Title Page[Submitted, accepted and published by Elsevier, Acta Tropica, 123: 1-7 (2012)]
Challenges for diagnosis and control of cystic hydatid disease
T. S. Barnes,1 P. Deplazes2, B. Gottstein,3 D.J.Jenkins,4A. Mathis2, M. Siles-Lucas,5 P. R. Torgerson,6 I. Ziadinov,2D. D. Heath,7
1 The University of Queensland, School of Veterinary Science, Gatton, QLD, Australia.()
2Vetsuisse-Faculty, University of Zurich, Institute of Parasitology, Zurich, Switzerland.( )
3Institute of Parasitology, University of Bern, Bern, Switzerland.( )
4School of Animal and Veterinary Science, Charles Sturt University, Wagga Wagga, NSW,Australia.()
5IRNASA, CSIC,Cordel de Merinas,40-52. 37008, Salamanca, Spain. ( )
6Vetsuisse-Faculty, University of Zurich, Institute for Epidemiology, Zurich, Switzerland.( )
7 Animal Health Division, AgResearch, Hopkirk Research Centre, Palmerston North, NewZealand. ()
Corresponding Author:
Tamsin Barnes,
School of Veterinary Science,
The University of Queensland,
Gatton Campus,
Gatton,
4343,
Australia
Tel: +61 7 5460 1965
Fax: +61 7 5640 1922
Email:
Abstract
This paper is based on the experience of the authors, with the aim to define the challenges for Echinococcus granulosus (E.g./CE) diagnosis and control for those countries that may now or in the future be contemplating control of hydatid disease. A variety of methods are available for diagnosis in humans but a world standard is lacking. Diagnosis in definitive hosts can avoid necropsy by the use of methods such as coproantigen detection but test performance is variable between populations. A sylvatic cycle adds challenges in some countries and the epidemiology of the parasite in these hosts is poorly understood. Control by solely administering praziquantel to dogs is not effective in developing countries where the disease is endemic. Additional avenues to pursue include the instigation of participatory planning, use of an existing vaccination for intermediate hosts and development of a vaccine and long-acting anthelmitic implants for definitive hosts. Promoting public acceptance of control of the dog population by humane euthanasia and reduced reproduction is also essential.
Key Words
Hydatid Disease; Echinococcus granulosus; Echinococcus multilocularis; Challenges; Control; Diagnosis in humans; Diagnosis in animals; Sylvatic hosts.
Introduction
Members of the genus Echinococcus have two-host life cycles; the definitive hosts are carnivores (canids or felids) and the intermediate hosts are herbivores or omnivores. Both hosts may be either domestic or sylvatic. Echinococcus granulosushas a worldwide distribution and E. multilocularis is endemic in the northern hemisphere (McManus et al., 2003). During the previous 50 years, control of hydatid disease caused by E.granulosus has been undertaken in several jurisdictions around the world, but these control programs have had varying degrees of success(Craig and Larrieu, 2006). There are ongoing challenges in diagnosing infection in different host species. Consequently it is difficult to assess prevalence and infection pressure in any host population. Although there are a variety of different control measures available they all have serious difficulties when applied at the population level.
Using the breadth of experience of the authors, this paper sets out to describe some of these difficulties. The first section focuses on difficulties in diagnosis in humans and domestic animals. The second section uses Australian wildlife as a case study to highlight the difficulties associated with infection in unmonitored sylvatic populations and the risks of spill over to humans and domestic animals. The final section reviews the past and existing control measures and suggests possible directions for the future.
1.1 Diagnosis in Humans
Humans may be infected with the larval stage of E. granulosus (CE, cystic echinococcosis) or E. multilocularis (AE, alveolar echinococcosis). Imaging procedures such as ultrasonography (US), CT and MRI are frequently used for diagnosis. An international classification of US images in CE has been released (Macpherson et al., 2003)and for AE a PNM classification system (P=parasitic mass in the liver, N=involvement of neighbouring organs, and M=metastasis) can be used to classify each patient according to clinical status(Kern, 2006). Immunodiagnostic tests for CE and AE are mostly ELISA based serological tests using either E. granulosus hydatid cyst fluid antigen or E. multilocularis crude vesicular fluid for primary screening(Brunetti et al., 2010). The diagnostic sensitivity for hepatic cases of CE ranges from 85 - 98%, depending on the product and the study. For pulmonary cyst localization, the diagnostic sensitivity is markedly lower (50 - 60%), for multiple organ localization higher (90 - 100%). For AE, the use of purified or recombinant E. multilocularis antigens (such as the Em2-antigen, recII/3-10, recEM10- or recEm18-antigen) exhibit high diagnostic sensitivities ranging between 91% and 100%, with overall specificities of 98 - 100%(Xiao et al., 2003). Most of the purified antigens allow discrimination between AE and CE(Gottstein et al., 1993; Sako et al., 2006). Positive test results may be confirmed by immunoblotting (Reiter-Owona et al., 2009) although this technique is not as widely available. Seronegative CE cases may arise because of an inadequate Th2 cell activation that is required for immunoglobulin expression, or because sequestration of antigens upon full encapsulation of pulmonary cysts minimises antigenic stimulation of the host. Several recombinant antigens have shown potential to overcome this and other problems associated with the clinical status of the CE patient(Hernandez-Gonzalez et al., 2008). Screening using serological methods has demonstrated a lower sensitivity and specificity compared to imaging methods (Moro et al., 2005) indicating that the use of this technique alone is not ideal for use in population studies. Parallel testing using imaging procedures and immunodiagnosis usually yieldsthe most reliable diagnosis, although in CE there are still cases where results are equivocal(Brunetti et al., 2004; Brunetti et al., 2010). Thus a challenge exists to develop a reliable world standard. Aspiration cytology can be helpful in the identification of atypical or non-conclusive lesions (Wahane et al., 2008) and labelled MAbs may allow the identification of the species ex vivo(DieboldBerger et al., 1997). Molecular techniques such as Echinococcus-specific PCR may be used(Siles-Lucas and Gottstein, 2001). Real-time PCR allows the assessment of the viability of parasite samples following chemotherapy or other treatment (Matsumoto et al., 2006). However, in a clinical setting the usefulness of this and other sophisticated techniques have yet to be proven.
One of the major problems of human CE and AE is the frequency of relapses. Markers can be used postoperatively for the rapid detection of newly growing or relapsing cysts. These include recP29(Ben Nouir et al., 2009)and recB2t antigen (coupled to conventional imaging procedures) for CE. For AE, PET-scan coupled to recII/3-10 (identical to recEm18) serology yields a relatively good indication (Ammann et al., 2004; Ishikawa et al., 2009)of efficient inactivation of the parasitic metacestode.
1.2 Diagnosis and Monitoring in Carnivores
Classical diagnostic methods for direct parasite detection in definitive hosts include necropsy and arecoline purgation. In addition it is possible to detect coproantigen by ELISA and copro-DNA by PCR. The ‘gold standard’ test is the sedimentation and counting technique. The test is performed at necropsy on intestinal material and has an estimated sensitivity of 96-100%. The intestinal scraping technique is a somewhat less laborious necropsy technique used in several laboratories and has a sensitivity of about 78% (Eckert et al., 2001). Arecoline hydrobromide purgation has been used for mass surveillance in E. granulosus control programs worldwide but has several serious limitations. A recent study suggests that sensitivity may be as low as 21% for E. multilocularis and 39 % for E. granulosus(Ziadinov et al., 2008). Furthermore, safety precautions in the field and in the laboratory are essential and time consuming. Arecoline can also cause serious adverse reactions in dogs requiring strict veterinary supervision, and this compound is not approved for the use for dogs in most countries. However the procedure has a specificity approaching 100% and is thus reliable in demonstrating the presence of the parasite(Eckert et al., 2001). It would be useful to develop a protocol combining a less traumatic purgative, such as magnesium sulphate, and praziquantel.
There are also limitations to diagnosis from faecal samples. The microscopical detection of eggs in faecal samples by routine coprological methods suffers from a low sensitivity and eggs of Echinococcus spp. cannot be differentiated morphologically from those of Taenia spp.(Deplazes et al., 2003). An ELISA for the detection of antigens in the faeces of definitive hosts was initially developed for the diagnosis of E. granulosus. These tests subsequently were shown to have cross-reactivity with E. multilocularis. However they lacked sensitivity for this species(Allan et al., 1992; Deplazes et al., 1992; Allan and Craig, 2006). ELISAs for the detection of E. multilocularis have subsequently become available. The sensitivity of coproantigen detection is generally good with moderate to high worm burdens (>100 worms), but less in animals with low burdens(Pierangeli et al., 2010). Coproantigen tests have the advantage that they can detect prepatent infections. Test performance and reproducibility are variable as crude rather than highly specific antigens are used to produce the antisera. It is also possible that variation in performance may also be associated with the strain of E. granulosus. However, coproantigen tests remain a useful procedure for population studies providing the potential pitfalls are fully understood when utilizing such tests. Coproantigen testing has been used successfully for diagnosis in various countries including Jordan(El-Shehabi et al., 2000), Peru(Lopera et al., 2003), Spain(Benito et al., 2006), Libya(Buishi et al., 2005), Argentina(Pierangeli et al., 2010)and Chile(Acosta-Jamett et al., 2010). Lopera et al. (2003) commented on the suitability of the technique for use in control programs in remote areas however cost and availability of the tests may prove to be an issue.
CoproPCR can also provide a validation test for coproantigen results (Lahmar et al., 2007b; Boufana et al., 2008; Nonaka et al., 2009). Species specific or strain specificPCR-based molecular tests have been developed by a number of groups for use directly on faeces or on eggs isolated from faeces to confirm the presence of Echinococcus infection (Dinkel et al., 2004). A recent study suggests that the sensitivity of egg isolation followed by PCR is 78% for E. granulosus and 50% for E. multilocularis infections in dogs (Ziadinov et al., 2008). Furthermore, microsatellite analyses with worm tissue or eggs may open new insights in the spatial and temporal genetic diversity of the parasite population (Knapp et al., 2009). However, PCR is a laborious and expensive technique and the requirement for extensive DNA isolation procedures to remove PCR-inhibitory components from faecal specimens limits the use of such tests for basic diagnosis (Armua-Fernandez et al., 2011). Finally, serum antibodies have been examined as a diagnostic method (Heath et al., 1985; Jenkins and Rickard, 1985; Gasser et al., 1990; Gasser et al., 1993; Gasser et al., 1994; Craig et al., 1995; Benito et al., 2006) and may be useful as an additional test (Varcasia et al., 2004; Torgerson and Deplazes, 2009).
Provided the features of the diagnostic tests are well understood many of these tests can be useful in population studies (Torgerson and Deplazes, 2009). Surveillance of infection in definitive hosts usually requires parallel testing or the use of additional confirmatory test(s) to improve diagnostic performance. The development of a cost-effective, reproducible and specific coproantigen test would greatly facilitate monitoring infection in definitive hosts.
1.3 Diagnosis and Monitoring in Domestic Intermediate Hosts
Diagnosis in non-human intermediate hosts is usually restricted to post-mortem findings(Eckert et al., 2001). Specificity using immunodiagnostic methods is low as a result of cross-reactions with antibodies to other taeniid cestodes such as Taenia hydatigena and T. ovis in sheep (Yong et al., 1978; Lightowlers et al., 1984). Natural infections in this species also produce relatively poor antibody responses compared to the high levels of Echinococcus-specific antibodies produced in human cases (Lightowlers et al., 1986; Kittelberger et al., 2002). This is thought to be due to antigen sequestration rather than immunological tolerance or non-specific immunosuppression. Attempts to detect circulating antigen have not been successful (Lightowlers and Gottstein, 1995). Work to develop immunodiagnostic tests in pigs, goats and cattle (Martinez Gomez et al., 1980), and buffalo and camels (Khan et al., 1990) have also proved unsuccessful. Ultrasound examination has also been used for diagnosis in sheep and goats (Sage et al., 1998; Lahmar et al., 2007a) but the sensitivity of this technique is low (54.36%). This is largely because pulmonary cysts cannot be detected unless they are close to the periphery of the organ due to high levels of attenuation of ultrasound in lung tissue (Bauld and Schwan, 1974). Thus, for surveillance of infection in intermediate hosts, monitoring infection in livestock killed in abattoirs combined with a reliable recording system remains the most practical option.
2 Quantifying Disease in Sylvatic Hosts
Where Echinococcus infection exists in wildlife as well as domestic species there are additional challenges before control methods can be instigated. This section uses the Australian situation as a case study. There, in addition to the widely recognised sheep-dog domestic cycle of Echinococcus granulosus, a sylvatic cycle exists in which macropodids (Family Macropodidae) and feral pigs (Sus scrofa) are the most common intermediate hosts (Kumaratilake and Thompson, 1982), while dingoes (Canis lupus dingo), dingo /domestic dog hybrids (Canis lupus dingoX Canis familiaris)) and red foxes (Vulpes vulpes) act as definitive hosts (Durie and Riek, 1952; Jenkins and Morris, 1991). These sylvatic hosts are reservoirs for infection of domestic species and humans (Jenkins, 2005). Such wildlife reservoirs present basic challenges such as estimating the extent of the host populations, assessing parasite distribution, prevalence and transmission dynamics, and the existence of possible preventive measures (Wobeser, 2007).
Although defining the susceptible population is fundamental to disease monitoring and control, this parameter can only be roughly estimated when the hosts are wildlife species(Wobeser, 2007). This difficulty is further exacerbated as E. granulosus has such a wide host range in Australia. There are 42 extant species in the family Macropodidae (Van Dyck and Strahan, 2008) for which rough distribution across the country is known. Population sizes of the commercially harvested species (red kangaroo (Macropus rufus), eastern grey kangaroo (M. giganteus), western grey kangaroo (M. fulginosus) and common wallaroo (M. robustus)) are estimated on an annual basis in those states where harvesting occurs. The 2009 estimate was 27 million (Department of the Environment, 2010). The feral pig population is concentrated in eastern Australia, with estimates of up to 23.5 million (Choquenot et al., 1996). The total wild dog and fox populations have not been estimated but they inhabit approximately 82.8% and 76% of Australia, respectively. Both species are common throughout most of their range (West, 2008). Thus, combined totals of potential intermediate host populations and definitive host populations are very high and the species are widespread across the country.
Methods for diagnosis of E. granulosus infection in sylvatic intermediate hosts are even more limited than for domestic intermediate hosts. Although radiographic techniques have been validated for lung cysts in small macropods (Barnes et al., 2007a), the requirement for general anaesthesia and radiographic equipment in the field limits the use of this technique. Attempts to develop serological tests have been unsuccessful (Barnes et al., 2008b). Most prevalence studies have very small sample sizes and results may be biased when the sampling method was the collection of road kill. These studies were also typically conducted over restricted geographic areas so their representativeness of the national populations is questionable. There are published data on presence and prevalence, or absence of infection in 19 macropodid species, feral pigs and wombats (summarised in Barnes (2007) with subsequent data in Barnes et al. (2007b; 2008a)). Sample sizes ranged from 1 – 2521 and infection was reported in 11/19 species. Only 14 studies had a sample size ≥ 40 per species. Among these, prevalence ranged from 0 – 28% and was typically higher in the medium sized macropods (black striped wallaby (M. dorsalis) and swamp wallaby (Wallabia bicolor)) and feral pigs. These studies have very limited geographic and host coverage and so give a poor indication of the true prevalence among wildlife intermediate hosts.
Diagnosis in wildlife definitive hosts can be by either necropsy or coproantigen detection(Jenkins and Morris, 1991; Jenkins et al., 2000). Survey data are even scarcer than for intermediate hosts (summarised in Barnes, (2007) with subsequent data in Jenkins et al., (2008) and have the same limitations. Prevalence in wild dogs is typically high: for studies with a sample size ≥40 prevalence range was 7 – 87%.
Although prevalence can be very high and spill over to humans and domestic species is known to occur (Jenkins, 2005), it is difficult to estimate the infection pressure from sylvatic hosts to other wildlife, domestic species and humans. The number of fertile cysts, number of protoscoleces per cyst and likelihood of cysts being eaten determine pressure on definitive hosts whereas number of mature tapeworms in the definitive hosts and egg distribution and survival in the environment determine pressure on intermediate hosts. Transmission dynamics in the macropod-wild dog cycle differ markedly from the sheep-dog cycle. Cyst growth rate is more rapid and cyst fertility occurs earlier (Barnes et al., 2007a). Unlike sheep, infection can be detrimental to macropodid hosts, increasing the likelihood of predation, thereby potentially shortening the life cycle and increasing the probability of transmission (Johnson et al., 1998; Barnes et al., 2007a; Barnes et al., 2008a). Wild dogs may also carry heavier burdens of the adult tapeworm compared to domestic dogs, (wild dogs: 12.5% > 100,000 worms, domestic dogs: 14.2% > 1,000 worms) resulting in increased infection pressure (Jenkins and Morris, 1991; Lahmar et al., 2001). The role of foxes in sylvatic transmission is debatable; although prevalence may be high and in some areas fox density may reach 7.2/km2, infected individuals usually carry < 50 worms (Obendorf et al., 1989; Jenkins and Craig, 1992).
The limited evidence available indicates that E. granulosus is well established in Australian sylvatic hosts but current data are not sufficient to identify risk factors for infection among the host species or across the geographic range. As a result it is not possible to identify areas in which to target possible control measures.
3 Existing Control Programs, Their Limitations and Future Ideas
The first descriptions of successful control were from Iceland (Beard et al.)and GreekCyprus (Polydorou, 1977). The progress in control programs from around the world have been analysed by a number of authors (Gemmell, 1978; Gemmell et al., 1987; Economides et al., 1998) but more recent analyses of hydatid control have been published by Larrieu et al. (2004), Ito et al.(2003), Torgerson and Heath(2003), Jenkins et al. (2005), Heath et al.(2006), Craig and Larrieu (2006), Pierangeli et al. (2007), Zhang et al. (2009) and McManus (2010). The most detailed analysis of the relative success or failure of all control programmes is by Craig and Larrieu (2006) and should be read by those contemplating future control of Echinococcus spp..