Principal authors

#### **References**


[9] Garippa G: Updates on Cystic Echinococcosis (CE) in Italy. Parassitologia 48: 57–59, 2006.

**Author details**

132 Current Topics in Echinococcosis

Stefano Puleo1

Catania, Italy

Principal authors

**References**

#

Antonella Agodi1 #, Martina Barchitta1

University of Catania, Italy

, Amy Giarrizzo1

co. Gazz San 9: 428-433, 1965.

eases 15: e611–e619, 2011.

475-494, 2003.

coccosis. Lancet Infectious Diseases 7: 385–394, 2007.

cyst.Ann Med Health Sci Res 204:447-522014.

www.who.int/echinococcosis/about\_disease/en/.

Neglected Tropical Diseases 5: article e893, 2011.

\*Address all correspondence to: dicataldoa@tiscali.it

Antonio Di Cataldo1 \* #, Giuseppe Petrillo2 #, Claudia Trombatore2 #, Stefano Palmucci2 #,

1 Department of Medical and Surgical Sciences, Advanced Technologies GF Ingrassia,

3 Department of Respiratory Diseases and Allergology, University of Catania, Italy

2 Radiodiagnostic and Radiotherapy Unit, University Hospital Policlinico-Vittorio Emanuele,

[1] Grassi G: Contributo allo studio di alcune localizzazioni rare delle cisti d'echinococ‐

[2] Craig PS, McManus DP, Lightowlers MW: Prevention and control of cystic echino‐

[3] Sachar S, Goyal S, Sangwan S: Uncommon locations and presentations of hydatid

[4] World Health Organization. About echinococcosis. Available online at: http://

[5] Hotez PJ, Gurwith M: Europe's neglected infections of poverty. Int J Infectious Dis‐

[6] Rojo-Vazquez FA, Pardo-Lledias J, Francos-Von Hunefeld M: Cystic echinococcosis in Spain: Current situation and relevance for other endemic areas in Europe, PLoS

[7] Polat P, Kantarci M, Alper F: Hydatid Disease from Head to Toe. Radiographics 23:

[8] European Centre for Disease Prevention and Control. Annual epidemiological report 2014—food and waterborne diseases and zoonoses. Stockholm: ECDC; 2014.

, Martina Calabrini1

, Annalisa Quattrocchi1

, Rosalia Latino1

, Nunzio Crimi3

, Silvia Fichera3

and Rosanna Portale1

,


[39] Nunnari G, Pinzone MR, Gruttadauria S, Celesia BM, Madeddu G, Malaguarnera G, Pavone P, Cappellani A, Cacopardo B: Hepatic echinococcosis: Clinical and thera‐ peutic aspects. World J Gastroenterol 18: 1448–1458, 2012.

[25] Szanto P, Goian I, Al Hajjar N, Badea R, Seicean A, Manciula D, Serban A: Hydatic cyst of the pancreas causing portal hypertension. Maedica (Buchar). 5(2): 139–41,

[26] Volders WK, Gelin G, Stessens RC: Hydatid cyst of the kidney: Radiologic-pathologic

[27] Ishimitsu DN, Saouaf R, Kallman C, Balzer BL: Best cases from the AFIP: Renal hyda‐

[28] Pickhardt PJ, Bhalla S: Unusual non-neoplastic peritoneal and subperitoneal condi‐

[29] Yuksel M, Demirpolat G, Sever A, Bakaris S, Bulbuloglu E, Elmas N: Hydatid disease involving some rare locations in the body: A pictorial essay. Korean J Radiol 8: 531–

[30] Yildirim M, Erkan N, Vardar E: Hydatid cyst with unusual localizations: Diagnostic and treatment dilemmas for surgeons. Ann Trop Med Parasitol 100: 137–142, 2006.

[31] Di Cataldo A, Latino R, Cocuzza A, Li Destri G: Unexplainable development of a hy‐

[32] Di Cataldo A, Puleo S, Li Destri G, Racalbuto A, Trombatore G, Latteri F, Rodolico G: Splenic trauma and overwhelming post-splenectomy infection. Br J Surg 74: 343–345,

[33] Guisantes JA, Vincente-Garcia F, Abril JM, Eraso E, Martinez J: Total and specific IgE levels in human hydatid disease determined by enzyme immunoassay: Serological

[34] Riganò R, Ioppolo S, Ortona E, Margutti P, Profumo E, Ali MD, Di Vico B, Teggi A, Siracusano A: Long-term serological evaluation of patients with cystic echinococcosis treated with benzimidazole carbamates. Clin Exp Immunol 129: 485–492, 2002.

[35] Boyano T, Moldenhauer F, Mira J, Joral A, Saiz F: Systemic anaphylaxis due to hepat‐

[36] Saenz de San Pedro B, Cazaña JL, Cobo J, Serrano CL, Quiralte J, Contreras J, Marti‐ nez F: Anaphylactic shock by rupture of hydatid hepatic cyst. Follow-up by specific

[37] Di Cataldo A, Lanteri R, Caniglia S, Santangelo M, Occhipinti R, Li Destri G: A rare complication of the hepatic hydatid cyst: Intraperitoneal perforation without anaphy‐

[38] Vuitton DA, Bresson-Hadni S, Lenys D, Flausse F, Liance M, Wattre P, Miguet JP, Capron A: IgE-dependent humoral immune response in Echinococcus multilocularis infection: circulating and basophil-bound specific IgE against Echinococcus antigens

in patients with alveolar echinococcosis. Clin Exp Immunol 71: 247–252, 1988.

ic hydatid disease. J Investig Allergol Clin Immunol 4: 158–159, 1994.

IgE serum antibodies. Allergy 47: 568–570, 1992.

laxis. Int Surg 90: 42–44, 2005.

follow-up after surgery. J Investig Allergol Clin Immunol 4: 301–304, 1994.

correlation. RadioGraphics 21: S255–S260, 2001.

tid disease. RadioGraphics 30: 334–337, 2010.

tions: CT findings. RadioGraphics 25: 719–730, 2005.

datid cyst. World J Gastroenterol 15: 3309–3311, 2009.

2010.

134 Current Topics in Echinococcosis

540, 2007.

1987.


## **Radiological Characteristics of Pulmonary Hydatid Cysts**

Dilek Emlik, Kemal Ödev, Necdet Poyraz and Hasan Emin Kaya

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60884

#### **Abstract**

[55] Zulfikaroglu B, Koc M, Ozalp N, Ozmen MM: A rare primary location of echinococ‐

[56] Dirican A, Unal B, Kayaalp C, Kirimlioglu V: Subcutaneous hydatid cysts occurring

[57] Polat P, Kantarci M, Alper F, Suma S, Koruyucu MB, Okur A: Hydatid disease from

[58] Tlili G, Bouassida K, Slama A, Tlili T, Ziadi S, Taher MA: Hydatid cyst of the scrotum

[59] Arora V, Nijjar IS, Gill KS, Singh G: Case report: Primary hydatid cyst of muscle—A

in the palm and the thigh: Two case reports. J Med Case Rep 2: 273, 2008.

cal disease: Report of a case. Ups J Med Sci 110:167–171, 2005.

miming a testicular tumor. J Case Reports 4: 151–154, 2014.

rare site. Indian J Radiol Imaging 16: 239–241, 2006.

head to toe. Radiographics 23: 475–494, 2003.

136 Current Topics in Echinococcosis

Hydatid disease is a parasitic infection caused by *Echinococcus granulosus* (EG), characterized by cystic lesions in the liver, lungs, and rarely in other parts of the body. Lungs and liver are the most frequent sites involved. Simultaneous lung and liver cysts are observed in less than 10% of the cases. Hydatid cysts are found more frequently in the lungs of children and adolescents than in their liver, while most cysts in adults are hepatic and relatively few are in the lungs. The hydatid serology results are often negative in patients with isolated pulmonary hydatidosis, and hence may not be helpful in problematic cases. Radiologic approach to the intact, complicated, or ruptured pulmonary hydatid cysts includes a CT scan following the chest radiograph. Thoracic CT may be supplemented with magnetic resonance (MR) imaging and occasionally with ultrasound (US) in clarifying a pleural-based hydatid cyst as extrapleural, pleural, or parenchymal.

**Keywords:** Thoracic hydatid disease, radiography, CT, MRI

#### **1. Introduction**

*Echinococcus granulosus* is a tapeworm of dogs and other carnivores. Eggs are passed in the feces of the dog and ingested by an intermediate host, commonly sheep or cattle, where they develop and grow into cystic structures [1]. After ingesting the cysts by carnivorous dogs, the life cycle is completed and numerous tapeworms develop in the intestine of the definitive host. Humans act as accidental intermediate hosts and harbor cysts that are most

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

commonly found in the liver and lung but echinococcal cysts may develop in almost any part of the body except hair, teeth, and fingernails [2]. The liver is the most common site of infection followed by the lung in 15%-25% of the cases, and other sites (spleen, kid‐ ney, brain, bone) in about 10% of the cases [3-5].

Hydatid disease, caused by EG, is endemic in some countries, particularly those where sheep and cattle are raised, such as Australia, New Zealand, the Mediterranean countries, the Middle East, and South America [5-7]. Hydatid disease affects not only those living in endemic regions but also those in regions with a high rate of immigration from endemic areas [8].

The purpose of this chapter is to show the spectrum of the pathogenesis, clinical manifestations and imaging findings of pulmonary hydatid cysts in adults and children based on our experience.

### **2. Pathogenesis and pathology**

Hydatid cyst may develop in almost any part of the body except hair, teeth, and fingernails [2]. Liver is the most frequently involved organ (60%-70%) [2, 5]. Thoracic involvement may occur via a transdiaphragmatic route (0.6%-16% of cases of hepatic disease) or by means of hema‐ togenous spread. The former results from the migration of the parasite from the liver to the pleural cavity. Pulmonary parenchymal involvement and chronic bronchial fistula can also be found. The lung is the second most common site of involvement in adults (10%-30% of cases) and the most frequent site of involvement in children and young adults [9-11]. Compressible organs, such as the lung or brain, facilitate the growth of the cyst and this has been proposed as a reason for the high prevalence of the disease in children [12, 13].

Echinococcal cysts have three layers composed of both host and parasite tissue. A nonruptured hydatid cyst is surrounded by the pericyst, a layer derived from compressed host tissue and chronic inflammatory cells. The true cyst wall is derived from the parasite and is arranged in two layers. The acellular outer laminated ectocyst is 1 mm-2 mm thick and is lined by a one-cell-thick germinal membrane, the endocyst [12].

#### **3. Clinical findings**

Adult patients with pulmonary hydatid cyst (PHC) present with nonspecific symptoms including cough, dyspnea, hemoptysis, pleurisy, and a bulge on the thoracic wall. The majority of the intact pulmonary cysts are known to produce no symptoms or are occasionally respon‐ sible for a non-productive cough or minimal hemoptysis. Pulmonary hydatid cyst usually remains asymptomatic until the time of rupture, and the clinic presentation in these patients is directly related to whether the cyst is intact or ruptured. The cyst may rupture spontaneously or due to antihelmintic treatment, percutaneous aspiration, or coughing and can lead to severe complications, such as massive hemoptysis and tension pneumothorax. Moreover, an acute hypersensitivity reaction or severe and life threatening problems can be encountered [14-17]. Compared with adults, PHCs in children frequently remain asymptomatic and cysts are often found by chance during physical examination or by imaging studies for other reasons. Moreover, pediatric patients may have symptoms such as chest pain, fever, purulent sputum, cough, and hemoptysis in the early period of the disease caused by compression of the surrounding tissue or perforation of the cyst [16].

#### **4. Laboratory tests**

commonly found in the liver and lung but echinococcal cysts may develop in almost any part of the body except hair, teeth, and fingernails [2]. The liver is the most common site of infection followed by the lung in 15%-25% of the cases, and other sites (spleen, kid‐

Hydatid disease, caused by EG, is endemic in some countries, particularly those where sheep and cattle are raised, such as Australia, New Zealand, the Mediterranean countries, the Middle East, and South America [5-7]. Hydatid disease affects not only those living in endemic regions

The purpose of this chapter is to show the spectrum of the pathogenesis, clinical manifestations and imaging findings of pulmonary hydatid cysts in adults and children based on our

Hydatid cyst may develop in almost any part of the body except hair, teeth, and fingernails [2]. Liver is the most frequently involved organ (60%-70%) [2, 5]. Thoracic involvement may occur via a transdiaphragmatic route (0.6%-16% of cases of hepatic disease) or by means of hema‐ togenous spread. The former results from the migration of the parasite from the liver to the pleural cavity. Pulmonary parenchymal involvement and chronic bronchial fistula can also be found. The lung is the second most common site of involvement in adults (10%-30% of cases) and the most frequent site of involvement in children and young adults [9-11]. Compressible organs, such as the lung or brain, facilitate the growth of the cyst and this has been proposed

Echinococcal cysts have three layers composed of both host and parasite tissue. A nonruptured hydatid cyst is surrounded by the pericyst, a layer derived from compressed host tissue and chronic inflammatory cells. The true cyst wall is derived from the parasite and is arranged in two layers. The acellular outer laminated ectocyst is 1 mm-2 mm thick and is lined

Adult patients with pulmonary hydatid cyst (PHC) present with nonspecific symptoms including cough, dyspnea, hemoptysis, pleurisy, and a bulge on the thoracic wall. The majority of the intact pulmonary cysts are known to produce no symptoms or are occasionally respon‐ sible for a non-productive cough or minimal hemoptysis. Pulmonary hydatid cyst usually remains asymptomatic until the time of rupture, and the clinic presentation in these patients is directly related to whether the cyst is intact or ruptured. The cyst may rupture spontaneously or due to antihelmintic treatment, percutaneous aspiration, or coughing and can lead to severe complications, such as massive hemoptysis and tension pneumothorax. Moreover, an acute hypersensitivity reaction or severe and life threatening problems can be encountered [14-17].

but also those in regions with a high rate of immigration from endemic areas [8].

as a reason for the high prevalence of the disease in children [12, 13].

by a one-cell-thick germinal membrane, the endocyst [12].

ney, brain, bone) in about 10% of the cases [3-5].

**2. Pathogenesis and pathology**

**3. Clinical findings**

experience.

138 Current Topics in Echinococcosis

Although the diagnosis of PHCs relies heavily on radiographic appearance and epidemio‐ logic setting, serologic tests [e.g., immunoelectrophoresis (IEP) or electrosyneresis, indi‐ rect immunofluorescence (IIF), enzyme-linked immunosorbent assay (ELISA) or hemagglutination] can provide indirect evidence of echinococcosis. They are all sensitive methods but are compromised by nonspecific cross reactivity with other helmints [51]. The hydatid serology results are often negative in patients with isolated pulmonary hydatido‐ sis and hence may not be helpful in problematic cases [15, 18]. Serologic tests have falsepositive or false-negative rates of 15%-20% and positive tests may not revert to normal until several years after cyst removal [4].

#### **5. Imaging findings**

A brief description of the morphological characterization of PHCs is essential for the under‐ standing of various conventional chest radiographic and CT appearances that will be dis‐ cussed. In adults, the typical hydatid cyst of the lungs, when discovered by chest radiography, usually presents as a large well-demarcated, spherical, homogeneous single mass (Figure 1a, 1b), multiple nodules (Figure 2a, 2b) or masses [5, 14]. The cysts may range between 1 cm and 20 cm in diameter. Large cysts can shift the mediastinum, and peripheral cysts can produce a pleural reaction, or cause atelectasis of adjacent parenchyma [5, 14, 19]. Radiographically, the closed or simple cyst (intact cyst) may simulate carcinoma of the lung, primary sarcoma of the lung, solitary metastasis, hematoma, arteriovenous aneurysm, granuloma of different etiology, benign tumors, inflammatory masses, solid or fluid-filled cysts (e.g., bronchogenic cyst, bronchiectatic cyst, dermoid cyst), and mesothelioma [5, 19]. When the cystic opacity is localized in the juxtamediastinal area, it may look like an aneurysm of the aorta, a tumor in the mediastinum or a huge left auricula. A cyst attached to the thoracic wall may resemble a tumor, a cold abscess or loculated pleural effusion. In fact, sharply circumscribed homogene‐ ous opacity in the lung is of great value in a country with endemic hydatid disease. Multiple cysts can be confused with pulmonary metastases. The imaging studies comprise only some of the diagnostic resources that must be used [15]. Calcifications of cysts in the liver and abdomen, and even in the rest of the body, are not uncommon but in the lung parenchyma they are extremely rare [4, 5]. However, on occasion they are found in the mediastinum including heart [5].

**Figure 1.** Intact simple cyst: (a) Chest radiograph shows a well-defined opacity located in the upper and middle zone of the right lung. (b) CT image shows that the mass has water attenuation.

**Figure 2.** (a) Chest radiograph shows multiple hydatid cysts located in both lungs as well-defined lesions. (b) CT scan shows multiple hydatid cysts in both lungs as well-demarcated cystic masses.

Unlike adults, in children the rate of growth of the cyst in the lung is progressive and more constant than in the liver due to elastic capability and low resistance of the lungs. This may explain the high incidence of pulmonary disease in children [21-23]. In the literature, it has been reported that giant PHCs (greater than 15 cm) were prevalent in children (Figure 3) [22].

(a) (b)

of the right lung. (b) CT image shows that the mass has water attenuation.

140 Current Topics in Echinococcosis

shows multiple hydatid cysts in both lungs as well-demarcated cystic masses.

**Figure 1.** Intact simple cyst: (a) Chest radiograph shows a well-defined opacity located in the upper and middle zone

(a) (b)

**Figure 2.** (a) Chest radiograph shows multiple hydatid cysts located in both lungs as well-defined lesions. (b) CT scan

Unlike adults, in children the rate of growth of the cyst in the lung is progressive and more constant than in the liver due to elastic capability and low resistance of the lungs. This may explain the high incidence of pulmonary disease in children [21-23]. In the literature, it has been reported that giant PHCs (greater than 15 cm) were prevalent in children (Figure 3) [22].

**Figure 3.** A 15-year-old boy. Chest radiograph shows a huge hydatid cyst located in the upper and mid-zone of the left lung causes displacement of the mediastinum to the right.

Computed tomography (CT) shows simple cysts to have smooth walls of variable thick‐ ness and homogenous internal contents of water or near-water density. The adventitious (pericyst), laminated, and germinal layers in an intact cyst are averaged together and seen as a single wall. On contrast enhanced CT, enhancement of the vascularized pericyst may not be a significant feature in intact cystic masses. However, in those cases exhibiting obvious enhancement, this finding has little or no diagnostic value [15, 18]. Chest CT can be of value in determining the presence of cysts in areas difficult to visualize with chest radiography, especially in the posterior and anterior costophrenic angles, as has been illustrated (Figure 4a, 4b) [18]. Also CT is superior to chest radiography in the cystic characterization of the parenchymal abnormality. Furthermore, determination of wall thickness is more accurate with CT, as compared with chest radiography. Multiple PHCs cause a diagnostic problem since they should be differentiated from metastatic disease, Wegener granulomatosis, and other granulomas [24]. Moreover, simple hydatid cyst cannot be differentiated from water-density lung cysts of different etiology in the basis of CT appearance alone. However, in endemic regions the CT demonstration of the cystic nature of a lung mass provides collaborative evidence in clinically suspected cases [18].

A patient with intact PHC is usually asymptomatic until the time of rupture and clinical presentation in these patients is directly related to whether the cyst is intact or ruptured. The cyst may rupture spontaneously due to trauma, degeneration by aging, or toxins. Moreover, infections, chemotherapy, or lack of nutrients may lead to the damage of the

**Figure 4.** (a) Chest radiograph shows well-defined mass in the lower zone of the left lung obscuring the left ventricular margin and costophrenic sinus. (b) Axial CT obtained through the postero-basal segment of the left lung shows a highdensity cystic lesion and also parenchymal consolidation adjacent to the HC.

cystic wall with an increased risk of rupture. As a consequence, the fragile parasite membranes may split and pressure necrosis may result in a communication with a bronchiole allowing air to dissect into the cyst wall. If air enters into the potential space between the pericyst and ectocyst (laminated membrane of the parasite), the local detach‐ ment of parasitic membranes from the pericyst is called 'the sign of detachment'. This segmental peripheral radiolucency is called 'the crescent' or the 'meniscus sign' (Figure 5). Some authors have stated that when the air enters the potential space between the pericyst and endocyst and separates the parasitic membranes, air meniscus or the crescent sign is formed [4, 14, 15]. This sign is highly reliable for hydatid disease but not pathognomonic [11, 20]. In non-endemic areas, cavities with fungus balls (mycetoma) are the most common cause of the meniscus sign, but blood clots, carcinoma, pulmonary gangrene, tuberculo‐ sis, sarcoma, and aircap within a tumor may also present with pulmonary meniscus sign [20, 25].

Expanding cysts sooner or later reach a bronchiolus and after erosion of the bronchiolus, a communication between the pericyst and bronchial tree is established. This condition causes a variety of more or less characteristic radiographic signs of ruptured PHCs, which may raise suspicion about the presence of hydatid cyst(s) or even allow a specific diagno‐ sis of the disease. In the literature, radiologic signs related to ruptured HC have been welldescribed [5, 14, 20, 26, 27]. Some of these signs are double-arch or cumbo sign (Figure 6a, 6b), iceberg sign, sign of the rising sun, serpent sign, and whirl sign (Figure 7). If more air is introduced to the parasitic membranes, the endocyst collapses and an air-fluid level is seen. If the parasitic membranes are floating on the fluid surface, this produces the 'water lily sign' or 'Camelot sign' resembling leaves of a water lily (Figure 8a, 8b). If all the parasitic

**Figure 5.** CT appearance of meniscus sign: A crescent shaped air is seen in the potential space between the pericyst and ectocyst of the cystic lesion.

cystic wall with an increased risk of rupture. As a consequence, the fragile parasite membranes may split and pressure necrosis may result in a communication with a bronchiole allowing air to dissect into the cyst wall. If air enters into the potential space between the pericyst and ectocyst (laminated membrane of the parasite), the local detach‐ ment of parasitic membranes from the pericyst is called 'the sign of detachment'. This segmental peripheral radiolucency is called 'the crescent' or the 'meniscus sign' (Figure 5). Some authors have stated that when the air enters the potential space between the pericyst and endocyst and separates the parasitic membranes, air meniscus or the crescent sign is formed [4, 14, 15]. This sign is highly reliable for hydatid disease but not pathognomonic [11, 20]. In non-endemic areas, cavities with fungus balls (mycetoma) are the most common cause of the meniscus sign, but blood clots, carcinoma, pulmonary gangrene, tuberculo‐ sis, sarcoma, and aircap within a tumor may also present with pulmonary meniscus sign

(a) (b)

**Figure 4.** (a) Chest radiograph shows well-defined mass in the lower zone of the left lung obscuring the left ventricular margin and costophrenic sinus. (b) Axial CT obtained through the postero-basal segment of the left lung shows a high-

density cystic lesion and also parenchymal consolidation adjacent to the HC.

Expanding cysts sooner or later reach a bronchiolus and after erosion of the bronchiolus, a communication between the pericyst and bronchial tree is established. This condition causes a variety of more or less characteristic radiographic signs of ruptured PHCs, which may raise suspicion about the presence of hydatid cyst(s) or even allow a specific diagno‐ sis of the disease. In the literature, radiologic signs related to ruptured HC have been welldescribed [5, 14, 20, 26, 27]. Some of these signs are double-arch or cumbo sign (Figure 6a, 6b), iceberg sign, sign of the rising sun, serpent sign, and whirl sign (Figure 7). If more air is introduced to the parasitic membranes, the endocyst collapses and an air-fluid level is seen. If the parasitic membranes are floating on the fluid surface, this produces the 'water lily sign' or 'Camelot sign' resembling leaves of a water lily (Figure 8a, 8b). If all the parasitic

[20, 25].

142 Current Topics in Echinococcosis

contents are evacuated and only the pericyst produced by the host remains, it may even be filled with air. This condition is called the 'empty cyst sign' (Figure 9) [4, 14].

**Figure 6.** Cumbo sign: Double air arc is seen in (a) chest radiography and (b) CT scan.

**Figure 7.** Whirl sign: CT scan (mediastinal window) shows floating detached membranes in the cystic cavity with min‐ imal pleural effusion.

**Figure 8.** Water lily or Camelot sign: (a) chest radiography shows a cavitary lesion with a germinative layer in the left lung. (b) On CT scan (mediastinal window), a cystic cavitary lesion with dependent wavy contour created by floated parasitic membranes is seen.

**Figure 9.** Empty cyst sign: CT scan of the chest shows an empty cavity with thin walls after complete evacuation of the hydatid membrane.

**Figure 10.** CT scan (mediastinal window) of the chest shows a collapsed cystic lesion.

**Figure 7.** Whirl sign: CT scan (mediastinal window) shows floating detached membranes in the cystic cavity with min‐

(a) (b)

**Figure 8.** Water lily or Camelot sign: (a) chest radiography shows a cavitary lesion with a germinative layer in the left lung. (b) On CT scan (mediastinal window), a cystic cavitary lesion with dependent wavy contour created by floated

imal pleural effusion.

144 Current Topics in Echinococcosis

parasitic membranes is seen.

Pathognomonic features of ruptured PHCs on CT are detached or collapsed endocyst mem‐ branes, collapsed daughter cyst membranes (Figure 10), and intact daughter cysts [18]. The most frequent complication of ruptured PHC is bacterial infection. Purulent sputum and fever are strong indicators of pneumonia or infected cyst. The presence of air pockets or air bubbles within the cyst and ring enhancement of the pericyst on contrast enhanced CT indicate either infection or communication with the bronchial tree (Figure 11a, 11b) [5, 27]. Because of the high density of infected hydatid cyst, the differentiation from a solid or fluid-filled cyts, abscess, or neoplasm is usually impossible [20, 28, 29].

**Figure 11.** Infected HC: (a) Chest radiograph and (b) enhanced CT scan show an infected cavitary lesion with adjacent parenchymal consolidation in the right lung.

Infected hydatid cyst may cause uncontrolled bacterial infection and hydatid lung abscess. Further disintegration of membranes and the purulent cystic content may produce an air-fluid level with no demonstrable floating membranes [14]. A hydatid cyst with such an appearance cannot be differentiated from ordinary pyogenic abscess by CT even in endemic regions and false-positive diagnosis is inevitable. Complicated PHCs, either ruptured or infected, may have higher CT attenuation numbers than that of water due to mucus, hemorrhagic content or infection (Figure 12a, 12b). These lesions are also difficult to differentiate from other cavitary lesions, such as infarctions, hemorrhage, carcinoma, benign tumors, inflammatory masses, fluid-filled cystic lesions, and active cavitary tuberculosis [24, 26, 28-31]. Thus, transthoracic aspiration or bronchoscopic biopsy can be attempted in PHCs with atypical or complicated radiologic appearances, if there is radiologic evidence of a coexisting mass [24, 30-32].

Pulmonary arteries are exceptional localizations for hydatid cysts [33]. Most frequent cause is embolism from primary cardiac locations [34, 35]. Another possibility is that the embryos of EG pass through the liver, into the inferior vena cava, and from there via the right cardiac chambers to the pulmonary arteries [33, 36]. On contrast enhanced CT, the cystic lesions within the pulmonary arteries show the typical hypodense appearance, quite similar to other cysts in the lung. These findings should be differentiated from other intraluminal filling defects such as pulmonary embolism and pulmonary artery tumors [33, 34, 37]. On MR imaging intra-arterial cyst is typical with low signal intensity on T1 weighted images and high signal intensity on T2 weighted images (Figure 13a, 13b).

Cardiac involvement of hydatid disease is very rare. It is known that cardiac involvement is approximately 0.02%-2% of all cases of human hydatidosis (Figure 14a, 14b) [11, 33, 34]. Rupture into the heart chambers may result in embolisation of hydatid tissue in the pulmonary arteries or organs of the major circulation (secondary metastatic hydatidosis) [15, 34, 38]. Infrequently, as a result of the rupture of the right ventricle and right atrium cysts into the pulmonary arteries, acute, subacute, or chronic recurrent embolization of hydatid cysts may be seen [34].

**Figure 12.** Abscesses: (a) Chest radiograph and (b) CT image show a thick-walled cystic lesion with air-fluid level lo‐ cated at the right paracardiac region with surrounding consolidation.

Infected hydatid cyst may cause uncontrolled bacterial infection and hydatid lung abscess. Further disintegration of membranes and the purulent cystic content may produce an air-fluid level with no demonstrable floating membranes [14]. A hydatid cyst with such an appearance cannot be differentiated from ordinary pyogenic abscess by CT even in endemic regions and false-positive diagnosis is inevitable. Complicated PHCs, either ruptured or infected, may have higher CT attenuation numbers than that of water due to mucus, hemorrhagic content or infection (Figure 12a, 12b). These lesions are also difficult to differentiate from other cavitary lesions, such as infarctions, hemorrhage, carcinoma, benign tumors, inflammatory masses, fluid-filled cystic lesions, and active cavitary tuberculosis [24, 26, 28-31]. Thus, transthoracic aspiration or bronchoscopic biopsy can be attempted in PHCs with atypical or complicated

**Figure 11.** Infected HC: (a) Chest radiograph and (b) enhanced CT scan show an infected cavitary lesion with adjacent

parenchymal consolidation in the right lung.

146 Current Topics in Echinococcosis

(a) (b)

radiologic appearances, if there is radiologic evidence of a coexisting mass [24, 30-32].

and high signal intensity on T2 weighted images (Figure 13a, 13b).

Pulmonary arteries are exceptional localizations for hydatid cysts [33]. Most frequent cause is embolism from primary cardiac locations [34, 35]. Another possibility is that the embryos of EG pass through the liver, into the inferior vena cava, and from there via the right cardiac chambers to the pulmonary arteries [33, 36]. On contrast enhanced CT, the cystic lesions within the pulmonary arteries show the typical hypodense appearance, quite similar to other cysts in the lung. These findings should be differentiated from other intraluminal filling defects such as pulmonary embolism and pulmonary artery tumors [33, 34, 37]. On MR imaging intra-arterial cyst is typical with low signal intensity on T1 weighted images

Cardiac involvement of hydatid disease is very rare. It is known that cardiac involvement is approximately 0.02%-2% of all cases of human hydatidosis (Figure 14a, 14b) [11, 33, 34]. Rupture into the heart chambers may result in embolisation of hydatid tissue in the pulmonary arteries or organs of the major circulation (secondary metastatic hydatidosis) [15, 34, 38]. Infrequently, as a result of the rupture of the right ventricle and right atrium cysts into the

**Figure 13.** (a) CT scan and (b) MRI of the chest show hydatid cyst with daughter cysts inside the left pulmonary artery.

Cysts that are localized in the chest wall, mediastinum, pericard, myocard, fissure, and pleura have been reported in the literature as intrathoracic extrapulmonary cysts [39, 40]. Intrathoracic extrapulmonary hydatid cysts have been reported in 7.4% of patients [39]. Primary pleural echinococcosis, including pleural fissure (Figure 15a, 15b, 15c) is relatively uncommon even in pastoral or domestic echinococcosis [41]. Rarely infection follows the primary hematoge‐ nous dissemination of larvae to the pleural tissues.

**Figure 14.** Primary pericardial multilocular HC. (a) Proton density weighted axial MR image shows high signal inten‐ sity of the multilocular pericardial cysts with a low signal intensity capsule in the pericardium. (b) The pericardial daughter cysts are best demonstrated with T2-weighted MR image.

**Figure 15.** Primary hydatid cyst in the pleural fissure (a, b) Enhanced CT images show a hypodense lobulated mass in the left upper lung parenchyma on the mediastinal and lung window settings. (c) Axial T2-weighted MR image show‐ ing a high signal intensity cyst with a low signal intensity capsule in the left major fissure (arrows).

Pleural hydatid cyst is rare and usually caused by the rupture of a pulmonary or hepatic cyst into the pleural space, but on rare occasions it may be primary (Figure 16) [11]. Hydatid cyst that perforates into the pleural cavity (secondary pleural hydatidosis or SPH) can cause pneumothorax, tension pneumothorax, hydropneumothorax, pleural effusions, or empyema [15, 16, 56]. The documented rate of simple pneumothorax in patients with PHC ranges from 2.4% to 6.2% [15, 42]. Secondary pleural hydatidosis may also occur after percutaneous transthoracic needle puncture performed for diagnostic purposes. Secondary pleural hydati‐ dosis may be due to hematogenous dissemination of the larvae of EG or by the rupture of neighboring hydatid cysts (multiple daughter cysts and scolices) along the pleura [41, 43]. This is a rare condition occurring in less than 10% of such cases. Although not frequent, involvement of the diaphragm (Figure 17) [44, 58], thoracic cavity or pleural space occurs in 0.6%-16% of cases of hepatic hydatid disease [13]. Transdiaphragmatic migration of hydatid disease from the posterior segment of the right hepatic lobe has been reported to be a common complication. This condition varies from simple adherence to the diaphragm to rupture into the pleural cavity [13, 45, 46]. Although ultrasound of the thorax and abdomen is useful for diagnosis of pulmonary hydatidosis [46], MR imaging is more useful in evaluation of cases with synchro‐ nous pulmonary and liver involvement and in depicting its close connection with thoracic lesions (Figure 18a, 18b, 18c) [47].

(a) (b)

**Figure 14.** Primary pericardial multilocular HC. (a) Proton density weighted axial MR image shows high signal inten‐ sity of the multilocular pericardial cysts with a low signal intensity capsule in the pericardium. (b) The pericardial

(a) (b)

(c)

**Figure 15.** Primary hydatid cyst in the pleural fissure (a, b) Enhanced CT images show a hypodense lobulated mass in the left upper lung parenchyma on the mediastinal and lung window settings. (c) Axial T2-weighted MR image show‐

ing a high signal intensity cyst with a low signal intensity capsule in the left major fissure (arrows).

daughter cysts are best demonstrated with T2-weighted MR image.

148 Current Topics in Echinococcosis

**Figure 16.** Ruptured primary pleural HC with diffuse pleural effusion. Coronal MRI shows a pleural HC after rupture. Detached germinal membranes are clearly seen as hypointense structures.

**Figure 17.** Primary diaphragmatic HC. Coronal and reformatted CT scans show a hydatid cyst in the right diaphragm.

Mediastinal hydatid cysts (MHCs) are very rare and are seen with an incidence ranging from 0% to 6% [11]. Several patterns of MHCs including, unilocular cyst or type 1, cyst with daughter cysts (multivesicular) or type 2 (Figure 19a, 19b), calcified cyst or type 3, and complicated cyst or type 4, have been described by using imaging techniques [47]. Characteristic finding of echinococcal cysts (e.g., floating membranes, daughter cysts, and vesicles) can usually help in establishing the diagnosis of MHCs [11, 47]. CT best demonstrates cyst wall calcification. MR imaging has the capacity of providing more information about anatomical location of MHCs. Type 2 and type 3 MHCs in the anterior mediastinum should be differentiated from cystic teratoma, thymoma, and necrotic neoplasms [11, 48].

Primary chest wall hydatid cyst is an exceptional entity. Chest wall disease presents with involvement of the anterior or lateral thoracic wall soft tissues. On contrast enhanced CT or MR imaging, the appearance of chest wall echinococcal cysts is characteristic as in other organ involvements. On contrast enhanced CT, chest wall involvement may occur as a multiloculated mass with daughter cysts in the chest wall. Multiloculated osteolytic lesion in the rib due to hydatid cyst may present as an extrapleural soft tissue mass and cause cortical expansion or destruction of the rib. This primary rib lesion slowly grows and may involve the adjacent structures such as vertebra, pleura, or subcutaneous soft tissues. The radiologic differential diagnosis includes round cell tumors, plasmacytoma, osteolytic metastases, neurofibromas, and other similar conditions that are associated with extrapleural soft tissue masses (Figure 20a, 20b) [49, 50].

**Figure 17.** Primary diaphragmatic HC. Coronal and reformatted CT scans show a hydatid cyst in the right diaphragm.

Mediastinal hydatid cysts (MHCs) are very rare and are seen with an incidence ranging from 0% to 6% [11]. Several patterns of MHCs including, unilocular cyst or type 1, cyst with daughter cysts (multivesicular) or type 2 (Figure 19a, 19b), calcified cyst or type 3, and complicated cyst or type 4, have been described by using imaging techniques [47]. Characteristic finding of echinococcal cysts (e.g., floating membranes, daughter cysts, and vesicles) can usually help in establishing the diagnosis of MHCs [11, 47]. CT best demonstrates cyst wall calcification. MR imaging has the capacity of providing more information about anatomical location of MHCs. Type 2 and type 3 MHCs in the anterior mediastinum should be differentiated from cystic

Primary chest wall hydatid cyst is an exceptional entity. Chest wall disease presents with involvement of the anterior or lateral thoracic wall soft tissues. On contrast enhanced CT or MR imaging, the appearance of chest wall echinococcal cysts is characteristic as in other organ involvements. On contrast enhanced CT, chest wall involvement may occur as a multiloculated mass with daughter cysts in the chest wall. Multiloculated osteolytic lesion in the rib due to hydatid cyst may present as an extrapleural soft tissue mass and cause cortical expansion or destruction of the rib. This primary rib lesion slowly grows and may involve the adjacent structures such as vertebra, pleura, or subcutaneous soft tissues. The radiologic differential diagnosis includes round cell tumors, plasmacytoma, osteolytic metastases, neurofibromas, and other similar conditions that are associated with extrapleural soft tissue masses (Figure

teratoma, thymoma, and necrotic neoplasms [11, 48].

20a, 20b) [49, 50].

150 Current Topics in Echinococcosis

(c)

**Figure 18.** Posterior mediastinal HC with transdiaphragmatic extension from the abdomen. (a) Enhanced CT scan shows a large cystic mass containing multiple daughter cysts in the posterior mediastinum. (b) Sagittal and (c) coronal T2-weighted MR images show a huge cystic mass extending through the diaphragm to the posterior mediastinum from abdomen.

(c) **Figure 19.** Mediastinal multilocular HC. (a) Enhanced CT scan shows a multilocular HC with mediastinal involvement. (b) T2-weighted MRI shows multiple high signal intensity daughter cysts with a low signal intensity capsule (arrows) in the mediastinum.

**Figure 20.** Chest wall HC. (a) Chest radiograph shows a bulging soft tissue mass extending to the thoracic cavity. (b) CT shows multivesicular cystic lesion involving right chest wall and adjacent ribs.

#### **6. Conclusion**

Primary diagnostic method in pulmonary hydatid disease is the plain radiograph. This method is helpful for the diagnosis of intact cysts, but it may be inadequate for the assessment of complicated cyst morphology. Computed tomography depicts certain details of the lesions and can detect others that are not visible by chest radiograph. CT examination can elucidate the cystic nature of the pulmonary lesion and provide accurate localization for planning of surgical treatment of complicated cysts. Multiplanar and multiparameter imaging features of MRI facilitate comprehensive evaluation of intrathoracic but extrapulmonary hydatid cysts (e.g., chest wall, mediastinal, pericardial, fissural, and pleural localization).

#### **Author details**

Dilek Emlik\* , Kemal Ödev, Necdet Poyraz and Hasan Emin Kaya

\*Address all correspondence to: drdemlik@hotmail.com

Necmettin Erbakan University, Meram Medicine School, Department of Radiology, Meram, Konya, Turkey

#### **References**

(a) (b)

CT shows multivesicular cystic lesion involving right chest wall and adjacent ribs.

**6. Conclusion**

in the mediastinum.

152 Current Topics in Echinococcosis

**Figure 20.** Chest wall HC. (a) Chest radiograph shows a bulging soft tissue mass extending to the thoracic cavity. (b)

(a) (b)

(c) **Figure 19.** Mediastinal multilocular HC. (a) Enhanced CT scan shows a multilocular HC with mediastinal involvement. (b) T2-weighted MRI shows multiple high signal intensity daughter cysts with a low signal intensity capsule (arrows)

Primary diagnostic method in pulmonary hydatid disease is the plain radiograph. This method is helpful for the diagnosis of intact cysts, but it may be inadequate for the assessment of complicated cyst morphology. Computed tomography depicts certain details of the lesions and can detect others that are not visible by chest radiograph. CT examination can elucidate the cystic nature of the pulmonary lesion and provide accurate localization for planning of surgical treatment of complicated cysts. Multiplanar and multiparameter imaging features of MRI facilitate comprehensive evaluation of intrathoracic but extrapulmonary hydatid cysts

(e.g., chest wall, mediastinal, pericardial, fissural, and pleural localization).


[29] Koul PA, Koul AN, Wahid A, et al. CT in pulmonary hydatid disease: Unusual ap‐ pearances. CHEST Journal. 2000;118(6):1645-7.

[13] Pedrosa I, Saiz A, Arrazola J, et al. Hydatid disease: radiologic and pathologic fea‐ tures and complications 1: (CME Available in print version and on RSNA Link). Ra‐

[14] Balikian JP, Mudarris FF. Hydatid disease of the lungs: a roentgenologic study of 50

[15] Ramos G, Orduña A, García-Yuste M. Hydatid cyst of the lung: Diagnosis and treat‐

[16] Kuzucu A, Soysal Ö, Özgel M, et al. Complicated hydatid cysts of the lung: Clinical and therapeutic issues. The Annals of Thoracic Surgery. 2004;77(4):1200-4.

[17] Balci AE, Eren N, Ülkü R. Ruptured hydatid cysts of the lung in children: Clinical review and results of surgery. The Annals of Thoracic Surgery. 2002.;74(3):889-892.

[18] Saksouk FA, Fahl MH, Rizk GK. Computed tomography of pulmonary hydatid dis‐

[19] Morar R, Feldman C. Pulmonary echinococcosis. European Respiratory Journal.

[20] von Sinner WN. New diagnostic signs in hydatid disease: Radiography, ultrasound, CT and MRI correlated to pathology. European Journal of Radiology. 1991;12(2):

[21] Karaoglanoglu N, Kurkcuoglu IC, Gorguner M, et al. Giant hydatid lung cysts. Euro‐

[22] Montazeri V, Sokouti M, Rashidi H. Comparison of pulmonary hydatid disease be‐

[23] Tantawy IM. Hydatid cysts in children. Annals of Pediatric Surgery. 2010;6(2):

[24] Gouliamos A, Kalovidouris A, Papailiou J, et al. CT appearance of pulmonary hyda‐

[26] Ramos L, Hernández-Mora M, Illanas M, et al. Radiological characteristics of perfo‐

[27] Odev K, Guler İ, Altinok T, et al. Cystic and cavitary lung lesions in children: Radio‐ logic Findings with pathologic correlation. Journal of Clinical Imaging Science.

[28] Köktürk O, Öztürk C, Diren B, et al. "Air bubble": A new diagnostic CT sign of per‐

forated pulmonary hydatid cyst. European Radiology. 1999;9(7):1321-3.

[25] Reeder MM. RPC 1 of the month from the AFIP 2. Radiology. 1970;95(2): 429-437.

rated pulmonary hydatid cysts 1. Radiology. 1975;116(3):539-42.

ease. Journal of Computer Assisted Tomography. 1986;10(2):226-232.

pean Journal of Cardio-thoracic Surgery. 2001;19(6):914-7.

tween children and adult. Tanaffos. 2007;6(1):13-8.

tid disease. CHEST Journal. 1991;100(6):1578-81.

cases. American Journal of Roentgenology. 1974;122(4):692-707.

ment. World Journal of Surgery. 2001;25(1):46-57.

diographics. 2000;20(3):795-817.

154 Current Topics in Echinococcosis

2003; 21(6):1069-1077.

150-159.

98-104.

2013;3.


## **Treatment and Prevention**

[44] Cattelani L, D'Ippolito R, Facciolongo N, et al. [Localization of hydatid cysts in the left hemidiaphragm. Description of a case]. Acta bio-medica de L'Ateneo parmense: organo della Societa di medicina e scienze naturali di Parma. 1987;59(1-2):41-7. [45] Gómez R, Moreno E, Loinaz C, et al. Diaphragmatic or transdiaphragmatic thoracic involvement in hepatic hydatid disease: surgical trends and classification. World

[46] Kilani T, El Hammami S, Horchani H, et al. Hydatid disease of the liver with thoracic

[47] Emlik D, Kiresi D, Sunam GS, et al. Intrathoracic extrapulmonary hydatid disease: Radiologic Manifestations. Canadian Association of Radiologists Journal. 2010;61(3):

[48] Ödev K, Arıbaş BK, Nayman A, et al. Imaging of cystic and cyst-like lesions of the mediastinum with pathologic correlation. Journal of Clinical Imaging Science. 2012;2.

[49] Bonakdarpour A, Zadeh YFA, Maghssoudi H, et al. Costal echinococcosis: Report of six cases and review of the literature. American Journal of Roentgenology.

[50] Ozdemir N, Akal M, Kutlay H, et al. Chest wall echinococcosis. Chest. 1994;105(4):

[51] Force L, Torres JM, Carrillo A, Buscá J. Evaluation of eight serological tests in the di‐ agnosis of human echinococcosis and follow-up. Clin Infect Dis. 1992;15:473-480.

Journal of Surgery. 1995;19(5):714-9.

170-6.

156 Current Topics in Echinococcosis

1277-9.

1973;118(2):371-7.

involvement. World Journal of Surgery. 2001;25(1):40-5.

## **Immunotherapy Can Enhance Anthelmintic Efficacy in Alveolar Echinococcosis**

Emília Dvorožňáková

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59732

#### **Abstract**

The immune response of the intermediate host with alveolar echinococcosis was investigated on mice intraperitoneally infected with *Echinococcus multilocularis* protoscoleces. The study was focused on cell-mediated immune response (dependent on interactions of T lymphocytes and macrophages), which is considered protective in alveolar echinococcosis. The immune response to *E. multilocularis* is regulated by Th1/Th2 cytokines produced by the CD4+ T lymphocyte subpopulation. Metacestode has been known for its ability to modify immune functions and suppress effective specific cell response to ensure its survival in host organism. The influence of immunomodulatory substances – muramyltripeptide (L-MTP-PE), glucan (GI), glucan with zinc (GIZn), and transfer factor (TF) – applied alone or combined with anthelmintic albendazole (ABZ) on regulative and effector components of immunity were tested and at the same time, antiparasitic efficacy of immunomodulators was evaluated.

**Keywords:** *Echinococcus multilocularis*, therapy, muramyltripeptide, glucan, transfer factor

#### **1. Introduction**

The larval stage of *Echinococcus multilocularis* causes alveolar echinococcosis, the serious helminthozoonosis with a high mortality in patients with late treatment [1]. The disease is characterized by an infiltrative, tumor-like growth of the *E. multilocularis*larval cysts, affecting

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

the liver of intermediate hosts such small mammals or man [2]. The therapy of this disease represents an important problem. The treatment with available anthelmintics (benzimida‐ zoles, praziquantel a.o.) is long term and relatively little effective. A surgical extirpation of cysts is not always successful owing to their ability to form metastatic foci in secondarily infected organs [3, 4]. The available treatment has only parasitostatic effect and is therefore performed over many years, often through the patient's life [5]. This brings the risk of adverse reactions of drugs, among which gastrointestinal disturbances, reversible alopecia, hepatitis, proteinuria, neurological symptoms are the most frequent [5, 6], apart from a strong terato‐ genic effect [7]. The disadvantage of benzimidazoles consists also in irresponsiveness to treatment in some patients and recurrence of the disease after intermission of the therapy [8]. There is still no consensus regarding the effective dosage and duration of treatment [9, 10]. Moreover, the immunological status of the host, host susceptibility, and actual stage of infection influence the results of treatment [11]. The generalized immunosuppression induced by *E. multilocularis* may complicate therapy efficacy in alveolar echinococcosis. *E. multilocula‐ ris* evades the host immune response by mechanisms that protect the parasite or modify the host immunity to ensure its long-lasting survival in the host organism [12, 13]. Application of immunomodulatory substances could improve host immune status during *E. multilocularis* infection and limit the growth of the parasite. Results of alternative therapeutic strategies with use of immunomodulatory substances seem to be promissing and might contribute to higher efficacy of the anti-*Echinococcus* treatment.

*E. multilocularis* induces parasite-specific cellular and humoral immune response in inter‐ mediate host [14]. The infection induces strong cellular immune response and production of all antibody isotypes, which are not effective in killing and elimination of parasites. Cellular immune response, leading to granulomatous infiltration of peri-parasite tissue, plays the dominant role in the fight with echinococcus [15]. Cell-mediated immune response depending on interaction of macrophages and T lymphocytes is regarded as protective against *E. multilocularis* infection [16]. On the contrary, *E. multilocularis* is able to evade host immune response or modify it to ensure its long-lasting survival in the organism of the intermediate host [12, 13]. Different activation of T cells subsets (CD4+, CD8+) and the combined Th1 and Th2 cytokine profile appear crucial for prolonged metacestode growth and survival. Regres‐ sive, as well as progressive course of the disease correlates with stadium-specific granuloma cell composition and antigen-specific T cell response [17]. An activation of Th1 CD4 T lym‐ phocytes is connected with the control of the infection [18] and a reversion to Th2 response may contribute to long-lasting manifestation of *E. multilocularis* infection in humans [12, 19]. The *E. multilocularis* metacestode can specifically manipulate the balance between Th1 and Th2 response leading to lowered effectiveness of immune response [20]. Macrophages on the periphery of the periparasitic granuloma can produce proinflammatory cytokines continually serving as mediators of acute phase of protein secretion and fibrogenesis [14]. Granuloma generation and fibrosis can restrict larval growth, but also reduces anthelmintic drug transport to the lesion and thus may be the reason for partial or total ineffectiveness of treatment [20]. Application of immunomodulatory substances could improve the host immune status during *E. multilocularis* infection and limit the growth of the parasite. New therapeutic approaches to alveolar echinococcosis use immunomodulators to overcome patient's immunosuppression (caused by *E. multilocularis* metacestode) that complicates benzimidazole therapy. The application of cytokine IFN-γ or IL-12 together with benzimidazoles stopped the progression of disease or limited the metacestode growth [17, 21], suggesting its usefulness in therapy. However, the use of recombinant cytokines in therapy is technically and financially demand‐ ing. Therefore, new ways to support a secretion of endogenous cytokines in patients are sought.

In our study, we focused on the activity of three immunomodulators (liposomized muramyl‐ tripeptide, glucan with zinc, and transfer factor) in enhancing of the host antiparasite defence and the efficacy of anthelmintic albendazole treatment in alveolar echinococcosis.

#### **2. Materials and methods**

the liver of intermediate hosts such small mammals or man [2]. The therapy of this disease represents an important problem. The treatment with available anthelmintics (benzimida‐ zoles, praziquantel a.o.) is long term and relatively little effective. A surgical extirpation of cysts is not always successful owing to their ability to form metastatic foci in secondarily infected organs [3, 4]. The available treatment has only parasitostatic effect and is therefore performed over many years, often through the patient's life [5]. This brings the risk of adverse reactions of drugs, among which gastrointestinal disturbances, reversible alopecia, hepatitis, proteinuria, neurological symptoms are the most frequent [5, 6], apart from a strong terato‐ genic effect [7]. The disadvantage of benzimidazoles consists also in irresponsiveness to treatment in some patients and recurrence of the disease after intermission of the therapy [8]. There is still no consensus regarding the effective dosage and duration of treatment [9, 10]. Moreover, the immunological status of the host, host susceptibility, and actual stage of infection influence the results of treatment [11]. The generalized immunosuppression induced by *E. multilocularis* may complicate therapy efficacy in alveolar echinococcosis. *E. multilocula‐ ris* evades the host immune response by mechanisms that protect the parasite or modify the host immunity to ensure its long-lasting survival in the host organism [12, 13]. Application of immunomodulatory substances could improve host immune status during *E. multilocularis* infection and limit the growth of the parasite. Results of alternative therapeutic strategies with use of immunomodulatory substances seem to be promissing and might contribute to higher

*E. multilocularis* induces parasite-specific cellular and humoral immune response in inter‐ mediate host [14]. The infection induces strong cellular immune response and production of all antibody isotypes, which are not effective in killing and elimination of parasites. Cellular immune response, leading to granulomatous infiltration of peri-parasite tissue, plays the dominant role in the fight with echinococcus [15]. Cell-mediated immune response depending on interaction of macrophages and T lymphocytes is regarded as protective against *E. multilocularis* infection [16]. On the contrary, *E. multilocularis* is able to evade host immune response or modify it to ensure its long-lasting survival in the organism of the intermediate host [12, 13]. Different activation of T cells subsets (CD4+, CD8+) and the combined Th1 and Th2 cytokine profile appear crucial for prolonged metacestode growth and survival. Regres‐ sive, as well as progressive course of the disease correlates with stadium-specific granuloma cell composition and antigen-specific T cell response [17]. An activation of Th1 CD4 T lym‐ phocytes is connected with the control of the infection [18] and a reversion to Th2 response may contribute to long-lasting manifestation of *E. multilocularis* infection in humans [12, 19]. The *E. multilocularis* metacestode can specifically manipulate the balance between Th1 and Th2 response leading to lowered effectiveness of immune response [20]. Macrophages on the periphery of the periparasitic granuloma can produce proinflammatory cytokines continually serving as mediators of acute phase of protein secretion and fibrogenesis [14]. Granuloma generation and fibrosis can restrict larval growth, but also reduces anthelmintic drug transport to the lesion and thus may be the reason for partial or total ineffectiveness of treatment [20]. Application of immunomodulatory substances could improve the host immune status during *E. multilocularis* infection and limit the growth of the parasite. New therapeutic approaches to alveolar echinococcosis use immunomodulators to overcome patient's immunosuppression (caused by *E. multilocularis* metacestode) that complicates benzimidazole therapy. The application of cytokine IFN-γ or IL-12 together with benzimidazoles stopped the progression

efficacy of the anti-*Echinococcus* treatment.

160 Current Topics in Echinococcosis

Experiments were performed on pathogen-free BALB/c mice, males, weighing 20–25 g. Mice were kept under a 12-h light/dark regime at room temperature (21±3°C) and 50–60% relative humidity on a commercial diet and water. The experimental protocols complied with the current Slovak ethics law.

**Figure 1.** Protoscoleces of *Echinococcus multilocularis*.

#### **2.1. Infection**

*E. multilocularis* metacestode (strain provided by Department of Medical Parasitology, Clinical Institute of Hygiene and Medical Microbiology, Medical University of Vienna, Austria) was passaged in our laboratory by intraperitoneal injection of Mongolian jirds *Meriones unguicu‐ latus*.

Parasite cysts were isolated 4 months post infection (p.i.) and cut into pieces in sterile RPMI 1640 medium (Sigma-Aldrich, Germany) supplemented with antibiotics, 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich, Germany), and passed through a Cell Dissocia‐ tion Sieve Tissue Grinder Kit using apertures ranging from 380 to 45,7 μm (Sigma-Aldrich, Germany).

Protoscoleces obtained after the last filtration were maintained in RPMI and counted for an infective dose.

#### **2.2. Efficacy of treatment** Protoscoleces obtained after the last filtration were maintained in RPMI and counted for an infective dose. Tissue Grinder Kit using apertures ranging from 380 to 45,7 m (Sigma-Aldrich, Germany). Protoscoleces obtained after the last filtration were maintained in RPMI and counted for an

*Infection* 

*Infection* 

The antiparasitic efficacy of immunotherapy was evaluated by the cyst development in infected mice. *E. multilocularis* cysts were isolated from sacrificed mice and parasite cysts were weighed and subsequently mean value ± S.D. was determined (n=3 or n=4). *Efficacy of treatment*  The antiparasitic efficacy of immunotherapy was evaluated by the cyst development in infected mice. *E. multilocularis* cysts were isolated from sacrificed mice and parasite cysts were weighed and subsequently mean value ± S.D. was determined (n=3 or n=4). infective dose. *Efficacy of treatment*  The antiparasitic efficacy of immunotherapy was evaluated by the cyst development in infected mice. *E. multilocularis* cysts were isolated from sacrificed mice and parasite cysts were weighed

and subsequently mean value ± S.D. was determined (n=3 or n=4).

*E. multilocularis* metacestode (strain provided by Department of Medical Parasitology, Clinical Institute of Hygiene and Medical Microbiology, Medical University of Vienna, Austria) was passaged in our laboratory by intraperitoneal injection of Mongolian jirds *Meriones unguiculatus*. Parasite cysts were isolated 4 months post infection (p.i.) and cut into pieces in sterile RPMI 1640

*E. multilocularis* metacestode (strain provided by Department of Medical Parasitology, Clinical Institute of Hygiene and Medical Microbiology, Medical University of Vienna, Austria) was passaged in our laboratory by intraperitoneal injection of Mongolian jirds *Meriones unguiculatus*.

medium (Sigma-Aldrich, Germany) supplemented with antibiotics, 100 U/ml penicillin and 100 g/ml streptomycin (Sigma-Aldrich, Germany), and passed through a Cell Dissociation Sieve

Tissue Grinder Kit using apertures ranging from 380 to 45,7 m (Sigma-Aldrich, Germany).

Figure 2. A – Mice infected with *E. multilocularis*. B – Cysts of *E. multilocularis* in the peritoneal cavity of an infected mouse (4th month after the infection). **Figure 2.** A – Mice infected with *E. multilocularis*. B – Cysts of *E. multilocularis* in the peritoneal cavity of an infected mouse (4th month after the infection). Figure 2. A – Mice infected with *E. multilocularis*. B – Cysts of *E. multilocularis* in the peritoneal cavity of an infected mouse (4th month after the infection).

**Figure 3.** A – The affected liver with *E. multilocularis* cysts. B – Cysts of *E. multilocularis* isolated from the peritoneal cavity of an infected mouse (4th month after the infection).

isolated from the peritoneal cavity of an infected mouse (4th month after the infection).

#### **2.3. T and B lymphocyte proliferation assay**

The spleen was aseptically homogenized in phosphate-buffered saline (PBS) (pH 7.2) to obtain cells. Cell suspension was washed twice with PBS and finally with RPMI 1640 medium (Sigma-Aldrich, Germany). Erythrocytes were removed by lysis in hypotonic solution (0.85 % NH4Cl) and lymphocytes were resuspended to a final concentration of 5 x 106 cells /ml in RPMI 1640 medium. The assay was performed in 96 wells plates (Nunc, Denmark) and cells were incubated in RPMI 1640 medium (100 μl) containing 10 % bovine fetal serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. Mitogens Concanavalin A (Con A) (T cells) and lipopolysaccharide (LPS) (B cells) (Sigma-Aldrich, Germany) were added in a dose 100 μl (concentration 10 μg/ml) to the cell suspensions and incubated at 37 °C in 5 % CO2 and 85 % humidity for 72 h. Then 20 μl of 3,4-dimethylthiazolyl 2,5-diphenyltetrazolium bromide (Sigma-Aldrich, Germany) (0.1 % solution) was added to the cell suspensions and incubated at 37 °C and 5 % CO2 for 4 h followed by centrifugation at 800 x *g* for 5 min. Reaction was terminated with dimethylsulfoxide (Sigma-Aldrich, Germany) (100 μl/cell sample) and read on ELISA reader (Multiskan Plus, Labsystem, Finland) at 540 and 630 nm. The stimulation indices (SI) were calculated according to the formula:

SI= E540 – E630 (stimulated cells) / E540 – E630 (unstimulated cells)

Proliferative responses were measured separately for lymphocytes isolated from each mouse per group.

#### **2.4. Number of CD4+ and CD8+ T cells**

**2.2. Efficacy of treatment**

162 Current Topics in Echinococcosis

mouse (4th month after the infection).

*Infection* 

*Infection* 

infective dose.

infective dose.

*Efficacy of treatment* 

*Efficacy of treatment* 

The antiparasitic efficacy of immunotherapy was evaluated by the cyst development in infected mice. *E. multilocularis* cysts were isolated from sacrificed mice and parasite cysts were

and subsequently mean value ± S.D. was determined (n=3 or n=4).

cavity of an infected mouse (4th month after the infection).

**Figure 2.** A – Mice infected with *E. multilocularis*. B – Cysts of *E. multilocularis* in the peritoneal cavity of an infected

**A B**

**Figure 3.** A – The affected liver with *E. multilocularis* cysts. B – Cysts of *E. multilocularis* isolated from the peritoneal

The spleen was aseptically homogenized in phosphate-buffered saline (PBS) (pH 7.2) to obtain cells. Cell suspension was washed twice with PBS and finally with RPMI 1640 medium (Sigma-Aldrich, Germany). Erythrocytes were removed by lysis in hypotonic solution (0.85 % NH4Cl) and lymphocytes were resuspended to a final concentration of 5 x 106 cells /ml in RPMI 1640

**A B**

cavity of an infected mouse (4th month after the infection).

**2.3. T and B lymphocyte proliferation assay**

**A B**

**A B**

cavity of an infected mouse (4th month after the infection).

*E. multilocularis* metacestode (strain provided by Department of Medical Parasitology, Clinical Institute of Hygiene and Medical Microbiology, Medical University of Vienna, Austria) was passaged in our laboratory by intraperitoneal injection of Mongolian jirds *Meriones unguiculatus*. Parasite cysts were isolated 4 months post infection (p.i.) and cut into pieces in sterile RPMI 1640 medium (Sigma-Aldrich, Germany) supplemented with antibiotics, 100 U/ml penicillin and 100 g/ml streptomycin (Sigma-Aldrich, Germany), and passed through a Cell Dissociation Sieve Tissue Grinder Kit using apertures ranging from 380 to 45,7 m (Sigma-Aldrich, Germany). Protoscoleces obtained after the last filtration were maintained in RPMI and counted for an

*E. multilocularis* metacestode (strain provided by Department of Medical Parasitology, Clinical Institute of Hygiene and Medical Microbiology, Medical University of Vienna, Austria) was passaged in our laboratory by intraperitoneal injection of Mongolian jirds *Meriones unguiculatus*. Parasite cysts were isolated 4 months post infection (p.i.) and cut into pieces in sterile RPMI 1640 medium (Sigma-Aldrich, Germany) supplemented with antibiotics, 100 U/ml penicillin and 100 g/ml streptomycin (Sigma-Aldrich, Germany), and passed through a Cell Dissociation Sieve Tissue Grinder Kit using apertures ranging from 380 to 45,7 m (Sigma-Aldrich, Germany). Protoscoleces obtained after the last filtration were maintained in RPMI and counted for an

The antiparasitic efficacy of immunotherapy was evaluated by the cyst development in infected mice. *E. multilocularis* cysts were isolated from sacrificed mice and parasite cysts were weighed

The antiparasitic efficacy of immunotherapy was evaluated by the cyst development in infected mice. *E. multilocularis* cysts were isolated from sacrificed mice and parasite cysts were weighed

Figure 2. A – Mice infected with *E. multilocularis*. B – Cysts of *E. multilocularis* in the peritoneal

Figure 2. A – Mice infected with *E. multilocularis*. B – Cysts of *E. multilocularis* in the peritoneal

Figure 3. A – The affected liver with *E. multilocularis* cysts. B – Cysts of *E. multilocularis* isolated from the peritoneal cavity of an infected mouse (4th month after the infection).

weighed and subsequently mean value ± S.D. was determined (n=3 or n=4).

and subsequently mean value ± S.D. was determined (n=3 or n=4).

Lymphocytes from the spleens and depleted of erythrocytes were resuspended in PBS (pH 7.2) at a final concentration of 1x 106 cells /ml. Monoclonal antibodies rat anti-mouse CD4 fluorescein isothiocyanate-conjugated and rat anti-mouse CD8 phycoerythrin-conjugated monoclonal antibodies (BD Biosciences PharMingen, Belgium) were used at the concentration of 0.4 μg/106 cells at 4°C for 30 min. After washing in PBS three times, cells were analyzed by the FACScan flow cytometer (Becton Dickinson Biosciences, Germany) and CellQuest software. Cells from each mouse per group were analyzed individually. The final numbers of both cell populations were calculated as proportion from the total isolated lymphocytes per spleen/mouse.

#### **2.5. Concentration of IFN-γ and IL-5 in serum**

The capture ELISA was employed to determine the concentration of cytokines IFN-γ and IL-5 in serum according to the method [22]. IFN-γ and IL-5 were used as marker cytokines for the Th1 and Th2 responses, respectively. Cytokine-specific monoclonal antibodies were used, for IFN-γ detection: pure anti-mouse IFN-γ (R4-6A2) and biotin anti-mouse IFN-γ (XMG1.2); for IL-5 detection: pure anti-mouse IL-5 (TRFK5) and biotin anti-mouse IL-5 (TRF4) (all BD Biosciences PharMingen, Belgium). Results were expressed at pg/ml using murine recombi‐ nant IFN-γ and IL-5 (BD Biosciences PharMingen, Belgium) as standards. The detection limit of the assay for the both cytokines was 40 pg/ml.

#### **2.6. Superoxide anion assay**

Production of superoxide anion (O2 - ) by peritoneal macrophages was detected as superoxide dismutase (SOD) – reduction of ferricytochrome C with and without stimulation with phorbol myristate acetate (PMA) [23]. Cells were obtained by peritoneal lavage and after washing in PBS were diluted at concentration of 1 x 106 cells/ml in RPMI 1640 (Sigma-Aldrich, Germany). Cell suspension (1 ml/well) was added to 24-well plate (Falcon, France) and incubated at 37 o C in 5 % CO2 and 85 % humidity for 2 h. Nonadherent cells were removed by washing with ice-cold Earls Balanced Salt Solution (EBSS) (pH 7.2). The reaction was carried out in 0.3 ml/ well of 160 μM ferricytochrome C (Sigma-Aldrich, Germany) in EBSS. In control, the reaction was immediately blocked by 300 μg SOD/10 μl in EBSS. The stimulation of cells was induced by 10 μl of PMA in ethanol. Cells were incubated at 37 o C in 5 % CO2 and 85 % humidity for 2 h. Supernatant from wells was centrifuged at 170 x *g* for 3 min at 4 <sup>o</sup> C. The optical density (OD) of supernatant was measured at 550 nm in a 96-well plate reader (Multiscan Plus, Labsystems, Finland). Bradford protein microassay was used to determine cell-protein concentration in each well with standard reagent and bovine serum albumin as the protein standard (BioRad, UK). The OD was read at 595 nm. The resulting value was used to calculate nmol of O2 produced according to the formula: nmol O2 - = (ODblocked by SOD - ODwithout SOD /6.3) x 100 and determinated for 1 mg of cell proteins.

#### **2.7. Statistical evaluation**

Statistical differences were assessed using Kruskal-Wallis ANOVA and post hoc Tukey's HSD test (a value of p<0.05 was considered significant) in the program Statistica 6.0 (Stat Soft, Tulsa, USA) statistical package.

### **3. Muramyltripeptide**

Muramylpeptides – components of bacterial cellular wall are classified as biological immu‐ nomodulators. Muramylpeptides primarily activate macrophages to a high production of oxygen radicals and a secretion of inflammatory cytokines, which activate neutrophils, T and B lymphocytes [24]. Muramyldipeptide (MDP) is the smallest bacterial structure with immu‐ nopotent activity. The positive effect of MDP on the host immune response was also observed in several parasitic infections [25–27]. Muramyltripeptide phosphatidylethanolamine (MTP-PE) is a lipofilic derivate of MDP. Liposome-encapsulated MTP-PE has an enhanced effect on macrophages to secrete pro-inflammatory cytokines, which results in increasing of cytotoxicity of these cells [28–30].

Affinity of liposomized forms of MTP-PE to the reticuloendotelial system, in particular to macrophages located in the liver and the spleen [30, 31], could be useful in therapy of alveolar echinococcosis, by which the parasite cysts primarily develop in the liver. The biological effect of MTP-PE takes place in Kuppfer cells in the liver (macrophage's equivalent) [32]. Macro‐ phages activated by liposomized MTP-PE stimulate Th1 subpopulation of lymphocytes and proinflammatory mediators via cytokine secretion [28, 29, 33, 34]. T lymphocytes therefore constitute an active component of immune reactions after immunomodulation with mura‐ mylpeptides.

The effect of muramyltripeptide phosphatidylethanolamine incorporated into multilamellar liposomes (L-MTP-PE) on immune response of intermediate host infected with *E. multilocula‐ ris* was examined.

#### **3.1. Experimental design**

Cell suspension (1 ml/well) was added to 24-well plate (Falcon, France) and incubated at 37

C in 5 % CO2 and 85 % humidity for 2 h. Nonadherent cells were removed by washing with ice-cold Earls Balanced Salt Solution (EBSS) (pH 7.2). The reaction was carried out in 0.3 ml/ well of 160 μM ferricytochrome C (Sigma-Aldrich, Germany) in EBSS. In control, the reaction was immediately blocked by 300 μg SOD/10 μl in EBSS. The stimulation of cells was induced

of supernatant was measured at 550 nm in a 96-well plate reader (Multiscan Plus, Labsystems, Finland). Bradford protein microassay was used to determine cell-protein concentration in each well with standard reagent and bovine serum albumin as the protein standard (BioRad, UK). The OD was read at 595 nm. The resulting value was used to calculate nmol of O2


Statistical differences were assessed using Kruskal-Wallis ANOVA and post hoc Tukey's HSD test (a value of p<0.05 was considered significant) in the program Statistica 6.0 (Stat Soft, Tulsa,

Muramylpeptides – components of bacterial cellular wall are classified as biological immu‐ nomodulators. Muramylpeptides primarily activate macrophages to a high production of oxygen radicals and a secretion of inflammatory cytokines, which activate neutrophils, T and B lymphocytes [24]. Muramyldipeptide (MDP) is the smallest bacterial structure with immu‐ nopotent activity. The positive effect of MDP on the host immune response was also observed in several parasitic infections [25–27]. Muramyltripeptide phosphatidylethanolamine (MTP-PE) is a lipofilic derivate of MDP. Liposome-encapsulated MTP-PE has an enhanced effect on macrophages to secrete pro-inflammatory cytokines, which results in increasing of cytotoxicity

Affinity of liposomized forms of MTP-PE to the reticuloendotelial system, in particular to macrophages located in the liver and the spleen [30, 31], could be useful in therapy of alveolar echinococcosis, by which the parasite cysts primarily develop in the liver. The biological effect of MTP-PE takes place in Kuppfer cells in the liver (macrophage's equivalent) [32]. Macro‐ phages activated by liposomized MTP-PE stimulate Th1 subpopulation of lymphocytes and proinflammatory mediators via cytokine secretion [28, 29, 33, 34]. T lymphocytes therefore constitute an active component of immune reactions after immunomodulation with mura‐

The effect of muramyltripeptide phosphatidylethanolamine incorporated into multilamellar liposomes (L-MTP-PE) on immune response of intermediate host infected with *E. multilocula‐*

C in 5 % CO2 and 85 % humidity for 2

= (ODblocked by SOD - ODwithout SOD /6.3) x 100 and

C. The optical density (OD)


by 10 μl of PMA in ethanol. Cells were incubated at 37 o

produced according to the formula: nmol O2

determinated for 1 mg of cell proteins.

**2.7. Statistical evaluation**

USA) statistical package.

**3. Muramyltripeptide**

of these cells [28–30].

mylpeptides.

*ris* was examined.

h. Supernatant from wells was centrifuged at 170 x *g* for 3 min at 4 <sup>o</sup>

o

164 Current Topics in Echinococcosis

Experiments were carried out on male BALB/c mice (n=150) weighing 20–25 g. Mice were randomly divided into five groups as follows:

Group 1 – uninfected and untreated (control)

Group 2 – infected intraperitoneally with 5000 *E. multilocularis* protoscoleces/mouse on Day 0 and no treatment

Group 3 – *E. multilocularis* infected (as Group 2) and treated with liposomized muramyltri‐ peptide phosphatidylethanolamine (L-MTP-PE) (Ciba-Geigy, Switzerland) intravenously (i.v.) twice a week at the dose of 1 mg/kg of body weight (b.w.) starting at weeks 5 and 6 post infection (p.i.)

Group 4 – *E. multilocularis* infected (as Group 2) and treated with albendazole (ABZ) (Sigma, Germany) per os (p.o.) twice a week at the dose of 10 mg/kg of b.w. starting at week 5 up to week 10 p.i.

Group 5 – *E. multilocularis* infected (as Group 2) and treated with combination of L-MTP-PE and ABZ as above.

Samples of blood, spleen, and peritoneal macrophages were obtained at the following weeks: 0 (prior infection), 2, 4, 8, 10, 12, 14, 18, 22, and 26 p.i. from all groups (3 mice per experimen‐ tal day).

#### **4. Results and discussion**

In our experiment, the *E. multilocularis* infection induced an inhibition of the proliferative activity of T and B lymphocytes almost in the course of the whole experiment (Figures 4, 5).

However, the application of L-MTP-PE to infected mice had a positive stimulatory effect on T and B lymphocytes, especially in combination of L-MTP-PE+ABZ, where proliferative activity of lymphocytes was increased for a long time, from week 8 p.i. (2 weeks after the end of the therapy) till week 14 p.i. (lasted almost 2 months). Muramylpeptides belongs to the polyclonal activators of B lymphocytes, inducing a high proliferation and production of polyclonal antibodies. Muramyldipeptide activates B cells particularly during a late phase of their differentiation because the specific receptors for MDP are expressed on B cell surface at a definitive stage of their maturation [35].

T lymphocytes play a major role in the control of immune response in intermediate host organism infected with *E. multilocularis* [36]. In alveolar echinococcosis CD4+ T cells represent the main T subpopulation, being abundant in the first phase of the periparasitic granuloma formation and later these cells are replaced by CD8+ T cells [17]. A long-term stimulation of CD4+ T cell subpopulation after the immunotherapy could participate in antiparasite defence (to form the periparasitic granuloma) and increase its efficacy. Both L-MTP-PE alone and

**Figure 4.** Proliferative activity of T lymphocytes in *E. multilocularis* infected mice after therapy L-MTP-PE+ABZ. \*(p<0.05); \*\*(p<0.01) statistically significant from infected mice.

**Figure 5.** Proliferative activity of B lymphocytes in *E. multilocularis* infected mice after therapy L-MTP-PE+ABZ. \*(p<0.05); \*\*(p<0.01) statistically significant from infected mice.

combination of L-MTP-PE+ABZ increased the presence of CD4+ T cells in the spleen of infected mice within 6 weeks after the immunotherapy (Figure 6).

Previous experimental studies have manifested that *E. multilocularis* infection can lead to an expansion of CD8+ T cell clones [18, 37, 38]. In our study (Figure 7), the CD8+ T cells occurrence was higher for a short time in mice after therapy with L-MTP-PE than in infected and non‐ treated mice. Combined therapy L-MTP-PE+ABZ induced high values of CD8+ T cells, which remained increased till the end of the experiment. Splenic suppressive CD8+ T cells in alveolar

**Figure 6.** Number of splenic CD4 T cells in *E. multilocularis* infected mice after therapy L-MTP-PE+ABZ. \*(p<0.05); \*\*(p<0.01) statistically significant from infected mice.

**Figure 7.** Number of splenic CD8 T cells in *E. multilocularis* infected mice after therapy L-MTP-PE+ABZ. \*\*(p<0.01) stat‐ istically significant from infected mice.

combination of L-MTP-PE+ABZ increased the presence of CD4+ T cells in the spleen of infected

**Figure 5.** Proliferative activity of B lymphocytes in *E. multilocularis* infected mice after therapy L-MTP-PE+ABZ.

**Figure 4.** Proliferative activity of T lymphocytes in *E. multilocularis* infected mice after therapy L-MTP-PE+ABZ.

Previous experimental studies have manifested that *E. multilocularis* infection can lead to an expansion of CD8+ T cell clones [18, 37, 38]. In our study (Figure 7), the CD8+ T cells occurrence was higher for a short time in mice after therapy with L-MTP-PE than in infected and non‐ treated mice. Combined therapy L-MTP-PE+ABZ induced high values of CD8+ T cells, which remained increased till the end of the experiment. Splenic suppressive CD8+ T cells in alveolar

mice within 6 weeks after the immunotherapy (Figure 6).

\*(p<0.05); \*\*(p<0.01) statistically significant from infected mice.

\*(p<0.05); \*\*(p<0.01) statistically significant from infected mice.

166 Current Topics in Echinococcosis

echinococcosis have a key role in modulation of immunosuppression and ensure the parasite survival in a host [20, 38]. Results in our experiment do not differentiate between cytotoxic and suppressor subtypes of CD8+ T cells and which one of them dominates. In alveolar echinococcosis, the CD8+ T splenocytes consist mostly of T suppressors with a low density of the CD8 antigen [37]. In our experiment, the immunotherapy suppressed the proliferation of *E. multilocularis* cysts and therefore we suppose L-MTP-PE could increase the occurrence of cytotoxic CD8+ T cells. Immunomodulator L-MTP-PE has also stopped a decrease of the CD4/ CD8 ratio and the CD4/CD8 index was stabilized over 2.00. This reflects that CD4+ T lympho‐ cytes were in the majority despite the increased values of CD8+ T lymphocytes. CD4+ T cells together with macrophages and IFN-γ actively participate in parasite's destruction [39].

In many parasitic infections, the clinical outcome of the disease is associated with Th1 or Th2 cell activation. *E. multilocularis* metacestode is able to direct a host immune response to a less efficient, "tolerant" Th2 profile [20, 40]. In [34, 41] it was found that liposomized immunomo‐ dulators induce a development of splenic Th1 lymphocyte subpopulation. The dominance of Th1 response is important in defensive reactions of a host infected with *E. multilocularis*. IFNγ is an important activator of macrophages and affects their energetic system [21]. In our study (Figure 8), the level of IFN-γ peaked after L-MTP-PE+ABZ therapy from weeks 8 to 18 p.i. (i.e., almost 3 months), which outlines an important credit of common administration of immuno‐ modulators and anthelmintic drugs for therapy of alveolar echinococcosis. The protective effect of combined therapy L-MTP-PE+ABZ was the most evident during the time with IFNγ stimulation (lasting 3 months after the therapy), manifesting in breaking of parasite's development (Figure 11).

**Figure 8.** Serum IFN-γ in *E. multilocularis*-infected mice after therapy L-MTP-PE+ABZ. \*(p<0.05); \*\*(p<0.01) statistically significant from infected mice.

Liposomized MTP-PE suppressed the Th2 response, associated with a progressive develop‐ ment of larval cysts in *E. multilocularis* infection. In our experiment, the Th2 response was suppressed in mice treated with L-MTP-PE; we recorded almost twofold reduction of IL-5 production (Figure 9) during the whole experiment. We observed a total IL-5 cytokine suppression in mice treated with combined therapy L-MTP-PE+ABZ. The cytokine IL-5 is related to eosinophilia, IgE production, and may induce allergic reactions of the disease [19, 42, 43]. The therapeutic regime of L-MTP-PE in our experiment was timed on the first stage of the *E. multilocularis* infection, which is characterized by secretion of Th1 cytokines IL-2 and IFN-γ [20]. In this early phase of the infection, Th2 response has not been activated in sub‐ stantial extent. Th2 response decreases a synthesis of proinflammatory Th1 cytokine IFN-γ by antagonistic IL-10 secretion and by inhibited IL-12 production in macrophages [44, 45]. Therefore, there is a higher possibility of an activation of protective Th1 response by immu‐ nomodulator before the stage when an expansive metacestode growth takes place (approx. from week 8 p.i.).

CD8 ratio and the CD4/CD8 index was stabilized over 2.00. This reflects that CD4+ T lympho‐ cytes were in the majority despite the increased values of CD8+ T lymphocytes. CD4+ T cells together with macrophages and IFN-γ actively participate in parasite's destruction [39].

In many parasitic infections, the clinical outcome of the disease is associated with Th1 or Th2 cell activation. *E. multilocularis* metacestode is able to direct a host immune response to a less efficient, "tolerant" Th2 profile [20, 40]. In [34, 41] it was found that liposomized immunomo‐ dulators induce a development of splenic Th1 lymphocyte subpopulation. The dominance of Th1 response is important in defensive reactions of a host infected with *E. multilocularis*. IFNγ is an important activator of macrophages and affects their energetic system [21]. In our study (Figure 8), the level of IFN-γ peaked after L-MTP-PE+ABZ therapy from weeks 8 to 18 p.i. (i.e., almost 3 months), which outlines an important credit of common administration of immuno‐ modulators and anthelmintic drugs for therapy of alveolar echinococcosis. The protective effect of combined therapy L-MTP-PE+ABZ was the most evident during the time with IFNγ stimulation (lasting 3 months after the therapy), manifesting in breaking of parasite's

**Figure 8.** Serum IFN-γ in *E. multilocularis*-infected mice after therapy L-MTP-PE+ABZ. \*(p<0.05); \*\*(p<0.01) statistically

Liposomized MTP-PE suppressed the Th2 response, associated with a progressive develop‐ ment of larval cysts in *E. multilocularis* infection. In our experiment, the Th2 response was suppressed in mice treated with L-MTP-PE; we recorded almost twofold reduction of IL-5 production (Figure 9) during the whole experiment. We observed a total IL-5 cytokine suppression in mice treated with combined therapy L-MTP-PE+ABZ. The cytokine IL-5 is related to eosinophilia, IgE production, and may induce allergic reactions of the disease [19, 42, 43]. The therapeutic regime of L-MTP-PE in our experiment was timed on the first stage of the *E. multilocularis* infection, which is characterized by secretion of Th1 cytokines IL-2 and IFN-γ [20]. In this early phase of the infection, Th2 response has not been activated in sub‐ stantial extent. Th2 response decreases a synthesis of proinflammatory Th1 cytokine IFN-γ by

development (Figure 11).

168 Current Topics in Echinococcosis

significant from infected mice.

**Figure 9.** Serum IL-5 in *E. multilocularis* infected mice after therapy L-MTP-PE+ABZ. \*(p<0.05); \*\*(p<0.01) statistically significant from infected mice.

Macrophages participate in the destruction of the parasite through synthesis of O2 and other free radicals and, together with T cells, control the parasite development [39]. In our experi‐ ment (Figure 10), therapy L-MTP-PE stimulated O2 - generation in infected mice from weeks 8 to 12 p.i. (i.e. for 1 month). The combination of L-MTP-PE+ABZ significantly increased the superoxide production from weeks 8 to 18 p.i. (almost for 3 months).

**Figure 10.** Macrophage's activity in *E. multilocularis* infected mice after therapy L-MTP-PE+ABZ. \*(p<0.05); \*\*(p<0.01) statistically significant from infected mice.

It documents a long-term and strong activation of macrophage's metabolism accompanied with a huge release of other biologically effective substances. In parallel, the proliferative activity of B cells was also increased, which could be related to macrophages' stimulation induced by B cell secrets [46]. Positive effect of L-MTP-PE on macrophages had been confirmed in immunosuppressed mice [25], in which the immunomodulator induced a macrophages restoration. An induction of cytotoxicity of liver macrophages against tumor cells after stimulation with L-MTP-PE was observed [28]. In addition, an increased tumoricidal activity of Kupffer cells in mice after application of L-MTP-PE was recorded [47]. Their results were later corroborated in the work [48], which described an activation of Kupffer cells with an increased production of superoxide anion and subsequent reduction of micrometastases in rats after MTP-PE therapy.

Macrophages' stimulation *in situ* is selectively influenced by a way of application of liposom‐ ized immunomodulator, and also by other biological substances. In [49], L-MTP-PE adminis‐ tered alone stimulated peritoneal macrophages with less effect than after coincubation of macrophages with L-MTP-PE and IFN-γ together, which doubled their cytotoxic activity. The synergetic and potentiating effect of IFN-γ in the immunomodulatory acting was also verified on macrophages infected with *Listeria monocytogenes* [50], in mice infected with *Klebsiella pneumoniae* [34]. These data confirmed an important role of T cells in immune activation of the host organism after the immunomodulator administration.

Potentiation of effector components of the immunity after the immunomodulation and anthelmintic therapy of *E. multilocularis* infected mice in our experiment resulted in a restric‐ tion of the parasite development and reduced larval cysts in a host organism (Figure 11). The immunomodulator L-MTP-PE administered alone slowed down *E. multilocularis* proliferation till week 14 p.i. and combination of L-MTP-PE+ABZ reduced the parasite development till week 22 p.i. (i.e., 3 months after the end of the experiment). A similar synergism of liposomized MDP and antiparasitic drug Glucantime was observed in visceral leishmaniasis [51].

**Figure 11.** Cyst's weight in *E. multilocularis* infected mice after therapy L-MTP-PE+ABZ. \*(p<0.05); \*\*(p<0.01) statistical‐ ly significant from infected mice.

In conclusion, our results show the possibility to increase antiparasitic efficacy of ABZ in *E. multilocularis* infection with L-MTP-PE, which properly stimulates the host immune response. Combined therapy L-MTP-PE+ABZ positively increased CD4+ T cells numbers and cytokine production that regulate the host immune response. The immunotherapy suppressed the Th2 response and *vice versa* activated the Th1 response with a restrictive impact on the parasite development. The immunomodulator L-MTP-PE stimulated macrophages' metabolism to produce reactive oxygen substances and together with IFN-γ increased an antiparasitic efficacy of albendazole.

#### **5. Glucan and zinc**

It documents a long-term and strong activation of macrophage's metabolism accompanied with a huge release of other biologically effective substances. In parallel, the proliferative activity of B cells was also increased, which could be related to macrophages' stimulation induced by B cell secrets [46]. Positive effect of L-MTP-PE on macrophages had been confirmed in immunosuppressed mice [25], in which the immunomodulator induced a macrophages restoration. An induction of cytotoxicity of liver macrophages against tumor cells after stimulation with L-MTP-PE was observed [28]. In addition, an increased tumoricidal activity of Kupffer cells in mice after application of L-MTP-PE was recorded [47]. Their results were later corroborated in the work [48], which described an activation of Kupffer cells with an increased production of superoxide anion and subsequent reduction of micrometastases in

Macrophages' stimulation *in situ* is selectively influenced by a way of application of liposom‐ ized immunomodulator, and also by other biological substances. In [49], L-MTP-PE adminis‐ tered alone stimulated peritoneal macrophages with less effect than after coincubation of macrophages with L-MTP-PE and IFN-γ together, which doubled their cytotoxic activity. The synergetic and potentiating effect of IFN-γ in the immunomodulatory acting was also verified on macrophages infected with *Listeria monocytogenes* [50], in mice infected with *Klebsiella pneumoniae* [34]. These data confirmed an important role of T cells in immune activation of the

Potentiation of effector components of the immunity after the immunomodulation and anthelmintic therapy of *E. multilocularis* infected mice in our experiment resulted in a restric‐ tion of the parasite development and reduced larval cysts in a host organism (Figure 11). The immunomodulator L-MTP-PE administered alone slowed down *E. multilocularis* proliferation till week 14 p.i. and combination of L-MTP-PE+ABZ reduced the parasite development till week 22 p.i. (i.e., 3 months after the end of the experiment). A similar synergism of liposomized

**Figure 11.** Cyst's weight in *E. multilocularis* infected mice after therapy L-MTP-PE+ABZ. \*(p<0.05); \*\*(p<0.01) statistical‐

MDP and antiparasitic drug Glucantime was observed in visceral leishmaniasis [51].

host organism after the immunomodulator administration.

rats after MTP-PE therapy.

170 Current Topics in Echinococcosis

ly significant from infected mice.

Glucans are β-(1,3)-D polymers of glucose occuring naturally as the basic component of the cell walls of bacteria, fungi, and yeast [52–54]. Glucans activate mainly nonspecific stimulation of the immune system [22], particularly proliferation and functional activity of phagocytic cells (macrophages, NK cells) as the part of innate immunity [55, 56], but also have an influence on specific immunity – increase of the T-lymphocyte activity and production of circulating antibodies [57–59]. The immunostimulative effect of glucan could be increased by its combi‐ nation with the other components – immunoglobulin G, zinc, or vitamin C [60, 61]. Zinc interacts with cytokines and proteases and indirectly influences the immune system [62]. Its immunostimulative effect on T lymphocytes, macrophages has been known [63–65] and so zinc could potentiate the immunostimulative effect of the glucan during therapy of alveolar echinococcosis. Also living *Echinococcus* needs zinc for growth and its reproduction [66]. An obvious association between serum zinc concentration and disease severity in patients with alveolar echinococcosis was confirmed in [67]. Decreased zinc concentration in progressive cases may be a consequence of enhanced immune activation and also consumption of zinc by the growing parasite. Zinc deficiency in the host may thus contribute to the observed immu‐ nosupression in alveolar echinococcosis.

The role of glucan immunomodulator (GI, soluble β-(1,3)-D glucan) and/or glucan immuno‐ modulator supplemented with zinc (GIZn) in stimulation of a host defence mechanism against *E. multilocularis* infection and the immunomodulator's effect on albendazole antiparasitic efficacy was observed.

#### **5.1. Experimental design**

Experiments were carried out on male BALB/c mice (n=220) weighing 20–25 g. Mice were divided randomly into five groups as folows:

Group 1 – uninfected and untreated (control)

Group 2 – infected intraperitoneally with 5000 *E. multilocularis* protoscoleces/mouse on Day 0 and no treatment

Group 3 – *E. multilocularis* infected (as Group 2) and treated with albendazole (ABZ) (Sigma, Germany) per os (p.o.) twice a week at the dose of 10 mg/kg of b.w. starting at week 5 up to week 10 p.i.

Group 4 – *E. multilocularis* infected (as Group 2) and treated with ABZ (as above) and glucan immunomodulator (GI) (Dimenzia, Slovak Republic) twice a week at the dose of 5 mg/kg b.w. at weeks 5 and 6 p.i.

Group 5 – *E. multilocularis* infected (as Group 2) and treated with ABZ (as above) with glucan supplied with zinc (GIZn) (Mevac, Slovak Republic) twice a week at the dose of 5 mg/kg b.w. at weeks 5 and 6 p.i.

Samples of blood, spleen, and peritoneal macrophages were obtained at the following weeks: 0 (prior infection), 2, 4, 6, 8, 10, 12, 14, 18, 22, and 26 p.i. from all groups (four mice per experimental day).

## **6. Results and discussion**

The *Echinococcus multilocularis* infection in our experiment inhibited the proliferative activity of T and B lymphocytes within 26 weeks of the experiment (Figures 12, 13), with the minimum at weeks 2 and 4 p.i., that is, in the early stage of infection, during the time important for the parasite establishment. These data correlate to results of other authors [17, 68] and it could be caused by immunosuppressive factors released by protoscoleces of *E. multilocularis*. The T cell proliferation (Figure 12) was stimulated in infected mice with GI+ABZ therapy from week 6 to 10 p.i., at the beginning of the progressive larval growth in infected mice without treatment. The GI stimulatory activity was prolonged for 4 weeks by zinc supplementation in GIZn immunomodulator. This confirms an important role for zinc in immune response, specifically lymphocyte proliferation induced by mitogens. T-cell division, maturation, and differentiation require an adequate zinc content [65, 69]. Even after ABZ therapy, the proliferative activity of T lymphocytes was increased during drug's administration, from week 6 to 12 p.i., similarly to another works [68, 70]. The modulatory effect of albendazole could be derived from the fact that a drug induces direct morphological and structural changes of parasite cysts walls and protoscoleces [71], which lead to the revealing of normally unexposed structural antigens, which are presented to T lymphocytes. It results in inducing a Th2 response (chronic antigen stimulation) or a Th1 response (small parasitic lesions or reduced antigen output/recognition) [72]. The positive synergistic effect of anthelmintic ABZ and immunomodultor GIZn resulted in a prolonged increased T cell proliferation for 8 weeks after the end of drug's administration.

Immunomodulators GI and GIZn did not induce such stimulative effect on B cell proliferation (Figure 13); they restored a suppressed proliferative activity of B cells only for a short time. The similar restoring effect of glucan was observed in other parasitic infections with *Ascaris suum* or *Toxocara canis*[60, 73–75]. The weaker influence of GIZn on B cells could be explained by the fact that severe zinc deficiency impairment of B cell function is rarely seen [76, 77] and also the humoral response of patients with progressive alveolar echinococcosis was not affected by zinc deficiency [67]. Both combined therapies GI+ABZ and GIZn+ABZ reinforced

Group 3 – *E. multilocularis* infected (as Group 2) and treated with albendazole (ABZ) (Sigma, Germany) per os (p.o.) twice a week at the dose of 10 mg/kg of b.w. starting at week 5 up to

Group 4 – *E. multilocularis* infected (as Group 2) and treated with ABZ (as above) and glucan immunomodulator (GI) (Dimenzia, Slovak Republic) twice a week at the dose of 5 mg/kg b.w.

Group 5 – *E. multilocularis* infected (as Group 2) and treated with ABZ (as above) with glucan supplied with zinc (GIZn) (Mevac, Slovak Republic) twice a week at the dose of 5 mg/kg b.w.

Samples of blood, spleen, and peritoneal macrophages were obtained at the following weeks: 0 (prior infection), 2, 4, 6, 8, 10, 12, 14, 18, 22, and 26 p.i. from all groups (four mice per

The *Echinococcus multilocularis* infection in our experiment inhibited the proliferative activity of T and B lymphocytes within 26 weeks of the experiment (Figures 12, 13), with the minimum at weeks 2 and 4 p.i., that is, in the early stage of infection, during the time important for the parasite establishment. These data correlate to results of other authors [17, 68] and it could be caused by immunosuppressive factors released by protoscoleces of *E. multilocularis*. The T cell proliferation (Figure 12) was stimulated in infected mice with GI+ABZ therapy from week 6 to 10 p.i., at the beginning of the progressive larval growth in infected mice without treatment. The GI stimulatory activity was prolonged for 4 weeks by zinc supplementation in GIZn immunomodulator. This confirms an important role for zinc in immune response, specifically lymphocyte proliferation induced by mitogens. T-cell division, maturation, and differentiation require an adequate zinc content [65, 69]. Even after ABZ therapy, the proliferative activity of T lymphocytes was increased during drug's administration, from week 6 to 12 p.i., similarly to another works [68, 70]. The modulatory effect of albendazole could be derived from the fact that a drug induces direct morphological and structural changes of parasite cysts walls and protoscoleces [71], which lead to the revealing of normally unexposed structural antigens, which are presented to T lymphocytes. It results in inducing a Th2 response (chronic antigen stimulation) or a Th1 response (small parasitic lesions or reduced antigen output/recognition) [72]. The positive synergistic effect of anthelmintic ABZ and immunomodultor GIZn resulted in a prolonged increased T cell proliferation for 8 weeks after the end of drug's administration. Immunomodulators GI and GIZn did not induce such stimulative effect on B cell proliferation (Figure 13); they restored a suppressed proliferative activity of B cells only for a short time. The similar restoring effect of glucan was observed in other parasitic infections with *Ascaris suum* or *Toxocara canis*[60, 73–75]. The weaker influence of GIZn on B cells could be explained by the fact that severe zinc deficiency impairment of B cell function is rarely seen [76, 77] and also the humoral response of patients with progressive alveolar echinococcosis was not affected by zinc deficiency [67]. Both combined therapies GI+ABZ and GIZn+ABZ reinforced

week 10 p.i.

at weeks 5 and 6 p.i.

172 Current Topics in Echinococcosis

at weeks 5 and 6 p.i.

experimental day).

**6. Results and discussion**

**Figure 12.** Proliferative activity of T lymphocytes in *E. multilocularis* infected mice after immunotherapy with GI or GIZn. \*(p<0.05); \*\*(p<0.01) statistically significant from infected mice.

**Figure 13.** Proliferative activity of B lymphocytes in *E. multilocularis* infected mice after immunotherapy with GI or GIZn. \*(p<0.05) statistically significant from infected mice.

a stimulatory activity of ABZ on B cell subpopulation from week 6 to 14 p.i., during the intensive cyst's growth in infected mice without treatment. The functional activity of T lymphocytes in cellular response to *E. multilocularis* infection is associated with two subpo‐ pulations of CD4+ and CD8+ T cells. A time-dependent supression of T cell proliferative response was observed in [78], and authors also recorded *in vitro* CD4 T cells' decrease and *vice versa* CD8 T cells' increase in splenocytes after activation with *E. multilocularis* protosco‐ leces.

**Figure 14.** Number of splenic CD4 T cells in *E. multilocularis* infected mice after immunotherapy with GI or GIZn. \*(p<0.05); \*\*(p<0.01) statistically significant from infected mice.

**Figure 15.** Serum IFN-γ in *E. multilocularis* infected mice after immunotherapy with GI or GIZn. \*(p<0.05); \*\*(p<0.01) statistically significant from infected mice.

CD4 T cells represent the main T subpopulation in the first phase of alveolar echinococcosis in granuloma formation around the parasite [17, 79]. In our experiment (Figure 14), the presence of CD4 T cells was stimulated from weeks 6 to 8 p.i. in the spleen of mice infected and treated with GI+ABZ. Supplementation of glucan with zinc could contributed to prolong an increase in the CD4 T cell subpopulation in mice treated with GIZn+ABZ from week 6 to 14 p.i. It has been known that zinc deficiency reduces the production of IL-4 cytokine acting

**Figure 16.** Serum IL-5 in *E. multilocularis* infected mice after immunotherapy with GI or GIZn. \*(p<0.05); \*\*(p<0.01) stat‐ istically significant from infected mice.

**Figure 14.** Number of splenic CD4 T cells in *E. multilocularis* infected mice after immunotherapy with GI or GIZn.

**Figure 15.** Serum IFN-γ in *E. multilocularis* infected mice after immunotherapy with GI or GIZn. \*(p<0.05); \*\*(p<0.01)

CD4 T cells represent the main T subpopulation in the first phase of alveolar echinococcosis in granuloma formation around the parasite [17, 79]. In our experiment (Figure 14), the presence of CD4 T cells was stimulated from weeks 6 to 8 p.i. in the spleen of mice infected and treated with GI+ABZ. Supplementation of glucan with zinc could contributed to prolong an increase in the CD4 T cell subpopulation in mice treated with GIZn+ABZ from week 6 to 14 p.i. It has been known that zinc deficiency reduces the production of IL-4 cytokine acting

\*(p<0.05); \*\*(p<0.01) statistically significant from infected mice.

174 Current Topics in Echinococcosis

statistically significant from infected mice.

as a growth factor for helper CD4 T cells [80]. The long-term stimulation of CD4 T cells after GIZn+ABZ therapy could increase antiparasite defence. Periparasitic granuloma in regressive alveolar echinococosis is composed of great numbers of CD4 T lymphocytes, macrophages, and myofibroblasts [81]. In patients with progressive disease, the CD8 T lymphocytes are dominant [82]. In our experiment, *E. multilocularis* infection did not induce serious changes in T cells. The CD8 T lymphocytes were not influenced by immunotherapy, except for a light growth in the CD8 T cell numbers at weeks 12 and 14 p.i. in mice infected and treated with GIZn+ABZ, which could be helpful in the development of immunosuppression of the host.

The Th1 and Th2 cytokine balance regulates the immune response to *E. multilocularis* [19]. The possible effect of the immunotherapy on the Th1/Th2 balance was evaluated according to serum levels of cytokines IFN-γ and IL-5. GIZn+ABZ therapy stimulated the level of serum IFN-γ with the strongest and longest effect in mice with *E. multilocularis* infection from week 8 to 22 p.i. (Figure 15). This result documents the positive effect of GIZn immunomodulator on preferential Th1 stimulation. Immunomodulators GI and GIZn decreased the serum level of IL-5 from week 6 p.i. (Figure 16). The strong Th2 downregulative effect of both glucan immunomodultors stopped the rise of IL-5 (recorded in mice infected and without the treatment) from week 14 p.i till the end of the experiment.

IFN-γ is widely recognized as a major priming signal for macrophages' activation, which have a key role in effector phase of immune response to *E. multilocularis.* Macrophages provide for the protoscoleces destruction by reactive oxygen metabolites production (superoxide anion, hydrogenperoxide)[20]. The activation of peritoneal macrophages in the course oftherapy was evaluated by generation and release of superoxide anion (O2 - ). Although the most increase in IFN-γserumlevelinour experiment was foundafterGIZntherapy,GIhadthe strongestimpact on O2 release (Figure 17). Both GI+ABZ and GIZn+ABZ therapies resulted in the huge genera‐ tion of O2 from week 12 to 18 p.i., but with lower values in mice treated with GIZn+ABZ.

**Figure 17.** Macrophages' activity in *E. multilocularis* infected mice after immunotherapy with GI or GIZn. \*(p<0.05); \*\*(p<0.01) statistically significant from infected mice.

A notable finding in the treatment efficacy was that GI's presence in GI+ABZ therapy partly inhibited the parasitostatic effect of ABZ (Figure 18).

It could be explained by GI's stimulatory effect on fibrosis in periparasitic granuloma, which could inhibit anthelmintic drug from penetrating to parasite vesicle. The glucan's profibrotic activity in metacestode infection *Mesocestoides corti* was found in [83], where glucan therapy stimulated mononuclear-phagocytic cells in the liver followed by increased fibrotic process around parasite's larvae. In our experiment, GIZn+ABZ therapy was evaluated as the most effective therapeutic approach to alveolar echinococcosis, because it stopped larval cyst's growth in the host till week 14 p.i. This coincides with the highest levels of IFN-γ in our experiment – the cytokine with an antifibrotic property [21]. Cytokine IFN-γ plays a key role in the regulation of collagen metabolism and might ameliorate liver fibrogenesis. Also in [84] it was documented that IFN-γ downregulated lysyl oxidase gene expression, an essential catalyst for the cross-linking of extracellular collagen and elastin.

In our experiment, the stimulation of IFN-γ production induced by GIZn could reduce the irreversible fibrosis in periparasitic granuloma and thus allow penetration of anthelmintics to larvocyst. IFN-γ has not only an antifibrotic property, but it is also engaged in Th1 immune reactions, which are protective against *E. multilocularis* and include fibrogenesis. A correlation of T cell immune response and hepatic fibrosis was found in [85]; serum IFN-γ levels were negatively correlated with serological markers of fibrosis. Another immunological component connected with fibrogenesis appear to be reactive oxygen metabolites produced by macro‐ phages. Superoxide anion, hydrogenperoxide, and hydroxyl radical are important stimuli to colagen gene induction of hepatic stellate cells (HSC) [86]. Collagen synthesis and subsequent cross-linking of collagen by pyridinoline, leading to irreversible fibrosis in periparasitic granuloma, is a substantial component of the host immune reaction in forming a resistance against parasite growth [87]. Therefore, the stimulation of fibrotic process could be beneficial

**Figure 18.** Cyst's weight in *E. multilocularis* infected mice after immunotherapy with GI or GIZn. \*(p<0.05); \*\*(p<0.01) statistically significant from infected mice.

for the host. On the contrary, overproduction of fibrous tissue may be harmful for host, as fibrosis results in many complications of alveolar echinococcosis in patients: vasculary and biliary obstructions in the liver [88]. In our experiment, the greatest inducer of O2 generation in peritoneal macrophages (connected with hepatic fibrosis) was GI. On the other hand, GI supplemented with zinc induced a short-time and a weaker production of this metabolite.

Zinc plays an important role as cofactor of enzymes involved in collagen synthesis [89] and zinc supplemention has favorable inhibitive effects on hepatic fibrosis [90, 91]. This fact can explain better the antiparasitic efficacy of treatment with GIZn and nearly twofold higher efficacy of GIZn+ABZ therapy in comparison with ABZ treatment (Figure 18).

According to host's immunosuppression and zinc deficiency in patients with alveolar echi‐ nococcosis [67], the combination of immunomodulator GIZn and anthelmintic drug ABZ achieved the most effective control of the parasite infection. The parasitostatic effect of therapy GIZn+ABZ lasted for the longest time; also after the end of the therapy it reached the greatest reduction of *E. multilocularis* cysts in the host. Zinc supplemention of glucan immunomodu‐ lator improved its immunostimulatory effects on cellular immunity, which activated Th1 response and significantly stimulated IFN-γ cytokine synthesis. We suppose zinc positively downregulated the liver fibrosis induced by glucan, because GIZn immunomodulator suppressed the generation of superoxide – an activator of hepatic stella cells involved in the fibrotic process.

#### **7. Transfer factor**

A notable finding in the treatment efficacy was that GI's presence in GI+ABZ therapy partly

**Figure 17.** Macrophages' activity in *E. multilocularis* infected mice after immunotherapy with GI or GIZn. \*(p<0.05);

It could be explained by GI's stimulatory effect on fibrosis in periparasitic granuloma, which could inhibit anthelmintic drug from penetrating to parasite vesicle. The glucan's profibrotic activity in metacestode infection *Mesocestoides corti* was found in [83], where glucan therapy stimulated mononuclear-phagocytic cells in the liver followed by increased fibrotic process around parasite's larvae. In our experiment, GIZn+ABZ therapy was evaluated as the most effective therapeutic approach to alveolar echinococcosis, because it stopped larval cyst's growth in the host till week 14 p.i. This coincides with the highest levels of IFN-γ in our experiment – the cytokine with an antifibrotic property [21]. Cytokine IFN-γ plays a key role in the regulation of collagen metabolism and might ameliorate liver fibrogenesis. Also in [84] it was documented that IFN-γ downregulated lysyl oxidase gene expression, an essential

In our experiment, the stimulation of IFN-γ production induced by GIZn could reduce the irreversible fibrosis in periparasitic granuloma and thus allow penetration of anthelmintics to larvocyst. IFN-γ has not only an antifibrotic property, but it is also engaged in Th1 immune reactions, which are protective against *E. multilocularis* and include fibrogenesis. A correlation of T cell immune response and hepatic fibrosis was found in [85]; serum IFN-γ levels were negatively correlated with serological markers of fibrosis. Another immunological component connected with fibrogenesis appear to be reactive oxygen metabolites produced by macro‐ phages. Superoxide anion, hydrogenperoxide, and hydroxyl radical are important stimuli to colagen gene induction of hepatic stellate cells (HSC) [86]. Collagen synthesis and subsequent cross-linking of collagen by pyridinoline, leading to irreversible fibrosis in periparasitic granuloma, is a substantial component of the host immune reaction in forming a resistance against parasite growth [87]. Therefore, the stimulation of fibrotic process could be beneficial

inhibited the parasitostatic effect of ABZ (Figure 18).

\*\*(p<0.01) statistically significant from infected mice.

176 Current Topics in Echinococcosis

catalyst for the cross-linking of extracellular collagen and elastin.

The dialysable leucocyte extract, a product of the immune system, is known as transfer factor (TF). Transfer factors are low molecular weight dialysable products extracted from immune cells which transmit the ability to express delayed-type hypersensitivity and cell-mediated immunity from sensitized donors to nonimmune recipients [92]. TF increases macrophage activation and IL-1, IL-2, and IFN-γ production *in vitro* and enhances leucocyte chemotaxis and natural killer function [93, 94]. The TF preparates activated the proliferation of splenocytes from animals with hypothyroidism [95] and stimulated hemopoiesis [96]. Transfer factor stimulated antigen-specific cell-mediated response in patients with various infections, and so administration of transfer factor has been recommended for patients with selective deficits in cell-mediated immunity such as refractory neoplasms and chronic infections [94]. Adminis‐ tration of dialysable leucocyte extract has been shown to be free of hypersensitivity, longlasting side effects or complications, except for transitory hyperpyrexia.

Nonspecific transfer factor (TF) from the blood leukocytes of immunized swine was studied as an appropriate candidate for immunotherapy, supplementing conventional ABZ treatment of alveolar echinococcosis.

#### **7.1. Experimental design**

Experiments were carried out on male BALB/c mice (n=165) weighing 20–25 g. Mice were randomly divided into five groups as follows:

Group 1 – uninfected and untreated (control)

Group 2 – infected intraperitoneally with 5000 *E. multilocularis* protoscoleces/mouse on Day 0 and no treatment

Group 3 – *E. multilocularis* infected (as Group 2) and treated with transfer factor (TF) (Imunor, ImunomedicA, Czech Republic) per os (p.o.) twice a week at the dose of 200 μg/kg of body weight (b.w.) starting at week 5 up to week 10 p.i.

Group 4 – *E. multilocularis* infected (as Group 2) and treated with albendazole (ABZ) (Sigma, Germany) per os (p.o.) twice a week at the dose of 10 mg/kg of b.w. starting at week 5 up to week 10 p.i.

Group 5 – *E. multilocularis* infected (as Group 2) and treated with combination of TF and ABZ as above

Samples of blood, spleen, and peritoneal macrophages were obtained at the following weeks: 0 (prior infection), 2, 4, 6, 8, 10, 12, 14, 18, 22, and 26 p.i. from all groups (3 mice per experimental day).

#### **8. Results and discussion**

Transfer factors (TFs) are proteins that transfer specific cellular immunity from an immune donor to a nonimmune recipient and possess a number of nonspecific activities, such as the ability to increase the numbers of immunocompetent cells, to stimulate phagocytosis, to induce the production of interferons and interleukins, to stimulate hemopoiesis, etc. TF includes a lot of molecules, acting as antigens (MW cca 5 kDa), or acting as immunomodulators (less than 3.5 kDa) [97]. Transfer factors are very efficient in diseases in which cellular immunity plays an important role in protection and control of the disease, for example, viral, bacterial, and parasite infections, as well as immunodeficiencies and some types of cancer [98].

cells which transmit the ability to express delayed-type hypersensitivity and cell-mediated immunity from sensitized donors to nonimmune recipients [92]. TF increases macrophage activation and IL-1, IL-2, and IFN-γ production *in vitro* and enhances leucocyte chemotaxis and natural killer function [93, 94]. The TF preparates activated the proliferation of splenocytes from animals with hypothyroidism [95] and stimulated hemopoiesis [96]. Transfer factor stimulated antigen-specific cell-mediated response in patients with various infections, and so administration of transfer factor has been recommended for patients with selective deficits in cell-mediated immunity such as refractory neoplasms and chronic infections [94]. Adminis‐ tration of dialysable leucocyte extract has been shown to be free of hypersensitivity, long-

Nonspecific transfer factor (TF) from the blood leukocytes of immunized swine was studied as an appropriate candidate for immunotherapy, supplementing conventional ABZ treatment

Experiments were carried out on male BALB/c mice (n=165) weighing 20–25 g. Mice were

Group 2 – infected intraperitoneally with 5000 *E. multilocularis* protoscoleces/mouse on Day 0

Group 3 – *E. multilocularis* infected (as Group 2) and treated with transfer factor (TF) (Imunor, ImunomedicA, Czech Republic) per os (p.o.) twice a week at the dose of 200 μg/kg of body

Group 4 – *E. multilocularis* infected (as Group 2) and treated with albendazole (ABZ) (Sigma, Germany) per os (p.o.) twice a week at the dose of 10 mg/kg of b.w. starting at week 5 up to

Group 5 – *E. multilocularis* infected (as Group 2) and treated with combination of TF and ABZ

Samples of blood, spleen, and peritoneal macrophages were obtained at the following weeks: 0 (prior infection), 2, 4, 6, 8, 10, 12, 14, 18, 22, and 26 p.i. from all groups (3 mice per experimental

Transfer factors (TFs) are proteins that transfer specific cellular immunity from an immune donor to a nonimmune recipient and possess a number of nonspecific activities, such as the ability to increase the numbers of immunocompetent cells, to stimulate phagocytosis, to induce the production of interferons and interleukins, to stimulate hemopoiesis, etc. TF includes a lot

lasting side effects or complications, except for transitory hyperpyrexia.

of alveolar echinococcosis.

178 Current Topics in Echinococcosis

**7.1. Experimental design**

and no treatment

week 10 p.i.

as above

day).

**8. Results and discussion**

randomly divided into five groups as follows:

Group 1 – uninfected and untreated (control)

weight (b.w.) starting at week 5 up to week 10 p.i.

The host cellular immunity plays a principal role in the control of the *E. multilocularis* prolif‐ eration, which was documented by experiments, in which mouse strains not developing cellular immune response do not control metacestode growth, while mice deficient in humoral immunity control parasite growth up to a certain level [99]. Furthermore, T-cell immunity significantly contributes to the control of alveolar echinococcosis in human patients, demon‐ strated by the rapid fatal outcome of the infection in an immunodeficient patient coinfected with human immunodeficiency virus (HIV) [100]. Upon restoration of CD4-immunocomp‐ tence in another AIDS-patient coinfected with *E. multilocularis*, the course of disease was positively influenced again [101].

In our work, both the therapy with TF or combination of TF+ABZ abolished a suppressory impact of *E. multilocularis* infection on T- and B-cell proliferation (Figures 19, 20) and the therapeutic stimulatory effect was recorded up to week 14 p.i., that is, one month after the end of the therapy. Immunomodulating activity of TF on lymphocyte functional status was also recorded by [95], who detected an increased proliferation of splenocytes from rats with hypothyroidism induced by TF. A significant stimulative effect of TF on T- and B-cell popu‐ lations was also confirmed in experimental ascariasis in pigs [63].

**Figure 19.** Proliferative activity of T lymphocytes in *E. multilocularis* infected mice after therapy TF+ABZ. \*(p<0.05); \*\*(p<0.01) statistically significant from infected mice.

T lymphocytes play a key role in the control of immune response in alveolar echinococcosis [18, 36]. In our study, the administration of TF alone or in combination of TF+ABZ caused the increase in numbers of CD4+ T cells in the spleen of infected mice up to week 14 p.i. (Figure 21). Patients with active alveolar echinococcosis showed increased numbers of CD8+ T lymphocytes [20] and also our results documented an increase in this subpopulation after *E.* *multilocularis* infection from weeks 4 to 8 p.i., that is, during the massive proliferation of larval cysts. The CD8+ T cells were not markedly influenced with TF immunomodulator, because all therapeutic approaches inhibited the CD8+ T cell numbers during drug administration, from weeks 6 to 10 p.i., but with no effect after the end of therapies. The splenic CD8+ T subpopu‐ lation in alveolar echinococcosis are mostly T suppressor cells [78]. Therefore, we deduce that TF therapy stopped the development of host immunosuppression only for a short time, during the drug administration. The increase in CD4+ T lymphocytes induced by TF was also observed in viral infection (herpes zoster) and CD8+ T lymphocytes showed less decrease [102], similar to our results.

**Figure 20.** Proliferative activity of B lymphocytes in *E. multilocularis* infected mice after therapy TF+ABZ. \*(p<0.05); \*\*(p<0.01) statistically significant from infected mice.

**Figure 21.** Number of splenic CD4 T cells in *E. multilocularis* infected mice after therapy TF+ABZ. \*(p<0.05) statistically significant from infected mice.

Active alveolar echinococcosis is related to Th2 response – dominance of IL-5 and IL-10 and week production of IFN-γ [20]. The superiority of Th1 response plays a key role in the host defence reactions against *E. multilocularis*. TF therapy selectively affects cytokine production in response to antigen stimulation, particularly by secreting IFN-γ [93]. In our experiment, the results of cytokine proteins detected by capture ELISA may not correlate with the levels of bioactive cytokine protein. ELISA may utilize anticytokine antibodies that cannot discriminate between the precursor (inactive) and mature (bioactive) forms of a cytokine protein. Moreover, an ELISA may detect partially degraded cytokine proteins, which have retained their immu‐ noreactive properties but may have lost their bioactivity [103, 104]. But capture ELISA is a helpful indicator of cytokine presence, although it does not reflect the biological potency of the detected cytokines.

*multilocularis* infection from weeks 4 to 8 p.i., that is, during the massive proliferation of larval cysts. The CD8+ T cells were not markedly influenced with TF immunomodulator, because all therapeutic approaches inhibited the CD8+ T cell numbers during drug administration, from weeks 6 to 10 p.i., but with no effect after the end of therapies. The splenic CD8+ T subpopu‐ lation in alveolar echinococcosis are mostly T suppressor cells [78]. Therefore, we deduce that TF therapy stopped the development of host immunosuppression only for a short time, during the drug administration. The increase in CD4+ T lymphocytes induced by TF was also observed in viral infection (herpes zoster) and CD8+ T lymphocytes showed less decrease [102], similar

**Figure 20.** Proliferative activity of B lymphocytes in *E. multilocularis* infected mice after therapy TF+ABZ. \*(p<0.05);

**Figure 21.** Number of splenic CD4 T cells in *E. multilocularis* infected mice after therapy TF+ABZ. \*(p<0.05) statistically

to our results.

180 Current Topics in Echinococcosis

\*\*(p<0.01) statistically significant from infected mice.

significant from infected mice.

The concentration of serum IFN-γ in mice infected with *E. multilocularis* and treated with TF or both TF+ABZ (Figure 22) was increased during therapy and also for one month after therapy (from weeks 6 to 14 p.i.). The stimulative effect of TF on Th1 response, particularly together with drug TF+ABZ, was observed in IFN-γ production *in vitro* [105], where high release of this cytokine from splenocytes was found till the end of the experiment. Similar results were achieved in mice with tuberculosis and treated with TF and antibiotics [98]. Th1-inducing effect of TF was also confirmed in antitumour immunotherapy [92]. The concentration of IL-5 in serum in mice infected with *E. multilocularis* and treated with TF or TF+ABZ in our work was suppressed till the end of the experiment. This is consistent with an inhibited production of IL-5 *in vitro* after immunotherapy with TF and TF+ABZ [105], which was significantly decreased two weeks after the end of the therapy. The intensive Th2 downregulative effect of TF was found by [93], who detected low splenocytes secretion of IL-10 and no IL-4. This cytokine IL-5 modulates eosinophilia, IgE production, and can enhance allergic reactions of the disease [20].

**Figure 22.** Serum IFN-γ in *E. multilocularis* infected mice after therapy TF+ABZ. \*(p<0.05); \*\*(p<0.01) statistically signifi‐ cant from infected mice.

**Figure 23.** Macrophages' activity in *E. multilocularis* infected mice after therapy TF+ABZ. \*(p<0.05); \*\*(p<0.01) statisti‐ cally significant from infected mice.

IFN-γ (that was stimulated after TF therapy) is an important activator of macrophages and affects their energetic system [21]. Immunotherapy with TF or TF+ABZ activated the respira‐ tory burst in macrophages with high generation of O<sup>2</sup> - for a long time, from weeks 8 to 18 p.i. (Figure 23). This is documented by a long-term stimulation of macrophages' metabolic activity accompanied with a high production of also other biologically effective substances. At the same time, the proliferation of B cells and the IFN-γ level were increased. B cells release stimulation factors for macrophages after activation with antigens from destroyed *Echinococ‐ cus* protoscoleces [46] and cytokine IFN-γ markedly affects the macrophage energetic system. In our study, high concentrations of IFN-γ time-matched with intensive O<sup>2</sup> production and slow parasite proliferation in infected mice treated with TF or TF+ABZ. Stimulative effect of TF on macrophages' metabolism had also been recorded by [106], who found the TF dialysis fraction (less than 3500 MW) activating the capacity of peritoneal macrophages to produce superoxide anion.

Activation of effector components of immune response after the immunotherapy of *E. multilocularis* infected mice in our experiment reduced the parasite growth and limited the development of larval cysts in the host (Figure 24). The immunomodulatory effect of TF suppressed the parasite proliferation till week 14 p.i. with the similar activity to anthelmintic drug albendazole and prolonged the parasite-reducing effect for one month, that is, till week 18 p.i. The TF-inhibiting effect on the parasite development was associated with the time of TF administration and a short time after it. The addition of TF to albendazole therapy resulted in a high antiparasitic effect with a significant cyst's reduction up to week 18 p.i. (2 months after the end of the therapy) in comparison to untreated group. Thereafter, the protective effect of anthelmintic therapy was lost.

**Figure 24.** Cyst's weight in *E. multilocularis* infected mice after therapy TF+ABZ. \*(p<0.05); \*\*(p<0.01) statistically signif‐ icant from infected mice.

In conclusion, the results of our study suggest that TF can have a positive effect on the host cellular immune response against the development of *E. multilocularis*. Nowadays, it is known that TF consists of a high quantity of low molecular weight proteins (> 200), and the exact chemical nature and molecular mechanisms of its action have not been defined yet. Low synthesis of IFN-γ induced by the *E. multilocularis*infection was restored with TF and exceeded the control concentration. The rise recorded in IFN-γ cytokine could be explained as a result of immunorestoration of CD4+ Th1 lymphocytes or due to the proliferation of another CD4 subpopulation. We think that regulation of immune homeostasis with TF therapy, which occurred between the CD4 cells and IFN-γ, played an important role in the immune response to *E. multilocularis* infection. Regulatory and effector mechanisms (CD4+ T cells, IFN-γ, and macrophages) which actively participate in the parasite destruction were stimulated with TF and supported antiparasitic effect of albendazole.

#### **9. Conclusion**

**Figure 23.** Macrophages' activity in *E. multilocularis* infected mice after therapy TF+ABZ. \*(p<0.05); \*\*(p<0.01) statisti‐

IFN-γ (that was stimulated after TF therapy) is an important activator of macrophages and affects their energetic system [21]. Immunotherapy with TF or TF+ABZ activated the respira‐

(Figure 23). This is documented by a long-term stimulation of macrophages' metabolic activity accompanied with a high production of also other biologically effective substances. At the same time, the proliferation of B cells and the IFN-γ level were increased. B cells release stimulation factors for macrophages after activation with antigens from destroyed *Echinococ‐ cus* protoscoleces [46] and cytokine IFN-γ markedly affects the macrophage energetic system.

slow parasite proliferation in infected mice treated with TF or TF+ABZ. Stimulative effect of TF on macrophages' metabolism had also been recorded by [106], who found the TF dialysis fraction (less than 3500 MW) activating the capacity of peritoneal macrophages to produce

Activation of effector components of immune response after the immunotherapy of *E. multilocularis* infected mice in our experiment reduced the parasite growth and limited the development of larval cysts in the host (Figure 24). The immunomodulatory effect of TF suppressed the parasite proliferation till week 14 p.i. with the similar activity to anthelmintic drug albendazole and prolonged the parasite-reducing effect for one month, that is, till week 18 p.i. The TF-inhibiting effect on the parasite development was associated with the time of TF administration and a short time after it. The addition of TF to albendazole therapy resulted in a high antiparasitic effect with a significant cyst's reduction up to week 18 p.i. (2 months after the end of the therapy) in comparison to untreated group. Thereafter, the protective effect

In our study, high concentrations of IFN-γ time-matched with intensive O<sup>2</sup>



production and

cally significant from infected mice.

182 Current Topics in Echinococcosis

superoxide anion.

of anthelmintic therapy was lost.

tory burst in macrophages with high generation of O<sup>2</sup>

Our results suggest the possible way to increase ABZ antiparasitic efficacy in *E. multilocula‐ ris* infection by stimulation of the host immune response.

L-MTP-PE was recognized as a strong macrophage activator in mice infected with *E. multilo‐ cularis*. This immunomodulator stimulated the superoxide anion production for 3 months. The activity of macrophages was supported by dominant Th1 protective response – considered the highest serum IFN-γ level in that time. The antiparasitic effect of combined L-MTP-PE +ABZ therapy was manifested by marked reduction of cysts growth for 3 months after the end of treatment.

GIZn+ABZ has been shown to be the most effective immunomodulatory and antiparasitic treatment, the greatest reduction of metacestode growth was observed as early as 2 weeks from the beginning of this therapy, lasting till the end of experiment (for 4 months). The addition of zinc to immunomodulatory glucan substance has greatly contributed to the reduction of intensive fibrosis after glucan treatment, which attenuated the antiparasitic effect of ABZ alone.

TF could correct the immune balance disturbance after the *E. multilocularis* infection, which is responsible for lymphocyte suppressor activity. Inhibited IFN-γ production after the *E. multilocularis* infection was restored and increased with TF, which could result from a resto‐ ration of CD4+ Th1 lymphocytes. TF administration prolonged the parasitostatic effect of ABZ for 2 months. Restoration of suppressed host immune mechanisms that are important for restriction of parasite expansive growth seems to be a promissing approach to *E. multilocula‐ ris* infection.

Immunomodulatory agents can be beneficial in the treatment of alveolar echinococcosis, where the developed Th1 immune response supports the improvement of the anthelmintic action of benzimidazoles. The type and development of the immune response are generally managed by the lymphocyte network and its immunoregulatory mechanism. Immunomodulators could regulate the immune disbalance after the *E. multilocularis* infection, which suppresses lym‐ phocytes' and macrophages' activities. Restoring of this immunosuppresion may be one promising way to overcome this situation and to control chronic alveolar echinococcosis.

### **Acknowledgements**

This study was supported by the Slovak VEGA agency, grants No. 2/0172/13 and No. 2/0081/15.

#### **Author details**

Emília Dvorožňáková\*

Address all correspondence to: dvoroz@saske.sk

Institute of Parasitology, Slovak Academy of Sciences, Košice, Slovak Republic

#### **References**

[1] Wilson JF, Rausch RL, Wilson FR. Alveolar hydatid disease. Review of the surgical experience in 42 cases of active disease among Alaskan Eskimos. *Ann Surg* 1995;221(3) 315-323.


GIZn+ABZ has been shown to be the most effective immunomodulatory and antiparasitic treatment, the greatest reduction of metacestode growth was observed as early as 2 weeks from the beginning of this therapy, lasting till the end of experiment (for 4 months). The addition of zinc to immunomodulatory glucan substance has greatly contributed to the reduction of intensive fibrosis after glucan treatment, which attenuated the antiparasitic effect of ABZ alone.

TF could correct the immune balance disturbance after the *E. multilocularis* infection, which is responsible for lymphocyte suppressor activity. Inhibited IFN-γ production after the *E. multilocularis* infection was restored and increased with TF, which could result from a resto‐ ration of CD4+ Th1 lymphocytes. TF administration prolonged the parasitostatic effect of ABZ for 2 months. Restoration of suppressed host immune mechanisms that are important for restriction of parasite expansive growth seems to be a promissing approach to *E. multilocula‐*

Immunomodulatory agents can be beneficial in the treatment of alveolar echinococcosis, where the developed Th1 immune response supports the improvement of the anthelmintic action of benzimidazoles. The type and development of the immune response are generally managed by the lymphocyte network and its immunoregulatory mechanism. Immunomodulators could regulate the immune disbalance after the *E. multilocularis* infection, which suppresses lym‐ phocytes' and macrophages' activities. Restoring of this immunosuppresion may be one promising way to overcome this situation and to control chronic alveolar echinococcosis.

This study was supported by the Slovak VEGA agency, grants No. 2/0172/13 and No. 2/0081/15.

[1] Wilson JF, Rausch RL, Wilson FR. Alveolar hydatid disease. Review of the surgical experience in 42 cases of active disease among Alaskan Eskimos. *Ann Surg*

Institute of Parasitology, Slovak Academy of Sciences, Košice, Slovak Republic

*ris* infection.

184 Current Topics in Echinococcosis

**Acknowledgements**

**Author details**

**References**

1995;221(3) 315-323.

Emília Dvorožňáková\*

Address all correspondence to: dvoroz@saske.sk


[29] Asano T, McWatters A, An T, Matsushima K, Kleinerman ES. Liposomal muramyl tripeptide up-regulates interleukin-1 alpha, interleukin-1 beta, tumor necrosis factoralpha, interleukin-6 and interleukin-8 gene expression in human monocytes. *J Phar‐ macol Exper Therap* 1994;268(2) 1032-1039.

[17] Emery I, Liance M, Deriaud E, Vuitton DA, Houin R, Leclerc C. Characterization of T-cell immune responses of *Echinococcus multilocularis*-infected C57BL/6J mice*. Paras*

[18] Manfras BJ, Reuter S, Wendland T, Boehm BO, Kern P. Impeded Th1 CD4 memory T cell generation in chronic-persisting liver infection with *Echinococcus multilocularis*.

[19] Sturm D, Menzel J, Gottstein B, Kern P. Interleukin-5 is the predominant cytokine produced by peripheral blood mononuclear cells in alveolar echinococcosis. *Infect*

[20] Vuitton DA. The ambiguous role of immunity in echinococcosis: protection of the

[21] Liance M, Ricard-Blum S, Emery I, Houin R, Vuitton DA. *Echinococcus multilocularis* infection in mice: in vivo treatment with a low dose of IFN-gamma decreases meta‐

[22] Šoltýs J, Quinn MT. Modulation of endotoxin- and enterotoxin-induced cytokine re‐ lease by in vivo treatment with beta-(1,6)-branched beta-(1,3)-glucan. *Infect Immun*

[23] Hrčková G, Velebný S. Effect of praziquantel and liposome-incorporated praziquan‐ tel on peritoneal macrophage activation in mice infected with *Mesocestoides corti* tet‐

[24] Pabst MJ, Beranova-Giorgianni S, Krueger JM. Effects of muramyl peptides on mac‐ rophages, monokines, and sleep. *Neuroimmunomodulation* 1999;6(4) 261-283.

[25] Adam A, Lederer E. Muramylpeptides as immunomodulators. *Atl Sci - Immunol*

[26] Hui GS, Tam LQ, Chang SP, Case SE, Hashiro C, Siddiqui WA, Shiba T, Kusumoto S, Kotani S. Synthetic low-toxicity muramyl dipeptide and monophosphoryl lipid A re‐ place Freund complete adjuvant in inducing growth-inhibitory antibodies to the *Plas‐ modium falciparum* major merozoite surface protein, gp195. *Infect Immun* 1991;59(5)

[27] Dvorožňáková E, Borošková Z, Dubinský P, Tomašovičová O, Hříbalová V, Mach‐ nicka B. Immunomodulative effect of muramyldipeptide in mice with larval toxoca‐

[28] Dieter P, Ambs P, Fityke E, Schwende H. Lipopolysaccharide and liposome-encapsu‐ lated MTP-PE-induced cytotoxicity and release of eicosanoids, tumor necrosis factoralpha and nitric oxide in liver macrophages. In: Honn KV, Marnett LJ, Nigam S, et al. (eds.) *Eicosanoids And Other Bioactive Lipids in Cancer, Inflammation, and Radiation In‐*

host or of the parasite? *Acta Tropica* 2003;85(2) 119-132.

rathyridia (Cestoda). *Parasitology* 1997;114(5) 475-482.

rosis. *Parasitol Res* 1999;85(12) 1034-1040.

*jury 3* Book Series: *Adv Exper Med Biol* 1997;Vol407 485-490.

cestode growth and liver fibrogenesis. *Parasite* 1998;5(3) 231-237.

*Immunol* 1996;18(9) 463-472*.*

186 Current Topics in Echinococcosis

*Int Immunol* 2004;16(1) 43-50.

*Immun* 1995;63(5) 1688-1697.

1999;67(1) 244-252.

1988;1(3-4) 205-214.

1585-1591.


[51] Adinolfi LE, Bonventre PF, Vanderpas M, Eppstein DA. Synergistic effect of glucan‐ time and a liposome-encapsulated muramyl dipeptide analog in therapy of experi‐ mental visceral *Leishmania*sis. *Infect Immun* 1985;48(2) 409-416.

[39] Harraga S, Godot V, Bresson-Hadni S, Mantion G, Vuitton DA. Profile of cytokine production within the periparasitic granuloma in human alveolar echinococcosis. *Ac‐*

[40] Hübner MP, Manfras BJ, Margos MC, Eiffler D, Hoffmann WH, Schulz-Key H, Kern P, Soboslay PT. *Echinococcus multilocularis* metacestodes modulate cellular cytokine and chemokine release by peripheral blood mononuclear cells in alveolar echinococ‐

[41] Sehra S, Chugh L, Gangal SV. Role of liposomes in selective proliferation of splenic

[42] Jenne L, Kilwinski J, Scheffold W, Kern P. IL-5 expressed by CD4+ lymphocytes from *Echinococcus multilocularis*-infected patients. *Clin Exper Immunol* 1997,109(1):90-97.

[44] Sher A, Fiorentino D, Caspar P, Pearce E, Mosmann T. Production of IL-10 by CD4+ T lymphocytes correlates with down-regulation of Th1 cytokine synthesis in hel‐

[45] Fiorentino D, Zlotnik A, Vieira P, Mosmann TR, Howard M, Moore KW, O'Garra A. IL-10 acts on the antigen-presenting cell to inhibit cytokine production by Th1 cells. *J*

[46] Šoltýs J, Turčeková L, de Rycke PH. The effect *Echinococcus* hydatid cyst fluid and protoscoleces on mouse peritoneal macrophages and spleen lymphocytes. *Helmintho‐*

[47] Xu Z, Fidler IJ. The *in situ* activation of cytotoxic properties in murine Kupffer cells by the systemic administration of whole *Mycobacterium bovis* organisms or muramyl

[48] Karpoff HM, Jarnagin W, Delman K, Fong Y. Regional muramyl tripeptide phospha‐ tidylethanolamine administration enhances hepatic immune function and tumor sur‐

[49] Tanguay S, Bucana CD, Wilson MR, Fidler IJ, von Eschenbach AC, Killion JJ. *In vivo* modulation of macrophage tumoricidal activity by oral administration of the lipo‐ some-encapsulated macrophage activator CGP 19835A. *Canc Res* 1994;54(22)

[50] Melissen PM, van Vianen W, Bidjai O, van Marion M, Bakker-Woundenberg IA. Free versus liposome-encapsulated muramyl tripeptide phosphatidylethanolamide (MTPPE) and interferon-γ (IFN-γ) in experimental infection with *Listeria* monocyto‐

[43] Vuitton DA. Echinococcosis and allergy. *Clin Rev Aller Immunol* 2004;26(2) 93-104.

cosis patients. *Clin Exper Immunol* 2006;145(2) 243-251.

lymphocytes. *Mole Cell Biochem* 1998;183(1-2) 133-139.

minth infection. *J Immunol* 1991;147(8) 2713-2716.

tripeptide. *Can Immunol Immunother* 1984;18(2) 118-122.

*Immunol* 1991;146(10) 3444-3451.

veillance. *Surgery* 2000;128(2) 213-218.

genes. *Biotherapy* 1993;6(2) 113-124.

*logia* 1999;36(1) 25-30.

5882-5888.

*ta Tropica* 2003;85(2) 231-236.

188 Current Topics in Echinococcosis


[77] Crea A, Guérin V, Ortega F, Hartemann P. Zinc and the immune system. *Annales de Médecine Interne* 1990;141(5) 447-451.

[63] Benková M, Borošková Z, Šoltýs J. Immunostimulatory effects of certain substances in experimental ascaridiasis in pigs. *Vet Med* (Prague) 1991;36(12) 717-724.

[64] Wellinghausen N, Kirchner H, Rink L. The immunobiology of zinc. *Immunol Today*

[65] Rink L, Kirchner H. Zinc-altered immune function and cytokine production. *J Nutr*

[66] Chowdhury N, Singh R. Distribution of zinc in parasitic helminths. *J Helminthol* 1989;

[67] Wellinghausen N, Jochle W, Reuter S, Flegel WA, Grunert A, Kern P. Zinc status in patients with alveolar echinococcosis is related to disease progression. *Paras Immunol*

[68] Borošková Z, Dvorožňáková E, Ševčíková Z. Cellular immune reactions of mice with alveolar echinococcosis after albendazole therapy. *Helminthologia* 2003;40(4) 187-194.

[69] Baum MK, Shor-Posner G, Campa A. Zinc status in human immunodeficiency virus

[70] Dvorožňáková E, Hrčková G, Borošková Z, Velebný S, Dubinský P. Effect of treat‐ ment with free and liposomized albendazole on selected immunological parameters and cyst growth in mice infected with *Echinococcus multilocularis*. *Parasitol Int*

[71] Pérez-Serrano J, Denegri G, Casado N, Rodríguez-Caabeiro F. *In vivo* effect of oral al‐ bendazole and albendazole sulphoxide on development of secondary echinococcosis

[72] Abbas AK, Murphy KM, Sher A. Functional diversity of helper T lymphocytes. *Na‐*

[73] Šoltýs J, Benková M, Borošková Z. Immunorestorative effect of glucan immunomo‐ dulator on guinea pigs with experimental ascariosis. *Vet Immunol Immunopathol*

[74] Borošková Z, Dvorožňáková E, Dubinský P, Velebný S, Tomašovičová O, Machnická B. Effect of free and liposomised albendazole on the immune responses in healthy

[75] Dvorožňáková E, Akao A. 2000: Th1 and Th2 response after glucan immunomodula‐ tion in murine larval toxocarosis. Reports of Czech Parasitological Society, Prague,

[76] Bach JF. The multi-faceted zinc dependency of the immune system. *Immunol Today*

and *Toxocara canis* infected mice. *Vet Med* (Prague) 1998;43(10) 293-300.

1997;18(11) 519-521.

63(2) 149-152.

190 Current Topics in Echinococcosis

1999;21(5) 237-241.

2004;53(4) 315-325.

*ture* 1996;383, 787-793.

1994;42(3-4) 379-388.

Czech Republic, 2000. S8:53.

1981;2(11) 225-227.

2000;130(Suppl) S1407-S1411.

infection. *J Nutr* 2000;130(Suppl) S1421-S1423.

in mice. *Int J Parasitol* 1997;27(11) 1341-1345.


[102] Zingg W, Renner-Schneiter EC, Pauli-Magnus C, Renner EL, vanOverbeck J, Schlap‐ fer E, Weber M, Weber R, Opravil M, Gottstein B, Speck RF. Swiss HIV Cohort Study. Alveolar echinococcosis of the liver in an adult with human immunodeficiency virus type-1 infection. *Infection* 2004;32(5) 299-302.

[90] Kojima-Yuasa A, Ohkita T, Yukami K, Ichikawa H, Takami N, Nakatani T, Opare Kennedy D, Nishiguchi S, Matsui-Yuasa I. Involvement of intracellular glutathione in zinc deficiency-induced activation of hepatic stellate cells. *Chemico-Biologic Interact*

[91] Kojima-Yuasa A, Umeda K, Ohkita T, Opare Kennedy D, Nishiguchi S, Matsui-Yuasa I. Role of reactive oxygen species in zinc deficiency-induced hepatic stellate cell acti‐

[92] Pineda B, Estrada-Parra S, Pedraza-Medina B, Rodriguez-Ropon A, Pérez R., Arrieta O. Interstitial transfer factor as adjuvant immunotherapy for experimental glioma. *J*

[93] Alvarez-Thull L, Kirkpatrick Ch. Profiles of cytokine production in recipients of

[94] Foschi FG, Marsigli L, Bernardi M, Salvi F, Mascalchi M, Gasbarrini G, Stefanini G. Acute multifocal cerebral white matter lesions during transfer factor therapy. *J Neu‐*

[95] Holeva OH, Paster IP, Liubchenko TA, Paster IeU, Kholodna LS, Zamotaierva HA, Hrodzinskyi DM. The immune reactivity transfer factor as a modulator of lympho‐

[96] Vacek A, Hofer M, Barnet K, Čech K, Pekárek J, Schneiderová H. Positive effects of dialyzable leukocyte extract (DLE) on recovery of mouse haemopoiesis suppressed by ionizing radiation and on proliferation of haemopoietic progenitor cells in vitro.

[97] Foschi FG, Marsigli L, Bernardi M, Salvi F, Mascalchi M, Gasbarrini G, Stefanini G. Acute multifocal cerebral white matter lesions during transfer factor therapy. *J Neu‐*

[98] Kirkpatrick CH. Activities and characteristics of transfer factors. *Biotherapy*

[99] Fabre RA, Pérez TM, Aguilar LD, Rangel MJ, Estrada-Garcia I, Hernández-Pando R, Estrada Parra S. Transfer factors as immunotherapy and supplement of chemothera‐ py in experimental pulmonary tuberculosis. *Clin Exp Immunol* 2004;136(2) 215-223.

[100] Dai WJ, Waldvogel A, Siles-Lucas M, Gottstein B. *Echinococcus multilocularis* prolifer‐ ation in mice and respective parasite 14-3-3 gene expression is mainly controlled by an alpha beta(+) CD4(+) T-cell-mediated immune response. *Immunology* 2004;112(3)

[101] Sailer M, Soelder B, Allerberger F, Zaknun D, Feichtinger H, Gottstein B. Alveolar echinococcosis of the liver in a six-year-old girl with acquired immunodeficiency

cyte functional activity in rats. *Fiziolohichnyĭ zhurnal* 2000;46(4) 58-65.

2003*;* 146(1) 89-99.

192 Current Topics in Echinococcosis

vation. *Free Rad Biol Med* 2005;39(5) 631-640.

transfer factors. *Biotherapy* 1996;9(1-3) 55-59.

*rol Neurosurg Psychiat* 2000;68(1) 114-115.

*Int J Immunopharmacol* 2000;22(8) 623-634.

*rol Neurosurg Psychiat* 2000;68(1) 114-115.

syndrome. *J Pediat* 1997;130(2) 320-323.

1996;9(1-3) 13-16.

481-488.

*Exp Clin Can Res* 2005;24(4) 575-583.


## **Hydatidosis and Intervention Strategies**

### YuRong Yang

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60838

#### **Abstract**

Human echinococcosis is a zoonotic infection caused by larval forms (metacestodes) of tapeworms of the genus *Echinococcus*. Among the recognised species, *Echinococcus granulosus* and *E. multilocularis* are of considerable medical importance, causing cystic and alveolar echinococcosis (AE and CE), respectively. The factors of immunology, host-genetic inherits, and Echinococcus genetic-diversity and adaption clearly influence infectious establishment and disease progression. However, subtle mechanisms between host and parasite interactions/relationships are still open to study for answers. Despite the global burden, echinococcosis remains a neglected zoonosis. The importance of environmental factors influencing the transmission intensity and distribution of *Echinococcus* species is increasingly being recognised. The intervention strategies for this public health threat have integrated host immunegenetic research, parasite adaptation, and genetic diversity analysis, as well as the transmission dynamic investigations; the limitations of current control programmes are clearly presented in this study that hampers the elimination of *Echinococcus* species worldwide. Continuous efforts by multidiscipline researches are needed.

**Keywords:** *Echinococcus* species, host immunology and genetics, *Echinococcus* genet‐ ic diversity and adaptation, intervention strategies

#### **1. Introduction**

The zoonotic disease of echinococcosis (hydatidosis) is one of the most important parasitic helminth diseases, with over three million people infected worldwide. The two major species infecting man are *Echinococcus multilocularis* that causes alveolar echinococcosis (AE) and

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Echinococcus granulosus* that causes cystic echinococcosis (CE). Both CE and AE are life threatening and are associated with severe morbidity. The global burden of human CE exceeds one million disability-adjusted life years (DALYs), resulting in a loss of at least US\$760 million per annum. Case series and clinical trials show a mortality rate of 2%–4% for CE, but this increase is marked with poor treatment and care. CE also impacts upon agriculture as *E. granulosus* infects domestic livestock, further adding to economic loss. There are 0.4 million cases of human AE, and survival analysis has shown that, if untreated or if limited treatment is provided, mortality exceeds 90%, 10–15 years post-diagnosis. There are approximately 18,000 new cases of AE annually, with a total annual burden of 666,434 DALYs.

#### **2. Transmission of** *Echinococcus* **species**

All *Echinococcus* species are transmitted to intermediate hosts via the ingestion of eggs and are transmitted to definitive hosts by means of eating infected cyst/lesion containing organs. The life cycle of *E. multiclocularis* involves carnivorous definitive hosts (foxes and dogs) and intermediate hosts (predominantly microtine rodents).

For *E. granulosus,* dogs are the major definitive hosts and sheep and other livestock are typical intermediate hosts. In Australia, however, the definitive hosts for *E. granulosus* also include wild dogs and dingoes, while sheep, kangaroos, and wallabies are common intermediate hosts.

Human echinococcosis occurs through the ingestion of *Echinococcus* eggs from environments that have been contaminated by the faeces of infected dogs or foxes. Mostly, following close contact with infected canines carrying *Echinococcus* eggs on their fur; by ingestion of eggcontaminated water or food; or by coprophagic flies serving as mechanical vectors for transmission. Once inside the body, the eggs release oncospheres in the intestine that then migrate through the circulatory system to various organs of the host, notably the liver and lungs. Generally, echinococcosis is associated with poverty, which impacts on sanitation and hygiene, and access to health services, particularly in livestock-raising communities.

#### **3. Epidemiology in Australia and China**

Cosmopolite distribution of *E. granulosus* in a few of countries is completely free from this parasite contamination, while *E. multilocularis* mainly occurs in the northern hemisphere; but it will be an emerging or re-emerging disease in certain countries as a result of ecological alternatives in modern life (Figure 1). AE and CE are of considerable public health concern, particularly in parts of Central and Eastern Europe, and especially Northwest China. While the annual incidence of AE may appear low in many endemic areas (0.03–1.2 per 100,000 inhabitants), it is estimated that many cases are undiagnosed. Human CE is endemic in many pastoral communities. The mortality rate from CE is lower than that from AE (about 2%–4%), but increases considerably if medical treatment is unavailable or inadequate.

**Figure 1.** The distribution of *Echinococcus* spp. in Eastern parts of world (Asian and Australia).

#### **3.1. In Australia**

*Echinococcus granulosus* that causes cystic echinococcosis (CE). Both CE and AE are life threatening and are associated with severe morbidity. The global burden of human CE exceeds one million disability-adjusted life years (DALYs), resulting in a loss of at least US\$760 million per annum. Case series and clinical trials show a mortality rate of 2%–4% for CE, but this increase is marked with poor treatment and care. CE also impacts upon agriculture as *E. granulosus* infects domestic livestock, further adding to economic loss. There are 0.4 million cases of human AE, and survival analysis has shown that, if untreated or if limited treatment is provided, mortality exceeds 90%, 10–15 years post-diagnosis. There are approximately

All *Echinococcus* species are transmitted to intermediate hosts via the ingestion of eggs and are transmitted to definitive hosts by means of eating infected cyst/lesion containing organs. The life cycle of *E. multiclocularis* involves carnivorous definitive hosts (foxes and dogs) and

For *E. granulosus,* dogs are the major definitive hosts and sheep and other livestock are typical intermediate hosts. In Australia, however, the definitive hosts for *E. granulosus* also include wild dogs and dingoes, while sheep, kangaroos, and wallabies are common intermediate hosts.

Human echinococcosis occurs through the ingestion of *Echinococcus* eggs from environments that have been contaminated by the faeces of infected dogs or foxes. Mostly, following close contact with infected canines carrying *Echinococcus* eggs on their fur; by ingestion of eggcontaminated water or food; or by coprophagic flies serving as mechanical vectors for transmission. Once inside the body, the eggs release oncospheres in the intestine that then migrate through the circulatory system to various organs of the host, notably the liver and lungs. Generally, echinococcosis is associated with poverty, which impacts on sanitation and

hygiene, and access to health services, particularly in livestock-raising communities.

but increases considerably if medical treatment is unavailable or inadequate.

Cosmopolite distribution of *E. granulosus* in a few of countries is completely free from this parasite contamination, while *E. multilocularis* mainly occurs in the northern hemisphere; but it will be an emerging or re-emerging disease in certain countries as a result of ecological alternatives in modern life (Figure 1). AE and CE are of considerable public health concern, particularly in parts of Central and Eastern Europe, and especially Northwest China. While the annual incidence of AE may appear low in many endemic areas (0.03–1.2 per 100,000 inhabitants), it is estimated that many cases are undiagnosed. Human CE is endemic in many pastoral communities. The mortality rate from CE is lower than that from AE (about 2%–4%),

18,000 new cases of AE annually, with a total annual burden of 666,434 DALYs.

**2. Transmission of** *Echinococcus* **species**

196 Current Topics in Echinococcosis

intermediate hosts (predominantly microtine rodents).

**3. Epidemiology in Australia and China**

*E. granulosus* occurs in Australia and is prevalent in wildlife. The true extent of human CE in Australia is not accurately known. It is estimated that 80–100 new cases of CE are diagnosed annually; however, CE is not a notifiable disease in Australia and has notoriously been underreported so the true prevalence of CE is likely to be considerably higher than described. Of few data that are available for rural Australian communities, it is apparent that some have an infection index as high as 23.5/100,000. Other data from Western Australia have shown that the incidence in Aboriginal people was 12.2 times higher than in the equivalent non-Aboriginal rural population.

#### **3.2. In China**

There are approximately 1.3 million people with echinococcosis in China, where the disease burden is greater than that of any other country. Of the 33 provinces, autonomous regions, and municipalities in China, at least 20 are considered to be endemic for *E. granulosus* and at least five for *E. multilocularis*. The provinces and autonomous regions with the highest risk of echinococcosis include Xinjiang, Qinghai, Sichuan, Gansu, and Ningxia Hui Autonomous Region (NHAR). These provinces/regions are co-endemic for both human CE and AE, with common cases of co-infections. In addition, significant numbers of CE cases occur in Tibet, Shaanxi, as well as Inner Mongolia, Heilongjiang Province. There are now 66 million people at risk of infection.

#### **4. Pathogenesis of human echinococcosis**

The initial phase of a primary infection is always asymptomatic for both AE and CE in humans and may remain so for a matter of months up to many (typically more than 10) years.

The metacestodes of *E. multilocularis* develop almost exclusively in the liver as a tumour-like, infiltrative growth. The rapidly growing cysts can grow up to 20 cm or more in diameter, often with a central necrotic cavity. A primary infection of *E. multilocularis* outside of the liver is rare, but the spread of parasitic larvae from the primary site in the liver to other organs by metastatic infiltration is not uncommon. AE is typically identified following the investigation of symp‐ toms such as fatigue and weight loss, hepatomegaly and abnormal US, or through routine laboratory examination.

In CE, *E. granulosus* metacestodes may develop in almost any organ. For the majority of patients, however, a single-organ infection site is generally observed with a solitary cyst localized to the liver or to the lungs. Ultrasound surveys have shown that, while some cysts may grow up to 50 mm per year, others may persist without change for many years. Liver cysts also appear to grow at a lower rate than lung cysts. Most CE cases remain asymptomatic until the cyst compresses or ruptures into neighbouring structures and organs by which time the disease is already advanced.

#### **5. Disease diagnosis and severity**

Patients with AE and CE are diagnosed based on clinical parameters including assessment of hepatomegaly, jaundice and upper abdominal complaints, as well as by imaging techniques such as ultrasound (US), computed tomography (CT) scans and magnetic resonance imaging (MR). ELISA-based detection of serum *Echinococcus* specific-antibodies and histological examination are also used to confirm diagnosis.

To assess the degree of hepatic involvement of the parasite mass, AE patients are classified according to PNM (P: hepatic localization of parasite; N: extrahepatic involvement of neigh‐ bouring organs; M: absence or presence of distant metastases) and staged as P1, P2, P3, or P4 as recommended by WHO guidelines and the European Network for concerted surveillance of alveolar echinococcosis classification. For CE patients, classification of disease is based on liver lesion type (CE1, CE2, CE3, CE4 and CE5) at initial diagnosis, as proposed by the WHO Informal Group on Echinococcosis.

#### **6. Susceptibility of human echinococcosis**

Despite its public health significance, the susceptibility of human echinococcosis is poorly understood. The general factors that may render an individual more susceptible include malnutrition, co-infection, and immuno-suppression caused by other diseases or through the use of immuno-suppressive drugs. Several reports of children with cystic echinococcosis were predominantly located in the lungs. The reason for lung hydatidosis in children may be explained by either the weaker immune capability in their respiratory system or the faster cyst growth rate (or both) in young ages than that in adults. Pregnancy has also been thought to increase risk of infection or aggravate the disease due to the impaired cellular immunity frequently observed in pregnant women.

#### **6.1. Host immunology**

The metacestodes of *E. multilocularis* develop almost exclusively in the liver as a tumour-like, infiltrative growth. The rapidly growing cysts can grow up to 20 cm or more in diameter, often with a central necrotic cavity. A primary infection of *E. multilocularis* outside of the liver is rare, but the spread of parasitic larvae from the primary site in the liver to other organs by metastatic infiltration is not uncommon. AE is typically identified following the investigation of symp‐ toms such as fatigue and weight loss, hepatomegaly and abnormal US, or through routine

In CE, *E. granulosus* metacestodes may develop in almost any organ. For the majority of patients, however, a single-organ infection site is generally observed with a solitary cyst localized to the liver or to the lungs. Ultrasound surveys have shown that, while some cysts may grow up to 50 mm per year, others may persist without change for many years. Liver cysts also appear to grow at a lower rate than lung cysts. Most CE cases remain asymptomatic until the cyst compresses or ruptures into neighbouring structures and organs by which time

Patients with AE and CE are diagnosed based on clinical parameters including assessment of hepatomegaly, jaundice and upper abdominal complaints, as well as by imaging techniques such as ultrasound (US), computed tomography (CT) scans and magnetic resonance imaging (MR). ELISA-based detection of serum *Echinococcus* specific-antibodies and histological

To assess the degree of hepatic involvement of the parasite mass, AE patients are classified according to PNM (P: hepatic localization of parasite; N: extrahepatic involvement of neigh‐ bouring organs; M: absence or presence of distant metastases) and staged as P1, P2, P3, or P4 as recommended by WHO guidelines and the European Network for concerted surveillance of alveolar echinococcosis classification. For CE patients, classification of disease is based on liver lesion type (CE1, CE2, CE3, CE4 and CE5) at initial diagnosis, as proposed by the WHO

Despite its public health significance, the susceptibility of human echinococcosis is poorly understood. The general factors that may render an individual more susceptible include malnutrition, co-infection, and immuno-suppression caused by other diseases or through the use of immuno-suppressive drugs. Several reports of children with cystic echinococcosis were predominantly located in the lungs. The reason for lung hydatidosis in children may be explained by either the weaker immune capability in their respiratory system or the faster cyst growth rate (or both) in young ages than that in adults. Pregnancy has also been thought to

laboratory examination.

198 Current Topics in Echinococcosis

the disease is already advanced.

**5. Disease diagnosis and severity**

examination are also used to confirm diagnosis.

**6. Susceptibility of human echinococcosis**

Informal Group on Echinococcosis.

Immuno-suppression is frequently observed in patients with severe AE or CE. High levels of circulating *Echinococcus* antigens can contribute to immune-suppression through polyclonal over-stimulation. The chronicity of *Echinococcus* infection results from persistent antigenic stimulation, polarization of T cell-subset populations and humoral immune responses. Whilst increased Th1 cell activity has been associated with degeneration of CE and AE lesions and successful chemotherapy, high Th2 cell activity is typically associated with active disease and a poor response to chemotherapy. In animals, CE induces local immune-suppression associ‐ ated with increased IL-10 and TGFβ production. Similarly, in humans, IL-10 production plays a key role in the immune response against *E. multilocularis*. Other studies show, however, a clear immunopathology-associated Th2 polarization in patients with progressive disease related to increased levels of IL-4 and IL-13. Th1/Th2 cytokines have shown association with susceptibility or resistance to both *Em* and *Eg* infection in *in vitro* studies. Immunological markers have proved useful for monitoring the natural course of echinococcosis. High IgG4/IgE levels are associated with active disease whereas IgG1, IgG2 and IgG3 responses occur when cysts became infiltrated and are destroyed by the host. This highlights the importance of measuring IgG subclasses individually for a more sensitive index of disease activity than total IgG levels. Measurement of both cytokine and antibody levels can provide a more sensitive diagnostic tool for studying the immunological mechanisms involved in echinococcosis.

#### **6.2. Host genetics**

Despite the high morbidity and mortality associated with echinococcosis, relatively few studies to date have investigated the genetics underlying human susceptibility to the disease.

The case-control studies of candidate genes that have been previously undertaken have identified a number of associations with susceptibility to human echinococcosis in the HLA region for both AE and CE. Of these, however, many have not been replicated, likely reflecting the complexities and diversities of host susceptibility in different ethnic populations, different environmental conditions, and exposure to different *Echinococcus* genotypes or strains. The majority of candidate gene studies have used small cohorts. Only two genes outside of the HLA loci have been investigated in terms of AE and CE susceptibility. These genes, *TAP1* and *TAP2,* belong to the MDR/TAP subfamily and have been previously implicated in autoimmune diseases such as ankylosing spondylitis, type 1 diabetes, and coeliac disease. As with other complex diseases, it is anticipated that host genetic influences on echinococcosis susceptibility are likely to comprise multiple additive loci. Indeed, a recent study has demonstrated that multiple loci exist that contribute to the susceptibility to echinococcosis in mice. This has been supported by recent evidence that implicated a large number of differentially expressed genes in murine AE. However, the relevant genes that determine susceptibility to human echino‐ coccosis and the clinical outcome from this severe disease remain largely unknown. It is clear that any gene variations that interfere with these interactions may alter the etiopathogenesis of disease. Given the highly diverse allelic variation within the MHC region observed between different geographic populations and racial groups, it is plausible that the race and origin of an individual can greatly affect their phenotype and the subsequent outcome of infection.

#### **7.** *Echinococcus* **adaptation/genetics**

*Echinococcus* species have developed their signalling systems for the microenvironment, encompassing the host insulin acting as a stimulant for larval *E. multilocularis* development and revealed an important factor in the pathology of alveolar echinococcosis predominantly in the host liver.

The extensive genetic variation of *E. granulises* comprises a number of strains (G1–G10) that differ in biological features of intermediate host specificity, with diverse viscera involvements, anitgenicity, transmission dynamics, and infectivity to humans. An abundant AgB in HCF is involved in the evasion of the immune response of the host due to its ability to inhibit elastase activity and neutrophils recruitment and to elicit an immunopathology-associated Th2 cell response. The significantly different expression levels of 14-3-3 proteins in larval *E.multilocu‐ laris* and *E.granulosus* provide different growth behaviours of AE and CE in the intermediate hosts, respectively.

#### **8. Public health threat**

To date, over five species are recognized in the genus and four species are already revealed to be involved in human diseases. The most common forms are *E. granulosis* and *E. multilocula‐ ris* responsible for CE and AE, respectively. Two other forms, namely *E. oligarthrus* and *E. vogeli* cause polycystic echinococcosis; two new species, *E. felidis* and *E. shiquicus* may also contribute to human infection, though little is known.

However, human behavioural changes with economic, technological development, and the spatial expansion of agriculture promoted encroachment into wildlife habits, driven by increasing human population, leading to ecosystem changes and bringing human, domestic animals into closer proximity to wildlife. Many recently emerged zoonoses originated in wildlife have been reported.

#### **9. Intervention strategies**

#### **9.1. Immunological/genetic research**

Many studies of echinococcosis have provided significant information on risk factors of infection, as well as on socio-economic influences and ecological determinants of parasite transmission. The new immunological/genetic research components for new therapeutic targets, in combination with standard imaging techniques, will enable rapid and efficient evaluation of echinococcosis patients. This will not only greatly assist in monitoring disease progression and treatment efficacy, but also in the development and deployment of new control strategies and disease surveillance fundamental to reducing morbidity caused by longterm chronic infection and at a low cost to the health care systems of areas where echinococcosis is endemic. Further, the genetic study aims that identification of the genes involved in disease susceptibility can provide valuable insight into the protective and pathogenic mechanisms involved in the different clinical outcomes of echinococcosis. Understanding these processes can provide novel therapeutic targets that are essential for the long-term control of the disease worldwide. A significant, but essential challenge will be to develop strategies for translating knowledge of novel susceptibility genes into improved patient outcomes from both AE and CE. Given the considerable inter-individual variation observed in susceptibility to the different clinical phenotypes and their associated clinical outcomes, it is anticipated that subtle manip‐ ulation of the host immune response will translate into clinical benefits. Genomics offers a powerful approach to dissect the relevant pathways and may offer novel therapeutic targets for new drugs against both AE and CE, which are urgently needed as the current albendazole treatment is far from satisfactory. Definition of the molecules and pathways that are important in individual patients may eventually lead to a personalised approach to care, with therapy tailored on the basis of an individual's genetic background.

#### **9.2. Animal host intervention**

that any gene variations that interfere with these interactions may alter the etiopathogenesis of disease. Given the highly diverse allelic variation within the MHC region observed between different geographic populations and racial groups, it is plausible that the race and origin of an individual can greatly affect their phenotype and the subsequent outcome of infection.

*Echinococcus* species have developed their signalling systems for the microenvironment, encompassing the host insulin acting as a stimulant for larval *E. multilocularis* development and revealed an important factor in the pathology of alveolar echinococcosis predominantly

The extensive genetic variation of *E. granulises* comprises a number of strains (G1–G10) that differ in biological features of intermediate host specificity, with diverse viscera involvements, anitgenicity, transmission dynamics, and infectivity to humans. An abundant AgB in HCF is involved in the evasion of the immune response of the host due to its ability to inhibit elastase activity and neutrophils recruitment and to elicit an immunopathology-associated Th2 cell response. The significantly different expression levels of 14-3-3 proteins in larval *E.multilocu‐ laris* and *E.granulosus* provide different growth behaviours of AE and CE in the intermediate

To date, over five species are recognized in the genus and four species are already revealed to be involved in human diseases. The most common forms are *E. granulosis* and *E. multilocula‐ ris* responsible for CE and AE, respectively. Two other forms, namely *E. oligarthrus* and *E. vogeli* cause polycystic echinococcosis; two new species, *E. felidis* and *E. shiquicus* may also

However, human behavioural changes with economic, technological development, and the spatial expansion of agriculture promoted encroachment into wildlife habits, driven by increasing human population, leading to ecosystem changes and bringing human, domestic animals into closer proximity to wildlife. Many recently emerged zoonoses originated in

Many studies of echinococcosis have provided significant information on risk factors of infection, as well as on socio-economic influences and ecological determinants of parasite

**7.** *Echinococcus* **adaptation/genetics**

in the host liver.

200 Current Topics in Echinococcosis

hosts, respectively.

**8. Public health threat**

wildlife have been reported.

**9. Intervention strategies**

**9.1. Immunological/genetic research**

contribute to human infection, though little is known.

Various methods for animal host interventions have been employed in echinococcosis endemic regions, for example, the culling of dogs/foxes for CE/AE, culling of rodents for AE, and the anthelminthic treatment (with praziquantel (PZQ)) of dog/foxes for CE/AE. Vaccine develop‐ ment has been ongoing and whilst a vaccine targeting the definitive dog/fox hosts could be a "magic bullet", all current candidates have low efficacy. A highly efficacious sheep vaccine for *E. granulosus* (Eg95) is currently under investigation but the evidence base, though growing, is not yet established for its incorporation into control programmes to date.

#### **9.3. Comprehensive intervention and limitation**

The use of geographic information system (GIS) have become more common as a tool for public health investigations due to the increase in number and quality of satellites used for terrestrial observation. These systems integrate the use of geographic positions sensors (GPS) tool for infectious disease studies, including *Echinococcus*. Spatial analysis provides an improvement in epidemiological analysis and prediction of future events. Using remote sensors for moni‐ toring *Echinococcus* transmission, particularly *E. multilocularis*, have produced information that has showed much importance in designing a control programme. Apart from the detection of animal host assembling linking transmission dynamics due to prey-predatory relations, in terms of geological and ecological environments, prediction mapping provides geographical identification of *Echinococcus* spp. risk areas, allowing for the allocation of resources in reasonable ways/location. Linking with a mathematical model has been done in many other parasitic disease controls. It is believed to be also useful for the determination of the optimal strategies for control and/or elimination of echinococcosis in specific locations. The optimal intervention strategies can be translated into policy and practice reducing the burden of this disease, leading to improved direct health outcomes. Modelling can also be used for future economic assessments of interventions that reduce the financial impact of the disease.

To date, only five islands (Iceland, New Zealand, Tasmania, Falkland Islands, and Cyprus) have been able to successfully control echinococcosis. Control programmes were predomi‐ nantly based on health education and control or elimination of home slaughter of sheep, with behaviour change, is central to their success.

Despite the range of intervention strategies, control in endemic regions of the world (especially the poor rural areas) has proved difficult, as demonstrated by the increasing number of cases over the last decade. This failure to effectively control echinococcosis can be attributed to a number of causes: 1) culling of animals has ethical challenges (e.g., use of rodenticides in NHAR resulted in the poisoning of domestic dogs); 2) PZQ is effective in killing adult *E. granulosus* and *E multilocularis* in definitive hosts, but it does not prevent reinfection in the definitive hosts; 3) existing health promotion materials are passive and may not be sufficiently engaging to bring about behaviour change at the population level; and 4) the lack of significant governmental support for control programmes.

#### **10. Conclusion**

Hydatid disease is a major cause of morbidity and mortality in many parts of the world. Although immunological research has provided important insight into the mechanisms of immunity in CE and AE, the genetic variants within the host-participating genes may be too subtle or too few to cause much effect on individual risk. The genotypic variation of *Echino‐ coccus* species reflects phenotypic differences with important consequences in terms of increased host infectivity by local *Echinococcus* strains. Such adaptations may also result in different sensitivity to drugs or increased virulence for hosts that will impede controls efforts and even affect vaccination strategies against *Echinococcus*. The environmental factors have been correlated with transmission to humans through changes in animal population, dynamics spatial overlap of competent hosts, and the creation of improved conditions for egg survival. Therefore, echinococcosis is a complex zoonosis with sparse evidence on the effectiveness of control strategies in diverse settings despite many efforts worldwide for decades. Identifying the environmental determinants of the transmission risk to humans will be vital for the design of accurate predictive models to guide preventative public health action against echinococco‐ sis. Mathematic modelling is a useful tool for simulating control packages under locally specific transmission conditions to inform optimal timing and frequency of phased interventions for cost-effective control of echinococcosis.

#### **Acknowledgements**

Fund support from the National Health Medical Research Councils (NHMRCs, APP1009539), Australia, and National Nature Science Foundation of China (NNSFC, 30960339).

#### **Author details**

YuRong Yang1,2

strategies for control and/or elimination of echinococcosis in specific locations. The optimal intervention strategies can be translated into policy and practice reducing the burden of this disease, leading to improved direct health outcomes. Modelling can also be used for future economic assessments of interventions that reduce the financial impact of the disease.

To date, only five islands (Iceland, New Zealand, Tasmania, Falkland Islands, and Cyprus) have been able to successfully control echinococcosis. Control programmes were predomi‐ nantly based on health education and control or elimination of home slaughter of sheep, with

Despite the range of intervention strategies, control in endemic regions of the world (especially the poor rural areas) has proved difficult, as demonstrated by the increasing number of cases over the last decade. This failure to effectively control echinococcosis can be attributed to a number of causes: 1) culling of animals has ethical challenges (e.g., use of rodenticides in NHAR resulted in the poisoning of domestic dogs); 2) PZQ is effective in killing adult *E. granulosus* and *E multilocularis* in definitive hosts, but it does not prevent reinfection in the definitive hosts; 3) existing health promotion materials are passive and may not be sufficiently engaging to bring about behaviour change at the population level; and 4) the lack of significant

Hydatid disease is a major cause of morbidity and mortality in many parts of the world. Although immunological research has provided important insight into the mechanisms of immunity in CE and AE, the genetic variants within the host-participating genes may be too subtle or too few to cause much effect on individual risk. The genotypic variation of *Echino‐ coccus* species reflects phenotypic differences with important consequences in terms of increased host infectivity by local *Echinococcus* strains. Such adaptations may also result in different sensitivity to drugs or increased virulence for hosts that will impede controls efforts and even affect vaccination strategies against *Echinococcus*. The environmental factors have been correlated with transmission to humans through changes in animal population, dynamics spatial overlap of competent hosts, and the creation of improved conditions for egg survival. Therefore, echinococcosis is a complex zoonosis with sparse evidence on the effectiveness of control strategies in diverse settings despite many efforts worldwide for decades. Identifying the environmental determinants of the transmission risk to humans will be vital for the design of accurate predictive models to guide preventative public health action against echinococco‐ sis. Mathematic modelling is a useful tool for simulating control packages under locally specific transmission conditions to inform optimal timing and frequency of phased interventions for

Fund support from the National Health Medical Research Councils (NHMRCs, APP1009539),

Australia, and National Nature Science Foundation of China (NNSFC, 30960339).

behaviour change, is central to their success.

governmental support for control programmes.

cost-effective control of echinococcosis.

**Acknowledgements**

**10. Conclusion**

202 Current Topics in Echinococcosis

Address all correspondence to: yangyurong@hotmail.com

1 Ningxia Medical University, Yinchuan, Ningxia Hui Autonomous Region, P.R. of China

2 Molecular Parasitology Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Australia

#### **References**


eastern Tibetan Plateau, northwest Sichuan/southeast Qinghai, China. *Acta tropica* 2010, 113(3):248–256.

[25] Craig PS, Li T, Qiu J, Zhen R, Wang Q, Giraudoux P, Ito A, Heath D, Warnock B, Schantz P *et al.*: Echinococcosis and Tibetan communities. *Emerging infectious diseases* 2008, 14(10):1674–1675.

[11] Jenkins DJ, Allen L, Goullet M: Encroachment of *Echinococcus granulosus* into urban

[12] Jenkins DJ: *Echinococcus granulosus* in Australia, widespread and doing well! *Parasitol*

[13] Gottstein B: [Epidemiology and systematics of cystic and alveolar hydatid disease].

[14] Yang YR, McManus DP, Huang Y, Heath DD: *Echinococcus granulosus* infection and options for control of cystic echinococcosis in Tibetan communities of Western Si‐

[15] Vuitton DA, Zhou H, Bresson-Hadni S, Wang Q, Piarroux M, Raoul F, Giraudoux P: Epidemiology of alveolar echinococcosis with particular reference to China and Eu‐

[16] Jimenez S, Perez A, Gil H, Schantz P, Ramalle E, Juste R: Progress in control of cystic echinococcosis in La Rioja, Spain: Decline in infection prevalences in human and ani‐

[17] Vuitton DA, Wang Q, Zhou HX, Raoul F, Knapp J, Bresson-Hadni S, Wen H, Girau‐ doux P: A historical view of alveolar echinococcosis, 160 years after the discovery of the first case in humans: Part 1. What have we learnt on the distribution of the dis‐

[18] Wang YH, Rogan MT, Vuitton DA, Wen H, Bartholomot B, Macpherson CN, Zou PF, Ding ZX, Zhou HX, Zhang XF *et al*.: Cystic echinococcosis in semi-nomadic pastoral communities in north-west China. *Trans R Soc Trop Med Hyg* 2001, 95(2):153–158. [19] Barnes TS, Goldizen AW, Morton JM, Coleman GT: Cystic echinococcosis in a wild population of the brush-tailed rock-wallaby (Petrogale penicillata), a threatened mac‐

[20] Jenkins DJ, Romig T, Thompson RC: Emergence/re-emergence of *Echinococcus* spp.—

[21] Jenkins DJ: Cystic Echinococcosis in Australia: The current situation. *Southeast Asian J*

[22] Budke CM, Jiamin Q, Qian W, Torgerson PR: Economic effects of echinococcosis in a disease-endemic region of the Tibetan Plateau. *The American journal of tropical medi‐*

[23] Yang YR, Craig PS, Sun T, Vuitton DA, Giraudoux P, Jones MK, Williams GM, McManus DP: Echinococcosis in Ningxia Hui Autonomous Region, northwest China. *Transactions of the Royal Society of Tropical Medicine and Hygiene* 2008, 102(4):319–328.

[24] Li T, Chen X, Zhen R, Qiu J, Qiu D, Xiao N, Ito A, Wang H, Giraudoux P, Sako Y *et al.*: Widespread co-endemicity of human cystic and alveolar echinococcosis on the

mal hosts and economic costs and benefits. *Acta Trop* 2002, 83(3):213–221.

ease and on its parasitic agent? *Chin Med J (Engl)* 2011, 124(18):2943–2953.

areas in eastern Queensland, Australia. *Aust Vet J* 2008, 86(8):294–300.

chuan Province, China. *PLoS neglected tropical diseases* 2009, 3(4):e426.

*Int* 2006, 55 Suppl:S203–206.

rope. *Parasitology* 2003, 127 Suppl:S87–107.

ropodid. *Parasitology* 2008, 135(6):715–723.

*Trop Med Public Health* 2004, 35(1).

*cine and hygiene* 2005, 73(1):2–10.

A global update. *Int J Parasitol* 2005, 35(11-12):1205–1219.

*Chirurg* 2000, 71(1):1–8.

204 Current Topics in Echinococcosis


[48] Rigano R, Buttari B, De Falco E, Profumo E, Ortona E, Margutti P, Scotta C, Teggi A, Siracusano A: *Echinococcus granulosus*-specific T-cell lines derived from patients at various clinical stages of cystic echinococcosis. *Parasite immunology* 2004, 26(1):45–52.

[36] Sailer M, Soelder B, Allerberger F, Zaknun D, Feichtinger H, Gottstein B: Alveolar echinococcosis of the liver in a six-year-old girl with acquired immunodeficiency

[37] Yang YR, Gray DJ, Ellis MK, Yang SK, Craig PS, McManus DP: Human cases of si‐ multaneous echinococcosis and tuberculosis—Significance and extent in China. *Para‐*

[38] Kern P, Bardonnet K, Renner E, Auer H, Pawlowski Z, Ammann RW, Vuitton DA, Kern P, European Echinococcosis R: European echinococcosis registry: Human alveo‐ lar echinococcosis, Europe, 1982-2000. *Emerging infectious diseases* 2003, 9(3):343–349.

[39] Matsaniotis N, Karpathios T, Koutoyzis J, Nicolaidou P, Fretzayas A, Papadellis F, Thomaidis T: Hydatid disease in Greek children. *The American journal of tropical medi‐*

[40] Romig T, Zeyhle E, Macpherson CN, Rees PH, Were JB: Cyst growth and spontane‐

[41] Srour SF, Sayfan J: Echinococcosis of the spleen during pregnancy. *The Israel Medical*

[42] Vuitton DA, Zhang SL, Yang Y, Godot V, Beurton I, Mantion G, Bresson-Hadni S: Survival strategy of *Echinococcus multilocularis* in the human host. *Parasitology interna‐*

[43] Kacprzak E, Stefaniak J: Evaluating the activity of liver cystic echinococcosis using the delayed-hypersensitivity skin reaction to common antigens. *Annals of tropical*

[44] Kolligs FT, Gerbes AL, Durr EM, Schauer R, Kessler M, Jelinek T, Loscher T, Bilzer M: [52-year-old patient with subcutaneous space-occupying lesion in immunosup‐

[45] Bonifacino R, Carter SD, Craig PS, Almeida I, Da Rosa D: Assessment of the immu‐ nological surveillance value of humoral and lymphocyte assays in severe human cystic echinococcosis. *Transactions of the Royal Society of Tropical Medicine and Hygiene*

[46] Rigano R, Profumo E, Ioppolo S, Notargiacomo S, Teggi A, Siracusano A: Cytokine patterns in seropositive and seronegative patients with *Echinococcus granulosus* infec‐

[47] Ortona E, Margutti P, Delunardo F, Nobili V, Profumo E, Rigano R, Buttari B, Carulli G, Azzara A, Teggi A *et al.*: Screening of an *Echinococcus granulosus* cDNA library with IgG4 from patients with cystic echinococcosis identifies a new tegumental pro‐ tein involved in the immune escape. *Clinical and experimental immunology* 2005, 142(3):

syndrome. *The Journal of pediatrics* 1997, 130(2):320–323.

*sites & vectors* 2009, 2(1):53.

206 Current Topics in Echinococcosis

*cine and hygiene* 1983, 32(5):1075–1078.

ous cure in hydatid disease. *Lancet* 1986, 1(8485):861.

*Association journal : IMAJ* 2001, 3(4):290–291.

*medicine and parasitology* 1995, 89(1):25–29.

pression]. *Der Internist* 2003, 44(6):740–745.

tion. *Immunology letters* 1998, 64(1):5–8.

*tional* 2006, 55 Suppl:S51–55.

2000, 94(1):97–102.

528–538.


[71] Siles-Lucas M, Nunes CP, Zaha A: Comparative analysis of the 14-3-3 gene and its expression in *Echinococcus granulosus* and *Echinococcus multilocularis* metacestodes. *Parasitology* 2001, 122(Pt 3):281–287.

[59] Al-Ghoury AB, El-Hamshary EM, Azazy AA, Hussein EM, Rayan HZ: HLA class II alleles: susceptibility or resistance to cystic echinococcosis in Yemeni patients. *Parasi‐*

[60] Aydinli B, Pirim I, Polat KY, Gursan N, Atamanalp SS, Ezer M, Donmez R: Associa‐ tion between hepatic alveolar echinococcosis and frequency of human leukocyte anti‐

[61] Eiermann TH, Bettens F, Tiberghien P, Schmitz K, Beurton I, Bresson-Hadni S, Am‐ mann RW, Goldmann SF, Vuitton DA, Gottstein B *et al.*: HLA and alveolar echino‐

[62] Li F, Shi Y, Shi D, Vuitton DA, Craig PS: HLA-DRB1 allele in 35 patients with alveo‐ lar echinococcosis in Gansu Province of China. *Chin Med J (Engl)* 2003, 116(10):

[63] Li F, Shi Y, Shi D: [Association of HLA-DRB1 allele and the susceptibility to alveolar echinococcosis in the west of China]. *Zhonghua Yi Xue Za Zhi* 2000, 80(6):414–416. [64] Kiper N, Gerceker F, Utine E, Yalcin E, Pekcan S, Cobanoglu N, Aslan A, Kose M, Dogru D, Ozcelik U *et al.*: TAP1 and TAP2 gene polymorphisms in childhood cystic

[65] Zhang S, Penfornis A, Harraga S, Chabod J, Beurton I, Bresson-Hadni S, Tiberghien P, Kern P, Vuitton DA: Polymorphisms of the TAP1 and TAP2 genes in human al‐

[66] Emery I, Leclerc C, Houin R, Vuitton DA, Liance M: Lack of H-2 gene influence on mouse susceptibility to secondary alveolar echinococcosis. *Int J Parasitol* 1997, 27(11):

[67] Nakao R, Kameda Y, Kouguchi H, Matsumoto J, Dang Z, Simon AY, Torigoe D, Sasa‐ ki N, Oku Y, Sugimoto C *et al.*: Identification of genetic loci affecting the establish‐ ment and development of *Echinococcus multilocularis* larvae in mice. *Int J Parasitol*

[68] Hemer S, Konrad C, Spiliotis M, Koziol U, Schaack D, Forster S, Gelmedin V, Stadel‐ mann B, Dandekar T, Hemphill A *et al.*: Host insulin stimulates *Echinococcus multilo‐ cularis* insulin signalling pathways and larval development. *BMC biology* 2014, 12:5.

[69] McManus DP, Thompson RC: Molecular epidemiology of cystic echinococcosis. *Para‐*

[70] Parkinson J, Wasmuth JD, Salinas G, Bizarro CV, Sanford C, Berriman M, Ferreira HB, Zaha A, Blaxter ML, Maizels RM *et al.*: A transcriptomic analysis of *Echinococcus granulosus* larval stages: implications for parasite biology and host adaptation. *PLoS*

gen class I and II alleles in Turkish patients. *Hepatol Res* 2007, 37(10):806–810.

*tol Res* 2010, 107(2):355–361.

1557-1560.

208 Current Topics in Echinococcosis

1433–1436.

2011, 41(11):1121–1128.

*sitology* 2003, 127 Suppl:S37-51.

*neglected tropical diseases* 2012, 6(11):e1897.

coccosis. *Tissue Antigens* 1998, 52(2):124–129.

echinococcosis. *Parasitol Int* 2010, 59(2):283–285.

veolar echinococcosis. *Eur J Immunogenet* 2003, 30(2):133–139.


echinococcosis in southern Ningxia, China. *PLoS neglected tropical diseases* 2008, 2(9):e287.


echinococcosis in southern Ningxia, China. *PLoS neglected tropical diseases* 2008,

[83] Graham AJ, Danson FM, Giraudoux P, Craig PS: Ecological epidemiology: landscape metrics and human alveolar echinococossis. *Acta tropica* 2004, 91(3):267–278.

[84] Giraudoux P, Raoul F, Afonso E, Ziadinov I, Yang Y, Li L, Li T, Quere JP, Feng X, Wang Q *et al*: Transmission ecosystems of *Echinococcus multilocularis* in China and

[85] Atkinson JA, Williams GM, Yakob L, Clements AC, Barnes TS, McManus DP, Yang YR, Gray DJ: Synthesising 30 years of mathematical modelling of *Echinococcus* trans‐

[86] Atkinson JA, Gray DJ, Clements AC, Barnes TS, McManus DP, Yang YR: Environ‐ mental changes impacting *Echinococcus* transmission: research to support predictive

[87] Craig PS, McManus DP, Lightowlers MW, Chabalgoity JA, Garcia HH, Gavidia CM, Gilman RH, Gonzalez AE, Lorca M, Naquira C *et a*l.: Prevention and control of cystic

[88] Heath D, Yang W, Li T, Xiao Y, Chen X, Huang Y, Yang Y, Wang Q, Qiu J: Control of

Central Asia. *Parasitology* 2013, 140(13):1655–1666.

mission*. PLoS neglected tropical diseases* 2013, 7(8):e2386.

surveillance and control. *Global change biology* 2013, 19(3):677–688.

echinococcosis. The Lancet Infectious diseases 2007, 7(6):385–394.

hydatidosis. Parasitology international 2006, 55 Suppl:S247–252.

2(9):e287.

210 Current Topics in Echinococcosis

## *Edited by Alfonso J. Rodriguez-Morales*

Echinococcosis remains an important cause of morbidity and mortality in certain areas of the world, tropical and non-tropical, particularly in rural settings. This book includes different topics with regard to the epidemiology, biology, clinical manifestations, treatment and prevention of the wide spectrum of diseases caused by the different species of Echinococcus involved in human and animal infection, with an aim to update the most significant research in many of them as well as to offer a multinational perspective on different aspects. The book has been organized into three major sections: (I) Epidemiology; (II) Biological and Clinical Aspects; and (III) Treatment and Prevention. Section I includes topics covering epidemiological studies in Colombia, Chile, Mexico and Tunisia, including molecular biology approaches to the study of parasite species. Section II includes topics covering the biology of some Echinococcus species affecting mainly animals, as also the human clinical manifestations in the central nervous system (CNS), genitourinary tract and other organic typical and atypical locations, as well as radiological manifestations of pulmonary disease. Section III includes topics on the usefulness of immunotherapy for antihelmintic treatment and intervention strategies.

Current Topics in Echinococcosis

Current Topics in

Echinococcosis

*Edited by Alfonso J. Rodriguez-Morales*

Photo by scubaluna / iStock