Cysticercus cellulosae antigens in the serodiagnosis of neurocysticercosis.

Neurocysticercosis (NCC) is difficult to diagnose clinically because of its varied clinical presentation. However, an accurate diagnosis is possible only after suspicion on epidemiological grounds, proper interpretation of the clinical data, analysis of the findings on imaging studies, and specific immunological tests on the serum and cerebrospinal fluid (CSF). The diagnosis of NCC by any single parameter thus continues to remain difficult. In the past, detection of NCC was based on autopsy studies and histological confirmation. In recent times, the advent of imaging methods such as computed tomography and magnetic resonance imaging have provided excellent non-invasive tools for easy detection of NCC. Nevertheless, an imaging technique of the brain, although useful, is not considered as a gold standard for the diagnosis of NCC. Serological tests are being increasingly used in adjunct with imaging techniques, to aid the diagnosis of NCC. Immunodiagnostic techniques include detection methods for specific antibodies and for circulating parasite antigens in the serum and CSF. Currently, many of the immunodiagnostic tests, including the enzyme-linked immunosorbent assay and enzyme immunotransfer blot, use purified native antigens for the immunodiagnosis of NCC. Nevertheless, the main problem with the use of native cysticercal antigens is that the native proteins often show cross reactions with sera from humans infected with other parasites. The preparation of native antigens also demand a constant supply of parasitic material from the intermediate host pig. In order to overcome the problems in using native antigens, the recombinant antigens or synthetic peptides, which can be produced under stable conditions, are being evaluated for the serodiagnosis of NCC.

INTRODUCTION

Neurocysticercosis (NCC) due to Taenia solium metacestode is an important cause of human morbidity and mortality, particularly in the developing countries.[1] This disease not only causes neurological morbidity, but also imposes economic hardships on impoverished populations. However, there are wide variations in the prevalence rates of NCC in different regions and different socioeconomic groups in the same country.[2]

Convulsions and / or seizures, intracranial hypertension, and psychiatric disturbances are the three most important manifestations of NCC, which may occur separately or in combination.[34] However, the clinical manifestations of NCC are highly variable and depend on the number, location, and viability of the cyst, stage of development of the cyst, whether young or mature, intact or degenerated, and the immune status of the host.[3] Moreover, the incubation period varies from a few months to several years.[5-7] Hence, the clinical diagnosis of the condition continues to remain a problem.

In the absence of specific clinical manifestation(s) of NCC, diagnosis of the condition depends more on laboratory procedures.[8] During the last two decades, tremendous progress has been made in many fields, which have a direct bearing on the diagnosis of NCC in humans. First, the advent of newer imaging methods such as computed tomography (CT) and magnetic resonance imaging (MRI) have revolutionized the diagnosis of space-occupying lesions caused by many parasitic conditions, including NCC. Second, emphasis has now shifted from traditional methods of detecting cysticercus antibodies, by NCC serology, to detecting cysticercus antigens in the serum, cerebrospinal fluid (CSF), urine or other body fluids. Imaging methods and immunological tests in combination are now extensively used to supplement the clinical diagnosis of NCC.[5]

The newer imaging techniques such as CT and MRI, although helpful in identification and characterization of cysticerci, are not available in most endemic areas or are often too expensive.[9] The atypical appearance of the visualized lesions, failure to provide information about the viability of the parasite, and difficulty in differentiating these lesions from abscesses or neoplasms are also some of the limitations of imaging methods.[10]

The improvement of immunodiagnostic tests has contributed immensely to the diagnosis of NCC and a better understanding of the prevalence and epidemiology of the infection. Immunological techniques used in the diagnosis of NCC include detection of specific antibodies and antigens in the serum, cerebrospinal fluid, and other body fluids.[9] Extensive studies have been made on the use and evaluation of a variety of serological tests for diagnosis of NCC, but no test on its own provides unequivocal proof. Occurrence of a variation in the sensitivity and specificity of various immunoassays for diagnosis of NCC is a recognized problem due to (i) host heterogenous immune response, (ii) use of a variety of Cysticercus antigens from different sources, and (iii) an antigenic variation or drift exhibited by the parasite.[11-13]

Infection with Cysticercus cellulosae results in a specific antibody response. These antibodies can be detected in the serum, in CSF, and other body fluids. Several techniques such as radioimmunoassay, hemagglutination, the complement fixation test, dipstick assay, latex agglutination, ELISA, and the immunoblot techniques have been described to detect antibodies to C. cellulosae infections in humans.[9]

Initially, antigens used in antibody-detection assays were the native parasite antigens such as cyst fluid, excretion-secretion (ES) products or crude homogenates from cysticerci, from either T. solium or its related parasites. However, tests using these unpurified native parasite antigens show moderate sensitivities and relatively poor specificities.[91415] In the recent past, improved protein-purification techniques and research on the antigenic properties of cyst fluid and surface proteins have led to the development of better serological tools.[16-18]

Classically, the serological tests for the detection of antibodies or antigens are based on the use of crude or purified antigen fractions. Of late, specific recombinant antigens and even synthetic peptide antigens are being used to obviate the difficulties associated with the traditional source of antigen material. This article seeks to review the various C. cellulosae antigens used in the serodiagnosis of NCC.

ANTIGENS OF THE CYSTICERCI

Native antigens of C. cellulosae

Serological tests in NCC using the C. cellulosae native antigen, prepared by conventional methods, had the inherent disadvantage of showing cross-reactions with related cestodes or other parasitic infections.[15] The cross reaction was partly due to the antigenic variation in the developmental stages of C. cellulosae and even strain variations in certain Taenia species. As the quality of the cysticercus antigen used in the methods played a major role in the specificity and sensitivity of an immunodiagnostic test, stress was placed on fractionation, purification, and characterization of the antigens.

The native antigens of C. cellulosae can be broadly classified into somatic Cysticercus antigens and metabolic Cysticercus antigens. Somatic Cysticercus antigens are the molecules comprising of the soma of the parasite, whereas, metabolic Cysticercus antigens are associated with molecules comprising of metabolic secretions and excretions, the excretory-secretory (ES) antigens.[19]

Somatic antigens of C. cellulosae

Various serological tests using crude preparations of somatic Cysticercus antigens have been employed in the diagnosis of NCC, particularly for the detection of antibodies in the serum and / or CSF, in many studies conducted earlier. These include protoscolex extract,[20-23] cystic fluid,[24-26] cyst wall antigen,[1314] and the whole cyst homogenate preparation.[2227-31] Heterologous T. crassiceps antigenic peptides have also been used for the serodiagnosis of NCC in humans.[223233]

Different tests such as the complement fixation test (CFT), the indirect hemagglutination test (IHA), counter current immuno-electrophoresis (CIEP), ELISA, and so on, using various somatic C. cellulosae antigens, have been evaluated in the diagnosis of NCC.[34-37] Of these, the ELISA, using purified antigenic fractions has been reported to be better than the ELISA using crude antigen, for obviating cross-reactions.[313839]

Various purified antigenic fractions of C. cellulosae have been reported to avoid cross-reactions, with varying degrees of success, although the desired specificity has not been achieved.[39] The ELISA using Sephadex G-200 purified antigen fraction (PAF-II) showed 95.23% sensitivity and 100% specificity.[40] Further analysis of Sephadex G-200 purified PAF-II of C. cellulosae revealed three highly immunoreactive and specific bands (18, 20, 24kDa) for diagnosis of NCC. The ELISA using these fractions has been evaluated in patients with NCC, particularly to assess the response to treatment with anti-cysticercal therapy.[41]

The EITB, with purified glycoprotein fraction of the cyst fluid appears to be sensitive and specific for the diagnosis of NCC.[42-44] GP50 is a glycosylated and GPI-anchored membrane protein, one of the diagnostic components of the lentil lectin purified glycoprotein (LLGP) antigens that have been used for the antibody-based diagnosis of NCC in an EITB, during the last 15 years.[45] The results of a study showed the affinity purified antigen fractions from the Taenia crassiceps Cysticercus vesicular fluid to be an important source of specific peptides, and to be useful in detecting Cysticercus antibodies.[33]

Excretory–secretory antigen of C. cellulosae

The ES antigens have been found to be better than the crude somatic antigen in the serodiagnosis of other parasitic diseases such as lymphatic filariasis,[46-49] onchocerciasis,[50] dirofilariasis,[51] schistosomiasis,[52] fascioliasis,[53] trichinellosis,[54] toxocariasis,[55] opisthorchiasis,[56] amoebiasis,[57] malaria,[58] and toxoplasmosis.[59] Hence, the ES antigens of many parasites are being increasingly used in many parasitic infections, as useful markers for detecting active infections.

C. cellulosae ES antigens have also been evaluated in the diagnosis of NCC.[316061] Many studies carried out earlier have reported the use of Cysticercus ES antigens, mostly sourced from heterologous parasites such as T. crassiceps, in the diagnosis of NCC.[28336062]

Studies using homologous ES antigens, however, are only few and have been reported recently.[316163] In a previous study conducted in our laboratory, the ELISA using a homologous ES antigen to detect the antibody in CSF showed a sensitivity of 64.28% and a specificity of 96.96%.[61] The ELISA using the homologous ES antigen detected Cysticercus antibodies in CSF with a sensitivity of 100% and a specificity of 97.7%, in a study conducted in Mexico.[27] In another study, the ELISA using homologous T. solium metacestode ES antigen detected Cysticercus antibodies in 22 out of 24 cases of active NCC.[63] The ELISA using homologous ES antigen showed a sensitivity of 63.2% and specificity of 76.8% for the detection of antibodies in the serum, in a study reported from Chandigarh.[31]

Both homologous and heterologous ES antigens of Cysticercus have also been evaluated, in porcine and bovine cysticercosis, for identification of infected pigs and cattle infected with C. cellulosae.[64-66] In an Indian study, the ELISA using ES antigens showed a sensitivity of 92% and specificity of 100%, when detecting C. cellulosae antibodies in the serum of pigs.[67]

Biochemical composition of cysticerci

The vesicular fluid, membrane, and the scolex of the cysticerci were found to contain 45.9, 20.1, and 34.9% proteins, respectively, and 49, 26.4, and 24.6% carbohydrates, respectively.[68] Pathak and Gaur (1985)[69] reported the cyst fluid to contain a pH of 6.9 – 7, ash (4.89%), dry matter (55.46%), organic substance (49.57%), and crude protein (1.89%). They reported the scolex to contain 8.20% ash, 65.26% dry matter, 57.20% organic substance, and 8.35% crude protein, while the membrane contained 6.69% ash, 63.36% dry matter, 53.30% organic substance, and 5.25% crude protein. In a related study, Gaur (1985)[70] reported 115.20 ± 0.32, 472.50 ± 2.65, and 85.10 ± 0.50 total lipids (mg / g), 128.73 ± 0.58, 501.52 ± 5.72, and 76.63 ± 3.96 cholesterol (mg / 100 ml) and 56.66 ± 2.65, 101.51 ± 2.70, and 38.06 ± 1.23 phospholipids (mg / 100 ml) in the scolex, cyst membrane, and cyst fluid, respectively.

The scolex, cyst membrane, and fluid were found to contain 9, 13, and 10 amino acids, respectively.[71] Pseudo cholinesterases and acetylcholine esterases were found to be localized in the vesicular membrane, whereas, acetylcholine esterase alone was found to be present in the scolex.[72] Martinez-Zedillo, et al.,[73] found that pseudo-cholinesterase activity in C. cellulosae varied between 0.15 and 1.50 IU / ml and the mean acetylcholine esterase activity in the intact larvae was 2.5 IU / ml. The isolated bladder walls had greater activity of both enzymes than the intact cysticerci. In vitro incubation of C. cellulosae had a mean value of protein, in the hatching fluid, of 5.75 mg / dl at 2 hours and 16.38 mg / dl at 24 hours. The mean value of uric acid was 0.34 mg / dl at 24 hours.[74]

Synthetic antigens of C. cellulosae

In recent times, the advent of synthetic peptides has opened a new field and perspectives in diagnostic serology, as the source of pure epitopes and molecules, for use as antigens in the diagnosis of a variety of parasitic infections.

The first synthetic peptides were identified and evaluated in the immunodiagnosis of NCC by screening of the cDNA library of T. crassiceps, and were synthesized by solid-phase peptide synthesis.[75] Subsequently, the synthetic polypeptides that represented the full-length mature protein sTS14 were assessed for serologic potential using ELISA for the diagnosis of NCC.[76] Since then, pure epitopes and molecules are being increasingly used as diagnostic antigens in a large number of parasitic infections including malaria,[77] Chagas’ disease,[78] leishmaniasis,[79] schistosomiasis,[80] cystic echinococcosis,[81] fascioliasis,[82] and cysticercosis.[83]

A few studies evaluating synthetic peptides have been carried out in NCC. For instance, the ELISA using a synthetic peptide SPACc showed a sensitivity of 93% and specificity of 94.3%, to detect a Cysticercus antibody in the serum for diagnosis of NCC.[84] The ELISA using a synthetic peptide Ts45W-1 showed a sensitivity of 85% and a specificity of 83.5% to detect the Cysticercus antibody in serum, in a study carried out in Venezuela.[83] In another study carried out in Mexico, the ELISA using a synthetic peptide KETc12 exhibited a sensitivity of 93.7% and a specificity of 100%, to detect the Cysticercus antigen in CSF.[85] The Diagnostic utility of five Taenia saginata onchosphere-derived synthetic peptides in T. solium NCC was evaluated with CSF samples, in Brazilian patients.[86] In that study, the indirect ELISA using the peptides either unconjugated or coupled to carrier proteins, showed a sensitivity of 93% and specificity of 85%, with peptides HP6-2 and Ts45W-1.[86]

Recombinant antigens of C. cellulosae

Recombinant antigens have been evaluated in the recent past for diagnosis of many parasitic diseases such as cystic echinococcosis;[87] Chagas’ disease;[88] fascioliasis[89] filariasis;[90] amoebiasis;[91] malaria[92] and leishmaniasis.[93]

Characterizing species-specific antigens of T. solium has led to the availability of several high quality antigens in the serodiagnosis of NCC. Now the research on the serodiagnosis has focused on the stable production of diagnostic antigens, using molecular techniques. Many studies have been carried out for the production and evaluation of recombinant antigens for the serodiagnosis of NCC.[94-100]

Bueno, et al,[97] evaluated ELISA using recombinant GP50 antigen and showed a sensitivity of 94.7% and a specificity of 100% for cysticercus antibodies in the serum and showed a sensitivity of 93.8% and specificity of 100% for cysticercus antibodies in the CSF. Similarly, Hancock, et al,[96] expressed the active recombinant GP50 in a baculovirus expression system, and the recombinant antigen, when used in ELISA, showed a sensitivity of 90% and specificity of 100% in the diagnosis of NCC. The EITB assay, using a T24 recombinant antigen, showed a sensitivity of 94% and a specificity of 98% with serum samples in patients with NCC.[98]

In a recent study, Handali, et al,[100] evaluated ELISA using six cysticercosis diagnostic proteins (rGP50, rT24H, sTsRS1, sTs18var1, sTsRS2var1, sTs14) of which rT24H performed well in detecting cases with two or more viable cysts in the brain, with a sensitivity of 97% and a specificity of 99.4%, with sera collected from defined cases of NCC. Ferrer, et al,[101] expressed the Ts8B2 (8 kDa antigen), which when used in ELISA showed a sensitivity of 96.8% and 67.7% for active and inactive cases of NCC, respectively.

The ELISA, using a recombinant TS14 antigen, showed 100% positivity with CSF and 97% positivity with serum samples from NCC patients, in a study carried out in Brazil.[99] The purified G-F18 fusion protein synthesized by antibody screening of T. solium metacestode cDNA library showed the best results when it was used in ELISA with sera from NCC patients.[95] In a study by Chung, et al[94] , conducted in Korea, the EITB analysis of sera using a 10 kDa recombinant protein, from patients with active NCC, showed a strong reactivity of 97%.

Screening of T. solium metacestode cDNA library identified four antigen candidate clones Ag1, Ag1V1, Ag2, and Ag2V1 in a study carried out in Japan. These clones, except Ag2V1, encoded a 7-kDa polypeptide, and Ag2 encoded a 10-kDa polypeptide and showed 53 - 94% similarity at the amino acid level. The difference between the polypeptide size predicted from a cDNA sequence and the native antigen size detected by immunoblot analysis suggested the occurrence of an N-linked glycosylation, as putative N-linked glycosylation sites existed in Ag1 and Ag1V1.[102] Obregon-Henao, et al,[103] demonstrated that purified 12, 16, and 28 kDa native GPs migrated as 7-kDa proteins after treatment of enzymatic deglycosylation, which indicated that the protein backbone of GPs was similar in size (7 kDa), but the extent of glycosylation was different. The sensitivity and specificity of ELISA and EITB using some of the C. cellulosae recombinant antigens are listed in Table 1.

Table 1

Different C. cellulosae recombinant antigens employed in ELISA and EITB with their sensitivity and specificity

Currently, routine immunodiagnosis of NCC still relies heavily on the use of native antigens such as complete somatic homogenate, metacestode cyst fluid, cyst wall antigen, protoscolex antigen, and excretory – secretory products. The only source for antigens is T. solium cysticerci, obtained from the parasites extracted from diseased pork meat. The amount of cysticerci obtained from pork varies widely in relation to the parasite burden.[76104] Moreover, in many cases, preparation of adequate antigen extracts in sufficient amounts is still linked to the detection of swine naturally infected with T. solium larvae, which is often difficult to obtain.[105] Parasitized pork is also difficult to obtain even in areas where the organism is endemic because farmers tend to hide sick animals to avoid confiscation; denying the occurrence of such infected pork and the animals are eventually sacrificed and sold clandestinely.[104]

It would be desirable to have an animal model for T. solium that could easily be maintained in the laboratory and could be used as an alternative source of parasites.The possibility of achieving such an animal model arises from the observation that the Taenia species share common antigens. The ORF strain of Taenia crassiceps reproduces in an asexual manner by intraperitoneal passage through female BALB/c mice, representing an important experimental model for possible use in experimental Taenia infection.[106107]

Difficulties in obtaining somatic cysticercal antigens in large quantities have prevented the standardization of immunodiagnostic tests and the access of many patients to these diagnostic studies. To overcome the problems with somatic cysticercal antigen, many studies have been carried out on the use of ES antigens in cysticercosis serology.[316163108] However, the main problem with the use of ES antigen is that the production of ES through in vitro cysticerci culture is labor-intensive, time-consuming, and expensive. The turnaround time from the collection of live cysticerci to the end of the culture harvest takes nearly about one month, and production of the crude ES proteins from each batch is of a very small quantity.[105] Second, because of the complexity of the crude ES proteins produced from the in vitro cultures, the ES antigen often cannot be used directly in tests such as ELISA and lateral flow (immunochromatographic strip tests), without further purification.[109] Moreover, the low protein yield from the in vitro cultured ES antigen limits further purification.[105]

Unfortunately, most of the diagnostic tests that employ these native cysticercus antigens, either somatic or ES antigens, suffer from poor sensitivity, specificity, and poor reproducibility. Cross-reactions with serum from patients with other parasitic infections such as cystic echinococcosis and alveolar echinococcosis, caused by larval Echinococcus granulosus and Echinococcus multilocularis, respectively, is a noted problem.[110111] Hence, in several studies carried out earlier, attempts have been made to refine these native antigen preparations further to enhance their specificity and to avoid cross reaction with other parasitic infections when used in various immunoassays in cysticercosis.

Effective immunodiagnosis of NCC depends largely on the identification and availability of well-defined specific antigens. The use of purified antigens from T. solium metacestodes in immunoassays allows a sensitive, but not a specific diagnosis of cysticercosis.[110112] Over the past two decades, many efforts have been directed toward purification and characterization of specific antigens of T. solium metacestode, either from whole worms or from cyst fluid.[162543111] Various biochemical methods had been evaluated to obtain the purified antigen from complex antigenic preparations of C. cellulosae. Serological tests for the diagnosis of NCC using crude or purified Cysticercus antigen, show varying specificity due to the immunological cross reactivity, because of the presence of carbohydrate epitopes.[111] Moreover, all these methods carried out to obtain purified antigen are laborious, expensive, and the material obtained after the purification is very little.[361]

As the isolation of purified antigens from cysticerci is limited by the paucity of materials, recombinant DNA technology that would yield sufficient quantities of pure antigens appears to be the best alternative available in this direction.[83] Production of antigens by recombinant DNA technology ensures a uniform, specific, pure, and continuous supply, which can be used in the serodiagnosis of an infectious disease.[99105]

CONCLUSION

Theoretically, a mixture of recombinant proteins with immunodiagnostic potential can help to solve these drawbacks of using native antigens. Unlimited amounts of antigen can be produced under controlled conditions, and it may even be possible to identify and remove the cross reactive epitopes, without losing the diagnostic efficiency. Such a permanent source of diagnostic antigens will also be useful in endemic countries where collection of T. solium cysticerci is difficult. The use of recombinant antigen will also overcome the major disadvantages of poor reproducibility of serodiagnostic methods between different laboratories, presumably due to variability in the nature and purity of the parasite antigen extracts employed. Reproducibility in diagnostic tests will be much more enhanced by the use of cysticercus recombinant antigens or synthetic peptides.