Indoor Content Delivery Solution for a Museum Based on BLE Beacons.

The digital transformation advancement enables multiple areas to provide modern services to their users. Culture is one of the areas that can benefit from these advances, more specifically museums, by presenting many benefits and the most emergent technologies to the visitors. This paper presents an indoor location system and content delivery solution, based on Bluetooth Low Energy Beacons, that enable visitors to walk freely inside the museum and receive augmented reality content based on the acquired position, which is done using the Received Signal Strength Indicator (RSSI). The solution presented in this paper was created for the Foz Côa Museum in Portugal and was tested in the real environment. A detailed study was carried out to analyze the RSSI under four different scenarios, and detection tests were carried out that allowed us to measure the accuracy of the room identification, which is needed for proper content delivery. Of the 89 positions tested in the four scenarios, 70% of the received signals were correctly received throughout the entire duration of the tests, 20% were received in an intermittent way, 4% were never detected and 6% of unwanted beacons were detected. The signal detection is fundamental for the correct room identification, which was performed with 96% accuracy. Thus, we verified that this technology is suitable for the proposed solution.

Demand Response Contextual Remuneration of Prosumers with Distributed Storage.

Prosumers are emerging in the power and energy market to provide load flexibility to smooth the use of distributed generation. The volatile behavior increases the production prediction complexity, and the demand side must take a step forward to participate in demand response events triggered by a community manager. If balance is achieved, the participants should be compensated for the discomfort caused. The authors in this paper propose a methodology to optimally manage a community, with a focus on the remuneration of community members for the provided flexibility. Four approaches were compared and evaluated, considering contextual tariffs. The obtained results show that it was possible to improve the fairness of the remuneration, which is an incentive and compensation for the loss of comfort. The single fair remuneration approach was more beneficial to the community manager, since the total remuneration was lower than the remaining approaches (163.81 m.u. in case study 3). From the prosumers' side, considering a clustering method was more advantageous, since higher remuneration was distributed for the flexibility provided (196.27 m.u. in case study 3).

A New Exergy Disaggregation Approach for Complexity Reduction and Dissipative Equipment Isolation in Thermoeconomics.

Thermoeconomics connects thermodynamic and economic concepts in order to provide information not available in conventional energy and economic analysis. Most thermoeconomicists agree that exergy is the most appropriate thermodynamic magnitude to associate with cost. In some applications, exergy disaggregation is required. Despite the improvement in result accuracy, the modeling complexity increases. In recent years, different exergy disaggregation approaches have been proposed, mostly to deal with dissipative components and residues, despite all of them also increasing the complexity of thermoeconomics. This study aims to present a new thermoeconomic approach based on exergy disaggregation, which is able to isolate dissipative components with less modeling complexity. This approach, called the A&F Model, splits the physical exergy into two terms, namely, Helmholtz energy and flow work. These terms were evaluated from a thermoeconomic point of view, through a cost allocation in an ideal Carnot cycle, and they were also applied and compared with the UFS Model, through a cost allocation analysis, in a case study with an organic Rankine cycle-powered vapor compression refrigeration system. The complexity and computational effort reduction in the A&F are significantly less than in the UFS Model. This alternative approach yields consistent results.

Towards circular economy in the textiles and clothing value chain through blockchain technology and IoT: A review.

The textile and clothing industry sector has today a big environmental impact, not only due to the consumption of water and the use of toxic chemicals but also due to the increasing levels of textile waste. One way to reduce the problem is to circularise the, currently linear, textile and clothing value chain, by using discarded clothes as raw material for the production of new clothes, transforming it into a model of circular economy. This way, while reducing the need to produce new raw materials (e.g. cotton), the problem of textile waste produced is also reduced, thus contributing to a more sustainable industry. In this article, we review the current approaches for traceability in the textile and clothing value chain, and study a set of technologies we deem essential for promoting the circular economy in this value chain - namely, the blockchain technology - for registering activities on traceable items through the value chain, and the Internet of Things (IoT) technology, for easily identifying the traceable items' digital twins.

Use of Sensors and Analyzers Data for Load Forecasting: A Two Stage Approach.

The increase in sensors in buildings and home automation bring potential information to improve buildings' energy management. One promissory field is load forecasting, where the inclusion of other sensors' data in addition to load consumption may improve the forecasting results. However, an adequate selection of sensor parameters to use as input to the load forecasting should be done. In this paper, a methodology is proposed that includes a two-stage approach to improve the use of sensor data for a specific building. As an innovation, in the first stage, the relevant sensor data is selected for each specific building, while in the second stage, the load forecast is updated according to the actual forecast error. When a certain error is reached, the forecasting algorithm (Artificial Neural Network or K-Nearest Neighbors) is trained with the most recent data instead of training the algorithm every time. Data collection is provided by a prototype of agent-based sensors developed by the authors in order to support the proposed methodology. In this case study, data over a period of six months with five-minute time intervals regarding eight types of sensors are used. These data have been adapted from an office building to illustrate the advantages of the proposed methodology.

Consumption Optimization in an Office Building Considering Flexible Loads and User Comfort.

This paper presents a multiperiod optimization algorithm that is implemented in a Supervisory Control and Data Acquisition system. The algorithm controls lights and air conditioners as flexible loads to reduce the consumption and controls a dishwasher as a deferrable load to implement the load shifting. Several parameters are considered to implement the algorithm for several successive periods in a real building operation. In the proposed methodology, optimization is done regarding user comfort, which is modeled in the objective function related to the indoor temperature in each room, and in the constraints in order to prevent excessive power reduction, according to users' preferences. Additionally, the operation cycle of a dishwasher is included, and the algorithm selects the best starting point based on the appliance weights and power availability in each period. With the proposed methodology, the building energy manager can specify the moments when the optimization is run with consideration of the operational constraints. Accordingly, the main contribution of the paper is to provide and integrate a methodology to minimize the difference between the actual and the desired temperature in each room, as a measure of comfort, respecting constraints that can be easily bounded by building users and manager. The case study considers the real consumption data of an office building which contains 20 lights, 10 ACs, and one dishwasher. Three scenarios have been designed to focus on different functionalities. The outcomes of the paper include proof of the performance of the optimization algorithm and how such a system can effectively minimize electricity consumption by implementing demand response programs and using them in smart grid contexts.

Shape responses in a macaque frontal area connected to posterior parietal cortex.

The primate dorsal visual stream processes object shape to guide actions involving an object, but the transmission of shape information beyond posterior parietal cortex remains largely unknown. To clarify the information flow between parietal and frontal cortex, we applied electrical microstimulation during functional Magnetic Resonance Imaging (fMRI) in a shape-selective patch in the posterior part of the Anterior Intraparietal area (pAIP) to chart its connectivity. Subsequently, we recorded single-unit responses to images of objects in the fMRI activation in prefrontal cortex, corresponding to area 45B, elicited by pAIP microstimulation. Neurons in area 45B had properties similar to neurons in pAIP, responding selectively to shape contours and to very small shape fragments measuring less than one deg at exceedingly short latencies. However, contrary to the prevailing view on the hierarchical organization of cortical areas, neurons in area 45B preferred even smaller shape fragments and had smaller receptive fields than neurons in pAIP. These findings provide the first evidence for ultra-fast shape processing in prefrontal cortex, and suggest that the pathway from pAIP to area 45B may not be important for object grasping.

WAGNER syndrome: anatomic, functional and genetic characterization of a Portuguese family.

PURPOSE: To report the clinical (anatomic and functional) and genetic findings of Wagner Syndrome (WS) in a Portuguese family. METHODS: Nine members of the family agreed to be examined. All had complete clinical eye examinations. The proband and selected patients underwent color fundus photography, spectral domain optical coherence tomography (SD-OCT), automatic static white-on-white computerized perimetry, and electrophysiology assessment (flash ERG, multifocal(mf) ERG and dark adaptometry). A pedigree was constructed based on interviews with known affected subjects. Genomic DNA samples derived from venous blood were collected from all affected family members examined. RESULTS: Twenty-eight family members are affected. This family has the typical features of Wagner Syndrome, namely an empty vitreous cavity with veils, mild myopia and cataract. Four examined patients underwent vitreoretinal surgery due to abnormal peripheral vitreoretinal adhesions with peripheral retinal traction (n = 3). Retinal detachment was observed in 5 of the examined subjects. Four of them occurred between the ages of 5 and 15 years. Chorioretinal atrophy is also a frequent finding which results in moderate to severe visual field and advanced rod-cone dystrophy from younger ages, also confirmed by absence of scotopic function on dark adaptation. The macular dysfunction on mfERG was profound and of early onset. A heterozygous mutation in intron 7 of the VCAN gene (c.4004-1G > A) was found. CONCLUSIONS: We described a rare autosomal dominant vitreoretinopathy with near complete penetrance in a Portuguese family. Abnormal peripheral vitreoretinal adhesions, retinal detachment and chorioretinal atrophy are present in most of the examined individuals at young ages. Early onset of advanced visual field and electrophysiologic abnormalities were observed in this family. We also added relevant information to the literature by reporting our experience in surgical management of Wagner Syndrome patients with, and at risk of, retinal detachment.

A Regression Model for Predicting Shape Deformation after Breast Conserving Surgery.

Breast cancer treatments can have a negative impact on breast aesthetics, in case when surgery is intended to intersect tumor. For many years mastectomy was the only surgical option, but more recently breast conserving surgery (BCS) has been promoted as a liable alternative to treat cancer while preserving most part of the breast. However, there is still a significant number of BCS intervened patients who are unpleasant with the result of the treatment, which leads to self-image issues and emotional overloads. Surgeons recognize the value of a tool to predict the breast shape after BCS to facilitate surgeon/patient communication and allow more educated decisions; however, no such tool is available that is suited for clinical usage. These tools could serve as a way of visually sensing the aesthetic consequences of the treatment. In this research, it is intended to propose a methodology for predict the deformation after BCS by using machine learning techniques. Nonetheless, there is no appropriate dataset containing breast data before and after surgery in order to train a learning model. Therefore, an in-house semi-synthetic dataset is proposed to fulfill the requirement of this research. Using the proposed dataset, several learning methodologies were investigated, and promising outcomes are obtained.

Frailty Syndrome in Association with Depressive Symptoms and Functional Disability among Hospitalized Elderly.

We sought to examine the frailty association with depression and functional disability in hospitalized older adults. In particular, we compared non-frail, pre-frail, and frail elderly hospitalized individuals. We performed a cross-sectional study with 255 hospitalized Brazilian elderly patients. We used a structured instrument to assess socio-economic data, the Fried frailty phenotype and used morbidity scales (Geriatric Depression; Katz; Lawton and Brody). The adjusted analysis revealed that frail elderly exhibit increased odds ratios (OR) for depressive symptoms (OR = 2.72, 95% CI: 1.12-6.62), disability related to basic activities (OR = 3.50, 95% CI: 1.26-9.60), and instrumental daily living (OR = 2.70, 95% CI: 1.12-6.44). Frailty in hospitalized older adults is associated with depressive symptomatology and functional disability.

Possible role for fundus autofluorescence as a predictive factor for visual acuity recovery after epiretinal membrane surgery.

PURPOSE: To study the potential association between fundus autofluorescence, spectral-domain optical coherence tomography, and visual acuity in patients undergoing surgery because of epiretinal membranes. METHODS: Prospective, interventional case series including 26 patients submitted to vitrectomy because of symptomatic epiretinal membranes. Preoperative evaluation consisted of a complete ophthalmologic examination, autofluorescence, and spectral-domain optical coherence tomography. Studied variables included foveal autofluorescence (fov.AF), photoreceptor inner segment/outer segment (IS/OS) junction line integrity, external limiting membrane integrity, central foveal thickness, and foveal morphology. All examinations were repeated at the first, third, and sixth postoperative months. The main outcome measures were logarithm of minimal angle resolution visual acuity, fov.AF integrity, and IS/OS integrity. RESULTS: All cases showing a continuous IS/OS line had an intact fov.AF, whereas patients with IS/OS disruption could have either an increased area of foveal hypoautofluorescence or an intact fov.AF, with the latter being associated with IS/OS integrity recovery in follow-up spectral-domain optical coherence tomography imaging. The only preoperative variables presenting a significant correlation with final visual acuity were baseline visual acuity (P = 0.047) and fov.AF grade (P = 0.023). CONCLUSION: Recovery of IS/OS line integrity after surgery, in patients with preoperative IS/OS disruption and normal fov.AF, can be explained by the presence of a functional retinal pigment epithelium-photoreceptor complex, supporting normal photoreceptor activity. Autofluorescence imaging provides a functional component to the study of epiretinal membranes, complementing the structural information obtained with optical coherence tomography.

Ocular severe involvement in oculofaciocardiodental syndrome: Description of a case series.

BACKGROUND: Oculofaciocardiodental (OFCD) syndrome is a rare genetic disorder affecting ocular, facial, dental, and cardiac systems, being an X-linked condition caused by pathogenic variants in the BCL-6 corepressor gene (BCOR). We report a case series of three female patients with OFCD syndrome with severe glaucoma. RESULTS: Three female patients with OFCD syndrome with different variants involving BCOR gene, in heterozygosity: a seven-years-old girl with an insertion (c.2037_2038dupCT), a nine years-old girl with a microdeletion in the X (p21.2-p11.4)) spanning the BCOR gene; and a 25 years-old female with a deletion (c.3858_3859del). Systemic involvement is variable among patients ranging from one patient mainly with ocular and dental involvement to one with associated intra-auricular and intra-ventricular defects. All the patients presented with congenital cataracts diagnosed in the first days of life. Cataract surgery was performed without incidents between 6 and 16 weeks of age in all the patients. Postoperatively, the three patients developed ocular hypertension and glaucoma with the need for surgical interventions, including trabeculectomy, Ahmed valve implantation, and cyclophotocoagulation. CONCLUSION: OFCD syndrome characterizes by a severe ocular involvement with glaucoma as a characteristic feature. Ocular hypertension after cataract surgery in these patients is challenging, almost always needing surgery during childhood. Therefore, we consider BCOR disruption may predispose to a higher incidence of glaucoma due to its aggressiveness and early onset on our case series. The awareness of these complications is crucial to an adequate follow-up of the patients.

Optical coherence tomography angiography based prognostic factors and visual outcomes in primary rhegmatogenous retinal detachment after pars plana vitrectomy.

PURPOSE: To evaluate the visual outcomes and changes in the retinal microcirculation in patients with primary rhegmatogenous retinal detachment (RRD) following successful pars plana vitrectomy (PPV). METHODS: Nine macula-on RRD and 23 macula-off RRD eyes were retrospectively evaluated. Clinical data was collected at admission and 3 months after PPV. Optical coherence tomography angiography (OCTA) was performed 3 months after PPV. Superficial vascular plexus data obtained with OCTA was compared between affected and fellow eyes and according to macular involvement. Quantitative measurements of the superficial retinal capillary associated with the preoperative and intraoperative factors were analyzed. RESULTS: Overall RRD inner vessel densities (IVD), full vessel densities (FVD), inner perfusion densities (IPD) and full perfusion densities (FPD) were significantly and positively correlated with best corrected visual acuity (BCVA)(p = 0.002, p = 0.006, p = 0.009, p = 0.023, respectively). In the macula-off RRD, IVD and FVD were significantly decreased compared with macula-on RRD (p = 0.014 and p = 0.034, respectively) and significantly correlated with a longer duration and larger extension of detachment. Higher differences of IVD and FVD between the fellow and affected eyes were significantly correlated with worse BCVA in the macula-off subgroup. CONCLUSION: Macula-off RRD presented worse OCTA superficial vascular parameters compared with the macula-on group and fellow eyes, which were correlated with a poorer visual outcome and exacerbated by a longer duration and larger extension of the detachment. Macula-off RRD causes not only retinal structural damage but also a reduction in retinal perfusion despite successful anatomical repair.

Fecal microbiota transplantation and its repercussions in patients with melanoma refractory to anti-PD-1 therapy: scope review.

INTRODUCTION: despite being extremely effective in some cases, up to 70% of patients with melanoma do not respond to anti-PD-1/PD-L1 (primary resistance) and many of the responders eventually progress (secondary resistance). Extensive efforts are being made to overcome this resistance through new strategies, especially aimed at modulating the intestinal microbiota. OBJECTIVE: to assess whether fecal microbiota transplantation (FMT), associated with immunotherapy, is beneficial in the clinical course of patients with refractory melanoma. METHODS: this is a scope review, based on studies collected on the MEDLINE, ScienceDirect, The Cochrane Library, Embase and BMJ Journals; using the terms: "Antibodies, Monoclonal"; "Drug Resistance, Neoplasm"; "Fecal Microbiota Transplantation"; "Host Microbial Interactions"; "Immunotherapy"; "Melanoma"; and "Microbiota". Clinical trials, in English, with relevant data on the subject and fully available were included. A cut-off period was not determined, due to the limited amount of evidence on the topic. RESULTS: crossing the descriptors allowed the identification of 342 publications and, after applying the eligibility criteria, allowed the selection of 4 studies. From the analyses, it was observed that a considerable part of those studied overcame resistance to immune checkpoint inhibitors after FMT, with better response to treatment, less tumor growth and increased beneficial immune response. CONCLUSION: it is noted that FMT favors the response of melanoma to immunotherapy, translated into significant clinical benefit. However, further studies are necessary for the complete elucidation of the bacteria and the mechanisms involved, as well as for the translation of new evidence to oncological care practice.

Intravitreal Fluocinolone Acetonide Implant (FAc, 0.19 mg, ILUVIEN®) in the Treatment of Patients with Recurrent Cystoid Macular Edema After Pars Plana Vitrectomy.

INTRODUCTION: Postoperative cystoid macular edema (PCME) is a complication of several ocular procedures, including pars plana vitrectomy (PPV), due to the activation of the inflammatory cascade. The purpose of this case series is to evaluate the effectiveness and safety of fluocinolone acetonide intravitreal implant (FAc, 0.2 µg/day; ILUVIEN®) in the treatment of refractory PCME after successful PPV. METHODS: This retrospective observational case series includes consecutive eyes of patients with recurrent PCME after PPV and treated with a single FAc implant at Centro Hospitalar Universitário de São João, Porto, Portugal. Previous treatments, best-corrected visual acuity (BCVA, ETDRS letters), central macular thickness (CMT, µm), intraocular pressure (IOP, mmHg), and IOP-lowering medication needed were recorded at baseline and during follow-up. Total macular edema resolution was defined as CMT less than 300 µm or a reduction of greater than 20%, and partial macular edema resolution was defined as a reduction of greater than 10%. RESULTS: Nine eyes from nine patients were included. Before FAc implant, all eyes received intravitreal short-action corticosteroids (triamcinolone and dexamethasone implant), with a good response but relapse 1-5 months later. At baseline, BCVA was 55.0 ± 10.6 letters, CMT was 514.9 ± 165.6 µm, and IOP was 15.4 ± 2.4 mmHg with four eyes under IOP-lowering medication. After FAc implant, all eyes achieved edema resolution (eight total and one partial) with a peak gain of 17.2 letters and a maximum decrease of 208.2 µm in CMT. During follow-up (44.0 ± 14.8 months), 66.7% of the eyes kept their macula dry and three showed recurrence after 11, 14, and 28 months, respectively. The maximum IOP registered was 17.0 ± 6.0 mmHg. IOP-lowering regimen was increased in one eye and two additional eyes started hypotensive drops. CONCLUSION: FAc implant can be considered a therapeutic alternative in PCME refractory to other therapies in vitrectomized eyes, reducing the need for repeated treatments.

Terson Syndrome - Clinical Presentation, Management, and Visual Outcomes in a Tertiary Centre.

Purpose: The purpose of this study was to characterize the clinical presentation, management strategy and visual outcomes of patients diagnosed with Terson syndrome and followed in a tertiary centre in Portugal. Patients and Methods: A single-centre retrospective study was performed, based on the survey review of the medical records of every consecutive patient diagnosed with Terson syndrome and followed from January 2018 to August 2021. The change in best-corrected visual acuity (BCVA) from baseline to the final evaluation was the primary outcome. Results: Fifteen eyes from 8 patients (50% female) were included. The mean age at diagnosis was 55±7 years. The neurological event was traumatic brain injury in 37.5% (n=3) and subarachnoid haemorrhage in 62.5% of the patients (n=5). Bilateral intraocular haemorrhage occurred in 875% (n=7) of the patients. Vitreous and preretinal haemorrhages occurred each in 66.7% (n=10), intraretinal in 30% (n=3) and subretinal in 13.3% (n=2) of the eyes. In 40% of the eyes (n=6), spontaneous resolution of intraocular haemorrhage occurred, while PPV was performed in the remaining 60% (n=9). Ocular haemorrhage detection occurred 58.47 ± 40.94 days after the neurological event (range 11 to 121 days). Baseline BCVA was 1.11 ± 1.01 logMAR and improved to 0.32 ± 0.69 logMAR in the follow-up period (p=0.004). A positive correlation was found between initial and final BCVA (Spearman's rho = 0.643, p=0.01). Baseline BCVA of eyes undergoing PPV was lower than of those conservatively managed (1.84±0.72 vs 0.20±0.28 logMAR, p<0.001). However, there were no statistically significant differences in final BCVA after surgery or observation (0.56 ± 0.90 vs 0.04 ± 0.04 logMAR, p=0.149). Longer periods between the neurological and the ophthalmological diagnosis were correlated with worse final BCVA (Spearman's rho = 0.688, p=0.005). Conclusion: Terson syndrome is a potential cause of irreversible visual loss. Diagnosis delay may affect visual prognosis. PPV is indicated when intraocular haemorrhage is dense and does not resolve spontaneously or when visual acuity at presentation is low, allowing for good visual outcomes with minimal complications.

Dataset of an energy community's generation and consumption with appliance allocation.

Energy data measured on-site from buildings can help describe the consumption behavior of end-users and can be used to examine and prove certain theorems and models, that require a large volume of data to be gathered. However, the direct extraction of this data can often be a lengthily and costly process. As a result, a dataset of a residential community was constructed based on real data, where sample consumption and photovoltaic generation profiles were attributed to 50 residential households and a public building (municipal library), a total of 51 buildings. In addition, the overall power consumption of these houses was desegregated into the consumption of 10 commonly used appliances using real energy profiles. First, several consumption and photovoltaic generation profiles, as well as a vast collection of appliance profiles, were gathered. These profiles were obtained from household readings in different locations, while the public building's profile was based on the consumption and photovoltaic production profiles of the research building GECAD. The profiles went through the process of normalization and new profiles were generated to complete the number of end-users needed. Moreover, these profiles were given a maximum consumption and production level at random before being accepted by one of the end-users. Therefore, fourteen of these households and the public building were randomly attributed with renewable solar energy. Finally, if possible, the tool created allocated, at random in previously determined intervals, the appliances' load profiles into each end-user's available consumption areas.

Canine rabies in Belford Roxo City, Rio de Janeiro, Brazil: a case report.

Rabies is a highly lethal disease and is considered one of the most important zoonoses worldwide. In Brazil, the rabies cycle in domestic animals is under control. However, there is feedback from the wildlife cycle as many bats are present in the cities and rural areas of the country. This paper reports a case of a dog receiving veterinary assistance that presented with misbehavior and other unusual signs. After clinical evaluation by two veterinarians and a period of hospitalization to perform tests and treatment, the dog died, and the Municipal Health Department was contacted due to the suspicion of rabies. After laboratory testing and sample analysis, the diagnosis of rabies was confirmed. This case demonstrates the importance of veterinarians' qualifications in performing clinical and laboratory diagnoses, such as their knowledge concerning surveillance measures and preventive steps before and after exposure. Reinforcing the significance of maintaining vaccination coverage for rabies and promoting public and private vaccination campaigns in areas that lack vaccination campaigns are useful.

A Raiva é uma doença de elevada letalidade e é considerada uma das mais importantes zoonoses do planeta. Seu ciclo urbano está praticamente controlado no Brasil, porém, é constantemente retroalimentado pelo ciclo silvestre devido à presença de morcegos tanto nas áreas rurais como urbanas. Este trabalho relata o caso de um cão que foi levado para atendimento veterinário apresentando comportamento e outros sinais incomuns. Após a avaliação de dois profissionais veterinários e uma internação para exames e tratamento, o animal foi a óbito e o serviço municipal de saúde foi acionado devido à suspeita de Raiva. Após os devidos exames laboratoriais, a Raiva foi confirmada nas amostras analisadas. Este caso demostrou a importância não só da capacitação dos profissionais veterinários para suspeitar, realizar o diagnóstico clínico e laboratorial da Raiva como o conhecimento acerca das medidas de vigilância, prevenção e controle e os esquemas de profilaxia da raiva humana pré e pós exposição. Ressalta-se ainda a importância da manutenção da cobertura vacinal contra a Raiva, seja ela realizada por campanhas públicas de vacinação, seja por iniciativa dos tutores onde as campanhas não mais ocorrem.

3'Nucleotidase/nuclease is required for Leishmania infantum clinical isolate susceptibility to miltefosine.

BACKGROUND: Miltefosine treatment failure in visceral leishmaniasis in Brazil has been associated with deletion of the miltefosine susceptibility locus (MSL) in Leishmania infantum. The MSL comprises four genes, 3'-nucleotidase/nucleases (NUC1 and NUC2); helicase-like protein (HLP); and 3,2-trans-enoyl-CoA isomerase (TEI). METHODS: In this study CRISPR-Cas9 was used to either epitope tag or delete NUC1, NUC2, HLP and TEI, to investigate their role in miltefosine resistance mechanisms. Additionally, miltefosine transporter genes and miltefosine-mediated reactive oxygen species homeostasis were assessed in 26 L. infantum clinical isolates. A comparative lipidomic analysis was also performed to investigate the molecular basis of miltefosine resistance. FINDINGS: Deletion of both NUC1, NUC2 from the MSL was associated with a significant decrease in miltefosine susceptibility, which was restored after re-expression. Metabolomic analysis of parasites lacking the MSL or NUC1 and NUC2 identified an increase in the parasite lipid content, including ergosterol; these lipids may contribute to miltefosine resistance by binding the drug in the membrane. Parasites lacking the MSL are more resistant to lipid metabolism perturbation caused by miltefosine and NUC1 and NUC2 are involved in this pathway. Additionally, L. infantum parasites lacking the MSL isolated from patients who relapsed after miltefosine treatment were found to modulate nitric oxide accumulation in host macrophages. INTERPRETATION: Altogether, these data indicate that multifactorial mechanisms are involved in natural resistance to miltefosine in L. infantum and that the absence of the 3'nucleotidase/nuclease genes NUC1 and NUC2 contributes to the phenotype. FUNDING: MRC GCRF and FAPES.