Tutoriales escritos y animados para favorecer el autoaprendizaje del alumno en asignaturas de grado y máster

Memoria ID-142. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2020-2021

Application of the LAMP Technique for the Detection of Loa Loa and Mansonella perstans

Filariases are endemic diseases of tropical regions caused by filiform nematodes transmitted by insect bites. They produce high morbidity. Loiasis (Loa Loa) and mansonellosis (Mansonella perstans) globally affect 10 and 100 million people, respectively. The diagnosis of certainty is parasitological, but it does not detect early infections or low microfilaeremia, and it is necessary to consider the periodicity of microphilaremias in blood. Molecular techniques, such as PCR, have great sensitivity and specificity, but they are expensive, technically complex and require infrastructure not available in endemic areas of scarce resources. LAMP technology (loop-mediated isothermal amplification) has advantages over PCR as faster, scarce equipment, more tolerant of inhibitors and the results can be observed colorimetrically. In this work, LAMP technology is applied and evaluated for the detection of DNA from Loa loa and M. perstans in 22 blood samples stored on filter paper from individuals living in Equatorial Guinea. The samples were analyzed microscopically, using qPCR and LAMP. The molecular methods were more sensitive than microscopy. LAMP resulted more sensitive than qPCR for the detection of DNA from Loa loa and M. perstans.Las filariosis son enfermedades endémicas de regiones tropicales ocasionadas por nematodos filiformes trasmitidos por la picadura de insectos. Producen elevada morbilidad. La loaosis (Loa Loa) y la mansonelosis (Mansonella perstans) afectan globalmente a 10 y 100 millones de personas, respectivamente. El diagnóstico de certeza es parasitológico, pero no detecta infecciones precoces o bajas microfilaremias y hay que considerar la periodicidad de las microfilarias en sangre. Las técnicas moleculares, como la PCR, tienen gran sensibilidad y especificidad, pero son caras, técnicamente complejas y requieren infraestructura no disponible en zonas endémicas de escasos recursos. La tecnología LAMP (loop-mediated isothermal amplification) presenta ventajas sobre la PCR como mayor rapidez, escaso equipamiento, más tolerante a inhibidores y los resultados pueden observarse colorimétricamente. En este trabajo se aplica y valora la tecnología LAMP para la detección de ADN de Loa loa y M. perstans en 22 muestras de sangre almacenadas en papel de filtro de individuos residentes en Guinea Ecuatorial. Las muestras se analizaron microscópicamente, mediante qPCR y LAMP. Los métodos moleculares resultaron más sensibles que la microscopía. El LAMP resultó más sensible que la qPCR para la detección de ADN de Loa loa y M. perstans

SMART-LAMP: A Smartphone-Operated Handheld Device for Real-Time Colorimetric Point-of-Care Diagnosis of Infectious Diseases via Loop-Mediated Isothermal Amplification

[ENG]Nucleic acid amplification diagnostics offer outstanding features of sensitivity and specificity. However, they still lack speed and robustness, require extensive infrastructure, and are neither affordable nor user-friendly. Thus, they have not been extensively applied in point-of-care diagnostics, particularly in low-resource settings. In this work, we have combined the loop-mediated isothermal amplification (LAMP) technology with a handheld portable device (SMART-LAMP) developed to perform real-time isothermal nucleic acid amplification reactions, based on simple colorimetric measurements, all of which are Bluetooth-controlled by a dedicated smartphone app. We have validated its diagnostic utility regarding different infectious diseases, including Schistosomiasis, Strongyloidiasis, and COVID-19, and analyzed clinical samples from suspected COVID-19 patients. Finally, we have proved that the combination of long-term stabilized LAMP master mixes, stored and transported at room temperature with our developed SMART-LAMP device, provides an improvement towards true point-of-care diagnosis of infectious diseases in settings with limited infrastructure. Our proposal could be easily adapted to the diagnosis of other infectious diseases

Detection of Schistosoma mansoni-derived DNA in human urine samples by loopmediated isothermal amplification (LAMP)

[EN]Background Schistosoma mansoni is the main species causing hepatic and intestinal schistosomiasis in Sub-Saharan Africa, and it is the only species in South America. Adult stages of the parasite reside in the mesenteric venous plexus of infected hosts, and eggs are shed in feces. Collecting patient stool samples for S. mansoni diagnostic purposes is difficult in large-scale field trials. Urine samples would be an alternative approach for molecular S. mansoni detection since they have several advantages over stool samples, including better handling, management and storage. Additionally, loop-mediated isothermal amplification (LAMP) technology is a powerful molecular diagnostic tool for infectious diseases, particularly under field conditions in developing countries. The present study aimed to assess the effectiveness of our previously developed LAMP assay (SmMIT-LAMP) for S. mansoni-specific detection in clinical urine samples. Methodology/Principal findings The sensitivity of SmMIT-LAMP in urine was established in simulated fresh human urine samples artificially spiked with genomic DNA from S. mansoni. LAMP for 120 min instead of 60 min improved the sensitivity, reaching values of 0.01 fg/μL. A set of well-defined frozen stored human urine samples collected from Sub-Saharan immigrant patients was selected from a biobank to evaluate the diagnostic validity of SmMIT-LAMP. The set included urine samples from patients with microscopy-confirmed infections with S. mansoni, S. haematobium and other nonschistosome parasites, as well as urine samples from patients with microscopy-negative eosinophilia without a confirmed diagnosis. The SmMIT-LAMP was incubated for 60 and 120 min. A longer incubation time was shown to increase the LAMPpositive results in patient urine samples. We also tested urine samples from mice experimentally infected with S. mansoni, and LAMP-positive results were obtained from the third week after infection. A real-time LAMP assay was also performed with three individual urine samples. Conclusions/Significance The SmMIT-LAMP could effectively detect S. mansoni DNA in mouse urine samples and produced promising results for human clinical samples. The detection of S. mansoni DNA in mouse urine samples from the third week after infection indicates that early diagnosis of active S. mansoni infection is possible using urine as a source of DNA. Further studies are still needed, but our method could be used as a promising molecular tool applicable to urine samples to diagnose human intestinal schistosomiasis caused by S. mansoni

Protocol for establishing primary human lung organoid-derived air-liquid interface cultures from cryopreserved human lung tissue.

Primary human lung organoid-derived air-liquid interface (ALI) cultures serve as a physiologically relevant model to study human airway epithelium in vitro. Here, we present a protocol for establishing these cultures from cryopreserved human lung tissue. We describe steps for lung tissue cryostorage, tissue dissociation, lung epithelial organoid generation, and ALI culture differentiation. We also include quality control steps and technical readouts for monitoring virus response. This protocol demonstrates severe acute respiratory syndrome coronavirus 2 infection in these cultures as an example of their utility. For complete details on the use and execution of this protocol, please refer to Diana Cadena Castaneda et al. (2023)

Progress in loop-mediated isothermal amplification assay for detection of Schistosoma mansoni DNA: towards a ready-to-use test

[ENG]Schistosomiasis is one of the most prevalent Neglected Tropical Disease, affecting approximately 250 million people worldwide. Schistosoma mansoni is the most important species causing human intestinal schistosomiasis. Despite significant efforts in recent decades, the global disease burden of schistosomiasis remains extremely high. This could partly be attributed to the absence of accurate diagnostic tools, primarily in endemic areas. Loop-mediated isothermal amplification (LAMP) is increasingly used in molecular diagnostics as a field-friendly alternative to many other complex molecular methods and it has been proposed as an ideal candidate for revolutionizing point-of-care molecular diagnostics. In a previous work, a LAMP-based method to detect S. mansoni DNA (SmMIT-LAMP) was developed by our research group for early diagnosis of active schistosomiasis in an experimental infection murine model. The SmMIT-LAMP has been further successfully evaluated in both human stool and snail samples and, recently, in human urine samples. In this study, we developed an important improvement for SmMIT-LAMP molecular assay, transforming it into a cold maintenance dry format suitable for potentially manufacturing as kit for ready-to-use for schistosomiasis diagnosis. This procedure could be applied to create dry LAMP kits for a laboratory setting and for diagnostic applications for other neglected tropical diseases

Colorimetric and Real-Time Loop-Mediated Isothermal Amplification (LAMP) for Detection of Loa loa DNA in Human Blood Samples

Loiasis, caused by the filarial nematode Loa loa, is endemic in Central and West Africa. Loa loa has been associated with severe adverse reactions in high Loa-infected individuals receiving ivermectin during mass drug administration programs for the control of onchocerciasis and lymphatic filariasis. Diagnosis of loiasis still depends on microscopy in blood samples, but this is not effective for large-scale surveys. New diagnostics methods for loiasis are urgently needed. Previously, we developed a colorimetric high-sensitive and species-specific LAMP for Loa loa DNA detection. Here, we evaluate it in a set of 100 field-collected clinical samples stored as dried blood spots. In addition, Loa loa-LAMP was also evaluated in real-time testing and compared with microscopy and a specific PCR/nested PCR. A simple saponin/Chelex-based method was used to extract DNA. Colorimetric and real-time LAMP assays detected more samples with microscopy-confirmed Loa loa and Loa loa/Mansonella perstans mixed infections than PCR/nested-PCR. Samples with the highest Loa loa microfilariae counts were amplified faster in real-time LAMP assays. Our Loa loa-LAMP could be a promising molecular tool for the easy, rapid and accurate screening of patients for loiasis in endemic areas with low-resource settings. The real-time testing (feasible in a handheld device) could be very useful to rule out high-microfilariae loads in infected patients.This research was funded by the Institute of Health Carlos III, ISCIII, Spain (www.isciii.es), grants: RICET RD16/0027/0018 (A.M.), RD16/0027/0000 (A.B.), FCSAI-ISCIII (P.N.) and PI19/01727 (P.F.-S.), European Union co-financing by FEDER (Fondo Europeo de Desarrollo Regional) ‘Una manera de hacer Europa’. We also acknowledge support by the Predoctoral Fellowship Program of Junta de Castilla y León co-financing by Fondo Social Europeo (BDNS (Identif.): 422058 and BDNS (Identif.): 487971), by the ISCIII-Sara Borrell contract CD17CIII/00018 financed by the Institute of Health Carlos III and Predoctoral Fellowship Program of University of Salamanca, and co-financing by Santander Bank.S

EducaFarma 9.0

Memoria ID-020 Ayudas de la Universidad de Salamanca para la innovación docente, curso 2020-2021

Educafarma 10.0

Memoria ID-030. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2021-2022

SMART-LAMP: A Smartphone-Operated Handheld Device for Real-Time Colorimetric Point-of-Care Diagnosis of Infectious Diseases via Loop-Mediated Isothermal Amplification

Nucleic acid amplification diagnostics offer outstanding features of sensitivity and specificity. However, they still lack speed and robustness, require extensive infrastructure, and are neither affordable nor user-friendly. Thus, they have not been extensively applied in point-of-care diagnostics, particularly in low-resource settings. In this work, we have combined the loop-mediated isothermal amplification (LAMP) technology with a handheld portable device (SMART-LAMP) developed to perform real-time isothermal nucleic acid amplification reactions, based on simple colorimetric measurements, all of which are Bluetooth-controlled by a dedicated smartphone app. We have validated its diagnostic utility regarding different infectious diseases, including Schistosomiasis, Strongyloidiasis, and COVID-19, and analyzed clinical samples from suspected COVID-19 patients. Finally, we have proved that the combination of long-term stabilized LAMP master mixes, stored and transported at room temperature with our developed SMART-LAMP device, provides an improvement towards true point-of-care diagnosis of infectious diseases in settings with limited infrastructure. Our proposal could be easily adapted to the diagnosis of other infectious diseases