Tecnología LAMP: diagnóstico en diferentes escenarios epidemiológicos

Tesis por compendio de publicaciones[ES]La aparición de las técnicas de amplificación de ácidos nucleicos hace unas décadas permitió perfeccionar la detección de agentes infecciosos y supuso una revolución en el diagnóstico microbiológico. Con la introducción de la PCR y sus distintas variantes surgió definitivamente el concepto de diagnóstico molecular, que ha permitido la detección rápida y sensible de microorganismos mediante la amplificación específica de determinadas regiones de su genoma. Actualmente, su uso ha pasado a ser rutinario para el diagnóstico de las enfermedades más habituales en los laboratorios de referencia, pero no así para un gran número de enfermedades importadas y, mucho menos, para aquellas enfermedades tropicales que ocurren en zonas endémicas de escasos recursos. La elección de la técnica diagnóstica dependerá de las características del laboratorio y el objetivo que persigamos en el diagnóstico. De este modo, un caso importado será generalmente estudiado en un laboratorio de referencia bien equipado y con personal especializado. Sin embargo, la detección de un caso en zona endémica, generalmente en zonas inaccesibles durante estudios de campo, requerirá de una prueba tradicional microscópica o una técnica rápida. En cualquier caso, a una técnica diagnóstica se le debe de exigir la máxima especificidad y sensibilidad, reproducibilidad, facilidad de ejecución y un bajo coste. Las técnicas de amplificación isotérmica de ácidos nucleicos son las que más se acercan a cumplir estos requisitos. Entre ellas, la tecnología de amplificación mediada por bucle (LAMP, del inglés Loop-mediated isothermal amplification) es actualmente la más utilizada y sobre la que más se ha trabajado para conseguir acercar el diagnóstico molecular realizado en un laboratorio de referencia a estudios de campo y diagnóstico point-of-care. De esta manera, la tecnología LAMP podría utilizarse en diferentes contextos epidemiológicos. Teniendo en cuenta lo anterior, nos planteamos como objetivo general de este trabajo la evaluación de la tecnología LAMP como método diagnóstico en diferentes situaciones epidemiológicas. Para ello, definimos una serie de objetivos específicos encaminados a la aplicación y desarrollo de esta tecnología para abordar nuevos estudios epidemiológicos, ante situaciones de emergencia sanitaria, o como diagnóstico point-of-care en áreas endémicas con escasos recursos. Estos objetivos se van presentando y alcanzando en los diferentes artículos de investigación que se presentan en esta Tesis Doctoral. [POR]O surgimento de técnicas de amplificação de ácidos nucleicos, há algumas décadas, levou a uma melhor detecção de agentes infecciosos e a uma revolução no diagnóstico microbiológico. Com a introdução da PCR e das suas diferentes variantes, surgiu definitivamente o conceito de diagnóstico molecular, que permite a detecção rápida e sensível de microrganismos através da amplificação específica de determinadas regiões do seu genoma. A sua utilização tornou-se rotina para o diagnóstico das doenças mais comuns em laboratórios de referência, mas não para um grande número de doenças importadas, e muito menos para as doenças tropicais que ocorrem em zonas endémicas com poucos recursos. A selecção da técnica de diagnóstico dependerá das características do laboratório e do objectivo do diagnóstico. Assim, um caso importado será geralmente estudado num laboratório de referência bem equipado e com pessoal especializado. No entanto, a detecção de um caso numa área endémica, geralmente em áreas inacessíveis durante os inquéritos de campo, exigirá um teste microscópico tradicional ou uma técnica rápida. Em qualquer caso, uma técnica de diagnóstico deve ter a máxima especificidade e sensibilidade, reprodutibilidade, facilidade de execução e baixo custo. As técnicas de amplificação isotérmica de ácidos nucleicos são as que mais se aproximam destes requisitos. Entre elas, a tecnologia de amplificação isotérmica mediada por laço (LAMP) é actualmente a mais utilizada e aquela em que mais se trabalhou para aproximar os diagnósticos moleculares realizados num laboratório de referência dos estudos de campo ou dos diagnósticos point-of-care. Desta forma, a tecnologia LAMP pode ser utilizada em diferentes contextos epidemiológicos. Tendo em conta o exposto, o objectivo geral deste trabalho é avaliar a tecnologia LAMP como um método de diagnóstico em diferentes situações epidemiológicas. Para isso, definimos uma série de objectivos específicos que visam a aplicação e o desenvolvimento desta tecnologia para abordar novos estudos epidemiológicos, em situações de emergência sanitária, ou como diagnóstico point-of-care em áreas endémicas com recursos escassos. Estes objectivos são apresentados e alcançados nos diferentes artigos de investigação apresentados nesta Tese de Doutoramento

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

First field and laboratory evaluation of LAMP assay for malaria diagnosis in Cubal, Angola

Angola; MalariaAngola; MalariaAngola; MalàriaBackground Malaria is a globally distributed infectious disease. According to the World Health Organization, Angola is one of the six countries that account for over half the global malaria burden in terms of both malaria cases and deaths. Diagnosis of malaria still depends on microscopic examination of thin and thick blood smears and rapid diagnostic tests (RDTs), which often lack analytical and clinical sensitivity. Molecular methods could overcome these disadvantages. The aim of this study was to evaluate, for the first time to our knowledge, the performance of a loop-mediated isothermal amplification (LAMP) for the diagnosis of malaria in an endemic area in Cubal, Angola, and to assess the reproducibility at a reference laboratory. Methods A total of 200 blood samples from patients attended at Hospital Nossa Senhora da Paz, Cubal, Angola, were analysed for Plasmodium spp. detection by microscopy, RDTs, and LAMP. LAMP assay was easily performed in a portable heating block, and the results were visualized by a simple colour change. Subsequently, the samples were sent to a reference laboratory in Spain to be reanalysed by the same colorimetric LAMP assay and also in real-time LAMP format. Results In field tests, a total of 67/200 (33.5%) blood samples were microscopy-positive for Plasmodium spp., 98/200 RDT positive, and 112/200 (56%) LAMP positive. Using microscopy as reference standard, field LAMP detected more microscopy-positive samples than RDTs (66/67; 98% vs. 62/67; 92.5%). When samples were reanalysed at a reference laboratory in Spain using both colorimetric and real-time assays, the overall reproducibility achieved 84.5%. Conclusions This is the first study to our knowledge in which LAMP has been clinically evaluated on blood samples in a resource-poor malaria-endemic area. The colorimetric LAMP proved to be more sensitive than microscopy and RDTs for malaria diagnosis in field conditions. Furthermore, LAMP showed an acceptable level of reproducibility in a reference laboratory. The possibility to use LAMP in a real-time format in a portable device reinforces the reliability of the assay for molecular diagnosis of malaria in resource-poor laboratories in endemic areas.The work was supported by the Institute of Health Carlos III, ISCIII, Spain (www.isciii.es) grant number PI22/01721 (PFS), European Union cofinancing 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 cofounded by Fondo Social Europeo: (BDNS Identif.: 422058, BFS) and (BDNS Identif: 487971, BCV)

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

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

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

Development of a Duplex LAMP Assay with Probe-Based Readout for Simultaneous Real-Time Detection of <i>Schistosoma mansoni</i> and <i>Strongyloides</i> spp. -A Laboratory Approach to Point-Of-Care

Loop-mediated isothermal amplification (LAMP) is the most popular technology for point-of-care testing applications due its rapid, sensitive and specific detection with simple instrumentation compared to PCR-based methods. Many systems for reading the results of LAMP amplifications exist, including real-time fluorescence detection using fluorophore-labelled probes attached to oligonucleotide sequences complementary to the target nucleic acid. This methodology allows the simultaneous detection of multiple targets (multiplexing) in one LAMP assay. A method for multiplexing LAMP is the amplification by release of quenching (DARQ) technique by using a 5′-quencher modified LAMP primer annealed to 3′-fluorophore-labelled acting as detection oligonucleotide. The main application of multiplex LAMP is the rapid and accurate diagnosis of infectious diseases, allowing differentiation of co-infecting pathogens in a single reaction. Schistosomiasis, caused among other species by Schistosoma mansoni and strongyloidiasis, caused by Strongyloides stercoralis, are the most common helminth-parasite infections worldwide with overlapping distribution areas and high possibility of coinfections in the human population. It would be of great interest to develop a duplex LAMP to detect both pathogens in the same reaction. In this study, we investigate the use of our two previously developed and well-stablished LAMP assays for S. mansoni and Strongyloides spp. DNA detection in a new duplex real-time eight-primer system based on a modified DARQ probe method that can be performed in a portable isothermal fluorimeter with minimal laboratory resources. We also applied a strategy to stabilize the duplexed DARQ-LAMP mixtures at room temperature for use as ready-to-use formats facilitating analysis in field settings as point-of-care diagnostics for schistosomiasis and strongyloidiasis