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From Balouz, V., Agüero, F., Buscaglia, C.A., 2017. Chagas Disease DiagnosticApplications: Present Knowledge and Future Steps. In: Rollinson, D., Stothard, J.R.

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CHAPTER ONE

Chagas Disease DiagnosticApplications: Present Knowledgeand Future StepsV. Balouz, F. Ag€uero, C.A. Buscaglia1Instituto de Investigaciones Biotecnol�ogicas e Instituto Tecnol�ogico de Chascom�us (IIB-INTECH),Universidad Nacional de San Martín (UNSAM) e Consejo Nacional de Investigaciones Científicas yTécnicas (CONICET), Buenos Aires, Argentina1Corresponding author: E-mail: [email protected]

Contents

1. Introduction 22. Trypanosoma cruzi, an ‘all-wheel drive’ parasite 4

2.1 Epidemiological features 42.2 Genetic and phenotypic variability 6

3. Diagnostic Applications for Chagas disease: Present Knowledge 83.1 Parasitological and clinical methods 83.2 Serological methods 123.3 Molecular methods 14

4. Diagnostic Applications for Chagas Disease: Pending Issues 164.1 Early diagnosis of congenital transmission 164.2 Rapid assessment of therapy efficacy 184.3 Indication/prediction of Chagas disease progression 194.4 Typing of parasite strains 214.5 Point-of-care diagnosis 22

5. Diagnostic Applications for Chagas Disease: The Road Ahead 246. Concluding Remarks 26Acknowledgement 27References 27

Abstract

Chagas disease, caused by the protozoan Trypanosoma cruzi, is a lifelong anddebilitating illness of major significance throughout Latin America and an emergentthreat to global public health. Being a neglected disease, the vast majority of Chagasicpatients have limited access to proper diagnosis and treatment, and there is only amarginal investment into R&D for drug and vaccine development. In this context,identification of novel biomarkers able to transcend the current limits of diagnosticmethods surfaces as a main priority in Chagas disease applied research. The expectation

Advances in Parasitology, Volume 97ISSN 0065-308Xhttp://dx.doi.org/10.1016/bs.apar.2016.10.001

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is that these novel biomarkers will provide reliable, reproducible and accurate resultsirrespective of the genetic background, infecting parasite strain, stage of disease, andclinical-associated features of Chagasic populations. In addition, they should be ableto address other still unmet diagnostic needs, including early detection of congenitalT. cruzi transmission, rapid assessment of treatment efficiency or failure, indication/prediction of disease progression and direct parasite typification in clinical samples.The lack of access of poor and neglected populations to essential diagnosticsalso stresses the necessity of developing new methods operational in point-of-care settings. In summary, emergent diagnostic tests integrating these novel andtailored tools should provide a significant impact on the effectiveness of currentintervention schemes and on the clinical management of Chagasic patients. In thischapter, we discuss the present knowledge and possible future steps in Chagas diseasediagnostic applications, as well as the opportunity provided by recent advances inhigh-throughput methods for biomarker discovery.

1. INTRODUCTION

Chagas disease or American Trypanosomiasis, caused by the parasiticprotozoan Trypanosoma cruzi (Kinetoplastida, Trypanosomatidae), is a life-long, neglected tropical disease and leading cause of cardiomyopathy inendemic areas (Rassi et al., 2010). With 8e10 million people alreadyinfected and up to 120 million individuals at risk of infection, Chagas diseaseconstitutes the most important parasitic disease in Latin America and one ofthe most common globally (Stanaway and Roth, 2015). Its exact burden is,however, difficult to assess due to several factors including the widespreadgeographic distribution of T. cruzi vectorborne transmission, the decades-long lag between infection and appearance of symptoms, certain pitfalls ofcurrent diagnostic methods, biased prevalence data and incomplete recogni-tion of Chagas disease-attributable symptoms (Stanaway and Roth, 2015).The most recent estimates indicate that Chagas disease is responsible forw550,000 disability-adjusted life years (DALYs), a measure that capturesboth premature mortality (w12,000 deaths per year) and nonfatal healthlosses (Stanaway and Roth, 2015). Despite this enormous toll, only twotrypanocydal drugs, benznidazole and nifurtimox, are currently availablefor chemotherapy. Both are nitroheterocyclic, oral compounds that requireprolonged administration, may display severe adverse effects, cannot be usedto treat pregnant women due to their uncertain teratogenic risks and, mostimportantly, show high efficacy solely if administered at the onset ofinfection (Carlier and Truyens, 2015; Rassi et al., 2010; Viotti et al.,2006). The prospects for the development of an effective vaccine for

2 V. Balouz et al.



prophylactic and/or therapeutic purposes, on the other hand, are stillclouded by substantial scientific and socioeconomic challenges (Beaumieret al., 2016; Bustamante and Tarleton, 2015).

T. cruzi transmission primarily occurs when humans are exposed to thecontaminated feces of infected, haematophagous triatomine vectors. Large-scale intervention schemes launched in different regions of Latin Americain the 1990s have successfully shrunk the geographic limits and prevalenceof vectorborne parasite transmission and led to an overall w40% reductionof disease prevalence (Schofield et al., 2006). However, different ecologicaland demographic issues converged in the last decades to shift the epidemio-logical landscape for this disease. For instance, recent outbreaks of acute casesin certain regions from Brazil and Venezuela were not strictly vectorborne butrather due to accidental ingestion of T. cruzi-tainted food and fluids (Alarconde Noya et al., 2010; Segovia et al., 2013). This ‘foodborne’ transmissionmode likely constitutes an ancient epidemiological trait, very important tothe zoonotic spreading of the parasite (Gurtler and Cardinal, 2015), andappears to be associated with increased virulence and a higher case-fatalityrate in humans (Alarcon de Noya et al., 2010; Segovia et al., 2013). Inaddition, migratory trends of infected populations from rural areas to urbancentres and/or to nonendemic regions along with changes in the ecogeo-graphical distribution of vector populations have led to the gradual urbaniza-tion and globalization of Chagas disease, which is now recognized as anemerging worldwide threat to public health (Eisenstein, 2016). Indeed, therisk of acquiring Chagas disease through infected blood transfusion and organtransplantation is becoming a major problem even in areas of nonendemicity,such as the United States, Australia and Europe (Requena-Mendez et al.,2015; Schmunis and Yadon, 2010). Moreover, the congenital route ofinfection, which constitutes the main transmission mode of T. cruzi innonendemic areas, is now estimated to be responsible for 22% of new annualinfections in endemic countries with active programs for home vectorinfestations control (Carlier and Truyens, 2015).

In this scenario, a strong and global partnership aimed to coordinateactions to control parasite transmission is urgently needed. In particular,we need to redouble our efforts to control home vector infestation, to screenblood supplies and to identify and subsequently treat T. cruzi-infectedpeople who are still in the early stages of the disease to avoid sequelae,morbidity and economic losses. As a major step towards these goals, weought to develop novel biomarkers able to overcome the limitations of cur-rent diagnostic applications. In this chapter, we critically appraise what has so

Diagnostic Applications for Chagas Disease 3



far been achieved in this area. We also discuss possible ways to proceed toaddress major and still unmet diagnostic demands and the opportunity pro-vided by recent advances in high-throughput methods (i.e., peptide synthe-sis technology, genomics and proteomics) in Chagas disease biomarkerdiscovery.

2. TRYPANOSOMA CRUZI, AN ‘ALL-WHEEL DRIVE’PARASITE

T. cruzi is a promiscuous parasite that traverses a complex life cycleinvolving extracellular proliferation and differentiation inside haematopha-gous insect vectors from different genera and intracellular proliferation anddifferentiation in a variety of vertebrate hosts (De Souza, 2002). Host switch-ing, immune pressure as well as constant transition from intracellular toextracellular niches (and vice versa) pose significant adaptationchallenges and are concomitantly accompanied by extensive remodelling ofdifferent aspects of T. cruzi such as intracellular transport, primarymetabolism, gene expression profiling and overall cellular architecture (DeSouza, 2002). This striking plasticity can be also readily recognized in thediverse genetic, phenotypic and epidemiological features displayed bydifferent strains and field isolates comprised within the T. cruzi taxon (Zingaleset al., 2012). In this first section, we outline some aspects that underlie thebiological flexibility of this ‘all-wheel drive’ parasite and that may be relevantin terms of biomarker discovery for Chagas disease diagnostic purposes.

2.1 Epidemiological featuresPotential T. cruzi vectors present a broad geographic distribution (fromcentral Argentina and Chile to southern USA) and include more than 140species of ‘kissing bugs’ from the subfamily Triatominae (Hemiptera,Reduviidae). Of these, only a few (i.e., Triatoma infestans, Triatoma dimidiata,Triatoma brasiliensis, Rhodnius prolixus and Pastrongylus megistus) have adaptedto live in domiciliary setting and to blood-feed on humans and/or domesticanimals and thus define the ‘domestic/peridomestic’ cycle of Chagas disease(Gurtler and Cardinal, 2015; Zingales et al., 2012). The ‘sylvatic’ cycle of T.cruzi, on the other hand, is actually an array of poorly understood cycles withdifferent ecoepidemiological properties, each one involving multiple sylvaticand/or synanthropic triatomine species, which in turn feed on a variablerange of animals. The latter include a variety of rodents, primates, carnivores,bats, marsupials (i.e., opossums) and xenarthrans (i.e., armadillos, sloths,

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anteaters) (Fig. 1) (Fernandes et al., 1999; Noireau et al., 2009; Zingaleset al., 2012). In general terms, and although not yet fully established, allmammals are considered susceptible, whereas birds and reptiles areconsidered refractory to T. cruzi. From an epidemiological standpoint, thesenonhuman hosts may play key roles as parasite reservoirs and/or as determi-nant factors affecting T. cruzi transmission dynamics in endemic areas(Gurtler and Cardinal, 2015; Noireau et al., 2009). Importantly, they mayalso work as complex selective systems leading to the emergence of novelparasite traits (Noireau et al., 2009).

Interestingly, distinct though partially overlapping sets of strains circulatein the ‘domestic/peridomestic’ and the ‘sylvatic’ cycles of T. cruzi (Gurtler

Figure 1 Schematic diagram showing the Trypanosoma cruzi life cycle and differentbiological features that contribute to ensure its transmission and the establishmentof multiple interactions with insect vectors and infected humans. Those features forwhich there is direct or indirect experimental evidence suggesting interstrain variabilityare denoted in italics.




and Cardinal, 2015; Noireau et al., 2009; Zingales et al., 2012). In the lastdecades, environmental alterations and demographic issues converged infavouring the intermingling of the two cycles. This translates into a steadyincrease of emergent transmission patterns involving ‘exotic’ T. cruzigenotypes, with the possible occurrence of atypical disease physiopathol-ogies (Coura et al., 2002; Zingales et al., 2012).

2.2 Genetic and phenotypic variabilityAs revealed for several pathogenic protozoa and fungi, T. cruzi displays abasically clonal reproduction mode, with occasional events of geneticexchange leading to the emergence of hybrid genotypes (Messenger andMiles, 2015). These features led to a complex population structure, madeup of multiple ‘clonal’ strains showing remarkable genetic diversity(Tibayrenc and Ayala, 2015). Interstrain variations may be grasped at thenucleotide level (Ackermann et al., 2012) but also structurally, in terms ofdosage/diversification of antigenic gene families (Campo et al., 2004;Cerqueira et al., 2008; Llewellyn et al., 2015; Urban et al., 2011), DNAcontent and overall genome architecture (Lewis et al., 2009a; Minninget al., 2011; Reis-Cunha et al., 2015; Souza et al., 2011). Importantly,biochemical and genetic typing schemes developed throughout the lastdecades converged in the delineation of six major T. cruzi evolutionarylineages or discrete typing units (DTUs) termed TcI to TcVI, with multiplestrains and even cryptic sublineages within each DTU (Tibayrenc and Ayala,2015; Zingales et al., 2012). A potential seventh lineage, termed TcBat, hasbeen recently identified in South and Central American bats (Marcili et al.,2009; Pinto et al., 2012). So far, and although all six (or seven, includingTcBat) T. cruzi DTUs are capable of infecting humans, certain DTUs suchas TcI, TcII, TcV, and TcVI are most frequently isolated from clinical samples(Ramirez et al., 2014; Zingales et al., 2012). The reasons for this skeweddistribution are unclear, although current evidence suggest that parasite strainsdetected in patients reflect the principal DTUs circulating among ‘domestic/peridomestic’ cycles in that geographical area (Messenger et al., 2015).

T. cruzi genotypic heterogeneity could also be grasped at the phenotypiclevel when different biological parameters are studied. These include, forinstance, the rate of epimastigote proliferation in the vector midgut (Castroet al., 2012; de Lana et al., 1998; Vieira et al., 2016); and the extent ofepimastigote differentiation into metacyclic trypomastigotes, the develop-mental form that bring the infection into vertebrates (Fig. 1) (da SilveiraPinto et al., 2000; de Lana et al., 1998). The molecular basis for these

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differences is not yet understood, but it might be related to the dissimilarresistance capacity of parasite strains to antimicrobial peptides or haemolyticfactors and/or to their differential interaction with receptor(s) inside the cropof triatomines (Castro et al., 2012; Gonzalez et al., 2013; Vieira et al., 2016).Importantly, these biological traits define both T. cruzi infectivity towardsinsect vectors and its potential transmissibility to vertebrate hosts (Fig. 1).These, together with differential eco-geographical distribution and certainpreference of triatomids for their blood source, are in turn major determi-nants of Chagas disease epidemiology (Gurtler and Cardinal, 2015; Noireauet al., 2009; Zingales et al., 2012).

Parasite genotypic heterogeneity also seems to modulate key aspects of itsinteraction with vertebrate hosts, including humans. For instance, the capacityof metacyclic trypomastigotes to resist the harsh conditions of the gastric milieuand to invade gastric epithelium following oral infection is largely dependenton the strain-specific glycoprotein composition of their surface coat(Camandaroba et al., 2002; Hoft et al., 1996; Maeda et al., 2016). On thesame lines, interstrain genetic variations underpin a variety of biological traitsinvolved in parasite infectivity and long-term persistence such as antigenicprofile, subversion of the immune system, host cell invasion capacity, intracel-lular growth rate and survival of amastigotes, and sensitivity to anti-Chagasicdrugs (Fig. 1) (Magalhaes et al., 2015; Martin et al., 2006; Moraes et al.,2014; Mortara et al., 2008; Nagajyothi et al., 2012; Ruiz et al., 1998; Toledoet al., 2003). In contrast, no clear association between a particular T. cruzigenotype and an eventual tropism for congenital transmission could be yetestablished. Even though distinct strains may display subtle differences in theirability to invade trophoblasts or chorionic villi explants in vitro (Castillo et al.,2013), genetic profiling experiments have conclusively shown that (1) the sameset of strains circulate in the bloodstream of transmitting and nontransmittingmothers and (2) nearly identical T. cruzi genetic signatures are recoveredfrom infected infants born to Chagasic mothers coursing concurrent,multistrain infections (Fig. 1) (Burgos et al., 2007; del Puerto et al., 2010;Virreira et al., 2006a). Overall, the actual consensus is that maternal parasiteload and human polymorphisms constitute the main risk factors for T. cruzicongenital transmission (Bua et al., 2012; Fabbro et al., 2014; Juiz et al.,2016; Kaplinski et al., 2015; Rendell et al., 2015).

Interestingly, certain (though not all) epidemiological studies have showna partial correlation between the prevalence of particular clinical manifesta-tions of Chagas disease and the genotype of the infecting strain (Andradeet al., 1983; D’Avila et al., 2009; Luquetti et al., 1986; Macedo and Pena,




1998; Virreira et al., 2006b; Zafra et al., 2011; Zingales et al., 2012). This maybe attributed in part to the genetic aspects and immune competence of localhuman populations (Ayo et al., 2013; Deng et al., 2013; Frade et al., 2013;Luz et al., 2016; Nogueira et al., 2012) and/or to parasite genetic heteroge-neities. The latter hypothesis finds support in animal studies (that do notstrictly recapitulate Chagas disease-associated physiopathologies), whichrevealed interstrain variations in complex phenotypes such as parasitaemia,virulence, tissue tropism/distribution and pathogenicity (Fig. 1) (Andradeet al., 1999; Andrade, 1990; Camandaroba et al., 2002; de Souza et al.,1996; Laurent et al., 1997;Monteiro et al., 2013; Revollo et al., 1998; Roelliget al., 2010). However, generalized conclusions are difficult to derive,particularly because these epidemiological studies might have been skewedby a number of intrinsic shortcomings. Briefly, (1) they often lacked detailedgenetic/clinical information on the studied populations; (2) the infectinggenotype has been in some cases inferred based on the prevailing parasitegenotypes circulating in the area and not typed directly from patients; (3)patients might have been coinfected with other coendemic pathogens thatimpact on the clinical presentation of Chagas disease (Salvador et al., 2016);(4) patients might have been infected with multiple parasite strains, whichis usually the case in endemic areas (Perez et al., 2014) and (5) these studiesmight have been biased due to parasite typing pitfalls [i.e., samples werecollected only from peripheral blood, which may not be representative ofthe situation within affected organs (Manoel-Caetano Fda et al., 2008;Vago et al., 2000)] and/or associations between local parasites and disease,making it difficult to determine whether the absence of a specific strain/DTU in patients with a given disease phenotype is due to parasite factorsor to lack of patient exposure to this DTU.

Overall, and although this issue may have major implications for Chagasdisease diagnosis and treatment, the existence of particular associationsbetween T. cruzi genotype and susceptibility to different clinical presenta-tions on Chagasic patients remains to be addressed (Messenger et al., 2015).

3. DIAGNOSTIC APPLICATIONS FOR CHAGASDISEASE: PRESENT KNOWLEDGE

3.1 Parasitological and clinical methodsUpon T. cruzi infection, patients undergo the acute phase of Chagas

disease, which extends for 40e60 days. Symptoms, if indeed occur, are

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usually very mild and atypical, thus often misleading its clinical recognition(Rassi et al., 2010). In rare cases of vectorborne transmission, a skin nodule(called ‘chagoma’) or painless prolonged eyelid oedema (called the‘Romanha’s sign’) may indicate the site of parasite inoculation. Due to thepatent parasitaemia verified at this initial phase, conventional microscopy(i.e., visualization of circulating trypomastigotes in peripheral blood filmsor buffy coat smears) remains the gold standard for diagnosis, both in acutecases and in newborns that were infected congenitally (Freilij and Altcheh,1995; Gomes et al., 2009). Either direct tests or concentration tests (i.e.,microhematocrit or Strout test) are routinely used for this purpose. Thesetechniques, however, present certain limitations in terms of sensitivity(w80e90%) and commonly require highly trained personnel (Table 1)(Freilij and Altcheh, 1995; Gomes et al., 2009).

Following the initial, acute phase, if untreated, patients enter the indeter-minate form of the chronic phase that may last for several years or persistindefinitely (Rassi et al., 2010). This phase is characterized by the absenceof relevant clinical symptoms and very low and intermittent or nullparasitaemia. During this phase, parasite replication is maintained in checkby the elicitation of a strong and parasite-specific B cell- and T cell-mediatedimmunity (Tarleton, 2015), being the latter the most important in terms ofcontrolling the infection. However, elaborate pathogen immune evasionsystems (Albareda et al., 2009; Giraldo et al., 2013; Padilla et al., 2009; Paivaet al., 2012; Vasconcelos et al., 2012) and their ability to quickly invade hostcells (Mortara et al., 2008; Nagajyothi et al., 2012) turn this immuneresponse only partially effective, and most patients maintain a subpatentinfection for life. T. cruzi reactivation in immunocompromised Chagasicpatients provides solid support to this hypothesis (Tarleton, 2015). DirectT. cruzi detection during the chronic phase requires biological amplificationmethods, such as hemoculture and xenodiagnosis (Brener, 1962), which arealso difficult, expensive, time-consuming and require special laboratorybiosecurity conditions. In the case of xenodiagnosis, in addition, it is notapplicable to certain patient populations. Most importantly, these methodsyield positive results in only a proportion of serologically positive patients,thus limiting their usefulness in diagnosis and/or in monitoring drug efficacy(Table 1).

Up to 20 years after the infection, w35% of patients develop patholog-ical signs characteristic of Chagas disease such as cardiomyopathy, peripheralnervous system damage or dysfunction of the digestive tract often leading tomegaesophagus and/or megacolon (Rassi et al., 2010). These pathological




Table 1 Overview of performance and features of available diagnostic applications for Chagas disease

Diagnostic Performance

Technique/ToolChronic

casesAcutecases

Congenitalcases

Drugefficacy

Diseaseprognosis

Parasitetyping

PoCsettingb Remarks

Pa

rasi

tolo

gic

al

Me

tho

ds

Direct bloodexamination tests N.A. N.A.

Microscopy-based methods, gold standard for diagnosis of acute and congenital cases

Hemoculture N.A. N.A. N.A.Improved sensitivity due to parasite amplification,time-consuming, expensive

Xenodiagnosis N.A. N.A. N.A. N.A.Improved sensitivity due to parasite amplification, time-consuming, expensive, not well tolerated

Se

rolo

gic

al

Me

tho

ds

Who

le P

aras

ite-

base

dIHAc N.A. N.A. Moderate specificity, trained personnel required

IFAc N.A. N.A.Moderate specificity, trained personnel and infrastructure required

ELISAc N.A. N.A.Method of choice for diagnosis of most clinical situations, cost-effective, easy to perform

CoML N.A. N.A. N.A. N.A. N.A.Measures antibodies that are cleared in parasitological cured hosts early after drug treatment

Para

site

frac

tion TESAd N.A. N.A. Secreted/Excreted Antigens from trypomastigotes

F2/3 ? ? N.A.Highly O-glycosylated trypomastigote mucins, expensive, low purification yields

Rec

ombi

nant

Ant

igen

s

Parental Repertoirec,e N.A. N.A.

High specificity but impaired sensitivity as compared to whole parasite-based methods.

SAPA ? ?Repetitive, surface antigen associated to enzymatically active trans-sialidases

Ag13/TcD ? ? ?Repetitive, surface antigen associated to enzymatically inactive trans-sialidases

F29/FCaBP/Tc-24/Tc-28/1F8 ? ? ? ? Flagellar Calcium Binding Protein

Ag1/JL7/FRA/H49 N.A. Repetitive, internal antigen

JL5 ? ? ? N.A.Ribosomal antigen that elicits 'auto-antibodies' able to cross-recognize endogenous receptors

TSSA ?Mucin-like, surface antigen showing polymorphisms among strains

Mo

lecu

lar

Me

tho

ds

PCR, qPCR N.A. High specificity, trained personnel required

Genotypification Markers f N.A. N.A. N.A. N.A. N.A.

High specificity, trained personnel required, manual technique , expensive

Biochemical Markersg N.A. ? ? ?

Low specificity and sensitivity, in most cases only 'indicative/predictive' of cardiomyopathy

a

aPerformance is arbitrarily indicated as appropriate (green boxes), nonappropriate (vermillion boxes) or intermediate/needs to be improved(yellow boxes) according to available data. N.A. and question marks (?) stand for nonapplicable or not enough experimental data available toassess the performance, respectively.

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bAdaptability to be deployed in PoC settings, which depends on specific features of the tool/technique and which is arbitrarily indicated as above.cCommercially available from different vendors.dTESA assays include diverse techniques that measure anti-TESA antibodies in serum samples (i.e., TESA-ELISA, TESA-dot blot, TESA-blot)and techniques that capture TESA antigens directly in urine samples (i.e., Chunap assays).eThe term ‘parental’ repertoire refers to Trypanosoma cruzi antigens identified in large-scale initiatives carried out in the 1980s andincludes Ag1/JL7/FRA/H49; Ag2/B13/TCR39/PEP-2; Ag10; Ag13/TcD; Ag19; Ag26; Ag30/CRA/JL8/TCR27; Ag36/JL9/MAP; Ag54;JL1; JL5; JL9; SAPA; B12; TcE; A13; F29/FCaBP/Tc-24/Tc-28/1F8; Tc-40; HSP70; HSP78 and FL-160, according to current nomenclature.These antigens were assayed for conventional diagnosis in different combinations using a variety of technological platforms (ELISA, LFIA,Western blot, dot blot), some of which are commercially available from different vendors. The individual performance of some of these antigensshowing particular features is indicated below.fInclude RFLP, PCR-RFLP, RAPD, Southern blot and other DNA hybridization methods, karyotyping methods and sequence-based methodseither using a single locus or MLST.gInclude TNF-a, ACE2, BNP, ANP, ET-1 and other biochemical markers indicated in text.ACE2, angiotensin-converting enzyme 2; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; CoML, complement-mediated lysis;ELISA, enzyme-linked immunosorbent assay; ET-1, endothelin-1; F2/3, purified highly O-glycosylated and antigenic trypomastigote mucins;HIA, haemagglutination inhibition assay; IFA, immunofluorescence assay; LFIA, lateral-flow immunochromatographic assays; MLST, multi-locus sequence typing assay; PCR, polymerase chain reaction; PCR-RFLP, PCR-based restriction fragment length polymorphism assay; PoC,point-of-care; RAPD, random amplification of polymorphic DNA; SAPA, shed acute-phase antigen; TESA, trypomastigote excreted/secretedantigens; TNF-a, tumour necrosis factor a; TSSA, trypomastigote small surface antigen.

Diagnostic

Applications

forChagas

Disease

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changes can be revealed by electrocardiogram clinical diagnosis, X-rays andultrasound. The nature and relative contribution of the multiple factorsinvolved in this quite broad pathogenic range has been the subject of intensedebate (Bonney and Engman, 2015; Machado et al., 2012; Teixeira et al.,2011). The current consensus states that a failure to downregulate theinflammatory response, which is maintained by parasite persistence in tissues,appears to play a predominant role. Other contributory factors include sex,age and genotypic features of the patient, route of infection, sustained vectorexposure, autoimmune responses and the existence of coinfections (Ayoet al., 2013; Bonney and Engman, 2015; Deng et al., 2013; Frade et al.,2013; Luz et al., 2016; Machado et al., 2012; Nogueira et al., 2012;Tarleton, 2015; Teixeira et al., 2011). As mentioned, a possible role ofparasite genotype on Chagas disease progression/outcome has beenproposed, although further research supported by novel and robust epidemi-ologic and diagnostic tools, and appropriate animal models are needed toaddress this issue.

3.2 Serological methodsConsidering that patients seroconvert early upon infection, detection of anti-T. cruzi antibodies remains the most effective method for demonstratingdirect exposure to the parasite (Gomes et al., 2009). At present, the mostwidely used serologic methods are indirect haemagglutination assay (IHA),indirect immunofluorescence assay (IFA), and enzyme-linked immunosor-bent assay (ELISA) (Table 1). First-generation ELISA tests were originallydeveloped using total parasite homogenates or, later on, using biochemicallypurified, parasite antigenic fractions. Among the latter, TESA (trypomastigoteexcreted/secreted antigens) and F2/3 (highly O-glycosylated trypomastigotemucins obtained by sequential solvent partitions) demonstrated the highestdiagnostic potential (Table 1) (Almeida et al., 1997; Umezawa et al.,1996b). In addition to ELISA, multiple techniques including dot blot andWestern blot were developed for the evaluation of these reagents.

In the late 1980s, the advent of recombinant DNA technology allowedthe generation of parasite DNA or cDNA expression libraries, which werethen coupled to high-throughput screenings using serum samples fromChagasic patients or experimentally infected animals. These approachesprovided the first glimpses into the genomic make-up of this parasite and,most importantly, led to a ‘golden era’ of T. cruzi antigen discovery. Indeed,by using these methods, several of the immunodominant T. cruzi antigensthat are still in use were identified and characterized (Burns et al., 1992;

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Cotrim et al., 1990; Hoft et al., 1989; Houghton et al., 1999; Ibanez et al.,1987, 1988; Lafaille et al., 1989; Levin et al., 1989). For a complete list ofthese antigens (henceforth the ‘parental repertoire’), see Table 1. The factthat many of them emerged from independent screenings carried out indifferent laboratories further supported their diagnostic potential but,unfortunately also led to a confusing nomenclature that still persist (Table 1)(reviewed in da Silveira et al., 2001; Frasch et al., 1991). Some of theantigens included in the T. cruzi ‘parental repertoire’ were extensivelyvalidated, in certain cases by way of multicentre trials (Caballero et al.,2007; Reithinger et al., 2010), and set the stage for the development ofsecond-generation Chagas disease diagnostic methods. A variety of antigenexpression procedures (i.e., using full-length or partially deleted recombi-nant, fusion proteins or synthetic peptides functionalized in differentways) and antigen display strategies (i.e., using mono- or multiepitopicmolecules) were evaluated before them being incorporated either individu-ally or in defined mixtures into in-house and/or commercial kits (Godselet al., 1995; Hernandez et al., 2010; Houghton et al., 2000; Krieger et al.,1992; Longhi et al., 2012; Oelemann et al., 1999; Pastini et al., 1994).Some of these tests have shown very good performances and were thusextensively used in basic research and/or as confirmatory tests in clinicalpractice up to this day.

The major advantage of recombinant antigens-based tests is that theyminimize the extent of specificity problems. As previously shown, serafrom individuals with other coendemic infections such as leishmaniasisand/or afflicted of certain autoimmune disorders cross-react with crudepreparations of T. cruzi antigens (Araujo, 1986; Gomes et al., 2009;Schnaidman et al., 1986). On the down side, recombinant antigen-basedtests present lower sensitivity than whole parasite-based techniques. Inaddition, it should be said that the proven success of this ‘parental reper-toire’, along with a shift in R&D priorities, converged in curbing theenthusiasm for subsequent large-scale antigen discovery efforts for Chagasdisease. Indeed, most of the additional antigens/biomarkers withmoderate-to-excellent diagnostic performance that emerged in the lastdecades were identified either as by-products of basic hypothesis-drivenresearch (Buchovsky et al., 2001; Di Noia et al., 2002; Martinez et al.,1991; Saborio et al., 1990) or using low-to-medium throughput antigenexpression/synthesis approaches (see later discussion). Most importantly,just a few of these novel antigens have been incorporated into the alreadyexisting diagnostic application platforms.




New generation tests displaying potentially improved accuracy such as thechemiluminescent microparticle immunoassay (CMIA) and its improvedversion, Architect Chagas (both from Abbott Laboratories, Wiesbaden,Germany) (Abras et al., 2016; Praast et al., 2011), or the Multi-cruzi test(InfYnity Biomarkers, Lyon, France) (Granjon et al., 2016) have beenrecently developed. Confirming the above-mentioned trend, both use a largepanel of T. cruzi antigens belonging to the ‘parental repertoire’ e thosediscovered in the 1980s e which in the case of the Multi-cruzi test issupplemented with TSSA (trypomastigote small surface antigen (Di Noiaet al., 2002)). Despite the virtual lack of antigen innovation, these testsincorporate a large degree of automation and highly sensitive detectionsystems (Abras et al., 2016) or major technical improvements, such as amultiplexed printing method inside ELISA microplates (Granjon et al., 2016).

Overall, currently available serodiagnostic tests are simple, affordable anddisplay quite good results in terms of large-scale population diagnosis.However, they all present certain minor concerns with regards to theirreproducibility, reliability, specificity (those using whole parasites) andsensitivity (those using parasite fractions and/or recombinant antigens)which in turn affect their overall performance. In addition, and likely dueto their biased antigenic composition, they also show suboptimal perfor-mance with particular Chagasic populations (i.e., acute and/or congenitalinfections) (Gomes et al., 2009) and often discordant results between assaysdepending on the geographic origin of the patients (Caballero et al., 2007;Guzman-Gomez et al., 2015). The latter may be attributed to variations inthe local human immune responses and/or, as stated earlier, to differences inthe antigenic constitution of T. cruzi DTUs that prevail in different endemicareas. Importantly, even after successive improvements, none of the availabletests emerged as the ‘gold standard’, i.e., able to showw100% specificity andsensitivity. In this context, current guidelines developed by the WorldHealth Organization advise the use of at least two serological tests basedon different principles for reaching a ‘conclusive’ diagnosis. In the case ofambiguous or discordant results, a third technique should be conducted.These guidelines thereby increase the cost of diagnosis and risk of patient‘loss’, particularly in endemic areas. Most importantly, these diagnosticlimitations delay the initiation of chemotherapy, thus limiting its efficacy.

3.3 Molecular methodsIn the late 1980s, DNA-based molecular methods emerged as an appealingalternative for the diagnosis of Chagas disease, mainly to overcome the low

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sensitivity of direct parasitological approaches (Moser et al., 1989; Sturmet al., 1989). The introduction of the recently developed polymerase chainreaction (PCR) assay, in particular, held promises of high sensitivity,specificity and high-throughput potential. The first targets for PCRamplification were focused on the major molecular component of themitochondrial DNA (also known as kinetoplast DNA or kDNA). In T. cruziand other kinetoplastid parasites, this component is present in the form ofcircular DNA of short length (minicircles), which add up to several thousandcopies per cell (Simpson, 1986). Later on, a large repertoire of kDNA ornuclear DNA targets including single- or multicopy genes and satellitesequences, as well as different multitarget strategies, has been explored(Schijman et al., 2011). More recently, a parasite concentration approachbased on short and stable RNA aptamers was developed to facilitatePCR-based detection methods and proposed as a potential alternative toolin monitoring parasite load in Chagasic patients (Nagarkatti et al., 2012,2014).

Major challenges for the clinical implementation of PCR-basedtechniques derive from a number of technical factors such as source (i.e.,cord blood, umbilical tissue, placental tissue), volume, conservation andtransportation of the samples, and underlying molecular biology protocols(i.e., DNA isolation, purification, pretreatment, thermocycling conditions,etc.) (Schijman et al., 2011). In addition, the reproducibility and overallperformance of PCR-based methods are significantly affected by the fluctu-ations in parasitaemia that characterize the chronic phase of Chagasdisease and by inter-DTU variations in dosage and/or sequence of thetargets of amplification (Lewis et al., 2009b). Despite these issues, severalPCR-based and, more recently, quantitative real-time PCR (qPCR)-basedprocedures have been developed. These allowed detection and quantifica-tion of parasite DNA from clinical samples with variable levels of reliability,complexity, selectivity and analytical sensitivity (Duffy et al., 2009, 2013;Piron et al., 2007; Qvarnstrom et al., 2012; Valadares et al., 2008). In arecent work, a multicentre trial optimized and evaluated in parallel twolast generation qPCR methods targeting either satellite or kDNA targets(Ramirez et al., 2015). These methods were tested using a large and hetero-geneous panel of blood samples from acute and chronic patients eitherasymptomatic or showing different clinical manifestations and infectedthrough different transmission modes (Ramirez et al., 2015). Though highlyspecific and reproducible, both methods showed clinical sensitivities ofw80%, which is still not good enough for their application as confirmatory




testing of blood donors or in clinical settings. In addition, it is worth notingthat DNA-based molecular methods are expensive and difficult to beimplemented in endemic areas, e.g., in point-of-care (PoC) sites withlimited infrastructure (Table 1).

4. DIAGNOSTIC APPLICATIONS FOR CHAGASDISEASE: PENDING ISSUES

As described earlier, current diagnostic methods and particularly thosebased on serology are highly accurate in detecting most of T. cruzi infectionsin humans. However, there are some clinical and/or epidemiologicalsituations, discussed in this section, in which their performance is signifi-cantly hampered by methodological and/or biological issues.

4.1 Early diagnosis of congenital transmissionAccording to epidemiological data, maternalefoetal transmission occurs inan average of 5% of infected mothers in endemic areas, thus leading tow15,000 congenital cases per year (Carlier and Truyens, 2015). Diagnosisin newborns is commonly based on the microscopic observation oftrypomastigotes in peripheral and/or umbilical cord blood, which is moreeffective by the microhematocrit concentration technique (Freilij andAltcheh, 1995). Considering the usually high parasitaemia at the initial stage,PCR-based analyses provide a valuable supporting tool to detect infectionand to evaluate treatment outcome in these patients (Table 1) (Duffyet al., 2009; Russomando et al., 1998; Schijman et al., 2003). Standardserodiagnostic methods, though routinely used, have low positive predictivevalue until 8e9 months after birth due to passive transfer of maternal IgGantibodies (Freilij and Altcheh, 1995). In this context, identification of novelantigens recognized by foetal IgM or IgA appeared as an appealing approach.However, and despite initial promising results (Antas et al., 2000; Betonicoet al., 1999; Corral et al., 1998; Lorca et al., 1995), these efforts have beendiscontinued due to the high number of false negatives (Blanco et al., 2000;Freilij and Altcheh, 1995; Gomes et al., 2009). It should be noted though,that a serious, high-content and unbiased screening for congenital IgMspecificities have not been yet undertaken.

As an alternative approach to discover surrogate markers of congenitalinfection, the group of Dr. Frasch in Argentina undertook the identificationof T. cruzi antigens exclusively and/or preferentially recognized byfoetal IgGs. This approach relied on the parallel evaluation of paired

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mother/newborn serum samples against a multiplexed antigen panelfollowed by the comparison between signatures of recognition. Using thisstrategy, they identified SAPA (shed acute-phase antigen) and, to a lesserextent, Ag13/TcD as suitable markers to detect congenital transmission(Table 1) (Reyes et al., 1990). Both antigens belonged to the above-mentioned T. cruzi ‘parental repertoire’ and, interestingly, both (particularlySAPA) had been previously found to display a biased, though not exclusiverecognition towards acute infection sera (Affranchino et al., 1989). This isconsistent with the assumption that congenital T. cruzi infection constitutesin fact an acute infection in newborns (Carlier and Truyens, 2015).

Further research disclosed that SAPA is a repetitive sequence displaying acomplex antigenic structure (Alvarez et al., 2001) and involved in improvingthe pharmacokinetics of trans-sialidase, a major T. cruzi virulence factor(Buscaglia et al., 1999; Dc-Rubin and Schenkman, 2012; Frasch, 2000).Up to this day, SAPA remains the only available serological marker of earlyinfection and has been widely used to diagnose recently acquired vector-borne infections and congenitally transmitted cases (Mallimaci et al., 2010;Russomando et al., 2010; Volta et al., 2015).

More sophisticated, laborious and difficult-to-interpret methods werealso developed. These include testing of the newborn sample byimmunoblot against the TESA fraction (Table 1) (Umezawa et al.,1996a) and, more recently, the Chunap (Chagas urine nanoparticle) test(Castro-Sesquen et al., 2014). In the latter method, instead of T. cruzi-specific antibodies, active infection is indirectly assessed by the captureand concentration of T. cruzi TESA antigens from patient’s urine, whichare then revealed using a monoclonal antibody directed to a parasitelipophosphopeptideglycan, included in the test (Castro-Sesquen et al.,2014). When evaluated on children living in endemic areas and tested atthe peak of parasitaemia (w1-month old), Chunap was able to accuratelydiagnose congenital infections (Castro-Sesquen et al., 2014) and isnow being evaluated for other diagnostic applications (Castro-Sesquenet al., 2016).

On a final note, it should be stressed that given the high efficacy oftrypanocidal drugs in infected newborns, their rapid diagnosis andsubsequent treatment is essential. Intensive screening for distinctive immuneresponses (i.e., those based on IgM and/or IgA antibodies) and/or forspecific molecular signatures that may translate into surrogate biomarkersfor early detection of congenital T. cruzi transmission should be thusconsidered a top-ranking priority in Chagas disease applied research.




4.2 Rapid assessment of therapy efficacyOverall, the best chemotherapy results have been achieved in acute or earlychronic infections. However, even in children the cure rate is up to 62% at2 years of follow-up, although this figure may vary according to populationand geographical location (Ribeiro et al., 2009). This variability may beattributed in part to differences in the prevalence of human-associatedgenotypes across endemic areas. Indeed, substantial interstrain variations indrug resistance have been ascertained both in in vitro systems and in animalinfection models (Fig. 1) (Moraes et al., 2014; Toledo et al., 2003).

During the chronic stage, the efficacy of drug treatment is lower andvariable and is also difficult to assess because most studies used different treat-ment regimens and outcome evaluation methods (variable assays, frequencyand duration of follow-up) (for a recent review, see Pinazo et al., 2014).Even in successful treatments, the gold standard for evaluating drug efficacy,which relies on consistent negative results using conventional parasitologicaland whole parasite-based serological tests, may take years to decades toassess, thus stressing the necessity of novel and improved therapeuticresponse markers.

Several therapeutic studies support the usefulness of PCR-basedstrategies to evaluate treatment outcome in congenital or acute cases ofChagas disease. Moreover, PCR-based methods may also assist, thoughwith suboptimal performance due to lower parasitemias, in evaluatingchemotherapy in chronic cases of Chagas disease (Britto et al., 1995; Galvaoet al., 2003; Munoz et al., 2013; Russomando et al., 1998; Schijman et al.,2003). In particular, PCR-based methods seem to be a rapid indicator of theparasite’s susceptibility to drugs, allowing early therapy modification in casesof resistance or reactivation of Chagasic infection (Lages-Silva et al., 2002;Pinazo et al., 2014; Schijman et al., 2000).

In 1982, Drs. Krettli and Brener in Brazil demonstrated the existence of aspecial type of antibodies, which they termed ‘antibodies against living bloodforms’, that were absent in parasitological cured hosts early after drug treat-ment (Krettli and Brener, 1982). These antibodies were shown to participatein resistance against T. cruzi and were solely detectable by complement-mediated lysis (CoML) assays (Krettli and Brener, 1982). Upon thesefindings, the authors proposed the inclusion of the CoML technique inthe context of cure criteria. Indeed, the CoML technique offered animportant contribution for posttherapeutic monitoring of Chagas diseasein clinical trials, mostly by significantly reducing the required follow-upperiods (Table 1) (Galvao et al., 1993). Numerous improvements in terms

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of the sensitivity and operational safety of the CoMLmethod have been lateron introduced (Alessio et al., 2014; Martins-Filho et al., 2002; Vitelli-Avelaret al., 2007). Most importantly, this concept of ‘nonconventional’ serolog-ical responses sets the basis for multiple screenings looking for parasitemolecules that could be the target of antibody responses showing differentqualitative, quantitative and/or kinetic properties. Several potentialbiomarkers have been thereby identified and evaluated for their postthera-peutic application (Andrade et al., 2004; Fernandez-Villegas et al., 2011;Gazzinelli et al., 1993; Laucella et al., 2009; Machado-de-Assis et al.,2012; Sanchez Negrette et al., 2008; Silva et al., 2002). They includeddifferent parasite fractions (i.e., TESA, F2/3), individual recombinantantigens belonging to the ‘parental repertoire’ such as F29/FCaBP/Tc-24/Tc-28/1F8 (Sosa Estani et al., 1998) and other molecules such asTSSA (our unpublished results) (Table 1). Overall, the best results wereobtained with the F2/3 fraction (Table 1) (Andrade et al., 2004; de Andradeet al., 1996). Interestingly, antibodies directed to a-galactosyl-containingepitopes (i.e.. the main antigenic determinant in the F2/3 fraction) werelater on shown to induce trypomastigote lysis independently of complement(Pereira-Chioccola et al., 2000).

More recently, the group of Dr. Tarleton explored the use of a multi-pronged approach based on the simultaneous evaluation of T. cruzi-specificB cell and T cell responses as an alternative way of measuring treatmentefficacy (Alvarez et al., 2016; Laucella et al., 2009). By doing so, a declinein the frequency of IFNg-producing T cells and in antibody titresmeasured by a previously developed recombinant multiplex serologicalassay (Cooley et al., 2008) were observed shortly after benznidazoletreatment and were thus proposed as surrogate markers for refining theposttherapeutic cure criterion (Albareda and Laucella, 2015; Alvarezet al., 2016; Laucella et al., 2009). Identification of novel biomarkers forearly evaluation of antitrypanocidal drug efficacy will fasten and improvethe assessment of current chemotherapy treatments in clinical trials and,importantly, will be instrumental for the development of novel andimproved treatments.

4.3 Indication/prediction of Chagas disease progressionAs mentioned, Chagas disease evolves into a wide range of pathologicalsymptoms, ranging from subclinical to potentially fatal megasyndromes(Rassi et al., 2010). Identification of diagnostic and/or predictive biomarkersof disease progression would therefore represent a major achievement




towards improving the clinical management of Chagasic patients. Someauthors proposed serological alternatives to tackle this issue, such asquantifying the antibody titres to Ag1/JL7/FRA/H49 to distinguishbetween Chagas disease-associated cardiac pathology and other non-Chagasic cardiological dysfunctions (Table 1) (Abraham and Derk, 2015;Bhattacharyya et al., 2014; Kaplan et al., 1997; Levin and Hoebeke, 2008;Thomas et al., 2012). On the other hand, antibodies to another ‘parentalrepertoire’ antigen, termed JL5, were shown to cross-recognize endogenousb1-adrenergic and M2 muscarinic receptors. Such ‘autoantibodies’ wereshown to be associated with arrhythmogenic anomalies, which maycontribute to cardiac alterations in Chagasic patients (Kaplan et al., 1997),and were thus proposed to be included in the context of disease prognosis(Table 1). More recently, a certain correlation between serological responsesto TSSA and electrocardiogram abnormalities was also noted (Table 1)(Bhattacharyya et al., 2014).

Alternatively, several groups attempted to identify biochemical markersof cardiac damage and/or inflammation such as TNF-a (Talvani et al.,2004a), angiotensin-converting enzyme 2 (ACE2) (Wang et al., 2010), brainand atrial natriuretic peptides (BNP and ANP, respectively) (Garcia-Alvarezet al., 2010; Heringer-Walther et al., 2005; Ribeiro et al., 2002) ascandidates for disease prognosis (Table 1). In particular, the concentrationsof BNP and ANP in serum were systematically studied as markers of heartdamage in Chagasic patients (Garcia-Alvarez et al., 2010; Heringer-Waltheret al., 2005; Ribeiro et al., 2002) since they had been previously related withcardiovascular diseases (Wang et al., 2006; Wondergem et al., 2001).Increased concentrations of BNP and ANP strongly correlated with theseverity of Chagas-associated cardiac damage, being BNP more sensitivethan ANP (Fernandes et al., 2007; Heringer-Walther et al., 2005; Talvaniet al., 2004b). Upon these findings, the authors proposed that BNP couldbe measured periodically in asymptomatic patients as screening test to detectincipient ventricular dysfunction (Heringer-Walther et al., 2005). On thesame lines, higher levels of plasma ACE2, which catalyses the conversionfrom angiotensin II to angiotensin 1e7 (Keidar et al., 2007), were shownto correlate with clinical severity and worsening echocardiographicparameters in Chagasis patients (Wang et al., 2010). More importantly,given that the combined determination of BNP concentration and ACE2activity had better positive predicted value than when separately analysed,authors encouraged the use of both markers to predict fatal outcomes(Wang et al., 2010).

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On the same lines, the role of endothelin-1 (ET-1) as a prognosticmarker for T. cruzi-induced pathogenesis has also been extensively studied,though in animal models. ET-1 is a vasoactive peptide synthesized by manycell types including cardiac myocytes and cardiac fibroblasts associated withvasospasm, vascular damage, cardiovascular remodelling and inflammation(Kedzierski and Yanagisawa, 2001). Mice infected with T. cruzi exhibitincreased levels of ET-1 and endothelin-converting enzyme (ECE), theenzyme responsible for the conversion of the precursor to ET-1, in plasma,in the vasculature and in T. cruzi-infected myocardial cells (Huang et al.,2000; Petkova et al., 2000). Interestingly, phosphoramidon, an inhibitorof ECE, ameliorated the pathology and reduced the extent of cardiacremodelling in these animals (Jelicks et al., 2002). Moreover, ET-1 KOmice showed certain protection against chronic Chagasic cardiomyopathy(Tanowitz et al., 2005).

Overall, and despite multiple and disparate attempts, none of theserological and/or biochemical markers that have been explored so fartranslated into a reliable, easy to assay and interpret marker to assess cardiacdamage and/or disease prognosis (recently reviewed in Pinazo et al., 2015).The complex and likely multifactorial nature of Chagas disease pathogenesistogether with intrinsic difficulties in establishing appropriate experimental/epidemiological models converge in turning this area as the most challengingin terms of T. cruzi biomarker discovery.

4.4 Typing of parasite strainsPioneer studies aimed at fingerprinting the infecting parasite strain(s) directlyin clinical samples were based on biochemical markers (i.e., MLEE,multilocus enzyme electrophoresis) (Tibayrenc and Ayala, 2015). Recentadvances in typing schemes based on RFLP (restriction fragment lengthpolymorphism), PCR-RFLP, RAPD (random amplification of polymor-phic DNA), DNA hybridization, karyotyping and, particularly, sequence-based markers either using a single locus or multiple loci have greatlyimproved parasite genotypic resolution in vitro (Table 1) (Tibayrenc andAyala, 2015). However, in vivo, these genotyping methods are time-consuming and costly and require parasite isolation and amplification or ahigh quantity of DNA, therefore necessitating invasive sampling withmedical risks. Moreover, the fact that concurrent infections with multipleT. cruzi strains appear to be the norm rather than the anomaly (Perezet al., 2014) and the discovery that the most prevalent T. cruzi genotypepresent in the bloodstream can differ from the strain(s) found sequestered




within organs (Manoel-Caetano Fda et al., 2008; Vago et al., 2000) furthercomplicate this task. In this context, serotyping methods emerge as anappealing alternative to overcome these limitations, as demonstrated in otherhuman infectious diseases (Dunbar et al., 2015; Maksimov et al., 2012).Serotyping is based on the use of specific antigens with qualitative and/orquantitative differences among parasite strains/DTUs to detect strain-specific antibody signatures.

TSSA (Di Noia et al., 2002) is a parasite adhesin displaying significantsequence homology to TcMUC, a huge family of polymorphic genes thatcode for the polypeptide backbones of the trypomastigote mucin molecules(Buscaglia et al., 2004; Campo et al., 2006), some of which are terminallydecorated with a-galactosyl residues and constitute the F2/3 antigenicfraction. Interestingly, detailed genetic characterization of the TSSA locusdisclosed minor sequence variations between TSSA variants expressed bydifferent parasite DTUs (Bhattacharyya et al., 2010; Di Noia et al., 2002).Some of these variations were shown to have major impact on TSSAbiological function and antigenicity, thereby leading to differential antibodyprofiles between variants (Balouz et al., 2015; Bhattacharyya et al., 2014;Canepa et al., 2012; De Marchi et al., 2011; Di Noia et al., 2002). So far,TSSA remains the only polymorphic antigen that has been successfullyused for the development of DTU-specific serology methods for T. cruziinfections of humans (Table 1) (Bhattacharyya et al., 2014; Bisio et al.,2011; Burgos et al., 2010; Longhi et al., 2014; Risso et al., 2011). Despitethis, the resolution and specificity of TSSA-based serotyping assays needto be improved. Discrimination between certain DTUs remains challengingas they possess identical or almost identical TSSA alleles or they are poorlyimmunogenic (Bhattacharyya et al., 2014; Canepa et al., 2012).

Serotyping could be a rapid, sensitive, cost-effective and relativelynoninvasive alternative to stringent T. cruzi genotyping in humans andmay also be used in animal reservoirs for epidemiological studies(Bhattacharyya et al., 2015; Cimino et al., 2011). Most importantly,development of novel serotyping tools will facilitate the unravelling ofpossible relationships between parasite genetic variability and clinicalfeatures, a major issue in Chagas disease applied research.

4.5 Point-of-care diagnosisPeople living in Chagas disease-endemic areas have restricted access tolaboratory facilities and/or to appropriate health centres. This, togetherwith the associated costs and expertise needed for conventional diagnostic

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methods, points to a number of technological and economic barriers thatfurther stress the need to deploy PoC tests for T. cruzi infection diagnosis.PoC devices, in addition, allow the patients to see the results for themselves,which contributes to a better working relationship between localcommunities and people carrying out the testing (e.g., during field surveys).Moreover, the need for follow-up visits to surveyed individuals and there-fore the operational costs and risks of possible attrition bias are reduced.From an epidemiological point of view, a reliable PoC test would allowintervention strategies to be implemented in situ, such as for serologicsurveillance, vaccine or clinical trials; as well as for rapid initiation of treat-ment of infected individuals during outbreaks of acute cases, such as thoserecently reported (Alarcon de Noya et al., 2010; Segovia et al., 2013).

Several rapid diagnostic and PoC tests to detect infection based onimmunochromatography, particle agglutination, immunofiltration,immunodot, lateral-flow immunochromatographic assays (LFIA) or DNAdetection are available for a variety of infectious diseases (Nair et al.,2016; Natoli et al., 2014; Teles and Fonseca, 2015). These may be eitherqualitative or semiquantitative and are characterized by the delivery of quickresults, in most cases without the need for electrical equipment. In the caseof Chagas disease, several LFIAs based on antigenic fractions or recombinantantigens and a single PoC test aimed at the capture and concentration ofT. cruzi TESA antigens in urine samples (Chunap, see earlier discussion)were developed (Table 1) (Castro-Sesquen et al., 2014; Houghton et al.,2009; Reithinger et al., 2010). Serological tests, however, show substantialvariations in their sensitivity in different geographical areas (Verani et al.,2009) and display suboptimal performances (Sanchez-Camargo et al.,2014). Unfortunately, and despite being widely used in different parasiticdiseases (Mondal et al., 2016; Sriworarat et al., 2015), PCR-based methodsapplicable in PoC settings such as those based on simple colorimetric loop-mediated isothermal amplification or recombinase polymerase amplification(RPA) techniques are not yet available for T. cruzi detection.

As a first step towards the development of alternative PoC devices fordiagnosis, a new diagnostic platform based on superparamagneticmicrobeads coated with recombinant antigens and fluorescent (FMBIA)or electrochemical (EMBIA) detection was recently developed (Cortinaet al., 2016). The EMBIA platform, including antigen-functionalizedmagnetic microbeads, disposable electrochemical cells-electrode cartridgesand a portable potentiostat, was evaluated for serodiagnosis of human andcattle infectious diseases. In the particular case of Chagas disease, a more




extensive validation was performed showing that the EMBIA platformdisplayed an excellent diagnostic performance almost indistinguishablefrom the well-established ELISA methods (Cortina et al., 2016).

5. DIAGNOSTIC APPLICATIONS FOR CHAGASDISEASE: THE ROAD AHEAD

As discussed throughout this chapter, parasite-specific immunesignatures provided a prime source of biomarker candidates for developmentof Chagas disease diagnostic applications (see Table 1). However, they maynow be reexplored using modern and powerful ‘omics’-based fingerprintapproaches. Indeed, the availability of complete T. cruzi genomes fromseveral strains together with a variety of recent genome mining exerciseswas performed to identify potential serodiagnostic reagents and vaccinecandidates (Bhatia et al., 2004; Carmona et al., 2012; Cooley et al., 2008;Goto et al., 2008; Reis-Cunha et al., 2014). Importantly, most of theantigens that emerged from these in silico-guided screenings were notincluded in the ‘parental repertoire’ (Table 1) and could thereby entail noveldiagnostic capabilities, such as early assessment of drug efficacy (Alvarezet al., 2016; Laucella et al., 2009). Genome-wide approaches could bealso used as a starting point to identify novel DNA-based DTU resolutionmarkers (Cosentino and Aguero, 2012) and/or novel serotyping reagents.As previously shown, genetic variation among T. cruzi DTUs translatesinto differential proteomes (Telleria et al., 2010) and therefore also likelyin distinct epitope collections (‘epitomes’) recognized during humaninfections. By carrying out genome-wide B cell epitope prediction onproteins derived from allelic pairs of the hybrid T. cruzi CL Brener genome,the group of Dr. Bartholomeu in Brazil was able to identify three novelpolymorphic epitopes potentially able to discriminate between parasiteDTUs (Mendes et al., 2013), although one of them turned out not to beDTU specific (Bhattacharyya et al., 2014). Unfortunately, this study andthose mentioned earlier were somehow limited by the use of low-to-medium throughput antigen expression/synthesis approaches and/or bythe use of nonhuman sources of serum samples.

Recent advances in computerized photolithography and photochem-istry, however, have allowed the development of a novel peptide chiptechnology where up to 1 million individual peptides are synthesized insitu on a glass slide (Buus et al., 2012). This in situ synthesis makes theproduction of the chip highly cost-effective and allows, for the first time,

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to interrogate complete proteomes. Recent work in our laboratoriesdemonstrated both the high technical reproducibility and epitope mappingconsistency of this platform when compared with earlier technologies(Balouz et al., 2015; Carmona et al., 2015). Most importantly, by screeningthe complete length of 457 parasite proteins (w7% of the T. cruzi deducedproteome) we were able to identify 2031 Chagas disease-specific peptidesand 97 novel parasite antigens (Carmona et al., 2015). Together withabove-mentioned studies, and besides emphasizing the huge potential ofgenomic/proteomic methods as major driving forces in antigen discovery,these findings indicate that a vast majority of the T. cruzi antigenic repertoireremains uncharacterized. We aim now at interrogating the entire T. cruzideduced proteome, including interstrain polymorphisms revealed bypreviously developed genetic diversity maps (Ackermann et al., 2012;Panunzi and Aguero, 2014). The use of samples collected from particularpatients populations such as infected with different parasite DTUs orshowing distinct Chagas disease-associated pathologies followed by a finalintegration of the emerging data will put us in position to discriminatebetween the common linear B cell ‘epitome’ of T. cruzi and sets of epitopesshowing differential recognition among distinct Chagas disease populations.

Considering the huge functional and diagnostic significance of carbohy-drates on T. cruzi biology (de Lederkremer and Agusti, 2009), it could behypothesized that similar high-throughput approaches carried out on glycanand/or lectin microarrays (Fernandez-Tejada et al., 2015) will have a majorimpact on Chagas disease biomarker discovery. These methodologies havebeen successfully explored in different parasitic infections (Anish et al.,2013; Aranzamendi et al., 2011; Martin et al., 2013).

From a wider perspective, high-throughput strategies may be alsopursued to develop novel biomarkers based on the detection of T. cruzi-derived molecules (Castro-Sesquen et al., 2014; de Titto and Araujo,1988) and/or parasite-induced modifications on host molecules and/or cells(Li et al., 2016; Mucci et al., 2002; Muia et al., 2010; Risso et al., 2007;Trocoli Torrecilhas et al., 2009). Theoretically, these strategies wouldhave the additional advantage to discriminate between active from pastinfections and/or to assist in monitoring disease progression, as validatedin other human diseases (Karsdal et al., 2010; Leeansyah et al., 2013; Trittenet al., 2014). In this context, it is important to keep in mind (1) that theextent of variability due to host genetic background on setting these putativebiochemical markers and (2) that T. cruzi-derived molecules, and particu-larly during the chronic phase of Chagas disease, are present in biological




fluids at extremely low concentrations and likely aggregated in membrane-coated vesicles (Bayer-Santos et al., 2013; Fernandez-Calero et al., 2015;Lantos et al., 2016; Trocoli Torrecilhas et al., 2009).

Nevertheless, modern ‘-omic’ technologies are offering alternativestrategies for such a difficult system biology exploration (Cantacessi et al.,2015; Hockl et al., 2016; Preidis and Hotez, 2015). In particular, massspectrometry (MS) methods provide a robust, versatile and sensitiveanalytical technology allowing high-throughput detection with massaccuracy, precise quantitation and verification of protein variants, spliceisoforms, metabolites and disease-specific posttranslational modifications(Crutchfield et al., 2016). Indeed, using MS-based approaches, globalchanges in metabolic profiles in Chagasic patients showing acute myocarditis(Girones et al., 2014) and serological biomarkers showing differencesbetween Chagasic and healthy subjects (Santamaria et al., 2014) wererecently identified. In the latter case, biomarker peaks with the best discrim-inatory power were further characterized by a range of proteomic andimmunological techniques, indicating that specific fragments derived fromproteolysis of apolipoprotein AeI and one fragment of fibronectin arespecifically upregulated in Chagasic patients (Santamaria et al., 2014). Inter-estingly, these biomarkers returned to normal values more rapidly thanwhole parasite-based serological tests in patients treated with nifurtimox,thus supporting their potential use for evaluation of therapeutic efficacy(Table 1) (Santamaria et al., 2014).

In addition to the obvious impact in the diagnostic application field, it isexpected that the utilization of these and other high-throughput, ‘-omic’techniques will provide a vast amount of putative biomarkers to be exploredin other Chagas disease research areas such as molecular epidemiology andprioritization of targets for vaccine development. Most importantly,together with appropriate animal models and robust bioinformatics,molecular and cellular tools (Aguero et al., 2008; Crowther et al., 2010;Katsuno et al., 2015; Lewis et al., 2015; Magarinos et al., 2012; Moraeset al., 2014), these biomarkers will be instrumental to screen, prioritizeand evaluate safety and efficacy of novel drugs/treatments.

6. CONCLUDING REMARKS

Over 100 years after its discovery, and despite its huge medical,economic and social burden, Chagas disease remains a major threat in several

26 V. Balouz et al.



countries of Latin America and an emergent global health problem. Greatefforts have been made and are still being made in Latin America and otherdeveloped countries to halt T. cruzi transmission. However, one of the keyissues concerning Chagas disease control remains that of diagnosis. Withoutaccessible and effective diagnostics tools and methods, infected individualscannot be timely identified and hence treated. Even if treated, the successof treatment cannot be efficiently assessed.

As discussed here, current diagnostic methods are highly accurate indetecting most of T. cruzi infections in humans. However, there are stillsome clinical and/or epidemiological situations in which their performanceis severely impaired. In addition, the complex epidemiological features ofChagas disease and the remarkable phenotypic diversity displayed by distinctT. cruzi strains, which may be associated to certain aspects of disease progres-sion/outcome, also stress the necessity of developing new methods able tobe deployed in PoC settings and capable of typing the infecting parasite(s)directly in clinical samples. The implementation of modern ‘-omic’, high-throughput and aggressive strategies constitutes an appealing alternative tomove on towards filling current diagnostic gaps. Emergent diagnostic testsintegrating these novel and tailored tools will provide a significant impacton the effectiveness of current intervention schemes and, most importantly,will improve the clinical management of Chagasic patients by providing theintervening physician with an accurate and integrated diagnosis.

ACKNOWLEDGEMENTWe express our gratitude to Dr. Carlos Frasch (IIB-INTECh) for critical reading of themanuscript and to Dr. Javier Di Noia (IRCM, Canada) for the enthusiasm and superb graphicart. We apologize to people whose work was not referenced due to limited space. Researchcarried out in our labs is supported by grants and contracts from the Agencia Nacional dePromoci�on Científica y Tecnol�ogica (ANPCyT, Argentina) (to FA and CAB), the NationalInstitutes of Health (NIAID/NIH, USA) (to FA), and Fundaci�on Bunge y Born (Argentina)(to CAB). VB holds a PhD fellowship, and FA and CAB are career investigators from theNational Research Council of Argentina (CONICET).

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