Advances in Serological Diagnosis of Chagas ’ Disease by Using Recombinant Proteins

Chagas’ disease is an infection caused by the parasite Trypanosoma cruzi, mainly occurring in American countries where the parasite vector bug, Triatoma infestans, is widespread. One hundred million individuals are currently under threaten of infection, as well as 16 million people are considered affected by the illness in Latin America alone.(Editorial, 2009) Considering indicators such as the disability-adjusted life years, DALY, and from a social point of view, Chagas’ disease accounts for the third most important tropical illness of the World, following malaria and schistosomiasis.(Bitran et al., 2009) Moreover, Chagas disease epidemiology nowadays impacts in non-endemic regions due to globalization, being the infection disseminated all over the world (Gascon et al. 2009). Certainly, the non-vectorial disease transmission (mother to child, transfusional and by organ transplantation) is the way the illness spreads in Europe, North America, Japan, and Australia, because many infected people have migrated from endemic regions to distant cities. (Bowling & Walter, 2009; Lescure et al., 2009; Yadon & Schmunis, 2009) The parasite distribution and living habits of rural Latin-American people have determined that the main transmission route is the vectorial one (via Triatoma infestans), leading to up 80% of human infection.(WHO, 2003) The main contagion way in urban areas arise from blood-transfusion, being responsible for 5-20% of the reported cases, while vertical transmission (mother-to-child) accounts for 2-10% of the infections.(Carlier & Torrico, 2003) The infection transmission by oral route because of consumption of contaminated food and drinks is lower than that reported for the previously-mentioned routes.(Dias et al., 2011) However, it has to be taken into account that the success of the non-oral infection prevention has increased the importance of the oral route of transmission.(Dias et al., 2011; Nóbrega et al., 2009) It is noteworthy that the acute outcome of the oral infection is particularly severe.(Bastos et al 2010). From the clinical point of view, the illness presents variable unspecific symptoms, depending on its stage: the acute one, shortly after primary infection, and the chronic stage, which may last many years if the individual is not treated. Human infection typically


Introduction
Chagas' disease is an infection caused by the parasite Trypanosoma cruzi, mainly occurring in American countries where the parasite vector bug, Triatoma infestans, is widespread.One hundred million individuals are currently under threaten of infection, as well as 16 million people are considered affected by the illness in Latin America alone.(Editorial, 2009) Considering indicators such as the disability-adjusted life years, DALY, and from a social point of view, Chagas' disease accounts for the third most important tropical illness of the World, following malaria and schistosomiasis.(Bitran et al., 2009) Moreover, Chagas disease epidemiology nowadays impacts in non-endemic regions due to globalization, being the infection disseminated all over the world (Gascon et al. 2009).Certainly, the non-vectorial disease transmission (mother to child, transfusional and by organ transplantation) is the way the illness spreads in Europe, North America, Japan, and Australia, because many infected people have migrated from endemic regions to distant cities. (Bowling & Walter, 2009;Lescure et al., 2009;Yadon & Schmunis, 2009) The parasite distribution and living habits of rural Latin-American people have determined that the main transmission route is the vectorial one (via Triatoma infestans), leading to up 80% of human infection.(WHO, 2003) The main contagion way in urban areas arise from blood-transfusion, being responsible for 5-20% of the reported cases, while vertical transmission (mother-to-child) accounts for 2-10% of the infections.(Carlier & Torrico, 2003) The infection transmission by oral route because of consumption of contaminated food and drinks is lower than that reported for the previously-mentioned routes.(Dias et al., 2011) However, it has to be taken into account that the success of the non-oral infection prevention has increased the importance of the oral route of transmission.(Dias et al., 2011;Nóbrega et al., 2009) It is noteworthy that the acute outcome of the oral infection is particularly severe.(Bastos et al 2010).From the clinical point of view, the illness presents variable unspecific symptoms, depending on its stage: the acute one, shortly after primary infection, and the chronic stage, which may last many years if the individual is not treated.Human infection typically performed parasitologic analysis in adults since more than 50 years, (Strout, 1962) and it has been reported about 95% sensitivity.(Freilij & Storino, 1994) The other commonly used concentration method is the pediatric, more recent version, named the direct micromethod or microhematocrit, which requires a lower blood volume than Strout method, and is mainly used to diagnose congenital Chagas' disease and acute infection in children.(Freilij et al., 1983;Freilij & Altcheh, 1995) However, newborn babies usually present low parasitemia, therefore making difficult to perform a proper conventional parasitologic analysis.It is then recommended to perform serologic tests to diagnose the congenital infection.The evaluation of specific anti-T.cruzi IgA and IgM is not recommended due to the high rate of false-negative results in neonates.(Moya et al., 2005) Considering that maternal specific anti-T.cruzi IgG antibodies are commonly present in newborn circulating blood, even up to the ninth month, it is not advisable to perform serologic IgG determinations as routine, in newborns younger than 9 months old.In this line, if the micromethod is negative or if it has not been performed during the first months of life of the newborn, then congenital infection should be serologically diagnosed using peripheral blood not before the child is 9 months old, once maternal antibodies have disappeared.(Gomes et al., 2009) Following, when specific IgG presence is negative after the ninth month of life, then vertical transmission is ruled out.Alternatively, during the first months of life of babies, it is possible to forego results using other non-standard, more expensive techniques such as the polymerase chain reaction, PCR.(Diez et al., 2008) This technology is particularly preferred when the health center counts with the supplies to carry out the methodology.Indirect parasitological methods are also used, mainly when the parasite is not easily found in samples.These methods are the hemoculture and xenodiagnosis, and consist of enriching the parasites present in the patient's blood sample, through allowing their replication.(Chiari et al., 1989) Both of these latter techniques are also used when diagnosing chronic infection.These methods demand long periods of time to arise to the results (weeks or months), together with other drawbacks.For example, xenodiagnostic method has the disadvantage of producing rather variable sensitivity results, 20-50%, alongside the requirement of a suitable building infrastructure and trained personnel to deal with insect breeding.Thus, this method is not commonly performed in basic health centers.(Luquetti & Schmunis, 2010) When searching for reappearance of acute infection in immunosuppressed individuals under risk, negative serological results are not always associated with absence of the infection.This is a consequence of the immunological status of the patient that shows difficulties to produce detectable amounts of specific IgG.As mentioned previously, in the particular, difficult cases, expensive PCR techniques are the recommended diagnostic method.

Serological diagnosis of chronic entities
The widely used serological assays to diagnose T. cruzi infection in present clinical practice are indirect haemagglutination (IHA), indirect immunofluorescence (IIF), and enzymelinked immunosorbent assay (ELISA).(WHO, 2002;Yadon & Schmunis, 2009) The analyst's choice of the particular technique depends on sanitary-authority recommendations, market impositions, and the lab-technician preference.This latter one is generally related to the methodology simplicity, and the personal confidence he/she has in a particular technique after having performed it for a long while.IHA is an inexpensive technique, which is easy to be performed and interpreted, and it has been used for more than 50 years, therefore being appropriately settled among lab technicians.Similarly, IIF was developed in the sixties and presents equivalent features to IHA though, more skillful technicians are required to perform the analysis and produce accurate readings, as well as it needs a fluorescence microscope.IIF is a very sensitive, specific and cheap alternative for those who have the equipment and the trained personnel.However, regular health centers do not count with both of them.ELISA is a more recent technique which was firstly described during 1975 to diagnose Chagas' disease, (Voller et al., 1975) and its usage was settled just at the ends of the eighties.This technique has the advantage of being widely extended as a diagnostic tool of many infections.Therefore, most of regular laboratories have the required equipment and trained personnel to appropriately perform the analysis.Contrarily to IHA and IIF, ELISA may be performed with automatic equipment at large health institutions.Moreover, even though ELISA is more expensive than the other two techniques, its notable performance in terms of sensitivity and specificity, has made of this the preferred methodology to diagnose T. cruzi infection.(Saez-Alquezar et al., 1997) Recently, one very fast technique with a different format has been developed namely, lateral chromatography.(Ponce et al., 2005;Barfield et al., 2011) This methodology uses small volume samples such as one serum drop, and allows acquiring results in 15 min, therefore being useful to perform the test in the field, without the need of refrigerator to preserve the reagents.Several multicenter studies have demonstrated that a commercial lateral chromatography kit show more than 92% sensitivity, whereas specificity is ca.96%.(Ponce et al., 2005;Brutus et al., 2008;Roddy et al., 2008;Sosa-Estani et al., 2008) The fundamental problems of T. cruzi infection serological diagnostic methods are the lack of reproducibility that sometimes occur, deficient immunological reaction specificity, what produces false-positive results, and the occasional insufficient sensitivity translated into false-negative outcomes.Chagasic infection serology tests may produce cross-reactions with antibodies produced during the course of other illnesses.In this line, unspecific reactivity has been described for infections caused by T. cruzi phylogenetically related microorganisms, such as T. rangeli and Leishmania sp.(Soto et al., 1996;Araujo, 1986;Saez-Alquezar et al., 2000) Moreover, other falsepositive results due to cross-reactions have been described when testing samples from patients with autoimmune diseases, (Reiche et al., 1996) or from individuals suffering from other acute infections or pregnant women who display an important, polyclonal unspecific response.(Konishi, 1993) The clinical practice often finds an important number of inconsistent results regarding reproducibility and confidence when diagnosing chagasic infection.The lack of reproducibility and confident results has also been reported in a multicenter study.(Saez-Alquezar et al., 1997) I n t h i s w o r k , i t w a s p r o v e d t h e d e f i c i e n c y of reagents standardization, what produced incongruent results when testing the same serum panel.Along the same direction, since the early nineties, several works accounted for the huge losses caused by disposal of whole blood reservoirs typified as undetermined for T. cruzi infection.(Carvalho et al., 1993;Salles et al., 1996;Saez-Alquezar et al., 2000) Taking into account tests discrepancies, one of WHO recommendations states that T. cruzi infection must be diagnosed when the sample produces positive results by two different serological methods, whereas the undetermined condition is established for samples rendering dissimilar outcomes.Traditionally, whole parasites, or extracts from laboratory strains of T. cruzi epimastigotes cultures, have been the source of antigens used for the serological infection diagnosis.However, this yields to complex protein mixtures of unknown composition, which display severe difficulties to be standardized, and additionally lead to false-positive results.The diagnostic problems arising from serology deficient specificity, as well as the deprived reagents standardization, can be resolved through the use of defined antigens, such as the proteins obtained by molecular biology technology procedures.(Saez-Alquezar et al., 2000;Umezawa et al., 2003;Umezawa et al., 2004;Aguirre et al., 2006) The following sections will be focused in this issue and the most important contributions that several research groups have recently made.

Use and prospects of recombinant DNA technology
Since the emergence of recombinant DNA technology, many protein molecules have been designed and prepared to eventually be assessed for serological diagnosis.The proteins obtained through this technology may be used as antigens to capture antibodies, to evaluate exclusively defined molecules, avoiding potential interferences from other components that usually occur when the antigens have been obtained by purifying native source proteins.(da Silveira et al., 2001) It follows that the usage of recombinant proteins as antigens to detect or quantify specific antibodies markers of a disease permits enhancing the specificity of the immunological reaction involved, therefore leading to more accurate diagnosis.(Aguirre et al., 2006;Camussone et al., 2009) In this methodology the proteins are usually prepared by heterologous expression, mainly in Escherichia coli cells.(da Silveira et al., 2001) Sequences of T. cruzi-protein codifying-DNA are inserted in a bacterial plasmid, which is transformed in competent bacteria.The proteins encoded by the plasmid are expressed in the bacterial culture, and are afterward purified into a highly pure product.The advantage of the proteins thus obtained is that they are an entirely characterized antigen, which may be evaluated individually for antibody determination in different clinical conditions.The prepared antigens can therefore be characterized considering the clinical information they provide, and may then be used to prepare specific diagnostic reagents.These proteins count with one desired feature of diagnostic reagents, as it is that their production and evaluation can be highly standardized.From another point of view, recombinant antigens do not require manipulation of the infective agent as do the antigens obtained by purification procedures from rough cultures.This has been a significant progress when considering the characteristics of viral infective agents, for which reagents production has substantially switched to that derived from recombinant DNA technology.Not less important is the major saving financial benefit of these reagents.Indeed, once bacteria are transformed into competent, protein producing strain, they can be used to prepare substantial amounts of antigen with low cost of production.Using this technology, many gene expression clones have been create, a fact that has made available the obtainment of massive amounts of highly pure, standardized T. cruzi proteins.(da Silveira et al., 2001) During the latest three decades, many parasite antigens have been cloned and characterized.The cloned antigens correspond to different parasite stages namely, the trypomastigote sanguineous, the amastigote intracellular and the epimastigote, which is the form found inside the vector bowel and that can be cultured.Several of these antigens were obtained by immunological tracing through expression of cDNA libraries from chagasic patient sera, as well as from immunized animals.(Lafaille et al., 1989;Affranchino et al., 1989;Levin et al., 1989;Cotrim et al., 1990;Gruber & Zingales, 1993) The antigen codifying genes have been identified from cDNA present in the libraries accomplished from epimastigote or trypomastigote forms.(Affranchino et al., 1989;Levin et al., 1989;Gruber & Zingales, 1993;Godsel et al., 1995) Lately, Da Rocha et al. have proposed using amastigote proteins since this is the intracellular parasite form, being these antigens more significant for serodiagnosis.(DaRocha et al., 2002) The usage of DNA technology brought into light the existence of many parasite antigens with repetitive sequences, a fact that had been previously described when cloning proteins of other parasites.(Hoft et al., 1989) Generally, these are the most immunogenic antigens, and are the mainly selected when performing immunological tracing in cDNA libraries cloned in phages.Therefore, it was initially stated that these were the most valuable antigens for diagnosis.(Frasch & Reyes, 1990) However, it was afterward proved that some nonrepetitive antigens have equivalent diagnostic value than repetitive ones.Certainly, a multicenter study evaluated in parallel 4 repetitive recombinants antigens (H49, JL7, B13, JL8) together with 2 non-repetitive ones (A13 y 1F8).(Umezawa et al., 1999) The results demonstrated that both type of antigens were similarly useful for T. cruzi infection diagnosis, and the authors suggested that if they were to be used together in a mixture, they could supplemented each other enhancing the sensitivity of the assay.This was afterwards proved by the same group, see Tables 1 A,B and C. (Umezawa et al., 2003) Once the complete genome sequence of Trypanosoma cruzi was annotated, (El Sayed et al., 2005) alternative antigenic candidates have been searched in the parasite genome.The studies have been supported by bioinformatic prediction of putative proteins and antigenicity predictors.(Goto et al., 2008;Cooley et al., 2008;Hernandez et al., 2010) Using these tools, it has been possible to choose antigens which display the lowest homology level with proteins of organisms related to T. cruzi.(Hernandez et al., 2010) Moreover, the bioinformatic analysis has allowed describing for the first time a specific antigen to type discrete typing units (DTUs).(Di Noia et al., 2002) The results published by many different laboratories point towards considering recombinant proteins as the chosen molecules to be used in immunoassays to diagnose T. cruzi infection.Moreover, the lack of specificity leading to false-positive results can be overcome by deleting sequence regions encoding for proteins which cross-react when analyzing negative sera, (Aguirre et al., 2006), or using recombinant proteins that are specific for anti-T.cruzi antibodies, yet keeping a high sensitivity.(Belluzo et al., 2011;Camussone et al., 2009) Indeed, the largest studies on the diagnosis reveal the convenience of using these antigens, regarding not only specificity but also the possibility of standardizing both, the methodology and the protein production.(Umezawa et al., 1999;Saez-Alquezar et al., 2000;Umezawa et al., 2003) The following table display the key recombinant antigens discussed in the present chapter, which were evaluated by several authors for T. cruzi infection diagnosis.Notice, that many of these antigens particularly named by one author have amino acid sequences, which may be very similar to those obtained by other authors who have named them differently (e.g.FRA, Ag1, JL7, H49).Identical or highly similar antigens were grouped in the same row.

Recombinant proteins use: Mixtures vs. fusion proteins
The first works dealing with a single recombinant protein for diagnostic purposes reported lack of sensitivity when using only one of those antigens.(Levin et al., 1991;Moncayo & Luquetti, 1990;Peralta et al., 1994) Consequently, most of these proteins have been evaluated not only alone and independently from others, but also together as part of mixtures or as fusion proteins, carrying several recombinant epitopes.(Umezawa et al., 1999;Umezawa et al., 2004;Camussone et al., 2009;Foti et al., 2009) Accordingly, a multicenter study evaluating 6 recombinant proteins separately with a serum panel composed by sera from patients of several countries, described that using the set of results of the 6 proteins together had yield a sensitivity and specificity compatible with the reference assays.(Umezawa et al., 1999) Later, the same group evaluated the mixture of the 6 proteins, supporting the use of the mixture to reach the same sensitivity and specificity.(Umezawa et al., 2003) Soon after, the reactivity of individual antigens vs. antigen mixtures was systematically assessed by ELISA.(Umezawa et al., 2004) This study confirmed that the results obtained with recombinant protein mixtures led to higher media values of optical densities, ODs, than the results produced when using the individual recombinant proteins.Moreover, sera rendering low ODs when examined with individual recombinant proteins produced higher ODs outcomes when using the protein mixtures.Along with this, several commercial ELISA kits with recombinant protein mixtures display equivalent or even higher sensitivities and specificities than those produced by kits with total parasite homogenate.(Gadelha et al., 2003;Pirard et al., 2005;Remesar et al., 2009;Caballero et al., 2007) These works have studied kits using Ag1, Ag2, Ag30, Ag13 together with Ag36 recombinant antigens (Chagatest Rec from Wiener lab, Argentina), and FRA and CRA recombinant antigens (Biomanguinhos, Friocruz, Brazil).However, another study reported that Chagatest Rec v3.0 (Wiener) displayed a rather low 95% sensitivity.(Ramirez et al., 2009) One of the strategies proposed to enhance reagents production standardization is to obtain multiepitope molecules, designed as a unique construction by fusing several relevant diagnostic antigens.(Houghton et al., 1999;Aguirre et al., 2006;Camussone et al., 2009) It has recently been proved that when using these constructions, the ODs of sera with low reactivity increases, as well as it had been reported for mixtures.(Camussone et al., 2009) Moreover, by this approach the attachment of the antigen turned out to be homogenous and reproducible when using different surfaces such as ELISA plaques, latex particles or bioelectrodes.(Camussone et al., 2009;Gonzalez et al., 2010;Belluzo et al., 2011) It has been proposed that when there is only one molecule exposed to the surface, competition for the active sites is prevented, therefore resulting in a uniform attachment.Furthermore, sensitivity may be increased because a higher number of freely accessible epitopes are available to capture the antibodies present in samples, as depicted in Fig. 1. (Camussone et al., 2009) A few articles report on the use of this strategy to produce commercial ELISA kits which have demonstrated to be highly satisfying.One of these works, analyzes the performance of the TcF antigen, previously described by Houghton et al in 1999, with which the Biolab Merieux reagent was prepared.(Ferreira et al., 2001) In this case, the recombinant protein used bears the PEP2, TcD, TcE and TcLo1.2 peptides.Recently, Abbot Laboratories have presented a new kit which uses a 4-antigen multiepitope protein containing TcF, FP3 -built up with TcR27 and FcaBP-, FP6 -with TcR39 and FRA-and FP10 -with SAPA and MAP-.(Praast et al., 2011) According to the authors, this kit performed even better than the Biolab Merieux one.

Acute phase diagnosis
As previously mentioned, during acute infection, serological methods are poorly sensitive.In the case of immunosuppressed individuals, as they do not develop an appropriate humoral immunological response, the direct method or molecular techniques are advisable.Serology is pertinent for in the neonatal congenital diagnostic when microhematocrit repeatedly shows negative results, or when diagnosis has not been made during the first month of life of the newborn.In these cases, it is necessary to serologically monitor congenital infection between 6 to 9 months, no matter if conventional reagents or recombinant ones are used.Nevertheless, the search for antibodies which are usually triggered during acute phase could enhance results.In the early nineties a shed acute phase antigen (SAPA) was proposed to discriminate between acute and chronic infection.(Reyes et al., 1990) This antigen was described when a panel of recombinant proteins obtained from a cDNA library was used to analyze the reactivity of IgG antibodies occurring in sera of chronic chagasic mothers and their newborns.The recognized IgGs against different T. cruzi antigens produced the same signal in sera from newborns and their respective mothers but SAPA antigen was recognized most frequently by antibodies from the infected newborns than it was by antibodies occurring in their mothers serum.Accordingly, the authors proposed it to be used to detect specific anti-T.cruzi IgG antibodies in neonates.Other works report that antibodies anti-SAPA allows the discrimination between acute and chronic T. cruzi infection because they were not present in the later stage of the infection.(Lorca et al., 1993) Nevertheless, later works described SAPA as reactive when assessed with sera from chronic infected individuals.(Breniere et al., 1997;Camussone et al., 2009) This apparent contradiction could be explained considering the significant differential reactivity of anti-SAPA antibodies generated during the different stages of the infection.Indeed, anti-SAPA antibodies are detected in almost all infected individuals but its reactivity is higher in the acute infection.It has recently been performed a study by following up of 2283 chagasic mothers, from which 209 transmitted the infection to the newborns.(Russomando et al., 2010) This work provides evidence on SAPA utility to serologically diagnose congenital infection before the third month of life, thus turning the protein into a promising inexpensive reagent to reduce the required time to detect the neonatal infection, and proceed to its early treatment.Although different reactivity patterns have been described in Western Blot assays which use native T. cruzi excretion antigens, to discriminate between acute and chronic infection (Umezawa et al., 1996) no other useful recombinant antigen different from SAPA has been described to diagnose the acute phase.

Chronic infection diagnosis
It has been already mentioned above that, when T. cruzi homogenate is used to perform ELISA tests, the assay sensitivity is high leading to a quite reliable result, therefore some authors have suggested that a single assay could be enough to test sera in blood banks.(Sosa Stani et al., 2008;Otani et al., 2009) Several multicenter studies carried out on samples from blood-banks, report that ELISA tests which use parasite homogenates perform similarly than those which used recombinant proteins.(Remesar et al., 2009;Otani et al., 2009) However, cross-reactivity of antibodies towards antigens from T. cruzi and Leishmania sp has been frequently informed, and can be explained considering the phylogenic proximity between both parasite species.(Chiller et al., 1990;Vexenat et al., 1996;Chiaramonte et al., 1996;Desquesnes et al., 2007;Aguirre et al., 2006) When sera from patients infected with Leishmania ssp parasites are included in the evaluations, specificity of recombinant proteins are higher (Umezawa et al., 1999;Ferreira et al., 2001;Aguirre et al., 2006;Caballero et al., 2007;Camussone et al., 2009) It has been recently reported that the antigen TSSA2 displays 87.8% sensitivity and 100% specificity to discriminate between chagasic and leishmaniasic patients.(Cimino et al., 2011) TSSA2 is the only reported recombinant antigen, which has displayed specificity to type T. cruzi genotypes DTUII, DTUV or DTUVI by specific antibodies from infected patients.(di Noia et al., 2002;Bhattacharyya et al., 2010) As these DTUs are those predominant in South America, the authors proposed using this antigen in confirmatory T. cruzi infection diagnostic tests, in regions which are co-endemic for both infections.It was also described that SAPA antigen could be specific and sensitive enough to be used when trying to distinguish between chronic T. cruzi and leishmaniasic infections, in regions where both illnesses are co-endemic.(Gil et al., 2011) It should be considered that it is difficult to discard T. cruzi infection in patients suffering from leishmaniasis, because these are co-endemic diseases.That is why antigens crossreactivity is normally assayed testing sera from patients who live in Chagas' disease nonendemic regions, because this allows ruling out T. cruzi infection from an epidemiological point of view.(Hernandez et al., 2010;Caballero et al., 2007;Aguirre et al., 2006) It therefore follows the need to discuss and define new criteria to study the performance of immunochemical tests at those regions.In this regard, the enhanced sensitivity displayed by PCR techniques should allow overcoming the mentioned drawback.The lack of cross-reactions of T. cruzi recombinant proteins towards samples from T. rangeli infected individuals was also described.(Caballero et al., 2007) However, these are only preliminary results, and large evaluations have not been still performed since T. cruzi and T. rangelli are co-endemic and mixed infections are difficult to exclude with conventional or epidemiologic analysis.The first studies reporting discrimination of both infections using molecular approaches have been published in the last year and will allow to compose a serum panel with samples from patients suffering from either only one or both infections.(Botero et al., 2010) In spite of the advantages yielded when using recombinant antigens in ELISA, it has also been described that sensitivity varies according to the antigen used to sensitize plaques and the geographical region.Thus, sensitivity obtained when performing tests in Colombia using recombinant antigens related to the predominant strain in South America is different from that obtained when assays are carried out in regions at the South of the continent.(Ramirez et al., 2009) The same holds true when examining samples from Panamá patients, where T. cruzi strains are very similar to those from Colombia.(Caballero et al., 2007) These works point out that serum level of antibodies in Panamanian patients were significantly lower than those from Brazilian individuals, from where the recombinant proteins were obtained using parasite genotypes isolated in Brazil.These data are in agreement with the serological differential reactivity produced by experimental infections in a mouse model, when strains representatives of different DTUs are inoculated.(dos Santos et al., 2009).

Laboratory treatment monitoring
The evaluation of treatment effectiveness is normally carried out through serological analysis.Direct parasitological techniques miss reliability because of the extremely low parasitemia, which after treatment diminishes more, even when total parasite elimination could have not been reached.Conventional serology turns into negative for more than 80% of the patients treated during the acute phase, once passed about 2 years after treatment.However, this percentage drops to less than 10% for patients who have been treated during the chronic phase, this taking several years.(Cancado, 1999) The patient status is classified, according to the laboratory tests results.Thus, patients are considered cured when parasitological tests and conventional serology are negative.When parasitological tests are negative and 2 of 3 conventional serological tests are positive, the patient is classified as dissociated.Patients are considered not to be cured when the 3 tests are positive.The serological test that demonstrated to be especially useful to monitor treatment effectiveness is the assessment of lytic antibodies.This test showed 100% correlation with parasitological cure, when lytic antibodies were evaluated in sera from patients who were confirmed to be cured.(Krettli et al., 1979) The drawback of this test is the need to count with in vivo trypomastigotes culture, thus not being available in clinical diagnostic laboratories.Consequently, several recombinant antigens to evaluate patient's treatment response have been proposed and assessed by ELISA as follows.The target antigen of lytic antibodies was identified as a 160 KDa molecule, a complement regulatory protein, usually named CRP.(Krettli, 2009) The assessment of antibodies against this protein displayed 100% correlation with that of lytic antibodies, when using both the native protein and the recombinant one.(Meira et al., 2004) Cruzipain and Tc24 are other recombinant proteins which were also evaluated to monitor patient's treatment, and displayed 70% and 80% correlation with the lytic antibodies method, respectively.(Gazzinelli et al., 1993;Krautz et al., 1995) F29, which is Tc24 homologous, was used to follow up the treatment in children younger than 12 years old, after 48 months of initiated the medication.(Sosa et al., 1998) This work reports that 67% of sera from treated children showed lack of anti-F29 antibodies whereas 100% of untreated children showed positive results for the specific antibodies.Therefore, the authors proposed to confirm the lack of anti-F29 antibodies as a serological marker of children cure.More recently, F29 was again evaluated as antigen of treatment monitoring in adults.(Fabbro et al., 2007) Results showed lack of the specific antibodies in 82.4% of sera from treated patients who still showed positive conventional serology.The same group has recently evaluated the levels of specific antibodies against the ribosomal protein TcP2β, as a cure marker.(Fabbro et al., 2011) Their results showed a significant decrease of specific anti-TcP2β in sera from treated patients, although no negative results occurred, which is a similar behavior than that displayed when performing conventional serology.Therefore, anti-TcP2β does not resemble to be an apparently good candidate to be used as an early marker of the infection cure.Another interesting study is the one where CRA and FRA antigens were evaluated, and displayed a 67% correlation with the reference technique.This makes CRA and FRA as quite promising antigens to be used for cure monitoring.The results are interesting considering that the Bio-manguinhos, Fiocruz commercial kit, commercialized in Brazil is manufactured with a single mixture of these two recombinant proteins.

Chronic infection monitoring
In the context of Chagas disease autoimmune hypothesis, during the nineties, it was proposed that different antigens contributed to the generation of autoantibodies, which could be used as illness evolution markers.(Leon & Engman, 2001) Among these antigens we should mention cruzipain, (Giordanengo et al., 2000;Goin et al., 1999;Duschak et al., 2001) sulfo-cerebrosides (Avila et al., 1993) and the ribosomal protein TcP2.(Levitus et al., 1991) Precursor works had described that this ribosomal antigen that shares the C terminal region with its homologous from humans, generated autoimmune antibodies, whose concentration was increased in patients who had developed chagasic cardiopathy.(Levin et al., 1991;Aznar et al., 1995).Our group evaluated the concentration of antibodies against cruzipain, sulfocerebrosides and ribosomal TcP2 in three different groups of patients: those classified as asymptomatic, those who only displayed electrocardiographic alterations and those who had evident cardiopathy.(Diez et al., 2006) In our experience, only those samples from patients with evident cardiopathy had increased specific anti-TcP2 concentration.However, these results have not yet been confirmed analyzing a larger number of patients.In another study, anti-TcP2 concentration was higher in sera from patients at indeterminate 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 marker of myocardic damage.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 different stages of the illness.(Tovar et al., 2009;Talvani et al., 2006) The difficulties to establish clear illness evolution markers lead to the present state, where we do not count yet with useful tools to evaluate Chagas' disease prognosis.

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)

Fig. 1 .
Fig.1.Illustration of the ELISA plaque sensitizing process and the exposure to the sample: left-hand side, when using a protein mixture of three recombinant peptides RP1+RP2+RP5; right-hand side, when using a multiepitope chimeric protein bearing the same peptides fused in a single protein, CP2.RP: recombinant peptide, CP: chimeric protein obtained by fusion of peptides RP1, RP2 and RP5 together in only one molecule.Reproduction fromCamussone et al 2009.