Eggs in transit

People who have ingested infected domestic animal livers (mainly cattle, sheep and pig) a short time before testing may reflect ‘false’ fascioliasis when the fluke eggs are found in their stools (Stork et al. Reference Stork, Venables, Jennings, Beesley, Bendezu and Capron1973; Ragab and Farag, Reference Ragab and Farag1978; Campo et al. Reference Campo, Marcen, Gonzalez, Milazzo, Pascual and Ponce1980). Such spurious infection may give rise to false positives. Although an expert microscopist may differentiate the somewhat degenerated aspect of ‘eggs in transit’ from ‘normal eggs’, to avoid confusion in such cases diagnosis requires placing the patient on a liver-free diet and performing repeated follow-up stool examinations.

Invasive and biliary periods of the disease

In humans, the incubation phase (from a few days to 2–3 months) is shorter than the prepatent period (at least 3–4 months), meaning that infected subjects may present clinical findings long before eggs could be found in the stools (Mas-Coma and Bargues, Reference Mas-Coma and Bargues1997), making egg finding in faeces as an early diagnosis tool impossible. Thus, egg finding techniques become useful only 3–4 months post-infection (p.i.). Serological tests allow for an earlier detection of infection and are therefore the appropriate techniques for patient diagnosis in the invasive phase. A serological test may allow for the detection of anti-Fasciola antibodies as early as 2 weeks p.i. although it does not differentiate between current or past infection, whereas a positive antigen-detecting test indicates an active infection although only from the 8th week p.i., that is around 2 months before beginning of egg shedding (see below).

Asymptomatic cases

Patients without symptoms have been reported, sometimes even in as relatively high percentages as 15% (Haseeb et al. Reference Haseeb, El-Shazly, Arafa and Morsy2003a). Several of these patients do moreover not present eggs in faeces (Arjona et al. Reference Arjona, Riancho, Aguado, Salesa and Gonzalez-Macias1995; Adachi et al. Reference Adachi, Kotani, Shimizu, Tanaka and Okada2005; Demir et al. Reference Demir, Ellidokuz, Degirmenci, Yücel and Gökçe2005). Eosinophilia, invasive techniques such as laparoscopy with liver biopsy, and non-invasive ones such as ultrasonography may be useful for guiding towards a confirmative diagnosis of such patients by means of a serological test and/or coproantigen test.

Egg output dynamics

In livestock the absence of a direct relationship between number of flukes present and number of eggs shed is well known. Egg production oscillations in trematodes in general and Fasciola in particular throughout their lifespan is also well known (Valero et al. Reference Valero, Panova, Comes, Fons and Mas-Coma2002). In humans, Fasciola egg output number and dynamics are unfortunately unknown. Different possibilities such as intermittent and very low egg shedding, especially in infection burdens of only one or a few fluke adults as well as in old infections, may not be ruled out and may be overlooked when analysing only one stool sample or when applying insufficiently sensitive techniques. The stimulation of the contraction of the gall bladder, where eggs accumulate, by administration of appropriate food (cholagogues) increases the appearance of eggs in stools thereafter. This has been useful in surveys of schoolchildren (R. Angles, La Paz, unpublished data). Significant intra-specimen variations in six Kato–Katz slides from a single stool sample were found, while the inter-specimen variation was almost negligible in daily exams for 5 consecutive days (El-Morshedy et al. Reference El-Morshedy, Shehab, Zaki and Farag2002). However, pronounced epg variations were found in children who were followed-up during a longer period in which no drug for treatment was available (unpublished data). Consequently, multiple and repeated follow-up stool examinations may be needed in such cases.

Biliary cases without egg shedding due to immature flukes or liver abscesses

Except in human fascioliasis endemic regions, humans are generally believed to be a non-suitable host for Fasciola. Consequently, sporadic or isolated case reports in animal endemic areas (Mas-Coma et al. Reference Mas-Coma, Bargues, Esteban and Dalton1999a) highlight the possibility of hepatic infections by flukes unable to attain maturity in human subjects. This may explain patients in whom eggs could never be found despite being in the biliary phase of the disease.

In many human infections, eggs are often not found in stools even after multiple faecal examinations (Hillyer, Reference Hillyer and Dalton1999). In Europe for instance, the diagnosis of human fascioliasis is frequently established using serological tests, because the detection of F. hepatica eggs in stools is not always possible. Thus, in the Limousin region, central France, egg detection in stools was positive in only 27·6% of a total of 711 subjects with fascioliasis (Rondelaud et al. Reference Rondelaud, Dreyfuss and Vignoles2006). In Asia, a similar situation is found in Vietnam, where cases have been ascribed to F. gigantica (De et al. Reference De, Murrell, Cong, Cam, Chau, Toan and Dalsgaard2003) and egg detection in stools was positive in only 16·9% of a total of 249 infected patients (De et al. Reference De, Nguyen, Dang, Nguyen, Le and Trieu2005). In a human fascioliasis mesoendemic area recently described in Mexico, where individual intensities in children were low (24–384 epg), eggs were only found in 14 out of a total of 50 children positive by coproantigen test (Zumaquero-Rios et al. Reference Zumaquero-Rios, Sarracent-Perez, Rojas-Garcia, Rojas-Rivero, Martinez-Tovilla, Valero and Mas-Coma2013).

Rare cases have also been reported in which a fluke reached maturity in the liver but its ‘encapsulation’ in a hepatic abscess did not allow eggs to reach the biliary canals and be shed with faeces (Fig. 3).

Fig. 3.

Eggs of Fasciola hepatica found in a liver abscess of a male adult Italian patient who was shedding no eggs in stools.

Re-infections and overlap of acute and chronic phases

Mainly in human fascioliasis endemic areas where the infection risk is high, but also in individual patients whose infection is related to the habit of usually consuming freshwater vegetables in animal fascioliasis endemic areas, subjects may be re-infected once already in the biliary period due to previous infection. In such cases, a relapse of the clinical characteristics of the invasive phase may appear and overlap with symptoms of the chronic period, as infected hosts do not develop resistance due to the rapid and potent ability of fasciolids to suppress the immune response (Girones et al. Reference Girones, Valero, Garcia-Bodelon, Chico-Calero, Punzon, Fresno and Mas-Coma2007; Dalton et al. Reference Dalton, Robinson, Mulcahy, O'Neill and Donnelly2013). In this group of patients, an increase of egg shedding may occur once the migrating juveniles reach the biliary canals, mature and start egg production.

Long-term relapses of the disease

Fascioliasis may have been overlooked due to milder symptomatology or misdiagnosis with patients going on to suffer a relapse including impressive manifestations and even death after many years of chronic infection (Ruggieri et al. Reference Ruggieri, Correa and Martínez1967; Correa et al. Reference Correa, Ruggieri and Martinez1969; Auer et al. Reference Auer, Kristoferitsch, Picher and Wessely1982; Kristoferitsch et al. Reference Kristoferitsch, Wessely, Auer and Picher1982). It should be considered that flukes may survive up to 9–13·5 years in the human host (Mas-Coma et al. Reference Mas-Coma, Bargues and Valero2005).

In Argentina, the long time elapsed between the appearance of symptoms and confirmation of infection by appropriate diagnosis has been emphasized in many patients: nearly four years in average, including patients having suffered from symptoms for 10 or more years without diagnosis (Mera y Sierra et al. Reference Mera y Sierra, Agramunt, Cuervo and Mas-Coma2011). This suggests either infected subjects had not sought professional diagnosis due to mild symptoms caused by low fluke burdens and/or misdiagnosis of patients due to the non-pathognomonic clinical picture, easily confused with other diseases when the patient attends a health centre not used to dealing with fascioliasis (La Pook et al. Reference La Pook, Magun, Nickerson and Meltzer2000).

The breakage of multiple old liver abscesses, aberrant specimens surviving as immature flukes for long periods, and hypersensitivity of the Arthus reaction type appear to underlie in these cases (Mas-Coma et al. Reference Mas-Coma, Agramunt and Valero2014). In such cases, eggs may be found in stools, but sometimes not when the flukes involved in the pathogenic picture are ectopic migrants. Neither do eggs appear in stools from patients presenting clinical disorders and biological abnormalities after years from the last date of fascioliasis treatment. Such symptoms appearing just after treatment and persisting afterwards up to 2–18 years were suggested to be due to the presence of persistent hepatic lesions following fascioliasis treatment (Rondelaud et al. Reference Rondelaud, Dreyfuss and Vignoles2006).

Ectopic infections

In fascioliasis, juvenile flukes sometimes deviate during migration from the intestine to the liver and enter other organs. Ectopic localization of the parasite may cause a confusing clinical presentation in both the acute and chronic infections. Ectopic lesions most frequently reported are in the gastrointestinal tract and subcutaneous tissue. Other ectopic locations described include: heart, blood vessels, the lung and pleural cavity; brain; dorsal spine; orbit; abdominal wall; stomach, caecum and appendix; pancreas; spleen; peritoneum; inguinal nodes; cervical node; blood vessels; skeletal muscle; and epididymis. Ectopic manifestations usually appear shortly after infection, although exceptions have been reported. Such ectopic flukes achieve maturity only very rarely (Mas-Coma et al. Reference Mas-Coma, Agramunt and Valero2014). No eggs in stools may be found when such ectopic lesions appear during the invasive phase, which is usually the case, but positive faecal samples may be found when in the biliary phase. Serological tests are thus the techniques of choice for such cases. Imaging techniques which may help guide towards fascioliasis diagnosis in several of the aforementioned localizations are unfortunately not usually available in remote poor rural areas of developing countries.

Different fasciolids

In areas of Europe, the Americas, Australia and the African Maghreb only F. hepatica is present and in western Africa there is only F. gigantica. The presence of only one of the two fasciolids is due to the lymnaeid species present in the aforementioned regions, only belonging to vector species specific for F. hepatica or F. gigantica, respectively (see details of this selective geographical distribution in Mas-Coma et al. Reference Mas-Coma, Valero and Bargues2009a). There is therefore no need for differential diagnosis when dealing with subjects inhabiting those regions, except in cases of immigrants from and travellers to endemic regions where the distribution of the two fasciolid species overlap.

However, there is an overlap of the geographical distribution of the two Fasciola species in eastern and southern Africa, as well as in many regions of Asia. In these regions, two different overlap situations can be distinguished (Mas-Coma et al. Reference Mas-Coma, Valero and Bargues2009a): (i) local overlap, in places where climatic characteristics throughout the year enable the coexistence of Galba and Radix species in the same locality, nearby water bodies and even sometimes the same water body. This allows transmission and consequent infection of livestock and humans living sedentarily in the locality by both F. hepatica and F. gigantica; (ii) Zonal overlap, in areas with different altitudes so that highlands offer the necessary cold-mild weather conditions for Galba truncatula and F. hepatica, and lowlands offer the warm-hot climate necessary for Radix species and F. gigantica. Thus, definitive hosts become co-infected by both fasciolids when moving from lowlands to neighbouring highlands and vice versa (animals because of interzonal transhumance, transportation and trade; humans when moving around for various reasons). Egypt is an example of local overlap, whereas Iran and Pakistan are examples of zonal overlap (see more details on local/zonal overlaps in Mas-Coma et al. Reference Mas-Coma, Valero and Bargues2009a).

In both kinds of overlaps, hybrid intermediate adult and egg forms are found, with higher and lower frequency in local and zonal overlap areas, respectively. Thus, techniques allowing for differential diagnosis are recommended for both local and zonal overlap areas given the different transmission characteristics, pathogenicity and control measures for one or other fasciolid species (Mas-Coma et al. Reference Mas-Coma, Valero and Bargues2009a). It should be considered here that immunological techniques (serological and coproantigen tests) do not differentiate between the two species, and that egg-finding coprological techniques may sometimes not be sufficient (Valero et al. Reference Valero, Perez-Crespo, Periago, Khoubbane and Mas-Coma2009a; Mas-Coma et al. Reference Mas-Coma, Agramunt and Valero2014). Only specific molecular techniques allow for such a differential diagnosis between F. hepatica, F. gigantica and hybrid intermediate forms. Morphological phenotyping of adult flukes may also be applied if parasites can be obtained with invasive methods, although this is also prone to difficulty and time-consuming, and may sometimes furnish inconclusive results if specimens are intermediate forms and their number too few (Mas-Coma et al. Reference Mas-Coma, Valero and Bargues2009a).

Situations in human fascioliasis endemic areas

Most of these situations are in developing countries, although they have also been infrequently described in impoverished remote rural areas of developed countries. Different prevalences and intensities are characteristic features in the human hyper-, meso- and hypoendemic areas (Mas-Coma et al. Reference Mas-Coma, Bargues, Esteban and Dalton1999a, Reference Mas-Coma, Esteban and Barguesb, Reference Mas-Coma, Bargues and Valero2005, Reference Mas-Coma, Valero and Bargues2009a). Whereas none or only very low differences may be obtained between prevalence results from immunological and direct coprological techniques in human hyperendemic areas with very high prevalences such as the Northern Bolivian Altiplano (Valero et al. Reference Valero, Periago, Perez-Crespo, Angles, Villegas, Aguirre, Strauss, Espinoza, Herrera, Terashima, Tamayo, Engels, Gabrielli and Mas-Coma2012a; Esteban et al. unpublished data), there may be differences between results from immunological and direct coprological techniques when in meso- and hypoendemic areas, and also hyperendemic areas with not very high prevalences. In the latter areas, immunological techniques, whether serological or coproantigen tests, furnish more accurate prevalence results (Espinoza et al. Reference Espinoza, Maco, Marcos, Saez, Neyra, Terashima, Samalvides, Gotuzzo, Chavarry, Huaman, Bargues, Valero and Mas-Coma2007; Valero et al. Reference Valero, Periago, Perez-Crespo, Angles, Villegas, Aguirre, Strauss, Espinoza, Herrera, Terashima, Tamayo, Engels, Gabrielli and Mas-Coma2012a; Zumaquero-Rios et al. Reference Zumaquero-Rios, Sarracent-Perez, Rojas-Garcia, Rojas-Rivero, Martinez-Tovilla, Valero and Mas-Coma2013).

Moreover, highly sensitive diagnostic techniques are needed for areas presenting low prevalences and intensities. Additionally, in human fascioliasis endemic areas there are adult subjects who may have been infected for many years (Esteban et al. Reference Esteban, Flores, Aguirre, Strauss, Angles and Mas-Coma1997a, Reference Esteban, Flores, Angles, Strauss, Aguirre and Mas-Comab, Reference Esteban, Flores, Angles and Mas-Coma1999). The adult flukes infecting them may be very old and consequently no longer shedding eggs whilst even eosinophilia may have become negligible.

In human fascioliasis endemic areas in general, children presenting first symptoms of the invasive phase do not usually attend a health centre for diagnosis, and if they do they are usually misdiagnosed because of absence of eggs in stools and lack of serological diagnostics, especially in rural health centres. Thus, children are the main target for epidemiological assessment and coprological methods should be used to avoid community disturbance due to blood extraction procedures. Inhabitants of remote rural endemic areas are usually reluctant to give blood samples, and if successful in convincing them for one occasion, this usually poses problems for subsequent studies.

Epidemics of human fascioliasis have also been described in human fascioliasis endemic areas. In such outbreaks involving a prevalence increase, epidemics are usually related to previous climatic conditions having favoured both the parasite and the snail life cycles (Mas-Coma et al. Reference Mas-Coma, Valero and Bargues2008, Reference Mas-Coma, Valero and Bargues2009b), and also to anthropogenic modifications of the environment (Afshan et al. Reference Afshan, Fortes-Lima, Artigas, Valero, Qayyum and Mas-Coma2014). Such epidemics can take place in hypoendemic, mesoendemic and hyperendemic areas (Mas-Coma et al. Reference Mas-Coma, Bargues, Esteban and Dalton1999a, Reference Mas-Coma, Esteban and Barguesb, Reference Mas-Coma, Bargues and Valero2005, Reference Mas-Coma, Valero and Bargues2009a) and may show a parallelism with outbreaks in livestock, early detection of which may help in forecasting the human epidemic.

Situations in animal fascioliasis endemic areas

This is the usual situation in developed countries, but can also appear in developing countries where human fascoliasis does not appear to be a serious public health problem. People usually present for diagnosis in hospitals and health centres as soon as the first symptoms appear. Therefore, patients are usually diagnosed in the invasive period or beginning of the biliary period. Consequently, there are usually no eggs in stools, but anamnesis and eosinophilia provide valuable evidence and the diagnosis should preferentially rely on serodiagnosis. An immunological test is also better due to the fact that there may be fluke strains not adapted to humans and therefore not able to reach maturity and produce eggs in the patients in given areas.

Patients are usually isolated and sporadic cases in animal fascioliasis endemic areas (Mas-Coma et al. Reference Mas-Coma, Bargues, Esteban and Dalton1999a, Reference Mas-Coma, Esteban and Barguesb, Reference Mas-Coma, Bargues and Valero2005, Reference Mas-Coma, Valero and Bargues2009a). However, infection reports of immigrants from endemic areas (Auer et al. Reference Auer, Kristoferitsch, Picher and Wessely1982; Pelletier et al. Reference Pelletier, Chosidow, Rogeaux, Lenoir, Piette, Frances and Herson1995; Graham et al. Reference Graham, Brodie and Weller2001; MacLean et al. Reference Mac Lean, Varghese and Graeme-Cook2002; Noyer et al. Reference Noyer, Coyle, Werner, Dupouy-Camet, Tanowitz and Wittner2002; Turrientes et al. Reference Turrientes, Saenz de Sta. Maria, Ceballos, Diaz, Bareno, Muro, Pardo and Lopez-Velez2004; Cheung et al. Reference Cheung, Enns, Romney, Reynolds and Amar2005; Channabasappa et al. Reference Channabasappa, Vettiankal, Riles and Attar2006; Fullerton et al. Reference Fullerton, Vitale and Vitale2006; Morse et al. Reference Morse, McLean and Bedi2006; Alatoom et al. Reference Alatoom, Sheffield, Gander, Shaw and Cavuoti2008; Chand et al. Reference Chand, Herman, Partridge, Hewitt and Chiodini2009; Novo-Veleiro et al. Reference Novo-Veleiro, Martín-Sánchez, Elcuaz-Romano, Muro, Afonso-Rodríguez, García-Bardeci, Bordes-Benítez, Carranza-Rodríguez, Hernández-Cabrera, Alvela-Suárez and Pérez-Arellano2012), and travellers (professionals, business people, tourists) and military personnel having visited endemic areas are not uncommon in developed countries.

The rarity of Fasciola infection in those areas makes diagnosis challenging and often unrecognized. It may also lead to misdiagnosis or late diagnosis when local physicians are not aware of fascioliasis entering into the diagnostic probabilities related to the clinical picture presented (La Pook et al. Reference La Pook, Magun, Nickerson and Meltzer2000; Mera y Sierra et al. Reference Mera y Sierra, Agramunt, Cuervo and Mas-Coma2011).

Human epidemics have also been described in animal fascioliasis endemic areas. These outbreaks appear in zones where previous human reports have always been isolated and sporadic. Such outbreaks usually concern a very few subjects infected from the same contaminated source (family or small group reports; contaminated wild, home-grown or commercially grown watercress or other metacercariae-carrying vegetables) (Mas-Coma et al. Reference Mas-Coma, Bargues, Esteban and Dalton1999a, Reference Mas-Coma, Esteban and Barguesb, Reference Mas-Coma, Bargues and Valero2005, Reference Mas-Coma, Valero and Bargues2009a). Outbreaks of this type usually appear following heavy rainfall in the previous years or seasons (Carme et al. Reference Carme, Niel, Danis and Gentilini1978; Perez Rodríguez et al. Reference Perez Rodríguez, Casero Valladares, Martinez Rodriguez and Perez Martin1986; Ripert et al. Reference Ripert, Tribouley, Luong Dinh Giap, Combe and Laborde1987; Cadel et al. Reference Cadel, Barbier, Duhamel and Georges1996). Sometimes seasonality is related to ingestion of infected plants. For example, in France most human cases occurred during the October–April watercress season (Ripert et al. Reference Ripert, Tribouley, Luong Dinh Giap, Combe and Laborde1987), with a maximum from November to February (Rondelaud et al. Reference Rondelaud, Amat-Frut and Pestre-Alexandre1982). Unexpected problems of contamination of watercress cultures due to, for example, disease spread by an introduced sylvatic reservoir animal such as the nutria (Houin et al. Reference Houin, Moquet, Czeher, Vallois, Mas-Coma, Bargues, Esteban and Valero2004) appear related to the recent emergence of human fascioliasis in particular areas (Mailles et al. Reference Mailles, Vaillant, Schepens, Ajana, Capek, Ilef, Fillebeen, Lefort, Therouanne, Volant, Flavigny and De Valk2003, Reference Mailles, Capek, Ajana, Schepens, Ilef and Vaillant2006). In rural areas of developing countries, the best method to differentiate between an outbreak in an animal endemic area and a human endemic situation is mass diagnostic screening of schoolchildren.

Different transmission patterns

Different disease seasonalities have been described in human and also in animal fascioliasis endemic areas. Mono- and bi-seasonalities and permanent transmission may be distinguished. Diagnostic results are unavoidably influenced by these different local transmission patterns, mainly when working with direct coprological techniques allowing for egg finding (Valero et al. Reference Valero, Periago, Perez-Crespo, Angles, Villegas, Aguirre, Strauss, Espinoza, Herrera, Terashima, Tamayo, Engels, Gabrielli and Mas-Coma2012a). Intensities (epg) are higher in seasons when human subjects are at the beginning of the egg-shedding period. Consequently these seasons should be preferred when implementing mass screenings (Esteban et al. Reference Esteban, Flores, Angles and Mas-Coma1999; Afshan et al. Reference Afshan, Fortes-Lima, Artigas, Valero, Qayyum and Mas-Coma2014) to avoid overlooking cases with very low burdens. Serological antibody-detecting tests may be less influenced by seasonality, because of the perseverance of antibodies. Antigen-detecting techniques may also be influenced by seasonality, as antigen amounts, both faecal and circulating, are related to the burden. Seasonal infection data in livestock inhabiting the same area may be useful in establishing the best season for human surveys (Mas-Coma et al. Reference Mas-Coma, Angles, Esteban, Bargues, Buchon, Franken and Strauss1999c).

In areas of zonal overlap where hybridization is rare (Mas-Coma et al. Reference Mas-Coma, Valero and Bargues2009a), transmission by pure F. hepatica may occur in different months than transmission of pure F. gigantica, due to their different temperature thresholds, F. hepatica preferring milder months and F. gigantica warmer months (Afshan et al. Reference Afshan, Fortes-Lima, Artigas, Valero, Qayyum and Mas-Coma2014). In areas of local overlap where hybridization is common (Mas-Coma et al. Reference Mas-Coma, Valero and Bargues2009a), the existence of two transmission peaks, each corresponding to one Fasciola species, may become diluted and human infection and subsequent beginning of egg shedding after the prepatent period appear continuous throughout more months.

Social habits and cultural traditions

Human diet and behaviour related to livestock (local animal management traditions) have to be considered in prevalence surveys as well as ethnic aspects facilitating the participation (or not) in surveys of the inhabitants of the endemic area. In the Northern Bolivian Altiplano, the highest human hyperendemic area known, the Aymara inhabitants do not allow blood sampling due to religious customs. Thus all surveys in these areas have been made with coprological techniques (Esteban et al. Reference Esteban, Flores, Angles and Mas-Coma1999; Mas-Coma et al. Reference Mas-Coma, Angles, Esteban, Bargues, Buchon, Franken and Strauss1999c). In initial studies, the acceptance of inhabitants of given localities to furnish blood samples was obtained (Hillyer et al. Reference Hillyer, Soler de Galanes, Rodriguez-Perez, Bjorland, Silva de Lagrava, Guzman and Bryan1992) although very few additional ones could later be further obtained (Strauss et al. Reference Strauss, Angles, Esteban and Mas-Coma1997; O'Neill et al. Reference O'Neill, Parkinson, Strauss, Angles and Dalton1998). Since then, Aymaras of the endemic area have remained reluctant to give blood, and as a result all subsequent serological studies were repeatedly using the same old, few samples obtained for the aforementioned interventions (ignoring this fact has given rise to several misunderstandings in recent reviews). Thus, the present WHO control initiative is based only on coprological techniques (Villegas et al. Reference Villegas, Angles, Barrientos, Barrios, Valero, Hamed, Grueningr, Ault, Montresor, Engels, Mas-Coma and Gabrielli2012; Valero et al. Reference Valero, Periago, Perez-Crespo, Angles, Villegas, Aguirre, Strauss, Espinoza, Herrera, Terashima, Tamayo, Engels, Gabrielli and Mas-Coma2012a).

A contrasting situation, however, is described in a human fascioliasis endemic area recently described in Argentina (Carnevale et al. Reference Carnevale, Cabrera, Cucher, di Risio, Malandrini, Kamenetzky, Alazraqui, Etchart, Pantano and Velasquez2013), where inhabitants are reluctant to give stool samples, so that diagnostic studies relied only on serological tests (Malandrini, personal communication). This situation will also, in turn, pose problems for the verification of treatment success.

Antigen capture with monoclonal antibodies

MoAbs have been described against antigens of both F. hepatica and F. gigantica. With regard to F. hepatica, they have been developed against somatic and tegumental antigens (Hanna and Trudgett, Reference Hanna and Trudgett1983; Hanna et al. Reference Hanna, Trudgett and Anderson1988) as well as against ES antigens (ES78 and MM3) (Espino et al. Reference Espino, Diaz, Perez and Finlay1998; Mezo et al. Reference Mezo, Gonzalez-Warleta and Ubeira2007). All have been shown to be excellent diagnostic reagents for detecting coproantigens in humans, sheep, cattle and wild boars with chronic infection (Espino et al. Reference Espino, Diaz, Perez and Finlay1998; Dumenigo et al. Reference Dumenigo, Espino, Finlay and Mezo2000; Mezo et al. Reference Mezo, Gonzalez-Warleta, Carro and Ubeira2004, Reference Mezo, Gonzalez-Warleta, Castro-Hermida, Manga-Gonzalez, Peixoto, Mas-Coma and Valero2013; Ubeira et al. Reference Ubeira, Muiño, Valero, Periago, Perez-Crespo, Mezo, Gonzalez-Warleta, Romaris, Paniagua, Cortizo, Llovo and Mas-Coma2009; Valero et al. Reference Valero, Ubeira, Khoubbane, Artigas, Muiño, Mezo, Perez-Crespo, Periago and Mas-Coma2009b, Reference Valero, Periago, Perez-Crespo, Angles, Villegas, Aguirre, Strauss, Espinoza, Herrera, Terashima, Tamayo, Engels, Gabrielli and Mas-Coma2012a). Two other MoAbs highly specific for FhSAP2 have recently been obtained (2F5 and 2F9), which do not cross-react with antigens from Schistosoma mansoni, Schistosoma japonicum or Clonorchis sinensis (Caban-Hernandez and Espino, Reference Caban-Hernandez and Espino2013).

With regard to F. gigantica, MoAbs have been produced against total extract (Fagbemi et al. Reference Fagbemi, Aderibigbe and Guobadia1997), against ES antigens (Estuningsih et al. Reference Estuningsih, Widjayanti, Adiwinata and Piedrafita2004, Reference Estuningsih, Spithill, Raadsma, Law, Adiwinata, Meeusen and Piedrafita2009; Demerdash et al. Reference Demerdash, Diab, Aly, Mohamed, Mahmoud, Zoheiny, Mansour, Attia and El Bassiouny2011), against partially purified native proteins derived from tegument (Chaithirayanon et al. Reference Chaithirayanon, Wanichanon, Vichasri-Grams, Ardseungneon, Grams, Viyanant, Upatham and Sobhon2002; Krailas et al. Reference Krailas, Panomsuk, Janecharat and Ukong2002; Anuracpreeda et al. Reference Anuracpreeda, Wanichanon, Chaithirayanon, Preyavichyapugdee and Sobhon2006, Reference Anuracpreeda, Wanichanon, Chawengkirtikul, Chaithirayanon and Sobhon2009b) and against recombinant proteins such as FABPs (Sirisriro et al. Reference Sirisriro, Grams, Vichasri-Grams, Ardseungneon, Pankao, Meepool, Chaithirayanon, Viyanant, Tan-Ariya, Upatham and Sobhon2002; Allam et al. Reference Allam, Bauomy, Hemyeda and Sakran2012), a SAPLIP member from F. gigantica (FgSAP2) (Kueakhai et al. Reference Kueakhai, Changklungmoa, Riengrojpitak, Chaichanasak, Meemon, Chaithirayan, Chantree, Sansri, Itagaki and Sobhon2013a, Reference Kueakhai, Changklungmoa, Chaithirayanon, Songkoomkrong, Riengro-Jpitak and Sobhonb) and cathepsin B3 protease (Anuracpreeda et al. Reference Anuracpreeda, Chawengkirtikul, Tinikul, Poljaroen, Chotwiwatthanakun and Sobhon2013).

The most widely used of the MoAbs for human fascioliasis diagnosis described to date are the ES78 (Marcet Sanchez et al. Reference Marcet Sánchez, Figueredo Pino, Núñez Fernández, Rojas Rivero and Sarracent Pérez2012) and MM3 (Muiño et al. Reference Muiño, Perteguer, Garate, Martinez-Sernandez, Beltran, Romaris, Mezo, Gonzalez-Warleta and Ubeira2011), although others have also been described (Demerdash et al. Reference Demerdash, Diab, Aly, Mohamed, Mahmoud, Zoheiny, Mansour, Attia and El Bassiouny2011; Caban-Hernandez and Espino, Reference Caban-Hernandez and Espino2013).

Coproantigen tests

Stool antigen detection techniques have been reported for both human and animal fascioliasis, most of them based on Fasciola ES products. The detection of these products by means of a sandwich-ELISA reflects the installation of flukes in the bile ducts and the presence of the biliary stage of the disease. The majority of methods based on antigen detection have been applied to F. hepatica infection (Hanna and Trudgett, Reference Hanna and Trudgett1983; Hanna et al. Reference Hanna, Trudgett and Anderson1988; Espino et al. Reference Espino, Diaz, Perez and Finlay1998; Dumenigo et al. Reference Dumenigo, Espino, Finlay and Mezo2000; Mezo et al. Reference Mezo, Gonzalez-Warleta, Carro and Ubeira2004, Reference Mezo, Gonzalez-Warleta and Ubeira2007, Reference Mezo, Gonzalez-Warleta, Castro-Hermida, Manga-Gonzalez, Peixoto, Mas-Coma and Valero2013; Ubeira et al. Reference Ubeira, Muiño, Valero, Periago, Perez-Crespo, Mezo, Gonzalez-Warleta, Romaris, Paniagua, Cortizo, Llovo and Mas-Coma2009; Valero et al. Reference Valero, Ubeira, Khoubbane, Artigas, Muiño, Mezo, Perez-Crespo, Periago and Mas-Coma2009b, Reference Valero, Periago, Perez-Crespo, Angles, Villegas, Aguirre, Strauss, Espinoza, Herrera, Terashima, Tamayo, Engels, Gabrielli and Mas-Coma2012a; Caban-Hernandez and Espino, Reference Caban-Hernandez and Espino2013) and have only in recent years started to be applied to F. gigantica infection (Arafa et al. Reference Arafa, Abaza, El-Shewy, Mohareb and El-Moamly1999; Chaithirayanon et al. Reference Chaithirayanon, Wanichanon, Vichasri-Grams, Ardseungneon, Grams, Viyanant, Upatham and Sobhon2002; Krailas et al. Reference Krailas, Panomsuk, Janecharat and Ukong2002; Sirisriro et al. Reference Sirisriro, Grams, Vichasri-Grams, Ardseungneon, Pankao, Meepool, Chaithirayanon, Viyanant, Tan-Ariya, Upatham and Sobhon2002; Estuningsih et al. Reference Estuningsih, Widjayanti, Adiwinata and Piedrafita2004; Anuracpreeda et al. Reference Anuracpreeda, Wanichanon, Chaithirayanon, Preyavichyapugdee and Sobhon2006, Reference Anuracpreeda, Wanichanon, Chawengkirtikul, Chaithirayanon and Sobhon2009b, Reference Anuracpreeda, Chawengkirtikul, Tinikul, Poljaroen, Chotwiwatthanakun and Sobhon2013; Kueakhai et al. Reference Kueakhai, Changklungmoa, Riengrojpitak, Chaichanasak, Meemon, Chaithirayan, Chantree, Sansri, Itagaki and Sobhon2013a, Reference Kueakhai, Changklungmoa, Chaithirayanon, Songkoomkrong, Riengro-Jpitak and Sobhonb).

Coproantigen tests have both positive and negative attributes (Table 3) and have been developed based on different antibodies. Besides the most used ES78 monoclonal antibody used for the FasciDIG test (Espino et al. Reference Espino, Dumenigo, Marcet, Reyes and Finlay1993, Reference Espino, Diaz, Perez and Finlay1998; Espino and Finlay, Reference Espino and Finlay1994; Marcet Sanchez et al. Reference Marcet Sánchez, Figueredo Pino, Núñez Fernández, Rojas Rivero and Sarracent Pérez2012) and MM3 monoclonal antibody used for the MM3-COPRO test (Ubeira et al. Reference Ubeira, Muiño, Valero, Periago, Perez-Crespo, Mezo, Gonzalez-Warleta, Romaris, Paniagua, Cortizo, Llovo and Mas-Coma2009; Valero et al. Reference Valero, Ubeira, Khoubbane, Artigas, Muiño, Mezo, Perez-Crespo, Periago and Mas-Coma2009b, Reference Valero, Periago, Perez-Crespo, Angles, Villegas, Aguirre, Strauss, Espinoza, Herrera, Terashima, Tamayo, Engels, Gabrielli and Mas-Coma2012a; Muiño et al. Reference Muiño, Perteguer, Garate, Martinez-Sernandez, Beltran, Romaris, Mezo, Gonzalez-Warleta and Ubeira2011), other monoclonal antibodies have also been described (Arafa et al. Reference Arafa, Abaza, El-Shewy, Mohareb and El-Moamly1999; El-Bassiouny et al. Reference El-Bassiouny, Mohamed, Salah, Zoheiry, Mansour, Diab, Rabia, Safwat and Demerdash2008; Demerdash et al. Reference Demerdash, Diab, Aly, Mohamed, Mahmoud, Zoheiny, Mansour, Attia and El Bassiouny2011; Allam et al. Reference Allam, Bauomy, Hemyeda and Sakran2012; Attallah et al. Reference Attallah, Bughdadi, El-Shazly and Ismail2013). Several are commercially available: (i) ‘Fasciola hepatica Antigen ELISA Kit Bio K 201’; (ii) ‘Bovine Fasciola hepatica Antigenic ELISA Kit’; (iii) ‘Fasciola hepatica Ag’; (iv) ‘FasciDIG IPK La Havana’.

Table 3.

Advantages and disadvantages of antigen-detecting techniques applied to stool samples for fascioliasis diagnosis in humans

To date, the only coproantigen test to be evaluated for community surveys in the field according to proposed standards is the MM3-COPRO (Valero et al. Reference Valero, Periago, Perez-Crespo, Angles, Villegas, Aguirre, Strauss, Espinoza, Herrera, Terashima, Tamayo, Engels, Gabrielli and Mas-Coma2012a). Test efficacy was evaluated in children within two human fascioliasis high-prevalence areas: one with an altiplanic transmission pattern in Bolivia (high epg and permanent transmission) and another with a valley transmission pattern in Peru (low-medium epg and seasonal transmission). Sensitivity and specificity were 94·68 and 98·48% using Kato–Katz as the preferred method in Bolivia and 94·73 and 93·58% using rapid sedimentation and Kato–Katz together in Peru. Of note in these studies is that six children were found to be shedding eggs despite showing a negative result with MM3-COPRO, including one child with a high egg count (1248 epg) (Valero et al. Reference Valero, Periago, Perez-Crespo, Angles, Villegas, Aguirre, Strauss, Espinoza, Herrera, Terashima, Tamayo, Engels, Gabrielli and Mas-Coma2012a).

This field study showed no correlation between coproantigen detection by optical density (OD) and infection intensity by epg of faeces in Cajamarca (low burden cases <400 epg), nor in Huacullani (high burden cases >400 epg). In contrast there was correlation in Huacullani regarding low burden cases (<400 epg) (Valero et al. Reference Valero, Periago, Perez-Crespo, Angles, Villegas, Aguirre, Strauss, Espinoza, Herrera, Terashima, Tamayo, Engels, Gabrielli and Mas-Coma2012a). Observed lack of correlation in two of these studies is in contrast to the positive correlation between epg counts and OD readings obtained by other authors (Espino and Finlay, Reference Espino and Finlay1994; Mezo et al. Reference Mezo, Gonzalez-Warleta, Carro and Ubeira2004; Demerdash et al. Reference Demerdash, Diab, Aly, Mohamed, Mahmoud, Zoheiny, Mansour, Attia and El Bassiouny2011; Attallah et al. Reference Attallah, Bughdadi, El-Shazly and Ismail2013), but agrees with results found in hospital patients (Ubeira et al. Reference Ubeira, Muiño, Valero, Periago, Perez-Crespo, Mezo, Gonzalez-Warleta, Romaris, Paniagua, Cortizo, Llovo and Mas-Coma2009). Results suggest that the OD/epg correlation found by other authors may be due to the analysis of stool from low to very low fluke burdens. Moreover, it must be kept in mind that (i) the pattern of egg shedding is not linear but fluctuates between maximum and minimum values (Valero et al. Reference Valero, Panova, Comes, Fons and Mas-Coma2002, Reference Valero, Panova, Perez-Crespo, Khoubbane and Mas-Coma2011), (ii) the kinetics of coproantigen release vs the kinetics of egg shedding show a similar pattern but with a 2-week time-lag in epg (Valero et al. Reference Valero, Ubeira, Khoubbane, Artigas, Muiño, Mezo, Perez-Crespo, Periago and Mas-Coma2009b) and (iii) the influence of the crowding effect on Fasciola egg shedding should also be taken into account in human hyperendemic areas (Valero et al. Reference Valero, De Renzi, Panova, Garcia-Bodelon, Periago, Ordoñez and Mas-Coma2006b). Therefore, the use of only a coproantigen technique appears to be insufficient to evaluate fascioliasis intensity.

Results from a study by Valero et al. (Reference Valero, Periago, Perez-Crespo, Angles, Villegas, Aguirre, Strauss, Espinoza, Herrera, Terashima, Tamayo, Engels, Gabrielli and Mas-Coma2012a) indicated that a coproantigen-detecting test such as the MM3-COPRO ELISA meets proposed criteria for control of human fascioliasis, including high sensitivity and specificity, rapid large mass screening capacity, detection in the acute phase, early detection of treatment failure or re-infection in post-treated subjects, and value for surveillance programmes. It provides a good tool to detect biliary fascioliasis, including a higher sensitivity than egg detection techniques, in areas where burdens are from low to very high.

The aforementioned field study highlights the benefits of such coproantigen tests for mass screening surveys prior to application of Kato–Katz, prioritizing coproantigen positive samples. Coproantigen positive subjects in which no eggs are found are recommended to receive treatment. Benefits include: (i) rapid response to communities surveyed with infected subjects treated within days of the survey and (ii) to distinguish re-infection from potential drug resistance. Coproantigen negative samples may finally be analysed later for the eventual detection and subsequent selected treatment of very few subjects shedding eggs (Valero et al. Reference Valero, Periago, Perez-Crespo, Angles, Villegas, Aguirre, Strauss, Espinoza, Herrera, Terashima, Tamayo, Engels, Gabrielli and Mas-Coma2012a).

Techniques for circulating antibody detection

Different circulating antibody-detecting techniques have been applied for human fascioliasis diagnosis. Several older methods are complicated to use and hence are no longer or only sporadically used, although the wider commercialization and availability of some of these methods (e.g. indirect haemagglutination) has led to their continued use in some countries. The following techniques are of note:

• – Precipitation tests. They include radial diffusion (RD), double diffusion (DD), immunoelectrophoresis (IEP) and/or counter-immunoelectrophoresis (CIEP), and metacercarial precipitin test (see review in Esteban et al. Reference Esteban, Bargues and Mas-Coma1998).

• – Agglutination tests. Indirect haemagglutination (IHA – commercialized: Fumouze Diagnostics) (see numerous references in Esteban et al. Reference Esteban, Bargues and Mas-Coma1998). The IHA test is a technique still used frequently in endemic areas, using purified adult F. hepatica F₁ as antigen (El-Shazly et al. Reference El-Shazly, Handousa, Gabr, Morsy, Ramadan and Morsy2002; Haseeb et al. Reference Haseeb, El-Shazly, Arafa and Morsy2003b; El-Ahl et al. Reference El-Ahl, El Shazly, El Shafei, Hegazi and El-Dardiry2007; Hammami et al. Reference Hammami, Hamed and Ayadi2007; Deveci et al. Reference Deveci, Oztürk and Ustün2011; Gökçe et al. Reference Gökçe, Sahiner, Unal, Parlakay, Oncel, Saçkesen, Kara and Gümrük2011; Kaya et al. Reference Kaya, Beştaş and Cetin2011; Sakru et al. Reference Sakru, Korkmaz, Demirci, Kuman and Ok2011; Tanir et al. Reference Tanir, Karaman, Tüfekçi, Erdogan, Tuygan and Ozkan2011; Karadağ-Öncel et al. Reference Karadağ-Öncel, Ozsürekçi, Ozkaya-Parlakay, Celik, Cengiz, Haliloğlu, Ceyhan and Kara2012; Yalav et al. Reference Yalav, Yağmur, Ülkü, Akcam and Sönmez2012).

• – Haemolysis tests. Complement fixation (CF) (see review in Esteban et al. Reference Esteban, Bargues and Mas-Coma1998).

• – Fluorescence tests. Immunofluorescence assay (IFA) (see review in Esteban et al. Reference Esteban, Bargues and Mas-Coma1998). This technique is still used frequently in laboratories, using parasite histological sections as antigen (Abdul-Fattah et al. Reference Abdul-Fattah, Yousef, Nasr, Fouad and Abdul-Wahab1992; Strauss et al. Reference Strauss, Angles, Esteban and Mas-Coma1997; Chand et al. Reference Chand, Herman, Partridge, Hewitt and Chiodini2009). Sensitivity is reported at 92–96% in the acute phase of the infection (Capron et al. Reference Capron, Wattre, Capron and Lefebure1973).

• – Radioisotope tests. Radioimmuno assays (RIST, RAST) (Sampaio Silva et al. Reference Sampaio-Silva, Vindimian, Wattre and Capron1985).

• – Anti-P ₁antibody tests. Automated assay of anti-P₁ antibodies (Ben-Ismail et al. Reference Ben-Ismail, Carme and Gentilini1978, Reference Ben-Ismail, Rouger, Carme, Gentilini and Salmon1980, Reference Ben-Ismail, Carme, Mogahed, Niel and Gentilini1982).

Nowadays, immunological techniques rely on numerous variants of enzymic tests (see many references in Esteban et al. Reference Esteban, Bargues and Mas-Coma1998):

Enzyme-linked immunosorbent assay (ELISA)

Reports including the use of ELISA for diagnosis of fascioliasis are numerous (Thi, Reference Thi2004; El-Ahl et al. Reference El-Ahl, El Shazly, El Shafei, Hegazi and El-Dardiry2007; Ozturhan et al. Reference Ozturhan, Emekdaş, Sezgin, Korkmaz and Altintaş2009; Karahocagil et al. Reference Karahocagil, Akdeniz, Sunnetcioglu, Cicek, Mete, Akman, Ceylan, Karsen and Yapici2011; Karadağ-Öncel et al. Reference Karadağ-Öncel, Ozsürekçi, Ozkaya-Parlakay, Celik, Cengiz, Haliloğlu, Ceyhan and Kara2012; Castillo Contreras and Frisancho Velarde, Reference Castillo Contreras and Frisancho Velarde2013). Due to its simplicity and functionality, researchers have mainly focused on improving the ELISA method, and a broad spectrum of antigens has been used so far. Nevertheless, human infections frequently occur in non-developed countries where access to diagnostic laboratories is not always possible. ELISA requires a well-equipped laboratory with a spectrophotometer and has to be carried out by trained personnel. Furthermore, there are currently non-standardized criteria to establish the cut-off limit between positivity and negativity for fascioliasis.

ELISA variants

They include the enzyme-linked immunofiltration assay (ELIFA) (Pailler et al. Reference Pailler, Puygauthier-Toubas, Bonnin, Marx-Chemla, Camerlynck, Thoannes and Pinon1990), the enzyme-linked immuno-electrotransfer blot (EITB) or western blot (Santiago and Hillyer, Reference Santiago and Hillyer1986; Hillyer and Soler De Galanes, Reference Hillyer and Soler De Galanes1988; Hillyer et al. Reference Hillyer, Soler de Galanes, Rodriguez-Perez, Bjorland, Silva de Lagrava, Guzman and Bryan1992; Silva et al. Reference Silva, Urarte, Mercado and Gorman1993; Shaker et al. Reference Shaker, Demerdash, Mansour, Hassanein, El-Baz and El-Gindy1994; Hammami et al. Reference Hammami, Ayadi, Camus and Dutoit1997; Marcos et al. Reference Marcos, Tagle, Terashima, Bussalleu, Ramirez, Carrasco, Valdez, Huerta-Mercado, Freedman, Vinetz and Gotuzzo2008; Trifina et al. Reference Trifina, Spenger, Zandieh, Haller, Auer, Österreicher, Klaushofer and Mikosch2011; Agnamey et al. Reference Agnamey, Fortes-Lopes, Raccurt, Boncy and Totet2012) and the Falcon™ assay screening test-enzyme linked immunosorbent assay (FAST-ELISA) (Hillyer and Soler De Galanes, Reference Hillyer and Soler De Galanes1988, Reference Hillyer and Soler de Galanes1991; Hillyer et al. Reference Hillyer, Soler de Galanes, Rodriguez-Perez, Bjorland, Silva de Lagrava, Guzman and Bryan1992; Bjorland et al. Reference Bjorland, Bryan, Strauss, Hillyer and McAuley1995; Rahimi et al. Reference Rahimi, Ashrafi, Koosha, Abdi and Rokni2011; Elsibaei et al. Reference Elsibaei, Ali, Ibrahim and Mohammed2013).

Among ELISA variant developments, four techniques merit mention because of their promising advantages, mainly to facilitate field work in community surveys in the cases of those tests having solved the problem of the usually low sensitivity of these techniques:

Micro-ELISA

This technique can be applied as a screening test when a large number of samples is involved, because of its low requirement of reagents (especially antigens and second-antibody conjugates). The conventional ELISA could be employed as a confirmatory test following micro-ELISA analysis (Daveau and Ambroise-Thomas, Reference Deveau and Ambroise-Thomas1982; Shaheen et al. Reference Shaheen, Kamal, Farid, Mansour, Boctor and Woody1989; Itoh and Sato, Reference Itoh and Sato1990; Kim et al. Reference Kim, Kim, Huh and Cho1995, Reference Kim, Lee, Yun, Kim, Choi, Oh, Song and Chu2010; Espino et al. Reference Espino, Padron, Dumenigo and Laferte1997; Carnevale et al. Reference Carnevale, Rodríguez, Santillán, Labbé, Cabrera, Bellegarde, Velásquez, Trgovcic and Guarnera2001; Dalimi et al. Reference Dalimi, Hadighi and Madani2004).

Dot-ELISA

The standard indirect ELISA format is less practical for field surveys and a dot-ELISA has been successfully applied for antibody screening (Shaheen et al. Reference Shaheen, Kamal, Farid, Mansour, Boctor and Woody1989, Reference Shaheen, Al Khafif, Farag and Kamal1994; Itoh and Sato, Reference Itoh and Sato1990). The dot-ELISA has some advantages over the indirect ELISA. Nitrocellulose papers spotted with antigen are stable for at least three months at −20 °C (Intapan et al. Reference Intapan, Maleewong, Nateeworanart, Wongkham, Pipitgool, Sukolapong and Sangmaneedet2003), all incubation steps are performed at room temperature, and the results can be read with the naked eye, thus an expensive spectrophotometer is not required. The test is applicable to diagnose in the field setting as well as in laboratories that are not well equipped. The dot–ELISA is simple and allows testing of multiple samples at the same time (Shaheen et al. Reference Shaheen, Kamal, Farid, Mansour, Boctor and Woody1989, Reference Shaheen, Al Khafif, Farag and Kamal1994; Itoh and Sato, Reference Itoh and Sato1990; Hassan et al. Reference Hassan, Matar, Mowafy, Saleh, El-Badrawy, Awad and Ramadan2002; Intapan et al. Reference Intapan, Maleewong, Nateeworanart, Wongkham, Pipitgool, Sukolapong and Sangmaneedet2003; Dalimi et al. Reference Dalimi, Hadighi and Madani2004; Maeda et al. Reference Maeda, Yamada, Akao, Iga, Endo, Koibuchi, Nakamura, Odawara, Iwamoto and Fujii2008).

Dipstick

Dipsticks require no expensive instruments and fewer reagents in comparison to other techniques for diagnosis. Moreover, dipstick assay preparation takes only 2 h compared with the long incubation period of ELISA, and the assay can be completed in 15 min with results obtained visually. Dipstick is a simple antigen and serum test requiring only micrograms of parasite antigen and 20 μL of the patient's serum. This test can be carried out while patients wait. Additionally, patients can see the results themselves, which may increase compliance rates (Al-Sherbiny et al. Reference Al-Sherbiny, Farrag, Fayad, Makled, Tawfeek and Ali2004). From an epidemiological point of view, a dipstick assay allows control measures to be implemented in situ. Dipsticks have the merit of allowing for screening of large numbers of blood samples. They also allow for an indirect although not specific measure of burden through determination of the end-point titration of an individual's serum. Antigens kept frozen on nitrocellulose strips at −20 °C retain antigenicity for at least 3 months (Pappas, Reference Pappas1988; Intapan et al. Reference Intapan, Maleewong, Nateeworanart, Wongkham, Pipitgool, Sukolapong and Sangmaneedet2003) and, hence, they are convenient for rapid, routine laboratory use. A dipstick assay may thus be valuable for the serodiagnosis of human fascioliasis thereby saving time and eliminating the need for special training. Such a rapid test could therefore be practically implemented in epidemiological surveys. A sensitivity of 100% and a specificity of 96·7% have been preliminarily noted (Ali, Reference Ali2012), although appropriate independent complete evaluation is still pending.

Lateral flow immunoassays or immunochromatographic tests

A new rapid lateral flow immunoassay (LFIA) test (SeroFluke) for human diagnosis in both invasive and biliary periods of the disease appears to be a useful step forward (Martinez-Sernandez et al. Reference Martinez-Sernandez, Muiño, Perteguer, Garate, Mezo, Gonzalez-Warleta, Muro, Correia da Costa, Romaris and Ubeira2011). This test was constructed with a recombinant cathepsin L1 from F. hepatica, and uses protein A and mAb MM3 as detector reagents in the test and control lines, respectively. In comparison with an ELISA test (MM3-SERO), the SeroFluke test showed the advantage of being applicable to both serum or whole blood samples, although maximum specificity and sensitivity values are still pending field evaluation. Its simplicity allows it to be used in major hospitals as well as in endemic/hyperendemic regions where point-of-care testing is required. The SeroFluke test requires no expensive instruments and fewer reagents in comparison to other techniques for diagnosis. The swiftness of LFIA tests make them suitable for endemic rural areas and district hospitals. Moreover, the SeroFluke test can be used by personnel with minimal training, and more importantly, it can be used for point-of-care testing, and thus is particularly useful in several countries where human fascioliasis is endemic. In such countries, the possibility of using whole blood samples for the SeroFluke test (obtained by e.g. taking a drop of blood by fingertip puncture with a lancet or even collected on filter paper) is also advantageous, as this saves the time and expense involved in obtaining serum samples. However, even considering only major laboratories in hypoendemic countries, the new LFIA may offer advantages over ELISA methods, as it allows individual testing of patients, whereas ELISA methods are designed to test several patients at once and some robustness may be lost when they are used only sporadically (Corstjens et al. Reference Corstjens, van Lieshout, Zuiderwijk, Kornelis, Tanke, Deelder and van Dam2008). Furthermore, the LFIAs can be produced in small quantities (e.g. in the range of 10–25 tests), which would reduce the costs associated with the shelf-life of the kits.

Antigens for circulating antibody detection

In animal models, the specificity of antibody responses to Fasciola varies along the course of infection (Bennett et al. Reference Bennett, Joshua and Hughes1982). Hitherto, several antigen groups have been described (whole-worm extract, ES products, extracted proteins from the adult fluke surface and recombinant proteins) and numerous test systems have been developed for the efficient diagnosis of human fascioliasis. Nevertheless, the use of complex antigens reduces the specificity of the tests due to cross-reactivity with parasites sharing similar immunogens (Hillyer and Serrano, Reference Hillyer and Serrano1983). Currently, the conventional use of complex mixtures of antigens such as ES products is being replaced by defined and purified antigens to minimize cross-reactions. However, the expression and purification of recombinant proteins is a tedious and time-consuming task, and this technology is not always accessible to all laboratories, especially to those in endemic areas of human fascioliasis and lacking adequate economic resources. Moreover, ES products are usually prepared using in vitro techniques that require a degree of sterility (Morales and Espino, Reference Morales and Espino2012).

Although a great deal of progress has been made with regard to immunodiagnosis of human fascioliasis, the persistent problem of selected antigens required for the development of efficient diagnostic methods, together with further evaluations on their sensitivity and specificity, is yet to be solved. The main problems of antibody-detecting techniques lie in their sensitivity (false serological negatives) and specificity (cross-reactions), which are dependent on the antigen type, and the lack of consensus concerning the optimal antigen to be used in the different tests (Stork et al. Reference Stork, Venables, Jennings, Beesley, Bendezu and Capron1973; Chen and Mott, Reference Chen and Mott1990; Hillyer, Reference Hillyer1993; Sampaio Silva et al. Reference Sampaio-Silva, Da Costa, Da Costa, Pires, Lopes, Castro and Monjour1996; Valero et al. Reference Valero, Periago, Perez-Crespo, Rodriguez, Perteguer, Garate, Gonzalez-Barbera and Mas-Coma2012b).

Earlier works on ELISA for the immunodiagnosis of human fascioliasis have been reviewed (Hillyer, Reference Hillyer1993, Reference Hillyer and Dalton1999). Specific ELISA methods developed using adult fluke whole worm crude extracts provide less than optimal specificity, as they may easily present cross-reactions with other helminthic infections. The use of partially purified somatic antigen from adult worms and excretory/secretory (E/S) products, among which cysteine proteinases are the most abundant, has improved the sensitivity and specificity of the tests significantly.

Purified antigens from ES products, including cysteine proteinases, among which mainly papain-like cysteine endopeptidases termed cathepsins, mainly cathepsin L1 (CL1), should be highlighted. The use of an ELISA with purified cathepsin antigen provides high sensitivity and higher specificity than tests using parasite somatic or ES antigens (O'Neill et al. Reference O'Neill, Parkinson, Strauss, Angles and Dalton1998, Reference O'Neill, Parkinson, Dowd, Strauss, Angles and Dalton1999; Rokni et al. Reference Rokni, Massoud, O'Neill, Parkinson and Dalton2002; Espinoza et al. Reference Espinoza, Timoteo and Herrera-Velit2005). Recombinant cathepsins overcome the problem of the complex and time-consuming production of sufficient quantities of pure cathepsin, and display similar performance characteristics to native antigens (Mezo et al. Reference Mezo, Gonzalez-Warleta, Carro and Ubeira2004, Reference Mezo, Gonzalez-Warleta and Ubeira2007, Reference Mezo, Gonzalez-Warleta, Castro-Hermida, Muiño and Ubeira2010; Valero et al. Reference Valero, Perez-Crespo, Periago, Khoubbane and Mas-Coma2009a) and also demonstrate their usefulness for human diagnosis (Martinez-Sernandez et al. Reference Martinez-Sernandez, Muiño, Perteguer, Garate, Mezo, Gonzalez-Warleta, Muro, Correia da Costa, Romaris and Ubeira2011). Good results have also been obtained with two B-cell epitopes of cathepsin L1 (Cornelissen et al. Reference Cornelissen, Gaasenbeek, Boersma, Borgsteede and van Milligen1999, Reference Cornelissen, Gaasenbeek, Borgsteede, Holland, Harmsen and Boersma2001), synthesized as single synthetic peptides (Intapan et al. Reference Intapan, Tantrawatpan, Maleewong, Wongkham, Wongkham and Nakashima2005; Tantrawatpan et al. Reference Tantrawatpan, Maleewong, Wongkham, Wongkham, Intapan and Nakashima2007).

Interesting results have also been obtained in attempts at utilizing both native and recombinant fatty-acid binding proteins (FABPs) (Sirisriro et al. Reference Sirisriro, Grams, Vichasri-Grams, Ardseungneon, Pankao, Meepool, Chaithirayanon, Viyanant, Tan-Ariya, Upatham and Sobhon2002), among which one has recently shown to be a valuable antigen for human fascioliasis diagnosis (Allam et al. Reference Allam, Bauomy, Hemyeda and Sakran2012). Other strategies include the use of the putative diagnostic value of leucine aminopeptidase (LAP) (Piacenza et al. Reference Piacenza, Acosta, Basmadjian, Dalton and Carmona1999) and recombinant saposin-like proteins (SAPLIPs) already shown to be good antigens for antibody detection in chronic human fascioliasis (Figueroa-Santiago et al. Reference Figueroa-Santiago, Delgado and Espino2011). Finally, the use of specific immunoreactive components of F. hepatica adult fluke surface (FhTA) has been emphasized because the preparation of FhTA is simpler and faster and does not require particular expertise or advanced technology (Morales and Espino, Reference Morales and Espino2012).

The antibody isotype profile

The antibody isotype profile varies depending on the characteristics of the immunogen and other factors such as cytokines. In natural infection with parasites, the host is dealing with a multiplicity of different antigens, resulting in polyspecific responses. In human sera, the predominant antibodies of isotypic responses are IgG1 and IgG4 against ES products, crude whole-worm extract, cathepsin-L1 and liver fluke tegumental extract (O'Neill et al. Reference O'Neill, Parkinson, Strauss, Angles and Dalton1998; Maher et al. Reference Maher, El Ridi, Elhoda, El-Ghannam, Shaheen, Shaker and Hassanein1999; Wongkham et al. Reference Wongkham, Tantrawatpan, Intapan, Maleewong, Wongkham and Nakashima2005; Morales and Espino, Reference Morales and Espino2012).

The human fascioliasis serodiagnostic capability has been increased by the detection of the IgG4 subclass antibody. Employing anti-IgG4 secondary antibodies led to an improved discrimination between seropositives and seronegatives compared with anti-total IgG using F. hepatica native CL1 as antigen, in samples obtained from the field in a blind study using Bolivian volunteers who undoubtedly harboured different intensities of infection where the cut-off is more difficult to determine (O'Neill et al. Reference O'Neill, Parkinson, Strauss, Angles and Dalton1998).

Opposite results were also found in clinically diagnosed fascioliasis patients with presumably high-level and long-term infection (Gonzales Santana et al. Reference Gonzales Santana, Dalton, Vasquez Camargo, Parkinson and Ndao2013), and may be explained by the fact that helminthic infections increase anti-IgG4 antibodies in correlation with intensity. The clear distinction between Fasciola positive/negative allowed for a robust calculation of the cut-off line. Different results may perhaps be obtained depending on the population of subjects examined. Nevertheless, both studies showed that using anti-total IgG provides sufficiently accurate results for it to be considered the most optimal secondary antibody (O'Neill et al. Reference O'Neill, Parkinson, Strauss, Angles and Dalton1998; Gonzales Santana et al. Reference Gonzales Santana, Dalton, Vasquez Camargo, Parkinson and Ndao2013).