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

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

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

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

Advances in Serological Diagnosis of Chagas' Disease by Using Recombinant Proteins 277

methodology simplicity, and the personal confidence he/she has in a particular technique

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.

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*

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* 

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

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

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) In this work, it was proved the deficiency 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

after having performed it for a long while.

infection.(Saez-Alquezar *et al.*, 1997)

false-negative outcomes.

response.(Konishi, 1993)

However, regular health centers do not count with both of them.

*al.*, 2005;Brutus *et al.*, 2008;Roddy *et al.*, 2008;Sosa-Estani *et al.*, 2008)

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.

#### **2.2 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

performed parasitologic analysis in adults since more than 50 years,(Strout, 1962) and it has

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

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 &

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

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

been reported about 95% sensitivity.(Freilij & Storino, 1994)

out the methodology.

Schmunis, 2010)

**2.2 Serological diagnosis of chronic entities** 

method.

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) In this work, it was proved the deficiency 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

Advances in Serological Diagnosis of Chagas' Disease by Using Recombinant Proteins 279

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

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

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

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.*,

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,

proved by the same group, see Tables 1 A,B and C.(Umezawa *et al.*, 2003)

discrete typing units (DTUs). (Di Noia *et al.*, 2002)

2000;Umezawa *et al.*, 2003)

serodiagnosis.(DaRocha *et al.*, 2002)

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.
