Human intestinal enteroids platform to assess the infectivity of gastroenteritis viruses in wastewater.

Fecal-orally transmitted gastroenteritis viruses, particularly human noroviruses (HuNoVs), are a public health concern. Viral transmission risk through contaminated water results underexplored as they have remained largely unculturable until recently and the robust measuring of gastroenteritis viruses infectivity in a single cell line is challenging. This study primarily aimed to test the feasibility of the human intestinal enteroids (HIE) model to demonstrate the infectivity of multiple gastroenteritis viruses in wastewater. Initially, key factors affecting viral replication in HIE model were assessed, and results demonstrated that the reagent-assisted disruption of 3D HIE represents an efficient alternative to syringe pass-through, and the filtering of HuNoV stool suspensions could be avoided. Moreover, comparable replication yields of clinical strains of HuNoV genogroup I (GI), HuNoV GII, rotavirus (RV), astrovirus (HAstV), and adenoviruses (HAdV) were obtained in single and multiple co-infections. Then, the optimized HIE model was used to demonstrate the infectivity of multiple naturally occurring gastroenteritis viruses from wastewater. Thus, a total of 28 wastewater samples were subjected to (RT)-qPCR for each virus, with subsequent testing on HIE. Among these, 16 samples (57 %) showed replication of HuNoVs (n = 3), RV (n = 5), HAstV (n = 8), and/or HAdV (n = 5). Three samples showed HuNoV replication, and sequences assigned to HuNoV GI.3[P13] and HuNoV GII.4[P16] genotypes. Concurrent replication of multiple gastroenteritis viruses occurred in 4 wastewater samples. By comparing wastewater concentrate and HIE supernatant sequences, diverse HAstV and HAdV genotypes were identified in 4 samples. In summary, we successfully employed HIE to demonstrate the presence of multiple infectious human gastroenteritis viruses, including HuNoV, in naturally contaminated wastewater samples.

Culture of Human Rotaviruses in Relevant Models Shows Differences in Culture-Adapted and Nonculture-Adapted Strains.

Rotavirus (RV) is the leading cause of acute gastroenteritis (AGE) in children under 5 years old worldwide, and several studies have demonstrated that histo-blood group antigens (HBGAs) play a role in its infection process. In the present study, human stool filtrates from patients diagnosed with RV diarrhea (genotyped as P[8]) were used to infect differentiated Caco-2 cells (dCaco-2) to determine whether such viral strains of clinical origin had the ability to replicate in cell cultures displaying HBGAs. The cell culture-adapted human RV Wa model strain (P[8] genotype) was used as a control. A time-course analysis of infection was conducted in dCaco-2 at 1, 24, 48, 72, and 96 h. The replication of two selected clinical isolates and Wa was further assayed in MA104, undifferentiated Caco-2 (uCaco-2), HT29, and HT29-M6 cells, as well as in monolayers of differentiated human intestinal enteroids (HIEs). The results showed that the culture-adapted Wa strain replicated more efficiently in MA104 cells than other utilized cell types. In contrast, clinical virus isolates replicated more efficiently in dCaco-2 cells and HIEs. Furthermore, through surface plasmon resonance analysis of the interaction between the RV spike protein (VP8*) and its glycan receptor (the H antigen), the V7 RV clinical isolate showed 45 times better affinity compared to VP8* from the Wa strain. These findings support the hypothesis that the differences in virus tropism between clinical virus isolates and RV Wa could be a consequence of the different HBGA contents on the surface of the cell lines employed. dCaco-2, HT29, and HT29M6 cells and HIEs display HBGAs on their surfaces, whereas MA104 and uCaco-2 cells do not. These results indicate the relevance of using non-cell culture-adapted human RV to investigate the replication of rotavirus in relevant infection models.

Recombinant Noroviruses Circulating in Spain from 2016 to 2020 and Proposal of Two Novel Genotypes within Genogroup I.

Noroviruses are the leading cause of sporadic cases and outbreaks of viral gastroenteritis. For more than 20 years, most norovirus infections have been caused by the pandemic genotype GII.4, yet recent studies have reported the emergence of recombinant strains in many countries. In the present study, 4,950 stool samples collected between January 2016 and April 2020 in Valencia, Spain, from patients with acute gastroenteritis were analyzed to investigate the etiological agent. Norovirus was the most frequently detected enteric virus, with a positivity rate of 9.5% (471/4,950). Among 224 norovirus strains characterized, 175 belonged to genogroup II (GII) and 49 belonged to GI. Using dual genotyping based on sequencing of the open reading frame 1 (ORF1)/ORF2 junction region, we detected 25 different capsid-polymerase-type associations. The most common GII capsid genotype was GII.4 Sydney 2012, followed by GII.2, GII.3, GII.6, and GII.17. A high prevalence of recombinant strains (90.4%) was observed among GII infections between 2018 and 2020. GII.4 Sydney[P16] was the predominant genotype from 2019 to 2020. In addition, GII.P16 polymerase was found harbored within six different capsid genes. GI.4 and GI.3 were the predominant genotypes in genogroup I, in which recombinant strains were also found, such as GI.3[P10], GI.3[P13], and GI.5[P4]. Interestingly, applying the criterion of 2 times the standard deviation, we found that 12 sequences initially classified as GI.3 may represent two new tentative genotypes in genogroup I, designated GI.10 and GI.11. This study shows the extensive diversity of recombinant noroviruses circulating in Spain and highlights the role of recombination events in the spread of noroviruses. IMPORTANCE Human noroviruses are the most common cause of viral diarrhea. There are no approved vaccines to prevent their infections yet, which would be very useful to protect infants, small children, and the elderly in residential institutions. These viruses are extremely contagious and can be transmitted by contaminated food and water as well as directly from person to person. Molecular surveillance and epidemiology of norovirus infections allow the identification of the most common viral strains in different geographical areas over time. Noroviruses show wide genetic variability due to a high rate of mutations but also due to genomic recombinations, as we demonstrate in this study. We have detected 25 different viral capsid-polymerase gene associations among 224 norovirus strains characterized in Spain between January 2016 and April 2020, including two tentative new capsid genotypes in genogroup I.

Evaluation of SARS-CoV-2 rapid antigen diagnostic tests for saliva samples.

Using saliva samples would facilitate sample collection, diagnostic feasibility, and mass screening of SARS-CoV-2. We tested two rapid antigen (RAD) immunochromatographic tests designed for detection of SARS-CoV-2 in saliva: Rapid Response™ COVID-19 Antigen Rapid Test Cassette for oral fluids and DIAGNOS™ COVID-19 Antigen Saliva Test. Evaluation of detection limit was performed with purified SARS-CoV-2 nucleocapsid protein and live SARS-CoV-2 virus. Sensitivity and specificity were further evaluated with reverse transcription quantitative PCR (RT-qPCR) positive and negative saliva samples from hospitalized individuals with COVID-19 (n = 39) and healthcare workers (n = 20). DIAGNOS showed higher sensitivity than Rapid Response for both nucleocapsid protein and live virus. The limit of detection of the saliva test from DIAGNOS was further comparable with the Abbott Panbio™ COVID-19 Ag Rapid Test designed for nasopharyngeal samples. DIAGNOS and Rapid Response detected nine (50.0%) and seven (38.9%), respectively, of the 18 RT-qPCR positive saliva samples. All RT-qPCR negative saliva (n = 41) were negative with both tests. Only one of the RT-qPCR positive saliva samples contained infectious virus as determined by cell culture and was also positive using the saliva RADs. The results show that the DIAGNOS may be an important and easy-to-use saliva RAD complement to detect SARS-CoV-2 positive individuals, but validation with a larger sample set is warranted.

Evaluation of intrafraction motion with an open immobilization mask for HyperArc treatment of multiple brain metastases.

Purpose: In the implementation of the use of EncompassTM partially open immobilization mask to perform SRS of multiple brain metastasis, the evaluation of patient's intrafraction motion (IM) is deemed convenient to verify that the margins applied to the GTV are able to ensure adequate dose coverage to each lesion. Methods: IM was determined by comparing the pre- and post-treatment CBCT images with respect to the simulation CT for a total of 23 fractions. The dosimetric impact on GTV coverage due to translational errors in patient positioning and rotational uncertainties of LINAC's performance was also evaluated. Results: The absolute magnitude of IM was less than 1 mm in all cases. The dosimetric difference on GTV coverage due to patient's IM was inferior to 5%. There was not found any significant correlation between the dosimetric impact of rotational uncertainties with the distance to the isocenter. Conclusion: The margins applied to the GTV are adequate when using EncompassTM immobilization device.

Impact of maternal antibodies and microbiota development on the immunogenicity of oral rotavirus vaccine in African, Indian, and European infants.

Identifying risk factors for impaired oral rotavirus vaccine (ORV) efficacy in low-income countries may lead to improvements in vaccine design and delivery. In this prospective cohort study, we measure maternal rotavirus antibodies, environmental enteric dysfunction (EED), and bacterial gut microbiota development among infants receiving two doses of Rotarix in India (n = 307), Malawi (n = 119), and the UK (n = 60), using standardised methods across cohorts. We observe ORV shedding and seroconversion rates to be significantly lower in Malawi and India than the UK. Maternal rotavirus-specific antibodies in serum and breastmilk are negatively correlated with ORV response in India and Malawi, mediated partly by a reduction in ORV shedding. In the UK, ORV shedding is not inhibited despite comparable maternal antibody levels to the other cohorts. In both India and Malawi, increased microbiota diversity is negatively correlated with ORV immunogenicity, suggesting that high early-life microbial exposure may contribute to impaired vaccine efficacy.

Feasibility and potential advantages using VMAT in SRS metastasis treatments.

BACKGROUND: Utilization of stereotactic radiosurgery (SRS) for brain metastases (BM) has become the technique of choice as opposed to whole brain radiation therapy (WBRT). The aim of this work is to evaluate the feasibility and potential benefits in terms of normal tissue (NT) and dose escalation of volumetric modulated arc therapy (VMAT) in SRS metastasis treatment. A VMAT optimization procedure has therefore been developed for internal dose scaling which minimizes planner dependence. MATERIALS AND METHODS: Five patient-plans incorporating treatment with frame-based SRS with dynamic conformal arc technique (DA) were re-planned for VMAT. The lesions selected were between 4-6 cm3. The same geometry used in the DA plans was maintained for the VMAT cases. A VMAT planning procedure was performed attempting to scale the dose in inner auxiliary volumes, and to explore the potential for dose scaling with this technique. Comparison of dose-volume histogram (DVH) parameters were obtained. RESULTS: VMAT allows a superior NT sparing plus conformity and dose scaling using the auxiliary volumes. The VMAT results were significantly superior in NT sparing, improving both the V10 and V12 values in all cases, with a 2-3 cm3 saving. In addition, VMAT improves the dose coverage D95 by about 0.5 Gy. The objective of dose escalation was achieved with VMAT with an increment of the Dmean and the Dmedian of about 2 Gy. CONCLUSIONS: This work shows a benefit of VMAT in SRS treatment with significant NT sparing. A VMAT optimization procedure, based on auxiliary inner volumes, has been developed, enabling internal dose escalation.

Evaluation of SARS-CoV-2 rapid antigen diagnostic tests for saliva samples

BackgroundThe COVID-19 pandemic has highlighted the need for rapid, cost effective and easy-to-use diagnostic tools for SARS-CoV-2 rapid antigen detection (RAD) for use in point of care settings or as self-tests, to limit disease transmission. Using saliva samples would further greatly facilitate sample collection, diagnostic feasibility, and mass screening. ObjectiveWe tested two rapid antigen immunochromatographic tests designed for detection of SARS-CoV-2 in saliva: Rapid Response COVID-19 Antigen Rapid Test Cassette for oral fluids (Rapid Response) and DIAGNOS COVID-19 Antigen Saliva Test (DIAGNOS). Evaluation of detection limit was performed with purified SARS-CoV-2 nucleocapsid protein and titrated live SARS-CoV-2 virus and compared to Abbott Panbio COVID-19 Ag Rapid Test (Panbio) designed for nasopharyngeal samples. Sensitivity and specificity were further evaluated on RT-qPCR positive and negative saliva samples from individuals hospitalized with COVID-19 (n=34); and asymptomatic health care personnel (n=20). ResultsThe limit of detection of the saliva test from DIAGNOS was comparable with the Panbio test and showed higher sensitivity than Rapid Response for both nucleocapsid protein and diluted live viruses. DIAGNOS and Rapid Response further detected seven (47%) and five (33%), respectively, of the 15 RT-qPCR positive saliva samples in individuals hospitalized with COVID-19. Of the 39 RT-qPCR negative samples, all were negative with both tests (specificity 100%; 95% c.i. 0.91-1.00). Only one of the RT-qPCR positive saliva samples (Ct 21.6) contained infectious virus as determined by cell culture and was also positive using the saliva RADs. ConclusionThe results show that the DIAGNOS test exhibit a similar limit of detection as the Panbio RAD and may be an important and easy-to-use saliva RAD complement to detect infectious individuals.

Phantom development for daily checks in electron intraoperative radiotherapy with a mobile linac.

PURPOSE: IORT with mobile linear accelerators is a well-established modality where the dose rate and, therefore, the dose per pulse are very high. The constancy of the dosimetric parameters of the accelerator has to be checked daily. The aim of this work is to develop a phantom with embedded detectors to improve both accuracy and efficiency in the daily test of an IORT linac at the surgery room. METHODS: The developed phantom is manufactured with transparent polymethyl methacrylate (PMMA), allocating 6 parallel-plate chambers: a central one to evaluate the on-axis beam output, another on-axis one placed at a fixed depth under the previous one to evaluate the energy constancy and four off-axis chambers to evaluate the flatness and symmetry. To analyse the readings a specific application has been developed. RESULTS: For all chambers and energies, the mean saturation and polarization corrections were smaller than 0.7%. The beam is monitored at different levels of the clinical beam. Output, energy constancy and flatness correlate very well with the correspondent values with the complete applicator. During the first six months of clinical use the beam dosimetric parameters showed excellent stability. CONCLUSIONS: A phantom has been developed with embedded parallel plate chambers attached to the upper applicator part of an IORT linac. The phantom allows a very efficient setup reducing the time to check the parameters. It provides complete dosimetric information (output, energy and flatness) with just one shot and using ionization chambers with minimum saturation effect, as this highly pulsed beam requires.

Inter-observer and intra-observer variability in reporting vaginal dose points for cervical cancer in high-dose-rate brachytherapy.

PURPOSE: The ICRU 89 recommends reporting a set of vaginal dose points for cervical cancer treatments in order to quantify the goodness of implant. This vaginal dose reporting method for combined external beam radiotherapy and brachytherapy has been adopted by the EMBRACE II study protocol. Large variations in dose between patients and centers have been reported. The aim of this study was to determine possible discrepancies with consensus observers from the same institution. Therefore, the inter- and intra-observer variability were analyzed. MATERIAL AND METHODS: For five patients, five experienced observers reported dose at the proposed vaginal points twice. The effect of inter- and intra-observer variations on total dose was analyzed by estimating biologically equivalent dose EQD2 (α/ß = 3 Gy). Coefficient of variation (CV) was used to provide a measure of data dispersion as a proportion to the mean. RESULTS: The maximum inter-observer deviation among all patients and all points ranged from 0.5 Gy to 24.1 Gy in EQD2. The higher inter-observer discrepancies were found at points at 3 o'clock and at 6 o'clock, with respect to ovoids. In case of the maximum intra-observer deviation, it ranged from 0.5 Gy to 14.2 Gy, with higher deviation points at 12 o'clock and 9 o'clock, with respect to ovoids. CONCLUSIONS: There is a need to ensure consistency in vaginal points reporting. The impact of the dosimetric inter- and intra-observer variability should also be considered when dealing with dose tolerances and limits due to the potential dose gradient.

Rotavirus symptomatic infection among unvaccinated and vaccinated children in Valencia, Spain.

BACKGROUND: Human group A rotavirus is the leading cause of severe acute gastroenteritis in young children worldwide. Immunization programs have reduced the disease burden in many countries. Vaccination coverage in the Autonomous Region of Valencia, Spain, is around 40%, as the rotavirus vaccine is not funded by the National Health System. Despite this low-medium vaccine coverage, rotavirus vaccination has substantially reduced hospitalizations due to rotavirus infection and hospital-related costs. However, there are very few studies evaluating symptomatic rotavirus infections not requiring hospitalization in vaccinated children. The objective of this study was to investigate symptomatic rotavirus infections among vaccinated children in the health area served by the Hospital Clínico Universitario of Valencia, Spain, from 2013 to 2015. METHODS: A total of 133 children younger than 5 years of age with rotavirus infection were studied. Demographic and epidemiological data were collected and informed consent from their caretakers obtained. Rotavirus infection was detected by immunological methods and G/P rotavirus genotypes were determined by RT-PCR, following standard procedures from the EuroRotaNet network. RESULTS: Forty infants (30.1%; 95% CI: 22.3-37.9) out of 133 were diagnosed with symptomatic rotavirus infection despite having been previously vaccinated, either with RotaTeq (85%) or with Rotarix (15%). Children fully vaccinated against rotavirus (24.8%), partially vaccinated (5.3%) and unvaccinated (69.9%) were found. The infecting genotypes showed high G-type diversity, although no significant differences were found between the G/P genotypes infecting vaccinated and unvaccinated children during the same time period. G9P[8], G12P[8] and G1P[8] were the most prevalent genotypes. Severity of gastroenteritis symptoms required 28 (66.6%) vaccinated and 67 (73.6%) unvaccinated children to be attended at the Emergency Room. CONCLUSION: Rotavirus vaccine efficacy in reducing the incidence of severe rotavirus infection has been well documented, but symptomatic rotavirus infection can sometimes occur in vaccinees.

Histo-Blood Group Antigens in Children with Symptomatic Rotavirus Infection.

Group A rotaviruses are a major cause of acute gastroenteritis in children. The diversity and unequal geographical prevalence of rotavirus genotypes have been linked to histo-blood group antigens (HBGAs) in different human populations. In order to evaluate the role of HBGAs in rotavirus infections in our population, secretor status (FUT2+), ABO blood group, and Lewis antigens were determined in children attended for rotavirus gastroenteritis in Valencia, Spain. During three consecutive years (2013-2015), stool and saliva samples were collected from 133 children with rotavirus infection. Infecting viral genotypes and HBGAs were determined in patients and compared to a control group and data from blood donors. Rotavirus G9P[8] was the most prevalent strain (49.6%), followed by G1P[8] (20.3%) and G12P[8] (14.3%). Rotavirus infected predominantly secretor (99%) and Lewis b positive (91.7%) children. Children with blood group A and AB were significantly more prone to rotavirus gastroenteritis than those with blood group O. Our results confirm that a HBGA genetic background is linked to rotavirus P[8] susceptibility. Rotavirus P[8] symptomatic infection is manifestly more frequent in secretor-positive (FUT2+) than in non-secretor individuals, although no differences between rotavirus G genotypes were found.

Endocarcinoma gástrico y embarazo reporte de dos casos y revisión de la literatura / Gastric carcinoma and pregnancy report of two clinical cases and review of the literature / Endocarcinoma gástrica e gravidez relatório de dois casos e revisão da literatura

RESUMEN La incidencia de cáncer durante la gestación es de 1: 1000 a 2000 embarazos, siendo los más frecuentes el de cérvix, mama, ovario, melanoma, tiroides, colon y hematológicos como linfoma y leucemia. A pesar de ser una entidad poco frecuente en el embarazo, se ha visto una tendencia a aumentar en los últimos años debido al incremento progresivo de la edad materna en el momento de la concepción. El carcinoma gástrico es una patología aún menos frecuente, siendo su incidencia durante la gestación de aproximadamente 0.016% a 0.026% en las series de casos de Japón, país con la mayor prevalencia de enfermedad. La cirugía es el tratamiento oncológico menos controversial durante la gestación. En los últimos años, ha cobrado importancia la quimioterapia adyuvante con antineoplásicos de tipo antimetabolito como el 5 fluoracilo (5 - FU), medicamento que ha demostrado mejoría de la supervivencia en pacientes gestantes con adenocarcinoma gástrico. Desafortunadamente, el 96.7% de los casos son diagnosticados en estadios avanzados, pues su presentación clínica suele confundirse con las manifestaciones propias de la primera mitad de la gestación como son las nauseas y el vomito. Presentamos dos casos clínicos, el de una gestante de 34 años con embarazo de 19 semanas y 3 días al momento del diagnóstico de un carcinoma gástrico Bormann III, llevada a laparotomía con intención curativa a las 21 semanas con hallazgo intraoperatorio de enfermedad metastásica avanzada y tumor ovárico izquierdo sugestivo de tumor de Krukenberg, a quien se le inicia a las 23 semanas quimioterapia paliativa con esquema FOLFIRI el cual recibió hasta la finalización de la gestación a las 33 semanas por restricción del crecimiento intrauterino y posteriormente durante el puerperio; y el de una gestante de 31 años con embarazo de 26 semanas y 6 días quien se ingresa para estudio de emesis persistente durante la gestación con confirmación diagnóstica de adenocarcinoma gástrico Bormann III asociado a Restricción del crecimiento intrauterino que fue llevada a cesárea a las 34 semanas y en el puerperio a gastrectomía total con vaciamiento ganglionar del area celiaca y reconstrucción en Y de Roux. El objetivo de realizar estos reportes surge de la carencia de guias de manejo respecto a dicha entidad durante la gestación debido a la poca frecuencia de presentación de la misma. Es frecuente encontrar discrepancias entre los conceptos en cuanto a conductas médicas se refiere. Disponemos de reportes y series de casos que orientan el manejo hacia un enfoque interdisciplinario dirigido a sopesar los riesgos y beneficios derivados de implementar o no un tratamiento durante la gestación siendo pocos los casos exitosos reportados en la literatura.

ABSTRACT Incidence of cancer during pregnancy is 1 in 1000. The most frequent carcinomas are the cervix, breast, ovaries, melanoma, thyroid, colon and hematological such as lymphoma and leukemia. Although cancer is not frequently associated with pregnancy, there has been a tendency to increase in recent years due to the progressive increase in maternal age at conception. Gastric carcinoma during pregnancy is a rare pathology, with an incidence of 0.016% to 0.026% in the series of cases from Japan, one of the countries with the greatest prevalence of this disease. Surgery is the least controversial type of oncologic treatment during pregnancy. In the last years, the adjuvant chemotherapy with antimetabolite drugs like the 5-fluoracile has gained relevance because it has demonstrated to improve survival in pregnant patients with gastric carcinoma. Unfortunately, the 96.7% of cases are diagnosed in advanced states because its symptoms can be misinterpreted as pregnancy induced nausea and vomiting characteristic of first half of pregnancy. We present two Clinical cases. The case of a 34 years old pregnant woman with 19 weeks and 3 days of gestation at the time of diagnosis of a gastric carcinoma in Bormann III stage, she was laparotomized with curative intentions at 21 weeks of gestation with intraoperative findings consistent with advanced metastatic disease and left-sided ovarian tumor suggestive of Krukenberg tumor. At 23 weeks palliative chemotherapy with FOLFIRI was initiated and it was continued until the end of gestation at 33 weeks and later in the puerperium; and the case of a 31 years old pregnant woman with 26 weeks and 6 days of gestation, she was admitted for the study of persistent vomiting during pregnancy with diagnostic confirmation of gastric adenocarcinoma Bormann III associated with intrauterine growth restriction, she was carried cesarean at 34 weeks. In the postpartum she was carried total gastrectomy with lymph node dissection of the celiac area and Roux-Y reconstruction. The objective of this report arises from the absence of clinical randomized studies about the management of this entity during pregnancy in consequence of the few cases available in the literature. This has been a cause of discrepancies between medical staff regarding therapeutics. There are some available literature series and case reports that guide management to a multidisciplinary approach in pursuit of more benefits and less risk in the context of the pregnant patient, with little cases reported as successful.

RESUMO A incidência de câncer durante a gravidez é de 1 em 1000. Os carcinomas mais freqüentes são o colo do útero, mama, ovários, melanoma, tireóide, cólon e hematológicos, como linfoma e leucemia. Embora o câncer não seja freqüentemente associado à gravidez, houve tendência de aumento nos últimos anos devido ao aumento progressivo da idade materna na concepção. O carcinoma gástrico durante a gravidez é uma patologia rara, com incidência de 0,016% a 0,026% na série de casos do Japão, um dos países com maior prevalência desta doença. A cirurgia é o tipo menos controverso de tratamento oncológico durante a gravidez. Nos últimos anos, a quimioterapia adjuvante com fármacos antimetabolitos como a 5-fluoracile ganhou relevância porque demonstrou melhorar a sobrevida em gestantes com carcinoma gástrico. Infelizmente, os 96,7% dos casos são diagnosticados em estados avançados porque seus sintomas podem ser mal interpretados, pois a náusea e os vômitos induzidos pela gravidez são característicos da primeira metade da gravidez. A presentamos dois casos clínicos. O caso de uma mulher grávida de 34 anos com 19 semanas e 3 dias de gestação no momento do diagnóstico de carcinoma gástrico no estádio Bormann III, foi laparotomizada com intenções curativas às 21 semanas de gestação com achados intraoperatórios consistentes com doença metastática avançada e tumor de ovário esquerdo sugestivo de tumor de Krukenberg. Às 23 semanas, iniciou-se a quimioterapia paliativa com FOLFIRI e continuou até o final da gestação às 33 semanas e mais tarde no puerpério; e o caso de uma mulher grávida de 31 anos com 26 semanas e 6 dias de gestação, foi admitida para o estudo de vômitos persistentes durante a gravidez com confirmação diagnóstica de adenocarcinoma gástrico Bormann III associado à restrição de crescimento intra-uterino, foi realizada cesariana às 34 semanas. No pós-parto foi transportada gastrectomia total com dissecção dos linfonodos da área celíaca e reconstrução Roux-Y. O objetivo deste relatório decorre da ausência de estudos clínicos randomizados sobre o manejo dessa entidade durante a gravidez em conseqüência dos poucos casos disponíveis na literatura. Esta foi uma causa de discrepâncias entre a equipe médica em relação à terapêutica. Existem algumas séries de literatura disponíveis e relatos de casos que orientam o gerenciamento para uma abordagem multidisciplinar em busca de mais benefícios e menos risco no contexto da paciente grávida, com pequenos casos relatados como bem sucedidos.

Novel simple templates for reproducible positioning of skin applicators in brachytherapy.

PURPOSE: Esteya and Valencia surface applicators are designed to treat skin tumors using brachytherapy. In clinical practice, in order to avoid errors that may affect the treatment outcome, there are two issues that need to be carefully addressed. First, the selected applicator for the treatment should provide adequate margin for the target, and second, the applicator has to be precisely positioned before each treatment fraction. In this work, we describe the development and use of a new acrylic templates named Template La Fe-ITIC. They have been designed specifically to help the clinical user in the selection of the correct applicator, and to assist the medical staff in reproducing the positioning of the applicator. These templates are freely available upon request. MATERIAL AND METHODS: Templates that were developed by University and Polytechnic Hospital La Fe (La Fe) and Hospital Clínica Benidorm (ITIC) in cooperation with Elekta, consist of a thin sheet made of transparent acrylic. For each applicator, a crosshair and two different circles are drawn on these templates: the inner one corresponds to the useful beam, while the outer one represents the external perimeter of the applicator. The outer circle contains slits that facilitate to draw a circle on the skin of the patient for exact positioning of the applicator. In addition, there are two perpendicular rulers to define the adequate margin. For each applicator size, a specific template was developed. RESULTS: The templates have been used successfully in our institutions for more than 50 patients' brachytherapy treatments. They are currently being used for Esteya and Valencia applicators. CONCLUSIONS: The template La Fe-ITIC is simple and practical. It improves both the set-up time and reproducibility. It helps to establish the adequate margins, an essential point in the clinical outcome.

Characterization of a Novel Conformational GII.4 Norovirus Epitope: Implications for Norovirus-Host Interactions.

UNLABELLED: Human noroviruses (NoVs) are the main etiological agents of acute gastroenteritis worldwide. While NoVs are highly diverse (more than 30 genotypes have been detected in humans), during the last 40 years most outbreaks and epidemics have been caused by GII.4 genotype strains, raising questions about their persistence in the population. Among other potential explanations, immune evasion is considered to be a main driver of their success. In order to study antibody recognition and evasion in detail, we analyzed a conformational epitope recognized by a monoclonal antibody (3C3G3) by phage display, site-directed mutagenesis, and surface plasmon resonance. Our results show that the predicted epitope is composed of 11 amino acids within the P domain: P245, E247, I389, Q390, R397, R435, G443, Y444, P445, N446, and D448. Only two of them, R397 and D448, differ from the homologous variant (GII.4 Den-Haag_2006b) and from a previous variant (GII.4 VA387_1996) that is not recognized by the antibody. A double mutant derived from the VA387_1996 variant containing both changes, Q396R and N447D, is recognized by the 3C3G3 monoclonal antibody, confirming the participation of the two sites in the epitope recognized by the antibody. Furthermore, a single change, Q396R, is able to modify the histo-blood group antigen (HBGA) recognition pattern. These results provide evidence that the epitope recognized by the 3C3G3 antibody is involved in the virus-host interactions, both at the immunological and at the receptor levels. IMPORTANCE: Human noroviruses are the main cause of viral diarrhea worldwide in people of all ages. Noroviruses can infect individuals who had been previously exposed to the same or different norovirus genotypes. Norovirus genotype GII.4 has been reported to be most prevalent during the last 40 years. In the present study, we describe a novel viral epitope identified by a monoclonal antibody and located within the highly diverse P domain of the capsid protein. The evolution of this epitope along with sequential GII.4 variants has allowed noroviruses to evade previously elicited antibodies, thus explaining how the GII.4 genotype can persist over long periods, reinfecting the population. Our results also show that the epitope participates in the recognition of host receptors that have evolved over time, as well.

A Complex Molecular Interplay of Auxin and Ethylene Signaling Pathways Is Involved in Arabidopsis Growth Promotion by Burkholderia phytofirmans PsJN.

Modulation of phytohormones homeostasis is one of the proposed mechanisms to explain plant growth promotion induced by beneficial rhizobacteria (PGPR). However, there is still limited knowledge about the molecular signals and pathways underlying these beneficial interactions. Even less is known concerning the interplay between phytohormones in plants inoculated with PGPR. Auxin and ethylene are crucial hormones in the control of plant growth and development, and recent studies report an important and complex crosstalk between them in the regulation of different plant developmental processes. The objective of this work was to study the role of both hormones in the growth promotion of Arabidopsis thaliana plants induced by the well-known PGPR Burkholderia phytofirmans PsJN. For this, the spatiotemporal expression patterns of several genes related to auxin biosynthesis, perception and response and ethylene biosynthesis were studied, finding that most of these genes showed specific transcriptional regulations after inoculation in roots and shoots. PsJN-growth promotion was not observed in Arabidopsis mutants with an impaired ethylene (ein2-1) or auxin (axr1-5) signaling. Even, PsJN did not promote growth in an ethylene overproducer (eto2), indicating that a fine regulation of both hormones signaling and homeostasis is necessary to induce growth of the aerial and root tissues. Auxin polar transport is also involved in growth promotion, since PsJN did not promote primary root growth in the pin2 mutant or under chemical inhibition of transport in wild type plants. Finally, a key role for ethylene biosynthesis was found in the PsJN-mediated increase in root hair number. These results not only give new insights of PGPR regulation of plant growth but also are also useful to understand key aspects of Arabidopsis growth control.

Antibodies against Lewis antigens inhibit the binding of human norovirus GII.4 virus-like particles to saliva but not to intestinal Caco-2 cells.

BACKGROUND: Human noroviruses (NoVs) are the main cause of gastroenteritis worldwide. The most commonly detected NoV strains belong to the genetically diverse GII.4 genotype, with new pandemic variants emerging periodically. Despite extensive efforts, NoV investigation has been hampered by the lack of an effective in vitro cell culture system. However, NoV-derived recombinant virus-like particles (VLPs) resembling empty capsids are good surrogates for analysing NoV antigenicity and virus-ligand interactions. NoV VLPs have been reported to bind to histo-blood group antigens (HBGAs). We have analysed the ability of NoV VLPs derived from GI.1 genotype and from three GII.4 genotype variants, GII.4-1999, GII.4-2004 and GII.4-2006b, to bind to porcine gastric mucin (PGM), human saliva and differentiated human intestinal Caco-2 cells (D-Caco-2 cells). RESULTS: Distinct patterns of saliva binding with the NoV GII.4 variant VLPs were observed, although they bound to D-Caco-2 cells independently of the expression of HBGAs. Monoclonal antibodies against Lewis antigens were able to block the binding of NoV VLPs to saliva, but not to D-Caco-2 cells. Blocking HBGAs on the surface of D-Caco-2 cells with specific monoclonal antibodies did not affect NoV VLP binding to cellular membranes. Co-localisation of Lewis y (Le(y)) and H-type 2 antigens with NoV VLPs was not observed by immunofluorescence assays. CONCLUSION: Although the binding of NoV VLPs of GII.4 genotype variants to human saliva samples occur with distinct HBGA binding patterns and can be blocked by antibodies against Lewis antigens, their attachment to D-Caco-2 cells can be mediated by other receptors, which still need further investigation.

Characterisation of a household norovirus outbreak occurred in Valencia (Spain).

BACKGROUND: Human noroviruses (NoVs) are the main cause of non-bacterial gastroenteritis worldwide. Several studies have linked human susceptibility to NoVs with the expression of histo-blood group antigens (HBGAs). In January 2012, a NoV gastroenteritis outbreak affected a household in Valencia, Spain, and the personal susceptibility to NoV was investigated. METHODS: To reach this aim 8 members of the affected household were recruited for this study and their secretor status, ABO and Lewis antigens were determined. NoV-specific saliva IgA and serum IgG antibody titers were analyzed. Their capacity to block viral binding to saliva receptors was analyzed, using virus-like particles (VLPs) of the NoV GII.4 genotype, 2006b variant, and saliva from a secretor O blood type donor. RESULTS: The most relevant finding was that an asymptomatic non-secretor individual shed NoVs in his stools. Interestingly, anti-NoV IgA antibody titers in saliva from secretor and non-secretor individuals showed no differences. On the contrary, high titers of NoV-specific IgG antibody were found in both convalescent sera and in sera collected 1 year post-infection, but only from secretor individuals. NoV GII.4-2006b VLP binding to receptors present in the saliva was efficiently blocked only by sera from secretor positive individuals. CONCLUSIONS: Despite the small number of individuals involved in this outbreak, this study reinforces the idea that susceptibility to human NoV is both dependent on the HBGA profile of the individuals as well as on the viral genotype and variant. We also show that the immunity to NoV lasts for at least 1 year after infection, demonstrating that symptomatic infections strongly stimulate immune responses.

Failure mode and effects analysis of skin electronic brachytherapy using Esteya® unit.

PURPOSE: Esteya® (Nucletron, an Elekta company, Elekta AB, Stockholm, Sweden) is an electronic brachytherapy device used for skin cancer lesion treatment. In order to establish an adequate level of quality of treatment, a risk analysis of the Esteya treatment process has been done, following the methodology proposed by the TG-100 guidelines of the American Association of Physicists in Medicine (AAPM). MATERIAL AND METHODS: A multidisciplinary team familiar with the treatment process was formed. This team developed a process map (PM) outlining the stages, through which a patient passed when subjected to the Esteya treatment. They identified potential failure modes (FM) and each individual FM was assessed for the severity (S), frequency of occurrence (O), and lack of detection (D). A list of existing quality management tools was developed and the FMs were consensually reevaluated. Finally, the FMs were ranked according to their risk priority number (RPN) and their S. RESULTS: 146 FMs were identified, 106 of which had RPN ≥ 50 and 30 had S ≥ 7. After introducing the quality management tools, only 21 FMs had RPN ≥ 50. The importance of ensuring contact between the applicator and the surface of the patient's skin was emphasized, so the setup was reviewed by a second individual before each treatment session with periodic quality control to ensure stability of the applicator pressure. Some of the essential quality management tools are already being implemented in the installation are the simple templates for reproducible positioning of skin applicators, that help marking the treatment area and positioning of X-ray tube. CONCLUSIONS: New quality management tools have been established as a result of the application of the failure modes and effects analysis (FMEA) treatment. However, periodic update of the FMEA process is necessary, since clinical experience has suggested occurring of further new possible potential failure modes.