Produção Nacional
Revisado por pares
Analytical Validation of Quantitative Real-Time PCR Methods for Quantification of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients
2015; Elsevier BV; Volume: 17; Issue: 5 Linguagem: Inglês
10.1016/j.jmoldx.2015.04.010
ISSN1943-7811
AutoresJuan Carlos Ramı́rez, Carolina Cura, Otacílio C. Moreira, Eliane Lages-Silva, Natalia Juiz, Elsa Velázquez, Juan David Ramírez, Anahí Alberti, Paula Pavía, María Flóres-Chávez, Arturo Muñoz-Calderón, Deyanira Pérez‐Morales, José Santalla, Paulo Marcos da Matta Guedes, Julie Péneau, Paula L. Marcet, Carlos Padilla, David Cruz‐Robles, Edward Valencia, Gladys Crisante, Gonzalo Greif, Inés Zulantay, Jaime A. Costales, Míriam J. Álvarez-Martínez, Norma E. Martínez, Rodrigo Villarroel, Sandro Villarroel, Zunilda Sánchez, Margarita Bisio, Rudy Parrado, Lúcia Maria da Cunha Galvão, Antônia Claudia Jácome da Câmara, Bertha Espinoza, Belkisyolé Alarcón de Noya, Concepción J. Puerta, Adelina Riarte, Patricio Diosque, Sergio Sosa‐Estáni, Felipe Guhl, Isabela Ribeiro, Christine Aznar, Constança Britto, Zaida E. Yadón, Alejandro G. Schijman,
Tópico(s)Viral Infections and Immunology Research
ResumoAn international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease. An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease. Chagas disease (CD), caused by the protozoan Trypanosoma cruzi, affects mostly the poor populations in 21 countries of the Americas, where close to 7 to 8 million people are infected, 25 million are at risk, and 10 thousand deaths are recorded annually (World Health Organization, www.who.int/mediacentre/factsheets/fs340/en, last accessed November 1, 2014).1Organización Panamericana de la Salud (OPS): Estimación cuantitativa de la enfermedad de Chagas en las Américas. OPS/HDM/CD/425-06. Montevideo, Uruguay: OPS, 2006Google Scholar In recent years, this neglected tropical disease is becoming a global concern because of the increasing migration from Latin America to nonendemic countries from Europe and North America.2Schmunis G.A. Yadon Z.E. Chagas disease: a Latin American health problem becoming a world health problem.Acta Trop. 2010; 115: 14-21Crossref PubMed Scopus (590) Google Scholar Complex interactions between the genetic background of the parasite and the host and environmental and epidemiologic factors determine the outcome of the infection. In the acute phase of CD the symptoms are variable, and in most cases resolve spontaneously after some weeks. Appropriate treatment can eliminate the parasite during this phase, but the infection is only recognized in 1% to 2% of infected persons during the acute phase. In the chronic phase, approximately 70% of seropositive persons are asymptomatic, whereas 30% ultimately develop serious cardiac and/or digestive disorders several years or decades later, and necrotizing inflammatory injuries in the central nervous system in cases of CD reactivation under immunodepression. Each year, 2% to 3% of symptomatic persons start to present manifestations that can rapidly evolve to sudden death. However, the factors that govern the progression of chronic CD remain unknown, and no prognostic markers are available.3Prata A. Clinical and epidemiological aspects of Chagas disease.Lancet Infect Dis. 2001; 1: 92-100Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar Accurate diagnostics tools and surrogate markers of parasitologic response to treatment are priorities in CD research and development.4WHO/TDR Disease Reference Group on Chagas Disease, Human African Trypanosomiasis and LeishmaniasisWHO Technical Report Series (No. 975). Research Priorities for Chagas Disease, Human African Trypanosomiasis and Leishmaniasis. WHO, Geneva, Switzerland2012Google Scholar To develop an accurate laboratory tool for diagnosis and treatment follow-up, several difficulties need to be addressed, such as the low and intermittent number of circulating parasites during the chronic phase of infection and parasite genotype diversity, because six discrete typing units (DTUs), TcI to TcVI, are unevenly distributed in different endemic regions.5Zingales B. Miles M.A. Campbell D.A. Tibayrenc M. Macedo A.M. Teixeira M.M. Schijman A.G. Llewellyn M.S. Lages-Silva E. Machado C.R. Andrade S.G. Sturm N.R. The revised Trypanosoma cruzi subspecific nomenclature: rationale, epidemiological relevance and research applications.Infect Genet Evol. 2012; 12: 240-253Crossref PubMed Scopus (604) Google Scholar Quantitative real-time PCR (qPCR)-based assays may fill these gaps, but their application in the clinical practice requires prior analytical and clinical validation studies.6Burd E.M. Validation of laboratory-developed molecular assays for infectious diseases.Clin Microbiol Rev. 2010; 23: 550-576Crossref PubMed Scopus (322) Google Scholar, 7Schijman A.G. Bisio M. Orellana L. Sued M. Duffy T. Mejia Jaramillo A.M. et al.International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients.PLoS Negl Trop Dis. 2011; 5: e931Crossref PubMed Scopus (264) Google Scholar So far, a few real-time PCR strategies have been developed for T. cruzi DNA detection and quantification in CD patients.8Duffy T. Bisio M. Altcheh J. Burgos J.M. Diez M. Levin M.J. Favaloro R.R. Freilij H. Schijman A.G. Accurate real-time PCR strategy for monitoring bloodstream parasitic loads in Chagas disease patients.PLoS Negl Trop Dis. 2009; 3: e419Crossref PubMed Scopus (207) Google Scholar, 9Duffy T. Cura C.I. Ramirez J.C. Abate T. Cayo N.M. Parrado R. Bello Z.D. Velazquez E. Munoz-Calderon A. Juiz N.A. Basile J. Garcia L. Riarte A. Nasser J.R. Ocampo S.B. Yadon Z.E. Torrico F. de Noya B.A. Ribeiro I. Schijman A.G. Analytical performance of a multiplex real-time PCR assay using TaqMan probes for quantification of Trypanosoma cruzi satellite DNA in blood samples.PLoS Negl Trop Dis. 2013; 7: e2000Crossref PubMed Scopus (178) Google Scholar, 10Piron M. Fisa R. Casamitjana N. Lopez-Chejade P. Puig L. Verges M. Gascon J. Gomez i Prat J. Portus M. Sauleda S. Development of a real-time PCR assay for Trypanosoma cruzi detection in blood samples.Acta Trop. 2007; 103: 195-200Crossref PubMed Scopus (281) Google Scholar, 11Qvarnstrom Y. Schijman A.G. Veron V. Aznar C. Steurer F. da Silva A.J. Sensitive and specific detection of Trypanosoma cruzi DNA in clinical specimens using a multi-target real-time PCR approach.PLoS Negl Trop Dis. 2012; 6: e1689Crossref PubMed Scopus (74) Google Scholar As part of the Small Grants Programme (joined initiative of Communicable Diseases Research/Pan-American Health Organization) and The Special Programme for Research and Training in Tropical Diseases/United Nations Development Program/United Nations Children’s Fund/World Bank/World Health Organization, an international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex qPCR strategies on the basis of TaqMan probes for detection and quantification of the parasite loads in blood samples of CD patients. The studies in which the samples were collected were approved by the ethical committees of the participating institutions, according to the principles expressed in the Declaration of Helsinki. Written informed consent forms were signed by the adult study subjects and from parents/guardians on behalf of all minor subjects. All samples were pre-existent at the time of this international study and were anonymized before being processed. Seronegative human blood samples were spiked with cultured epimastigotes of Sylvio X10 and CL-Brener stocks (TcId and TcVI, respectively) and were immediately mixed with one volume of guanidine hydrochloride 6 mol/L EDTA 0.2 mol/L buffer, pH 8.00 (GE). Peripheral blood samples from 156 CD patients were distributed into eight groups according to their geographic origin, as follows. Group 1 (G1) included samples from four seropositive patients from Mexico, two patients with acute CD (G1a) and two patients with asymptomatic chronic CD (G1b). Group 2 (G2) included samples from two patients from French Guiana with acute CD acquired by oral transmission. One patient was positive and the other patient was negative for IgG serologic studies. Both patients experienced cardiac symptoms and were infected with TcI. Group 3 (G3) included samples from five seropositive patients from Bolivia; two patients with acute CD acquired by oral transmission (G3a) and three patients with asymptomatic chronic CD acquired from vectors or congenitally (G3b). Group 4 (G4) included samples from five seropositive patients from Venezuela with acute CD acquired by oral transmission. Two of these patients were infected with TcI. Group 5 (G5) included samples from 13 seropositive patients from Colombia with asymptomatic chronic CD acquired from vectors or congenitally. Five of these patients were infected with TcI. Group 6 (G6) included samples from 21 Bolivian seropositive patients, resident in Spain, with chronic CD acquired from vectors or congenitally. One patient experienced digestive symptoms and the others were asymptomatic. Group 7 (G7) included samples from 31 seropositive patients from Brazil with chronic CD and the following clinical manifestations: asymptomatic (n = 5), cardiac (n = 17), digestive (n = 2), and cardiodigestive (n = 7). Thirteen patients were infected with TcII. Group 8 (G8) included samples from 75 seropositive patients from Argentina with chronic CD and the following clinical manifestations: asymptomatic (n = 27), cardiac (n = 34), digestive (n = 1), and cardiodigestive (n = 13). Fifty-one patients were infected with TcV or TcVI or combinations of TcII, TcV, and TcVI. In addition, samples from 50 persons from Argentina with negative serology for T. cruzi were included as negative controls to address the specificity of the procedures. The blood samples were obtained and immediately mixed with an equal volume of GE (GEB). After 48 to 72 hours at room temperature GEB samples were boiled for 15 minutes (except for G3 and G5 groups and seronegative samples) and stored at 4°C for DNA extraction and PCR analysis. GEB samples were processed with the High Pure PCR Template Preparation kit (Roche Diagnostics Corp., Indianapolis, IN) as described in Duffy et al.9Duffy T. Cura C.I. Ramirez J.C. Abate T. Cayo N.M. Parrado R. Bello Z.D. Velazquez E. Munoz-Calderon A. Juiz N.A. Basile J. Garcia L. Riarte A. Nasser J.R. Ocampo S.B. Yadon Z.E. Torrico F. de Noya B.A. Ribeiro I. Schijman A.G. Analytical performance of a multiplex real-time PCR assay using TaqMan probes for quantification of Trypanosoma cruzi satellite DNA in blood samples.PLoS Negl Trop Dis. 2013; 7: e2000Crossref PubMed Scopus (178) Google Scholar Those samples with cycle threshold (Ct) values lower than the Ct values for the most concentrated point of the standard curve were properly diluted in seronegative human blood treated with GE, and DNA extraction and qPCR procedures were repeated. To build the standard curves for quantification of parasitic loads, DNA from spiked blood samples were obtained in the same way as reported for the clinical samples. The DNA eluate was stored at −20°C until use in qPCR analysis. Two duplex qPCR procedures were compared, Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR assays. The former targets the satellite sequence of the nuclear genome of the parasite and the sequence of an internal amplification control (IAC) as described in Duffy et al,9Duffy T. Cura C.I. Ramirez J.C. Abate T. Cayo N.M. Parrado R. Bello Z.D. Velazquez E. Munoz-Calderon A. Juiz N.A. Basile J. Garcia L. Riarte A. Nasser J.R. Ocampo S.B. Yadon Z.E. Torrico F. de Noya B.A. Ribeiro I. Schijman A.G. Analytical performance of a multiplex real-time PCR assay using TaqMan probes for quantification of Trypanosoma cruzi satellite DNA in blood samples.PLoS Negl Trop Dis. 2013; 7: e2000Crossref PubMed Scopus (178) Google Scholar and the latter is a modification of the method reported by Qvarnstrom et al11Qvarnstrom Y. Schijman A.G. Veron V. Aznar C. Steurer F. da Silva A.J. Sensitive and specific detection of Trypanosoma cruzi DNA in clinical specimens using a multi-target real-time PCR approach.PLoS Negl Trop Dis. 2012; 6: e1689Crossref PubMed Scopus (74) Google Scholar which targets the conserved region of the minicircle parasite sequences with the addition of primers and TaqMan probe for the IAC.9Duffy T. Cura C.I. Ramirez J.C. Abate T. Cayo N.M. Parrado R. Bello Z.D. Velazquez E. Munoz-Calderon A. Juiz N.A. Basile J. Garcia L. Riarte A. Nasser J.R. Ocampo S.B. Yadon Z.E. Torrico F. de Noya B.A. Ribeiro I. Schijman A.G. Analytical performance of a multiplex real-time PCR assay using TaqMan probes for quantification of Trypanosoma cruzi satellite DNA in blood samples.PLoS Negl Trop Dis. 2013; 7: e2000Crossref PubMed Scopus (178) Google Scholar Both reactions were performed with 5 μL of re-suspended DNA, using FastStart Universal Probe Master Mix (Roche Diagnostics GmbHCorp., Mannheim, Germany) in a final volume of 20 μL. Optimal cycling conditions for both qPCR assays were a first step of 10 minutes at 95°C, followed by 40 cycles at 95°C for 15 seconds and 58°C for 1 minute. The amplifications were performed using Rotor-Gene 6000 (Corbett Life Science, Cambridgeshire, United Kingdom) or ABI7500 (Applied Biosystems, Foster City, CA) devices. Standard curves were plotted with 1/10 serial dilutions of total DNA obtained from a GEB-seronegative sample spiked with 105 parasite equivalents per milliliter of blood (par. eq./mL). TcId- and TcVI-DNA–based standard curves were used to quantify parasitic loads in G1a, G1b, G2, G3a, G4, and G5 and in G3b, G6, G7, and G8 samples, respectively. On the basis of the MICROVAL protocol,12European Committee for StandardizationMicrobiology of food and animal feeding stuffs. Protocol for the validation of alternative methods (EN ISO 16140). European Committee for Standardization, Paris, France2002Google Scholar the analytical validation of both qPCR methods included the following parameters. i) Selectivity is defined as a measure of the degree of response from target and nontarget microorganisms and comprises inclusivity and exclusivity. Inclusivity is the ability of an alternative method (each qPCR assay in this case) to detect the target pathogen from different strains (DTUs in this case), and exclusivity is the lack of response from closely related but nontarget strains (other trypanosomatids in this case). ii) Reportable range is a set of values of measurands for which the error of a measuring instrument is intended to lie within specified limits. iii) Limit of detection (LOD) is the smallest amount that the method can reliably detect to determine the presence or absence of an analyte. iv) Precision is the closeness of agreement between independent test/measurement results obtained under stipulated conditions. v) Limit of quantification (LOQ) is the smallest amount that the method can reliably measure quantitatively. The above-mentioned parameters were evaluated in the framework of the international study as described in the sections below. Both qPCR assays were tested with genomic DNA obtained from a panel of T. cruzi stocks belonging to the six different DTUs, plus TcI stocks representative of three different TcI Spliced Leader Intergenic Region (SL-IR)–based groups (TcIa, TcId, and TcIe), in concentrations that ranged from 0.0625 to 10 fg/μL tested on duplicates: TcI [K98 (TcIa SL-IR–based group), G (TcId group), and SE9V (TcIe group) stocks]13Cura C.I. Mejia-Jaramillo A.M. Duffy T. Burgos J.M. Rodriguero M. Cardinal M.V. Kjos S. Gurgel-Goncalves R. Blanchet D. De Pablos L.M. Tomasini N. da Silva A. Russomando G. Cuba C.A. Aznar C. Abate T. Levin M.J. Osuna A. Gurtler R.E. Diosque P. Solari A. Triana-Chavez O. Schijman A.G. Trypanosoma cruzi I genotypes in different geographical regions and transmission cycles based on a microsatellite motif of the intergenic spacer of spliced-leader genes.Int J Parasitol. 2010; 40: 1599-1607Crossref PubMed Scopus (117) Google Scholar, 14Falla A. Herrera C. Fajardo A. Montilla M. Vallejo G.A. Guhl F. Haplotype identification within Trypanosoma cruzi I in Colombian isolates from several reservoirs, vectors and humans.Acta Trop. 2009; 110: 15-21Crossref PubMed Scopus (98) Google Scholar, 15Herrera C. Bargues M.D. Fajardo A. Montilla M. Triana O. Vallejo G.A. Guhl F. Identifying four Trypanosoma cruzi I isolate haplotypes from different geographic regions in Colombia.Infect Genet Evol. 2007; 7: 535-539Crossref PubMed Scopus (114) Google Scholar, 16Herrera C. Guhl F. Falla A. Fajardo A. Montilla M. Adolfo Vallejo G. Bargues M.D. Genetic variability and phylogenetic relationships within Trypanosoma cruzi I isolated in Colombia based on miniexon gene sequences.J Parasitol Res [Internet]. 2009; 2009 (http://dx.doi.org/10.1155/2009/897364)Google Scholar; TcII (Tu18 stock), TcIII (M5361 stock), TcIV (CanIII stock), TcV (PAH265 stock), and TcVI (CL-Brener stock).17Zingales B. Andrade S.G. Briones M.R. Campbell D.A. Chiari E. Fernandes O. Guhl F. Lages-Silva E. Macedo A.M. Machado C.R. Miles M.A. Romanha A.J. Sturm N.R. Tibayrenc M. Schijman A.G. Second Satellite MeetingA new consensus for Trypanosoma cruzi intraspecific nomenclature: second revision meeting recommends TcI to TcVI.Mem Inst Oswaldo Cruz. 2009; 104: 1051-1054Crossref PubMed Scopus (757) Google Scholar Serial dilutions of purified DNAs from Trypanosoma rangeli and Leishmania major, Leishmania mexicana and Leishmania amazonensis that ranged from 0.1 fg/μL to 1000 pg/μL were assayed on duplicates by both qPCRs. A panel of GEB samples spiked with Sylvio X10 (TcId) and CL-Brener (TcVI) stocks, spanning 105 to 0.0625 par. eq./mL was prepared. After DNA purification, each dilution was amplified on triplicate by both qPCR assays. Assigned (theoretical) versus measured values were converted to Log10 par. eq./10 mL and plotted for linear regression analysis. The LOD was calculated as the lowest parasitic load that gives ≥95% of qPCR detectable results, according to the Clinical and Laboratory Standards Institute guidelines.18National Committee flor Clinical Laboratory Standards (NCCLS)Protocols for Determination of Limits of Detection and Limits of Quantification: Approved Guideline. NCCLS, Wayne, Pennsylvania2004Google Scholar It was measured from two panels of GEB samples spiked with the CL-Brener stock; one panel was boiled for 15 minutes before preparing serial dilutions,7Schijman A.G. Bisio M. Orellana L. Sued M. Duffy T. Mejia Jaramillo A.M. et al.International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients.PLoS Negl Trop Dis. 2011; 5: e931Crossref PubMed Scopus (264) Google Scholar and the other panel was diluted without prior boiling. For both panels, eight replicates from GEB dilutions that contained 0.125, 0.25, 0.5, and 1 par. eq./mL for SatDNA qPCR and 0.0625, 0.125, 0.25, and 0.5 par. eq./mL for kDNA qPCR were purified and amplified during five consecutive days. The LOD was determined by Probit regression analysis with Minitab 15 Statistical Software (Minitab Inc., State College, PA). Precision experiments were performed with spiked GEB samples at concentrations of 0.5, 10, and 103 par. eq./mL (0.699, 2, and 4 Log10 par. eq./10 mL) for SatDNA qPCR and 0.25, 10, and 103 par. eq./mL (0.398, 2, and 4 Log10 par. eq./10 mL) for kDNA qPCR, assayed on duplicates during 20 consecutive experiments, one run per day, according to the Clinical and Laboratory Standards Institute guidelines.19National Committee flor Clinical Laboratory Standards (NCCLS)Evaluation of Precision Performance of Quantitative Measurement Methods: Approved Guideline-Second Edition. NCCLS, Wayne, Pennsylvania2004Google Scholar The estimates of within-laboratory precision SDs (St) were calculated with the following formula: St = [B2 + (N−1)/N × Sr2]1/2, where B is the SD of the daily means and Sr is the estimate of repeatability SD (within-run precision). For both qPCR methods, the LOQ was derived from a 20% threshold value for the CV of measurements obtained in the precision experiments, as described in Schwarz et al.20Schwarz G. Baumler S. Block A. Felsenstein F.G. Wenzel G. Determination of detection and quantification limits for SNP allele frequency estimation in DNA pools using real time PCR.Nucleic Acids Res. 2004; 32: e24Crossref PubMed Scopus (49) Google Scholar Assuming an exponential decrease in CV, a curve for the relation between CV and Log10 par. eq./10 mL was fitted with SigmaPlot 10.0 (Systat Software Inc., San Jose, CA). A negative control and two positive controls that contained different concentrations of T. cruzi DNA (namely a high-positive control and a low-positive control near the limit of detection) were included in every run as recommended.6Burd E.M. Validation of laboratory-developed molecular assays for infectious diseases.Clin Microbiol Rev. 2010; 23: 550-576Crossref PubMed Scopus (322) Google Scholar A pZErO-2 recombinant plasmid that contains an inserted sequence of Arabidopsis thaliana aquaporin was used as a heterologous extrinsic IAC.8Duffy T. Bisio M. Altcheh J. Burgos J.M. Diez M. Levin M.J. Favaloro R.R. Freilij H. Schijman A.G. Accurate real-time PCR strategy for monitoring bloodstream parasitic loads in Chagas disease patients.PLoS Negl Trop Dis. 2009; 3: e419Crossref PubMed Scopus (207) Google Scholar To check for DNA integrity, clinical samples were tested with TaqMan RNase P Control Reagents Kit (Applied Biosystems) in an ABI7500 Real-Time PCR device. SatDNA qPCR quantifiable samples collected from laboratories that did not perform T. cruzi DTU typing (G1, G3, and G6) were genotyped with PCR-based strategies targeted to nuclear genomic markers, as described in Burgos et al.21Burgos J.M. Altcheh J. Bisio M. Duffy T. Valadares H.M. Seidenstein M.E. Piccinali R. Freitas J.M. Levin M.J. Macchi L. Macedo A.M. Freilij H. Schijman A.G. Direct molecular profiling of minicircle signatures and lineages of Trypanosoma cruzi bloodstream populations causing congenital Chagas disease.Int J Parasitol. 2007; 37: 1319-1327Crossref PubMed Scopus (176) Google Scholar The 15 samples provided by the laboratory of Disciplina de Parasitología, Universidade Federal do Triângulo Mineiro (Uberaba, Brazil), were evaluated by hemoculture. The assay was performed according to the method described in Chiari et al.22Chiari E. Dias J.C. Lana M. Chiari C.A. Hemocultures for the parasitological diagnosis of human chronic Chagas' disease.Rev Soc Bras Med Trop. 1989; 22: 19-23Crossref PubMed Scopus (115) Google Scholar Immediately after collection, 30 mL of blood was centrifuged at 4°C to remove plasma. The packed cells were washed by centrifugation at 4°C in liver infusion tryptose medium. The sedimented erythrocytes were resuspended in 30 mL of liver infusion tryptose medium and uniformly distributed in six test tubes. Cultures were maintained at 28°C and homogenized weekly. The culture was microscopically examined 30, 60, and 90 days after culture in 10-μL aliquots of suspension. The Cohen κ coefficient23Landis J.R. Koch G.G. The measurement of observer agreement for categorical data.Biometrics. 1977; 33: 159-174Crossref PubMed Scopus (51320) Google Scholar was used to compare the clinical sensitivity of SatDNA and kDNA qPCRs for the detection of T. cruzi DNA in samples from chronic CD patients. The unpaired t-test was used to compare the means of parasitic loads of quantifiable samples from acute versus chronic CD patients and asymptomatic versus symptomatic chronic CD patients for both qPCR methods. In addition, nonparametric analysis of variance was used to compare the parasitic loads of quantifiable samples grouped according to their T. cruzi genotypes for each qPCR assay. Bland-Altman bias plot6Burd E.M. Validation of laboratory-developed molecular assays for infectious diseases.Clin Microbiol Rev. 2010; 23: 550-576Crossref PubMed Scopus (322) Google Scholar was used to analyze the closeness of the agreement between the quantifiable results of both qPCR methods. Finally, the paired t-test was used to compare the means of IAC Ct values of both qPCR assays, and the Tukey criterion24Burns M.J. Nixon G.J. Foy C.A. Harris N. Standardisation of data from real-time quantitative PCR methods - evaluation of outliers and comparison of calibration curves.BMC Biotechnol. 2005; 5: 31Crossref PubMed Scopus (130) Google Scholar was used to detect samples with outlier Ct values of IAC (Ct > 75th percentile + 1.5× interquartile distance of median Ct), which indicated PCR inhibition or material loss during sample DNA extractions. The analytical validation results obtained for both qPCR assays are shown in Table 1. For the inclusivity, qPCR methods were assayed with DNA from strains that represented the six T. cruzi DTUs, plus TcI stocks representative of three different TcI SL-IR–based groups (TcIa, TcId, and TcIe).Table 1Analytical Validation Results Obtained for qPCR AssaysValidation ParametersSatDNA qPCR∗Data for SatDNA qPCR were taken from Duffy et al.9kDNA qPCRInclusivity (detectable qPCR), fg/μL TcIa0.06250.0625 TcId0.250.0625 TcIe10.0625 TcII0.06250.0625 TcIII0.06250.0625 TcIV0.250.0625 TcV0.06250.0625 TcVI0.06250.0625Exclusivity (nondetectable qPCR), pg/μL T. rangeli10.001 L. major10001000 L. mexicana10001000 L. amazonensis10001000Reportable range, par. eq./mL TcId105–1105–1 TcVI105–0.25105–0.25Limit of detection, par. eq./mL Boiled0.460.16 Nonboiled0.700.23Precision, CV % 0.25 par. eq./mLND31.98 0.5 par. eq./mL46.60ND 10 par. eq./mL6.008.79 1000 par. eq./mL1.722.92Limit of quantification, par. eq./mL1.530.90kDNA, kinetoplastid DNA; ND, not done; par. eq./mL, parasite equivalents in 1 mL of blood; qPCR, quantitative real-time PCR; SatDNA, Satellite DNA.∗ Data for SatDNA qPCR were taken from Duffy et al.9Duffy T. Cura C.I. Ramirez J.C. Abate T. Cayo N.M. Parrado R. Bello Z.D. Velazquez E. Munoz-Calderon A. Juiz N.A. Basile J. Garcia L. Riarte A. Nasser J.R. Ocampo S.B. Yadon Z.E. Torrico F. de Noya B.A. Ribeiro I. Schijman A.G. Analytical performance of a multiplex real-time PCR assay using TaqMan probes for quantification of Trypanosoma cruzi satellite DNA in blood samples.PLoS Negl Trop Dis. 2013; 7: e2000Crossref PubMed Scopus (178) Google Scholar Open table in a new tab kDNA, kinetoplastid DNA; ND, not done; par. eq./mL, parasite equivalents in 1 mL of blood; qPCR, quantitative real-time PCR; SatDNA, Satellite DNA. kDNA qPCR gave the same analytical sensitivity of 0.0625 fg/μL DNA, the lowest concentration tested, for all T. cruzi stocks analyzed. SatDNA qPCR gave an analytical sensitivity of 0.0625 fg/μL DNA for stocks representative of TcIa, TcII, TcIII, TcV, and TcVI; 0.25 fg/μL DNA for stocks belonging to TcId and TcIV; and 1 fg/μL DNA for the TcIe stock. Exclusivity was assayed in T. rangeli and Leishmania spp. Both qPCR methods were nondetectable when up to 1000 pg/μL DNA, the highest concentration tested, from Leishmania stocks was analyzed. In the case of T. rangeli, 10 fg/μL DNA could be amplified by kDNA qPCR, whereas SatDNA qPCR required an input of at least 10 pg/μL T. rangeli DNA to obtain a detectable PCR result. The reportable range was determined with 10 spiked GEB samples that contained serial dilutions of TcId- and TcVI-cultured epimastigotes. Linear regression analysis yielded the equation y = 1.013x − 0.058 (R2 = 0.992) and y = 1.001x − 0.005 (R2 = 0.998) for SatDNA qPCR, and y = 0.813x + 0.824 (R2 = 0.969) and y = 1.011x − 0.048 (R2 = 0.984) for kDNA qPCR and for TcId and TcVI representative stocks, respectively. Accordingly, the reportable range was from 105 to 1 par. eq./mL for TcId stock and from 105 to 0.25 par. eq./mL for TcVI stock, for both qPCR methods. The LOD was determined for TcVI DTU. It was 0.16 par. eq./mL (95% CI, 0.13–0.24 par. eq./mL) and 0.23 par. eq./mL (95% CI, 0.18–0.35 par. eq./mL) for kDNA qPCR in bo
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