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

Analytical Performance of a Multiplex Real-Time PCR Assay Using TaqMan Probes for Quantification of Trypanosoma cruzi Satellite DNA in Blood Samples

Background: The analytical validation of sensitive, accurate and standardized Real-Time PCR methods for Trypanosoma cruzi quantification is crucial to provide a reliable laboratory tool for diagnosis of recent infections as well as for monitoring treatment efficacy. Methods/Principal Findings: We have standardized and validated a multiplex Real-Time quantitative PCR assay (qPCR) based on TaqMan technology, aiming to quantify T. cruzi satellite DNA as well as an internal amplification control (IAC) in a single-tube reaction. IAC amplification allows rule out false negative PCR results due to inhibitory substances or loss of DNA during sample processing. The assay has a limit of detection (LOD) of 0.70 parasite equivalents/mL and a limit of quantification (LOQ) of 1.53 parasite equivalents/mL starting from non-boiled Guanidine EDTA blood spiked with T. cruzi CLBrener stock. The method was evaluated with blood samples collected from Chagas disease patients experiencing different clinical stages and epidemiological scenarios: 1- Sixteen Venezuelan patients from an outbreak of oral transmission, 2- Sixty three Bolivian patients suffering chronic Chagas disease, 3- Thirty four Argentinean cases with chronic Chagas disease, 4- Twenty seven newborns to seropositive mothers, 5- A seronegative receptor who got infected after transplantation with a cadaveric kidney explanted from an infected subject. Conclusions/Significance: The performing parameters of this assay encourage its application to early assessment of T. cruzi infection in cases in which serological methods are not informative, such as recent infections by oral contamination or congenital transmission or after transplantation with organs from seropositive donors, as well as for monitoring Chagas disease patients under etiological treatment.Fil: Duffy, Tomas. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones En Ingeniería Genética y Biología Molecular; ArgentinaFil: Cura, Carolina Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Ramírez, Juan C.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Abate, Teresa. Universidad Central de Venezuela. Instituto de Medicina Tropical; VenezuelaFil: Cayo, Nelly M.. Universidad Nacional de Jujuy. Instituto de Biologia de la Altura; ArgentinaFil: Parrado, Rudy. Universidad San Simón; BoliviaFil: Diaz Bello, Zoraida. Universidad Central de Venezuela. Instituto de Medicina Tropical; VenezuelaFil: Velazquez, Elsa Beatriz. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud. Instituto Nacional de Parasitología; ArgentinaFil: Muñoz Calderón, Arturo. Universidad Central de Venezuela. Instituto de Medicina Tropical; VenezuelaFil: Juiz, Natalia Anahí. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Basile, Joaquín. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Garcia, Lineth. Universidad San Simón; BoliviaFil: Riarte, Adelina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud. Instituto Nacional de Parasitología; ArgentinaFil: Nasser, Julio Rubén. Universidad Nacional de Salta. Facultad de Ciencias Naturales; ArgentinaFil: Ocampo, Susana B.. Universidad Nacional de Jujuy. Instituto de Biologia de la Altura; ArgentinaFil: Yadon, Zaida E.. Pan-American Health Organization; Estados UnidosFil: Torrico, Faustino. Universidad San Simón; BoliviaFil: Alarcón de Noya, Belkisyole. Universidad Central de Venezuela. Instituto de Medicina Tropical; VenezuelaFil: Ribeiro, Isabela. Drugs and Neglected Diseases Initiative; SuizaFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentin

ANTICUERPOS ANTI-RECEPTORES β1 ADRENÉRGICOS EN PACIENTES CON ENFERMEDAD DE CHAGAS: ESTANDARIZACION DE LA PRUEBA (ELISA) CON EL PEPTIDO SINTETICO DE LA SEGUNDA ASA EXTRACELULAR DEL RECEPTOR β1 ADRENÉRGICO

La Enfermedad de Chagas (ECh), es causada por el agente etiológico Trypanosomacruzi (T. cruzi) y es el principal causante de cardiomiopatías crónicas en América Latina.Probablemente el daño cardiaco es la consecuencia no solamente de la persistencia delparásito en el tejido cardiaco sino también de una regulación defectuosa de la respuestainmunitaria (teoría autoinmune)derivada de la auto-reactividad contra antígenos propios.Se ha demostrado la presencia de anticuerpos dirigidos contra la segunda asa extracelulardel receptor β1 adrenérgico en suero de pacientes conECh. La estandarización de la pruebaes necesaria para garantizar la obtención de resultados confiables y comparativos en ladeterminación de la presencia de anticuerpos anti-β1 adrenérgicos en pacientes en fasestempranas de la infección y vigilar de cerca su evolución clínica. Se ensayaron diferentesconcentraciones del antígeno (30μg/ml, 50μg/ml y 70 μg/ml), diluciones de los sueros (1/20,1/50, 1/60) y del conjugado (anti-IgG humano, acoplado a fosfatasa alcalina) en placas de96 pozos con sueros de personas con ECh con alta, media y baja reactividad en el ELISAde diagnóstico así como de personas negativas a fin de fijar el punto de corte de la prueba.Las condiciones evaluadas para la estandarización de la prueba resultaron apropiadas para ladetección de los anticuerpos anti-β1 adrenérgicos en pacientes con Enfermedad de Chagas.ANTIBODIES ANTI - RECEPTOR β1 ADRENERGIC CHAGASDISEASE PATIENTS: STANDARIZATION TEST (ELISA) WITHSYNTHETIC PEPTIDE OF THE SECOND EXTRACELLULARRECEIVER ASA β1 ADRENERGICAbstract: Chagas disease is caused by the etiologic agent tripanosomacruzi (T. cruzi)and is the maincause of chronic cardiomyopathy in Latin America. Probably the heart damage is a resultnotonly the persistence of the parasite in the cardiac tissuebut also of a defective regulation ofthe immune response (autoimmune theory). It has demonstrated the presence of antibodiesdirected against the second extracellular loop β1 adrenergic receptor in serum of patientswith Ech. The standarization of testing is necessary to ensure obtaining reliable resultsand comparative, the objective study was performed to standarize the test (ELISA) for thedetection of anti-β1 adrenergic antibodies in patients with Ech,were determined optimalconcentrations of antigen (30μg/ml, 50μg/ml y 70 μg/ml),optimal dilution of sera(1/20, 1/50,1/60) and the optimal conjugate title (anti - human IgG, coupled to alkaline phosphatase) in96-well plates with serums of high, medium and low reactivity. To discriminate positivesera from negative the cutoff was determined. It was selected as the optimal concentration ofantigen50μg/mldilutions of serum 1/50 and conjugate1/500the cut that was set at D.O 0,278nm. The conditions evaluated for standardized testing, were appropriate for the detection ofanti-β1 adrenergic antibodies in patients with Chagas disease

Loop-Mediated Isothermal Amplification of Trypanosoma cruzi DNA for Point-of-Care Follow-Up of Anti-Parasitic Treatment of Chagas Disease

A loop-mediated isothermal amplification assay was evaluated as a surrogate marker of treatment failure in Chagas disease (CD). A convenience series of 18 acute or reactivated CD patients who received anti-parasitic treatment with benznidazole was selected—namely, nine orally infected patients: three people living with HIV and CD reactivation, five chronic CD recipients with reactivation after organ transplantation and one seronegative recipient of a kidney and liver transplant from a CD donor. Fifty-four archival samples (venous blood treated with EDTA or guanidinium hydrochloride-EDTA buffer and cerebrospinal fluid) were extracted using a Spin-column manual kit and tested by T. cruzi Loopamp kit (Tc-LAMP, index test) and standardized real-time PCR (qPCR, comparator test). Of them, 23 samples were also extracted using a novel repurposed 3D printer designed for point-of-care DNA extraction (PrintrLab). The agreement between methods was estimated by Cohen’s kappa index and Bland–Altman plot analysis. The T. cruzi Loopamp kit was as sensitive as qPCR for detecting parasite DNA in samples with parasite loads higher than 0.5 parasite equivalents/mL and infected with different discrete typing units. The agreement between qPCR and Tc-LAMP (Spin-column) or Tc-LAMP (PrintrLab) was excellent, with a mean difference of 0.02 [CI = −0.58–0.62] and −0.04 [CI = −0.45–0.37] and a Cohen’s kappa coefficient of 0.78 [CI = 0.60–0.96] and 0.90 [CI = 0.71 to 1.00], respectively. These findings encourage prospective field studies to validate the use of LAMP as a surrogate marker of treatment failure in CD

Anticipated reportable range and linearity of qPCR method.

<p>Multiplex TaqMan qPCR strategy was carried out with spiked GEB samples containing parasite stocks belonging to TcI and TcVI in ten concentrations spanning 10⁶ to 0.625 par. eq./10 mL, tested in triplicate. Assigned values were plotted on the <i>x</i> axis versus measured values (converted to log₁₀) on the <i>y</i> axis using SigmaPlot 10.0 for Windows (SPSS, Chicago, IL). Linear regression analysis rendered the equation y = 1.013x+0.058 (R² = 0.992) for TcI, and y = 1.001x+0.005 (R² = 0.998) for TcVI.</p

Inclusivity assay for <i>T. cruzi</i> DTUs.

<p>Results are shown as mean Ct obtained from duplicates of each DNA concentration.</p>a<p>Only one replicate was detected. TcIa: K98; TcId: G; TcIe: SE9V; TcII: Tu18; TcIII: M5361; TcIV: CanIII; TcV: PAH265; TcVI: CL Brener. Ct: threshold cycle; Undet.: not detectable.</p

Estimation of IAC amplification in blood specimens from different clinical groups.

a<p>Sample PCC 331: qPCR positive, 0.69 log₁₀ par. eq./mL, IAC Ct 19.20.</p><p>Nb: Newborn; UCB, umbilical cord blood; PB: peripheral blood.</p

Sequences and concentrations of primers and probes used for the Multiplex Taqman qPCR assay.

<p>IAC: Internal amplification control. S: C/G.</p

Follow-up of <i>T. cruzi</i> infected patients using qPCR.

<p>A. Follow-up of orally infected cases from Chacao, Caracas, Venezuela. Years pos-treatment (ys pos-T) are represented in the <i>x</i>-axis. Parasite equivalents (par. eq.) were estimated using a Silvio X-10 (TcI) calibration curve. Case 1- Pre-T: 5.23 log₁₀ par. eq./10 mL; 2 ys pos-T: 1.88 log₁₀ par. eq./10 mL. Case 2- Pre-T: 3.78 log₁₀ par. eq./10 mL; 2 ys pos-T: 1.83 log₁₀ par. eq./10 mL. Case 3- Pre-T: 2.94 log₁₀ par. eq./10 mL; 2 ys pos-T: 1.88 log₁₀ par. eq./10 mL. B. A 42 year-old seronegative man received kidney transplantation from a seropositive cadaveric donor. Progression of parasitic load after transplantation is shown as well as post-treatment follow-up. The quantification was estimated using a Cl-Brener (TcVI) calibration curve. Days pos-Transplantation (Tx) are represented in the <i>x</i>-axis. The number of par. eq./10 mL of blood is represented in the <i>y-</i>axis, in a log-scale. Arrow marks initiation of Benznidazole treatment. ND: not detectable. The line indicates LOQ (1.185 log₁₀ par. eq./10 mL) derived from analysis of CL-Brener (TcVI) spiked samples. Discontinued line: parasitic loads in Chacao patients were estimated with Silvio X-10 (TcI) calibration curves.</p