400 words Public Health Discussion (Special Study Designs)

RESEARCH ARTICLE

Molecular Diagnosis of Chagas Disease in Colombia: Parasitic Loads and Discrete Typing Units in Patients from Acute and Chronic Phases CarolinaHernández1,2,ZulmaCucunubá1,2,CarolinaFlórez1,2,MarioOlivera1,2, CarlosValencia1,2,PilarZambrano2,CieloLeón1,2,JuanDavidRamírez3*

1 RedChagasColombia,InstitutoNacionalde Salud,Bogotá,Colombia,2 Grupode Parasitología, InstitutoNacionalde Salud,Bogotá,Colombia,3 Grupode InvestigacionesMicrobiológicas-UR(GIMUR), Programa de Biología,Facultad de CienciasNaturalesy Matemáticas, Universidad el Rosario,Bogotá, Colombia

* [email protected]

Abstract

Background The diagnosis of Chagas disease is complex due to the dynamics of parasitemia in the clini-

cal phases of the disease. The molecular tests have been consideredpromissorybecause

they detect the parasite in all clinicalphases.Trypanosoma cruzipresentssignificant geneticvariabilityand is classified into six DiscreteTyping Units TcI-TcVI(DTUs)with the

emergenceof foreseen genotypes withinTcI as TcIDomand TcI Sylvatic. The objective of

this study was to determinethe operatingcharacteristicsof molecular tests (conventional

and Real Time PCR) for the detectionofT.cruziDNA, parasitic loads and DTUs in a large cohortof Colombianpatients fromacute and chronicphases.

Methodology/PrincipalFindings Sampleswere obtainedfrom708 patients in all clinical phases. Standarddiagnosis (direct

and serological tests)and molecular tests (conventional PCR and quantitativePCR) target-

ing the nuclear satelliteDNA region.The genotypingwas performedby PCR using the

intergenicregionof the mini-exon gene, the 24Sa, 18S and A10 regions.The operating

capabilitiesshowed thatperformanceof qPCR was highercomparedto cPCR. Likewise,

the performance of qPCR was significantlyhigher in acute phase comparedwith chronic

phase. The median parasitic loads detectedwere 4.69 and 1.33 parasite equivalents/mLfor

acute and chronicphases. The main DTU identifiedwas TcI (74.2%).TcIDomgenotype

was significantlymorefrequent in chronicphase comparedto acute phase (82.1%vs

16.6%).The medianparasitic load for TcIDomwas significantlyhighercomparedwith TcI

Sylvatic in chronic phase (2.58vs.0.75 parasite equivalents/ml).

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OPEN ACCESS

Citation: Hernández C, Cucunubá Z, Flórez C, Olivera M, Valencia C, Zambrano P, et al. (2016) Molecular Diagnosis of Chagas Disease in Colombia: Parasitic Loads and Discrete Typing Units in Patients from Acute and Chronic Phases. PLoS Negl Trop Dis 10(9): e0004997.doi:10.1371/journal.pntd.0004997

Editor: Alain Debrabant, US Food and Drug Administration, UNITED STATES

Received:April 15, 2016

Accepted: August 22, 2016

Published: September 20, 2016

Copyright: © 2016 Hernández et al. This is an open access article distributedunder the terms of the Creative Commons AttributionLicense, which permits unrestricteduse, distribution,and reproduction in any medium, provided the original author and source are credited.

Data Availability Statement: All relevant data are within the paper and its SupportingInformationfiles.

Funding: This work was supportedby Departamento Administrativo Nacional de Ciencia y Tecnologíade Colombia ‘‘Francisco José de Caldas – COLCIENCIAS’’ and ‘‘Unión Temporal Programa Nacional de Investigación para la prevención,control y tratamiento integral de la enfermedadde Chagas en Colombia’’, Grant Number 380- 2011, code 5014- 537-30398.The funders had no role in study design, data collection and analysis, decision to publish, or preparationof the manuscript.

Conclusions/Significance The molecular tests are a precise tool to complement the standarddiagnosis of Chagas dis-

ease, specifically in acute phase showing high discriminativepower. However, it is neces-

saryto improve the sensitivityof molecular tests in chronic phase. The frequencyand

parasitemiaof TcIDomgenotype in chronic patientshighlight its possible relationshipto the

chronicityof the disease.

Author Summary

Chagas disease is a neglected tropical disease caused by the parasite Trypanosoma cruzi that shows tremendous genetic diversity evinced in at least six Discrete Typing Units and massive genetic diversity within TcI. Two clinical phases exist where acute phase shows high parasitemia and chronic phase shows low and intermittent parasite dynamics. One particularity of the disease is the diagnosis, because the parasitemia is highly variable dur- ing the phases of the disease. Molecular tests allow detecting DNA of the parasite in all clinical phases. Herein, we determined the operating characteristics of two molecular tests (cPCR and qPCR) to evaluate the performance of these tests for diagnosis of Chagas dis- ease in 708 Colombian patients. We determined the parasitic loads and DTUs to assess how is the behaviour of these characteristics in relation to the clinical phases. We found that the performance of qPCR was higher compared to cPCR and the molecular tests are a precise tool for diagnostic of Chagas disease, mainly in the acute phase. The parasitemia was higher in the acute phase compared to chronic phase and the DTU predominant in Colombian patients was TcI. The behaviour of TcIDom genotype in the chronic phase patients evidenced possible relationship with the chronicity of the disease.

Introduction Chagas disease is a zoonotic parasitic disease caused by the protozoan Trypanosoma cruzi. It is considered a public health problem in Latin-America, where approximately 6 million people are currently infected [1]. The acute phase of the disease is characterised by usually mild fever that in a small proportion of cases can be accompanied by myocarditis and other lethal compli- cations. Most of the patients continue through the chronic phase that is initially characterised by an asymptomatic clinical course during two or three decades, and about 30% of the infected patients will develop heart or digestive complications afterwards [2].

T. cruzi parasite shows significant genetic variability and classified into at least six Discrete Typing Units TcI-TcVI (DTUs), that present associations with the geographical distribution, epidemiological transmission cycles, insect vectors and clinical manifestations of Chagas dis- ease [3–5]. Recent studies suggest the occurrence of an emerging clade within TcI named TcI- Dom which is distributed in the Americas and associated with domestic cycles of transmission and human infection [6–10]. Recently, a genotype detected in anthropogenic bats and named as TcBat has been described in Panama, Ecuador, Colombia and Brazil including a case of human infection in Colombia [11–14].

The diagnosis of Chagas disease is complex due to the dynamics of parasitemia in the phases of the disease. During the acute phase the parasitemia is high, therefore the diagnosis is per- formed by direct parasitological tests [15,16]. Nevertheless, direct parasitological tests are not

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Competing Interests: The authors have declared that no competinginterests exist.

useful in the chronic phase due to the low and intermittent parasitemias. Therefore, the diag- nosis of Chagas disease in the chronic phase is determined by serological tests such as ELISA: enzyme-linked immunosorbent assay, IFA: indirect immunofluorescence assay or HAI: Hem- agglutination Inhibition Test [17–19]. Recently, molecular techniques such as cPCR (conven- tional PCR) and qPCR (quantitative real-time PCR) have been considered as supportive diagnostic tests due to their ability to determine parasitic loads of T. cruzi in all clinical phases of the disease [20–22]. The operating characteristics of molecular tests for diagnosis of T. cruzi infection have varied according to clinical phase and technical specifications. Sensitivity for identifying chronic infection with cPCR has ranged between 22 and 75% [23,24] and in both cases with a specificity of 100%. Contrastingly, for qPCR, sensitivity has ranged between 60 and 80% [22,25,26] in chronic phase and between 88% and 100% for acute phase [25,26], whereas specificity is between 70–100% [26–28]. Sampling methods have not been always clearly stated and the role of these techniques for diagnosis of Chagas disease in the different clinical phases still remains poorly understood.

The objective of this work was to determine the operating capabilities of qPCR and cPCR targeting the satellite nuclear DNA region, compared with standard diagnosis methods for acute and chronic Chagas disease. Additionally, we evaluated the plausible associations between parasitic load and DTUs in Colombian patients from the acute and chronic phases to untangle the natural course of T. cruzi infection in terms of parasite dynamics.

MaterialsandMethods

Participants All patients who attended the Colombian National Health Institute (Overall 985 individuals) seeking diagnostic tests for Chagas disease in acute (113 patients) or chronic phase (872 patients) between 2004 and 2015 were considered as potential participants. Inclusion criteria were: i. Clinical or epidemiological suspect of Chagas disease in acute or chronic phase ii. Not having received aetiological treatment for Chagas disease iii. Positive serological tests for Cha- gas disease (IFA, ELISA and/or HAI) iv. Adequate blood and serum samples available for per- forming diagnostic tests according to the clinical phase. v. Acceptance to participate and sign the informed consent.

Ethicalstatement The Technical Research Committee and Ethics Research Board at the National Health Institute in Bogotá, Colombia approved the study protocol CTIN-014-11. Participation was voluntary and patients were asked for informed written consent authorising to take blood and serum samples and access information on their clinical records.

Samplesize calculationand samplingmethods The total sample size (N) was calculated for test binary outcomes and separately for each clini- cal phase: acute and chronic. Considering, n = Z2 S (1−S) d2, where for a confidence level of 95% (1- α, with α = 0.05) Z is inserted by 1.96, and a maximum marginal error of estimate, d, is a desired value for precision based on researchers judgment, and S is a pre-determined value of sensitivity [29]. Based in previous studies, for the acute phase S was pre-established at 92% and with d at 8% [25,26], whereas for chronic phase S was pre-established at 60% with d at 5% [22– 26]. Then, N = n /P, being P the estimated prevalence in this specific population under study. Given this is a selected population, composed of patients with some suspicion of the infection and remitted to a reference centre, P was specified at 60% in suspected cases for both acute and

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chronic phases. This value was obtained as an approximation based on the laboratory records at the NHI (Bogota, Colombia). The minimum total sample sizes were then calculated as N = 74 and N = 615 for suspected cases in acute and chronic phases respectively. The tests were performed to all subjects without knowing their previous clinical status. Clinical evalua- tion was conducted simultaneously to all individuals as part of the study to determine health status and then to the confirmed cases to evaluate heart complications. The inclusion of partici- pants was conducted retrospectively for the period 2004 to 2012, and prospectively between 2013 and 2015. At the end, a total of 86 suspected acute patients and 622 suspected chronic patients were included in the study (Table 1).

Clinicalclassification Acute phase. a suspected case was defined as an individual with > 7 days of fever accom-

panied or not by hepatomegaly or splenomegaly. The patient was considered with acute Chagas disease if additionally to symptoms tested positive by parasitological tests (Strout, micro-strout, blood thick smear, or hemoculture) [15] or presented positive results to two serological tests over the course of the following weeks [30,31]. The patients were classified as negative to Cha- gas disease otherwise noted.

Chronic phase. individuals without criteria for acute phase but with clinical or epidemio- logical suspicion of Chagas disease. The patients were confirmed as positive T. cruzi infection when tested positive to two serological tests (IFA, ELISA and/or HAI). It was then classified as chronic undetermined (when no evidence of signs or symptoms of heart complications were evinced) or chronic determined otherwise.

The risk factors classification was conducted through a survey applied to each of the patients included in the study. A series of questions were asked such as the place of birth, knowledge of vector insects, blood donations and/or organ transplantation, housing type and presence of cardiac symptoms based on previous evaluated questionnaires (Survey 1) [32]. Patients whose serological tests were negative were classified into two groups according to the presence or absence of risk factors. The patients, who had one or more risk factors, were categorized as "negative with risk factors" and those patients that did not have any risk factors were catego- rized as "negative without risk factors".

Laboratorytests Parasitological methods. The direct parasitological methods were performed (Strout,

micro-strout, blood thick smear, or hemoculture) according to the methodology described by

Table 1. Generalcharacteristicsofpatientsincludedinthestudy.

General characteristics Acutephaseb N = 86 Chronicphasec N = 622

Positive Negative Positive Negatived

Patientsnumber(N) 708 71 15 481 141

Age,median(Q1-Q3)a 48 (47–49) 31 (26–35) 27 (23–30) 51 (50–53) 37 (39–41)

Sex, n (%)

Female 428 (60.4) 26 (36.6) 8 (53.3) 313 (65.1) 60 (42.5)

Male 280 (39.6) 45 (63.4) 7 (46.7) 168 (34.9) 81 (52.4)

a Age in years b Positive patientswere those who had positive directparasitological tests, symptomatology and/orserological tests. Negative patientscomprise a group of

febrilepatientswith negative serologyfor Chagas disease and diagnosedwith dengue. c Positive patientswere those who had two positive serological tests and negative patientswere those with two negative serological tests. d Twenty-nine were negative without risk factor and 112 negative with risk factor

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Freilij et al., 1983 [33]. The results were considered positive when morphology compatible with the T.cruzi was observed. All samples were analysed without knowledge of the clinical status or other test.

Serological tests. Enzyme-linked immunosorbent assay (ELISA), immunofluorescence antibody assay (IFA) or hemaglutination-inhibition assay (HAI) were originally standardized at the National Health Institute [34] with T. cruzi strains belonging to TcI. All serological tests were conducted in duplicate and positive and negative controls were used for each assay. ELISA test was considered as positive when absorbance was �0.300, IFA when titres were �1/ 32 and HAI when titres were �1/32.) (S1 Appendix). All samples were analysed without knowledge of the clinical status or other tests. The indeterminate results in the serology tests (ELISA and IFI) were resolved by use of HAI test.

Molecular diagnostic tests. 10 mL of blood samples were collected and stored with equal volume of Guanidine Hydrochloride 6M, EDTA 0.2 M buffer, pH 8.00 (GEB) and subsequently stored at 8°C. 5mL of serum was frozen at -70°C as described elsewhere [25,35]. 300 μL aliquots of GEB were employed and 5μL of IAC plasmid (40pg/μL) were added as internal control. The samples were submitted to DNA extraction using the High Pure PCR Template Roche kit according to Duffy et al., 2013. Conventional PCR (cPCR) and multiplex quantitative PCR (qPCR) for detection of satellite DNA of T. cruzi and IAC plasmid DNA were performed as reported elsewhere [23,26]. The qPCR test was considered positive when the amplification exceeded the threshold of fluorescence 0.01 and cPCR when was observed a DNA fragment of 166 bp in the electrophoresis. The positive samples for satellite nuclear PCR (qPCR and cPCR), were confirmed by kPCR. Parasitic loads by qPCR were measured as parasite equivalents per mL according to Moreira et al., 2013, using a TcI strain as standard curve (MHOM/CO/01/DA] [22]. All samples were analysed without knowledge of the clinical status or other tests (S1 Appendix).

DTUs discrimination. PCR was performed using five different molecular markers aimed at detecting the six DTUs and the two subdivisions of TcI previously described by other authors (TcIDom and TcI sylvatic) as recommended elsewhere [36–41] (S1 Appendix and S1 Fig).

Statisticalanalysis Operating characteristics of the molecular tests were estimated by comparing against standard diagnosis (described above). Sensitivity, specificity, positive (+LR) and negative likelihood ratio (LR-), predictive values (PV), diagnostic precision (DP), Area under the curve (AUC), and Kappa index (K) were estimated for each phase of the disease (acute and chronic), the clinical stage of chronic patients (determined and undetermined) and according to DTUs and TcI genotypes identified (TcI sylvatic/TcIDom) (S2 Appendix). Results are presented as percent- ages, with corresponding 95% confidence intervals (95% CI). Additionally, operational capabil- ities in chronic patients were calculated in two ways: the first including negative patients without risk factors since they are the true negative and the second including all the negative patients (with and without risk factors). Due to over dispersion of parasitic loads, medians and quartiles are presented. Comparisons are based on Mann-Whitney test between clinical phases, chronic clinical stages and the different T. cruzi DTUs and genotype groups identified. A p value at <0.05 was considered as statistically significant. All analysis was performed in Stata: Data Analysis and Statistical Software version 12.

Results

Generalcharacteristicsof the patients included in the study Overall, 985 patients were included, 872 suspected of chronic and 113 of acute infection. Gen- eral demographic characteristics are shown in Table 1. Out of the initial potential participants,

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27 and 129 were excluded for incomplete samples to perform all analysis from the acute and chronic groups, respectively and 121 from the chronic group due to absence of clinical informa- tion (Fig 1). The inclusion of patients was prospective, whereas the sample collection was both ret- rospective (for the period 2004–2011) and prospective (for the period 2012–2015). This means that for the retrospective component the samples were part of the repository. The repository con- sists of 144 samples, collected between 2004 and 2011, and corresponds to serum samples stored at (-80°C). In these samples, serological tests were repeated and it was found that the results were the same that they had been reported at the time of collection of samples and molecular tests were performed. The prospective component consists of 564 samples, collected in the period between 2012 and 2015, and maintained in guanidine hydrochloride solution until processing.

In patients from the acute phase, the qPCR test was positive in 95.7% of the patients and cPCR in 84.5%. In patients from the undetermined chronic phase, qPCR was positive in 68.0% of the cases and in 55.4% by cPCR. In the cardiac chronic phase, qPCR positivity was 59.1% and 58.6% by cPCR. The positive samples for satellite nuclear PCR (qPCR and cPCR), were confirmed by kPCR. In patients that were negative by serology but with risk factors cPCR (2.6%) and qPCR (3.6%) were positive. In febrile and negative patients without risk factors both tests were negative in all samples. In all samples analyzed we detected the internal amplifi- cation control for both cPCR and qPCR, the average Ct value in all samples tested was 21.

Fig1. Algorithmfor selectionandclassificationofpatients.Therewere selected 708 patients,71 in acutephase, 15 febrilenegatives, 481 in chronic phaseand 141 negatives. *RF:Risk Factor.

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Operatingcharacteristicsof molecularmethodsvs standarddiagnostic tests The operating characteristics including all negatives patients of chronic phase (Negatives with and without risk factors) are presented in Tables 2 and 3.

Performance of qPCR was higher compared to cPCR in both acute (AUC 0.98 vs 0.92) and chronic phase including only negatives with risk factors (0.82 vs 0.78) (Fig 2). Likewise, the per- formance was significantly higher in acute compared with chronic phase and in overall a speci- ficity higher than sensitivity particularly in chronic phase (Tables 2, 4 and 5).

T.cruziparasitic loads and clinicalphase Parasitic loads were determined in samples that tested positive by qPCR. Significantly different median values were detected in acute (4.69 parasite equivalents/mL) versus chronic phase (1.33 parasite equivalents/mL). A statistically median difference was also found between determined and undetermined chronic phase (Fig 3).

Table 3. Operatingcharacteristicsofmoleculartestsinchronicphases(undeterminedanddetermined)ofChagasdisease includingallnegatives patients(withandwithoutriskfactors).

Operatingcharacteristics ChronicundeterminedphaseN = 278/419 Chronicdetermined phaseN = 203/344

qPCR(95%CI) cPCR (95%CI) qPCR(95%CI) cPCR(95%CI)

Sensitivity 67.9 (62.3–73.1) 55.4 (49.5–61.1) 59.1 (52.2–65.6) 58.6 (51.7–65.1)

Specificity 97.2 (92.9–98.8) 97.9 (93.9–99.2) 97.2 (92.9–98.8) 97.9 (93.9–99.2)

PPV 97.9 (94.8–99.2) 98.0 (94.5–99.3) 96.7 (92.0–98.7) 97.5 (93.0–99.1)

NPV 60.6 (54.1–66.7) 52.7 (46.6–58.6) 62.2 (55.7–68.4) 62.1 (55.6–68.2)

DP 77.8 (73.6,81.5) 69.6 (65.1–73.9) 74.7 (69.86–79.0) 74.7 (69.9–79.0)

LR+ 24.0 (14.6–39.3) 26.0 (13.4–50.5) 20.8 (12.6–34.4) 27.5 (14.2–53.5)

LR- 0.33 (0.32–0.33) 0.45 (0.44–0.46) 0.42 (0.41–0.43) 0.42 (0.41–0.43)

K 0.57 (0.48–0.65) 0.44 (0.36–0.52) 0.52 (0.42–0.61) 0.51 (0.42–0.61)

AUC 0.83 (0.79–0.86) 0.77 (0.72–0.80) 0.77 7(0.73–0.82) 0.77 (0.73–0.82)

PPV: Positive predictive value; NPV: Negative predictive value; DP: diagnostic precision; LR+: positive likelihood ratio;LR-: negative likelihood ratio. N =

(Positive gold standard/ total assayed).

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Table 2. Operatingcharacteristicsofmoleculartestsinacuteandchronicphasesincludingallnegativepatients(withandwithoutriskfactors).

Operatingcharacteristics AcutephaseN = (71/86) ChronicphaseN = (481/622)

qPCR(95%CI) cPCR(95%CI) qPCR(95%CI) cPCR(95%CI)

Sensitivity 95.7 (88.3–98.5) 84.5 (74.3–91.2) 64.2 (59.8–68.4) 56.8 (52.3–61.1)

Specificity 100.0(79.6–100.0) 100.0(79.6–100.0) 97.1 (92.9–98.8) 97.9 (93.9–99.2)

PPV 100.0(94.6–100.0) 100.0(93.9–100.0) 98.7 (96.7–99.5) 98.9(96.8–99.6)

NPV 83.3 (60.8–94.2) 57.69(38.9–74.5) 44.3 (38.9–49.9) 39.8 (34.9–45.1)

DP 96.5 (90.2–98.8) 87.2 (78.5–92.8) 71.7 (68.0–75.1) 66.0 (62.3–69.6)

LR+ Undefined 22.6 (13.82–37.09) 26.68(13.8–51.5)

LR- 0.04 (0.02–0.1) 0.15 (0.13–0.18) 0.37 (0.36–0.37) 0.44 (0.43–0.45)

Kappaindex 0.89 (0.7–1.1) 0.62 (0.5–0.9) 0.43 (0.36–0.49) 0.36 (0.29–0.42)

AUC 0.98 (0.91–0.99) 0.92 (0.88–0.96) 0.81 (0.77–0.84) 0.77 (0.74–0.81)

PPV: Positive predictive value; NPV: Negative predictive value; DP: diagnostic precision; LR+: positive likelihood ratio;LR-: negative likelihood ratio. N =

(Positive gold standard/ total assayed). When the specificity is 100% the positive likelihood ratio is undefined.

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Fig2. ROC curvesofmoleculartestsinclinicalphasesofChagasdisease.A. AcutephaseB. ChronicphaseC. Chronicundetermined phase D. Chronicdeterminedphase.

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Molecular testsperformanceaccordingtoT.cruziDTUs and clinical phase In samples that tested positive (n = 407) by cPCR, the DTUs TcI-TcVI and TcI (TcI Dom, TcI Sylvatic) were evaluated. The distribution of DTUs was 74.2% for TcI, 17.2% for TcII, 1.48% for TcIII, 0.5% for TcV and 6.7% for mixed infections. For the latter seven different combina- tions were identified: TcIDom/TcII/TcV, TcIDom/TcII, TcIDom/TcISylv , TcIDom/TcISylv/ TcII, TcIDom/TcISylv/TcIII, TcIDom/TcIV, TcISylv/TcII. With respect to TcI, the genotyping was feasible in 290/302 samples. Out of them, 28.7% were classified as TcI Sylvatic and 71.4% as TcIDom. The median load parasitic value for TcII (4.68 parasite equivalents/mL) was signif- icantly different to the one for TcI (2.87 parasite equivalents/mL) and TcIII (1.72 parasite equivalents/mL) (Fig 4).

The genotype distribution according to clinical phase evidenced that TcIDom was sig- nificantly more frequent in chronic phase compared with acute phase (Table 6). The oper- ating characteristics of molecular tests for the different genotypes were calculated, observing that the sensitivity for identifying TcII was slightly higher than for TcI, mainly for qPCR (S1 Table). The median parasitic load for TcIDom was significantly higher (2.58 parasite equivalents/ml) compared with TcI Sylvatic (0.76 parasite equivalents/ml) in chronic phase (Fig 5).

Discussion

Operatingcharacteristicsof molecularmethodsagainststandard diagnostic tests The main limitation involved in this study is the fact that there is not a gold standard test for all clinical phases of Chagas disease. Particularly for chronic phase, the best comparators are serological tests but these techniques measure the immune response and not the relative pres- ence of the parasite. This particular situation impacts the evaluation of new diagnostic tests. This is reflected mainly in the kappa index (Tables 2 and 4) that presented very low values in the undetermined and determined chronic phases. Unfortunately, it has not a simple solution and more understanding of the course of the infection is still needed.

Table 4. Operating characteristicsofmolecular testsinchronicphaseofChagasdiseaseincluding onlynegativeswithoutriskfactors.

Operatingcharacteristics ChronicphaseN = (481/510)

qPCR(95%CI) cPCR(95%CI)

Sensitivity 64.2 (59.9–58.4) 56.8 (52.3–61.1)

Specificity 100.0(88.3–100.0) 100 (88.3–100.0)

PPV 100.0(98.8–100.0) 100.0(98.6–100.0)

NPV 14.4 (10.2–19.9) 12.2 (8.7–17.0)

DP 66.3(62.1–70.2) 59.2 (54.9–63.4)

LR+ Undefined

LR- 0.36 (0.35–0.36) 0.43 (0.42–0.44)

Kappaindex 0.17(0.1213–0.218) 0.13 (0.09–0.17)

PPV: Positive predictive value; NPV: Negative predictive value; DP: diagnosticprecision; LR+: positive

likelihood ratio;LR-: negative likelihood ratio. When the specificity is 100% the positive likelihood ratio is

undefined. N = (Positive gold standard/ totalassayed)

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Table 5. Operatingcharacteristicsofmoleculartestsinchronicphases(undeterminedanddetermined)ofChagasdisease includingonlynega- tiveswithoutriskfactors.

Operatingcharacteristics ChronicundeterminedphaseN = 278/307 Chronicdetermined phaseN = 203/232

qPCR(95%CI) cPCR(95%CI) qPCR(95%CI) cPCR(95%CI)

Sensitivity 68.0 (62.3–73.2) 55.4 (49.5–61.1) 59.1 (52.2–65.7) 58.6 (51.8–65.2)

Specificity 100 (88.3–100) 100.0(88.3–100.0) 100 (88.3–100) 100.0(88.3–100.0)

PPV 100 (98.0–100) 100.0(97.6–100.0) 100 (96.9–100) 100.0(96.8–100.0)

NPV 24.6 (17.7–37.1) 18.9 (13.5–25.9) 25.9 (18.7–34.7) 25.6 (18.5–34.4)

DP 71.0 (65.7–75.8) 59.6 (54.0–64.9) 64.2 (57.9–70.1) 63.6 (57.2–69.6)

LR+ Undefined

LR- 0.32 (0.31–0.33) 0.44 (0.43–0.45) 0.41 (0.39–0.41) 0.41 (0.40–0.43)

K 0.29 (0.21–0.36) 0.19(0.12–0.25) 0.26(0.17–0.35) 0.26 (0.17–0.34)

PPV: Positive predictive value; NPV: Negative predictive value; DP: diagnostic precision; LR+: positive likelihood ratio;LR-: negative likelihood ratio. When

the specificity is 100%the positive likelihood ratio is undefined.

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Fig3. Comparativeanalysisofparasitic loadsinpatientswithChagasdisease.Distributionof parasitic load and medianson the basis of theclinicalphases. Theoutlierswere removed fromthe graph for convenience. * p < 0.05 ** p<0.01 *** p < 0.001.

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Fig4. Comparativeanalysisofparasitic loadsforDTUs.Distributionof parasitic load and medianson thebasis of theT.cruziDTUs. For convenience the outlierswere removed for the graph. * p < 0.05 ** p<0.01 *** p < 0.001.

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Table 6. FrequencyofDTUsandTcIgenotypesfromclinicalphasesofChagasdiseasepatients.

DTUs Clinicalphases P value

Acute Chronic

N = 68 N = 332

N (%) (95%CI) n (%) (95%CI)

TcI 54 79.4 69.3 88.9 241 72.6 67.8 77.4 0.25

TcII 6 9.0 2.0 15.9 64 19.3 15.0 23.5 0.04

TcIII 4 6.0 0.2 11.7 2 0.6 0.2 1.4 <0.001 TcV - - - - 2 0.6 0.2 1.4 0.53

Mixed 4 6.0 0.2 11.7 23 6.9 4.2 9.7 0.15

TcI Genotypes N = 48 N = 235

TcI Sylvatic 40 85.1 74.8 95.4 42 17.9 12.9 22.8 <0.001 TcI Dom 8 16.6 4.6 25.2 193 82.1 77.2 87.0 <0.001

DTU: Discrete Unit Typing; bold text: p value at <0.05

doi:10.1371/journal.pntd.0004997.t006

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The results obtained for the molecular diagnosis in acute phase were optimal in terms of sensitivity for both qPCR (95.7%; 95%CI: 88.3–98.5) and cPCR sensitivity (84.5%; 95%CI: 74.3–91.2), and same specificity. Although the results are showing a potential superior perfor- mance of the sensitivity of qPCR compared with cPCR, this difference needs a cautious inter- pretation. This might be explained due to the fact that detection by qPCR increases the sensitivity and specificity because of the hybridization of the Taqman probe in the amplicon, whereas in the case of the cPCR it requires a considerable amount of amplicon so that it can be observed in agarose gels [25,26] In addition, the confidence intervals were slightly overlapped, meaning that there is some indication of this difference but it is not statistically significant, so not definitive. The performance of the molecular tests in the acute phase is explained because there are large numbers of parasites, for example in cases of reactivation in immunosuppressed patients and in oral outbreaks. The values obtained for LR evinced the high probability that positive results correspond to diseased patients (LR+) and the low probability that the diseased patients present negative results (LR-). In addition, the DP was very optimal specifically for qPCR test confirming that this molecular test is very useful for the diagnosis in the acute phase, considering that the direct diagnosis is complex when the parasitemia is low (As is the case of the acute patients detected more than a month after the infection where the parasitemia nor- mally begins to decrease due to the control of the immune response) and are required many tests for the confirmation of the acute cases (direct tests, serology tests and clinical informa- tion). Regarding the predictive power of molecular tests in the acute phase, these tests are very good predictors of the disease presence when positive results are obtained (PPV) but their per- formance as predictors of absence of the disease are less (NPV). However, it is worth noting that the predictive values depend on disease prevalence in the evaluated population.

The analysis of operational capabilities in the chronic phase was conducted in the first instance including only negative patients without risk factors or true negatives. For the chronic

Fig5. Comparativeanalysisofparasitic loadsforTcIGenotypesintheclinicalphases.Distribution of parasitic load and medianson the basis of theTcIgenotypesin theacuteand chronicphases. For convenience the outlierswere removed for thegraph. * p < 0.05 ** p<0.01 *** p < 0.001.

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phase, qPCR sensitivity was 64.2% and 56.8% for cPCR and in concordance with previous reports obtained by qPCR that have shown sensitivity ranging from 60–80% and 20–70% for cPCR [22–24,26,28,42]. These sensitivity results may be due to low and intermittent parasitic loads during chronic phase.

The performance of qPCR was better than cPCR in the chronic undetermined phase, while that was very similar between the two tests in the determined chronic phase (Tables 3 and 5). The discriminative power of the two molecular tests was acceptable in the chronic phase. For qPCR, the AUC and DP values obtained (Tables 3 and 5) were better for the undetermined phase than for determined phase. The differences between undetermined and determined phases for qPCR of the chronic phase can be explained by the natural course of the disease, in which the parasitic load decreases while increases the infection time. This is supported by sev- eral studies showing that there is no relationship between the evolution of the cardiac form of the disease and parasitemia but it declines with time as observed in this study [43,44]. Also, some studies show that cardiac form is mainly related to different types of strains, increased parasitemia, reinfection or immune system disorders in chronic patients [45,46]. In the cPCR AUC values were the same for both phases, while the value of DP was best for the determined phase. Possibly, this is because the detection limit of the cPCR is lower than qPCR, for this rea- son the cPCR behaves similarly in the two phases. In the two stages of the chronic phase, there is a high probability that patients with negative results in the molecular tests have the disease (LR-) and these tests are not good predictors of the absence of the disease (NPV) (Table 5). Therefore, the use of molecular methods as diagnostic tests is not appropriate due to the better performance displayed by serology. The probability that the results are positive is high in dis- eased individuals with respect to healthy individuals (LR +) and the molecular tests are excel- lent predictors of the presence of disease (PPV). Thus, these tests could be used in situations in which the diagnosis is doubtful, allowing the confirmation of the parasite in diseased patients, which is of great importance for example when monitoring etiological treatment. However, it is necessary to improve the sensitivity, which can be performed by analysing serial samples for each patient as seen in some studies in which such sensitivity improved from 69.2% to 85.2% with the addition of a second sample or conducting DNA extraction from a larger volume of the sample [47,48].

In addition, the operating capabilities of patients in chronic phase were calculated including all negatives by serology with and without risk factors (Table 1, N = 141). It was observed in the group of negative patients with risk factors a positivity of 2.6% (3 patients) by cPCR and 3.6% (4 patients) by qPCR, possibly due to an immunosuppression issue in these patients pre- venting the detection of antibodies or infection. Three patients are from the department of Casanare, which is an endemic area, and five patients had less than 24 years of age suggesting a recent infection. Also, all patients reported to know the vectors and have lived during his/her childhood in homes with features such as thatched or ‘barheque’, floor or wood and/or tread walls of earth, wood or ‘barheque’. Two of the seven patients that were negative by serology and had risk factors, whose ages were 36 and 51 showed the presence of symptoms at cardiac level. In this group of 7 patients, 4 presented the ELISA absorbance values greater than 0.200 and 4 detectable titles in the IFA (1/8 and 1/16).

As the operating capabilities calculated including all negative patients, a small percentage of decreased specificity in the two platforms was observed (S3 Appendix). The positivity of these serologically negative patients that generated the decrease can probably be explained because cases of recent infection or patients with some form of immunosuppression that has generated the absence of detectable antibodies. In fact, in the group of acute patients, 4 patients whose serology was negative showed positive PCR, in these patients the detection was achieved by direct parasitological methods. Regarding the molecular techniques, given that in all PCR runs

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were included negative controls including reagents controls, a plausible contamination with parasite DNA is discarded. Significantly, the DP and AUC values showed no obvious changes unlike the values obtained for the NPV and the Kappa index, in which there was a marked increase. However, the changes obtained do not change the interpretation of the usefulness of the test in the clinical setting, but can show that there are few cases where serological tests may have false negatives as noted previously using cPCR by Ramirez et al., 2009 [23]. Even though serological tests are considered the best current option for the diagnosis of Chagas disease, in a meta-analysis of high quality tests their sensitivity has been estimated at 90% [49]. Given this, we believe that an improvement of diagnostic tests for Chagas disease is needed for both serol- ogy and PCR techniques. An appropriate use of the comparator as gold standard and the inclu- sion of different phases of the disease are crucial to understand the utility of different diagnostic tests.

To our knowledge, this is the first study to include statistical calculation of the sample, which allowed the analysis of operating characteristics of the molecular tests in all clinical phases of Chagas disease. In addition, this study is the first in analysing the two PCR platforms (qPCR and PCR) for the same target (stDNA) in patients from all clinical phases of Chagas dis- ease. The conventional technique was included, due to the vast use of this technique in the diagnosis and its ease implementation in laboratories with restricted equipment (a Real Time PCR machine is not available) [23,24,28]. Lastly, acute patients had a less median age than chronic phase patients and in turn the largest number of acute cases are male. This possibly is because economic activity in endemic areas is developed by males that assist to the field and this facilitates direct patient contact with the vector and therefore with the parasite. On the other hand, females ratio and the median age were higher in chronic phase patients that are usually detected by screening blood banks or present cardiac abnormalities in chronic phase, then the detection occurs at a greater age. Additionally, in Colombia most blood donors are women facilitating their diagnosis.

T.cruziparasitemia,DTUs and clinicalphases Regarding the parasitemia, it is observed that the median parasitemia was higher in acute patients compared to chronic phase, which is expected given the dynamics of parasitemia in the disease [25,26]. As for the group of chronic patients, the herein reported median of parasi- temia is similar to those previously reported for Colombia [22,26]. In addition, the difference in medians between cardiac chronic and undetermined chronic stages was statistically signifi- cant, being higher in the undetermined chronic phase unlike the findings described by Ramirez et al, 2015 [26], in which statistically significant difference was not detected. However, our results are in accordance with the natural history of the disease where parasitic loads decrease with the chronicity of the infection and this is probably associated with the type of strain and/ or the immune response [2].

The DTU with highest frequency was TcI, both in acute and chronic patients, consistent with findings previously reported in Colombia [8,39,50,51]. Followed by TcII most often detected in chronic than acute patients. These findings are congruent due to the predominance of TcII in domestic cycles of transmission for the case of Colombia [50]. Regarding the parasitic loads of the DTUs detected, we observed that TcII had higher median parasitemia than other DTUs, consistent with the number of copies that has been reported in the DNA nuclear satel- lite region being higher for TcII than for TcI [52–54]. These findings highlight the importance of using the most representative DTU to generate the standard curves for quantification [22,25,26]. In addition, in murine models TcII shows higher parasitemias than TcI when per- forming individual and mixed infections [55].

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In this study, acute cases are likely caused by vector transmission and possible oral route. In most of the cases TcI (TcI sylvatic), TcII and TcIII infection was observed. These findings are consistent with previously documented reports for acute patients where DTUs associated with the sylvatic cycle of transmission were depicted [4,5,40,51,56–61]. An interesting finding was the detection of TcV in the patients surveyed. This DTU has been already reported in dogs and Rhodnius prolixus from eastern Colombia but this would be the first report of TcV human infection in the country [50]. It is necessary to conduct further studies to understand the host- parasite associations of this foreseen DTU in patients from northern areas of the continent. It is well known that TcV infection is endemic in Bolivia, Brazil and Argentina but in Colombia is a novel case that requires further investigation; in fact high-resolution markers have been applied to the few isolates of Colombian TcV showing a tailored hybrid profile suggesting a Pan-American import from south America [62]. The DTU TcVI, is mainly detected in the South Cone of Latin America. Normally associated with megavisceral syndromes and some cases of congenital heart disease [4]. In Colombia, TcVI has been very rare and almost infre- quent. In fact it is limited to a report in which was detected in humans and R. prolixus isolates (4% and 1.4% respectively). In addition, in different studies with a considerable number of patients conducted in Colombia it was not detected, confirming the low prevalence of the DTU in the country [39,51,63].

Recently, it has been highlighted the emergence of a genotype named as TcIDom and associ- ated to human infection and domestic transmission cycles via different molecular markers [5,6,8,64–66]. Other studies have shown the presence of TcI Sylvatic genotype in tissue and TcI- Dom in bloodstream of patients with Chagas cardiomyopathy [41]. In murine models was observed that TcIDom produced high parasitemia and low tissue invasion, a process that allows an adaptation to the host prolonging its permanence and likely generation of chronicity, opposite process to what happened with the TcI sylvatic strains [67]. In accordance with these previous findings, our results show that in chronic patients the frequency and parasitemia of TcIDom geno- type were significantly higher in chronic patients than in acute patients, supporting the hypothesis that this genotype may be related to chronicity in patients with Chagas cardiomyopathy.

In conclusion, the molecular diagnostic tests are becoming a precise tool to complement the standard diagnostic methods for Chagas disease. This study shows that in general qPCR has a better performance than cPCR. Also, the results confirm that PCR is highly specific for both acute and chronic clinical phases, whereas sensitivity is acceptable for acute phase but still very low for chronic patients. This situation could be partially explained by the higher parasitic loads detected in acute phase and the intermittent nature of the parasite release to the blood- stream in chronic phase. We explored for the first time in a large cohort of Chagas disease patients the DTU parasitemia and the natural course of infection. This type of studies is required in Latin-America for a better understanding of disease progression and molecular epi- demiology of Chagas disease. This makes PCR a potential tool for its use in acute phase diagno- sis in a routine basis, and potentially for determining aetiological treatment failure when tests positive but not substantially useful when tests negative and these results must be interpreted cautiously as in the clinical trials previously published [21,68]. Further research is needed to improve the sensitivity of this test and the mandatory deployment of new diagnostic tests.

SupportingInformation S1 Fig. Algorithm for genotyping of T. cruzi DTUs. Molecular characterization of T.cruzi by five molecular markers and genotyping of TcI DTU in two genotypes TcI Dom and �TcI Sylv: TcI Sylvatic. (JPG)

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S1 Table. Operating characteristics of molecular test for DTUs and genotypes TcI. (DOC)

S1 Appendix. Methodology of reference tests employed in the study. (DOCX)

S2 Appendix. Detailed methodologyof the molecular tools employed in the study. (DOC)

S3 Appendix. Sensitivity and specificity calculations. (DOC)

S4 Appendix. STARD. Standards for Reporting of Diagnostic Accuracy Checklist. (DOCX)

S5 Appendix. Research protocol. Project: Characterization of a cohort of patients with Chagas disease, its etiological treatment, adverse events and therapeutic response. (PDF)

Acknowledgments Parasitology group and Red Chagas Colombia, National Institute of Health, Colombia.

AuthorContributions

Conceived and designed the experiments: CH ZC JDR.

Performed the experiments: CH CF CL.

Analyzed the data: CH ZC MO CV PZ JDR.

Contributed reagents/materials/analysis tools: ZC JDR.

Wrote the paper: CH ZC JDR.

References 1. WHO. Weekly epidemiological record—Chagas diseases in LatinAmerica:an epidemiological update

basedon 2010estimates.2015p. 5–13.Available: http://www.who.int/wer/2015/wer9006.pdf

2. BernC. Chagas’Disease. N Engl J Med.2015;373(5):456–66. Available: doi:http://www.nejm.org/doi/ abs/10.1056/NEJMra1410150PMID:26222561

3. ZingalesB, Andrade SG, Briones MRS, Campbell DA, ChiariE, Fernandes O, et al. A new consensus for Trypanosoma cruzi intraspecific nomenclature: Secondrevision meetingrecommendsTcI to TcVI. MemInst Oswaldo Cruz.2009;104(7):1051–4.PMID:20027478

4. ZingalesB, MilesMA,Campbell DA, TibayrencM,MacedoAM,Teixeira MMG,et al.The revised Try- panosomacruzisubspecific nomenclature:Rationale, epidemiological relevance and researchapplica- tions. Infect GenetEvol. 2012;12(2):240–53. Available: doi:http://dx.doi.org/10.1016/j.meegid.2011. 12.009PMID:22226704

5. MessengerLA, MilesMA,BernC. Betweena bug and a hardplace:Trypanosomacruzigeneticdiver- sity and theclinicaloutcomesof Chagasdisease. ExpertRev Anti Infect Ther. 2015;13(8):995–1029. Available: doi: http://informahealthcare.com/doi/abs/10.1586/14787210.2015.1056158 PMID: 26162928

6. Llewellyn MS, MilesMA,CarrascoHJ, Lewis MD, Yeo M, Vargas J, et al.Genome-Scale Multilocus Microsatellite Typing of Trypanosoma cruziDiscreteTyping Unit I Reveals Phylogeographic Structure and SpecificGenotypes Linked to HumanInfection.PLoS Pathog. 2009;5(5):e1000410. Available: doi: http://dx.plos.org/10.1371/journal.ppat.1000410 PMID:19412340

7. Zumaya-EstradaF, MessengerL, Lopez-Ordonez T, Lewis MD, Flores-Lopez C, Martínez-IbarraAJ, et al. NorthAmerican import?Chartingtheoriginsof an enigmaticTrypanosoma cruzidomesticgeno- type. Parasit Vectors. 2012;5(1):226.

Diagnosis and Genotyping ofT.cruzi in Acuteand ChronicPhases

PLOS NeglectedTropical Diseases | DOI:10.1371/journal.pntd.0004997 September 20,2016 16 / 20

8. RamírezJD, Tapia-calle G, GuhlF. Geneticstructureof Trypanosomacruzi in Colombia revealed by a High-throughputNuclearMultilocus Sequence Typing (nMLST)approach. BMC Genet.2013;14:2–10.

9. Segovia M,CarrascoHJ, MartínezCE, MessengerLA,Nessi A, Londoño JC, et al.Epidemiologic SourceTracking of OrallyTransmittedChagasDisease,Venezuela. Epidemiol SourceTrack Chagas Dis. 2013;19(7):1098–101.

10. LeónCM,Hernández C, MontillaM,RamírezJD. Retrospective distribution of Trypanosoma cruzi I genotypesin Colombia.MemInstOswaldo Cruz.2015;110(3):387–93. Available: http://www.scielo.br/ scielo.php?script=sci_arttext&pid=S0074-02762015000300387&lng=en&nrm=iso&tlng=en doi:10. 1590/0074-02760140402 PMID:25946157

11. RamírezJD, Tapia-Calle G, Muñoz-CruzG,Poveda C, RendónLM,HincapiéE, et al.Trypanosome species in neo-tropical bats:Biological,evolutionaryand epidemiological implications. Infect Genet Evol. 2014;22:250–6. doi: 10.1016/j.meegid.2013.06.022PMID:23831017

12. Marcilia, LimaL, Cavazzana M,Junqueira a C V, Veludo HH,MaiaDa Silva F, et al. A new genotypeof Trypanosoma cruziassociated withbats evidenced by phylogeneticanalysesusingSSU rDNA,cyto- chromeb and HistoneH2B genesand genotypingbasedon ITS1rDNA.Parasitology. 2009;136 (6):641–55.doi:10.1017/S0031182009005861PMID:19368741

13. LimaL, Espinosa-Álvarez O, OrtizPA, Trejo-Varón JA, CarranzaJC, PintoCM,et al. Geneticdiversity of Trypanosoma cruzi in bats, and multilocus phylogeneticand phylogeographical analysessupporting Tcbatas an independent DTU(discretetypingunit).Acta Trop. 2015;151(1):166–77.Available: http:// dx.doi.org/10.1016/j.actatropica.2015.07.015

14. RamírezJ, HernándezC, MontillaM,ZambranoP, FlórezAC, Parra E, et al.FirstReportof HumanTry- panosomacruzi InfectionAttributedto TcBatGenotype. Zoonoses Public Health.2013;1–3.

15. Feilij H, MullerL, GonzalezCappaSMM,CappaSMG.DirectMicromethod for Diagnosis of Acuteand Congenital Chagas ‘ Disease. J ClinMicrobiol.1983Aug; 18(2):327–30. PMID:6413530

16. FloresMA,Trejos A, Paredes AR. Strout’s concentrationmethodin the diagnosisof acuteChagas dis- ease. Bol ChilParasitol. 1966;21:38–9.PMID:4958150

17. Da Silveira JF, Umezawa ES, LuquettiAO. Chagasdisease: recombinantTrypanosoma cruziantigens for serologicaldiagnosis.Trends Parasitol.2001;17(6):286–91. PMID:11378036

18. Umezawa ES, BastosSF, CouraJR, Levin MJ, GonzalezA, Rangel-Aldao R, et al. An improved sero- diagnostic test for Chagas’diseaseemployinga mixtureof Trypanosomacruzi recombinant antigens. Transfusion.2003;43(1):91–7.Available:http://www.ncbi.nlm.nih.gov/pubmed/12519436PMID: 12519436

19. BrittoC, CardosoMA,Vanni C, Hasslocher-MorenoA, Xavier SS, Oelemann W, et al.Polymerase chainreactiondetectionof Trypanosoma cruzi in humanblood samplesas a tool for diagnosisand treatmentevaluation. Parasitology.1995;110(3):241–7.

20. ISGlobal-CRESIB.VIII tallersobre la enfermedad de Chagas importada:Avances en el tratamiento antiparasitario. 2012;30–35. Available:https://issuu.com/isglobal/docs/viii_taller_de_chagas_2012/4

21. MorilloCA, Marin-NetoJA, Avezum A, Sosa-Estani S, RassiA, RosasF, et al. Randomized Trial of Benznidazole for ChronicChagas’ Cardiomyopathy. N Engl J Med.2015;373(14):1–12.Available: http://www.nejm.org/doi/full/10.1056/NEJMoa1507574#t=article

22. MoreiraOC, RamírezJD, Velázquez E, MeloMF, Lima-FerreiraC, GuhlF, et al. Towards theestablish- mentof a consensus real-time qPCR to monitorTrypanosoma cruziparasitemiain patientswithchronic Chagasdiseasecardiomyopathy: A substudyfromtheBENEFITtrial.Acta Trop. 2013;125(1):23–31. doi:10.1016/j.actatropica.2012.08.020PMID:22982466

23. RamírezJD, GuhlF, Umezawa ES, MorilloCA, Rosas F, Marin-Neto JA., et al. Evaluation of adult chronicChagas’heartdiseasediagnosisby molecular and serological methods.J ClinMicrobiol.2009 Dec;47(12):3945–51. doi:10.1128/JCM.01601-09PMID:19846646

24. DuarteL, FlorezO, RinconG, GonzalezC. Comparison of seven diagnostic tests to detectTrypano- somacruzi infection in patients in chronicphaseof Chagas disease.ColombMed.2014;45(2):61–6. PMID:25100890

25. DuffyT, Cura CI,RamirezJC, AbateT, Cayo NM,Parrado R, et al. AnalyticalPerformance of a Multi- plex Real-TimePCR Assay Using TaqMan Probesfor Quantificationof Trypanosoma cruziSatellite DNA in BloodSamples.PLoS NeglTrop Dis. 2013;7(1):e2000.Available: doi:http://journals.plos.org/ plosntds/article?id=10.1371%2Fjournal.pntd.0002000PMID:23350002

26. RamírezJC, Cura CI,MoreiraC, Lages-silva E, Juiz N, Velázquez E, et al.AnalyticalValidation of Quantitative Real-TimePCR Methodsfor Quanti fi cationof Trypanosoma cruziDNA in BloodSamples fromChagasDiseasePatients. J Mol Diagnostics. 2015;17(5).

Diagnosis and Genotyping ofT.cruzi in Acuteand ChronicPhases

PLOS NeglectedTropical Diseases | DOI:10.1371/journal.pntd.0004997 September 20,2016 17 / 20

27. PironM,Fisa R, CasamitjanaN, López-ChejadeP, Puig L, Vergés M,et al. Development of a real-time PCR assay for Trypanosoma cruzidetectionin blood samples.Acta Trop. 2007;103(3):195–200. PMID:17662227

28. SchijmanAG, BisioM,OrellanaL, Sued M,DuffyT, MejiaJaramilloAM,et al. International study to evaluate PCR methodsfor detectionof Trypanosoma cruziDNA in blood samples fromChagasdis- ease patients. PLoS NeglTrop Dis. 2011;5(1):e931.Available: doi:http://journals.plos.org/plosntds/ article?id=10.1371/journal.pntd.0000931 PMID:21264349

29. Hajian-Tilaki K. Samplesize estimationin diagnostic teststudiesof biomedical informatics. J Biomed Inform.2014Apr;48:193–204. doi:10.1016/j.jbi.2014.02.013PMID:24582925

30. INS. Protocolode vigilancia en saludpublica Chagas.2014p. 28.Available: http://www.ins.gov.co/ temas-de-interes/Chagas/01%20Protocolo%20Chagas.pdf

31. Shikanai-Yasuda MA,CarvalhoNB. Oral transmission of chagasdisease.Clin Infect Dis. 2012;54 (6):845–52.doi:10.1093/cid/cir956PMID:22238161

32. Castellanos-Dominguez YZ, Cucunubá ZM,OrozcoLC, Valencia-HernandezCA, LeónCM,Florez AC. Risk factors associatedwithChagasdisease in pregnantwomenin Santander, a highlyendemic Colombianarea.2016;21(1):140–8. doi:10.1111/tmi.12634PMID:26578246

33. Feilij H, MullerL, GonzalezCappaSM.Directmicromethodfor diagnosisof acuteand congenital Cha- gas’ disease.J Clin Microbiol. 1983;18(2):327–30. PMID:6413530

34. LópezMC, DuqueBeltránS, OrozcoVargas LC, CamargoD, GualdrónLE, CáceresE, et al. Inmuno- diagnóstico de la infección chagásicaporELISA.Biomédica. 1999;19(2):159–63.

35. MeloMF, MoreiraOC, Tenório P, LorenaV, Lorena-Rezende I, Júnior WO, et al. Usefulness of real timePCR to quantifyparasite load in serumsamplesfromchronicChagasdiseasepatients.Parasit Vectors. 2015; 8:154.Available: doi: http://parasitesandvectors.biomedcentral.com/articles/10.1186/ s13071-015-0770-0PMID:25890282

36. SoutoRP, FernandesO, MacedoAM,Campbell D a.,ZingalesB. DNAmarkersdefinetwo majorphylo- genetic lineagesof Trypanosoma cruzi.MolBiochemParasitol. 1996;83(2):141–52.PMID:9027747

37. LiarteDB, MurtaSMF, SteindelM, Romanha AJ. Trypanosoma cruzi:MultiplexPCR to detectand clas- sify strainsaccordingto groupsI and II.Exp Parasitol.2009;123(4):283–91.Available: doi:http://dx.doi. org/10.1016/j.exppara.2008.12.005PMID:19133262

38. BrisseS, Verhoef J, TibayrencM.Characterisationof largeand smallsubunit rRNAand mini-exon genesfurthersupportsthe distinction of six Trypanosomacruzi lineages. IntJ Parasitol. 2001;31 (11):1218–26. PMID:11513891

39. RamírezJD, GuhlF, Rendón LM,RosasF, Marin-Neto JA, MorilloC a. ChagasCardiomyopathy Mani- festations and TrypanosomacruziGenotypes Circulating in ChronicChagasic Patients. PLoS Negl Trop Dis. 2010;4(11):e899.Available: doi:http://dx.plos.org/10.1371/journal.pntd.0000899 PMID: 21152056

40. VillaLM,GuhlF, ZabalaD, Ramírez JD, UrreaDA, HernándezDC, etal.The identification of two Trypa- nosomacruzi I genotypes fromdomesticand sylvatic transmission cycles in Colombiabasedon a sin- gle polymerasechainreactionamplificationof the spliced-leader intergenicregion.MemInstOswaldo Cruz.2013;108(7):932–5. doi:10.1590/0074-0276130201 PMID:24037107

41. BurgosJM,DiezM,ViglianoC, BisioM,RissoM,DuffyT, etal.Molecular IdentificationofTrypanosoma cruziDiscreteTyping Units in End�Stage ChronicChagasHeartDisease and Reactivation afterHeart Transplantation.Clin Infect Dis. 2010;51(5):485–95.Available: doi:http://cid.oxfordjournals.org/ lookup/doi/10.1086/655680 PMID:20645859

42. Castroa M,Luquettia O, Rassia, Rassi GG,ChiariE, GalvãoLMC. Bloodcultureand polymerase chainreactionfor thediagnosisof thechronicphaseof humaninfection withTrypanosomacruzi.Para- sitolRes. 2002;88:894–900. PMID:12209329

43. Pereira J, Wilcox H, CouraJR. The evolution of chronicchagasiccardiopathy. I. The influence of para- sitemia.Rev Soc Bras MedTrop. 1992;25(2):101–8.PMID:1308935

44. CastroC, PrataA, MacedoV. The influenceof the parasitemiaon theevolution of the chronicChagas’ disease.Port Rev Soc Bras Med.2005;36:1–6.

45. MateusJ, Lasso P, Pavia P, RosasF, RoaN, Valencia-Hern??ndez CA, et al.Low Frequency of Circu- latingCD8+ T StemCellMemoryCells in ChronicChagasicPatients withSevere Formsof theDisease. PLoS NeglTrop Dis. 2015;9(1):e3432.Available:doi:http://journals.plos.org/plosntds/article?id=10. 1371/journal.pntd.0003432 PMID:25569149

46. BustamanteJM,Novarese M,Rivarola HW, Lo PrestiMS, FernándezAR, EndersJE, et al.Reinfec- tionsand Trypanosoma cruzistrainscan determinetheprognosisof thechronicchagasiccardiopathy in mice.Parasitol Res. 2007;100(6):1407–10.PMID:17372764

Diagnosis and Genotyping ofT.cruzi in Acuteand ChronicPhases

PLOS NeglectedTropical Diseases | DOI:10.1371/journal.pntd.0004997 September 20,2016 18 / 20

47. LanaM De, LopesL a, MartinsHR,BahiaMT, Machado-de-AssisGF, Wendling AP, et al.Clinicaland laboratorystatusof patientswithchronicChagasdisease living in a vector-controlledarea in Minas Gerais, Brazil,before and nineyears afteraetiologicaltreatment.MemInstOswaldo Cruz.2009;104 (8):1139–47. PMID:20140375

48. LacunzaCD, NegretteOS, MoraMC, GarcíaBustosMF, Basombrío MÁ.Uso de la reacciónencadena de la polimerasa para el control terapéuticode la infección crónicaporTrypanosoma cruzi.Rev Patol Trop. 2015;44(1):21–32. Available: http://www.revistas.ufg.br/index.php/iptsp/article/view/34815

49. Afonso AM,Ebell MH,Tarleton RL. A SystematicReview of HighQualityDiagnostic Tests for Chagas Disease.PLoS NeglTrop Dis. 2012;6(11).Available: doi:http://journals.plos.org/plosntds/article?id= 10.1371/journal.pntd.0001881

50. GuhlF, RamírezJD. Retrospective molecular integratedepidemiology of Chagasdisease in Colombia. Infect GenetEvol. 2013;20:148–54.Available: doi:http://dx.doi.org/10.1016/j.meegid.2013.08.028 PMID:24012949

51. RamírezJD, MontillaM,CucunubáZM,FlorézAC, ZambranoP, GuhlF. Molecular Epidemiology of HumanOralChagasDisease Outbreaksin Colombia. PLoS NeglTrop Dis. 2013;7(2):1–7.doi: http:// journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0002041

52. Elias MCQB, Vargas NS, ZingalesB, Schenkman S. Organization of satelliteDNA in the genomeof Trypanosoma cruzi.MolBiochemParasitol.2003;129(1):1–9. PMID:12798501

53. GonzalezA, Prediger E, HuecasME,NogueiratN, LizardiPM,NogueiraN, et al.Minichromosomal repetitiveDNA in Trypanosoma cruzi: its use in a high-sensitivity parasitedetectionassay. ProcNatl Acad Sci U S A. 1984;81:3356–60. PMID:6427769

54. MartinsC, BaptistaCS, IenneS, Cerqueira GC, Bartholomeu DC, ZingalesB. Genomicorganization and transcription analysisof the 195-bpsatelliteDNA in Trypanosomacruzi.MolBiochemParasitol. 2008;160(1):60–4. Available: doi:http://linkinghub.elsevier.com/retrieve/pii/S0166685108000674 PMID:18440654

55. Sales-Campos H, KappelHB, Andrade CP, LimaTP, de CastilhoA, GiraldoLER,et al. Trypanosoma cruziDTUTcIIpresentshigherblood parasitism thanDTU TcI in an experimental modelof mixed infec- tion.ActaParasitol. 2015;60(3):435–41. Available: doi:http://www.degruyter.com/view/j/ap.2015.60. issue-3/ap-2015-0060/ap-2015-0060.xmlPMID:26204180

56. NichollsRS, Cucunubá ZM,KnudsonA, FlórezAC, MontillaM, PuertaBula CJ, et al.Enfermedadde Chagasagudaen Colombia,una entidadpoco sospechada. Informede 10 casos presentados en el periodo2002a 2005.Biomédica, Rev del Inst Nac Salud.2007;27(supl.1):8–17.

57. MarciliA, Valente VC, Valente SA, Junqueira ACV, Silva FMda, PintoAY das N, et al. Trypanosoma cruzi in BrazilianAmazonia: Lineages TCIand TCIIa in wild primates, Rhodniusspp. and in humans withChagasdiseaseassociatedwithoral transmission. IntJ Parasitol. 2009;39(5):615–23.Available: doi:http://linkinghub.elsevier.com/retrieve/pii/S0020751908004086PMID:19041313

58. Valente SA, da CostaValente V, das Neves PintoAY, de Jesus BarbosaCésarM,dos SantosMP, MirandaCOS, et al. Analysisof an acuteChagasdiseaseoutbreakin theBrazilianAmazon:human cases, triatomines, reservoirmammalsand parasites.Trans R Soc Trop MedHyg.2009;103(3):291– 7. doi:10.1016/j.trstmh.2008.10.047PMID:19118852

59. BlanchetD, BrenièreSF, SchijmanAG, BisioM,SimonS, Véron V, et al.First reportof a family out- breakof Chagasdisease in French Guianaand posttreatment follow-up. Infect GenetEvol. Elsevier B. V.; 2014;28:245–50. Available: doi:http://www.ncbi.nlm.nih.gov/pubmed/25448161PMID:25448161

60. SotoH, Tibaduiza T, MontillaM,Triana-Chávez O, SuárezDC, Torres Torres M,et al. Investigación de vectores y reservorios en brotede Chagasagudoporposible transmisión oral en Aguachica,Cesar, Colombia.Cad SaúdePública. 2014;30(4):746–56. Available: doi:http://www.scielo.br/pdf/csp/v30n4/ 0102-311X-csp-30-4-0746.pdfPMID:24896050

61. HernándezC, CucunubáZ, Parra E, Toro G,ZambranoP, Ramírez JD. Chagasdisease(Trypano- somacruzi)and HIV co-infection in Colombia.IntJ Infect Dis. 2014;26:146–8.Available: http:// linkinghub.elsevier.com/retrieve/pii/S1201971214014969doi:10.1016/j.ijid.2014.04.002PMID: 25080354

62. MessengerLA, RamirezJD, Llewellyn MS, GuhlF MM.Importation of hybrid human-associatedTrypa- nosomacruzistrains of southernSouthAmerican origin,Colombia.EmergInfect Dis. 2016;22(8).

63. BianchiF, CucunubáZ, GuhlF, GonzálezNL, Freilij H, NichollsRS, et al. Follow-up of an Asymptom- atic ChagasDiseasePopulation of ChildrenafterTreatment withNifurtimox(Lampit) in a Sylvatic EndemicTransmission Areaof Colombia. PLoS NeglTrop Dis. 2015;9(2):e0003465.Available: doi: http://dx.plos.org/10.1371/journal.pntd.0003465 PMID:25723465

64. RamírezJD, GuhlF, MessengerL a.,Lewis MD, MontillaM,CucunubaZ, et al. Contemporarycryptic sexuality in Trypanosoma cruzi.MolEcol. 2012;21(17):4216–26. doi:10.1111/j.1365-294X.2012. 05699.xPMID:22774844

Diagnosis and Genotyping ofT.cruzi in Acuteand ChronicPhases

PLOS NeglectedTropical Diseases | DOI:10.1371/journal.pntd.0004997 September 20,2016 19 / 20

65. GuhlF, RamírezJD. Trypanosomacruzi I diversity:Towards the needof geneticsubdivision?Acta Trop. 2011;119(1):1–4. Available: doi:http://dx.doi.org/10.1016/j.actatropica.2011.04.002 PMID: 21510916

66. RamírezJD, DuqueMC, MontillaM,Cucunubá Z, GuhlF. Naturaland emergentTrypanosoma cruzi I genotypesrevealed by mitochondrial (Cytb)and nuclear (SSUrDNA)geneticmarkers.Exp Parasitol. 2012;132:487–94. doi:10.1016/j.exppara.2012.09.017PMID:23031800

67. CruzL, Vivas A, MontillaM,HernándezC, FlórezC, Parra E, et al. Comparative studyof thebiological propertiesof Trypanosoma cruzi I genotypes in a murineexperimental model. Infect GenetEvol. 2015; 29:110–7.Available: http://linkinghub.elsevier.com/retrieve/pii/S1567134814004158doi:10.1016/j. meegid.2014.11.012PMID:25461848

68. MolinaI, Gómezi Prat J, Salvador F, Treviño B, SulleiroE, SerreN, et al.RandomizedTrial of Posaco- nazole and Benznidazole for ChronicChagas’ Disease.N Engl J Med.2014;370(20):1899–908.Avail- able: doi:http://www.nejm.org/doi/abs/10.1056/NEJMoa1313122PMID:24827034

Diagnosis and Genotyping ofT.cruzi in Acuteand ChronicPhases

PLOS NeglectedTropical Diseases | DOI:10.1371/journal.pntd.0004997 September 20,2016 20 / 20