Trypanosoma cruzi loop-mediated isothermal amplification (Trypanosoma cruzi loopamp) kit for detection of congenital, acute and chagas disease reactivation

A Trypanosoma cruzi Loopamp kit was recently developed as a ready-to-use diagnostic method requiring minimal laboratory facilities. We evaluated its diagnostic accuracy for detection of acute Chagas disease (CD) in different epidemiological and clinical scenarios. In this retrospective study, a convenience series of clinical samples (venous blood treated with EDTA or different stabilizer agents, heel-prick blood in filter paper or cerebrospinal fluid samples (CSF)) from 30 infants born to seropositive mothers (13 with congenital CD and 17 noninfected), four recipients of organs from CD donors, six orally–infected cases after consumption of contaminated guava juice and six CD patients coinfected with HIV at risk of CD reactivation (N = 46 patients, 46 blood samples and 1 CSF sample) were tested by T. cruzi Loopamp kit (Tc LAMP) and standardized quantitative real-time PCR (qPCR). T. cruzi Loopamp accuracy was estimated using the case definition in the different groups as a reference. Cohen’s kappa coefficient (κ) was applied to measure the agreement between Tc LAMP (index test) and qPCR (reference test). Sensitivity and specificity of T. cruzi Loopamp kit in blood samples from the pooled clinical groups was 93% (95% CI: 77–99) and 100% (95% CI: 80–100) respectively. The agreement between Tc LAMP and qPCR was almost perfect (κ = 0.92, 95% CI: 0.62–1.00). The T. cruzi Loopamp kit was sensitive and specific for detection of T. cruzi infection. It was carried out from DNA extracted from peripheral blood samples (via frozen EDTA blood, guanidine hydrochloride-EDTA blood, DNAgard blood and dried blood spots), as well as in CSF specimens infected with TcI or TcII/V/VI parasite.Fil: Besuschio, Susana Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Picado, Albert. Foundation For Innovative New Diagnostics; SuizaFil: Muñoz Calderon, Arturo Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Wehrendt, Diana Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Fernández, Marisa. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas "Dr. Francisco Javier Muñiz"; Argentina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán". Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben"; ArgentinaFil: Benatar, Alejandro Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Diaz Bello, Zoraida. Universidad Central de Venezuela; VenezuelaFil: Irurtia, Cecilia. Hospital Nacional Profesor Alejandro Posadas; ArgentinaFil: Cruz Mata, Israel. Foundation for Innovative New Diagnostics; SuizaFil: Ndungu, Joseph M.. Foundation for Innovative New Diagnostics; SuizaFil: Cafferata, María L.. Instituto de Efectividad Clínica y Sanitaria; ArgentinaFil: Montenegro, Graciela. Hospital Nacional Profesor Alejandro Posadas; ArgentinaFil: Sosa-Estani, Sergio Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Epidemiología y Salud Pública. Instituto de Efectividad Clínica y Sanitaria. Centro de Investigaciones en Epidemiología y Salud Pública; Argentina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán". Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben"; ArgentinaFil: Lucero, Raúl H.. Universidad Nacional del Nordeste. Instituto de Medicina Regional; ArgentinaFil: Alarcón de Noya, Belkisyole. Universidad Central de Venezuela; VenezuelaFil: Longhi, Silvia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples

Background: Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI–TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR).Methods/Principal Findings: The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm.Conclusions/Significance: Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production.This work received financial support from the Ministry of Science and Technology of Argentina [PICT 2011-0207 to AGS] and the National Scientific and Technical Research Council in Argentina (CONICET) [PIP 112 2011-010-0974 to AGS]. Work related to evaluation of biological samples was partially sponsored by the Pan-American Health Organization (PAHO) [Small Grants Program PAHO-TDR]; the Drugs and Neglected Diseases Initiative (DNDi, Geneva, Switzerland), Wellcome Trust (London, United Kingdom), SANOFI-AVENTIS (Buenos Aires, Argentina) and the National Council for Science and Technology in Mexico (CONACYT) [FONSEC 161405 to JMR]

Utilización de la macrofauna bentónicos como indicador de calidad ambiental en la desembocadura del rio Anchicayá, Pacífico Colombiano.(Report)

Se evaluó la relación existente entre la cantidad de sedimentos que se encuentran en la desembocadura del cauce del río Anchicayá y el crecimiento de la macrofauna bentónica entre las zonas de Taparal y Soldado, Bahía de Buenaventura. Las muestras de macrofauna fueron recopiladas en agosto del 2003, empleando una draga Peterson de 225 [cm.sup.2] de área. Todas las muestras de macrofauna se preservaron en alcohol (concentración 70%) y formol (concentración 10%). Se aplicaron los índices de dominancia, densidad, diversidad y equidad. La fauna fue organizada en grupos tróficos: alimentadores de depósito de superficie (ADS), alimentadores de depósito de sub-superficie (ADSS), filtradores (FF), carnívoros (CAR) y omnívoros (OMN). También se empleó el Análisis Factorial de Correspondencia (AFC) para determinar su variación espacial. Se colectaron 309 individuos con 80 especies distribuidos en 7 grupos (Poliquetos, Amphipodos, Crustáceos, Gastrópodos, Bivalvos, Insectos y Sipunculas).En Taparal dominaron los ADS (amphipodos), mientras que en Soldado fueron los ADSS (poliquetos). Se encontró una mayor estructura comunitaria en la región de Soldado con un aumento de los índices ecológicos. La abundancia de poliquetos Capitellidae, se relacionan con la alta carga de sedimentos registrada y transportada por el río que incrementa el contenido de materia orgánica del sistema, lo cual puede servir para desarrollar bioindicadores de erosión en la cuenca del río Anchicayá

<i>T</i>. <i>cruzi</i>, <i>T</i>. <i>rangeli</i> and <i>Leishmania</i> spp. isolates used to evaluate the analytical performance of the multiplex real-time PCR genotyping assays.

<p>References: ^a[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref006" target="_blank">6</a>]; ^b[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref026" target="_blank">26</a>]; ^c[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref027" target="_blank">27</a>]; ^d[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref028" target="_blank">28</a>]; ^e[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref029" target="_blank">29</a>]; ^f[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref030" target="_blank">30</a>]; ^g[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref031" target="_blank">31</a>]; ^h[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref032" target="_blank">32</a>]; ⁱ[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref033" target="_blank">33</a>] ^j[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref034" target="_blank">34</a>]; ^k[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref022" target="_blank">22</a>]; ^l[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref023" target="_blank">23</a>]; ^m[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref035" target="_blank">35</a>]; ⁿ[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref036" target="_blank">36</a>]; ^o[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref037" target="_blank">37</a>]; ^p[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref038" target="_blank">38</a>]; ^q[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref039" target="_blank">39</a>]; ^r[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref040" target="_blank">40</a>]; ^s[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref041" target="_blank">41</a>]. DTU, Discrete Typing Unit; nd, no data.</p><p><i>T</i>. <i>cruzi</i>, <i>T</i>. <i>rangeli</i> and <i>Leishmania</i> spp. isolates used to evaluate the analytical performance of the multiplex real-time PCR genotyping assays.</p

Multiplex real-time PCR flowchart for identification of <i>Trypanosoma cruzi</i> DTUs in biological samples.

<p>SL-IR, spliced leader intergenic region; 18S, 18S-ribosomal ADN; COII, cytochrome oxidase II; 24Sα, 24Sα-ribosomal DNA; MTq, multiplex TaqMan Real-Time PCR.</p

Sequences and concentrations of primers and probes used in the multiplex real-time PCR assays.

<p>^a[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref044" target="_blank">44</a>]; ^b[<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003765#pntd.0003765.ref045" target="_blank">45</a>]; SL-IR, spliced leader intergenic region; 18S, 18S-ribosomal ADN; COII, cytochrome oxidase II; 24Sα, 24Sα-ribosomal ADN; MTq, multiplex Real-Time PCR; BHQ, Black Hole Quencher. The + in front of the nucleotide indicates an LNA (Locked Nucleic Acid) monomer substitution.</p><p>Sequences and concentrations of primers and probes used in the multiplex real-time PCR assays.</p

Inclusivity and specificity assays for the multiplex real-time PCR genotyping algorithm.

<p>Cycle threshold (Ct) values obtained for each TaqMan probe in the analysis of <i>T</i>. <i>cruzi</i>, <i>T</i>. <i>rangeli</i> and <i>Leishmania</i> sp. stocks and human DNA. 0.1–10 ng of each <i>T</i>. <i>cruzi</i> strain and 2–10 ng of <i>T</i>. <i>rangeli</i> and <i>Leishmania</i> spp. stocks were used in the reaction tube.</p><p>DTU, Discrete Typing Unit; neg, negative; nd, not done.</p><p>Inclusivity and specificity assays for the multiplex real-time PCR genotyping algorithm.</p

Linear range and analytical sensitivity of the second round multiplex real-time PCR tests.

<p><b>A.</b> 18S-COII MTq PCR assay for reference stocks representing <i>T</i>. <i>cruzi</i> DTUs TcII, TcV and TcVI. Detection of TcII stock is shown for both TaqMan probes 18S-FAM and COII-Cy5. <b>B.</b> 24Sα MTq PCR for reference stocks representing <i>T</i>. <i>cruzi</i> DTUs TcIII, TcIV-SA and TcIV-NA. X-axis represents serial dilutions of whole genomic DNA from each stock and Y-axis represents the obtained Ct value. Linear regression analysis, equation and R² are shown for each graph. TcII, strain Tu18; TcV, strain PAH265; TcVI, strain CL Brener; TcIII, strain M5631; TcIV-SA (TcIV from South America), strain CanIII; TcIV-NA (TcIV from North America), strain Griffin.</p