Ivermectin Used in Percutaneous Drug Injection Method for the Treatment of Liver Hydatid Disease in Sheep

GASTROENTEROLOGY 2002;122:957 962 Ivermectin Used in Percutaneous Drug Injection Method for the Treatment of Liver Hydatid Disease in Sheep MURAT HOKELEK,* BEKIR AHMET DEGER, EMIN DEGER, EDIZ TUTAR, and MUSTAFA SUNBUL *Department of Microbiology and Clinical Microbiology, and Department of Clinical Microbiology and Infectious Diseases, Ondokuz Mayis University School of Medicine, Samsun, Turkey; Department of Radiology, Private Hayat Hospital, Antalya, Turkey; Department of Radiology, Private Fair Hospital, Afyon, Turkey; Department of Pathology, Corum SSK Government Hospital, Corum, Turkey Background & Aims: Ivermectin is a macrocyclic lactone (avermectins) produced by the actinomycete Streptomyces avermitilis. In this experimental study, the effectiveness of intracystic injection of ivermectin was studied as a new approach of percutaneous treatment of cystic echinococcosis. Methods: Twelve naturally infected sheep were selected and divided into 2 subgroups: treatment group (n 9) and control group (n 3). In the treatment group, approximate volume of ivermectin solution needed to achieve an intracystic concentration of 10 g/ml was injected into cysts, but in the control group, sterile distillated water was applied. No reaspiration was performed at all. Results: In the following period of 6 months, repeated sonography revealed a significant decrease in cyst sizes and progressive solidification of the cysts in the treatment group. In the control group, volumes of the cysts were increased. No major complications occurred during or after the procedure. After 6 months, all sheep were killed and examined for macroscopic and microscopic changes. Pathologic examination in the treatment group showed pericyst hyalinization, inflammatory cells in the cyst wall, degeneration of laminated and germinal membrane, and necrotic material in the cyst cavity. No viable protoscolices or daughter cysts were observed. Conclusions: Percutaneous treatment of cystic echinococcosis with ivermectin as a scolicidal agent seems to be effective in this animal model. Hydatid disease is still endemic in developing countries and remains an important cause of morbidity. 1 Surgical treatment of hydatid disease is the traditional method but carries high risk compared with other methods including: percutaneous treatments, systemic medical treatments, or combinations of both. 2 6 Percutaneous treatment (PT) has become a widely used procedure in recent studies, which showed its safety and effectiveness in selected cases. 3,7,8 PT can be performed in surgically compromised patients and in inaccessible cysts, with the advantages of short duration of hospitalization and cost-effectiveness compared with surgery. 9,10 PT is usually performed with the percutaneous aspiration-injection-reaspiration (PAIR) method. The PAIR method includes mainly the puncture and evacuation of the content of the cyst, injection of the scolicidal agents such as hypertonic saline solution or 95% ethanol, and reaspiration of the content of the cyst. 10,11 A new method, percutaneous drug injection method (PEDIM), for percutaneous treatment of hydatid diseases was described previously by Deger et al. 12 PEDIM differs from PAIR and other similar techniques in 2 aspects. First, instead of a hypertonic solution, a chemotherapeutic agent such as albendazole sulphoxide is injected into the cyst cavity, and, secondly, no reaspiration is performed. 12 In this experimental study, we decided to use ivermectin as another drug in PEDIM. Ivermectin is a semisynthetic, anthelmintic agent for oral administration. It is derived from the avermectins, a class of highly active broad-spectrum antiparasitic agents isolated from the fermentation products of Streptomyces avermitilis. 13 Ivermectin is also an effective drug against Echinococcus granulosus. 14 17 PEDIM seems to be easier to perform and is accomplished in a short duration of time. The aim of this study was to evaluate the effectiveness of intracystic injection of ivermectin in liver hydatid diseases. Materials and Methods All experiments were performed according to European Community rules of animal care. 18 Twelve sheep naturally infected with liver hydatid diseases were identified with portable ultrasound (US) (SIUI CTS-285, Guangdong, China) for the present study. The sheep were divided into 2 subgroups; the treatment (n 9) and the control group (n 3). Abbreviations used in this paper: PAIR, percutaneous aspirationinjection-reaspiration; PEDIM, percutaneous drug injection method; PT, percutaneous treatment. 2002 by the American Gastroenterological Association 0016-5085/02/$35.00 doi:10.1053/gast.2002.32404

958 HOKELEK ET AL. GASTROENTEROLOGY Vol. 122, No. 4 Figure 1. (A) Serial sonograms of sheep 2; anechoic cyst before percutaneous treatment (between the cursors). (B) The needle is evident on sonogram obtained during PEDIM (arrow). (C) After 6 months, the sonography showed solid appearance of the cyst remnant (between arrows). Blood samples were taken for serum glutamic oxalacetic transaminase, serum glutamic pyruvic transaminase, -glutamyltransferase, alkaline phosphatase, and total bilirubin to test liver functions before the procedure. One cyst was selected for manipulation in each sheep. Selection criteria were easy access and type of cyst. All studied cysts were classified as type 1 according to the classification of Gharbie et al. (Figures 1A and 2A). 19 The diameter of the cysts varied between 22 70 mm (Tables 1 and 2). Ivermectin (I 8898; Sigma-Aldrich, St. Louis, MO) dissolved in sterile 1% dimethyl sulphoxide was added to sterile distilled water to obtain stock ivermectin solution. The puncture site was anesthetized locally by using prilocine 0.5%. An 18-g seldinger (Dispomedica GmbH 22041; Hamburg, Germany) needle was inserted under US guidance via the transhepatic route into the cyst (Figure 1B). The approximate volume of ivermectin solution needed to achieve an intracystic concentration of 10 g/ml, which was previously determined as an effective concentration in an in vitro study, 20 were calculated for each cyst, and determined volume of cystic fluid was aspirated before the drug injection. Immediate parasitologic examination was performed for this material under light microscope. Viability of protoscolices was assessed by staining with 0.1% eosin. Cystic fluid was examined for the presence of bilirubin to determine any biliary fistulization with urinary sticks (Chemstrip-Iris Urine Test strips, IRIStrips; Boehringer Mannheim, GmbH, Mannheim, Germany). After cyst puncture, the procedure was continued only if hydatid cyst fluid contained laminated membranes, scoleces, or hooklets on immediate microscopy. The estimated amount of ivermectin emulsion was injected into the cystic cavity without changing the cyst volume and intracystic pressure. In the control group, distilled water was applied into cysts instead of ivermectin. No reaspirations of cystic contents were performed at all. After injecting ivermectin, the needle was withdrawn immediately. The total procedure was finished within approximately less than 5 minutes. Follow-up was performed with US examination daily in the first week, then weekly during the next 6 months. The

April 2002 PEDIM FOR TREATMENT OF LIVER HYDATID DISEASE 959 Figure 2. (A) Serial sonograms of sheep 8; pure anechoic cyst before treatment in the right lobe of liver (between cursors). (B) Six months after intracystic ivermectin injection; cyst cavity completely solidified (between cursors). changes in cyst size, morphologic changes in cyst contents and cyst wall, and local recurrence or secondary dissemination were evaluated at each visit. Decreases in cyst sizes and progressive solidifications of cyst contents were regarded as positive criteria for healing. Blood samples were obtained at the end of the first week and monthly thereafter. At the end of 6 months, all sheep were killed with ketamine anesthesia and livers were completely excised for histopathologic evaluation. In histopathologic examinations, 5- m thick sections of formalin-fixed and paraffin-embedded tissues were stained with H&E and evaluated under light microscope. Wilcoxon signed rank test was used for statistical analyses. Results Cyst punctures were performed successfully without any complications in all 12 sheep. Results of parasitologic examinations were positive with viable protoscolices for echinococcosis in all cysts. Aspirated fluids were negative for bilirubin in all sheep. In the treatment group, the earliest change was detachment, which occurred within 3 days of the laminated membranes from the pericyst. US evaluations showed a significant decrease in diameters and volumes of cysts (P 0.05) (Table 1). The cysts filled with echogenic materials, progressive solidification, and increases in cyst wall thickness were observed in the follow-up period (Figures 1C and 2B). Serum glutamic oxalacetic transaminase, serum glutamic pyruvic transaminase, -glutamyltransferase, alanine transaminase, and total bilirubin levels were normal before the procedure and remained within normal limits during the follow-up period in both groups. Macroscopic appearance of treated cysts differed significantly from untreated cysts. Treated cysts were filled with amorphous solid gray material. Microscopic examination showed pericyst hyalinization, lymphocyte predominant inflammatory cells in the cyst wall, degeneration of laminated and germinal membranes, dystrophic calcifications, and necrotic material filling the cyst cavity. In all histologic sections there were no viable protoscolices or daughter cysts in the treated group (Figure 3). There was no ductal epithelial proliferation, ductal dilatation, or fibrosis in sections of portal areas. Diameters and volumes of cysts increased in the control group (Table 2). Viable protoscolices and daughter cysts were observed in histologic sections of the control group (Figure 4). Discussion Hydatid disease is still an important health problem not only in endemic areas in developing countries, but also in developed countries because of the increasing number of immigrations. 1,21 Minimally invasive attempts are more favorable than radical surgical treatment and systemic chemotherapy in the treatment protocols of cyst hydatid diseases. Radical surgical treatment is the most effective treatment, but it has considerable limitations, such as anesthetic contraindications for some patients, recurrent diseases, and in inaccessible cysts located centrally in the liver. 2,4 6 Systemic chemotherapy may interfere with liver functions because of the need for long-term use of drugs and may not be effective particularly in large cysts. The response to the chemothera-

960 HOKELEK ET AL. GASTROENTEROLOGY Vol. 122, No. 4 Table 1. Changes Observed on US Scans in the Treatment Group During Follow-Up Sheep no. Size before treatment (mm) Final US pattern Final cyst size (mm) Volume reduction (%) 1 44 38 38 Solid, inhomogeneous 28 26 25 71 2 44 32 35 Solid, homogeneous 32 16 19 80 3 33 28 27 Partially cystic 24 22 22 54 4 70 40 35 Solid, inhomogeneous 42 24 20 79 5 42 33 37 Partially cystic 26 23 24 73 6 55 45 40 Partially cystic 38 34 32 59 7 26 22 22 Solid, inhomogeneous 17 16 15 67 8 35 27 30 Solid, homogeneous 22 18 20 72 9 46 36 30 Partially cystic 27 25 25 66 peutic approach varies with the cyst conditions and the drugs used. 2,3,22,23 The PAIR method has become widely used as a minimally invasive procedure in the treatment of cystic echinococcosis. Scolicidal agents such as hypertonic saline solution or 95% ethanol are used in PAIR. 9,10 PAIR is contraindicated in cases of biliary fistulization. Cysts larger than 6 cm that are treated with catheterization are also disadvantages of PAIR or similar methods because of the increased complication rate. However, PEDIM, which was first described by Deger et al. 12 asamodification of PAIR, has some advantages over the PAIR method especially in suspected biliary fistulization and in large cysts that need catheterizations. In PEDIM, an effective scolicidal drug was injected into the cyst percutaneously instead of catheterization and the cyst volume and intracystic pressure remain unchanged. It is also superior to PAIR according to shorter duration of application time and low risk for sclerosing cholangitis. The drugs used in PEDIM such as albendazole sulphoxide may not be toxic to the liver and biliary tract, therefore, even if the patient has a biliary fistula, PEDIM may be performed without any biliary complications. PEDIM is also cost-effective compared with surgery. In addition, general anesthesia is not needed in contrast to surgery. In our experimental study, the classifications of various appearances of the liver hydatid cysts were performed according to US findings. Percutaneous treatments of type 1 and 2 cysts, according to Gharbie classification, Table 2. Volume Changes and Final US Patterns of Cysts in the Control Group During Follow-Up Sheep no. Size before treatment (mm) Final US pattern Final cyst size (mm) Volume changes 1 47 35 30 Pure 48 35 33 12% increase anechoic 2 27 24 25 Pure anechoic 28 26 26 17% increase 3 39 33 37 Pure anechoic 40 35 37 9% increase are the most appropriate types for the PT. 19,24 Therefore, we studied type 1 cysts in naturally infected sheep. Naturally infected sheep have 2 major advantages; first, it is difficult to create an experimental hydatid disease in the liver, and both human subjects and sheep share the similarity of being intermediate hosts of the parasites. 12,24 Scolicidal solutions are essential in the treatment of hydatid cyst disease. Properties of an ideal solution would be a rapid and complete scolicidal effect, absence of local and systemic side effects, and inexpensiveness. However, no ideal solution has been described yet and either the low scolicidal activity or the complications especially at the biliary system of the presently used solutions need investigation for more ideal agents. 12 The caustic sclerosing cholangitis is a well-known complication of chemical scolicidal agents used in both surgery and percutaneous treatment. 25 Ivermectin, a macrocyclic lacton, is widely used as an antiparasitic agent. It is also effective in echinococcus granulosus. Ivermectin shows effects faster than levamisole and causes contraction and paralysis of protoscolices. 15 Ivermectin or combinations with albendazole and albendazole sulphoxide inhibit the development of protoscoleces. 16 Ivermectin was also used with direct injection method at laparotomy into cysts. 14 Incubation with ivermectin causes damages to the protoscolices as indicated by viability loss, decreased protein content, and alteration of hsp60 and hsp70 levels. 17 A dose of 10 g/ml ivermectin was also found to be a strong scolicidal agent after the incubation period of 15 minutes. 20 Therefore, we used ivermectin in PEDIM as an effective drug. In the present study we obtained significant reduction in cyst sizes and solidification of the cysts. Pathologic examination also confirmed the effectiveness of the method with no remaining viable scolices. There were no changes in liver function tests and pathologic examination showed no ductal epithelial proliferation, ductal dilatation, or fibrosis in sections of portal areas. These

April 2002 PEDIM FOR TREATMENT OF LIVER HYDATID DISEASE 961 Figure 3. Histologic examination of sheep 3 of treatment group. Degeneration of laminated membrane and germinal membrane, pericyst hyalinization, and lymphocyte infiltration are seen. There are no viable protoscolices or daughter cysts (H&E; original magnification 100 ). findings suggest ivermectin used in PEDIM such as albendazole sulphoxide may also have no toxic effect on the liver and biliary system. However, further in vitro and in vivo studies for determination of the safety of ivermectin injection on hepatic and biliary system in animals and formal Phase 1 and Phase 2 trials in humans must be conducted before it can be recommended for human use. Intracystic ivermectin application seems to be effective and safe. It may be used successfully to treat cysts of hydatid disease without significant adverse effects. The advantages of PEDIM are time saving, cost-effectiveness, and probable low risk for sclerosing cholangitis. The result of this study on sheep is encouraging, but further studies should be performed for determining the applicability to human patients. Figure 4. Histologic examination of sheep 1 of control group. Protoscolices, intact laminated and germinal membrane are seen (H&E; original magnification 100 ).

962 HOKELEK ET AL. GASTROENTEROLOGY Vol. 122, No. 4 References 1. Ammann RW, Eckert J. Cestodes. Echinococcus. Gastroenterol Clin North Am 1996;25:655 689. 2. El Mufti M. Surgical management of hydatid disease. London, Butterworth, 1989. 3. Akhan O, Ozmen MN, Dincer A, Sayek I, Gocmen A. Liver hydatid disease: long term results of percutaneous treatment. Radiology 1996;198:259 264. 4. Di Matteo, Bove A, Chiarini S, Capuano LG, De Antoni E, Lanzi G, Lippolis G, Campana FP. Hepatic Echinococcus disease: our experience over 22 years. Hepatogastroenterology 1996;43: 1562 1565. 5. Leese T, Bismuth H. Surgical management of space-occupying lesions in the liver. Baillieres Clin Gastroenterol 1989;3:253 277. 6. Giordano G, Grimaldi F, Carrassa G, Ialongo P, D Abbicco D, Ventolone R, Cafagna L, Carbonara G. The rationale in surgery of hepatic echinococcosis: total pericystectomy and resections. Personal experience. G Chir 1995;16:213 218. 7. Al Karavi MA, el-shiekh Mohomed AR, Yasawy MI. Advances in diagnosis and management of hydatid disease. Hepatogastroenterology 1990;37:327 331. 8. Bret PM, Fond A, Bretagnolle M, Valette PJ, Thiesse P, Lambert R, Labadie M. Percutaneous aspiration and drainage of hydatid cysts in the liver. Radiology 1988;168:617 620. 9. Bastid C, Azar C, Doyer M, Sahel J. Percutaneous treatment of hydatid cysts under sonographic guidance. Dig Dis Sci 1994;39: 1576 1580. 10. Khuroo MS, Zarger SA, Mahajan R. Echinococcus granulosus cysts in the liver: management with percutaneous drainage. Radiology 1991;180:141 145. 11. Mueller PR, Dawson SL, Ferrucci JT Jr, Nardi GL. Hepatic echinococcal cyst: successful percutaneous drainage. Radiology 1985; 155:627 628. 12. Deger E, Hokelek M, Deger BA, Tutar E, Asil M, Pakdemirli E. A new therapeutic approach for the treatment of cystic echinococcosis: percutaneous albendazole sulphoxide injection without reaspiration. Am J Gastroenterol 2000;95:248 254. 13. Koch H. Ivermectin: novel systemic antiparasitic agent. Pharm Int 1984;5:55 56. 14. Ochieng -Mitula PJ, Burt MD. The effects of ivermectin on the hydatid cyst of Echinococcus granulosus after direct injection at laparotomy. J Parasitol 1996;82:155 157. 15. Casado N, Rodriguez-Caabeiro F, Jimenez A, Criado A, de Armas C. In vitro effects of levamisole and ivermectin against Echinococcus granulosus protoscoleces. Int J Parasitol 1989;19:945 947. 16. Casado N, Perez-Serrano J, Denegri G, Rodriguez-Caabeiro F. Development of truncated microtriches in Echinococcus granulosus protoscolices. Parasitol Res 1994;80:355 357. 17. Martinez J, Perez-Serrano J, Bernadina WE, Rodriguez-Caabeiro F. Echinococcus granulosus: in vitro effects of ivermectin and praziquantel on hsp60 and hsp70 levels. Exp Parasitol 1999;93:171 180. 18. Waldegrave W. Directive CEE 86/609. J Officiel Communautés Européennes. 1986;L358:1 28. 19. Gharbi HA, Hassine W, Brauner MW, Dupuch K. Ultrasound examination of the hydatid liver. Radiology 1981;139:459 463. 20. Hokelek M, Deger E, Erzurumlu K, Uyar Y. Ivermektin in Echinococcus granulosus protoskoleksleri uzerine in vitro etkisi. Turkiye Parazitoloji Dergisi 2000;24:43 46. 21. King CH. Cestodes (tapeworms). In: Mandell GL, Bennett JE, Dolin R, eds. Principles and practice of infectious diseases (Vol. 2, 5th ed.). Philadelphia: Churchill Livingstone, 2000:2962 2963. 22. Todorov T, Vutova K, Mechkov G, Tonchev Z, Georgiev P, Lazarova I. Experience in the chemotherapy of severe, inoperable echinococcosis in man. Infection 1992;20:19 24. 23. Anadol D, Ozcelik U, Kiper N, Gocmen A. Treatment of hydatid disease. Paediatr Drugs 2001;3:123 135. 24. Akhan O, Ozmen MN. Percutaneous treatment of liver hydatid cysts. Eur J Radiol 1999;32:76 85. 25. Castelano G, Moreno-Sanchez D, Gutierrez J, Moreno-Gonzalez E, Colina F, Solis-Herruzo JA. Caustic sclerosing cholangitis. Report of four cases and a cumulative review of literature. Hepatogastroenterology 1994;41:458 470. Received November 5, 2001. Accepted December 20, 2001. Address requests for reprints to: Emin Deger, M.D., Department of Radiology, Fair Hospital, 03001, Afyon, Turkey. e-mail: emindeger@hotmail.com; fax: (90) 272-215-7343.