Opisthorchiasis-associated biliary stones: light and scanning electron microscopic study

Abstract

Aim: Biliary stones are frequently encountered in areas endemic for opisthorchiasis in Thailand. The present study was to describe the prevalence and pathogenesis of these stones.

Methods: Gallstones and/or common bile duct stones and bile specimens from 113 consecutive cholecystectomies were included. Bile samples, including sludge and/or microcalculi, were examined for Opisthorchis viverrini eggs, calcium and bilirubin. The stones were also processed for scanning electron microscopic (SEM) study.

Results: Of the 113 cases, 82 had pigment stones, while one had cholesterol stones. The other 30 cases had no stones. Most of the stone cases (76%, 63/83) had multiple stones, while the remainder had a single stone. Stones were more frequently observed in females. Bile examination was positive for O. viverrini eggs in 50% of the cases studied. Aggregates of calcium bilirubinate precipitates were observed in all cases with sludge. Deposition of calcium bilirubinate on the eggshell was visualized by special staining. A SEM study demonstrated the presence of the parasite eggs in the stones. Numerous crystals, morphologically consistent with calcium derivatives and cholesterol precipitates, were seen.

Conclusion: Northeast Thailand has a high prevalence of pigment stones, as observed at the cholecystectomy, and liver fluke infestation seems involved in the pathogenesis of stone formation.

Figure 1

Histochemical staining of biliary sludge for bilirubin (A) and calcium (B). Calcium appears as dark deposits on the Opisthorchis eggshell (arrow) and bilirubin precipitates are demonstrated in the outer layer (arrowhead). Normal parasite eggs without deposition are shown in the same field. (A = Fouchet stain, B = von Kossa stain).

Figure 2

Typical pictures showing the cascades of Opisthorchis egg-associated stone formation starting from aggregation of the eggs admixed with mucin (A), deposition of calcium bilirubinate on the eggshells (B), and formation of tiny stones (C & D). Original magnification, × 100 (A & C) and × 200 (B & D).

Figure 3

SEM micrographs of gallstones showing Opisthorchis eggs with typical musk-melon-eggshell surface in the nidi of the stones. Several crystalline structures consistent with calcium (Ca), bilirubin derivatives (Bi) and cholesterol (Ch) could be noted (A, B). Higher magnification with highlighting calcium bilirubinate deposition on Opisthorchis eggshell and mucus is shown in Figure 3B.

Figure 4

Diagram showing the proposed pathogenesis of Opisthorchis-associated biliary stone.