**5.1 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.

#### **5.2 Chronic infection diagnosis**

284 Current Topics in Tropical Medicine

CP2 fusion chimera

RP1 RP2 R 5P

*washout*

RP1+RP2+RP5 peptide assortment

*+ Abs*

Camussone et al 2009.

**5.1 Acute phase diagnosis** 

recombinant ones are used.

**entities** 

Well binding site

*washout*

*+ Abs*

CP2

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 from

**5. Recombinant antigens use for the diagnosis of the different clinical** 

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

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 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)

Advances in Serological Diagnosis of Chagas' Disease by Using Recombinant Proteins 287

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

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

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

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

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

been proposed and assessed by ELISA as follows.

an early marker of the infection cure.

**5.4 Chronic infection monitoring** 

with a single mixture of these two recombinant proteins.

native protein and the recombinant one.(Meira *et al.*, 2004)

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).

#### **5.3 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.

#### **5.4 Chronic infection monitoring**

286 Current Topics in Tropical Medicine

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

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

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

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

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

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

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

PCR techniques should allow overcoming the mentioned drawback.

of different DTUs are inoculated.(dos Santos *et al*., 2009).

**5.3 Laboratory treatment monitoring** 

could have not been reached.

several years.(Cancado, 1999)

are positive.

infections.(Botero *et al.*, 2010)

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

Advances in Serological Diagnosis of Chagas' Disease by Using Recombinant Proteins 289

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

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

Affranchino,J.L.; Ibanez,C.F.; Luquetti,A.O.; Rassi,A.; Reyes,M.B.; Macina,R.A.; Aslund,L.;

Araujo,F.G. (1986) Analysis of *Trypanosoma cruzi* antigens bound by specific antibodies and by antibodies to related trypanosomatids. *Infect. Immun.* 53(1), 179-185. Avila,J.L.; Rojas,M. & Carrasco,H. (1993) Elevated levels of antibodies against sulphatide are

Aznar,C.; Lopez-Bergami,P.; Brandariz,S.; Mariette,C.; Liegeard,P.; Alves,M.D.;

Barfield,C.A.; Barney,R.S.; Crudder,C.H.; Wilmoth,J.L.; Stevens,D.S.; Mora-Garcia,S.;

Pettersson,U. & Frasch,A.C. (1989) Identification of a Trypanosoma cruzi antigen that is shed during the acute phase of Chagas' disease. *Mol. Biochem. Parasitol.* 34(3), 221-228. Aguirre,S.; Silver,A.M.; Brito,M.E.F.; Ribone,M.E.; Lagier,C.M. & Marcipar,I.S. (2006)

Design, Construction and Evaluation of a Specific Chimeric Antigen to Diagnose

present in all chronic chagasic and dilated cardiomyopathy sera. *Clin. Exp.* 

Barreiro,E.L.; Carrasco,R.; Lafon,S.; & Kaplan,D. (1995) Prevalence of anti-R-13 antibodies in human *Trypanosoma cruzi* infection. *FEMS Immunol. Med. Microbiol.*

Yanovsky,M.J.; Weigl,B.H. & Yanovsky,J. (2011) A highly sensitive rapid diagnostic test for Chagas disease that utilizes a recombinant Trypanosoma cruzi antigen.

markers that are useful for cure monitoring and cardiopathy prognosis.

standards of sensitivity and specificity than ELISA.(Belluzo *et al.*, 2011)

Chagasic Infection. *J. Clin. Microbiol.* 44, 1043-1046.

*Immunol.* 92(3), 460-465.

*IEEE Trans. Biomed. Eng*. 58(3), 814-817.

12(3-4), 231-238.

**7. References** 

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.
