Article Hydatid Liver Cyst An 11-Year Experience of Treatment With Percutaneous Aspiration and Ethanol Injection Antonio Giorgio, MD, Luciano Tarantino, MD, Giorgio de Stefano, MD, Giampiero Francica, MD, Nicola Mariniello, MD, Nunzia Farella, MD, Anna Perrotta, MD, Vincenza Aloisio, MD, Francesco Esposito, MD Objective. To report an 11-year experience of treatment of hydatid liver cysts with double percutaneous aspiration and injection of alcohol. Methods. Of the 129 patients with 174 hydatid liver cysts admitted to our department between January 1988 and January 1999, 79 patients with 119 vital hydatid liver cysts were selected for double percutaneous aspiration and injection of alcohol. Under ultrasonographic guidance, cystic cavities were first drained through fine needles, and then 95% sterile ethanol was injected and left in situ. The same procedure was repeated 3 days later without reaspiration of the injected alcohol. General anesthesia without endotracheal intubation was performed in 21 selected cases. Results. Double percutaneous aspiration and injection of alcohol was completed in 78 patients with 118 hydatid liver cysts. In 1 case the procedure could not be accomplished because of an intracystic hemorrhage. A total of 254 punctures were performed, and the ethanol injected per session ranged between 12 and 250 mL. The mean hospital stay was 2.9 days (range, 2–7 days). The overall median follow-up was 48 months (range, 6–122 months). At the last ultrasonographic examination, 45.8% of the treated hydatid liver cysts had a solid pattern, 47.4% were no longer appreciable, and 6.8% had a minimal liquid component. Intracystic relapse occurred in 5% of the patients. In no case were any new cysts observed either in different hepatic segments or in any extrahepatic location. The morbidity rate was 9%, and 1 death occurred (mortality rate, 1.3%). Conclusions. Over a long period, double percutaneous aspiration and injection of alcohol proved to be a substantially safe, effective, and low-cost procedure for hydatid liver cyst treatment. Key words: hydatid disease; liver; ultrasonography; interventional procedure; ethanol injection. Abbreviations D-PAI, double percutaneous aspiration and injection; HLC, hydatid liver cyst; IHA, indirect hemagglutination assay; WHO, World Health Organization Received December 27, 2000, from the Servizio di Ecografia Interventistica, Ospedale “D. Cotugno,” Naples, Italy. Revision requested January 17, 2001. Revised manuscript accepted for publication February 20, 2001. Address correspondence and reprint requests to Antonio Giorgio, MD, Viale Colli Aminei 491, 80131 Naples, Italy. L iver hydatidosis in humans is a parasitic disease caused by the larval form of a tapeworm, Echinococcus granulosus.1 The disease develops when the parasite’s eggs are ingested and the embryonic forms are released into the intestinal lumen and cross the mucosa, reaching the hepatic sinusoids via the portal circulation. The hydatid liver cyst (HLC) represents the full-blown expression of the vital parasite,1 whereas a number of other morphologic features that are finely depicted by ultrasonography2,3 represent various stages in the parasite’s life cycle. The most recent World Health Organization (WHO) classification of liver hydatidosis considers all morphologic features that contain liquid (unilocular with or without membrane detachment © 2001 by the American Institute of Ultrasound in Medicine • J Ultrasound Med 20:729–738, 2001 • 0278-4297/01/$3.50 Hydatid Cyst Treatment With Percutaneous Aspiration and Ethanol Injection and multilocular) as signs of living worms, whereas solid and calcific forms are indicative of the parasite’s death.4 According to this classification, only those patients with vital cysts are suitable for therapy. Although liver hydatidosis is considered a benign disease, it is generally accepted that the severity and frequency of complications of vital HLCs, especially in endemic areas, make therapy mandatory, with surgery being the standard form of treatment.5,6 Surgery may be radical (i.e., total pericystectomy or liver resection in the case of multiple cysts localized in 1 lobe) or conservative (fenestration or simple tube drainage).5,6 Recently, laparoscopic surgery has been introduced to remove HLCs in selected cases.7 Over the last decade, high costs, lack of surgical facilities, or both in those countries where the disease is most prevalent,5 together with an increase in the number of human cases of echinococcosis detected in population-based studies and resulting from widely available ultrasonographic examinations5 and the development of minimally invasive procedures such as percutaneous treatment of a variety of inflammatory and neoplastic liver and abdominal diseases,8,9 have prompted the search for alternative methods of treatment for liver hydatidosis. Medical therapy with an imidazole derivative is rarely curative,5,6,10,11 and side effects occur in as many as 35% of patients,11 with a 30% relapse rate also being recorded.12 Percutaneous injection of a scolicidal agent (absolute alcohol and hypertonic saline) has been widely used in purely cystic hydatid disease, especially in Mediterranean and Asian countries, with excellent results in terms of the disappearance of liver cysts and low morbidity and mortality rates.13–21 Moreover, 2 randomized trials by Khuroo et al22,23 showed that the percutaneous approach was superior to medical therapy alone and had a cure rate of liver cysts equal to that of surgical cyst drainage but at lower costs and with a reduced complication rate. Some of the most relevant questions about the long-term efficacy and safety of percutaneous treatment have been addressed by recent studies24–26 in which no case of either spillage of the echinococci into the abdomen or intrahepatic or extrahepatic diffusion of the hydatid disease was reported. The involutional changes after percutaneous treatment appeared stable during the follow-up period, with a mean observation time of 19 to 37 months (range, 1–72 months). 730 In 1992 we reported our initial experience with double percutaneous aspiration and injection (D-PAI) of alcohol,19 and the impressive results achieved encouraged us to pursue that therapeutic approach over the ensuing years. In this article, we report our 11-year experience with D-PAI in the treatment of HLCs and the long-term follow-up data. Materials and Methods Study Population Between January 1988 and January 1999, 129 patients (68 male and 61 female; mean age, 42 years; age range, 13–80 years) with 174 HLCs were examined at our institution. In 90 cases the hydatid origin of liver cysts was suggested by positive serologic text results (immunohemagglutination >1:128); in the remaining patients, diagnosis was based on clinical (known exposure to dogs or abdominal pain)5 or ultrasonographic findings consistent with hydatidosis, or both.2,3 In Table 1 the number, size, site, and ultrasonographic patterns according to the WHO classification4 of HLC are shown. There were 25 symptomatic patients, all with type I to III HLCs. Thirteen patients had right upper quadrant pain usually associated with a palpable mass; 6 had disease recurrence after surgical fenestration of the cysts; and 6 had jaundice caused by mild dilatation of the bile ducts near their HLCs. Extrahepatic disease was absent in all cases. The D-PAI Technique As reported elsewhere,19 we used a protocol that entailed puncturing the cyst with a fine needle twice every 3 days and injecting sterile absolute ethanol without reaspiration as follows. Under aseptic conditions and after administration of local anesthesia at the point of puncture, a 15- or 20-cm-long fine needle (22, 20, or 18 gauge) was inserted into the cystic cavity by a transhepatic route under ultrasonographic guidance, with every effort being made to avoid direct puncture of the cyst. After as much as 90% of the fluid content was aspirated, 95% sterile ethanol was injected into the cyst and left in situ to replace 50% to 60% of the amount drained. Immediately after aspiration, direct fresh microscopic examination was carried out for detection of fragments of laminated membranes, hooklets, and scolices, J Ultrasound Med 20:729–738, 2001 Giorgio et al Table 1. Characteristics of HLC Series WHO Class* Type Type Type Type Type Type I II III IV V VI No. of Patients No. of HLCs 14† 45† 13 7† 37 13 18 58 26 17 42 13 Site, Size [Mean Right Lobe/Left Lobe Diameter (Range)], cm 12/6 40/18 20/6 10/7 30/12 10/3 2.8 (1.5–4) 8.5 (4.3–20) 4.7 (2.5–7) 5.2 (4–7) 3.7 (2–5) 3.3 (2–4.2) IHA Titer, Range 1:256–4,000 1:64–1:16,800 1:256–1:2,000 1:64–1:516 1:64–1:1,024 1:64–1:512 *Type I indicates unilocular cyst smaller than 4 cm; type II, unilocular cyst larger than 4 cm; type III, multiloculated; type IV, detached membrane; type V, solid heterogeneous; and type VI, calcific. †In 4 patients there were multiple HLCs (3 or 4) with different patterns (I, II, and IV). and staining with 0.1% neutral red for vital scolices and 0.03% methylene blue for dead scolices was performed. To exclude the presence of a biliary fistula, the bilirubin level in the aspirated fluid was determined by a semiquantitative method (Combur-Test; Roche Diagnostics, Indianapolis, IN) within a few seconds before alcohol injection at each PAI. The same procedure was repeated after 3 days without reaspiration of the injected alcohol; scolex survival was also reassessed. To reduce the risk of intraperitoneal seeding of scolices possibly spilled during the interventional procedure, a brief course of mebendazole (3 g/d in the first 10 cases) or albendazole (800 mg/d in all remaining cases) 1 week before and 3 weeks after D-PAI was completed in all patients but 2 (1 pregnant woman and 1 patient with chronic liver disease). No other form of therapy was carried out. The procedure was always done on an inpatient basis with an anesthesiologist present. General anesthesia without endotracheal intubation (using atropine, 0.5 mg intravenously; induction with propofol, 1.0–1.5 mg/kg intravenously; maintenance with propofol, 9.0– 12 mg ⋅ kg–1 ⋅ h–1; and fentanyl citrate, 50 mg intravenously) was administered in 21 cases (anxious patients, very large cysts, and difficult technical approaches). The procedure was approved by our Institutional Ethical Committee. Informed written consent was obtained from all patients. Follow-up After the interventional procedure, all patients underwent clinical, laboratory, and ultrasonographic examinations every month for the first 3 months and every 6 months thereafter. Indirect hemagglutination assay (IHA) titers were deterJ Ultrasound Med 20:729–738, 2001 mined monthly during the first 6 months and then twice a year. Chest radiography was scheduled every 6 months, and a total body computed tomographic scan was scheduled every 2 years. Results Double percutaneous aspiration and injection of alcohol was accomplished in 78 of 79 patients with 118 HLCs (group I); in 1 case aspiration of fresh blood due to intracystic hemorrhage forced us to give up the interventional procedure (see “Complications”). In 37 of 50 patients with ultrasonographic pattern V, ultrasonographically guided aspiration yielded a creamy orange substance (“orange juice-like”) without vital scolices when further examined. All patients with type V HLCs and 13 patients with a calcific pattern (type VI) were not treated (group II). In group I, 254 punctures were performed, and ethanol injected per session ranged between 12 and 250 mL. Seventy-four patients had 1 course of D-PAI; 3 patients had 3 procedures; and 1 patient had 4 procedures. These latter 4 patients had multiloculated cysts, which represented the only cases of relapsing disease after the initial percutaneous treatment (see below). The durations of the interventional procedures ranged between 15 and 45 minutes, and the mean hospital stay was 2.9 days (range, 2–7 days). In all type I cysts, detachment of the inner membrane was observed just after the first aspiration and ethanol injection. Type II cysts had further detachment and folding of inner membranes. Generally speaking, hydatid cysts regained their initial volume almost entirely within 24 hours after the first PAI. In symptomatic patients, loss of cyst tension caused 731 Hydatid Cyst Treatment With Percutaneous Aspiration and Ethanol Injection immediate disappearance of abdominal tumefaction and abdominal pain. The overall median follow-up was 48 months (range, 6–122 months). Twenty-seven patients (34.6%) had follow-up of 1 to 4 years; 31 patients (38.9%) were followed for 5 to 8 years; and 20 patients (25.6%) were followed for more than 9 years. The follow-up durations for individual types of hydatid disease and the ultrasonographic outcomes of the treated cysts at the last observations are shown in Table 2. of evolution was never observed in multiloculated cysts, whereas it occurred without any significant differences among the other cyst types (55.5% in type I, 63% in type II, and 59% in type IV). The time taken to attain the final ultrasonographic pattern varied considerably. Generally speaking, the smaller, pure cysts (type I) took a short time for ultrasonographic pattern changes to become evident (15–70 days), whereas the largest, complex cysts took longer (up to 2 years). However, the type of the final ultrasonographic pattern was not strongly correlated to either initial HLC volume or follow-up length. As far as the latter point is concerned, in most of the patients with a healed pattern, an intermediate phase of solidification was observed but proved to be independent of the follow-up time. Similarly, even patients with long follow-up durations (>2 years) and a solid or liquid pattern had no changes over time. In patients with initial high IHA titers (>1:1024), levels of antibodies decreased over time and in 17 patients returned to normal. In Figures 1 and 2, two examples of the ultrasonographic aspects of the interventional procedure and the final ultrasonographic appearance are shown. Group II (untreated) patients did not have any change in size or ultrasonographic pattern during follow-up. Four patients (5%) were treated more than once because of relapse; they had multiloculated cysts, which after 6, 12, 15, and 24 months had new, vital daughter cysts inside the posttreatment heterogeneous masses. Three of these patients underwent another 2 sessions of PAI for their large cysts (6, 7, and 7.5 cm), and the last observed ultrasonographic pattern was a solid mass. The fourth patient had 3 multiloculated Ultrasonographic Findings At the last ultrasonographic examination, 3 ultrasonographic patterns were observed: 1. A liquid pattern characterized by detached and folded membranes floating in hydatid fluid. The 8 cysts with this pattern (6.8%) had fine-needle aspiration to verify parasite survival. In all cases, an orange juice-like substance was obtained consisting of leukocytes and necrotic debris without vital scolices. 2. A solid pattern (hypoechoic or hyperechoic mass). This pattern was observed in 54 (45.8%) of 118 cysts. It is worth noting that this was the final ultrasonographic aspect in all patients with multiloculated cysts, being significantly different (P < .01) from all other types of HLC, in which such a pattern occurred in about 30% of cases. In these 2 first patterns, a constant decrease in cyst size of about 50% to 80% of the initial volume was observed in the course of follow-up. 3. A healed pattern. Fifty-six (47.4%) of 118 liver cysts were undetectable, with complete reconstitution of liver parenchyma. This kind Table 2. Ultrasonographic Patterns of HLCs After Percutaneous Treatment WHO Class No. of Patients No. of HLCs Follow-up [Median (Range)], mo Type I 14 18 58 Type II 44 57 60 (12–122) Type III Type IV 13 7 26 17 38 (12–84) 44 (6–96) 732 (9–112) Sonographic Pattern at Last Examination, No. of Cases Healed Liquid Solid Healed Liquid Solid Solid Healed Liquid Solid (10) (3) (5) (36) (3) (18) (26) (10) (2) (5) J Ultrasound Med 20:729–738, 2001 Giorgio et al cysts of 5, 4.5, and 2.5 cm, and all were treated with 3 additional courses of D-PAI. In this case, a solid pattern was also observed at the last ultrasonographic examination. In no case were any new cysts observed either in different hepatic segments or in any extrahepatic location (namely, the thorax and abdomen). that, in all patients a short course of betamethasone (1.5 mg 3 times per day 2 days before and 1 day after the procedure) was administered, and the interventional procedure was carried out under general anesthesia in selected cases (see “Materials and Methods”). Discussion Complications As stated above, in 1 case D-PAI could not be accomplished because of an intracystic hemorrhage, which supervened just after insertion of an 18-gauge needle. Blood transfusions and emergency surgery were not required. Double percutaneous aspiration and injection of alcohol was postponed, but 2 weeks later a new puncture yielded fresh blood again, and the patient had to undergo surgery. This case represented the only failure of the interventional procedure in our series. Minor side effects were nausea and vomiting (1 case) and abdominal pain lasting 15 minutes (2 cases). Major complications were recorded in 2.5% of the treated patients. Hepatic abscesses developed inside 2 treated cysts (2 patients) 15 and 60 days after D-PAI; in both cases the abscess cavities were drained percutaneously, and at the last ultrasonographic scan, reconstitution of liver parenchyma was observed without any focal lesion. A biliary fistula was diagnosed in 1 case when bile-stained fluid was collected during the second aspiration; given the patient’s stable clinical condition, no other diagnostic or therapeutic procedures were carried out. The second ethanol injection was postponed until 6 months after spontaneous closure of the fistula. We had 1 case of procedure-related death (mortality, 1.3%), which occurred in a 54-year old man with a 9-cm uniloculated liver cyst in the seventh segment. Rash, dyspnea, and hypotension occurred just after aspiration of 5 mL of hydatid liquid through a 20-gauge Chiba needle (Ecojekt, Hospital Service, Tokyo, Japan). Despite every attempt to resuscitate him, the patient died 3 hours after the inception of the procedure. We hypothesized that a large amount of hydatid fluid had gained access to the general circulation through the liver parenchyma because of ruptures in the laminated membrane, which were caused by unintentional movement of the patient, who had difficulty tolerating the procedure. That occurrence in June 1998 caused us to change our methodological approach. After J Ultrasound Med 20:729–738, 2001 Surgery is still regarded as the standard form of treatment for liver hydatidosis caused by E granulosus.5,6 The 2 surgical approaches (i.e., radical and conservative) may be chosen on the basis of local expertise and the characteristics of each individual case (e.g., number, size, and location of the cysts).5 Generally speaking, the more radical the intervention, the higher the intraoperative risk but the fewer late complications; moreover, radical interventions, albeit the most desirable, are the least frequently performed, representing only 21% of the 677 patients in a recent surgical series.27 On the other hand, the more conservative the approach, the lower the interventional risk but the more common the late complications, which include fistula formation, bacterial infections, sepsis, and local cyst recurrence. On the whole, hydatid recurrences may be wide ranging depending on the diagnostic tool used to detect them (serology, laparoscopy, ultrasonography, or computed tomography), ranging from 1% to 30%.28–30 The morbidity rates of surgery may be as high as 19% to 84%,5,28–30 with mortality ranging between 1% and 60%.5,28–30 More recently the laparoscopic approach has been introduced to treat selected cases of HLCs, but the conversion rate has been reported as high as 23% in a recent series,7 and to date only short-term follow-up results have been available. Over the last decade there has been growing evidence that percutaneous treatment of uncomplicated hydatid liver disease may be a valid alternative to surgery, because it offers an effective and inexpensive therapeutic tool, particularly in those regions where the disease is endemic and medical resources are limited.13–26 Two recent randomized trials by Khuroo et al22,23 showed that percutaneous treatment of HLCs proved to be a superior form of medical treatment when used with albendazole and proved as effective as open surgical drainage but at a lower cost and with a more reduced complication rate than surgery. 733 Hydatid Cyst Treatment With Percutaneous Aspiration and Ethanol Injection Figure 1. Double percutaneous aspiration and injection of alcohol in hydatid cyst (type II). A, Purely liquid cyst (c) shown in the left lobe of the liver. B, The fine needle (arrow) is shown inside the cyst during aspiration. C, Ultrasonographic aspect at the end of aspiration. The needle tip (A) is clearly shown in the small residual cavity before ethanol injection. D, After injection of 125 mL of ethanol, the cyst cavity regained initial volume; it appears hyperechoic (arrows). E, Three days later another PAI was performed; the needle tip (white arrow) is shown in the cyst. Note the detached inner membranes after the first PAI. F (opposite page), One month later. The cyst is decreased in size and shows a solid pattern. G (opposite page), Two months after D-PAI. Ultrasonography shows the restitution of the liver parenchyma. G indicates gallbladder; and L, normal left lobe. A 734 B C D E J Ultrasound Med 20:729–738, 2001 Giorgio et al F G Our long-term experience with D-PAI confirms the good results in terms of the feasibility and effectiveness of this ultrasonographically guided interventional procedure, in keeping with the findings of other authors.15,16,18,20,21,23–26 In only 1 patient were we unable to carry out D-PAI, because of an intracystic hemorrhage. In the 78 cases in which D-PAI was performed, the 3 patterns we observed on the last ultrasonographic follow-up examinations (resolved in 47.4%, liquid in 6.8%, and solid in 45.8%) were all expressions of therapeutic success. Even in those cases with a residual liquid component, the aspirated fluid yielded an orange juice-like substance made up of necrotic debris. The most notable exception was represented by the multiloculated cysts. Only in patients bearing that type of HLC was complete healing of liver parenchyma never observed, and intracystic relapse occurred in 30% of this group (i.e., in 5% of the entire series), which required further cycles (up to 4) of D-PAI. This kind of cyst may necessitate more aggressive treatment; i.e., all cystic cavities should be punctured and injected with ethanol, and stricter follow-up should be instituted to recognize early relapse. The results for the multiloculated cysts may be explained by the reported high resistance of daughter cysts to scolicidal agents, as shown in an experimental setting.31 As far as side effects are concerned, it should be stressed that the complication rate was acceptably low (≈9% when both minor and major complications were considered), nor was it much different from that reported in other series dealing with a percutaneous treatment, and it proved to be lower than that reported in surgical series, ranging between 18% and 84%.6,23,25,30 It should be noted that the major complications were managed without resorting to surgery: in 2 cases percutaneous evacuation of superimposed abscesses was required, whereas the biliary fistula resolved spontaneously. In all those cases, D-PAI was accomplished, and the final outcome was uneventful. We had 1 death (mortality rate, 1.3%), to our knowledge the second so far reported in series dealing with percutaneous treatment of HLC, the other being a case reported by Men et al.25 In our opinion, the death in our series was attributable to rupture of a large cyst with massive absorption of hydatid fluid in the systemic circulation, which was caused by movement of an anxious patient who did not tolerate the procedure well at the time of needle insertion. After that incident, we decided in selected cases (very anxious patients, large cysts, and difficult technical approaches) to administer neuroleptanalgesia with or without tracheal intubation. That a fatal complication may occur after percutaneous treatment of HLCs emphasizes the point that such an interventional technique must be performed in specialized centers with surgical and anesthesiologic resources at hand. Our high success rate along with low morbidity and mortality rates may by explained by certain methodological choices we made at the beginning of our experience. First, we decided to use alcohol because of its well-proven scolicidal effect by dehydrating vital organisms13,15; indeed, 95% ethanol is considered the scolicidal agent with the strongest inflammatory effects J Ultrasound Med 20:729–738, 2001 735 Hydatid Cyst Treatment With Percutaneous Aspiration and Ethanol Injection Figure 2. Double percutaneous aspiration and injection of alcohol in a multilocular hydatid liver cyst. A, Multiseptated cyst shown in the seventh hepatic segment. B, One of the cavities has been punctured. A indicates needle tip. C, The cavity is filled with ethanol. D, The needle (arrow) is inserted in another cavity. E, Ultrasonographic appearance 3 months after completion of the procedure. This pattern remained unchanged after 4 years. A 736 B C D E J Ultrasound Med 20:729–738, 2001 Giorgio et al and has been shown to be highly effective in destroying the germinative membrane where living protoscolices can usually be found.26 Second, we left injected alcohol in situ without reaspiration in the belief that longer contact between the parasite and the scolicidal agent would have a greater likelihood of killing the scolices. Indeed, early in our experience we found vital parasites in 2 patients 3 days after the first aspiration and injection.19 Further support for our approach has recently come from an experimental study in which scolicidal agents (alcohol and hypertonic saline) proved unable to kill daughter cysts when brought into contact with them for 24 h.31 Moreover, partially refilling the cyst, along with the strong inflammatory response elicited by alcohol, appears to hamper leaks into the biliary tree through small fissures in the pericyst, thereby explaining the lower rate of biliary fistula formation (1.2%) when compared with that of about 6% reported by Ustunsoz et al24 and Men et al.25 It is worthy of note that no case of sclerosing cholangitis, one of the most serious complications of scolicidal agents,32 was observed in any of our patients. Criticisms about the long-term efficacy of PAI have been expressed,33 the most important question being the long-term prognosis of these patients with special regard to relapse of the disease in other liver segments or elsewhere in the body, namely the peritoneum and thorax, perhaps as a consequence of spillage of hydatid fluid. As in other recent series,24–26 in none of our patients (65% of whom were followed for >4 years) did such an event occur. Systemic prophylaxis with albendazole might have played a contributory role, because it has been shown to reduce relapse of hydatid disease after surgery.6 The point should be stressed that in our experience and that of others,24–26 not only did the observed posttreatment changes indicate cure of the disease over a long period, but also the relapse rate was very low (in our series only for multiloculated cysts), and extrahepatic diffusion was never observed. Another favorable characteristic of the percutaneous treatment modality was its costeffectiveness. The hospital stay in our series, as in other series,23–26 turned out to be far shorter than that after surgery, especially if more radical procedures were performed.26 J Ultrasound Med 20:729–738, 2001 In conclusion, percutaneous ethanol injection for HLCs is a cost-effective procedure not only in the short term (rapid reduction in size, low complication rate, and short hospital stay) but also in the long term, because intracystic relapse never occurred, except in a few multiloculated cysts, nor did intrahepatic or extrahepatic dissemination supervene. However, although very low in number, life-threatening complications make it necessary for this kind of therapeutic modality to be reserved for centers where resuscitation measures and surgical facilities are at hand. References 1. Rausch RL. Echinococcus granulosus: biology and ecology. In: Andersen FL, Ouhelli H, Kachani M (eds). 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