schantz, P.M., Evans, C.A.W., Flisser, A., Correa, D., Botero, D., Allan, J.C., Sarti, E., Gonzalez, A.E., Gilman, R.H., Garcia, H.H., 2001. Proposed diagnostic criteria for neurocysticercosis. Neurology 57, 177–183. Espindola, N.M., Vaz, A.J., Pardini, A.X., Fernandes, I., 2002. Excretory/secretory antigens (ES) from in vitro cultures of Taenia crassiceps cysticerci, and use of an anti-ES monoclonal antibody for antigen detection in samples of cerebrospinal fluid from patients with neurocysticercosis. Ann. Trop. Med. Parasitol. 96, 361–368. Fleury, A., Gomez, T., Alvarez, I., Meza, D., Huerta, M., Chavarria, A., Carrillo Mezo, R.A., Lloyd, C., Dessein, A., Preux, P.M., Dumas, M., Larralde, C., Sciutto, E., Fragoso, G., 2003. High prevalence of calcified silent neurocysticercosis in a rural village of Mexico. Neuroepidemiology 22, 139–145.

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