Acta Tropica 184 (2018) 59–66

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Acta Tropica
journal homepage: www.elsevier.com/locate/actatropica

Molecular diagnosis of Trypanosoma cruzi T

Alejandro G. Schijman
Laboratorio de Biología Molecular de la Enfermedad de Chagas, Instituto de Investigaciones en Ingeniería Genética y Biología Molecular “Dr. Hector Torres” (INGEBI-
CONICET), Ciudad de Buenos Aires, Argentina

A R T I C LE I N FO A B S T R A C T

Keywords: Chagas disease, caused by the kinetoplastid protozoan Trypanosoma cruzi, affects millions of people, most of
Chagas disease them neglected populations. The different phases of the disease, the transmission mode and the high genetic
Trypanosoma cruzi variability of the parasite determine that molecular detection methods display different degree of success.
Quantitative real time PCR Molecular diagnostic tests may be employed during epidemiological surveys of transmission, for early diagnosis
Loop-mediated isothermal amplification
of congenital transmission and acute infections due to oral transmission, transfusion or transplantation routes,
Molecular diagnosis
reactivation due to immunosuppression and monitoring of treatment response in chronically infected patients
Discrete typing units
receiving trypanocidal chemotherapy. This manuscript summarizes the most widely used molecular tools to
detect T. cruzi infection in different epidemiological and clinical scenarios.

1. Introduction 2. Molecular detection and T. cruzi gene diversity

Chagas disease (CD), caused by Trypanosoma cruzi is most likely “the
most neglected of the neglected diseases” (WHO, 2012). It has been
treated as an endemic disease in tropical and subtropical areas of
Southern and Central America, Mexico and Southern United States
(Garcia et al., 2017), and is an emerging global distress in non-endemic
areas (Schmunis and Yadon, 2010). Once vectorial and transfusional
control have been achieved, perpetuation of infection occurs mainly
through congenital transmission in endemic and non-endemic areas
whereas in rural zones, outbreaks of oral infection are more significant
(Alarcón de Noya et al., 2010; Shikanai-Yasuda and Carvalho, 2012).
The infection traverses an acute phase, evolving to an asymptomatic or
symptomatic chronic phase, with different degrees of progression and
severity (Rassi et al., 2010).
Clinical molecular diagnosis of CD is important for: (i) early diag-
nosis of congenital transmission in newborns when presence of ma-
ternal anti-T.cruzi antibodies may deliver false positive results and
microscopic observation lacks sensitivity, (ii) diagnosis of oral infec-
tions, (iii) early detection of infection in receptors of organs from CD
donors, (iii) monitoring of reactivation in chronically infected patients
immune-suppressed due to transplantation or AIDS and (iv) evaluation
of treatment response, because detection of serological negative con-
version in treated patients with a favorable outcome may take many
years to occur.
The genetic structure of T. cruzi populations is mainly a con-
sequence of clonal propagation with rare events of genomic exchange
(Tibayrenc et al., 1986; Sturm and Campbell, 2010). Biological, bio-
chemical and molecular markers demonstrated genetic polymorphism
(Macedo et al., 2004; Miles et al., 2009). Nowadays, natural popula-
tions are classified into six discrete typing units (DTUs TcI to TcVI),
composed of sets of stocks genetically closer to one another than to any
other one (Zingales et al., 2012). In addition, Tcbat has been recently
proposed as an independent DTU (Lima et al., 2015). DTUs are iden-
tifiable by specific molecular markers, they depict particular geo-
graphical distribution, harbor different DNA content and gene dosage
and may have preferential tropism for vector and reservoir species as
well as tissue tropism within an infected host (Burgos et al., 2010, 2005;
Miles et al., 2009; Lewis et al., 2009; Telleria et al., 2006; Vargas et al.,
2004). DTUs I to VI are all causative of CD (Zingales et al., 2012). This
genetic diversity must be taken into account when developing mole-
cular diagnostic tests for worldwide applications.
3. Nucleic acid amplification methods
3.1. Polymerase chain reaction
Since the nineties, the Polymerase Chain Reaction (PCR) was pro-
posed as the molecular tool of choice for sensitive detection of T. cruzi
infection and monitoring of trypanocidal chemotherapy (Avila et al.,
1993; Britto et al., 1995; Moser et al., 1989).

E-mail address: schijman@dna.uba.ar.

https://doi.org/10.1016/j.actatropica.2018.02.019
Received 24 July 2017; Received in revised form 5 February 2018; Accepted 14 February 2018
Available online 21 February 2018
0001-706X/ © 2018 Elsevier B.V. All rights reserved.
A.G. Schijman
Different combinations of molecular targets, sets of primers and
probes, DNA extraction methods and amplification platforms have been
reported with variable degrees of sensitivity, specificity and accuracy,
which depended on the epidemiological and clinical groups and type
and volume of tested samples, among other factors (Ramírez et al.,
2009; Brasil et al., 2010; Russomando et al., 1998; Schijman et al.,
2003; Diez et al., 2007; Murcia et al., 2010; Piron et al., 2007). To
identify the best performing methods, the Special Programme for Re-
search and Training in Tropical Diseases (TDR-WHO) supported a
comparative international study of PCR for detection of T. cruzi in
peripheral blood samples. This project challenged 48 PCR procedures
against a blind panel containing DNAs from different parasite stocks,
spiked seronegative blood samples and peripheral blood obtained from
seropositive patients and seronegative controls. Upon analysis of sen-
sitivity and specificity, four best performing methods were selected for
standardization and intra-laboratory validation; these were all based on
highly repetitive satellite DNA (SatDNA) or minicircle DNA (kDNA)
sequences, whereas methods targeted to other nuclear sequences, such
as 18S rDNA, 24S rDNA or spliced-leader intergenic regions did not
reach enough sensitivity for diagnostics applications in human samples
(Schijman et al., 2011). Later on, Real Time PCR was developed with
the possibility of parasitic load quantification (Piron et al., 2007; Duffy
et al., 2009, 2013; Moreira et al., 2013). Upon standardization and
analytical validation, Sybr Green Real Time PCR or duplex TaqMan
qPCR procedures directed to nuclear satellite DNA (SatDNA) or the
minicircle molecule (kDNA) – plus an internal amplification control
have been mostly used, in particular for treatment monitoring (Duffy
et al., 2013; Moreira et al., 2013; Ramírez et al., 2015). Duplex qPCR
using hydrolysis probes has the advantage of allowing internal control
of DNA degradation and/or PCR inhibition in the same tube reaction,
which can not be accomplished using SybrGreen or other DNA inter-
calating dyes. In standardized duplex qPCR assays, analytical sensitivity
of kDNA qPCR was somewhat higher than that of SatDNA qPCR, with
limits of detection of 0.234 and 0.698 parasite equivalents/mL, re-
spectively. High concordance was observed between both methods in
proficiency panels and clinical specimens (Ramírez et al., 2015). Ana-
lytical sensitivity was more uniform among different DTUs for kDNA
qPCR than for SatDNA qPCR, being the latter less sensitive for some TcI
and TcIV strains, indicative of a lower gene dosage in their genomes
(Ramírez et al., 2015; Duffy et al., 2009). In regions where T. rangeli
might be the cause of confounding diagnosis with T. cruzi (Guhl and
Vallejo, 2003), kDNA qPCR could lead to false positive results due to T.
rangeli because the minicircle region annealing with primers and probe
is highly conserved and repeated in both trypanosomatid species. In
fact, T. rangeli has been detected in infants in endemic areas for CD
(Saldana et al., 2005). Therefore, in these regions, a standardized qPCR
assay based on SatDNA sequences could be recommended, because T.
rangeli presents very low copy numbers of this sequence.
The Table 1 summarizes examples of PCR studies of clinical sensi-
tivity and specificity carried out using Satellite and/or kinetoplastid
DNA repetitive sequences as molecular targets from blood specimens of
at least 100 subjects with suspicion or previous diagnosis of T.cruzi
infection. Duplex Sat-DNA qPCR performed in peripheral blood samples
from patients residing in Colombia, where Tc I is the prevailing DTU,
showed high specificity in both acute and chronic Chagas disease pa-
tients and high sensitivity in acute patients, whereas in chronic patients
clinical sensitivity reached 64.2%, probably due to the lower parasitic
loads and intermittent release of trypomastigotes to bloodstream during
chronic infection (Table 1; Hernández et al., 2016a,b). This degree of
PCR positivity to detect circulating T.cruzi in chronically infected pa-
tients has been also observed in many other studies (Table 1). Ac-
cording to a recent work by Seiringer and coworkers (Seiringer et al.,
2017) the use of two methods, one targeting kDNA and the other Sat-
DNA, should be performed for more accurate diagnosis. This has been
done to enroll and follow-up chronic Chagas patients in a prospective
study of intermittent benznidazole chemotherapy (Álvarez et al., 2017).
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Acta Tropica 184 (2018) 59–66
If one of the two tests is positive, the patient has to be considered as
PCR-positive. Infection with a strain that has a low copy number or
specific mutations of Sat-DNA, which cannot be detected when tar-
geting Sat-DNA, is one possible explanation for such divergent results.
Sequence polymorphism of SatDNA due to DTU differences must be
considered to improve SatDNA PCR sensitivity, especially in Tc I and Tc
IV infected cases (Ramírez et al., 2017b).
3.2. Loop mediated amplification
Thanks to a complex design of primers, auto-strand displacement
DNA synthesis and Bst DNA polymerase, LAMP is able to amplify large
amounts of DNA or RNA within 30–60 min of incubation (Adams et al.,
2010; Hayashida et al., 2015). The reaction occurs between 60 and
65 °C, so it can be done without the use of a thermocycler (Mori et al.,
2001; Notomi et al., 2000). Visualization of amplification can be ad-
dressed by the naked eye and followed in real-time by measuring tur-
bidity or fluorescence using intercalating dyes. In-tube visualization
may be achieved using manganese loaded calcein which starts fluor-
escing upon complexation of manganese by pyrophosphate during DNA
synthesis. LAMP reagents are stable at room temperatures up to 37 °C,
avoiding the need of a cold chain (Njiru et al., 2008; Poon et al., 2006).
A first LAMP procedure was based on the 18s rDNA gene with an
analytical sensitivity of 100 fg of DNA per test and cross reactivity with
Leishmania sp and was evaluated in triatomine feces (Thekisoe et al.,
2010). The use of this target in a LAMP protocol tested in human blood
reached a detection level of 50 parasites/mL, which allowed detection
of congenitally infected patients (Rivero et al., 2017). A novel prototype
kit for detection of T.cruzi satDNA in human blood samples developed
by Eiken Company containing dried reagents on the inside of the caps in
microtubes reached high analytical accuracy, without cross-reactivity
with Leishmania sp or T. rangeli (Besuschio et al., 2017). Analytical
sensitivity was 1 × 10−2 fg/μL of CL Brener (Tc VI) and Sylvio X10 (Tc
I) DNAs and detected all DTUs, with some variations in the “time to
threshold”, due to the heterogeneity in copy numbers of satellite re-
peats (Duffy et al., 2009). It also detected 1 × 10−2 parasite equiva-
lents/mL in spiked EDTA blood extracted with commercial columns or
rapid Boil & Spin method and 1 × 10−1 par.eq/mL in spiked hepar-
inized blood using fiberglass columns for DNA extraction. It rendered
high sensitivity in acute, congenital and reactivated patients and less
sensitivity in chronic Chagas disease, in high concordance with qPCR
performed in the same samples (Besuschio et al., 2017).
4. Clinical specimens used for molecular diagnosis of CD
The type of clinical sample for diagnosis depends on the clinical
setting. Although most nucleic acid amplification SOPs were developed
for whole peripheral blood samples treated with Guanidine hydro-
chloride-EDTA stabilizing buffer, PCR based on frozen EDTA blood
alone or mixed with different stabilizing agents, PAXgene Blood DNA
tubes, blood spots in filter paper have been reported (Moreira et al.,
2013; Sánchez et al., 2016; Wei et al., 2016; Braz et al., 2008). In ad-
dition, heparinized blood was evaluated for LAMP (Besuschio et al.,
2017). Blood clots and serum samples showed adequate sensitivity in
some settings (Melo et al., 2015; Moreira et al., 2016; Russomando
et al., 1998). Umbilical cord blood and tissue and heel prick blood are
also candidate specimens for early diagnosis of congenital transmission
(Mora et al., 2005). Clinical samples other than blood have been tested
too. In the acute experimental model of Guinea pigs, DNA was detected
in urine, suggesting it could be valuable for acute human detection
(Castro-Sesquen et al., 2013). PCR detected parasite DNA in amniotic
fluid from an infected mother with a premature delivery (Nilo et al.,
2000). In chronic CD, T. cruzi DNA was amplified using different PCR
methods in a proportion of gum samples from cases with diverse de-
grees of gingival inflammation (Añez et al., 2011). In HIV coinfection,
reactivation can be detected in cerebral spinal fluid or brain biopsies
 Table 1
Clinical sensitivity and specificity of PCR assays based on Satellite and/or kinetoplastid DNA sequences carried out in blood samples from at least 100 subjects.
A.G. Schijman

Reference Country Study population Molecular target Clinical Sensitivity Clinical Specificity

Messenger et al. Bolivia Screening of 487 infants born to 476 SatDNA qPCR in cord blood 68.6% 99.1%
(2017) seropositive women: 38 Congenital CD patients
from 35 mothers
Hernández et al. Colombia 708 subjects: 86 with suspected Acute CD, 622 SatDNA PCR and Duplex Acute 71/86 pts: qPCR 95.7% (88.3–98.5), SatDNA Acute: qPCR and SatDNA 100% (79.6-100);
(2016a,b) with suspected Chronic CD TaqMan Sat DNA qPCR 84.5%(74.3–91.2); Chronic CD 481/622 pts: qPCR Chronic group qPCR 97.1 (92.9–98.8), SatDNA
64.2% (59.8–68.4), SatDNA 56.8% (52.3–61.1) 97.9 (93.9–99.2)
Ramírez et al. Endemic Countries Chronic CD (145): 70 asymptomatic, 75 with Duplex TaqMan Sat-DNA qPCR 80.69% (117/145) SatDNA; 84.14% (122/145) kDNA Not determined
(2015) cardiopathy or digestive megasyndromes and Duplex TaqMan kDNA qPCR
Lucero et al. (2016) Northern 308 Chronic CD: 238 from aboriginal kDNA PCR 42.15% aboriginal communities; 65.71% Creole Not determined
Argentina communities (Wichis, Pilagas, Mocoit) 70 from localities
Creole localities
Moreira et al. Brazil, Argentina,C 150 seropositive adult Chronic CD patients kDNA PCR and SatDNA SatDNA qPCR 71.3%, kDNA-PCR 80% 100% (30 SatDNA & qPCR negative/30
(2013) olombia SybrGreen qPCR seronegative)
Hidron et al. (2010) Bolivia 251 seropositive cardiopathy patients kDNA PCR 43% (109/251) 99.3% (1 PCR positive/143 seronegative)
Murcia et al. (2010) Spain Immigrants 181 seropositive treated patients with chronic kDNA PCR 68% (123/181) (100% in young patients, 72.1% in Not reported
CD: 64% asymptomatic and 36% symptomatic adults and 48.9% in seniors.
del Puerto et al. Bolivia 306 seropositive chronic CD patients kDNA PCR and SatDNA PCR 64.1% (196/306); 77.8% (238/306) Not determined
(2010)
Alarcón de Noya Venezuela 150 people potentially at risk of acute oral kDNA PCR 79.5% (35/44 serologically confirmed cases) 100% (106 PCR negative/106 seronegative)
et al. (2010) infection randomly chosen
Bern et al. (2009) Bolivia 154 seropositive mothers and their newborns kDNA PCR Mothers: 63% (97/154) Newborns First month: 6.5% 99.3% (137 PCR neg/138 samples from
(10/154). Follow up: 75% (27/36 specimens collected uninfected infants). Cord blood: 100% (109/109
prior to treatment). Cord blood: 67% (6/9). PCR neg).
Fernandes et al. Brazil 240 seropositive patients kDNA PCR Buffy-coat −70 °C: 86.7% (208/240); Buffy-coat 100% (50 PCR negative/50 seronegative)

61
(2009) Guanidine EDTA buffer: 71.7% (172/240); Whole blood
Guanidine EDTA buffer: 69.2% (156/240)
Ramírez et al. Colombia 100 chronic CD patients with cardiopathy kDNA PCR Solvent Extraction: 64%; DNA extraction kit: 54% 100% (10 PCR negative/10 seronegative)
(2009)
Fitzwater et al. Bolivia 520 Seropositive pregnant women (520 whole kDNA PCR Clot: 60.1% (89/148). Buffy coat: 46.5% (33/71); Whole 100%
(2008) blood, 516 clot, 208 buffy coat samples) blood: 40% (60/150)
Virreira et al. Bolivia 311 samples of neonatal blood previously tested SybrGreen SatDNA qPCR 18/18 congenital CD 0 PCR positive/30 control newborns of uninfected
(2003) with parasitological method mothers, 1 PCR positive/263 parasitologically
negative born to infected mothers
Galvão et al. (2003) Brazil 127 Tcruzi infected school children kDNA PCR plus Slot-blot 84.3% (95CI 77.1–89.8) Not determined
hybridization
Schijman et al. Argentina 152 children born to seroreactive mothers, kDNA PCR + southern PCR Group A: 100%; Group B: 73.8% Group A: 97%; Group B: 100%,
(2003) Group A: 50 infants aged 0–6 months; Group B: hybridization
102 children aged 7 months to 17 years
Brenière et al. Bolivia 372 subjects: 209 with chronic CD, kDNA PCR 83.8% Not determined
(2002) asymptomatic and cardiopaths
Solari et al. (2001) Chile Seropositive asymptomatic children (67) and kDNA PCR + southern Children: 100% (67/67). Adults: 69.3% (52/75) 100% (78 PCR neg/78 seronegative)
adults (75) hybridization
Ribeiro-dos-Santos Brazil Blood donors, 105 seropositive by all three SatDNA, KDNA, Nested kDNA Seropositive (by 3 techniques) SatDNA PCR 3.8% (2/ Inconclusive Serodiagnosis: SatDNA PCR 0/7;
et al. (1999) techniques, 70 seropositive by one or two 52); kDNA 4.5% (4/88), Nested kDNA 25.7% (27/105). kDNA 0/69; Nested kDNA 3/70
techniques (Inconclusive diagnosis)
Wincker et al. Bolivia 113 T. cruzi infected children kDNA PCR 93.8% 1 PCR positive/115 non-infected children
(1997)
Junqueira et al. Brazil 101 chronic CD patients kDNA PCR 59.4% Not reported
(1996)
Avila et al. (1993) Brazil 114 chronic CD patients kDNA PCR + southern 100% 18 PCR negative/18 seronegative blood donors
hybridization from non-endemic region, 2 PCR positive/3
visceral Leishmaniasis
Acta Tropica 184 (2018) 59–66
A.G. Schijman
(Burgos et al., 2005; Bern, 2012) and in transplanted patients re-
activation can be detected in endomyocardial biopsies or skin cha-
gomas (Diez et al., 2007; Burgos et al., 2010). Nevertheless, for diag-
nostic purposes, whole blood persists as the sample of choice.
5. Quality control assurance in molecular diagnosis of CD
Quality controls are fundamental for reliable molecular diagnosis.
False negative results can arise because of PCR inhibitors in the sam-
ples, which require the use of internal controls to detect inhibition and
enable discriminating true from false negative results. On the other
side, inadequate laboratory conditions may favor carry-over con-
tamination leading to false positive results. A first External Quality
Assurance system has been recently implemented to evaluate the per-
formance of molecular biology laboratories involved in qPCR based
follow-up in clinical trials of chronic Chagas disease cohorts (Ramírez
et al., 2017a). Proficiency testing panels containing seronegative blood
samples spiked with 1, 10 and 100 par. eq./mL of four T. cruzi stocks,
belonging to different DTUs, as well as negative controls were analyzed
simultaneously, blinded to sample distribution, at 4-month intervals in
different laboratories. Moreover, randomly selected blood samples from
patients were sent to the reference laboratory for retesting analysis.
Laboratories applied a same SOP (Duffy et al., 2013) with a high degree
of agreement, within and between laboratories, of qualitative results of
proficiency testing panels for all T. cruzi stocks. No significant differ-
ences were found between qualitative and quantitative qPCR results,
when clinical samples were retested (Ramírez et al., 2017a,b).
6. Application of nucleic acid amplification in epidemiological
and clinical scenarios
6.1. Oral infection
Oral transmission of T. cruzi has been associated mainly with the
consumption of food contaminated with triatomine feces or didelphid
secretions, leading to high morbidity and mortality (Silva-Dos-Santos
et al., 2017). It is the most important route in Brazilian Amazon and
Venezuela (Shikanai-Yasuda and Carvalho, 2012; Noya et al., 2015).
Other South American countries have also reported outbreaks asso-
ciated with food consumption (Ramírez et al., 2013; Blanchet et al.,
2014; Hernández et al., 2016a,b). In most outbreaks molecular methods
were fundamental for specific diagnosis and genotyping of the re-
sponsible strains. A real-time PCR method has been also developed for
detection of T. cruzi in açai pulp, to determine innocuity of this food
regarding oral transmission (de Souza Godoi et al., 2017).
6.2. Congenital infection
Current assays for early detection of congenital cases fail to diag-
nose more than half of infected neonates and 10 month follow-up for
serological diagnosis is poor. Molecular strategies in newborns/neo-
nates could enable earlier diagnosis and circumvent loss to follow-up
(Besuschio et al., 2017; Cura et al., 2017; Bua et al., 2013; Schijman
et al., 2003; Mora et al., 2005). A recent study carried out in Bolivia
showed a good cumulative sensitivity of qPCR done in cord blood and
peripheral blood at one month of age and revealed that infants with
clinical signs had higher parasite loads (Messenger et al., 2017). It is
still necessary to test standardized real-time procedures using lower
volumes of peripheral blood or using cord or heel prick blood, ideally
collected in solid supports such as filter paper. A kit prototype based on
duplex TaqMan Real-Time PCR (qPCR) that starts from 1 mL of cord or
peripheral blood mixed with a DNA stabilizer solution has been built
and is currently under field validation (Schijman et al. unpublished
results).
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Acta Tropica 184 (2018) 59–66
6.3. Prediction of congenital transmission in chronically infected pregnant
women
Treating infected women of childbearing age prevents congenital
Chagas disease (Sosa-Estani et al., 2009; Fabbro et al., 2014; Moscatelli
et al., 2015; Álvarez et al., 2017). A prospective study involving 144
seropositive pregnant women demonstrated that 18.8% of mothers with
a positive PCR result transmitted the infection (16 infected children out
of 85 pregnancies), while uninfected children were detected among 74
pregnancies when maternal PCR was negative (Murcia et al., 2017). Out
of a cohort of treated mothers, 92.1% had negative PCR results, com-
pared with 32.2% of untreated ones. No infected children were detected
from previously treated mothers, compared with 13.2% among un-
treated ones. Thus, PCR screening of T. cruzi-infected pregnant women
is a useful tool for predicting the risk of congenital transmission.
6.4. Acute infection by organ transplantation
Early detection of T. cruzi transmission allows prompt treatment of
donor-derived infections (McCormack et al., 2012; Cura et al., 2013).
The use of organs from seropositive donors could potentially be ex-
panded if nucleic acid amplification-based monitoring of recipients is
established in organ transplant units, with a substantial impact in
shortening the waiting period on the transplant lists. PCR was able to
detect acute infection in transplanted recipients between 13 and
224 days earlier than conventional serological assays and between 28
and 47 days earlier than the “Strout” method (Cura et al., 2013). LAMP
also detected parasite DNA in samples from transplanted patients
(Besuschio et al., 2017).
6.5. Chronic chagas disease
Different PCR and qPCR tests with adequate analytical performance
were used to evaluate sensitivity and specificity in peripheral blood of
chronic Chagas disease patients (Table 1; Schijman et al., 2011;
Ramírez et al., 2015, 2009; Moreira et al., 2013; Hernández et al.,
2016a,b; Brasil et al., 2010) and in all cases sensitivity was poor for
diagnostic purposes. This is likely due to the low bloodstream parasitic
loads and unpredictable intermittency of T. cruzi parasitaemia in the
chronic phase. A strategy to circunvect this limitation has been to
collect serial blood samples allowing increase the probability of DNA
detection in at least one of the samples and report such a case as PCR
positive (Seiringer et al., 2017, Torrico et al., 2018; Ramírez JC, Ph
Thesis, UBA, 2017). Nevertheless, up to date serological methods dic-
tate CD diagnosis in the chronic phase.
6.6. Treatment monitoring
Different PCR methods, mostly based on sat-DNA and kDNA targets
were used to evaluate treatment efficacy, with a high degree of varia-
bility in the level of detection at baseline and during post-treatment
follow-up. This is likely due, not only to the efficacy of the drugs per-se,
but also to several other factors, such as the phase of disease, the en-
demic region under study, the possibility of re-infections and the lack of
standardization and validation of the PCR procedures (Lana et al.,
2009; Fernandes et al., 2009; Lacunza et al., 2006; Fabbro et al., 2007;
Aguiar et al., 2012; Sguassero et al., 2015; Pinazo et al., 2013; Padilla
et al., 2017; Viotti et al., 2014; Schijman et al., 2011). Implementation
of standardized methods was then, crucial, to enable more accurate
measurements of parasitic response, and quantification, which has been
possible since the implementation of Real Time PCR was another ele-
ment towards the establishment of reliable measurements (Ramírez
et al., 2015).
In order to minimize adverse effects of Benznidazol, intermittent
treatment has been recently evaluated in a cohort of chronic patients,
using paired Real Time kDNA and SatDNA based Real Time procedures,
A.G. Schijman
with high agreement between both techniques, and promising findings
regarding response (Álvarez et al., 2017)
6.6.1. Monitoring of treatment with nitroheterocyclic compounds
The nitroheterocyclic compounds benznidazole and nifurtimox were
developed over 4 decades ago and showed to be highly effective in
acute, congenital and early chronic (pediatric) Chagas disease, but
observational studies in chronically infected adults showed a lower
efficacy with frequent adverse effects. In house as well as standardized
blood based PCR and qPCR techniques have being consistently used to
detect therapeutic response or failure with Benznidazole (Morillo et al.,
2015, 2017; Riarte et al., 2005; Moreira et al., 2013; Murcia et al.,
2017) and Nifurtimox (Muñoz et al., 2013; Jackson et al., 2013; Bianchi
et al., 2015). A Systematic Review of Follow-Up Studies of clinical trials
in chronic patients included 54 studies (six randomized clinical trials
and 48 cohort studies) of Benznidazole or Nifurtimox out of 2136 ci-
tations screened (Sguassero et al., 2015). Positivity of PCR was char-
acterized by a sharp decrease at twelve months post-treatment,
reaching 40% of positive findings in the long-term. Randomized trials
were judged as low risk of bias in all domains. The main sources of bias
identified across cohort studies were the lack of control for confounding
and attrition biases. The BENEFIT study (Marin-Neto et al., 2008;
Morillo et al., 2015, 2017),that was the first randomized, double-blind,
placebo controlled, multi-centric trial evaluated the efficacy of benz-
nidazole on the clinical evolution of chronic Chagas’ cardiac disease
patients showed that the drug was able to reduce parasitic loads
without significant effect on clinical progression through 5 years of
follow-up. Further analysis s showed differences in the parasitological
and clinical efficacy of the drug among the geographical procedence of
the patientścohorts (Moreira et al., 2013; Morillo et al., 2015, 2017).
The TRAENA trial (Riarte Adelina, unpublished results), a randomized,
double blind, placebo controlled trial aiming at evaluating the para-
sitological and clinical efficacy of benznidazole in chronic asympto-
matic and symptomatic patients from Argentina indicated a significant
reduction of parasitic loads up to 12–14 months post-treatment, mea-
sured by a standardized duplex TaqMan Real Time PCR (Duffy et al.,
2013), but no significant effect on the clinical evolution of patients,
with a mean follow up of 7 years.
6.6.2. Monitoring of treatment with ergosterol biosynthesis inhibitors
Recent studies evaluated the safety and efficacy of posaconazole in
chronic patients in monotherapy (CHAGASAZOL, 2014) and in com-
bination with benznidazole (STOP CHAGAS, 2017). Their findings
showed that the drug was much better tolerated than benznidazole but
was unable to induce a sustained parasitological response at the end of
one year follow-up period (Molina et al., 2014). Another study, the first
one that evaluated the safety and efficacy of E1224, a prodrug of ra-
vuconazole, in chronic patients from Bolivia indicated that 30% of
patients treated with the dose of 400 mg/week for 8 weeks had sus-
tained parasitological response at 12 months post-treatment period.
Interestingly, pharmacodynamic models derived from the study data
suggested that parasitological response can reach around 80% if the
treatment period is extended to 12 weeks or if it is combined with
benznidazole for 4–6 weeks (Torrico et al., 2018).
Nevertheless, conclusions regarding the efficacy of these novel
drugs could be somewhat biased because of pharmacodynamical issues
(Urbina, 2017). Besides, clearance of parasitic loads exerted by drugs
can be transient and lead to misleading conclusions when follow-up is
performed at the short term, such it has been observed in some studies
(Santos et al., 2016; Torrico et al., 2018). Ideally, molecular methods
used for monitoring of chronic patients should be performed for several
years after treatment to confirm or discard available data.
6.7. Chagas disease reactivation due to organ transplantation
The use of immunosuppressive drugs in organ transplantation has
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Acta Tropica 184 (2018) 59–66
increased clinical significance and predispose patients to reactivation of
chronic infection (Pinazo et al., 2011; Pinazo et al., 2013; Bern, 2012).
Among transplanted organs, heart transplantation leads to a higher
proportion of reactivation cases than other ones. Molecular methods
performed in peripheral blood and endomiocardial biopsies allow an-
ticipating clinical signs of CD reactivation by several months (Diez
et al., 2007; Burgos et al., 2010: da Costa et al., 2017).
6.8. Chagas disease reactivation due to HIV coinfection
Quantitative PCR distinguished groups of HIV/T. cruzi coinfected
patients with and without Chagas reactivation. Indeed the highest
parasitemia was observed in coinfected patients with clinical reactiva-
tion (median 1428.90 T. cruzi/mL), followed by coinfected patients
without reactivation (median 1.57 T. cruzi/mL) and patients with
Chagas disease without HIV (median 0.00 T. cruzi/mL) (de Freitas et al.,
2011). Therefore, this strategy could be used as a criterion for re-
commending pre-emptive therapy in patients with chronic CD with HIV
infection or immunosuppression (Martinez-Perez et al., 2014; Perez-
Molina et al., 2011a,b; Almeida et al., 2011).
7. T. cruzi DNA persistence and clinical implications
In situ polymerase chain reaction analysis was used in the murine
model to disclose a correlation between the persistence of parasites and
the presence of disease in muscle tissue (Zhang and Tarleton, 1999;
Schijman et al., 2003). Recently, molecular amplification of parasitic
mRNAs was set up to address persistence of active parasites in tissues,
which may aid in unraveling parasitic tissue tropism and efficacy of
trypanocidal drugs (Juiz et al., 2017).
8. Final remarks
Estimation of the biological significance of molecular based out-
comes, in particular showing clearance of bloodstream parasitic loads
after treatment has been seldom explored. Due to the intracellular live
forms of the parasite, the predictive value of a negative bloodstream-
based PCR or LAMP outcome to assess cure still remains to be de-
termined. Although trypanocidal treatment aim should be pathogen
eradication, it is difficult to assert if in CD, particularly in the adult
chronic phase, this will be the case. Although posttreatment reduction
of the pathogen burden in the chronic phase could not yet be associated
to a better clinical outcome, it is clear than it prevents vertical trans-
mission.
Target product profiles (TPPs) for molecular diagnosis of CD have
been proposed and focused to acute and congenital transmission,
chronic phase and assessment of response to anti-parasitic treatment
(Pinazo et al., 2014; Porras et al., 2015). These TPPs considered
minimal and optimal needs related to patientśepidemiological and
clinical groups, assay clinical sensitivity and specificity, sampling vo-
lume and types of clinical specimens, conservation, shipping and sto-
rage conditions, infrastructure needed and operators‘ technical skills,
usefulness of qualitative or quantitative reports and genotyping.
The need of point-of-care diagnostic tests has been highlighted; POC
assays should contain stable materials for sample collection and the
ability to perform the assays in prevailing climatic conditions. For
public health applications, diagnostic assays should include low cost,
simple manufacturing and distribution procedures, sustainable pro-
duction and supply requirements. Disposal should be in conformity
with biosafety standards and suitability by health care and target po-
pulations.
9. Conclusions
Standardized and validated PCR and LAMP methodologies are now
available laboratory tools with high sensitivity and specificity that can
A.G. Schijman
improve current diagnosis of CD, especially in the following scenarios:
(i) early diagnosis of congenital, transfusional, oral infections, mon-
itoring of reactivation in immunosuppressed T.cruzi infected patients
and follow-up of treatment response. The development of commercial
kits based on these standardized methods is in progress; their evalua-
tion in prospective field studies will allow predict their potential in
improving diagnosis in the clinical practice and benefit public health
diagnostics polices.
Acknowledgement
ERANET LAC HD 328, PICT 2014-1188 and PICT 2015-0074 to
AGS. AGS is member of the “Carrera del Investigador Científico”, from
the Consejo Nacional de Investigaciones Científicas y Técnicas de
Argentina (CONICET).
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