Diseño, desarrollo y aplicación de la tecnología LAMP para el diagnóstico de la esquistosomosis: del laboratorio al campo

Tesis por compendio de publicaciones[POR]Esquistossomose, uma doença parasitária causada por trematódeos dogênero Schistosoma, é uma das principais doenças tropicais desantendidas.Atualmente não há um método diagnóstico adequado, como técnicas parasitológicase sorológicas têm problemas de sensibilidade e especificidade. O diagnósticomolecular não é rotineiramente utilizada, particularmente em áreas endémicas epobre. A técnica LAMP é uma amplificação de ADN é realizada sob condiçõesisotérmicas. Tem as vantagens de ser rápida, sensível, específica, permite que osresultados de discriminação visual e pode ser aplicada como um diagnóstico point-ofcare.Neste trabalho foram concebidos, desenvolvidos e aplicados métodosmoleculares baseados na tecnologia LAMP para o diagnóstico de Schistosomamansoni e Schistosoma haematobium, e para detectar caracóis infectados com S.mansoni. Em primeiro lugar, uma técnica foi desenvolvida para a amplificação de ADNde S. mansoni, chamada SmMIT-LAMP, o qual provou ser eficaz para a detecção doparasita na fase aguda da infecção. A sua aplicação em amostras humanas e decaracois numa área endémica de baixa transmissão no Brasil tem mostrado altasensibilidade em relação às técnicas convencionais de diagnóstico e sua utilidadepara identificar focos de transmissão da doença. Estes estudos são detalhados notrabalho 1 e 5. Em seguida, outro método LAMP (Sh-LAMP) para a detecção de S.haematobium foi concebido. Ele foi otimizado em amostras clínicas de urina no laboratório com e sem extracção de ADN (Rapid-Heat LAMPellet method) e,subsequentemente, avaliadas com sucesso numa zona de transmissão em Angola.Além disso, Sh-LAMP mostrou elevada reprodutibilidade no laboratório dereferência. Estes estudos são recolhidos no trabalho 2 e 4. Finalmente, Biompha-LAMP mostrou-se útil para a detecção de S. mansoni emcaracóis infectados experimentalmente. Este estudo é o trabalho detalhado 3.[ES]La esquistosomosis, una enfermedad parasitaria producida por trematodos del género Schistosoma, es una de las principales enfermedades tropicales desantendidas. En la actualidad no se dispone de un método de diagnóstico adecuado ya que las técnicas parasitológicas y serológicas presentan problemas de sensibilidad y especificidad. El diagnóstico molecular no se utiliza de forma rutinaria, particularmente en zonas endémicas y de bajos recursos. El LAMP (loop-mediated isothermal amplification) es una técnica de amplificación de ADN que se realiza en condiciones isotérmicas. Presenta las ventajas de ser rápida, sensible, específica, permite la discriminación visual de los resultados y puede ser aplicada como diagnóstico point-of-care. En este trabajo se diseñaron, desarrollaron y aplicaron métodos moleculares basados en la tecnología LAMP para el diagnóstico de Schistosoma mansoni y Schistosoma haematobium, así como para la detección de caracoles infectados con S. mansoni. En primer lugar, se desarrolló una técnica de amplificación de ADN de S. mansoni, denominada SmMIT-LAMP, que ha mostrado su efectividad para la detección del parásito en la fase aguda de la infección. Su aplicación en muestras humanas y de caracoles en un área endémica de baja transmisión en Brasil ha demostrado una alta sensibilidad en comparación con las técnicas de diagnóstico clásicas, así como su utilidad para la identificación de focos de transmisión de la enfermedad. Estos estudios se detallan en los trabajos 1 y 5. A continuación se diseñó otro método LAMP (Sh-LAMP) para la detección de S. haematobium. Se puso a punto en muestras clínicas de orina en el laboratorio con y sin extracción previa de ADN (Rapid-Heat LAMPellet method) y posteriormente se evaluó con éxito en una zona de alta transmisión en Angola. Además, Sh-LAMP mostró una elevada reproducibilidad en un laboratorio de referencia. Estos estudios se encuentran recogidos en los trabajos 2 y 4. Finalmente, Biompha-LAMP mostró ser una herramienta útil para la detección de S. mansoni en caracoles infectados experimentalmente. Este estudio se encuentra detallado en el trabajo 3

Role of DNA-detection–based tools for monitoring the soil-transmitted helminth treatment response in drug-efficacy trials

[EN] More than 1 billion people have been reported to be infected with at least one soil-transmitted helminth (STH) worldwide, according to the last published report of the World Health Organization (WHO) [1]. WHO guidelines for STH control mainly encompass periodic administration of benzimidazoles (albendazole or mebendazole) to at-risk people of the endemic areas [1]. However, extended use of benzimidazoles could entail a great selection pressure for parasitic-resistant strains. In veterinary medicine, anthelmintic resistance in gastrointestinal nematodes has been developed in response to their excessive use, and it is currently considered a serious threat to livestock health and welfare [2, 3]. In humans, the estimated efficacy of albendazole and mebendazole against Trichuris trichiura has been observed to significantly decrease over time [4]. This observed decrement in drug efficacy could be due to the development of anthelmintic resistance (among other reasons such as drug quality and administration, the increasing of drug-efficacy studies, improvements in sensitivity of diagnostic tools after treatment, etc) after years of mass drug-administration campaigns, which is one of the major oncerns in STH controlSIThe Stopping Transmission Of intestinal Parasites (STOP) project is part of the EDCTP2 programme supported by the European Union (grant number RIA2017NCT-1845 — STOP). JG is personally supported by Ramon Areces Foundation, Spain; MMV by the Spanish ‘Ramo´n y Cajal’ Programme, Ministry of Economy and Competitiveness (RYC-2015-18368); and SK is supported by DELTAS Africa Initiative grant # DEL- 15-011 to THRiVE-2. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscrip

Teladorsagia circumcincta beta tubulin: the presence of the E198L polymorphism on its own is associated with benzimidazole resistance

[EN] BACKGROUND: Benzimidazole resistance is associated with isotype-1 β-tubulin gene F200Y, E198A and F167Y SNPs. In this study, the recently described polymorphism E198L was reported and analysed in Teladorsagia circumcincta. METHODS: The benzimidazole phenotypic resistance was measured by the faecal egg count reduction test (FECRT) and the egg hatch test (EHT) using a discriminating dose (DD) in 39 sheep flocks. Around 1000 larvae collected before and after treatment were used for DNA extraction. The resistant species identified in all flocks was T. circumcincta. The resistance alleles frequencies were measured for F200Y and E198A. A 371-bp fragment of the isotype-1 β-tubulin gene was analysed, including the three codons of interest, and a new pyrosequencing assay was designed for testing E198L. RESULTS: The percentage of resistant flocks was 35% by FECRT or 26% by EHT; however, F200Y and E198A SNPs were absent in T. circumcincta. The amplification of a 371-bp fragment confirmed the absence of F167Y and F200Y in 6 resistant flocks. Regarding codon 198, all samples after treatment carried a leucine (CTA). A pyrosequencing assay analysed the allele frequencies for the first two bases at codon 198 independently, G/C and A/T. The correlation between C and T frequencies was almost 1 (r = 0.929, P  0.720, P < 0.0001), although negatively associated with the FECRT and positively with the EHT. According to multivariate linear regression analysis, the T frequency was the most significant variable influencing the phenotypic resistance (FECRT or EHT; P < 0.0001). In the EHT, 67.1% of the phenotypic variability is associated with the T frequency but in the FECRT only 33.4%; therefore, the EHT using a DD seems to detect the genotypic resistance more accurately than the FECRT. CONCLUSIONS: The E198L polymorphism can confer BZ resistance on its own in T. circumcinctaSIThis study was funded by the Spanish “Ramón y Cajal” Programme of the Ministry of Economy and Competitiveness (MMV, RYC‑2015‑18368), and the Cooperativa Bajo Duero, COBADU. EVG was funded by FPU16/03536, JG by Ramon Areces Foundation, VCGA by Junta de Castilla y León and Fondo Social Europeo (LE082‑18), MCP by the Stopping Transmission Of intestinal Parasites (STOP) project (EDCTP2 programme; RIA2017NCT‑1845) and MMV by the Spanish “Ramon y Cajal” Programme (RYC‑2015‑18368

A Loop-Mediated Isothermal Amplification (LAMP) Assay for Early Detection of <i>Schistosoma mansoni</i> in Stool Samples: A Diagnostic Approach in a Murine Model

<div><p>Background</p><p>Human schistosomiasis, mainly due to <i>Schistosoma mansoni</i> species, is one of the most prevalent parasitic diseases worldwide. To overcome the drawbacks of classical parasitological and serological methods in detecting <i>S. mansoni</i> infections, especially in acute stage of the disease, development of cost-effective, simple and rapid molecular methods is still needed for the diagnosis of schistosomiasis. A promising approach is the loop-mediated isothermal amplification (LAMP) technology. Compared to PCR-based assays, LAMP has the advantages of reaction simplicity, rapidity, specificity, cost-effectiveness and higher amplification efficiency. Additionally, as results can be inspected by the naked eye, the technique has great potential for use in low-income countries.</p><p>Methodology/Principal findings</p><p>A sequence corresponding to a mitochondrial <i>S. mansoni</i> minisatellite DNA region was selected as a target for designing a LAMP-based method to detect <i>S. mansoni</i> DNA in stool samples. We used a <i>S. mansoni</i> murine model to obtain well defined stool and sera samples from infected mice with <i>S. mansoni</i> cercariae. Samples were taken weekly from week 0 to 8 post-infection and the Kato-Katz and ELISA techniques were used for monitoring the infection. Primer set designed were tested using a commercial reaction mixture for LAMP assay and an <i>in house</i> mixture to compare results. Specificity of LAMP was tested using 16 DNA samples from different parasites, including several <i>Schistosoma</i> species, and no cross-reactions were found. The detection limit of our LAMP assay (SmMIT-LAMP) was 1 fg of <i>S. mansoni</i> DNA. When testing stool samples from infected mice the SmMIT-LAMP detected <i>S. mansoni</i> DNA as soon as 1 week post-infection.</p><p>Conclusions/Significance</p><p>We have developed, for the first time, a cost-effective, easy to perform, specific and sensitive LAMP assay for early detection of <i>S. mansoni</i> in stool samples. The method is potentially and readily adaptable for field diagnosis and disease surveillance in schistosomiasis-endemic areas.</p></div

Specificity and sensitivity assessment of the LAMP assay for <i>S. mansoni</i>.

<p>(A) Specificity assessment performed with SmMIT-LAMP is shown. Identical results were obtained using <i>Loopamp DNA amplification Kit</i>. A ladder of multiple bands of different sizes could be only observed in <i>S. mansoni</i> DNA sample. Lane M, 50 bp DNA ladder (Molecular weight marker XIII, Roche); lanes Sm, Sh, Sj, Si, Fh, Dd, Hd, Cd, Ll, Bp, As, Ts, Tt, Eg, Cp, Gd, Eh, <i>S. mansoni</i>, <i>S. haematobium</i>, <i>S. japonicum</i>, <i>S. intercalatum</i>, <i>Dicrocoelium dendriticum</i>, <i>Hymenolepis diminuta</i>, <i>Calicophoron daubneyi</i>, <i>Loa loa</i>, <i>Brugia pahangi</i>, <i>Anisakis simplex</i>, <i>Trichinella spiralis</i>, <i>Taenia taeniformis</i>, <i>Echinococcus granulosus</i>, <i>Cryptosporidium parvum</i>, <i>Giardia intestinalis</i> and <i>Entamoeba histolytica</i> DNA samples (1 ng/each), respectively; lane N, negative control (no DNA template). (B) Sensitivity assessment performed with the <i>Loopamp DNA amplification kit</i> at 63°C for 1 h using serial dilutions of <i>S. mansoni</i> genomic DNA by the addition of SYBR Green I (up) or by visualization on agarose gel (down). (C) Sensitivity assessment performed with SmMIT-LAMP at 63°C for 1 h using serial dilutions of <i>S. mansoni</i> genomic DNA by the addition of SYBR Green I (up) or by visualization on agarose gel (down). Lane M: 50 bp DNA ladder (Molecular weight marker XIII, Roche); lanes Sm: genomic DNA from <i>S. mansoni</i> (1 ng); lanes 10⁻¹–10⁻⁹: 10-fold serially dilutions; lanes N: negative controls (no DNA template).</p

LAMP detection of <i>S. mansoni</i> genomic DNA samples using SmMIT-LAMP or the <i>Loopamp DNA amplification kit</i> at 63°C for 1 h.

<p>(A) The turbidity of the reaction mixture was inspected by the naked eye. (B) The LAMP amplification results were also visually detected by adding the fluorescent dye SYBR Green I to the reaction tubes. A successful LAMP reaction would turn to green; otherwise, it would remain orange (C) LAMP products were also monitored using 2% agarose gel electrophoresis stained with ethidium bromide. Lane M, 50 bp DNA ladder (Molecular weight marker XIII, Roche); lanes Sm: <i>S. mansoni</i> DNA (1 ng); lane N, negative control (no DNA template).</p

Lamp primer set targeting the selected sequence (GenBank Accesion. No. L27240) for mitochondrial <i>S. mansoni</i> minisatellite DNA region amplification.

<p>(A) The location of the LAMP primers within the selected sequence is shown. Arrows indicate the direction of extension. (B). Sequence of LAMP primers: F3, forward outer primer; B3, reverse outer primer; FIP, forward inner primer (comprising F1c and F2 sequences); BIP, reverse inner primer (comprising B1c and B2 sequences).</p

Examination of stool samples weekly obtained from mice infected with 200 <i>S. mansoni</i> cercariae.

<p>(A) By using the <i>Loopamp DNA amplification kit</i>. (B) By using the SmMIT-LAMP developed in this study. Figure shows the results obtained in feces samples weekly obtained from week 0 p.i. to week 8 p.i. from an infected mouse randomly selected. Identical results were obtained in all infected mice. Lanes M, 50 bp DNA ladder (Molecular weight marker XIII, Roche); lanes Sm, <i>S. mansoni</i> DNA, as positive control (1 ng); lanes 0–8, weeks 0, 1, 2, 3, 4, 5, 6, 7 and 8 p.i., respectively; lanes N; DNA mix from pooled DNA samples obtained from feces from non-infected mice, as negative control.</p

PCR verification, detection limit and specificity using outer primers F3 and B3.

<p>(A) PCR verification of expected 206 bp target length amplicon. Lane M, 50 bp DNA ladder (Molecular weight marker XIII, Roche); lane Sm, <i>S. mansoni</i> DNA (1 ng); lane N, negative control (no DNA template). (B) Detection limit of PCR. Lane M, 50 bp DNA ladder (Molecular weight marker XIII, Roche); lane Sm: <i>S. mansoni</i> DNA (1 ng); lanes 10⁻¹–10⁻⁹: 10-fold serially dilutions of <i>S. mansoni</i> DNA; lane N, negative control (no DNA template). (C) Specificity of PCR. Lane M, 50 bp DNA ladder (Molecular weight marker XIII, Roche); lanes Sm, Sh, Sj, Si, Fh, Dd, Hd, Cd, Ll, Bp, As, Ts, Tt, Eg, Cp, Gd, Eh, <i>S. mansoni</i>, <i>S. haematobium</i>, <i>S. japonicum</i>, <i>S. intercalatum</i>, <i>Dicrocoelium dendriticum</i>, <i>Hymenolepis diminuta</i>, <i>Calicophoron daubneyi</i>, <i>Loa loa</i>, <i>Brugia pahangi</i>, <i>Anisakis simplex</i>, <i>Trichinella spiralis</i>, <i>Taenia taeniformis</i>, <i>Echinococcus granulosus</i>, <i>Cryptosporidium parvum</i>, <i>Giardia intestinalis</i> and <i>Entamoeba histolytica</i> DNA samples (1 ng/each), respectively; lane N, negative control (no DNA template).</p