Desarrollo de herramientas para el diagnóstico temprano de las infecciones verticales por T. cruzi en recien nacidos

Name: Desarrollo de herramientas para el diagnóstico temprano de las infecciones verticales por T. cruzi en recien nacidos
Keywords: CHAGAS VERTICAL

Datos de investigación

Desarrollo de herramientas para el diagnóstico temprano de las infecciones verticales por T. cruzi en recien nacidos

Autores: Peverengo, Luz María Icon

; Pujato, Nazarena Icon

; Marcipar, Iván Sergio Icon

; Peretti, Leandro Ezequiel Icon

Colaboradores: Altcheh, Jaime Marcelo Icon

; Moscatelli, Guillermo Icon

Publicador: Consejo Nacional de Investigaciones Científicas y Técnicas

Fecha de depósito: 21/08/2024

Fecha de creación: 2021/2024

Clasificación temática:

Enfermedades Infecciosas

Resumen

La enfermedad de Chagas es causada por la infección del parásito hemoflagelado Trypanosoma cruzi (T. cruzi). Afecta a unos 8 millones de personas principalmente en América Latina, donde es endémica, pero ya ha alcanzado relevancia a nivel mundial. En la actualidad, la transmisión del parásito de la madre al hijo durante el embarazo o el parto (transmisión vertical, vCH) se ha convertido en la principal vía de contagio debido al complejo escenario que implica su abordaje. La transmisión vertical sucede sólo en un 5% de los casos, aproximadamente, es evitable si las madres reciben tratamiento previo al embarazo y, en caso de que ocurra la infección el tratamiento es altamente eficaz y seguro durante los primeros años de vida. A pesar de estos puntos a favor, las diferentes barreras de accesibilidad a los servicios de salud y las limitaciones del algoritmo diagnóstico que demoran la confirmación de los casos, dificultan el control de la transmisión materno-infantil. En Argentina, las estimaciones sugieren que sólo entre 20-50% de los niños nacidos de madres infectadas recibe diagnóstico y tratamiento a tiempo. En consecuencia, estos pacientes pierden su oportunidad de ser tratados y la enfermedad progresa a un estadio crónico, pudiendo sufrir cardiopatías severas y lesiones viscerales. Por ello, lograr el diagnóstico certero lo antes posible, especialmente mientras el recién nacido permanece en el hospital, es clave para asegurar la futura calidad de vida de los pacientes infectados. Considerando que se trata de una enfermedad desatendida, con mayor ocurrencia en contextos de recursos limitados y gran vulnerabilidad social, se hace evidente que sería ideal un método diagnóstico de baja complejidad, con buena sensibilidad y especificidad. En este contexto, nuestro grupo de trabajo desarrollo dos pruebas para el diagnóstico serológico del vCH, basado en la detección de anticuerpos IgM específicos contra el parásito: un ELISA-IgM y una inmunocromatografía de flujo lateral IFL-IgM. Se aportan los datos correspondientes a las muestras que se emplearon para evaluar estos tests (tiempo, resultados en el marco del algoritmo diagnóstico estándar aplicado en Argentina para la detección de la enfermedad y resultados con las dos pruebas desarroladas). También se incluyen los datos para el cálculo de los parámetros de desempeño diagnóstico de las pruebas e imágenes de los resultados obtenidos con el test IFL-IgM para todo el panel de muestras.

Métodos

1.METHODS 1.1.Serum samples Samples used corresponded to sera from neonates and infants around 3 months old, diagnosed as infected or non-infected cases for cCD according to the current diagnostic algorithm in Argentina. Patients were assisted and diagnose at Servicio de Parasitología, Hospital de Niños “Ricardo Gutiérrez”, Buenos Aires, Argentina. The samples were preserved at - 20◦C with coding and adequate protection of personal data. Sera samples were classified as: •Infected (cCD = 11): sera from infants born to women with positive serology for chronic CD, presenting positive MH or positive T. cruzi qPCR at birth or positive serology tests after their 10 months of age. •Non-Infected (NI = 17): sera from infants born to women with positive serology for chronic CD, presenting negative MH or qPCR at birth or negative serology after their 10 months of age. •Group to test cross-reactivity: children with acquired toxoplasmosis (Tx+ = 5) or syphilis (Siph+ = 5). 1.2.Antigen The T. cruzi CP4 chimeric antigen was prepared. Briefly, bacteria expressing CP4 were cultured in Luria-Bertani (LB) medium supplemented with kanamycin, under agitation until OD500nm ~ 0.5-0.6 and induced at room temperature with 0.1 mM IPTG during 16 hours. Cells were harvested by centrifugation at 5000 rpm for 5 min, then were resuspended in distilled water and disrupted by sonication on ice (Vibre-Cell, Sonic &Material Inc) until culture clarification. The suspension was centrifuged at 10,000 rpm for 5 minutes and the supernatant was conditioned with phosphate buffer (PBS)-Imidazole 20 mM, pH=7,4. The solution was loaded on a previously equilibrated HisTrap column (Ni-NTA Agarose R901-15, Invitrogen). The recombinant protein was eluted with a linear gradient from 20 to 500 mM imidazole. The purity of the recombinant protein was analyzed using 15% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and staining with Coomassie brilliant blue. Protein content was determined by bicinchoninic acid assay (BCA assay). 1.3.LFA assay for T. cruzi IgM antibodies 1.3.1.Labelling reagents based on gold nanoparticles (GNPs) 1.3.1.1. Synthesis of gold nanoparticles (GNPs) Gold nanoparticles (GNPs) were prepared by reducing gold chloride (Sigma-Aldrich) with sodium citrate (Anedra). To obtained the 30-40 nm in diameter GNPs, trisodium citrate was added under stirring to final concentration 0,015% w/v into a boiling solution of 0.01% w/v AuCl3·HCl·4H2O (in Millipore-Q water) and the reaction was allowed to progress for 30 min. Quality and size of the GNPs were controlled by UV/vis spectrophotometer, Dynamic light scattering (DLS, Litesizer TM 500, Anton Paar) and transmission electron microscopy (TEM). 1.3.1.2.Conjugation of GNPs CP4 or an IgG antibody made in rabbit (IgGrab, Sigma-Aldrich) were alternative bound to the obtained GNPs. Five ml of CP4 (0.01 mg/ml) or IgGrab (0.003 mg/ml) in borate buffer (20 mM, pH= 9) was added to 50 ml of the GNPs and incubated 30 min at room temperature and gentle shaking. Success of the reaction was assessed by UV/vis spectrophotometer comparing spectra from GNPs and CP4 alone and the final product after conjugation (IgGrab/GNPs). Nanoparticles surface was blocked with BSA (1% final concentration) for 30 min. Finally, unbounded reagents were removed by centrifugation at 5000 x g, 4°C, during 30 min and the pellet was resuspended in conjugation buffer consisting in borate buffer supplemented with 5% sucrose (Merck), 0.2% Tween-20 (Sigma-Aldrich), 0.2 M NaCl (Cicarelli) and 0,1% sodium azide (Sigma-Aldrich). A mix of both conjugates are used in the conjugate pad. CP4/GNPs enables the development of the specific reaction on the test line (TL), while IgGrab/GNPs facilitates the development of the control line (CL), serving as an internal control. Both conjugates were stored protected from light at 4°C until use. 1.3.2.Design and preparation of LFA strips Each porous substrate constituting the LFA platform was preconditioned before being disposed on the strip test. Sample pad (CF3, Whatman, GE Life Sciences) was conditioned by 5 min immersion in buffer PBS containing 0.05% SDS, 0.1 M NaCl and 0.1% sodium azide followed by drying at 50°C for 30 min. A mix of CP4/GNPs and IgGrab/GNPs was dispensed on the conjugate pad (Standard 17, GE Life Sciences) and dried for 15 min at 50°C. Test lines (TLs) and (CLs) on the reaction pad (FF120HP, GE Life Sciences) were obtained by dispensing the adequate quantity of anti-IgMhu (Sigma-Aldrich) or anti-IgGrab (KPL), respectively, and further drying at room temperature for 30 min. Each conditioned pad was adhered onto an adhesive, overlapping 2 mm with each other. A scheme of the test device is shown in Fig. 1. Each bare strip was placed in a plastic cassette for easy handling. A batch of strip tests were prepared using the selected conditions described below (2.4.4). They were kept at room temperature in a sealed plastic bag until use. 1.3.3.Optimization of the test The optimal quantities of reagents to use in the LFA device were determined by a chessboard titration assay using two pools of samples prepared as follow: •Pool of cCD: mixing equal volume of 5 samples of children with cCD. •Pool of non-infected: mixing equal volume of 5 samples of children non-infected. The selected conditions were: 1 µg of anti-IgMhu (Sigma-Aldrich) and 0.05 µg of anti-IgGrab (KPL) immobilized on the TL and CL, respectively, and a mixture of 10 µl of CP4/GNPs (diluted 1/2) and 10 µl of IgGrab/GNPs on the conjugate pad. The determination consisted of dispensing 5 µL of sera onto the conjugate pad, immediately followed by the addition of 100 µL of sample buffer onto the sample pad. After 15 minutes, the results were interpreted as follows: i) the sample is considered positive for cCD when both TL and CL are observed; ii) the sample is considered non-infected if only the CL appears; iii) the test is considered invalid if the CL is not observed. 1.3.4.Accuracy of IgM-LFA The panel of sera was assessed using IgM-LFA in a double-blinded assay. The results were contrasted with the final case definition and classified as True positives (TP), True Negative (TN), False positive (FP) and False negative (FN). Also, the panel of samples was assessed using the IgM-ELISA previously reported by Peverengo et al. (2021) with the aim of comparing it with the IgM-LFA. Additionally, the strength of agreement between the results of the IgM serological techniques (IgM-LFA and IgM-ELISA) and conventional parasitological techniques (qPCR and MH) was evaluated. Finally, the IgM-LFA was assessed for cross-reactions using samples with confirmed diagnoses for Tx+ and Siph+. 1.4.Data analysis Diagnostic parameters were estimated using the Diagnostic Tests module of Epidat 3.1 software: Sensibility (Se), Specificity (Sp), Youden´s index (J), negative and positive Likelihood ratio (LR) and Cohen’s kappa coefficient (κ) with their 95% confidence interval (IC95%). LRs values were interpreted according to the clinical utility as follow: poor clinical utility (LR+ ˂ 2 or LR- > 0.5); regular clinical utility (LR+ = 2-5 or LR- = 0.2- 0.5); good clinical utility (LR+ = 5- 10 or LR- = 0.1- 0.2) and high clinical utility (LR+ > 10 or LR- ˂ 0.1). The κ values were interpreted as follows: poor (κ = 0), slight (0 < κ ≤ 0.20), fair (0.21 < κ ≤ 0.40), moderate (0.41 < κ ≤ 0.60), substantial (0.61 < κ ≤ 0.80), and almost perfect (0.81 < κ ≤ 1.0) agreement.

Palabras clave: CHAGAS VERTICAL, DIAGNÓSTICO TEMPRANO, ELISA-IGM, TEST RÁPIDO, CHAGAS CONGÉNITO, INMUNOCROMATOGRAFÍA DE FLUJO LATERAL, TRYPANOSOMA CRUZI

Alcance geográfico

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Identificador del recurso

URI: http://hdl.handle.net/11336/242909

Colecciones

Datos de Investigación(CCT - SANTA FE)
Datos de Investigación de CTRO.CIENTIFICO TECNOL.CONICET - SANTA FE

Datos de Investigación(IMIPP)
Datos de Investigación de INSTITUTO MULTIDISCIPLINARIO DE INVESTIGACIONES EN PATOLOGIAS PEDIATRICAS

Datos de Investigación(INTEC)
Datos de Investigación de INST.DE DES.TECNOL.PARA LA IND.QUIMICA (I)

Citación

Peverengo, Luz María; Pujato, Nazarena; Marcipar, Iván Sergio; Peretti, Leandro Ezequiel; (2024): Desarrollo de herramientas para el diagnóstico temprano de las infecciones verticales por T. cruzi en recien nacidos. Consejo Nacional de Investigaciones Científicas y Técnicas. (dataset). http://hdl.handle.net/11336/242909

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