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Introduction: Chagas disease is caused by the parasite Trypanosoma cruzi. It affects 7 million people, mainly in Latin America. Diagnosis is usually made serologically, but at some clinical scenarios serology cannot be used. Then, molecular detection is required for early detection of congenital transmission, treatment response follow up, and diagnosis of immune-suppression reactivation. However, present tests are technically demanding and require well-equipped laboratories which make them unfeasible in low-resources endemic regions. Areas covered: Available molecular tools for detection of T. cruzi DNA, paying particular attention to quantitative PCR protocols, and to the latest developments of user-friendly molecular diagnostic methodologies. Expert commentary: In the absence of appropriate biomarkers, molecular diagnosis is the only option for the assessment of treatment response. Besides, it is very useful for the early detection of acute infections, like congenital cases. Since current Chagas disease molecular tests are restricted to referential labs, research efforts must focus in the implementation of easy-to-use diagnostic tools in order to overcome the access to diagnosis gap.
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Expert Review of Molecular Diagnostics
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Molecular diagnostics for Chagas disease: up to
date and novel methodologies
Julio Alonso-Padilla , Montserrat Gallego, Alejandro G. Schijman & Joaquim
Gascon
To cite this article: Julio Alonso-Padilla , Montserrat Gallego, Alejandro G. Schijman & Joaquim
Gascon (2017): Molecular diagnostics for Chagas disease: up to date and novel methodologies,
Expert Review of Molecular Diagnostics, DOI: 10.1080/14737159.2017.1338566
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REVIEW
Molecular diagnostics for Chagas disease: up to date and novel methodologies
Julio Alonso-Padilla
a
, Montserrat Gallego
a,b
, Alejandro G. Schijman
c
and Joaquim Gascon
a
a
Barcelona Institute for Global Health (ISGLOBAL), Barcelona Centre for International Health Research (CRESIB), Hospital Clínic - Universitat de
Barcelona, Barcelona, Spain;
b
Section of Parasitology, Department of Biology, Healthcare and the Environment, Faculty of Pharmacy, Universitat de
Barcelona, Barcelona, Spain;
c
Laboratory of Molecular Biology of Chagas Disease (LaBMECh), Instituto de Investigaciones en Ingeniería Genética y
Biología Molecular Dr Hector Torres(INGEBI-CONICET), Buenos Aires, Argentina
ABSTRACT
Introduction: Chagas disease is caused by the parasite Trypanosoma cruzi. It affects 7 million people,
mainly in Latin America. Diagnosis is usually made serologically, but at some clinical scenarios serology
cannot be used. Then, molecular detection is required for early detection of congenital transmission,
treatment response follow up, and diagnosis of immune-suppression reactivation. However, present
tests are technically demanding and require well-equipped laboratories which make them unfeasible in
low-resources endemic regions.
Areas covered: Available molecular tools for detection of T. cruzi DNA, paying particular attention to
quantitative PCR protocols, and to the latest developments of user-friendly molecular diagnostic
methodologies.
Expert commentary: In the absence of appropriate biomarkers, molecular diagnosis is the only option
for the assessment of treatment response. Besides, it is very useful for the early detection of acute
infections, like congenital cases. Since current Chagas disease molecular tests are restricted to refer-
ential labs, research efforts must focus in the implementation of easy-to-use diagnostic tools in order to
overcome the access to diagnosis gap.
ARTICLE HISTORY
Received 30 December 2016
Accepted 1 June 2017
KEYWORDS
Chagas disease;
Trypanosoma cruzi;
molecular detection;
quantitative PCR;
point-of-care; isothermal
amplification
1. Introduction
Chagas disease is a neglected tropical disease caused by the
protozoan parasite Trypanosoma cruzi (T. cruzi; order
Kinetoplastida; family Trypanosomatidae). The WHO estimates
that there are 7 million people infected worldwide, most of
them in Latin America where triatomine vectors (order
Hemiptera; family Reduviidae) that transmit the infection are
endemic [1]. Several other infection routes have been
described, like consumption of parasite-contaminated food,
from mother to child, through blood transfusion, and by
organ transplant [1]. The three latter routes are of relevance
also in non-endemic regions (e.g. Europe, Canada, Australia,
and Japan) where disease has been globalized in the last
decades with population flows from endemic regions [2]. In
the last report from the WHO, it is estimated that there are
~30,000 new vector-related cases and ~8600 new congenital
transmission cases per year [3]. Despite its impact in public
health, there is no available vaccine, yet there are two drugs to
treat Chagas disease: benznidazole and nifurtimox [1].
Unfortunately, both have severe side effects due to long-
term dosages and reduced parasitological efficacy in the
advanced chronic stage [1,4]. In contrast, therapy is more
effective and less toxic in younger patients, which highlights
the paramount importance of an accurate and timed diagnosis
[5]. However, Chagas disease remains largely underdiagnosed,
and as a consequence of its invisibility, chemotherapy barely
reaches 1% of the infected people [5].
The featured silence of Chagas disease is rooted to its clinical
progression characteristics [2]. Two disease stages can be distin-
guished, and the methodologies to be applied for disease diag-
nosis are stage dependent. Firstly, a short acute stage occurs.
Parasitemia is patent along it, and direct detection can be
achieved by parasitological techniques like parasite microscopic
observation in blood smears or microhematocrit, xenodiagnoses,
and hemoculture [6]. These methods may involve long culture
protocols and often entail poor sensitivities [6]. Bloodstream
parasite presence can also be detected by molecular amplifica-
tion of their genetic material by polymerase chain reaction (PCR)
or real-time quantitative PCR (qPCR) methods with higher sensi-
tivity than the aforementioned techniques [6,7]. However, in a
majority of cases, acute symptomatology is nonexistent or
courses as a mild flu and thus the infection mostly goes undiag-
nosed at this stage.
Surpassed the acute phase, the disease enters in an indeter-
minate chronic period that may span decades. In ~70% of
patients, no further clinical symptoms will ever manifest, but in
the remaining ~30%, severe anomalies will disrupt their heart
and/or gastrointestinal tract, potentially leading to death if
untreated [2]. Throughout the chronic phase, parasite blood-
stream presence is intermittent and low, which hampers direct
detection. Diagnosis is then made by means ofserological assays,
like indirect hemagglutination, indirect immunofluorescence, or
enzyme-linked immunosorbent assays. According to the WHOs
guidelines, at least two serological tests based on distinct anti-
gen sets must agree to establish a conclusive diagnosis due to
CONTACT Julio Alonso-Padilla julio.a.padilla@isglobal.org; Joaquim Gascon jgascon@clinic.ub.es
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS, 2017
https://doi.org/10.1080/14737159.2017.1338566
© 2017 Informa UK Limited, trading as Taylor & Francis Group
the wide antigenic variability of the parasite [1]. Nonetheless, the
use of a single technique has been recently postulated due to the
commercialization of increasingly sensitive and specific tests, like
the Architect Chagas (Abbott Laboratories) [8].
As it is in the chronic phase that symptomatology appears,
Chagas disease diagnosis is largely made by serological tests.
Nevertheless, molecular diagnosis is useful for (1) improving
early detection of congenital transmission in newborns when
presence of anti-T. cruzi immunoglobulins from the mother
can confound serological testing [7,9]; (2) follow-up of parasite
reactivation in immunosuppressed patients, be it T. cruzi-HIV
coinfected [10], advanced Chagas cardiomyopathy patients
receiving heart transplant [11], or noninfected patients that
have received an organ from a T. cruzi-positive donor [12,13];
and (3) the evaluation of new treatments in clinical trials
where serological negative conversion of treated seropositive
patients cannot be used because it is impractical from a study
time perspective [14,15]. Besides, molecular detection has also
been applied in the preclinical setting to assess the anti-T.
cruzi performance of drugs, like posaconazole, and the protea-
some inhibitor GNF6702 [16,17].
Molecular-based detection of T. cruzi is also very important
for the study of the parasite eco-epidemiology. More than 100
species of triatomines can spread the infection (e.g. Triatoma
infestans,Triatoma dimidiata,orRhodnius prolixus)[6]. A similar
number of susceptible mammalian hosts can get infected (e.g.
armadillos, opossums, raccoons, and domestic dogs) [6], and
for the vast majority of them there are no of specifically
designed serological tools available. As a result, molecular
detection is then required for the understanding of the para-
site domestic, peri-domestic, and sylvatic biological cycles as
well as of their overlapping or nonoverlapping nature, which
may ultimately translate in a better implementation of vector
control programs (insecticide spray of housing and surround-
ings and dog collars) [18,19]. In addition, development of T.
cruzi-tailored molecular tools has provided major insight on
the parasite genetic diversity [20]. This is organized in discrete
typing units (DTUs; TcI to TcVI), which have been distinctively
associated to disparate ecologies and geographical distribu-
tions [21]. Even further diversity has been acknowledged
within TcI genotype leading to its subdivision (TcIa-TcIe) [22].
Conclusive studies to address the relation between parasite
genotype and disease pathogenesis are to be done, but there
is certainly a geographical variation in the prevalence of its
cardiac, digestive, and/or cardiodigestive clinical forms, likely
related to distinct virulence of the circulating parasite strains
and to the genetic traits of the human populations of each
region [12,23,24]. In any case, occurrence of coinfections and
coexistence of various genotypes in the same patient over
time seems to be a common fact [12,2527]. This has been
mainly looked upon Argentinian and Bolivian patients, and the
most common combination of T. cruzi genotypes detected
was that of TcII/V/VI [12,25,27]. Hence, same as it has been
proposed for T. cruzi drug discovery programs [28], disease
diagnostics must encompass the parasite diversity.
As it happens with Chagas disease conventional serolo-
gical tests, currently available molecular techniques require
of equipped labs and trained personnel for their perfor-
mance. Such demands are frequently unattainable in low-
resource countries. In response, serum- or whole blood-
based immunological rapid diagnostic tests (RDTs) were
developed to be implemented in those regions in substitu-
tion of conventional serological assays [29]. RDTs are user-
friendly immune-chromatographic tests amenable to be per-
formed at point-of-need locations for disease surveillance
and diagnosis screening [29]. Likewise, easy-to-use point-of-
care (POC) molecular diagnostics would be of great aid for
the performance of early diagnosis of congenital transmis-
sion and follow-up of parasite reactivation in immunosup-
pressed patients at ill-equipped laboratories. Methodologies
based on isothermal amplification of nucleicacids amplifica-
tions that do not require thermocyclers or imaging equip-
ment for results readout [30,31], or low-cost technological
solutions to substitute expensive and energy-demanding
current apparatus are being investigated in order to facil-
itate accurate molecular diagnosis in low-resource
areas [32].
A recent article by a group of experts conveyed the
desired Target Product Profiles (TPPs) for the development
of Chagas disease diagnostics at three distinct scenarios: (1)
POC acute-phase diagnosis; (2) POC diagnosis of chronic-
phase patients; and (3) monitoring of antiparasitic treatment
response [33]. Indeed, establishing TPPs for much-needed
diagnostics is a very important first step. Biomarkers, espe-
cially for the assessment of drug treatment response, should
be considered too, and there are in fact several research
groups working on this matter [34]. However, there are still
no validated biomarkers in the market for the diagnosis,
prognosis, and treatment response assessment for Chagas
disease [34]. Indeed, there is a lot of work to be done. In
regard to molecular-based diagnostics, it must begin with
the standardization of currently available procedures and
the development of POC methodologies amenable to be
implemented in low-resource settings.
1.1. Structure and methods
A primary aim of the present article is to review recent
developments of Chagas disease molecular diagnostics,
mainly covered in its first part (Section 2). Nonetheless,
currently available methodologies, such as qPCR, are
unfeasible in many laboratories from endemic regions
that should be fitted with molecular tools to diagnose
acute and congenital infections. Therefore, the article also
covers late advancements in easy-to-use molecular diag-
nostics, which could have a profound impact on Chagas
disease control (Section 3).
Publications addressing Chagas disease molecular diagno-
sis were retrieved from PubMed/MEDLINE using the following
keywords: Chagas disease OR Trypanosoma cruzi AND mole-
cular diagnosis OR molecular detection OR polymerase chain
reaction. Searches for novel molecular methodologies were
made in PubMed/MEDLINE typing neglected tropical diseases
OR Chagas disease OR Trypanosoma cruzi AND isothermal
amplification detection OR loop isothermal amplification. In
all cases, secondary searches were made following the first
and/or last authors link as well as PubMed/MEDLINE-provided
articles indexed in Similar articlesand Cited bysections.
2J. ALONSO-PADILLA ET AL.
2. Clinical molecular diagnostics update
2.1. T. cruzi DNA detection, efforts to homogenize a
very heterogeneous landscape
Many diverse PCR assays have been developed for Chagas
disease diagnosis since a first protocol was released in 1989
[35], including conventional PCR, nested PCR, as well as simple
and multiplex qPCR [9,36,37]. Such diversity has resulted in a
heterogeneous set of techniques that often precludes compar-
ison of results between different studies and/or laboratories
[38]. The factors that contribute to the variable levels of sensi-
tivity and specificity encountered are the sample processing,
sample preservation conditions, the DNA purification methods
used, the distinct T. cruzi sequences targeted for amplification,
the primers and amplification reagents used, and the thermo-
cycling programs followed. With the objective of selecting the
best-performing procedures, a multisite collaborative work led
by Alejandro Schijman laboratory (at INGEBI-CONICET,
Argentina; coauthor of this article) evaluated the performance
of up to 48 PCR and qPCR protocols, present in 26 labora-
tories, over three standard sets of samples (A, B, and C) [38].
Sets were conformed so that their analysis would (A) inform
on the limit of detection (LOD) of strains from three distinct
lineages (DTUs I, IV, and VI); (B) decipher the influence of the
DNA extraction method followed; and (C) assess assay sensi-
tivity and specificity over well-characterized blood clinical
samples from distinct origins (Argentina, Bolivia, Paraguay,
and Brazil) and disease stages (immunosuppressed heart
transplanted and indeterminate and chronic symptomatic
patients) [38]. Out of this multinational effort, four procedures
were flagged as best performing, coded as LbD/2, LbD/3, LbF/
1, and LbQ in the article [38]. All four targeted multi-copy
genes: three of them the nuclear satellite DNA (satDNA) and
the remaining one the kinetoplastid minicircle (kDNA). Three
relied on solvent DNA extraction while one employed a com-
mercial kit. Two were conventional PCR procedures and the
other two qPCR methods (summarized in Table 1). In compar-
ison to serological status of patients, the retrieved sensitivity
levels were between 63% and 74%, and all four methods
reported 100% specificity [38].
All blood samples were treated with guanidine-HCl 6M
EDTA 0.2 M (pH = 8.0) buffer 1:1 (buffer:blood) to yield GEB.
DNA was extracted from 200 µl of GEB. In regard to the DNA
extraction method, despite three of the four best performers
selected relied on solvent extraction [38], the use of commer-
cial kits would be preferred as they favor reproducibility and
homogeneity of the procedure in comparison to solvent
extraction protocols [38].
In comparison to conventional PCR, qPCR techniques pro-
vide a quantitative output in shorter turnaround and are
better suited to scale up because they save the gel electro-
phoresis and gel visualization steps. Despite a slightly more
complex equipment is required and each diagnostic determi-
nation has a higher cost, the homogeneity, reproducibility,
and quantitative output make of qPCR the preferred molecular
diagnostic, in particular to assess treatment response in drug
clinical trials [14,15]. In the absence of reliable biomarkers,
consecutive negative results in the detection of T. cruzi DNA
stands as the surrogate of treatment response [14,15]. In the
clinical diagnosis setting, the use of qPCR over conventional
PCR is liaised to the availability of the required equipment,
and the cost of real-time thermocyclers is much higher than
that of conventional ones.
2.2. Real-time qPCRs, the preferred option
Among the four best-performing methods flagged in Schijman
et al. [38], the qPCR technique that used a commercial DNA
extraction kit [36] was taken forward for improvement in other
studies. Moreira and coworkers coupled it to SYBR green detec-
tion instead of its original TaqMan fluorogenic probe [44]. SYBR
green has the advantage of being cheaper for amplification of a
single target, and the cruzi1/cruzi2 primer set dissociation curves
indicated high specificity on their own. The study reported
equivalent LODs (0.4 parasites equivalents per ml) and sensitivity
(~70%) as had been shown before [36,38]. However, if accord-
ingly to the International Standard Organization (ISO), an internal
amplification control (IAC) must be carried along for accredited
standardization [45], then two test tubes should be arranged per
reaction. In contrast, if TaqMan probes are used, the target
reaction and its IAC can be multiplexed in a single tube/well
easing up the process and saving costs [37].
Duffy and coworkers took Piron et al. TaqMan qPCR [36]a
step further by including a previously described IAC [39]. This
IAC is a linearized plasmid containing an Arabidopsis thaliana
sequence that was spiked in the tubes as part of the reaction
[37,39]. The multiplex satDNA qPCR performance was thor-
oughly analyzed following ISO 16140 guideline [46,47]. The
method showed to be appropriately selective for T. cruzi (LOD
<1 fg/µl for all DTUs except TcIV), since no Leishmania spp.
was amplified at all, and cross-reaction with the closely related
Trypanosoma rangeli occurred only when 10 pg/µl of its DNA
was used as template [39].
Recently, the best satDNA and kDNA qPCRs from [38] were
IAC upgraded, and their performance validated in an interna-
tional study [40]. The duo of TaqMan multiplexed qPCR meth-
ods targeted to kDNA and satDNA and carrying their
corresponding A.thaliana-derived IAC were analyzed on the
basis of the parameters of the ISO 16140 guideline [45].
Comparison of both methods in the same lab using the
same DNA extraction protocols, amplification reagents, ther-
mocyclers, and quality controls showed that kDNA-based
qPCR achieved better sensitivity due to its lower LODs and
quantification [40]. SatDNA LODs of lineages TcI and TcIV were
reduced due to their lower satellite gene content [40].
Nonetheless, kDNA qPCR still presented the issue of potential
cross-reactivity to T. rangeli DNA as 10 fg/µl sufficed for its
detection. Thus, particular attention to potential false positives
must be paid in regions where both parasites prevail if only
kDNA method is used (Guatemala, Panama, Colombia,
Venezuela, and certain regions of Brazil [40]).
Clinical sensitivity of both methods in detection of acute
cases was 100% (11/11), and all these samples were quantifi-
able but one by satDNA [40]. The best results of the satDNA
qPCR described by Duffy et al. had been as well achieved with
the detection of acute samples derived from an oral
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS 3
Table 1. Polymerase chain reaction (PCR) and quantitative PCR (qPCR) procedures that have been analytically validated in multicenter international studies.
Primers
Coded name
a
Extraction method Target PCR Names Sequences Mastermix LOD
b
LOQ
b
Sensitivity
c
(%) Specificity
c
(%) Refs.
LbD/2 Solvent SatDNA RT TCZ-F GCTCTTGCCCACAMGGGTGC Quantitec 0.05 ND 69 100 [38]
TCZ-R CCAAGCAGCGGATAGTTCAGG Sybr-Green (kit)
LbD/3 Solvent SatDNA C TCZ-F GCTCTTGCCCACAMGGGTGC In-House 0.05 N/A 63 100 [38]
(182 bp) TCZ-R CCAAGCAGCGGATAGTTCAGG
LbF/1 Roche kit SatDNA RT cruzi1 ASTCGGCTGATCGTTTTCGA Roche (kit) 0.46 1.53 63 100 [36,3840]
cruzi2 AATTCCTCCAAGCAGCGGATA
cruzi3 CACACACTGGACACCAA
LbG/3 Qiagen kDNA RT 32f TTTGGGAGGGGCGTTCA Applied 0.16 0.90 78 40 [40,41]
Dneasy 148r ATATTACACCAACCCCAATCGAA Biosystems
Tissue kit 71P CATCTCACCCGTACATT (kit)
LbL/2
d
Qiagen DNA SatDNA C Tc-Sat-F CACTCTCTGTCAATGTCTGTTTGCGTG OligoC-TesT Coris 0.5 N/A 72 60 [42,43]
blood mini kit (81 bp) Tc-Sat-R GAAATTCCTCCAAGCAGCGGATA BioConcept (kit)
e
LbQ Solvent kDNA C 121 AAATAATGTACGGGKGAGATGCATGA In-house 0.5 N/A 63 100 [38]
(330 bp) 122 GGTTCGATTGGGGTTGGTGTAATATA
a
As labeled in [38].
b
LOD is limit of detection and LOQ is limit of quantification, as determined in [38] for T. cruzi CL Brener (TcVI)-spiked guanidine HCl-EDTA blood boiled, except LbF/1 and LbG/3 that were calculated according to the National
Committee for Clinical Laboratory Standards (NCCLS) guidelines as stated in [40].
c
As reported in [38] in comparison to patient serological status.
d
The only test that has ever been commercialized for molecular diagnosis of Chagas disease.
e
Sequences for the detection and internal control probes are provided in [43].
The two methods in boldface have been multiplexed with internal amplification controls (IAC) to meet the European Standardization Committee guidelines of standardization for PCR procedures. IAC primer sequences and VIC-
TaqMan probes are shown in [39] and [40]. In the RT protocols, the third primer corresponds to the probe sequence
C: conventional PCR; RT: real-time qPCR; ND: not determined; N/A: not applicable.
4J. ALONSO-PADILLA ET AL.
transmission outbreak in Venezuela and due to congenital
transmission with, respectively, 87.5% (11/16) and 100% (3/3)
sensitivity compared to serology and microhematocrit in each
case [39]. In contrast, sensitivity of qPCR methods to diagnose
chronic patients, either asymptomatic or symptomatic, had
been shown to be below 60% in comparison to serological
assays [38]. Reported levels of sensitivity for chronic-stage
diagnosis by Ramirez et al. were, respectively, 80.7% (117/
145) and 84.1% (122/145) for satDNA and kDNA qPCR meth-
ods, which managed to quantify 32.5% and 45.9% of those
detected samples [40]. Clinical specificity was not an issue for
the best-performing qPCR methods in [38] and the multi-
plexed satDNA and kDNA qPCRs from [40]. It was 100% in all
cases as no amplification was achieved from seronegative
samples [38,40].
2.3. How to circumvent the lack of sensitivity (for the
chronic stage)?
Due to the disease characteristics, presence of bloodstream
circulating parasites in the chronic stage is scarce [1,2]. In a
prospective study with chronically infected pregnant women
attending the service of obstetrics in a hospital at Buenos
Aires (Argentina), time-spaced (at least 4 weeks apart) serial
sampling and performance of two to three PCR detections was
shown to increase the sensitivity of a kDNA-targeted techni-
que [48]. It jumped from 75.6% sensitivity with one sample
detection to 95.6% when the output of three serial samples
was considered [48]. In the referred study, blood was obtained
during pregnancy follow-up and up to three qPCR detections
were made. In most endemic regions, such an approach would
be unfeasible in terms of required infrastructure and dedi-
cated costs.
Another feature to consider is T. cruzi wide genetic diversity
and the fact that some lineages are more prevalent in certain
regions than in others [12,23,24]. This might involve that the
same qPCR procedure performs differently depending on the
origin of the specimen [40]. In an attempt to increase the
probability of DNA amplification, the combination of various
qPCR protocols has been proposed to overcome poor sensi-
tivities and accuracy issues of using a single determination
method [40,41]. A diagnostic algorithm that included three
distinct qPCR techniques was suggested by Qvarnstrom and
coworkers [41]. The chosen methods were the best-perform-
ing satDNA protocol (LbF/1 in [38]; Table 1), the best qPCR of
all those targeting kDNA (labeled LbG/3 in [38]; Table 1), and a
very specific (though poorly sensitive) protocol targeted to the
18S-rRNA region (coded LbS/3 in [38]). As expected, kDNA
qPCR showed higher sensitivity. However, cross-reactivity
with T. rangeli DNA has been highlighted to potentially impact
on the specificity of kDNA-targeted qPCR methods due to the
homology of the amplified region between this parasite and T.
cruzi [41]. Although the diagnostic outcome indicated a better
performance than using a single method alone, the algorithm
did not remarkably improve a single use of the methodolo-
gies. Furthermore, this kind of diagnostic algorithm based on
three qPCRs can only be achieved in well-equipped labs that
process a very low number of samples, but it is unfeasible in
most labs dealing with Chagas disease diagnosis.
Very recently, a new qPCR assay able to detect very low
levels of parasite DNA (0.005 fg/µl for TcI strain K98 and 0.01
fg/µl for TcVI strain CL-Brener) has been described [49]. The
assay was specifically developed for the assessment of drug
treatment in the clinical trial STOP CHAGAS [50] using as
sample blood collected with PAXgene tubes [49]. It is based
on a previously published kDNA qPCR [41] that has been
multiplexed to include the A. thaliana IAC [49]. Several mod-
ifications have been made to the original kDNA algorithm in
order to improve its sensitivity, such as increasing the propor-
tion of lysis buffer to blood in the specimens processing,
redesigning the TaqMan probe to optimize its sequence and
fluorophore, and using new kits for the DNA purification
(Quick-gDNA Blood MiniPrep kit, by Zymo Research) and
qPCR amplification (1x TaqMan Universal Master Mix II with
UNG, by Thermo Fisher Scientific) [49].
2.4. Sample processing and the inclusion of quality
controls
All the steps required to go from the patient to a diagnosis
outcome must be taken into account to try to achieve the best
possible performance of the procedure. Beforehand the PCR,
the molecular diagnostic path includes blood sampling, blood
specimen processing, and DNA extraction (usually by a com-
mercial kit with or without modifications).
For Chagas disease diagnosis, blood is obtained by venous
puncture in adults and newborns from 1 month of age
onwards. Collected volume differs from the averaged 10 ml
of the adults to the 1 to 2 ml obtained from newborns [7,9,37].
Anyhow, from the moment blood specimens are collected,
start the differences between protocols (Table 2). Some collect
the blood in EDTA tubes and store it frozen [36,41]. Others mix
it 1:1 (blood:buffer) with guanidine-HCl 6 M/EDTA 0.2 M
(pH = 8.0) to yield GEB, which can be kept at 4ºC for months
without compromising results [38,40]. Besides allowing refri-
gerated storage, guanidine-HCl/EDTA (GE) buffer de-structures
the DNA, facilitating its subsequent amplification. More
recently, the use of PAXgene blood collection tubes has also
been described [49]. These are easy handling and may provide
enhanced workflow efficiency when used with its homon-
ymous blood DNA purification kit. In the procedure developed
by Wei et al., PAXgene blood tube-collected specimens were
mixed 1:1 with a commercial lysis buffer (GE is made in house)
before further purifying the DNA [49]. Dried blood spots in
Flinders Technology Associates (FTA) filter paper-based cards
have also been used for Chagas disease diagnosis, but blood
collection in this format has just been applied to serological
detection of T.cruzi-specific immunoglobulins [51].
Diagnostic algorithms also differ in the volume of treated
blood used for the DNA extraction, as well as in relation to the
commercial purification kits used for it (Table 2). Furthermore,
some attach to the manufacturersinstructions, whereas
others have introduced slight modifications to them, like
Moreira et al. that do not apply the proteinase K digestion
step and eluate the DNA in half of the kit´s instructed volume
[44]. Noteworthy, an increased sensitivity of satDNA and kDNA
qPCRs has been described if DNA is extracted from blood
buffy coat, which is rich in nucleated blood cells [41]. It follows
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS 5
the same parasite concentration principle as the parasitologi-
cal microhematocrit method [52]. Buffy coat is obtained upon
centrifugation of the blood specimen at 2500gfor 10 min
which segregates the plasma from the cellular blood fraction.
The former is removed and the DNA is extracted from the cells
[41,53]. Nonetheless, the use of buffy coat as sample for the
DNA extraction has not been generalized because it involves
an additional step. The simpler it is the manipulation of the
sample, the lesser will be the chances to make a mistake and
suffer cross-contaminations leading to false-positive results.
Something similar occurs with the boiling of the GEB speci-
mens. Despite increased analytical and clinical sensitivities
have been observed when using boiled samples [39], such
boiling is not advised, particularly when testing a big amount
of samples like in a clinical trial, as it entails a risk of contam-
ination of the negative samples.
Independently of the featured steps in each procedure, qual-
ity controls must be included to limit the risks of reporting false-
negative results. Some studies for quantification of T. cruzi para-
sitic loads have included a host DNA sequence, e.g. RNase P
human gene, as IAC [36,40,44]. Despite this is useful for qualita-
tive purposes, the use of a heterologous intrinsic IAC such as
RNase P should not be recommended. This is because the con-
tent of human blood cells can be highly variable between sam-
ples as it depends on the nutritional, metabolic, and
immunologic status of the patients [39]. The use of a heterolo-
gous extrinsic IAC like the linearized pZErO-2 recombinant plas-
mid with an inserted sequence of A. thaliana aquaporin is
preferred [37]. Besides serving as IAC, by spiking a normalized
amount of the plasmid in the samples before doing the DNA
extractions, the whole procedure can be monitored [37,40,49]. In
comparison to homologous extrinsic or heterologous intrinsic
controls, with a heterologous extrinsic IAC, there will not be
competition with the target sequence, nor will potentially over-
abundant host genetic materials shade any inhibitory effects on
parasite DNA amplification, plus the variability of host DNA con-
tent between samples will be avoided [39].
As it can be observed, present methodologies are complex
and expensive. Indeed they are useful for the evaluation of drug
treatment response in clinical trials and for the performance of
clinical diagnosis in well-equipped referential laboratories in
endemic and non-endemic regions. However, their complexity
and cost preclude their implementation to service the molecu-
lar diagnosis of the disease in vast areas of endemic regions
that are low resourced and endure poor investments.
3. New tools for Chagas disease POC molecular
diagnosis
A first attempt to ease up molecular diagnosis of Chagas
disease was based on the oligochromatographic OligoC-TesT
technology [43]. Although still relying on the above depicted
series of sequential events (blood DNA extraction and thermo-
cycler sequence amplification), its strips layout permitted
naked eye visualization of results in a quick disposable format
rather than by tedious agarose gel imaging or through the
more expensive real-time thermocyclers [43]. Initially designed
to target T. cruzi satellite DNA, a kDNA-based OligoC-TesT was
later on described for increased sensitivity [42]. So far, an
OligoC-TesT assay has been the only commercially available
molecular tool for Chagas disease diagnosis (Coris BioConcept,
Gembloux, Belgium; marked with
d
in Table 1). All required
reagents for PCR amplification as well as running buffers and
strips were included in the kit, but its production had to be
discontinued due to unfavorable market response (Coris
BioConcept Department ClientCare communication). Its
dependence on conventional thermal cycling equipment
might have been the cause behind OligoC-TesT commerciali-
zation failure.
In low-resource settings that lack the infrastructure, equip-
ment, and technical skills to support the use of PCR or qPCR as
molecular diagnostics, new isothermal molecular technologies
would be particularly amenable [31]. Among them, loop-
mediated isothermal amplification (LAMP; Eiken Co., Japan)
and recombinase polymerase assay (RPA; Alere, USA; and
TwistDx, UK) stand out due to their low-performance tempera-
tures and fast turnaround of results [31].
LAMP of T. cruzi DNA has been researched [54]. LAMP does
not require electrically demanding expensive thermocyclers
but a simple water bath or heat block device, and results
can be visualized by naked eye within an hour time. It is
based on Bacillus stearothermophilus (Bst) DNA polymerase
large fragment and a set of four to six primers that allow
Table 2. Variety of blood specimen-processing methods and DNA extraction protocols used in T. cruzi DNA quantitative detection algorithms.
DNA extraction
Ref. Specimen processing
Treated blood
vol. (µl) Commercial kit Kit modifications
DNA vol. for PCR
(µl)
[36] EDTA collection tubes and stored frozen 100 High Pure PCR Template Preparation
(Roche)
N/A 5
[44] GEB
a
boiled 200 QIAamp DNA Mini kit (Qiagen) No proteinase K and elution
in 50 µl
2
[39] GEB boiled and not boiled 300
b
High Pure PCR Template Preparation
(Roche)
N/A 5
[49] PAXgene tubes + Genomic Lysis Buffer (Zymo
Research)
c
400
d
Quick-gDNA Blood Mini Prep (Zymo
Research)
Elution in 50 µl 2
a
GEB states for 1:1 (vol:vol) guanidine-HCl 6 M/EDTA 0.2 M (at pH = 8.0) mix with blood. GEB samples are stored at 4ºC.
b
300 µl of GEB were mixed with 100 µl of the kit binding solution and 5 µl IAC and treated with 40 µl proteinase K.
c
PAXgene-collected blood was mixed 1:1 (vol:vol) with Genomic Lysis Buffer (Zymo Research), and allowed a 10-min lysis step at room temperature (RT) before
storage at 80ºC.
d
400 µl of lysed blood were further mixed with 600 µl of lysis buffer and 5 µl IAC and let 10 min at RT before DNA extraction.
PCR: polymerase chain reaction; N/A: not applicable; IAC: internal amplification control.
6J. ALONSO-PADILLA ET AL.
highly specific, rapid, and efficient DNA amplification at an
isothermal 65ºC step [55]. These characteristics make an ideal
POC diagnostic methodology of it, and as such it is being
developed for a plethora of tropical infectious diseases
[30,5661]. The technology has been thoroughly studied for
the diagnosis of human African trypanosomiasis (HAT) and
Leishmaniasis, respectively, caused by kinetoplastid parasites
Trypanosoma brucei (gambiense or rhodensiense) and
Leishmania spp, closely related to T. cruzi [62]. Recently, a
LAMP assay with dried reagents stabilized in a single tube
with long shelf life capable of specifically amplifying T. brucei
gambiense and T. brucei rhodensiense DNA directly from deter-
gent-lysed blood samples was described [61]. This LAMP
detection system has been refined to allow bedside diagnosis
and field surveillance by adding to it a portable battery system
to power a transilluminator for improved performance. In a
recent case report, LAMP blood detection of T. brucei rhoden-
siense was shown [63]. Upon larger-field studies, this metho-
dology could definitely be a major breakthrough towards HAT
control [61]. Several studies are also applying LAMP for
Leishmaniasis clinical diagnosis [56,64,65]. A recent work
with clinical samples (blood, saliva, and tissue) from just two
patients showed that the method could be used with crude
samples uncompromising sensitivity compared to qPCR, as far
as samples were boiled previous to LAMP [64]. Sample boiling
preparatory step has been described for Schistosoma haema-
tobium LAMP assay too [60].
In contrast, the only reference of a LAMP method for
Chagas disease diagnosis is Thekisoe and coworkers published
LAMP method, which was developed to discriminate between
T. cruzi and T. rangeli infections in field collected Rhodnius
pallescens vectors [54]. The designed primers targeted the
18S rRNA and the small nucleolar RNA (snoRNA) genes,
respectively, of T. cruzi and T. rangeli. They showed parasite-
specific DNA amplification with respect to human- or vector-
derived DNA and a sensitivity of 100 fg and 1 pg per reaction,
respectively, for T. cruzi and T. rangeli DNA [54]. In comparison
to the abovementioned qPCR methods, the T. cruzi-LAMP
sensitivity was >100-fold poorer which may be due to the
selected target (18S rRNA gene). Despite it was published in
2010, no further references to the application of LAMP in
Chagas disease clinical diagnosis could be retrieved from
PubMed. Nonetheless, an LAMP test for Chagas congenital
transmission diagnosis would be a much desired target as
due to its characteristics will come to fill a diagnostic gap in
congenital Chagas disease transmission [33,66]. Currently,
based on Eiken Co. LAMP technology design, FIND and colla-
borators, among which is Alejandro Schijman laboratory at
INGEBI, set up a T. cruzi LAMP assay targeted to the highly
repetitive satDNA sequence [67]. The amplification reaction
takes 40 min at 65ºC and a subsequent 5 more minutes at
80ºC to inactivate the enzyme. Prototype assay microtubes
already contain the required reagents dried in their caps,
and for direct naked eye visualization, calcein was used [67].
The assay showed very good inclusivity and selectivity as DNA
from T. cruzi stocks belonging to the six DTUs was detected
(Figure 1)[67]. The test sensitivity was analytically assessed in
comparison to its counterpart satDNA qPCR on serial dilutions
of T. cruzi DNA samples, as well as on EDTA and heparinized
blood samples that were spiked with known amounts of T.
cruzi epimastigotes. In terms of clinical diagnosis, LAMP assay
detected congenital and immunosuppressed Chagas disease
samples, but chronic patientssamples were only detectable
by qPCR (with Ct values below the limit of quantification) [67].
Further details of this assay will (hopefully) be available soon
as the article describing them is currently under review
(Alejandro Schijmans communication).
On the other hand, RPA couples isothermal enzymatically
driven primer targeting with strand-displacement DNA synthesis
[68]. It provides faster turnaround and works at lower
Figure 1. T. cruzi loop-mediated isothermal amplification (LAMP) assay. (a) three views of the assay micro-tubes that contain the required reagents dried inside their
caps; (b) detection of amplified products directly with the naked eye or using a fluorimeter; (c) assay detected DNA from T. cruzi stocks representative of DTUs I to VI (TcI
to TcVI in the figure). +, indicates positive samples; , indicates negative control tubes (no parasite DNA). T. cruzi-LAMP kit is a prototype by Eiken Chemical Co. (Japan).
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS 7
temperature than LAMP [31]. Furthermore, its assay design is
also less complex than that of LAMP, and the results readout can
be linked to lateral flow visualization of the amplification as it
has been described for the detection of Leishmania infantum
DNA in dogs [69]. Sample DNA extraction would still pose a
conundrum to surmount for bedside diagnosis under field con-
ditions. For that, a mobile lab based on RPA that also considers
the DNA extraction process has been designed for point-of-need
diagnosis of Leishmania donovani human infections [70].
Contained in two suitcases, one for DNA extraction with a fast
commercial method (SpeedXtract, Qiagen) and the other for the
performance of the amplification reaction, the system can be
powered by a portable generator and a solar panel to recharge
it [70].
Other technical solutions could serve as an alternative to
isothermal amplification reactions, like recent works developed
by Wong and collaborators at AI Biosciences Inc. (College
Station, TX, USA). Among the inventions they have devised
that would be useful for field molecular diagnosis, there is an
inexpensive thermocycler (less than $200) built up with thermos,
in which the performance has been shown to match that of
commercial thermocyclers at a fraction of their cost [32]. PCR
tubes are wire-held from an arm that is coupled to a micro-servo
controlled by a programmable microcontroller (Figure 2). The
system is fed with a small portable battery and overall consumes
a lot less electricity per run than a classic thermocycler which
redounds in a cost save. The invention, named thermos thermal
cycler (TTC), was shown to be specific and sensitive and provide
results within 30 min for target sequences up to 1.5 Kb long [32].
In a subsequent article, TTC capability to detect Chlamydia
trachomatis DNA extracted from positive urine samples was
proved [71]. TTC system managed so well with temperature
stability that it also permitted performance of RNA detection
from human serum samples spiked with Ebola virus, HIV, or
Dengue virus RNAs and even outdid the commercial reverse-
transcription qPCR methods brought along for comparison [71].
Amplification reagents, reactions set up, and tubes required are
the same as the ones required for traditional thermocycler-
based protocols. TTC could therefore allow molecular detection
in low-resource settings with currently available reagents as far
as it gets linked to low-cost detection technologies like nucleic
acid lateral flow or smartphone-mediated fluorescence detec-
tion [72]. Nonetheless, DNA extraction from the samples would
still be an issue. In another article, AI Biosciences Inc. team
provided a solution for this in the form of a repurposed 3D-
printer modified for automated DNA extraction [73]. Even the
printers heated bed heat can be redirected to perform nucleic
acid amplification reactions [73].
The clinical samples detected by TTC were urine of people
infected with C.trachomatis, which is a bacteria species that
causes urogenital infections. Detection of T. cruzi DNA in urine
of infected individuals is less likely, as it does not damage the
kidney or the urinary tract, and cell-free circulating DNA frag-
ments that may cross the trans-renal barrier towards being
secreted in the urine have been described to be 150200 bp in
size [74]. Indeed, the use of urine or saliva as samples for POC
diagnosis was agreed in the Chagas disease TPP document
[33]. In this regard, current efforts undertaken relate to direct
detection of parasite antigens in those samples [75,76].
4. Expert commentary
Molecular tests should not be ordered for the clinical man-
agement of Chagas disease chronic patients [38,77]. It is not
just the parasite genetic diversity that complicates a sensitive
detection, but also the inherent limitation imposed by the
biological behavior of the infection with low and intermittent
parasitemias in its chronic phase. However, molecular detec-
tion is the only method currently available to be used as
surrogate marker of treatment success or failure in drug trials
[14,15]. Furthermore, molecular tests have proved highly
sensitive in the detection of acute infections, like those
occurring by congenital or oral T. cruzi transmission, as well
as in anticipating disease reactivation in immunosuppressed
patients [6]. More emphasis must be given to its implemen-
tation in these particular scenarios, and standardized wide-
spread protocols must be made available to the health-care
community. Ideally, the definitive qPCR method should be a
single assay rather than a combination of methods to mini-
mize costs. However, if TPP guidelines for Chagas disease
diagnostics are considered, qPCR techniques, single or
Figure 2. Thermos thermal cycler (TTC) setup with three thermos for amplifica-
tion rounds that require three distinct temperatures. Components shown in the
picture include three thermoses, a pan-and-tilt servo to motion the PCR tubes
between them, the Arduino electronic controller, a breadboard, and a battery
pack. In order to reduce costs, the pan-and-tilt setup is constructed using a soup
can and a wood stick, and the PCR tubes holder is made with metal wire.
Figure reproduced from reference [71] under the terms of the Creative
Commons Attribution License © 2016 Chan et al.
8J. ALONSO-PADILLA ET AL.
multiplexed, would not be desirable at all as they are not
simple, nor cheap, cannot be performed at POC site, and
require preceding sample preparation steps [33]. In other
words, they might be applicable at main reference labora-
tories and hospitals in large urban areas but difficult to
implement in primary health centers with insufficiently
equipped labs. Therefore, easy-to-use molecular diagnostics
to be performed at POC sites by trained personnel (not
necessarily molecular biology specialists) should be pursued.
Involvement of the industry in the development of commer-
cial and standardized tests is expected taking into account
the high and widespread impact of Chagas disease . These
POC tests have to be easy to implement, should be evalu-
ated independently by reference laboratories, and must be
cheaply acquired over the counter.
Field deployment and implementation of POC molecular
diagnostics would mean a major breakthrough, especially for
congenital transmission control. Although it varies geographi-
cally, it is estimated that ~5% of newborns to women with
Chagas disease are infected [78]. Now that blood banks are
screened and vector transmission is receding in many regions,
congenital route is doomed with ~25% of new infections
[48,78]. Since drug treatment within the first year of age is
90100% effective, early T. cruzi diagnosis of pregnant women
and their newborns becomes crucial and must be included in
the standard of care in endemic regions or whenever there is
any suspicion of T. cruzi infection in the mother [2,7,9,78]. In
the case of molecular diagnosis in umbilical cord blood sam-
ples collected at birth, the detection of T. cruzi DNA from
maternal origin giving rise to a false-positive result cannot
be discarded [78]. Therefore, a confirmatory test should be
made a month later. By then, an increase in the parasitic load
will ease the detection, now made upon peripheral blood
sample from the newborn [7]. Nowadays in endemic areas,
sampling and testing at birth by micromethod is generally
performed to ensure the adherence of the mothers to the
health follow-up protocol. Nonetheless, micromethod has
been shown to be less sensitive than PCR and to provide a
slower results turnaround [7,9]. Even though being made at
first month of age, generalization of molecular diagnostics
would still reduce the time-to-treatment window and thus
increasing the chances to positively respond to it. Definitely,
early diagnosis of congenital transmission could be simplified
using POC molecular methods.
5. Five-year view
With currently available tools, Chagas disease diagnosis and
treatment is barely reaching a fraction of those infected.
Diagnosis algorithms must take into account the distinct clin-
ical settings and the field conditions found in many Chagas
disease-endemic regions. Therefore, in order to make diagno-
sis (and treatment) available to more people, low-cost POC
diagnostics, amenable in low-resource settings, have to be
widely implemented in the territory. Importantly, these meth-
odologies should involve minimal interventions and ideally
work with POC samples, such as urine [33]. Unfortunately,
use of this type of sample is still largely unexplored and
today limits to the detection of parasite antigens in it
[75,76]. More research efforts should be placed on the matter.
Molecular diagnosis has been shown to provide an earlier
diagnosis of congenital infection than current methods [7,9],
but its use has not been implemented in the health systems of
endemic countries due to a lack of resources. Current guide-
lines for congenital Chagas disease diagnosis rely on parasito-
logical detection by micromethod and serological assessment
>8 months after birth [78]. By the time a serological diagnosis
is achieved, a precious time for treatment may have been lost.
Therefore, congenital transmission is definitely a diagnostic
scenario where easy-to-use molecular tools, such as LAMP or
RPA, could play a major role and deserve to be investigated.
In comparison to the serological diagnosis of chronic
patients where several RDTs are now available [29], the land-
scape of POC molecular diagnostics for Chagas disease looks
flat. Nonetheless, given the complexity and costs of present
PCR methods, their arrival is impatiently expected. Then,
towards the establishment of the best diagnostic strategy,
population-based studies will need to be performed to show
that these alternative methodologies work at least as good a
currently available impractical molecular diagnostics.
Key issues
Chagas disease affects 7 million people worldwide and
there are two drugs available against it: benznidazole and
nifurtimox. They have toxic side effects and show dimin-
ished efficiency the longer the infection, but are well toler-
ated by children and have high efficacy in acute and early
diagnosed cases.
The disease is largely underdiagnosed because of a mostly
asymptomatic acute stage and a long lasting indeterminate
phase. As a result barely 1% of infected people receive
treatment. Furthermore, diagnosis often arrives when
symptoms are advanced and available drugs are less effi-
cient or useless.
Diagnosis of chronic Chagas disease is performed serologi-
cally. Molecular diagnostics are very useful for early detec-
tion of congenital infection, assessment of infection
reactivation in immune-suppressed patients, and assess-
ment of drug response. In all cases, seropositive status of
patients precludes the use of serological diagnostics.
Standardization of currently available molecular procedures
is paramount for reliable comparison of study results.
Recent international multicenter efforts have been con-
ducted to canalize diversity into a few best performing
assays.
Molecular diagnosis of congenital infection transmission is
not yet widely distributed despite it is faster and more
sensitive than current parasitological detection methods.
Nonetheless, the complexity and costs of presently avail-
able molecular methodologies preclude their generalized
use in endemic regions.
Rapid, low-cost diagnostics and a reliable access to treat-
ments are instrumental towards the control of Chagas dis-
ease, and will definitely result in a health status
improvement of large segments of the population in Latin
America.
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS 9
Towards an affordable field deployment of molecular diag-
nostics, the use of new methodological approaches should
be implemented. LAMP, RPA or TTC represent potential
alternatives to currently impractical methodologies. The
hacked 3D printer by AI Biosciences Inc. could be coupled
to LAMP, RPA or TTC to provide them with purified DNA
and support its amplification within the same low-cost
apparatus.
Funding
Research by J. Alonso-Padilla, J. Gascon and M. Gallego is funded by the
Departament dUniversitats i Recerca de la Generalitat de Catalunya, Spain
[AGAUR; grant 2014SGR26], and by Instituto de Salud Carlos III RICET
Network for Cooperative Research in Tropical Diseases [RD12/0018/0010
ISCIII; MICINN, Spain] awarded to J. Gascon. The authors have also
received support from the Generalitat de Catalunya CERCA Programme.
A.G. Schijman research is funded by the Argentinian National Agency of
Science and Technology [PICT 2014-1188 and PICT V 2015-0074] and by
European Commission funded ERANET-LAC HD 328.
Declaration of interest
J. Gascon, M. Gallego and A.G. Schijman are members of NHEPACHA
scientific network. J. Gascon is a member of the scientific communities
of CEADES (Bolivia) and ISGLOBAL. J. Alonso-Padillas position at ISGLOBAL
is funded by Instituto de Salud Carlos III RICET Network for Cooperative
Research in Tropical Diseases (RD12/0018/0010 ISCIII; MICINN, Spain). M.
Gallego is Professor at the Faculty of Pharmacy of the University of
Barcelona and Associate Researcher at ISGLOBAL. Currently, M. Gallego is
supervisor in the EU funded Euroleish training network. A.G. Schijman is
director of LABMECH and member of the Directory of INGEBI. The authors
have no other relevant affiliations or financial involvement with any
organization or entity with a financial interest in or financial conflict
with the subject matter or materials discussed in the manuscript apart
from those disclosed.
ORCID
Julio Alonso-Padilla http://orcid.org/0000-0003-4466-7969
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12 J. ALONSO-PADILLA ET AL.
... Esta infección en mujeres en edad fértil predispone a una presentación congénita (2); donde el 60 a 90% de los neonatos son asintomáticos con una progresión a una enfermedad crónica grave, que supone un subregistro y una limitante para el diagnóstico. Cuando se desarrolla una presentación sintomática, esta suele aparecer en los primeros 30 días de vida sin un cuadro patognomónico (3), teniendo manifestaciones clínicas variadas que incluyen bajo peso al nacer, prematuridad, distrés respiratorio, hepatoesplenomegalia, ictericia (2,8), edema generalizado e incluso hidrops fetalis y muerte (3,8), razón por la cual, la Enfermedad de Chagas congénito es considerado como un problema de salud pública (6). ...
... Se sabe que las técnicas moleculares pueden ofrecer un diagnóstico temprano de la infección congénita que los métodos actuales (6), especialmente la PCR en tiempo real (qPCR) por sus características de sensibilidad (3), resultado cuantitativo (carga parasitaria) e información de interés epidemiológico en la determinación de linajes del parásito (11) en áreas endémicas como no endémicas (12), además que la inclusión de esta técnica en el algoritmo diagnóstico en zonas endémicas podría reducir el periodo de seguimiento del recién nacido y supondría un tratamiento oportuno (7). ...
... Igualmente, Cura et al., mencionan que la elección del tipo de PCR (convencional, multiplex, en tiempo real, entre otras), el origen biológico de la muestra y la diana biológica para la amplificación, ha demostrado un papel predictivo en el diagnóstico congénito, encontrando una mayor sensibilidad clínica cuando se amplificó el ADNk pero menor especificidad por la pre-sencia de falsos positivos con Trypanosoma rangeli (49) requiriendo de la estandarización de la técnica junto con la investigación de nuevas tecnologías moleculares isotérmicas (46) que permitan una entrega oportuna y certera de resultados acortando el periodo diagnóstico (6,50). En Sudamérica aún no hay un flujograma unificado sobre el diagnóstico de la infección por T. cruzi y las técnicas diagnósticas a usar. ...
Article
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Introducción: El presente artículo describe aspectos relevantes entorno de la Enfermedad de Chagas congénita, tales como epidemiología, sintomatología, revisión de casos clínicos y las técnicas diagnósticas. Métodos: Se realizó una revisión de la literatura por medio de bases de datos bibliográficas como PubMed, Science direct, Scopus, Plos One, SciELO, teniendo como criterio de inclusión las publicaciones artículos o comprendidos entre enero de 2013 y enero del año 2022 en idioma español e inglés. Resultados: Se determinó que la prevalencia de la Enfermedad de Chagas congénita aún es un problema de salud pública en áreas endémicas y no endémicas, siendo la serología materna indispensable para dar seguimiento oportuno a los casos. Conclusiones: Los seguimientos diagnósticos actuales difieren en los países endémicos y se están aplicando tamizajes en zonas no endémicas donde migran mujeres procedentes de áreas de trasmisión activa de la Enfermedad Chagásica.
... Continued advancements in diagnostic tools are essential to overcoming the existing limitations, ensuring more accurate disease detection and effective treatment monitoring. This progress will ultimately lead to improved patient outcomes and more effective public health interventions [22]. Thus, in the present study, we validate using an automated magnetic bead-based DNA extraction method compared to the traditional silica-based column method. ...
... These results indicate that the MBs method yields a higher concentration of DNA and significantly improved purity compared to the SC method. health interventions [22]. Thus, in the present study, we validate using an automated magnetic bead-based DNA extraction method compared to the traditional silica-based column method. ...
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Chagas disease, caused by Trypanosoma cruzi, remains a significant global health challenge, particularly in the molecular diagnostics of low parasitemia during the chronic phase. This highlights the critical need for enhanced diagnostic methodologies. In response, this study evaluates the effectiveness of an automated magnetic beads-based DNA extraction method in improving the molecular diagnosis of Chagas disease compared to the traditional silica column-based extraction. Accordingly, this research seeks to enhance the DNA yield, purity, and sensitivity of real-time PCR (qPCR) assays for detecting T. cruzi satDNA. Blood samples spiked with guanidine-EDTA solution and varying concentrations of T. cruzi were used to compare the two extraction methods. The results indicated that the magnetic bead-based method outperformed the silica column in terms of DNA concentration, purity, and earlier detection of T. cruzi satDNA. Although both methods had similar limits of detection at a 95% confidence interval, the magnetic bead-based approach demonstrated higher sensitivity and reproducibility, particularly in low-parasitemia samples. The findings suggest that the magnetic beads-based DNA extraction method offers a more reliable, faster, and more sensitive alternative for diagnosing chronic Chagas disease, potentially improving clinical outcomes by enabling more accurate and earlier parasite detection.
... Looking more specifically into the ratio of patients with the different outcomes in each group, PCR data show sustained parasitological clearance in more patients than MultiCruzi. This could be explained by the limitations of the PCR method as the level of parasites in the bloodstream does not reflect the level of parasites in the tissues; the very low and sporadic parasitemia during the chronic phase of the disease makes direct detection of the parasite intrinsically difficult 39 negative PCR results does not necessarily prove the absence of the parasite. Some longer follow-up studies for example have shown positive PCR in patients after 4 or 5 years of follow-up while during the first years a sustained negative PCR was observed 42 . ...
Article
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Chagas disease following infection with Trypanosoma cruzi is a major public health issue, with the disease spreading beyond endemic regions and becoming more global due to the migration of infected individuals. The currently available anti-parasitic drugs, nifurtimox and benznidazole, remain insufficiently evaluated for their efficacy in adult patients. A key challenge is the lack of markers for parasitological cure, which also precludes the development of new treatments. Consequently, there is a critical need for a practical method to assess drug performance within a short timeframe. In this retrospective analysis of the phase 2 randomized controlled BENDITA trial (ClinicalTrials.gov: NCT03378661), we report the potential of a serological multiplex method (MultiCruzi), combined with advanced statistical analytical methods, to measure the response to anti-parasitic treatment of adult Chagas patients. Applying this approach to serum samples from adult patients in the indeterminate chronic stage of Chagas disease, treated with different benznidazole regimens and combinations, we predict treatment efficacy after just 6 months of follow-up, in sharp contrast to data obtained with conventional and recombinant T. cruzi ELISA tests. The obtained results are also compared with the PCR data. We propose integrating MultiCruzi as a serological method endpoint in proof-of-concept clinical trials for Chagas disease.
... In general, these studies found a high sensitivity and specificity, as well as a high degree of agreement between the different alternatives, supporting their use over microscopy-based methods [25]. The different primers and TaqMan probes used for the detection of T. cruzi DNA have been reviewed elsewhere [4,34]. However, despite having obvious advantages over classic parasitological direct detection methods, the cost of reagents, their supply chain, and the expensive equipment necessary to process the samples remain important limitations to their deployment in Chagas disease-endemic countries. ...
Article
Introduction: Chagas disease, caused by parasite Trypanosoma cruzi, is the most important neglected tropical disease in the Americas. Two drugs are available for treatment, but access to them is challenging, in part due to complex diagnostic algorithms. These are stage-dependent, involve multiple tests, and are ill-adapted to the reality of vast areas where the disease is endemic. Molecular and serologic tools are used to detect acute and chronic infections, with the performance of the latter showing geographic differences. Breakthroughs in the development of new diagnostic tools include the validation of a loop-mediated isothermal amplification assay for acute infections (T. cruzi-LAMP), and the regional validation of several rapid diagnostic tests (RDTs) for chronic infection, which simplify testing in resource-limited settings. The literature search was carried out in the MEDLINE database until 1 August 2023. Areas covered: This review outlines existing algorithms, and proposes new ones focused on point-of-care testing. Expert opinion: Integrating point-of-care testing into existing diagnostic algorithms in certain endemic areas will increase access to timely diagnosis and treatment. However, additional research is needed to validate the use of these techniques across a wider geography, and to better understand the cost-effectiveness of their large-scale implementation.
... Finally, while detecting circulating parasites through their DNA by molecular amplification techniques like quantitative polymerase chain reaction (qPCR) may help determine treatment failure, a negative qPCR result cannot be considered a surrogate of cure. 10 Seronegatization is the best therapeutic indicator for monitoring the condition during the chronic phase of the disease, it can be challenging to identify the causative agent as symptoms can take several years to appear. While PCR detection of T. cruzi may indicate treatment failure, it is not a reliable indicator of damage to heart tissue. ...
Article
Chagas disease is caused by the Trypanosoma cruzi parasite and is transmitted by infected triatomine bugs. This infection affects approximately 8 million people in the Americas, and due to globalisation and displacement, it is becoming increasingly common to find infected patients worldwide. Diagnosis of the disease in its acute form is relatively simple, as the parasite can be detected in peripheral blood smears, and symptoms are visible. However, in its chronic condition, the parasite is almost undetectable , and indirect tests are necessary to determine the presence of antibodies in infected patients. It is important to note that a single test is not enough to confirm the disease in this phase, as a second serological test should confirm the diagnosis. If the results are contradictory, a third test should be performed to confirm or discard the disease. Unfortunately, laboratories may not have access to all necessary tests in many rural areas where the disease is more frequent. Rapid tests to diagnose this disease present problems, such as significant variations in sensitivity and specificity in different countries. Therefore, searching for new biomarkers that allow for optimal correlation is essential. In this work, we have searched scientific literature from the last 10 years for mentions of novel biomarkers for diagnosis, treatment follow-up, and prediction of cardiac complications in Chagas disease in its chronic phase.
... Additionally, measuring seroconversion through conventional tests may not be feasible as it may take years or even decades for a patient with chronic disease to revert serologically. Finally, while detecting circulating parasites through their DNA by molecular amplification techniques like quantitative polymerase chain reaction (qPCR) may help determine treatment failure, a negative qPCR result cannot be considered a surrogate of cure (10) Seronegatization is the best therapeutic indicator for monitoring the condition During the chronic phase of the disease, it can be challenging to identify the causative agent as symptoms can take several years to appear. While PCR detection of T. cruzi may indicate treatment failure, it is not a reliable indicator of damage to heart tissue. ...
Preprint
Full-text available
Chagas disease is caused by the Trypanosoma cruzi parasite and is transmitted by infected triatomine bugs. This infection affects approximately 8 million people in the Americas, and due to globalization and displacement, it is becoming increasingly common to find infected patients worldwide. Diagnosis of the disease in its acute form is relatively simple, as the parasite can be detected in peripheral blood smears, and symptoms are visible. However, in its chronic condition, the parasite is almost undetectable, and indirect tests are necessary to determine the presence of antibodies in infected patients. It is important to note that a single test is not enough to confirm the disease, as a second serological test should confirm the diagnosis. If the results are contradictory, a third test should be performed to solve the problem. Unfortunately, laboratories may not have access to all necessary tests in many rural areas where the disease is more frequent. Rapid tests to diagnose this disease present problems, such as significant variations in sensitivity and specificity in different countries. Therefore, searching for new biomarkers that allow for optimal correlation is essential. In this work, we have searched scientific literature from the last years for mentions of novel biomarkers for diagnosis, treatment follow-up, and prediction of cardiac complications in Chagas disease in its chronic phase.
... In addition, indirect parasitological tests, such as xenodiagnoses or blood culture, have low sensitivity varying between 20% and 50% and require assistance from other methods, such as the concentration test, to confirm the result. The enzyme-linked immunosorbent assay (ELISA) and Polymerase Chain Reaction (PCR) tests are indicated for use during the chronic phase because they are more sensitive and specific than parasitological tests [18]. Despite its high sensitivity and specificity, PCR is an expensive test and is difficult to standardize [19]. ...
Article
Full-text available
Chagas disease remains a neglected disease that is considered to be a public health problem. The early diagnosis of cases is important to improve the prognosis of infected patients and prevent transmission. Serological tests are the method of choice for diagnosis. However, two serological tests are currently recommended to confirm positive cases. In this sense, more sensitive and specific serological tests need to be developed to overcome these current diagnosis problems. This study aimed to develop a new recombinant multiepitope protein for the diagnosis of Chagas disease, hereafter named rTC. The rTC was constructed based on amino acid sequences from different combinations of Trypanosoma cruzi antigens in the same polypeptide and tested using an enzyme-linked immunosorbent assay (ELISA) to detect different types of Chagas disease. rTC was able to discriminate between indeterminate (IND) and cardiac (CARD) cases and cross-reactive diseases, as well as healthy samples, with 98.28% sensitivity and 96.67% specificity, respectively. These data suggest that rTC has the potential to be tested in future studies against a larger serological panel for the diagnosis of Chagas disease.
... For example, higher Leishmania loads in seriously-diseased dogs can result in overall higher sensitivity of parasitological and DNA-based diagnostic procedures [21,22]. In addition, and importantly, test-performance estimates derived from laboratory studies done under highlycontrolled conditions (with, e.g., samples spiked with parasites or their DNA) often differ substantially from those derived from field studies [25][26][27][28]. A key reason for this difference is that, in laboratory studies, test-performance metrics are usually computed based on samples of known infection status, whereas in field studies the samples may, or may not, contain the target for detection [11,12]. ...
Article
Full-text available
Background Domestic dogs are primary reservoir hosts of Leishmania infantum, the agent of visceral leishmaniasis. Detecting dog infections is central to epidemiological inference, disease prevention, and veterinary practice. Error-free diagnostic procedures, however, are lacking, and the performance of those available is difficult to measure in the absence of fail-safe “reference standards”. Here, we illustrate how a hierarchical-modeling approach can be used to formally account for false-negative and false-positive results when investigating the process of Leishmania detection in dogs. Methods/Findings We studied 294 field-sampled dogs of unknown infection status from a Leishmania-endemic region. We ran 350 parasitological tests (bone-marrow microscopy and culture) and 1,016 qPCR assays (blood, bone-marrow, and eye-swab samples with amplifiable DNA). Using replicate test results and site-occupancy models, we estimated (a) clinical sensitivity for each diagnostic procedure and (b) clinical specificity for qPCRs; parasitological tests were assumed 100% specific. Initial modeling revealed qPCR specificity < 94%; we tracked the source of this unexpected result to some qPCR plates having subtle signs of possible contamination. Using multi-model inference, we formally accounted for suspected plate contamination and estimated qPCR sensitivity at 49–53% across sample types and dog clinical conditions; qPCR specificity was high (95–96%), but fell to 81–82% for assays run in plates with suspected contamination. The sensitivity of parasitological procedures was low (~12–13%), but increased to ~33% (with substantial uncertainty) for bone-marrow culture in seriously-diseased dogs. Leishmania-infection frequency estimates (~49–50% across clinical conditions) were lower than observed (~60%). Conclusions We provide statistical estimates of key performance parameters for five diagnostic procedures used to detect Leishmania in dogs. Low clinical sensitivies likely reflect the absence of Leishmania parasites/DNA in perhaps ~50–70% of samples drawn from infected dogs. Although qPCR performance was similar across sample types, non-invasive eye-swabs were overall less likely to contain amplifiable DNA. Finally, modeling was instrumental to discovering (and formally accounting for) possible qPCR-plate contamination; even with stringent negative/blank-control scoring, ~4–5% of positive qPCRs were most likely false-positives. This work shows, in sum, how hierarchical site-occupancy models can sharpen our understanding of the problem of diagnosing host infections with hard-to-detect pathogens including Leishmania.
Article
Full-text available
Background Cardiac complications, including heart failure and arrhythmias, are the leading causes of disability and death in Chagas disease (CD). CD, caused by the Trypanosoma cruzi parasite, afflicts 7 million people in Latin America, and its incidence is increasing in non-endemic countries due to migration. The cardiac involvement is explained by parasite-dependent, immune-mediated myocardial injury, microvascular abnormalities, and ischemia. Current treatment of early CD includes the administration of nifurtimox and benznidazole. However, their efficacy is low in the chronic phase and may induce severe adverse events, forcing therapy to halt. Therefore, finding innovative approaches to treat this life-threatening tropical disease is of utmost importance. Thus, improving the efficacy of the current antichagasic drugs by modifying the inflammatory response would render the current treatment more effective. It has been reported that, in mice, simvastatin decreases cardiac inflammation and endothelial activation, and improves cardiac function, effects that require clinical confirmation. Objective The study aims to analyze whether two doses of Atorvastatin, administered after CD treatment is completed, are safe and more efficacious than the antiparasitic drugs alone in reducing general inflammation and improving endothelial and cardiac functions in a proof-of-concept, placebo-controlled phase II trial. Methods 300 subjects will be recruited from four Chilean hospitals with an active Program for the Control of Chagas Disease. 40 or 80 mg/day of atorvastatin or placebo will be administered after completion of the antichagasic therapy. The patients will be followed up for 12 months. Efficacy will be determined by measuring changes in plasma levels of anti-inflammatory and pro-inflammatory cytokines, soluble cell adhesion molecules, BNP, and cTnT. Also, the resting 12-lead ECG and a 2D-echocardiogram will be obtained to evaluate cardiac function. Trial registration ClinicalTrials.gov Identifier: NCT04984616.
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