Validación de una prueba de PCR en tiempo real para el diagnóstico de la enfermedad de Chagas

Publicación a texto completo no autorizada por el autorSe estandariza la prueba de PCR en tiempo real (Q-PCR) para el diagnóstico de la enfermedad y evaluar las estimaciones de validación. La prueba está dirigida a la amplificación del ADN satelital del parásito T. cruzi y presenta un límite de detección de 0,005 equivalentes genómicos de parásito. Se evalúa el método con un panel de muestras de sangre de pacientes con diagnóstico serológico conocido (utilizado como gold estándar), pero de estadio clínico no determinado. El ensayo presenta una sensibilidad clínica de 83% y una especificidad de 100%, determinando una buena concordancia. Asimismo, la prueba es capaz de detectar ADN de parásito en muestras de heces de triatominos. La prueba demuestra ser eficiente para el diagnóstico de la enfermedad de Chagas y podría ser aplicada en la fase aguda y crónica, incluso cuando el nivel de parasitemia en sangre es bajo. Además, cuando los métodos convencionales no son concluyentes, la prueba de Q-PCR permite una detección temprana y oportuna de la infección por Trypanosoma cruzi.Tesi

A faster and less costly alternative for RNA extraction of SARS-CoV-2 using proteinase k treatment followed by thermal shock.

One of the biggest challenges during the pandemic has been obtaining and maintaining critical material to conduct the increasing demand for molecular tests. Sometimes, the lack of suppliers and the global shortage of these reagents, a consequence of the high demand, make it difficult to detect and diagnose patients with suspected SARS-CoV-2 infection, negatively impacting the control of virus spread. Many alternatives have enabled the continuous processing of samples and have presented a decrease in time and cost. These measures thus allow broad testing of the population and should be ideal for controlling the disease. In this sense, we compared the SARS-CoV-2 molecular detection effectiveness by Real time RT-PCR using two different protocols for RNA extraction. The experiments were conducted in the National Institute of Health (INS) from Peru. We compared Ct values average (experimental triplicate) results from two different targets, a viral and internal control. All samples were extracted in parallel using a commercial kit and our alternative protocol-samples submitted to proteinase K treatment (3 μg/μL, 56°C for 10 minutes) followed by thermal shock (98°C for 5 minutes followed by 4°C for 2 minutes); the agreement between results was 100% in the samples tested. In addition, we compared the COVID-19 positivity between six epidemiological weeks: the initial two in that the Real time RT-PCR reactions were conducted using RNA extracted by commercial kit, followed by two other using RNA obtained by our kit-free method, and the last two using kit once again; they did not differ significantly. We concluded that our in-house method is an easy, fast, and cost-effective alternative method for extracting RNA and conducing molecular diagnosis of COVID-19