The process of residual calcification following antiparasitic treatment in the pig model of neurocysticercosis is dynamic

Abstract

Background: Calcified neurocysticercosis (NCC), the end stage of brain cysts of the pork tapeworm Taenia solium is a common cause of epilepsy. Calcified NCC lesions are not inert and represent potential epileptogenic foci. Understanding the mechanisms of residual calcification in NCC is hindered by the difficulty of accessing human brain biopsies. Since cyst degeneration can be induced by antiparasitic treatment (APT) in NCC-infected pigs, this study assessed the residual calcification process in this model at three time points after APT.

Methods/principal findings: Fifteen naturally infected pigs with viable NCC confirmed by magnetic resonance imaging received APT with albendazole and praziquantel and were sacrificed after 4, 8, and 12 months (n = 5 each). The pigs' brains were removed and processed by ex vivo CT scan to assess the proportion of cysts that calcified by post-treatment time points using risk ratios (RR) from Poisson regression. Radiodensity levels (Hounsefield units) of calcified lesions were also measured and compared using linear coefficients from log-transformed values in generalized linear models. The overall proportion of residual calcification on CT scan was 63.9% (156 calcified lesions/244 viable cysts), being statistically higher in treated NCC pigs at 4 months (83.3% [50/60], RR = 2.61, P < 0.001) and 8 months (82.8% [77/93], RR = 2.59, P < 0.001) versus 12 months (31.9% [29/91]). At 8 months after APT, calcifications were more dense (100.6 ± 3.6 HU) compared to 12 months (74.4 ± 3.6 HU, β = 0.37, P = 0.010) and marginally higher compared to 4 months (85.2 ± 3.8 HU, β = 0.24, P = 0.096), and were also larger and more frequently found on histopathology.

Conclusion/significance: Calcification in NCC is a dynamic process that can be induced and monitored in naturally infected pigs. Eight months after treatment seems to be an optimal time point for assessing residual calcification.

Fig 1. Basal brain MRI exam showing viable cysts (A); ex vivo CT scan showing calcified lesions with measurements of radiodensity (HU) and areas (cm²) (B); apparent calcified lesion identified on gross exam (C).

Fig 2. Microscopic visualization of calcium aggregates by Alizarin–Red (AR) stain in calcified granulomas from treated NCC pigs at 4, 8.

and 12 months (A, B, and C).

Fig 3. Inflammatory and fibrotic responses in calcified granulomas from treated NCC pigs at 4 (A–C), 8 (D–F), and 12 (G-I) months.

H&E (A, D, G) and Masson’s Trichrome (B, E, H) stains. (C) Silhouette of a cysticercus with a central scolex is observed and partially effaced by dystrophic mineralization (blue arrow). Surrounding the degenerate metacestode is a semi dense and tortuous band of mature fibrous connective tissue (black arrow). (F) The cysticercus is completely effaced by large amounts of dystrophic mineralization and cellular necrotic debris surrounded by numerous lymphocytes (arrowhead), plasma cell and some histiocytes embedded in in a disperse and immature fibrous connective tissue (black arrow). The adjacent blood vessels are reactive and have lymphocytic perivascular cuffing (red arrow). (I) The metacestode is surrounded by some lymphocytes and plasma cells embedded in an immature fibrous connective tissue and externally sequestered by a dense, thick, and disorganized mature fibrous connective tissue (arrow). (A, B, D, E, G, H, 5X magnification) (C, F, I, 20X magnification).

Fig 4. Bar plots showing the mean

± standard error of element composition of calcified granulomas (expressed as weight percentages [Wt%]) by scanning–electron microscopy (SEM) in treated NCC pigs at 4, 8, and 12 months. Abbreviations: CK (carbon); OK (oxygen); NaK (sodium); MgK (magnesium); PK (phosphorus); CaK (calcium). Bars represent means and whiskers represent standard errors. * indicates P < 0.05 for comparisons by post-treatment time points.