**1. Introduction**

272 Current Topics in Tropical Medicine

Stark, C., B. J. Breitkreutz, T. Reguly, L. Boucher, A. Breitkreutz & M. Tyers (2006).

Sundar, S. & J. Chakravarty (2010). "Antimony toxicity." *Int J Environ Res Public Health* 7(12):

Taylor, V. M., D. L. Munoz, D. L. Cedeno, I. D. Velez, M. A. Jones & S. M. Robledo (2010).

Thangudu, R. R., M. Tyagi, B. A. Shoemaker, S. H. Bryant, A. R. Panchenko & T. Madej

Travi, B. L., Y. Osorio, P. C. Melby, B. Chandrasekar, L. Arteaga & N. G. Saravia (2002).

Uetz, P., L. Giot, G. Cagney, T. A. Mansfield, R. S. Judson, J. R. Knight, D. Lockshon, V.

Varela, M. R., D. L. Munoz, S. M. Robledo, B. K. Kolli, S. Dutta, K. P. Chang & C. Muskus

Weniger, B., S. Robledo, G. J. Arango, E. Deharo, R. Aragon, V. Munoz, J. Callapa, A.

Wiese, M. (1998). "A mitogen-activated protein (MAP) kinase homologue of Leishmania

Wiese, M. & I. Gorcke (2001). "Homologues of LMPK, a mitogen-activated protein kinase

Xenarios, I., D. W. Rice, L. Salwinski, M. K. Baron, E. M. Marcotte & D. Eisenberg (2000). "DIP: the database of interacting proteins." *Nucleic Acids Res* 28(1): 289-291. Yu, H., P. M. Kim, E. Sprecher, V. Trifonov & M. Gerstein (2007). "The importance of

Zanzoni, A., L. Montecchi-Palazzi, M. Quondam, G. Ausiello, M. Helmer-Citterich & G.

Zhang, R. & Y. Lin (2009). "DEG 5.0, a database of essential genes in both prokaryotes and

eukaryotes." *Nucleic Acids Res* 37(Database issue): D455-458.

GFP for screening of antileishmanial drug." *Exp Parasitol* 122(2): 134-139. von Mering, C., L. J. Jensen, B. Snel, S. D. Hooper, M. Krupp, M. Foglierini, N. Jouffre, M. A.

Saccharomyces cerevisiae." *Nature* 403(6770): 623-627.

hamsters infected with Leishmania spp." *Infect Immun* 70(5): 2288-2296. Trott, O. & A. J. Olson (2010). "AutoDock Vina: improving the speed and accuracy of

34(Database issue): D535-539.

*BMC Bioinformatics* 11: 365.

*Comput Chem* 31(2): 455-461.

33(Database issue): D433-437.

*Ethnopharmacol* 78(2-3): 193-200.

dynamics." *PLoS Comput Biol* 3(4): e59.

*Immunol* 190(1-2): 19-22.

agents." *Exp Parasitol* 126(4): 471-475.

4267-4277.

"BioGRID: a general repository for interaction datasets." *Nucleic Acids Res*

"Leishmania tarentolae: utility as an in vitro model for screening of antileishmanial

(2010). "Knowledge-based annotation of small molecule binding sites in proteins."

"Gender is a major determinant of the clinical evolution and immune response in

docking with a new scoring function, efficient optimization, and multithreading." *J* 

Narayan, M. Srinivasan, P. Pochart, A. Qureshi-Emili, Y. Li, B. Godwin, D. Conover, T. Kalbfleisch, G. Vijayadamodar, M. Yang, M. Johnston, S. Fields & J. M. Rothberg (2000). "A comprehensive analysis of protein-protein interactions in

(2009). "Leishmania (Viannia) panamensis: an in vitro assay using the expression of

Huynen & P. Bork (2005). "STRING: known and predicted protein-protein associations, integrated and transferred across organisms." *Nucleic Acids Res*

Lobstein & R. Anton (2001). "Antiprotozoal activities of Colombian plants." *J* 

mexicana is essential for parasite survival in the infected host." *EMBO J* 17(9): 2619-2628.

from Leishmania mexicana, in different Leishmania species." *Med Microbiol* 

bottlenecks in protein networks: correlation with gene essentiality and expression

Cesareni (2002). "MINT: a Molecular INTeraction database." *FEBS Lett* 513(1): 135-140.

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

Advances in Serological Diagnosis of Chagas' Disease by Using Recombinant Proteins 275

The infection diagnosis is not a simple task because the features of the illness development as well as the immunological response of the host must be taken into account when performing the laboratory diagnosis. Different diagnostic methods are used, depending on

The alternative ways to get acute Chagas' infection are mainly vectorial and vertical transmission, and the reactivation of the chronic disease in immunosuppressed individuals. Therefore, diagnosis at this stage limits to: *i)* the uncommon cases of symptomatic patients, *ii)* newborns delivered from chagasic mothers, and *iii)* immunosuppressed patients that

In the above mentioned cases, parasites are usually, easily found in blood, therefore being their direct microscopic observation the chosen method for a safe diagnosis.(WHO, 2002;Rosenblatt, 2009) In contrast, indirect serological techniques present low sensitivity in recently infected individuals because humoral immune response is delayed.(Zuniga *et al.*, 2000) Moreover, even specific IgM immunoglobulin has a window period that requires up to several weeks to be produced at detectable concentrations. Other pitfalls such as the lack of IgM anti-*T. cruzi* commercial kits prevents from choosing serology as the infection diagnostic method. This methodology is only used when the parasite is not found in fresh blood smears, by Struot and/or microhematocrit methods.(Luquetti & Schmunis, 2010) During the chronic phase, the *T. cruzi* infection diagnosis is required, *i)* as pregnancy or occupational routine control analysis -in countries where according legislation is in force-, *ii)*  when in presence of a Chagas' compatible cardiopathy, *iii)* during transfusion and transplantation screening, and *iv)* to achieve a reliable illness prognosis of patients who were already diagnosed. One particular case among the latter ones is monitoring the

Conversely to what happens in the acute phase, during the chronic infection, a significant humoral response is found in immunocompetent individuals, along with low parasite concentration in blood. Under these circumstances, indirect techniques are highly sensitive, while the direct ones fail. Hence, Chagas' disease serologic diagnoses intend to verify the occurrence of several specific antibodies against *T. cruzi*. However, conventional serology presents different sensitivity and selectivity, depending on the immunological technique used to perform the determination, and mainly in the nature of the antigen used to capture

In this Chapter, we will describe the most recent contributions of our and other groups to improve the analytical tools available to properly and safely diagnose Chagas' disease using

Direct parasite detection in whole blood is the simplest, regular procedure used to diagnose acute infection whereas, indirect serological tests are the chosen ones to diagnose the

The direct microscopic observation of parasites from patients peripheral blood is the elected methodology to confirm acute infection. The Strout concentration method is the routinely

recombinant proteins, in each one of the clinical entities mentioned above.

**1.1** *T. cruzi* **infection diagnostic during the different stages of the illness** 

the illness stage and the particular clinical entity of the patient.

have been previously diagnosed as infected.

antiparasitic treatment effectiveness.

the specific antibodies.(Belluzo *et al.*, 2011)

**2. Conventional diagnosis of** *T. cruzi* **infection** 

undetermined, chronic state.(Rosenblatt, 2009;WHO, 2002)

**2.1 Parasitological and serological diagnosis of acute entities** 

appears with an incubation period of 4-10 days, which is generally asymptomatic. Afterwards, the infection may advance to the short acute phase, followed by the long-lasting chronic stage, which may occur in a syntomatic or asymptomatic way.

The acute stage of the infection usually lasts 2-4 months. When symptoms appear, these are often light and atypical. The infection proceeds generally unnoticed, and this prevents its diagnosis. Most of the patients recover within 3-5 months. However, global mortality during this phase reaches 5-10%,(WHO, 2002) and may be higher in children.(Pinto *et al.*, 2004) Deaths can be caused by complications related to myocarditis and/or meningoencephalitis,(Pittella, 2009) which has been proved by the presence of the amastigote form of parasites in cardiac, skeletal, glial and soft tissue cells.(da Silveira *et al.*, 2007)

The undetermined, chronic phase generally begins 8-10 weeks after the acute infection, and may last several years or even the whole life of the individual. The illness overtakes asymptomatically, and generally, infected individuals keep their full working capacity without being aware of the infection. However, during this stage, patients display positive serology for specific IgG antibodies and low parasitemia.(Coura, 2007) Over 50% of the infected individuals show themselves healthy and do not develop serious outcomes. In the other cases, the infection is often detected once its sequels appear, after 10-20 years of the first parasite entry. The illness features that mainly develop are cardiac, digestive and/or neurological damages. Chagas' pathologic alterations can be summarized as chronic myocarditis, 27%, esophageal or colonic expansion, 5%, and abnormalities of central nervous system, ca. 3%.(Teixeira *et al.*, 2006) The most severe impact of the illness occurs during chronic phase, and after many years that the infection has been established, leading to high mortality rate among people who have develop cardiac pathology.(Rassi, Jr. *et al.*, 2007)

Undoubtedly, the most effective alternative to prevent Chagas' disease spreading is to control vectorial and transfusional ways of transmissions. Nevertheless, once the individual has been infected by *T. cruzi*, he/she may be treated. Two main aspects are covered by therapy, aiming to eliminate the parasite with trypanocidal medication, and/or medicating to relief symptoms of the several clinical outlines of the illness. Nowadays, two chemicals are used as trypanocidal agents namely, nifurtimox (Lampit®, Bayer; 5-nitrofuran 3-methyl-4-(5'-nitrofurfurylideneamine) tetrahydro-4H-1,4-tiazine-1,1 dioxide) and benznidazole (Rochagan® and Radanil®, Roche; N-benzyl-2-nitroimidazole acetamide). When infection is treated during the acute stage, it has been reported that parasitemia disappeared in 60% of the cases and serology turned into negative. Nevertheless, the outcome after therapy is finished shows variable effectiveness percentages, depending mainly on the age of the individual and the geographical region.(Perez-Molina *et al.*, 2009)

Currently, studies trying to expand the indicators on treatment effectiveness are in progress and will be accomplished by 2012.(Marin-Neto *et al.*, 2008) Though these studies have not yet finished, there is a consensus on treating Chagas' disease, taking into account the clearcut results when using trypanocidal medication with children, during the indeterminate phase of the infection.(Lescure *et al.*, 2010;Perez-Molina *et al.*, 2009;Sosa-Estani & Segura, 2006) It therefore follows that it is highly advisable to count with reliable methods to early diagnose the disease, since this may accelerate the patient treatment, helping to reduce the serious consequences that the long term infection may cause.

appears with an incubation period of 4-10 days, which is generally asymptomatic. Afterwards, the infection may advance to the short acute phase, followed by the long-lasting

The acute stage of the infection usually lasts 2-4 months. When symptoms appear, these are often light and atypical. The infection proceeds generally unnoticed, and this prevents its diagnosis. Most of the patients recover within 3-5 months. However, global mortality during this phase reaches 5-10%,(WHO, 2002) and may be higher in children.(Pinto *et al.*, 2004) Deaths can be caused by complications related to myocarditis and/or meningoencephalitis,(Pittella, 2009) which has been proved by the presence of the amastigote form of parasites in cardiac,

The undetermined, chronic phase generally begins 8-10 weeks after the acute infection, and may last several years or even the whole life of the individual. The illness overtakes asymptomatically, and generally, infected individuals keep their full working capacity without being aware of the infection. However, during this stage, patients display positive serology for specific IgG antibodies and low parasitemia.(Coura, 2007) Over 50% of the infected individuals show themselves healthy and do not develop serious outcomes. In the other cases, the infection is often detected once its sequels appear, after 10-20 years of the first parasite entry. The illness features that mainly develop are cardiac, digestive and/or neurological damages. Chagas' pathologic alterations can be summarized as chronic myocarditis, 27%, esophageal or colonic expansion, 5%, and abnormalities of central nervous system, ca. 3%.(Teixeira *et al.*, 2006) The most severe impact of the illness occurs during chronic phase, and after many years that the infection has been established, leading to high mortality rate among people who have develop

Undoubtedly, the most effective alternative to prevent Chagas' disease spreading is to control vectorial and transfusional ways of transmissions. Nevertheless, once the individual has been infected by *T. cruzi*, he/she may be treated. Two main aspects are covered by therapy, aiming to eliminate the parasite with trypanocidal medication, and/or medicating to relief symptoms of the several clinical outlines of the illness. Nowadays, two chemicals are used as trypanocidal agents namely, nifurtimox (Lampit®, Bayer; 5-nitrofuran 3-methyl-4-(5'-nitrofurfurylideneamine) tetrahydro-4H-1,4-tiazine-1,1 dioxide) and benznidazole (Rochagan® and Radanil®, Roche; N-benzyl-2-nitroimidazole acetamide). When infection is treated during the acute stage, it has been reported that parasitemia disappeared in 60% of the cases and serology turned into negative. Nevertheless, the outcome after therapy is finished shows variable effectiveness percentages, depending mainly on the age of the individual and the geographical

Currently, studies trying to expand the indicators on treatment effectiveness are in progress and will be accomplished by 2012.(Marin-Neto *et al.*, 2008) Though these studies have not yet finished, there is a consensus on treating Chagas' disease, taking into account the clearcut results when using trypanocidal medication with children, during the indeterminate phase of the infection.(Lescure *et al.*, 2010;Perez-Molina *et al.*, 2009;Sosa-Estani & Segura, 2006) It therefore follows that it is highly advisable to count with reliable methods to early diagnose the disease, since this may accelerate the patient treatment, helping to reduce the

chronic stage, which may occur in a syntomatic or asymptomatic way.

skeletal, glial and soft tissue cells.(da Silveira *et al.*, 2007)

cardiac pathology.(Rassi, Jr. *et al.*, 2007)

region.(Perez-Molina *et al.*, 2009)

serious consequences that the long term infection may cause.
