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Expert Review of Molecular Diagnostics
ISSN: 1473-7159 (Print) 1744-8352 (Online) Journal homepage: http://www.tandfonline.com/loi/iero20
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 WHO’s
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 [12,25–27]. 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 author’s link as well as PubMed/MEDLINE-provided
articles indexed in ‘Similar articles’and ‘Cited by’sections.
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 [36,38–40]
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 manufacturers’instructions, 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
[30,56–61]. 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 patients’samples 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 Schijman’s 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
printer’s 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 150–200 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
90–100% 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 d’Universitats 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-Padilla’s 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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