**6. Future prospects**

The number of recombinant proteins assessed and proposed as candidates to be used as tools for *T. cruzi* infection diagnosis is quite high. However, there is no serum panel to be used as international reference. Therefore, the reports on results produced by using these proteins can hardly be compared. Usually, serum panels, previously typified with other validated methods are used to appraise new serology reagents. These already standardized serological reagents have their own sensitivity and specificity, which may lead to a bias when typifying the panel.

Currently, several diagnostic tests have been proposed as the reference one, such as immunofluorescence or different versions of Western blot.(Otani *et al.*, 2009;Caballero *et al.*, 2007) However, no consensus exists among researchers and regulatory agencies on which tests are preferable.

Another inconvenience is that there is evidence on the absence of humoral response in some patients of endemic regions, whose infection was proved by parasitological techniques or by tests evaluating the cellular immune response.(Salomone *et al.*, 2003;Olivera *et al.*, 2010) These reports alert us on a potential overestimation of the sensitivity and specificity underestimation of the immunochemical assays when they are assessed with serum panels typified by conventional serology.

Conventional serology is still a crucial tool to diagnose the different entities studied during the chronic phase of the infection. The main multicenter studies carried out in regions where leishmaniasis is not endemic have shown that ELISAs using both parasite extractive antigens, as well as the recombinant ones, display optimal sensitivity and specificity. Although both kind of antigens perform similarly in those leishmaniasis-free places, when determinations are carried out where Chagas' disease and leishmaniasis are co-endemic, ELISAs using recombinant proteins have demonstrated to be the most useful.

In the same line, presently, several authors who have evaluated ELISA commercial kits with plaques sensitized with parasite homogenate or with recombinant antigens, have shown that it is not mandatory to carry out 2 different tests. Certainly, when performing only one ELISA, it is feasible to diagnose the infection, if the result is positive. This is acknowledged because results obtained with both kinds of ELISA correlate appropriately. (Remesar *et al.*, 2009;Otani *et al.*, 2009) However, considering the poor sensitivity of IHA, this latter one would not be recommended as a second test. (Remesar *et al.*, 2009)

Even though some recombinant proteins have been used to monitor Chagas´ disease treatment, it could not be still demonstrated that these proteins give diagnostic information to evaluate cardiopathy diagnosis and prognosis. During recent years, the description of the whole genome of *T. cruzi* has prompt to systematically analyze new antigens, some of which have been described as putative antigens, but has not yet confirmed. This is being evaluated nowadays by different research groups which, it is expected will suggest new interesting markers that are useful for cure monitoring and cardiopathy prognosis.

Lately, several research works on infection diagnostic tools have reported on the development of latex particle agglutination and amperometric biosensors to diagnose *T. cruzi* infection.(Gonzalez *et al.*, 2010;Belluzo *et al.*, 2011;Ribone *et al.*, 2006) Latex particle vs. conventional agglutination has the advantage of allowing particle sensitization with recombinant proteins, what leads to a more reproducible, standardized reagents production.(Gonzalez *et al.*, 2010) Biosensors technology admits reutilization of the device, potentially yielding to automation, thus facilitating laboratory operation. Moreover, the simplicity of the equipment required, permits the analysis to be performed in the field, which is an important attribute because infected people generally live in the countryside and do not attend health centers.(Belluzo *et al.*, 2011) The electrochemical biosensor technology developed follows the same ELISA format, exchanging the colorimetric signal readout by the amperometric one.(Belluzo *et al.*, 2011) Although no commercial device is yet available, the results of our studies are quite promising. This methodology could allow reducing costs and time of analysis in the near future, keeping the same or even higher standards of sensitivity and specificity than ELISA.(Belluzo *et al.*, 2011)

### **7. References**

288 Current Topics in Tropical Medicine

stage than in sera from symptomatic individuals.(Breniere *et al.*, 2002) In a longitudinal evaluation of asymptomatic and cardiac groups of patients, we described that only the individuals who evolved to a more severe clinical status increased specific anti-TcP2 concentration in late stages of the infection.(Fabbro *et al.*, 2011) However, the transversal comparison of the sera from patients with and without cardiopathy revealed that anti-TcP2 concentration between both groups was not significantly different. The discrepant results mentioned above show that it is not still clear if anti-TcP2 can be used as a serological

Also, muscarinic acetylcholine receptor subtype II, in this case a host antigen, has shown to be quite auspicious to monitor the chronic infection.(Goin *et al.*, 1999) Nevertheless, recent studies suggest that it is not apparent that this protein is useful to discriminate between

The difficulties to establish clear illness evolution markers lead to the present state, where

The number of recombinant proteins assessed and proposed as candidates to be used as tools for *T. cruzi* infection diagnosis is quite high. However, there is no serum panel to be used as international reference. Therefore, the reports on results produced by using these proteins can hardly be compared. Usually, serum panels, previously typified with other validated methods are used to appraise new serology reagents. These already standardized serological reagents have their own sensitivity and specificity, which may lead to a bias

Currently, several diagnostic tests have been proposed as the reference one, such as immunofluorescence or different versions of Western blot.(Otani *et al.*, 2009;Caballero *et al.*, 2007) However, no consensus exists among researchers and regulatory agencies on which

Another inconvenience is that there is evidence on the absence of humoral response in some patients of endemic regions, whose infection was proved by parasitological techniques or by tests evaluating the cellular immune response.(Salomone *et al.*, 2003;Olivera *et al.*, 2010) These reports alert us on a potential overestimation of the sensitivity and specificity underestimation of the immunochemical assays when they are assessed with serum panels

Conventional serology is still a crucial tool to diagnose the different entities studied during the chronic phase of the infection. The main multicenter studies carried out in regions where leishmaniasis is not endemic have shown that ELISAs using both parasite extractive antigens, as well as the recombinant ones, display optimal sensitivity and specificity. Although both kind of antigens perform similarly in those leishmaniasis-free places, when determinations are carried out where Chagas' disease and leishmaniasis are co-endemic,

In the same line, presently, several authors who have evaluated ELISA commercial kits with plaques sensitized with parasite homogenate or with recombinant antigens, have shown that it is not mandatory to carry out 2 different tests. Certainly, when performing only one ELISA, it is feasible to diagnose the infection, if the result is positive. This is acknowledged because results obtained with both kinds of ELISA correlate appropriately. (Remesar *et al.*, 2009;Otani *et al.*, 2009) However, considering the poor sensitivity of IHA, this latter one

ELISAs using recombinant proteins have demonstrated to be the most useful.

would not be recommended as a second test. (Remesar *et al.*, 2009)

different stages of the illness.(Tovar *et al.*, 2009;Talvani *et al.*, 2006)

we do not count yet with useful tools to evaluate Chagas' disease prognosis.

marker of myocardic damage.

**6. Future prospects** 

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**18** 

*Spain* 

**Echinococcosis/Hydatidosis** 

*University Hospital Joan XXIII of Tarragona* 

Antoni Soriano Arandes and Frederic Gómez Bertomeu

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

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

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 first part of this chapter 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

echinococcosis (PE) caused by *Echinococcus vogeli* or *Echinococcus oligarthrus*.

reported to the European Food Safety Authority (EFSA).

the redaction of this chapter.

**2. Echinococcosis** 

**1. Introduction** 

