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

Strong-LAMP: A LAMP Assay for Strongyloides spp. Detection in Stool and Urine Samples. Towards the Diagnosis of Human Strongyloidiasis Starting from a Rodent Model

[EN]Background: Strongyloides stercoralis, the chief causative agent of human strongyloidiasis, is a nematode globally distributed but mainly endemic in tropical and subtropical regions. Chronic infection is often clinically asymptomatic but it can result in severe hyperinfection syndrome or disseminated strongyloidiasis in immunocompromised patients. There is a great diversity of techniques used in diagnosing the disease, but definitive diagnosis is accomplished by parasitological examination of stool samples for morphological identification of parasite. Until now, no molecular method has been tested in urine samples as an alternative to stool samples for diagnosing strongyloidiasis. This study aimed to evaluate the use of a new molecular LAMP assay in a well-established Wistar rat experimental infection model using both stool and, for the first time, urine samples. The LAMP assay was also clinically evaluated in patients´ stool samples. Methodology/Principal Findings: Stool and urine samples were obtained daily during a 28-day period from rats infected subcutaneously with different infective third-stage larvae doses of S. venezuelensis. The dynamics of parasite infection was determined by daily counting the number of eggs per gram of feces from day 1 to 28 post-infection. A set of primers for LAMP assay based on a DNA partial sequence in the 18S rRNA gene from S. venezuelensis was designed. The set up LAMP assay (namely, Strong-LAMP) allowed the sensitive detection of S. venezuelensis DNA in both stool and urine samples obtained from each infection group of rats and was also effective in S. stercoralis DNA amplification in patients´ stool samples with previously confirmed strongyloidiasis by parasitological and real-time PCR tests. Conclusions/Significance: Our Strong-LAMP assay is an useful molecular tool in research of a strongyloidiasis experimental infection model in both stool and urine samples. After further validation, the Strong-LAMP could also be potentially applied for effective diagnosis of strongyloidiasis in a clinical setting. © 2016 Fernández-Soto et al

Zoonotic implications of onchocerca species on human health

The genus Onchocerca includes several species associated with ungulates as hosts, although some have been identified in canids, felids, and humans. Onchocerca species have a wide geographical distribution, and the disease they produce, onchocerciasis, is generally seen in adult individuals because of its large prepatency period. In recent years, Onchocerca species infecting animals have been found as subcutaneous nodules or invading the ocular tissues of humans; the species involved are O. lupi, O. dewittei japonica, O. jakutensis, O. gutturosa, and O. cervicalis. These findings generally involve immature adult female worms, with no evidence of being fertile. However, a few cases with fertile O. lupi, O. dewittei japonica, and O. jakutensis worms have been identified recently in humans. These are relevant because they indicate that the parasite's life cycle was completed in the new host-humans. In this work, we discuss the establishment of zoonotic Onchocerca infections in humans, and the possibility of these infections to produce symptoms similar to human onchocerciasis, such as dermatitis, ocular damage, and epilepsy. Zoonotic onchocerciasis is thought to be an emerging human parasitic disease, with the need to take measures such as One Health Strategies, in order to identify and control new cases in humans

Power minimization based robust OFDM radar waveform design for radar and communication systems in coexistence.

This paper considers the problem of power minimization based robust orthogonal frequency division multiplexing (OFDM) radar waveform design, in which the radar coexists with a communication system in the same frequency band. Recognizing that the precise characteristics of target spectra are impossible to capture in practice, it is assumed that the target spectra lie in uncertainty sets bounded by known upper and lower bounds. Based on this uncertainty model, three different power minimization based robust radar waveform design criteria are proposed to minimize the worst-case radar transmitted power by optimizing the OFDM radar waveform, which are constrained by a specified mutual information (MI) requirement for target characterization and a minimum capacity threshold for communication system. These criteria differ in the way the communication signals scattered off the target are considered: (i) as useful energy, (ii) as interference or (iii) ignored altogether at the radar receiver. Numerical simulations demonstrate that the radar transmitted power can be efficiently reduced by exploiting the communication signals scattered off the target at the radar receiver. It is also shown that the robust waveforms bound the worst-case power-saving performance of radar system for any target spectra in the uncertainty sets

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.

BACKGROUND NlmCategory: BACKGROUND content: "Benzimidazole resistance is associated with isotype-1 \xCE\xB2-tubulin gene F200Y, E198A and F167Y SNPs. In this study, the recently described polymorphism E198L was reported and analysed in Teladorsagia circumcincta." - Label: METHODS NlmCategory: METHODS content: "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 \xCE\xB2-tubulin gene was analysed, including the three codons of interest, and a new pyrosequencing assay was designed for testing E198L." - Label: RESULTS NlmCategory: RESULTS content: "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\xE2\x80\x89=\xE2\x80\x890.929, P\xE2\x80\x89<\xE2\x80\x890.0001) and the mean value of both was calculated to measure the leucine frequency; this value ranged between 10.4-80.7% before treatment, and 82.3-92.8% after treatment. High and similar correlations were reported between the genotypic variables (C frequency, T frequency or mean of both frequencies) and phenotypic resistance (r\xE2\x80\x89>\xE2\x80\x890.720, P\xE2\x80\x89<\xE2\x80\x890.0001), 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\xE2\x80\x89<\xE2\x80\x890.0001). 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." - Label: CONCLUSIONS NlmCategory: CONCLUSIONS content: The E198L polymorphism can confer BZ resistance on its own in T. circumcincta

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

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

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

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 concerns in STH control [5]. Monitoring anthelmintic efficacy trials have been traditionally done by microscopic approaches, although it is well known that microscopy's sensitivity may be insufficient in this context [6, 7]. We think that DNA-detection-based tools represent an accurate alternative to parasitological methods, and they should be evaluated and validated not only for monitoring worm burden before and after treatment but also for detecting genetic markers related to anthelmintic resistance

Improving stool sample processing and pyrosequencing for quantifying benzimidazole resistance alleles in Trichuris trichiura and Necator americanus pooled eggs

Background: There is an urgent need for an extensive evaluation of benzimidazole efcacy in humans. In veterinary science, benzimidazole resistance has been mainly associated with three single-nucleotide polymorphisms (SNPs) in the isotype-1 β-tubulin gene. In this study, we optimized the stool sample processing methodology and resistance allele frequency assessment in Trichuris trichiura and Necator americanus anthelmintic-related SNPs by pyrosequenc‑ ing, and standardized it for large-scale benzimidazole efcacy screening use. Methods: Three diferent protocols for stool sample processing were compared in 19 T. trichiura-positive samples: fresh stool, egg concentration using metallic sieves with decreasing pore size, and egg concentration followed by fotation with saturated salt solution. Yield of each protocol was assessed by estimating the load of parasite DNA by real-time PCR. Then, we sequenced a DNA fragment of the β-tubulin gene containing the putative benzimidazole resistance SNPs in T. trichiura and N. americanus. Afterwards, resistant and susceptible-type plasmids were produced and mixed at diferent proportions, simulating diferent resistance levels. These mixtures were used to compare previ‑ ously described pyrosequencing assays with processes newly designed by our own group. Once the stool sample processing and the pyrosequencing methodology was defned, the utility of the protocols was assessed by measur‑ ing the frequencies of putative resistance SNPs in 15 T. trichiura- and 15 N. americanus-positive stool samples. Results: The highest DNA load was provided by egg concentration using metallic sieves with decreasing pore size. Sequencing information of the β-tubulin gene in Mozambican specimens was highly similar to the sequences previ‑ ously reported, for T. trichiura and N. americanus, despite the origin of the sample. When we compared pyrosequenc‑ ing assays using plasmids constructs, primers designed in this study provided the most accurate SNP frequencies. When pooled egg samples were analysed, none of resistant SNPs were observed in T. trichiura, whereas 17% of the resistant SNPs at codon 198 were found in one N. americanus sample