Introduction
Echinococcosis is a zoonotic infection caused by adult or larval (metacestode) stages of cestodes belonging to the genus Echinococcus and the family Taeniidae. Life cycles imply two mammal hosts. Definitive hosts are carnivores containing adult forms in the gut. The infection is acquired by the intermediate hosts and humans after the ingestion of eggs from the feces of carnivorous definitive hosts, which harbour the adult egg-producing stage in the intestine. Eggs ingested by infected human develop into the infective metacestode stage causing various forms of Echinococcosis. The disease in humans and intermediate hosts is called Hydatidosis and is characterized according to the morphologic features of the larval stages: cystic echinococcosis (CE) caused by Echinococcus granulosus and related organisms, alveolar echinococcosis (AE) caused by Echinococcus multilocularis, and polycystic echinococcosis (PE) caused by Echinococcus vogeli or Echinococcus oligarthrus. This disease is becoming an important public health problem in many parts of the world where dogs are used for cattle breeding. Control measures are unable to be implemented everywhere, and where control programs were initiated the success of them have been incomplete generating a re-emergence of the disease. This has also lead to the interruption of control measures excluding Echinococcosis from the list of notifiable diseases. As a consequence of this the incidence and prevalence of CE in Mediterranean countries in humans and animals are not known (Dakkak, 2010). Also, there are a number of factors that contribute to the increase of prevalence and to the spreading of CE in the Mediterranean Region. Cyprus is the only country where an eradication programme has been successfully implemented. However, CE has obtained important developments in the last decade, in the epidemiology, in the diagnosis of canine infection, in strain characterisation and in immune strategies against CE in animals. This scientific progress, together with effective health education programmes, will likely improve control programmes and reduce the time required to achieve significant decreases in prevalence or eradication. Thus, European authorities recognized, through the directive 2003/99/CE, Echinococcosis as a disease to be reported to the European Food Safety Authority (EFSA).
The "WHO/OIE Manual on Echinococcosis in Humans and Animals: a Public Health Problem of Global Concern", published in 2001, has been used as a fundamental issue for the redaction of this topic.
Echinococcosis
The first part of this topic is dedicated to explain the etiology to know the taxonomy and the life transmission cycles which perpetuate the agent in nature. The biology of the causative agents of various forms of Echinococcosis is faced because it can help to understand the maintaining of these parasites in different geographic areas. Following to this part the epidemiology and clinical presentation forms are assessed. Finally, diagnosis, treatment and prevention are developed and emphasis is given to the identification of species and strains within the genus as an essential prerequisite to the establishment of local control programmes.
Etiology and life cycles
Echinococcus presents certain unique characteristics that set it apart from the other major genus in the family, Taenia. The adult form is only few millimetres long, has no gut and all metabolic interchange takes place across the syncytial outer covering, the tegument. Scolex is the anterior part of the Echinococcus becoming an attachment organ with four muscular suckers and two rows of hooks. The body is the strobila and is segmented in a different number of reproductive units called proglottids. The adult worm is hermaphrodite with reproductive ducts opening at a common, lateral, genital pore, the position of which may vary depending on species and strain. The uterus dilates after fertilisation, eventually occupying most of the terminal segment when the eggs are fully developed. The eggs are ovoid, consisting of a hexacanth embryo surrounded by several envelopes and are morphologically indistinguishable to those of other tapeworms of the genus Taenia. The metacestode is the second larval stage and consists of a bladder with an outer acellular laminated layer and an inner nucleated germinal layer. Protoscoleces arise from the inner wall of the brood capsules (figure 1). The structure and development of the metacestode differs between the four species of Echinococcus.
Fig. 1. Representation of the metacestode of Echinococcus granulosus (WHO/OIE publication: Manual on echinococcosis in humans and animals)
The life cycle of Echinococcus spp. requires two mammalian hosts for its completion. Gravid proglottids containing eggs or free eggs are passed in the faeces of the definitive host, a carnivore. These eggs are ingested by an intermediate host, in which the metacestode stage and protoscoleces develop. The cycle is completed if such an intermediate host is eaten by a suitable carnivore. Eggs are highly resistant to the environmental factors being infective for months at lower ranges of temperatures (from +4°C to +15°C). However, they are very sensitive to desiccation and to high temperatures as 60-80°C.
Intermediate hosts are represented by a wide range of mammals which acquire the infection by the ingestion of eggs. The oncosphere is released from the keratinised embryophore in the stomach and small intestine. Bile activates the oncosphere which penetrates the wall due to the hook movements and secretions and arrives to the liver where some of them are retained (figure 2). All mammals (including man) in which metacestodes of Echinococcus species develop after infection with eggs may be referred to as ‘intermediate hosts’. However, man is an aberrant host because metacestode stages do not become fertile in this host or because does not interact in the transmission cycle. Once the oncosphere has reached its final location, it develops into the metacestode stage. Time of development is variable and it may take several months before protoscoleces are produced (fertile metacestode). There may be several thousand protoscoleces within a single cyst of E. granulosus or an aggregation of vesicles of E. multilocularis. Each single protoscolex is capable of developing into a sexually mature adult worm. Not all metacestodes produce protoscoleces (sterile metacestode).
Fig. 2. Life-cycle of Echinococcus (WHO/OIE publication: Manual on echinococcosis in humans and animals)
Strain identification is possible for all four species of Echinococcus using morphological and biological features and/or molecular techniques, such as sequence comparison of a 366 bp-fragment of the mitochondrial cytochrome oxidase subunit 1 DNA (CO1) and a 471 bp-region in the mitochondrial NADH dehydrogenase gene 1 (ND1), by analysis of a ribosomal (r) DNA fragment (1ST2) or by the random amplified polymorphic DNA-PCR (RAPD-PCR). Recent genetic studies have principally confirmed the concept of strain diversity within the species E. granulosus, previously based on morphological and biological features. Several molecular techniques are now available which would quite easily allow the identification of certain E. granulosus strains using genetic markers. To prepare the identification of the strain using molecular techniques protoscoleces are collected from E. granulosus cysts being washed several times in physiological saline solution and preserved in 70% ethanol. The material needs to be examined by an experienced laboratory. The rDNA ITS1 (internal transcribed spacer) region has been shown to be a potentially very useful genetic marker for distinguishing strains and species of Echinococcus and small quantities of Echinococcus material can be characterised using a PCR-RFLP ‘fingerprinting’ technique (Bowles & McManus, 1993). Other method is the single strand conformation polymorphism (SSCP) which is technically simple and has high resolution capacity under optimised conditions. The utility of SSCP has been demonstrated for the categorisation of different Echinococcus genotypes (Gasser et al., 1998). The different strains of Echinococcus have an epidemiological significance for the prognosis in infected patients. Epidemiological studies have evidenced that the sylvatic strain of E. granulosus in northern North America is causing a benign infection with low pathogenicity, and affecting the lungs. Also, these epidemiological observations have been demonstrated in the People’s Republic of China. In contrast, in parts of Kenya and Libya, it has been suggested that there are local virulent strains of E. granulosus (Thompson, 1995). Isoenzyme and molecular studies have confirmed that sheep strain is infective to humans (Bowles & McManus, 1993). Developmental differences between species and strains of Echinococcus are likely to be a limiting factor in control programmes which employ regular adult cestocidal treatment of definitive hosts for breaking the cycle of transmission (Thompson, 1995).
Clinical forms of Echinococcosis
The metacestodes of all four recognised Echinococcus species can infect humans and cause various forms of echinococcosis (Table 1). Among these forms cystic and alveolar echinococcosis are of special medical importance.
|
Forms of Echinococcosis |
Causative agent |
Disease synonyms |
|
Cystic |
Echinococcus |
Hydatid disease, Hydatidosis, E. |
|
Echinococcosis |
granulosus |
granulosus echinococcosis |
|
Alveolaar |
Echinococcus |
Alveolar hydatid disease, E. multilocularis |
|
Echinococcosis |
multilocularis |
echinococcosis |
|
Polycystic Echinoccosis |
Echinococcus vogeli |
E.vogeli echinococcosis |
|
Polycystic Echinococcosis |
Echinococcus oligarthrus |
E. oligarthrus echinococcosis |
Table 1. Forms of Echinococcosis
Primary echinococcosis is established when metacestodes develop in various sites of the human body from oncospheres liberated from ingested eggs of Echinococcus spp. In CE liver and lung are the most frequently affected organs.
Secondary echinococcosis occurs when metacestode material spreads from primary site to adjacent or distant organs and proliferates. Regarding CE this form occurs after release of viable parasite material during invasive treatment procedures.
Cystic echinococcosis (CE)
The causative agent of CE is the metacestode of Echinococcus granulosus, becoming a cystic structure filled with a clear fluid. Most of the cysts grow slowly in size and become surrounded by host tissue (pericyst) encompassing the endocyst of metacestode origin. The endocyst consists of the outer laminated layer and the inner cellular germinal layer, which may form brood capsules and protoscoleces. The minimum time required for the development of protoscoleces in cysts in humans is not exactly known, but it is expected to be 10 months or longer after infection (Pawlowski, 1997). Fertile (with protoscoleces) and sterile (without protoscoleces) cysts may coexist in the same patient. Frequently, smaller daughter cysts are formed within a larger mother cyst. If these smaller cysts are growing in close proximity to each other forming clusters the appearance of "polycystic" needs to be distinguished from AE or PE.
The initial phase of primary infection is always asymptomatic remaining as this for many years or permanently. However, the infection may become symptomatic when cysts press adjacent tissues or induce other pathological events. When symptoms appear suddenly a spontaneous or traumatic cyst rupture has to be suspected. Spontaneous cure is possible but improbable, and is due to the collapse and resolution of cysts or due to the cyst rupture into the bile duct or the bronchial tree. The fatality rate is highly dependent on the severity of the infection and on facilities for treatment.
The age of the symptomatic infected patients can vary from below 1 year of age to over 75 years old. In a study from Madrid (Spain), over 1,473 patients admitted to a children’s hospital, 2%were <1 year old, 21% between 1 and 4 years and 77% between 5 and 14 years (Utrilla et al., 1991). Many patients (about 40% up to 80%) with CE have a single organ involved and harbour a solitary cyst.
Clinical presentation of CE
Clinical symptoms of CE are variable and depend on the organ involved, the size of the cysts, the interaction between the expanding cysts and the adjacent organ structures, and the complications related to the cyst rupture and bacterial infection. CE involving the liver can remain asymptomatic for more than ten years (Frider et al., 1999). Liver and lungs are the two more frequent organ sites involved. Complications affecting the biliary tract are the most common and include the cystic rupture into bile ducts. Other complications are bacterial infection of the cyst, intraperitoneal rupture, and lung involvement.
Diagnosis of CE
Diagnosis of CE is done through different steps as follows:
a. Clinical suspect or screening
b. Confirmation by imaging and identification of suspicious cyst structures
c. Confirmation by detection of specific antibodies with immunodiagnostic tests
d. If doubt diagnostic puncture may be considered
e. Material obtained by biopsy puncture or surgery is examined.
Ultrasonography (US) is used for the diagnosis of the cystic structure and portable units are suitable to take into account in field situations. Immunodiagnostic tests for detecting specific antibodies are commonly used for the aetiological confirmation of the findings of imaging examinations.
Protoscoleces or hooks of E. granulosus are found in aspirated fluid samples. This technique is not performed frequently because the material can only be available after a surgical intervention, therapeutic puncture (PAIR) or diagnostic puncture. Direct diagnosis can also be made by macroscopic identification of the E. granulosus obtained by surgery or biopsy. Other methods include the identification of specific E. granulosus antigen (antigen 5) in the fluid from sterile cysts or DNA markers in the cysts fluid or parasite tissue (e.g. by PCR). Imaging techniques for diagnosis:
• Standard radiology: chest radiography detects uncomplicated cyst structures displaying a homogeneous shadow that indicates a fluid-filled space. Calcification in lung imaging is rare and cysts may be located anywhere as solitary or multiple. For differential diagnosis, cysts filled with clear fluid, with an air shadow or with water-lily sign are pathognomonic. If a rounded parenchymatous opacity is seen, it is necessary to consider tuberculoma, a tumour or pulmonary sequestration. A fluid and air shadow will lead to consideration of a bacterial, fungal or amoebic abscess.
• Ultrasonography (US): an expert committee of the WHO Working Group on echinococcosis presented an internationally agreed classification of US images in hepatic CE in 2001 (WHO, 2001), according to the use of PAIR (Puncture, Aspiration, Injection, Re-aspiration). This technique was proposed in 1986 by the Tunisian team that first used it in a prospective study. PAIR is a minimally invasive therapeutic option for percutaneous drainage of echinococcal cysts located in the abdomen, complementing or replacing surgery in most of the settings. PAIR also helps the use of benzimidazoles (albendazole and mebendazole) for the treatment of CE. The drainage is performed with a fine needle or a catheter, followed by the killing of the protoscolices remaining in the cyst cavity by a protoscolicide agent. If numerous and large daughter cysts are present, an alternative percutaneous technique "Percutaneous Puncture with Drainage and Curettage" (PPDC) may be used.
Fig. 3. Image from a computed tomography study of the abdomen of a patient.
• Computed tomography (CT) can detect small cysts, and it also facilitates differential diagnosis of lesions caused by Echinococcus metacestodes from non-parasitic lesions (figure 3) (Soriano Arandes et al., 2010). CT is only indicated when US diagnosis is uncertain, mainly in cysts CE4 or CE5. However, CT is the principal method for diagnosis of cerebral cysts showing a spherical cyst with a thin wall, not enhanced after injection of contrast medium, without perilesional oedema the adjacent structures.
WHO classification of CE
The WHO classification of CE cysts (WHO, 2001) is done according to the US images:
Cystic lesion (CL): Unilocular, cystic lesion (s) (CL) with uniform anechoic content, not clearly delimited by a hyperechoic rim (= cyst wall not visible).
• Normally round but may be oval.
• Size: variable but usually small. CLs (< 5.0 cm), CLm (5 – 10 cm), CLl (> 10cm).
• Status: If CE – active. If these cystic lesions are caused by CE at an early stage of development then usually these cysts are not fertile.
• Ultrasound does not detect any pathognomonic signs.
• Differential diagnosis of these cystic lesions requires further diagnostic techniques. CE1: Unilocular, simple cyst with uniform anechoic content. Cyst may exhibit fine echoes due to shifting of brood capsules which is often called hydatid sand ("snow flake sign") (figure 4).
Fig. 4. Hydatid sand containing a protoscolex of Echinococcus granulosus seen by light microcopy.
• Cyst wall is visible.
• Normally round or oval.
• Size variable: CE1s (< 5.0 cm), CE1m (5 – 10 cm), CE1l (> 10cm)
• Status: active.
• Usually fertile.
• Pathognomonic signs include visible cyst wall and snow flake sign.
CE2: Multivesicular, multiseptated cysts; cysts septations produce "wheel-like" structures, and presence of daughter cysts is indicated by "rosette-like" or "honeycomb-like" structures. Daughter cysts may partly or completely fill the unilocular mother cyst.
• Cyst wall normally visible.
• Normally round or oval.
• Size variable: CE2s (< 5.0 cm), CE2m (5 – 10 cm), CE2l (> 10cm).
• Status: active.
• Usually fertile.
• Ultrasound features are pathognomonic.
CE3: Unilocular cyst which may contain daughter cysts. Anechoic content with detachment of laminated membrane from the cyst wall is visible as floating membrane or as "water-lily sign" which is indicative of wavy membranes floating on top of remaining cyst fluid.
• Cyst form may be less rounded because of decrease of intra-cystic fluid pressure.
• Size variable: CE3s (< 5.0 cm), CE3m (5 – 10 cm), CE3l (> 10cm).
• Status: transitional.
• Transitional stage. Cyst which may degenerate further or may give rise to daughter cysts.
• Ultrasound features are pathognomonic.
CE4: Heterogenous hypoechoic or hyperechoic degenerative contents. No daughter cysts.
• May show a "ball of wool" sign which is indicative of degenerating membranes.
• Size variable: CE4s (< 5.0 cm), CE4m (5 – 10 cm), CE4l (> 10cm).
• Status: inactive.
• Most cysts of this type are not fertile.
• US features are not pathognomonic and further diagnostic tests are required to ascertain a diagnosis.
CE5: Cysts characterized by thick calcified wall that is arch shaped, producing a cone shaped shadow.
• Degree of calcification varies from partial to complete.
• Size variable: CE5s (< 5.0 cm), CE5m (5 – 10 cm), CE5l (> 10cm).
• Status: inactive.
• Cyst not fertile in most cases.
• Diagnosis is uncertain. Features are not pathognomonic but highly suggestive for E. granulosus.
Laboratory findings of CE
Routine laboratory tests show non-specific results. Patients with rupture of a cyst into the biliary tree can show transient elevations of GGT or alkaline phosphatase concentrations, often associated with hyperamylasaemia and eosinophilia (>500/μ!). When cyst is ruptured eosinophilia achieves higher concentrations.
Immunodiagnosis of CE
Immunodiagnosis procedures for serum antibody detection are used for the aetiological confirmation of imaging structures suggestive for CE. Certain proportion of patients with echinococcosis is unable to be diagnosed with highly sensitive diagnostic tests such as IgG-ELISA and false-negative results are obtained. Cysts in the brain or eye and calcified cysts often induce low or no antibody titres. Antibody response may also be low in certain human population groups and in young children. False positive results may also occur, especially in patients with other helminthic diseases. Approaches to the diagnosis of CE using immune methods are specified in table 2.
|
|
Fisrt step: Primary antibody test |
|
|
Test for serum antibody detection: IgG-ELISA or IgE-ELISA with E. Granulosus antigen A combination of two or more primary tests may increase sensitivity |
||
|
|
Subsequent steps |
|
|
Seronegative samples + No suggestive images for CE |
Seronegative samples + Suggestive images for CE Asymptomatic cases |
Seropositive samples + With or without suggestive images for CE |
|
Extended and/or advanced imaging and repeated serological examinations, including differential diagnosis for AE* |
||
|
Asymptomatic and symptomatic cases |
||
|
Secondary antibody test: Arc 5 test IgG4-ELISA |
||
|
‘Wait and observe’ approach with repeated serological examinations |
||
|
No further serological follow-up or further steps for differential diagnosis |
||
|
Immunoblot for antibodies reactive with subunits of E. granulosus antigens |
||
|
Symptomatic cases |
Serological differential diagnosis for AE (ELISA-Em2plus, immunoblot) |
|
|
Consideration of cyst puncture |
||
|
Consideration of surgical intervention and/or chemotherapy without further serological examinations |
||
Table 2. Approaches for immunodiagnosis of CE
IgG-ELISA is the preferable test used as a primary test for detecting anti-Echinococcus serum antibodies. Most of the routine laboratory test systems or commercialized test kits are based on crude or semi-purified preparations of E. granulosus antigens. The use of the two major hydatid cyst fluid antigens, antigen 5 (thermolabile) and antigen B (thermostable), is predominantly restricted to scientific applications, and these antigens are not generally available. Secondary tests for antibody detection are used to increase specificity and these are: arc 5, identification of IgG subclasses, and immunoblotting which demonstrates the reactivity of serum antibodies with subunits of E. granulosus antigens (Craig, 1997; Di Felice, 1986; Ioppolo, 1996; Leggatt & McManus, 1994; Leggatt, 1992; Ligthowlers & Gottstein, 1995; Profumo, 1994; Sheperd & McManus, 1987; Siracusano & Vuitton, 1997; Wen & Craig, 1994). Generally, these tests are less sensitive, but more specific than primary test systems. Putative hydatid cyst fluid samples obtained by puncture or after surgical intervention can be tested for the presence or absence of Echinococcus antigen through binding of enzyme-labelled anti-Echinococcus (hydatid cyst fluid) antibodies in an ELISA with a monoclonal antibody against antigen 5 (Ag5) that may be useful in confirmation of the Echinococcus nature of the fluid (Paul & Stefaniak, 1997).
Treatment of CE
Surgery is still the treatment that has the potential to remove E. granulosus cysts and lead to complete cure (WHO, 1996). Up to 90% of the patients can be treated surgically if a cyst does not have a risky localisation or if the disease is not too far advanced. However, surgery may be impractical in patients with multiple cysts localised in several organs and if surgical facilities are inadequate. Chemotherapy and PAIR offer an attractive option for treatment, especially in inoperable patients and for cases with a high surgical risk.
