The production of second generation biofuels -- cellulosic ethanol from renewable lignocellulosic biomass has the potential to lead the bioindustrial revolution necessary to the transition from a fossil fuel-based economy to a sustainable carbohydrate economy. Effective release of fermentable sugars through biomass pretreatment followed by enzymatic hydrolysis is among the most costly steps for emerging cellulosic ethanol biorefineries. In this project, two pretreatment methods (dilute acid, DA, and cellulose solvent- and organic solvent-lignocellulose fractionation, COSLIF) for corn stover were compared. It was found that glucan digestibility of the corn stover pretreated by COSLIF was much higher, along with faster hydrolysis rate, than that by DA- pretreated. This difference was more significant at a low enzyme loading. Quantitative measurements of total substrate accessibility to cellulase (TSAC), cellulose accessibility to cellulase (CAC), and non-cellulose accessibility to cellulase (NCAC) based on adsorption of a non-hydrolytic recombinant protein TGC were established to find out the cause. The COSLIF-pretreated corn stover had a CAC nearly twice that of the DA-pretreated biomass. Further supported by qualitative scanning electron microscopy images, these results suggested that COSLIF treatment disrupted microfibrillar structures within biomass while DA treatment mainly removed hemicelluloses, resulting in a much less substrate accessibility of the latter than of the former. It also concluded that enhancing substrate accessibility was the key to an efficient bioconversion of lignocellulose. A simple method for determining the adsorbed cellulase on cellulosic materials or pretreated lignocellulose was established for better understanding of cellulase adsorption and desorption. This method involved hydrolysis of adsorbed cellulase in the presence of 10 M of NaOH at 121oC for 20 min, followed by the ninhydrin assay for the amino acids released from the hydrolyzed cellulase. The major lignocellulosic components (i.e. cellulose, hemicellulose, and lignin) did not interfere with the ninhydrin assay. A number of cellulase desorption methods were investigated, including pH adjustment, detergents, high salt solution, and polyhydric alcohols. The pH adjustment to 13.0 and the elution by 72% ethylene glycol at a neutral pH were among the most efficient approaches for desorbing the adsorbed cellulase. For the recycling of active cellulase, a modest pH adjustment to 10.0 may be a low-cost method to desorb active cellulase. More than 90% of cellulase for hydrolysis of the pretreated corn stover could be recycled by washing at pH 10.0. This study provided an in-depth understanding of biomass saccharification for the production of cellulosic ethanol for cellulose hydrolysis and cellulase adsorption and desorption. It will be of great importance for developing better lignocellulose pretreatment technologies and improving cellulose hydrolysis by engineered cellulases. / Master of Science

[ES] El objetivo de la presente tesis doctoral es el estudio, desarrollo y validación de diferentes métodos inmunoquímicos que permitan determinar contaminantes químicos en alimentos de origen vegetal y en agua, de manera que contribuyan a mejorar su calidad y por ende la seguridad del consumidor. Spirotetramat es un plaguicida de nueva generación altamente eficiente, comercializado mundialmente para su uso como insecticida en multitud de cultivos agrícolas. Tiene propiedades sistémicas, ya que después de la absorción se transloca tanto a través del xilema como del floema, gracias a que es transformado por la planta en spirotetramat-enol, mucho más polar. En consecuencia, la definición de residuo de este insecticida en alimentos de origen vegetal para fines analíticos incluye también dicho metabolito. Por otro lado, anatoxina-a es un alcaloide secundario con neurotoxicidad aguda que se pueden encontrar en agua dulce. Esta toxina es producida por siete géneros diferentes de cianobacterias, y se ha detectado en lagos y otras fuentes de agua de todos los continentes. El análisis de sustancias como spirotetramat y anatoxina-a se lleva a cabo actualmente mediante métodos cromatográficos como HPLC-MS. Estas técnicas presentan una elevada sensibilidad y fiabilidad; sin embargo, requieren personal altamente cualificado y un equipamiento caro y no portable. Una opción complementaria son los métodos inmunoquímicos, como el ELISA (Enzyme-Linked ImmunoSorbent Assay) o el inmunoensayo de flujo lateral (LFIA, Lateral Flow ImmunoAssay), ya que son métodos de análisis rápidos y económicos, y además son muy versátiles permitiendo adaptarlos a necesidades analíticas particulares, como los ensayos de cribado de numerosas muestras o los ensayos portátiles con lectura visual de los resultados. A partir de una colección de bioconjugados y de anticuerpos de spirotetramat y de anatoxina-a se caracterizó la afinidad y especificidad de los inmunorreactivos con el fin de seleccionar parejas conjugado/anticuerpo aptas para el desarrollo de inmunoensayos tipo ELISA y LFIA competitivos. Se optimizaron las condiciones de ensayo, y se llevó a cabo un estudio de la influencia de diferentes factores fisicoquímicos sobre los parámetros analíticos de los ensayos seleccionados. Posteriormente se evaluó la influencia de la matriz alimentaria, particularmente uva, zumo de uva y vino, así como de aguas de diferente procedencia, sobre la señal y la sensibilidad de los inmunoensayos. La diferente afinidad de los anticuerpos hacia spirotetramat y spirotetramat-enol nos llevó a optimizar el tratamiento de muestra, incluyendo una etapa de hidrólisis para transformar spirotetramat en spirotetramat-enol de manera controlada, rápida y cuantitativa. De este modo se hizo posible aportar resultados en forma de suma de la concentración de ambos compuestos en la muestra, tal y como exige la legislación vigente. Además, para la extracción de residuos de este insecticida a partir de muestras de uva se puso a punto un procedimiento empleando la tecnología QuEChERS, y para la reducción de interferencias de vinos y zumos se utilizó polivinilpolipirrolidona. En el caso de las aguas, se aplicó una simple filtración para eliminar partículas en suspensión. Los inmunoensayos enzimáticos en microplaca optimizados para determinar de manera competitiva residuos de spirotetramat presentaron valores de IC50 para spirotetramat-enol en torno a 0.1 ng/mL, y límites de detección alrededor de 0.02 ng/mL. El estudio de la precisión y exactitud del método empleando muestras de alimentos dopados reflejó límites de cuantificación de 2.5 ng/mL para uva, zumos de uva y vinos, tanto blancos como tintos, muy por debajo de los límites máximos de residuos autorizados en la Unión Europea para este insecticida en dichos alimentos. / [EN] The aim of this doctoral thesis is the study, development and validation of different immunochemical methods for determining chemical contaminants in produce and water, in a way that they may contribute to improving food quality and therefore to assure consumer safety. Spirotetramat is a highly efficient new-generation pesticide, marketed worldwide for use as insecticide in many agricultural crops. It has systemic properties, since short after absorption it translocates through both the xylem and the phloem, thanks to the fact that it is transformed by the plant into the much more polar spirotetramat-enol. Consequently, the residue definition for this insecticide in foods of plant origin with analytical purposes also includes said metabolite. On the other hand, anatoxin-a is a secondary alkaloid with acute neurotoxicity that can be found in fresh water. This toxin is produced by seven different genera of cyanobacteria, and has been detected in lakes and other water resources on all continents. The analysis of substances like spirotetramat and anatoxin-a is currently carried out by chromatographic methods like HPLC-MS. These techniques are highly sensitive and reliable; however, they require highly qualified personnel and expensive, non-portable equipment. Nowadays, the immunochemical methods, such as the ELISA (Enzyme-Linked ImmunoSorbent Assay) or the LFIA (Lateral Flow ImmunoAssay), constitute excellent complementary analytical options to instrumental strategies, since they are fast and inexpensive analytical methods, and are also very versatile so they can be adapted to particular analytical needs, such as screening assays for large numbers of samples or portable tests with visual reading of the results. The antibody affinity and specificity from a collection of spirotetramat and anatoxin-a immunoreagents was characterized in order to select conjugate/antibody pairs suitable for the development of competitive ELISA and LFIA tests. The assay conditions were optimized, and a study of the influence of different physicochemical factors over the analytical parameters of the selected immunoassays was carried out. Subsequently, the influence of the food matrix, particularly grape, grape juice and wine, as well as water from different sources, over the assay signal and sensitivity was evaluated. The different affinity of the available antibodies towards spirotetramat and spirotetramat-enol led us to optimize the sample treatment procedure, so a hydrolysis step to transform spirotetramat into spirotetramat-enol in a controlled, rapid and quantitative way, was included. Thus, it was possible to provide results in the form of the sum of the concentration of both compounds in the sample, as required by current legislation. In addition, a procedure using QuEChERS technology was developed to extract residues of this insecticide from grape samples, and polyvinylpolypyrrolidone was used to reduce interferences from wines and juices. In the case of waters, a simple filtration was applied to remove suspended particles. Microplate enzyme immunoassays that were optimized to competitively determine spirotetramat residues showed IC50 values for spirotetramat-enol around 0.1 ng/mL, and limits of detection around 0.02 ng/mL. Precision and accuracy studies with these immunoassays using fortified food samples reflected limits of quantification of 2.5 ng/mL for grapes, grape juices and wines, both white and red, well below the maximum residue limits authorized by the European Union for this insecticide in such foodstuffs. Finally, a comparative study with HPLC-MS/MS validated the studied immunoassay for analyzing spirotetramat residues in grape samples within a wide range of concentrations. / [CA] L’objectiu de la present tesi doctoral és l’estudi, desenvolupament i validació de diferents mètodes immunoquímics que permeten determinar contaminants químics en aliments d’origen vegetal i en aigua, de manera que contribuïsquen a millorar la seua qualitat i per tant la seguretat dels consumidors. Spirotetramat és un plaguicida de nova generació altament eficient, comercialitzat mundialment per a l’ús com insecticida en multitud de cultius agrícoles. Té propietats sistèmiques, ja que en ser absorbit es transloca tant a través del xilema com del floema, gràcies a que és transformat per la planta en spirotetramat-enol, molt més polar. Conseqüentment, la definició de residu d’aquest insecticida en aliments d’origen vegetal amb finalitats analítiques inclou també l’esmentat metabòlit. D’una altra banda, anatoxina-a és un alcaloide secundari amb neurotoxicitat aguda que es pot trobar en aigua dolça. Aquesta toxina és produïda per set gèneres de cianobacteris diferents, i s’ha detectat en llacs i altres fonts d’aigua de tots els continents. L’anàlisi de substàncies com spirotetramat i anatoxina-a es duu a terme actualment mitjançant mètodes cromatogràfics, com HPLC-MS. Aquestes tècniques presenten una elevada sensibilitat i fiabilitat; tanmateix, requereixen personal altament qualificat i un equipament car i no portable. Una opció complementària són els mètodes immunoquímics, com l’ELISA (Enzyme-Linked ImmunoSorbent Assay) o l’immunoassaig de flux lateral (LFIA, Lateral Flow Immunoassay), ja que són mètodes d’anàlisi ràpids i econòmics, i a més són molt versàtils, la qual cosa permet adaptar-los a necessitats analítiques particulars, com són els assaigs per destriar nombroses mostres o els assaigs portàtils amb lectura visual dels resultats. A partir d’una col·lecció de bioconjugats i d’anticossos de spirotetramat i d’anatoxina-a es va caracteritzar l’afinitat i especificitat dels immunorreactius amb la finalitat de seleccionar parelles conjugat/anticòs aptes per desenvolupar immunoassaigs tipus ELISA i LFIA competitius. S’optimitzaren les condicions d’assaig, i es va dur a terme un estudi de la influència de diferents factors fisicoquímics sobre els paràmetres analítics dels assaigs seleccionats. Posteriorment, es va avaluar la influència de la matriu alimentària, particularment raïm, suc de raïm i vi, així com d’aigües de diferent procedència, sobre el senyal i la sensibilitat dels assaigs. La diferent afinitat dels anticossos cap a spirotetramat i spirotetramat-enol ens va dur a optimitzar el tractament de mostra mitjançant la inclusió d’una etapa d’hidròlisi per transformar spirotetramat en spirotetramat-enol de manera ràpida, controlada i quantitativa. D’aquesta manera es va fer possible aportar resultats en forma de suma de la concentració d’ambdós composts en la mostra, tal i com exigeix la legislació vigent. A més a més, per extraure residus d’aquest insecticida a partir de mostres de raïm es va posar a punt un procediment emprant la tecnologia QuEChERS, i per reduir interferències de vins i sucs es va utilitzar polivinilpolipirrolidona. En el cas de les aigües, es va aplicar una simple filtració per eliminar partícules en suspensió. Els immunoassaigs enzimàtics en microplaca optimitzats per determinar de manera competitiva residus de spirotetramat presentaren valors d’IC50 per spirotetramat-enol al voltant de 0.1 ng/mL, i límits de detecció propers a 0.02 ng/mL. L’estudi de la precisió i exactitud del mètode emprant mostres d’aliments dopats va reflectir límits de quantificació de 2.5 ng/mL per raïm, sucs de raïm i vins, tant blancs com negres, molt per sota dels límits màxims de residus autoritzats per la Unió Europea per a aquest insecticida en els esmentats aliments. Finalment, un estudi comparatiu amb HPLC-MS/MS va validar l’immunoassaig estudiat per analitzar residus de spirotetramat en mostres de raïm en un ampli rang de concentracions. En el cas d’anatoxina-a, es van optimitzar dos immunoassaigs tipus ELISA competitiu, els valors d’IC50 dels quals van estar entre 0.5 i 1.0 ng/mL, amb límits de detecció per davall de 0.1 ng/mL. L’anàlisi de diferents tipus d’aigües fortificades amb anatoxina-a ens va revelar que els immunoassaigs desenvolupats permeten quantificar aquesta cianotoxina entre 0.5 i 500 ng/mL. Addicionalment es van optimitzar i caracteritzar assaigs immunocromatogràfics, tipus tires reactives, tant per spirotetramat com per anatoxina-a, vàlids com a tècniques portables i ràpides per determinar semi-quantitativament aquestes substàncies tòxiques en vi i aigües, respectivament. Seguint la normativa europea per a mètodes ràpids front a petites molècules orgàniques, es va determinar el senyal indicatiu del llindar per distingir les mostres positives, que superen la concentració de destriament establerta, de les negatives. En el cas de spirotetramat, el mètode desenvolupat permet el triatge, tant instrumental com visual, amb un interval de confiança del 99%, de mostres de vi amb una concentració de residu de 1000 ng/mL, equivalent al límit màxim de residus, expressada como la suma de spirotetramat més spirotetramat-enol. Per anatoxina-a, les tires immunocromatográfiques desenvolupades van poder detectar mostres d’aigua amb 2 ng/mL de l’esmentada cianotoxina amb una fiabilitat del 99%, i mostres amb 1 ng/mL amb una probabilitat del 40%, mentre que el límit de detecció visual va ser de 3 ng/mL. / A la Secretaría Nacional de Educación Superior, Ciencia, Tecnología e Innovación (SENESCYT), del gobierno de la República de Ecuador que al adjudicarme la beca bajo el “PROGRAMA DE BECAS PARA DOCTORADO (PHD) PARA DOCENTES DE UNIVERSIDADES Y DE ESCUELAS POLITÉCNICAS 2015”, permitió formarme como persona y profesional en mis estudios de doctorado. / Cevallos Cedeño, RE. (2020). Desarrollo de métodos inmunoquímicos para la determinación de sustancias tóxicas en alimentos y aguas [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/149570

Using a behavioural and genetic approach, we employed single-gene mutations and photoreceptor cell ablations to study the molecules and cell-types underlying larval response to light Drosophila larvae modulate their locomotion in response to light In the Checker Assay the response to light is defined as an increased residence time in dark quadrants versus lit. In the ON/OFF Assay, it is in part defined as a decreased path length in the light. Previously, mutations in genes, which function in the adult phototransduction cascade were found to abolish fight perception as defined by a reduction in path length. This response to light was reported to be mediated by rhodopsins, other than Rh1, via a pathway similar to the one present in the adult visual system. After undertaking a similar genetic approach in the Checker Assay, the response to light measured in this assay appears also to be mediated through a similar pathway. Mutations in sine oculis (so), a homeobox gene necessary for proper visual system development, and targeted expression of the cell death gene head involution defective (hid), to larval photoreceptor neurons, abolished light response as measured in the Checker Assay. Thus, mutations affecting development oflarval visual system suggest that this response to light is also housed in the larva’s main photoreceptor organ, the BO. The modular GAL4 system was used to target expression of cell death genes, rpr and hid, to Rh5 and Rh6 expressing larval photoreceptor cells. In strains tested in the ON/OFF Assay, in which Rh5 cells are missing, the response to light is abolished, as measured by both decreased path length and increased head swinging behaviour in the light. In a strain in which Rh6 photoreceptor cells are ablated, this response to light is not abolished. This suggests that Rh5 mediates responses in the ON/OFF Assay, which were previously abolished by mutations in genes operating in the adult phototransduction cascade. Thus Rh5, not Rh6, appears to be necessary in mediating the response to light carried out via a pathway similar to the operating in adult phototransduction. In both ablated strains, Rh5-gal4xUAS-rpr and Rh5-gal4xUAS-hid, the integrity of remaining photoreceptor cells is not compromised, and in the latter strain, the extent of ablation appears to be complete. Previously in the ON/OFF assay, mutations and ablations of cell-types were found to disrupt only a subset of behaviours associated with the larval perception of light. Based upon this evidence it was surmised that Rh1 mediates a basic independent visual system, which operates in the larva. However, uncovering the possible roles in this system was hindered, as parental control strains did not respond. / Thesis / Master of Science (MS)

No / Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) are produced in the skin under the influence of UV radiation. These compounds are highly reactive and can induce DNA lesions in epidermal cells. Melanin is considered to protect human skin against DNA damage by absorbing UV radiation. We have investigated whether melanin can, in addition, offer protection against the effects of H2O2 in human melanocytes and HaCaT keratinocytes. In the present study, it was shown that 40 and 100 μM H2O2 increased the number of DNA strand breaks as measured using the comet assay, in melanocytes of Caucasian origin. In melanocytes of the same origin in which melanin levels were increased by culturing in presence of 10 mM NH4Cl and elevated l-tyrosine, H2O2-induced DNA damage was reduced compared to that in control melanocytes. Similarly, HaCaT cells that were loaded with melanin were better protected against H2O2-induced DNA strand breaks than control HaCaT cells. These protective effects of melanin were mimicked by the intracellular Ca2+-chelator BAPTA. Thus, BAPTA reduced the level of H2O2-induced DNA strand breaks in melanocytes. Like BAPTA, melanin is known to be a potent chelator of Ca2+ and this was confirmed in the present study. It was shown that melanin levels in melanocytic cells correlated directly with intracellular Ca2+ binding capacity and, in addition, correlated inversely with H2O2-induced increases in intracellular Ca2+. Our results show that melanin may have an important role in regulating intracellular Ca2+ homeostasis and it is suggested that melanin protects against H2O2-induced DNA strand breaks in both melanocytes and keratinocytes and through its ability to bind Ca2+.

No / Numerous factors may influence the incidence of diabetes in the population. The production of reactive oxygen species (ROS) is elevated in diabetes patients. Based on the reported involvement of reactive species and nitrate/nitrite in diabetes, this present study has examined in the alkaline Comet assay, the effect of different levels of NaNO2 in the presence of the oxygen radical generating agent, hydrogen peroxide (H2O2). Peripheral lymphocytes from diabetic and non-diabetic Caucasians and Asians of both sexes were studied in vitro. Endogenous factors (e.g., sex, age, body mass index-BMI) and exogenous factors (lifestyle factors e.g., smoking and drinking habits, diet) were taken into account. A preliminary study in two individuals showed that DNA damage remained constant over a wide dose range of NaNO2 (1-75 mM), but when H2O2 was added at a constant concentration of 50 ¿M per dose of NaNO2, there was an increase in DNA damage corresponding with the varying levels of NaNO2 investigated. This was also seen with the 44 individuals (non-diabetic, n = 24; type 1 diabetic, n = 11; type 2 diabetic, n = 9) investigated. NaNO2 was capable of inducing a significant level of DNA damage in lymphocytes (p<0.001), but only with the addition of H2O2. When levels of DNA damage were analysed in terms of the different variables there were few significant differences in damage between diabetic and non-diabetic subjects, or other sub-population groups, and no statistically significant differences in susceptibility were observed between subject covariates using regression techniques.

No / Telomerase-targeted strategies have aroused recent interest in anti-cancer chemotherapy, because DNA-binding drugs can interact with high-order tetraplex rather than double-stranded (duplex) DNA targets in tumour cells. However, the protracted cell-drug exposure times necessary for clinical application require that telomerase inhibitory efficacy must be accompanied by both low inherent cytotoxicity and the absence of mutagenicity/genotoxicity. For the first time, the genotoxicity of a number of structurally diverse DNA-interactive telomerase inhibitors is examined in the Ames test using six Salmonella typhimurium bacterial strains (TA1535, TA1537, TA1538, TA98, TA100, and TA102). DNA damage induced by each agent was also assessed using the Comet assay with human lymphocytes. The two assay procedures revealed markedly different genotoxicity profiles that are likely to reflect differences in metabolism and/or DNA repair between bacterial and mammalian cells. The mutational spectrum for a biologically active fluorenone derivative, shown to be mutagenic in the TA100 strain, was characterised using a novel and rapid assay method based upon PCR amplification of a fragment of the hisG46 allele, followed by RFLP analysis. Preliminary analysis indicates that the majority (84%) of mutations induced by this compound are C→A transversions at position 2 of the missense proline codon of the hisG46 allele. However, despite its genotoxic bacterial profile, this fluorenone agent gave a negative response in the Comet assay, and demonstrates how unwanted systemic effects (e.g., cytotoxicity and genotoxicity) can be prevented or ameliorated through suitable molecular fine-tuning of a candidate drug in targeted human tumour cells. / CAEB, Balearic Islands and Yorkshire Cancer Research

Yes / Genotoxic compounds have induced DNA damage in male germ cells and have been associated with adverse clinical outcomes including enhanced risks for maternal, paternal and offspring health. DNA strand breaks represent a great threat to the genomic integrity of germ cells. Such integrity is essential to maintain spermatogenesis and prevent reproduction failure. The Comet assay results revealed that the incubation of isolated germ cells with n-ethyl-n-nitrosourea (ENU), 6-mercaptopurine (6-MP) and methyl methanesulphonate (MMS) led to increase in length of Olive tail moment and % tail DNA when compared with the untreated control cells and these effects were concentration-dependent. All compounds were significantly genotoxic in cultured germ cells. Exposure of isolated germ cells to ENU produced the highest concentration-related increase in both DNA damage and gene expression changes in spermatogonia. Spermatocytes were most sensitive to 6-MP, with DNA damage and gene expression changes while spermatids were particularly susceptible to MMS. Real-time PCR results showed that the mRNA level expression of p53 increased and bcl-2 decreased significantly with the increasing ENU, 6-MP and MMS concentrations in spermatogonia, spermatocytes and spermatids respectively for 24 hr. Both are gene targets for DNA damage response and apoptosis. These observations may help explain the cell alterations caused by ENU, 6-MP and MMS in spermatogonia, spermatocytes and spermatids. Taken together, ENU, 6-MP and MMS induced DNA damage and decreased apoptosis associated gene expression in the germ cells in vitro. / Libyan Government

Yes / Regular use of non-steroidal anti-inflammatory drugs (NSAIDs) may be protective against tumours, including breast cancer. We have studied the effects of ibuprofen and aspirin on DNA damage in lymphocytes obtained from breast cancer patients and healthy female controls. Both nanoparticle (NPs) and bulk formulations were used in the comet and micronucleus (MN) assays. Non-toxic doses (250 ng/ml ibuprofen; 500 ng/ml aspirin) were tested. Aspirin, both bulk and nano formulations, significantly reduced DNA damage measured with the comet and micronucleus assays; the nano formulation was more effective. Ibuprofen was not effective in the comet assay but showed a significant reduction in MN frequency, with the nano formulation being more effective. NPs may have better penetration through the nuclear membrane relative to the bulk formulation. NSAIDs such as aspirin and ibuprofen may have a promising role in cancer prevention and treatment. / LIBYAN GOVERNMENT

Yes / Our previous case-control study observed isolated lymphocytes from 208 individuals and determined the differences in the sensitivity to genomic damage of lymphocytes derived from cancer patients, pre/suspect cancer patients and healthy volunteers using the Comet assay (Anderson et al, 2014). We adapted the LGS technique using a slightly different method and examined 700 more blood samples from 598 patients with cancer or suspected cancer and 102 healthy individuals. To help increase the sensitivity of the test and detect cancer at the level of each individual, we joined with the IMSTAR team who analysed our cells with their fully automated Pathfinder™ cell reader-analyser system. With this reading and analysis system 4,000 to 10,000 cells were able to be read per slide. The new test which is called TumorScan is a highly sensitive test to detect any cancer at an early stage through the response of the white blood cells to UV treatment. These patient blood samples have also been collected at the stage before confirming diagnosis and treatment. There were four of these individuals with cancer who had received anti-cancer treatment. The results from these patients showed a reverse pattern compared to non-treated cancer patients and followed the pattern seen in healthy individuals. The results are consistent with the early results as reported in the above 2014 paper. Given the results from these samples were in a particularly challenging subgroup, whose cancer status was difficult to distinguish, the data suggest that the technique using the TumorScan system could exceed the area under the ROC curve >93% obtained in the earlier study on a group basis, whereas this present study was to detect cancer at an early stage in each individual. / Department of Research and Knowledge Transfer at the University of Bradford, Bradford, UK

Yes / Background: The Xpert MTB/RIF (Xpert) assay is used globally to rapidly diagnose tuberculosis and resistance to rifampicin. We investigated the frequency and predictors of false-positive findings of rifampicin resistance with Xpert. Methods: We did a prospective, observational study of individuals who were enrolled in a Rwandan nationwide diagnostic cohort study (DIAMA trial; NCT03303963). We included patients identified to have rifampicin resistance on initial Xpert testing. We did a repeat Xpert assay and used rpoB Sanger and deep sequencing alongside phenotypic drug susceptibility testing (pDST) to ascertain final rifampicin susceptibility status, with any (hetero)resistant result overriding. We used multivariable logistic regression to assess predictors of false rifampicin resistance on initial Xpert testing, adjusted for HIV status, tuberculosis treatment history, initial Xpert semi-quantitative bacillary load, and initial Xpert probe. Findings: Between May 4, 2017, and April 30, 2019, 175 people were identified with rifampicin resistance at initial Xpert testing, of whom 154 (88%) underwent repeat Xpert assay. 54 (35%) patients were confirmed as rifampicin resistant on repeat testing and 100 (65%) were not confirmed with resistance. After further testing and sequencing, 121 (79%) of 154 patients had a final confirmed status for rifampicin susceptibility. 57 (47%) of 121 patients were confirmed to have a false rifampicin resistance result and 64 (53%) had true rifampicin resistance. A high pretest probability of rifampicin resistance did not decrease the odds of false rifampicin resistance (adjusted odds ratio [aOR] 6·0, 95% CI 1·0–35·0, for new tuberculosis patients vs patients who needed retreatment). Ten (16%) of the 64 patients with true rifampicin resistance did not have confirmed rifampicin resistance on repeat Xpert testing, of whom four had heteroresistance. Of 63 patients with a very low bacillary load on Xpert testing, 54 (86%) were falsely diagnosed with rifampicin-resistant tuberculosis. Having a very low bacillary load on Xpert testing was strongly associated with false rifampicin resistance at the initial Xpert assay (aOR 63·6, 95% CI 9·9–410·4). Interpretation: The Xpert testing algorithm should include an assessment of bacillary load and retesting in case rifampicin resistance is detected on a paucibacillary sputum sample. Only when rifampicin resistance has been confirmed on repeat testing should multidrug-resistant tuberculosis treatment be started. When rifampicin resistance has not been confirmed on repeat testing, we propose that patients should be given first-line anti-tuberculosis drugs and monitored closely during treatment, including by baseline culture, pDST, and further Xpert testing. / The European & Developing Countries Clinical Trials Partnership 2 programme, and Belgian Directorate General for Development Cooperation.