Advances in Serological Diagnosis of Taenia solium Neurocysticercosis in Korea

Abstract

Cysticercosis, a parasitic disease caused by Taenia solium metacestode (TsM), has a major global public health impact in terms of disability-adjusted life years. The parasite preferentially infects subcutaneous tissue, but may invade the central nervous system, resulting in neurocysticercosis (NC). NC is an important neglected tropical disease and an emerging disease in industrialized countries due to immigration from endemic areas. The prevalence of taeniasis in Korea declined from 0.3%-12.7% during the 1970s to below 0.02% since the 2000s. A survey conducted from 1993 to 2006 revealed that the percentage of tested samples with high levels of specific anti-TsM antibody declined from 8.3% to 2.2%, suggesting the continuing occurrence of NC in Korea. Modern imaging modalities have substantially improved the diagnostic accuracy of NC, and recent advances in the molecular biochemical characterization of the TsM cyst fluid proteome also significantly strengthened NC serodiagnosis. Two glycoproteins of 150 and 120 kDa that induce strong antibody responses against sera from patients with active-stage NC have been elucidated. The 150 kDa protein showed hydrophobic-ligand binding activities and might be critically involved in the acquisition of host-derived lipid molecules. Fasciclin and endophilin B1, both of which play roles in the homeostatic functions of TsM, showed fairly high antibody responses against calcified NC cases. NC is now controllable and manageable. Further studies should focus on controlling late-onset intractable seizures and serological diagnosis of NC patients infected with few worms. This article briefly overviews diagnostic approaches and discusses current issues relating to NC serodiagnosis.

Keywords: Republic of Korea; Taenia solium; immunodiagnosis; neurocysticercosis; proteome.

Fig. 1.

(A) Scattergram of specific antibody levels in sera from patients with NC, NC crossreacted to the sparganum extracts and sera from patients with sparganosis. Serum samples from NC cases showed the highest levels of reactivity against TsM CF. Some sparganosis sera revealed cross-reactions against TsM parenchymal extracts, but showed no crossreactions against TsM CF. (B) Immunoblot analysis of three different antigens (TsM CF, TsM parenchymal extracts, and sparganum extracts). Lanes a–c, sera from NC patients; lanes d–f, sera from sparganosis patients; lanes g–i, sera from NC cases cross-reacted with the sparganum extracts, lanes j–l, negative controls. NC, neurocysticercosis; TsM, Taenia solium metacestode; CF, cyst fluid; M_r, molecular weights in kDa.

Fig. 2.

Partial purification of 120 and 150 kDa macromolecular proteins from TsM CF. (A) A 1.6 × 60 cm long FPLC packed with Superdex 200 prep grade was equilibrated with 20 mM Tris-HCl (pH 8.0) containing 150 mM NaCl. A total of 3 mL (10 mg) of TsM CF was applied to the column with a flow rate of 0.5 mL/min. Eighty-five fractions (1.5 mL each) were analyzed for their absorbance at 280 nm, as monitored by UNICORN (v3.0). (B) Analysis of the eluted group III and IV proteins by native PAGE. (C) Western blot analysis of the fraction III and IV proteins probed with anti-CF antibody. TsM, Taenia solium metacestode; CF, cyst fluid; FPLC, fast-performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; Mr, molecular weights in kDa; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.

Fig. 3.

Biophysical properties and phylogenetic relationship of the 120 and 150 kDa proteins of the TsM CF. (A) The 120 kDa protein (fraction IV) was separated in a pH range of 6–11, after which it was further resolved by 15% SDS-PAGE. The subunit proteins of 14, 16, 18, 22, and 28 kDa, which were identified by proteome analysis, are seen. (B) Phylogenetic analysis demonstrated that the subunits of the 120 kDa macromolecule were clustered into two groups of 14 and 18 kDa lineages. (C) 2DE analysis of the 150 kDa protein (fraction III). The protein was isoelectrically focused using IPGphor (pH 6–11), after which it was further separated by 15% SDS-PAGE. Each protein was subjected to MALDI-TOF analysis. (D) Phylogenetic analyses of the subunit proteins. The representative amino acid sequences were aligned, and optimized with ClustalW and GeneDoc. The phylogram was constructed with the neighborjoining algorithm (PHYLIP). The statistical significance of each branching node was evaluated with 1,000 random samples. TsM, Taenia solium metacestode; CF, cyst fluid; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; 2DE, two-dimensional electrophoresis; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; M_r, molecular weights in kDa.

Fig. 4.

Immune response of the representative recombinant proteins derived from the 120 and 150 kDa subunits and chimera against sera from active-stage NC patients. Each protein was separated by 12% SDS-PAGE and transblotted to a PVDF membrane. The blots were incubated with 1:200 diluted individual NC sera overnight. The blots were further incubated with horseradish peroxidase–conjugated anti-human IgG and developed with 4-chloro-1- naphthol as a chromogen. NC, neurocysticercosis; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride.

Fig. 5.

Changing patterns of specific IgG antibody levels in serum and CSF samples from the NC cases after medical treatment shown by individual patients. Acute encephalitic attacks in each patient are indicated by red arrows. Significant changes of specific IgG antibody levels in either serum or CSF were not observed during the 22-month observation period. CSF, cerebrospinal fluid; NC, neurocysticercosis.