Silk fibroin (SF) and konjac glucomannan (KGM) are promising materials in the biomedical field due to their low toxicity, biocompatibility, biodegradability and low immune response. Beads of these natural polymers are interesting scaffolds for biomedical applications, but their fabrication is a challenge due to their low stability and the necessary adaptation of their chemical and mechanical properties to be successfully applied. In that sense, this study aimed to synthesize a blend of silk fibroin and konjac glucomannan (SF/KGM) in the form of porous beads obtained through dripping into liquid nitrogen, with a post-treatment using ethanol. Intermolecular hydrogen bonds promoted the integration of SF and KGM. Treated beads showed higher porous size, crystallinity, and stability than untreated beads. Characterization analyses by Fourier-transform infrared spectroscopy (FTIR), thermogravimetric (TGA), and X-ray diffraction (XDR) evidenced that ethanol treatment allows a conformational transition from silk I to silk II in SF and an increase in the KGM deacetylation. Those chemical changes significantly enhanced the mechanical resistance of SF/KGM beads in comparison to pure SF and KGM beads. Moreover, samples showed cytocompatibility with HaCaT and BALB/c 3T3 cells. Global challenges in scaffolds technology offer opportunities for further research that involves new types of polymers in different forms such as films, membranes, and beads. In the last decades, natural polymers have been investigated to be used as cell scaffolds . Howeverd-mannose and d-glucose monomers linked by β(1→4) bonds. KGM is a hydrophilic polymer with high viscoelasticity and mechanical resistance. Over the last years, it was mainly used as a bioactive polymer. KGM, combined with other polymers, can be used as a controlled delivery system for drugs, and antimicrobial materials [One of the most promising materials to be used as cell scaffolds, among other applications in biomedicine is silk fibroin (SF), which is a biodegradable polymer, and biocompatible with human tissues. Its chemical composition consists of simple amino acids, such as glycine, alanine, serine, and tyrosine ,4. SF isaterials ,8.In spite of the mentioned properties of SF and KGM, their mechanical properties could be improved in order to be used as a scaffold. Blending two or more polymers is a usual technique to overcome challenges in polymer properties and resulting in the new materials. Several studies of SF-based blends have been reported in the literature. Park et al. 1999) verified that the mechanical tensile strength of SF and chitosan (CHI) membranes depended on CHI concentration. Moreover, the resistance of membranes presented higher value than pure polymers, due to the increase in β-sheet conformational transition 99 verifi. Lee et In that sense, this study was focused on the synthesizes and characterization of porous blend beads obtained from two natural polymers, SF and KGM, with suitable characteristics to be applied to tissue engineering or drug release field . A surfa2CO3 1 g/L solution at 85 °C for 30 min. Afterward, the obtained SF fibers were rinsed with distilled water to remove Na2CO3 residues and dried at room temperature (25 °C) for 24 h. A ternary solvent of CaCl2:ethanol:H2O in a molar ratio of 1:2:8 was used to dissolve the SF fibers to 10% (w/v) at 85 °C for 1.5 h. The SF salt solution was dialyzed by a cellulose membrane  against distilled water for 72 h at 4 °C, with changes every 24 h [w/v), which was determined by casting the solution in a petri dish and weighing the dried mass after solvent evaporation. Finally, the dialyzed solution was diluted in deionized water to a concentration of 2.5% (w/v).Bombyx mori silkworm cocoons were supplied by Fiação Bratac . Cocoons were degummed three times by soaking in Naery 24 h . The obt2  was added at a concentration of 1.5 mmol/L to pre-stabilize KGM [KGM  was dissolved in deionized water to a concentration of 1 g/L. After homogenization, Ca(OH)lize KGM ,16,17. Cw/v) into KGM 1% (w/v) at a weight ratio of 50% (w/w) under continuous stirring, and this maintained for 5 min for complete homogenization.The blends were prepared by adding the solution of SF 2.5%  engaged in a silicone hose  and a needle . Each solution was pumped to the needle and dripping into liquid nitrogen (fast freezing) for the formation of beads. SF, KGM, and SF/KGM beads were prepared by using a peristaltic pump  at room temperature for 24 h. Ethanol was used to induce SF structural transformation and KGM deacetylation [To study the effect of a post-treatment with ethanol, SF, KGM, and SF/KGM beads were treated with ethanol 95% .The surface tension of solutions was determined by a tensiometer  using the ring method . The sizThe morphology of the surface beads was examined using a scanning electron microscope  with a current and voltage of 50 pA and 10 kV, respectively. The average pore size of the beads was determined from SEM images by ImageJ® software.The samples were fixed on aluminum supports using double-sided carbon tape, and in sequence, the samples were metallized using a 200 Å thick gold layer. Metallization was performed in a vacuum chamber  under argon flow for 15 min. After metallization was completed, the supports containing the metalized samples were placed under a microscope, and the samples were photographed for recording purposes.−1 in the range of 650–4000 cm−1. Deconvolution of FTIR spectra was obtained by a Gaussian function using the Fityk 0.9.8 software . The percentage of β-sheet conformation was evaluated by integrating the curve area of the whole studied region.Attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) was used for the chemical characterization of the samples. Analyses were carried out with an ATR-FTIR-6100  spectrometer. Each measurement was acquired in transmittance mode by an accumulation of 128 scans at a resolution of 4 cm2 flow of 50 mL/min. The glass transition temperature (Tg) was obtained by a differential scanning calorimeter . All measurements were carried out from 25 to 300 °C under a nitrogen atmosphere using a heating rate of 10 °C/min.Thermogravimetric characterization was performed by TGA equipment  in a temperature range of 25–600 °C with a ramp rate of 10 °C/min, and an NThe crystallinity of beads was studied by X-ray diffraction  with Cu-Kα radiation (λ = 1.54056 Å). The X-ray source was operated at 40 kV and 40 mA. The measurements were performed in a range of 2θ = 5–80° using a scanning speed of 0.033°/s with a step size of 0.02°.The mechanical properties of freeze-dried beads were measured by confined compression test using a TA.XT2 texturometer  with a 50 N load cell at room temperature. The speed test was 0.2 mm/s. The beads were subjected to a compressive force of up to 50%.2) with Roswell Park Memorial Institute (RPMI) 1640 medium, supplemented with 10% inactivated fetal bovine serum (FBS) and 1% antibiotic solution (penicillin and streptomycin). The cultures were incubated at 37 °C in an atmosphere containing 5% CO2. The medium was changed every 48 h, and when the culture reached confluence, the subculture was treated with trypsin-EDTA, until complete release of the cells. The released cells were transferred to a new plastic flask or 24-well plates.Normally established cell lines HaCaT (human keratinocytes) and BALB/c 3T3 (murine fibroblast) were used. The cells were grown in plastic flasks -2,5-diphenyltetrazolium bromide (MTT) solution (0.5 mg/mL in FBS free culture medium) was added to each well. After incubation for 2 h at 37 °C, the MTT solution was removed and the formazan crystal was solubilized in 1 mL of ethanol. The plate was shaken for 5 min and the absorbance of each well was read in the spectrophotometer . The measured absorbance at λ = 570 nm was normalized to % of control [HaCaT and BALB/c 3T3 cells were distributed in 24-well plates using a density of the 4 × 10 control . This vap-value ≤ 0.05.Each experiment was carried out in triplicate unless otherwise specified. All results are presented as the mean ± standard deviation (SD). The experimental data from all the studies were analyzed using Analysis of Variance (ANOVA). Statistical significance was set to 50/KGM50 presented a decrease of 8.9%, 7.6% and 23.4%, respectively. In the case of SF50/KGM50 beads, they showed an intermediate size between both pure natural polymers beads, due to the intermolecular interactions between both substances. According to the literature, higher surface tensions should produce higher diameters of drops, because the drop increases its size adding mass until the surface tension cannot keep it together [6 Da [together . Howevertogether , while Ker [6 Da .50/KGM50 exhibited a rough and homogeneous surface with pores of 0.5 ± 0.2 μm. On the contrary, the treated beads presented large pores of 64.2 ± 13.4 μm. According to studies of Teimouri et al. (2015), the range of the pore size should be from 50 to 150 μm for successfully grown fibroblast cells and keratinocytes, since this size favors the transport of nutrients and oxygen [50/KGM50 treated with ethanol should provide the requirements for the cellular growth of fibroblasts and keratinocytes.d oxygen . In the d oxygen . Then, i−1), amide II (1535–1540 cm−1) and amide III (1230–1240 cm−1). The amide bands I, II and III are attributed to C=O stretching, N–H deformation and O–C–N folding, respectively [−1), amide II (1515–1530 cm−1) and amide III (1250–1260 cm−1), which is a more stable conformation of the SF [−1: at 1024 (C–O and C–OH), 1066 (C–H), 1153 (C–O), and 1247 cm−1 (CH2OH and C(=O)O). The main difference in the treated bead appears in the 1643 cm-1 band, which is attributed to the deformation in the plane of the water molecule due to crystallization. This is an intermolecular hydrogen bonding interaction originated by treatment in an ethanol and alkali agent [−1 (C=O) was faded, which is related to an increase of deacetylation, causing an increase of hydrogen bonds [50/KGM50 beads, previously discussed bands corresponding to SF and KFM were identified. In fact, as the case of SF, SF50/KGM50 spectra exhibited a shift of higher energies: from amides of silk I to amides of silk II. The ethanol treatment made a more stable conformation of beads. Yang et al. (2000) reported the same tendency for SF/cellulose films [50/KGM50, which is usually accompanied by the formation of hydrogen bonds between polymers [To chemically characterize the synthesized beads, FTIR-ATR analyses were performed . Figure ectively . After tII 1250–10 cm−1, wf the SF  in the rli agent . Finallyen bonds . As expese films . Also, av/v)  b. As KGMpolymers . Accordipolymers . Authorsup of SF ,34,35.50/KGM50 beads, the loss of mass was very similar between untreated and treated samples: 43% and 46%, respectively. Regarding the decomposition temperatures ranges, both cases were similar but with a slight increase in the maximum decomposition temperature: 233–299 °C for untreated beads, and 233–303 °C for treated beads.Thermal stability of the beads was studied by thermogravimetric analysis A. The exTg) of the beads, which were obtained from the DSC . For all cases (pure polymer beads and different blends) the ethanol treatment significantly improved the strength of samples. Regarding the SF component, the organic solvent dehydrated its chains favoring hydrogen bonds and Van der Waals forces. It, thus, changes to a more stable structural conformation [ormation . Accordiormation ,43, the ormation , facilitormation ,42. Thisormation ,31,44, pPark et al. 1999) found that the mechanical tensile strength of SF/CHI membranes depended on CHI content. In fact, blends presented higher resistance than pure polymers, possibly due to the increase in the conformational transition β-sheet 99 found .The cytotoxicity of solutions  and beadKGM showed a tendency towards low cytotoxic profile for HaCaT in the concentration of 1 mg/mL since it is within the limit of the recommended in ISO 10993-5 (2009) . Based oIn general, the decrease in cytotoxicity at the highest concentration tested was approximately 30% for HaCaT and 20% for BALB/c 3T3. When the cells were exposed to the combination of both natural polymers, it did not show cytotoxicity to the studied strains when compared to MTT reduction. Thus, the obtained beads could be applied in the field of tissue engineering.50/KGM50 reached the highest values of cell viability: 136% ± 0.1% for HaCaT, and 135% ± 1% for BALBc/3T3, both at 120 h.Regarding the beads , in geneSF, KGM, and SF/KGM beads were obtained by a viable protocol that produced porous beads by dripping into liquid nitrogen, with a post-treatment using ethanol. Ethanol treatment reduced the diameter of beads but significantly increased the size of surface pores; making them viable for cell adhesion and growth. Physicochemical analysis of samples showed that SF/KGM beads were integrated due to the formation of hydrogen bonds between both polymers. Also, beads treated with ethanol exhibited more crystallinity and thermal stability, which were generated by a conformational transition from silk I (α-helix or random coil) to silk II (β-sheet) in SF, and an increase in the deacetylation of KGM. Due to these chemical changes, treated SF/KGM beads showed higher mechanical resistance than pure SF and KGM beads under compression tests. Finally, all beads did no present cytotoxicity effect for HaCaT and BALBc/3T3 cells up to 120 h and treated SF/KGM beads reached the highest values of cell viability. In general, the obtained porous blend beads presented improved characteristics compared to pure polymer beads, which could be used in the field of biomedicine.


In women with abnormal uterine bleeding, fibroids are a frequent finding. In case of heavy menstrual bleeding and presence of submucosal type 0–1 fibroids, hysteroscopic resection is the treatment of first choice, as removal of these fibroids is highly effective. Hysteroscopic myomectomy is currently usually performed in the operating theatre. A considerable reduction in costs and a higher patient satisfaction are expected when procedural sedation and analgesia with propofol (PSA) in an outpatient setting is applied. However, both safety and effectiveness – including the necessity for re-intervention due to incomplete resection – have not yet been evaluated.This study is a multicentre randomised controlled trial with a non-inferiority design and will be performed in the Netherlands. Women > 18 years with a maximum of 3 symptomatic type 0 or 1 submucosal fibroids with a maximum diameter of 3.5 cm are eligible to participate in the trial. After informed consent, 205 women will be randomised to either hysteroscopic myomectomy using procedural sedation and analgesia with propofol in an outpatient setting or hysteroscopic myomectomy using general anaesthesia in a clinical setting in the operating theatre.Primary outcome will be the percentage of complete resections, based on transvaginal ultrasonography 6 weeks postoperatively. Secondary outcomes are cost effectiveness, menstrual blood loss , quality of life, pain, return to daily activities/work, hospitalization, (post) operative complications and re-interventions. Women will be followed up to one year after hysteroscopic myomectomy.This study may demonstrate comparable effectiveness of hysteroscopic myomectomy under procedural sedation and analgesia versus general anaesthesia in a safe and patient friendly environment, whilst achieving a significant cost reduction.NTR5357. Registered 11th of August 2015.Dutch trial register, number The online version of this article (10.1186/s12905-019-0742-1) contains supplementary material, which is available to authorized users. In women with abnormal uterine bleeding, fibroids are a frequent finding. In case of heavy menstrual bleeding and presence of submucosal type 0–1 fibroids, hysteroscopic resection is the treatment of first choice, as removal of these fibroids is highly effective . The widIn the last decades, there has been a trend in moving hysteroscopic surgery from a clinical setting with general or spinal anaesthesia to an outpatient setting. Especially hysteroscopic surgery of smaller diameter polyps (< 2 cm) and low grade adhesions are eligible for resection in an outpatient setting –10. EvenProcedural sedation and analgesia (PSA) is used for a wide variety of interventional procedures in multiple settings outside the operating theatre. In gynaecology, the use of PSA has also become more popular since technical and instrumental improvements have significantly increased the feasibility and acceptability of hysteroscopy in outpatient setting , 13. UntA considerable cost reduction is expected when PSA will be applied for hysteroscopic resection of fibroids: the use of PSA means that hysteroscopic myomectomy can be moved away from the operating theatre to an outpatient setting. Hence less dedicated personnel is needed. Other potential advantages of hysteroscopic fibroid resection with PSA are avoidance of general anaesthesia and its associated risks, a shorter recovery time – resulting in a shorter hospital stay –, faster return to mobility, full fitness and normal activities. Finally, waiting lists for major surgery will be reduced by averting the need for the operating theatre for minor procedures.Due to the abovementioned factors higher patient satisfaction is also expected, as both hospital stay and time-to-work are shorter and side effects of general anaesthesia such as nausea are reduced.The use of PSA seems to be feasible and well-tolerated in gynaecological surgery –16. HoweThe aim of this study is to compare hysteroscopic resection of symptomatic type 0 and type I submucosal fibroids under procedural sedation and analgesia with propofol in an outpatient setting with hysteroscopic myomectomy in an inpatient clinical setting using general anaesthesia. It is hypothesised that hysteroscopic myomectomy with PSA is non-inferior to the same procedure carried out under general anaesthesia. This study will also compare the cost effectiveness, pain, menstrual blood loss , quality of life, return to daily activities/work, hospitalization, (post) operative complications and re-interventions.This study is a multicentre randomised controlled trial with a non-inferiority design. A cost-effectiveness study will be performed together with the clinical study. The study is embedded in the Dutch Consortium for Healthcare Evaluation and Research in Obstetrics and Gynaecology. Participating hospitals are university and teaching hospitals in the Netherlands. Women will be randomised for hysteroscopic myomectomy either under general anaesthesia or procedural sedation and analgesia with propofol.A minimum age of 18 yearsA maximum of 3 symptomatic type 0 and type 1 submucosal fibroidsA maximum diameter of 3.5 cm American Society of Anaesthiologists (ASA) class 1 or 2Sufficient knowledge of Dutch or English language to fully understand the study and complete the questionnairesThe following women will be included:Presence of clotting disordersSevere anemia (Hb under 5.0 mmol/l)ASA class 3 or 4According to guidelines from the Health Care Inspectorate (IGZ) and Dutch Institute for Healthcare Improvement (CBO) Non-Anaesthesiologist Administered Propofol (NAAP) sedation is given and monitored by a qualified sedation practitioner , 18. NonAn experienced surgeon performs the hysteroscopic resection by standard procedure with the use of a resectoscope or morcellation device in an office based setting. Patients are observed after the procedure by qualified personnel and discharged as soon as all the discharge criteria are met, normally within 1 to 1.5 h.General anaesthesia can be volatile based or total intravenously, with the use of a laryngeal mask or tube. Postoperatively, patients will be observed in the recovery room and discharged home from clinic when all the discharge criteria are met. Hysteroscopic myomectomy is performed by standard procedure.Primary outcome will be the percentage of complete resections, evaluated by transvaginal ultrasonography (TVU) (contrast sonography if TVU is inconclusive) 6 weeks postoperatively. The ultrasonography will be performed by an independent gynaecologist or ultrasonographer blinded for the treatment arm and judgment of completeness by the surgeon who performed the hysteroscopic myomectomy. This TVU should be conducted 6 weeks postoperatively. A complete resection means that there are no signs of an intracavitary remaining of the fibroid (s) resected during hysteroscopic myomectomy. This follow-up visit and TVU is part of the current usual care, so no extra ultrasonography is needed for study purposes.Secondary outcomes are cost effectiveness, pain, menstrual blood loss (PBAC score), quality of life, return to daily activities/work, hospitalization, (post) operative complications, re-interventions. These secondary parameters will be assessed by several questionnaires.The gynaecologist participating in the network will inform women about the study and refer the eligible women to dedicated research nurses if available. These nurses or the local investigator will counsel women, ask for written informed consent and perform randomisation to either PSA or general anaesthesia. The investigators will also organize the required treatments, depending on the result of the randomisation. Subjects can leave the study at any time for any reason if they wish to do so without any consequences. The investigator can decide to withdraw a subject from the study for urgent medical reasons.Randomisation will be performed web based with the use of a block design, with a variable block size.The study will not be double-blinded, as it is impossible to blind the health care workers and women involved for the strategy to which the woman is allocated. The person performing the transvaginal ultrasonography at 6 weeks follow up, however, will be blinded for the study arm and judgement of completeness by the surgeon during the procedure.Women will be asked to complete questionnaires online, through a link they receive by e-mail (it is possible to complete the questionnaires on paper when women prefer to do so).A website dedicated to studies in the Dutch Consortium for Healthcare Evaluation and Research in Obstetrics and Gynaecology will be used for data collection. Research nurses in each of the participating centres will perform the data collection. A computer generated numeric code – to which the key is only available to the local investigator or local research nurse–will be attributed to each participant, enabling anonymous data management.In accordance with the guidelines of the Dutch Federation of University Medical Centers (NFU) the data will be preserved for 15 years.Baseline characteristicsWomen will be followed from baseline (pre-operatively) up to 1 year after hysteroscopic myomectomy , operating time, intravasation/fluid deficit, recovery time (time from end of procedure until completely awake and communicative), use of pain medication, pain intensity measured by NRS scores, duration of hospitalization (hours).Short questionnaire on side effects 24 h after surgery. See Additional files The EuroQoL (EQ-5D-5 L) questionnaire (15 questions) to assess the quality of life .Pictorial Blood Assessment Chart (PBAC) scores to assess the amount of menstrual blood loss. See Additional files Uterine Fibroid Symptoms – Quality of Life (UFS-QoL) questionnaire (37 questions) to assess the quality of life focussing on women suffering from uterine fibroids . See AddRecovery Index (RI-10) questionnaire (10 questions): to assess a patients’ recovery process .Medical Consumption Questionnaire (iMCQ): for cost effectiveness analysis .Productivity Cost Questionnaire (iPCQ) to assess productivity loss .Short questionnaire on recurrence and re-interventions at 12 months after surgery. See Additional files Participating women will fill in questionnaires at baseline, which will be given before the surgical procedure. During follow-up, women will fill in questionnaires at 24 h, 2 weeks, 8 weeks, 6 and 12 months after randomisation (Table An independent Data Safety Monitoring Board (DSMB) will be asked to monitor the progress of the study and the safety of its participants. Accumulating data on serious adverse events will be sent to the DSMB each time data of serious adverse effects in 20 women has been received. In addition, an overview of reported SAEs will be send to the DSMB after each 50 women included and operated during the study. The DSMB will meet as required to review any expected adverse events and may ask to review outcomes or other data that may have an impact on the trial.No formal interim analysis or efficacy will be done.The study is designed as a non-inferiority study, in which we aim to investigate if hysteroscopic myomectomy under procedural sedation with propofol is non-inferior to the same surgical procedure under general anaesthesia. With 205 women randomized we have 90% power to demonstrate non-inferiority, based on an estimated 2.5% incomplete resections in both groups, with an non-inferiority upper limit of 10% incomplete resections defined as non-inferior (i.e. a delta of 7.5%), an alpha of 0.025 and a drop-out rate of 10%.Data will be analysed according to the intention-to-treat principle. We will also perform a per protocol analysis, given the non-inferiority design of the study, where crossover between the groups will increase the chances of concluding non-inferiority, if in reality the treatment is not inferior. We will present the percentage of complete resections at 6 weeks in both groups, with according relative risks and 95% confidence interval. Differences will be tested with the Chi-square test, or, if the expected cell count is low using the Fisher exact test. We will also calculate the relative risk, adjusted for the resection technique which was used. Stratification will be performed by resection technique (resection or morcellation). Complications during and after surgery, and re-interventions will be categorized. The Data and Safety Monitoring Board will evaluate serious adverse events.Pain intensity after the procedure and at discharge will be reported as means with SD, risk differences between both groups will be calculated with according 95% confidence interval. Time to recovery and pain will be visualized in a graph, and analyzed using a mixed model, that can take into account repeated measures in the same woman over time. The quality of life and PBAC scores will be analyzed according the developed algorithms. We will perform a subgroup analysis for fibroid size (< 2 cm versus ≥2 cm) and for parity (nulliparous versus multiparous women).This economic evaluation aims to link the difference in societal and healthcare costs to the difference in clinical effects between PSA and general anaesthesia. A cost-effectiveness and cost-utility analysis will be carried out with a time horizon of 1 year. Thus, discounting is not necessary. Costs will be measured from a societal perspective using internet questionnaires based on the iMCQ after 8 weeks, 6 and 12 months of follow-up. Direct costs consist of costs of primary and hospital care as well as costs of complementary care and home care. Indirect costs involve costs caused by being absent from paid and unpaid work, or by presenteeism. Estimation of indirect costs will be done by using the friction cost approach. The standard prices presented in the Dutch costing guidelines  will be Societal costs will be related to the following effect measures in the economic evaluation: percentage of complete resections and quality-adjusted life-years (QALYs) based on the Dutch tariff for the EuroQol (EQ-5D-5 L) , 27. TheFibroids are a frequent finding in women with abnormal uterine bleeding. The Dutch Guideline ‘Heavy menstrual bleeding’ recommends hysteroscopic myomectomy as treatment of first choice in case of symptomatic submucous fibroids . In the Additional file 1:Questionnaire on side effects 24 h after surgery. (PDF 129 kb)Additional file 2:Questionnaire on side effects 24 h after surgery. (PDF 109 kb)Additional file 3:Pictorial Blood Assessment Chart (PBAC). (PDF 134 kb)Additional file 4:Pictorial Blood Assessment Chart (PBAC). (PDF 78 kb)Additional file 5:Uterine Fibroid Symptoms – Quality of Life (UFS-QoL) questionnaire. (PDF 265 kb)Additional file 6:Questionnaire on recurrence and re-interventions 12 months after surgery. (PDF 76 kb)Additional file 7:Questionnaire on recurrence and re-interventions 12 months after surgery. (PDF 78 kb)


We evaluated the benefit of whole blood versus plasma to detect acute Zika virus infections. Comparison of Zika virus quantitative reverse transcription PCR results in single timepoint whole blood–plasma pairs from 227 patients with suspected Zika virus infection resulted in confirmation of 8 additional patients with Zika virus infection. Flavivirus) has become a major public health concern. Zika virus infections are linked to congenital malformations in neonates from mothers infected during pregnancy and to neurologic disorders in adults  results for 249 EDTA–whole blood and EDTA–plasma pairs submitted for laboratory testing from 227 patients with suspected Zika virus infection during July 2016–May 2017. These patients were those with a Zika virus diagnostic request in this period from whom both plasma and whole blood could be collected. In line with previous observations in our laboratory (g) to collect plasma. We stored the samples at −80°C until use. For testing, we spiked the samples with an internal control and extracted total nucleic acids from a 500-μL sample in 100 μL of eluate using the MagNAPure 96 DNA and Viral NA large volume kit and Viral NA Universal LV 2.0 protocol , according to the manufacturer’s instructions. Extraction was followed by an ISO15189:2012-validated laboratory-developed Zika virus qRT-PCR, as described previously , as described by the manufacturer. Using a standard EDTA blood collection tube, we aliquoted 600 μL of whole blood before the centrifugation step  of 249 whole-blood samples and in 23 (74.2%) of the 31 corresponding plasma samples. The 31 positive whole-blood samples were collected from 31 individual patients. This comparison indicated that 8 additional Zika virus–positive patients would have been identified if whole blood had been used routinely instead of plasma . This fiStandard practice in international guidelines on diagnostic algorithms for Zika virus is to combine molecular testing of plasma with molecular testing of urine, along with serology, to come to an accurate Zika virus diagnosis. However, confirmation of cases based on serology only is usually limited to expert Biosafety Level 3 laboratories being able to perform virus neutralization tests comparing Zika virus titers with titers of other flaviviruses (https://www.euroimmun.com) for the presence of Zika virus–specific IgM and IgG, as described by the manufacturer. For 3 of the 8 additional patients, Zika virus infection had already been confirmed on the basis of the presence of Zika virus RNA and IgM in an earlier plasma sample. For the remaining 5 patients, only a status of probable case was achieved without the whole-blood testing  to confirm cases based on serology. Therefore, we have implemented whole-blood RT-PCR testing for Zika virus diagnostic requests in our routine diagnostic setup. Further studies in larger cohorts, including dengue and chikungunya virus testing, as well to address the often multiplex settings in endemic countries, are needed to demonstrate the general usefulness of our observations.


We conducted a survey on Zika virus perceptions and behaviors during the 2016 outbreak in Miami-Dade County, Florida, USA. Among women, Zika knowledge was associated with having a bachelor’s degree. Among men, knowledge was associated with knowing someone at risk. Interventions during future outbreaks could be targeted by sex and education level. Aedes spp. mosquitoes, such as Zika virus, can lead to misplaced reactions and affect local public health officials’ abilities to contain outbreaks  telephone survey with a random sample of adults in late spring , when the Zika virus outbreak began in Florida. We applied the basic concepts of the Health Belief Model (HBM) in an attempt to understand perceptions of Zika virus risk and prevention practices in Miami-Dade County, Florida, the epicenter of the 2016 Zika virus outbreak  by using multivariate logistic regression with a log-link function adjusted for demographics  and all other variables of the HBM. We presented data as adjusted odds ratios (aORs) with 95% CIs. A low score (0–7 points) on the Zika virus knowledge test indicates the participant correctly answered 0–7 questions and suggests the respondent had simply heard of Zika and knew that mosquitoes could transmit Zika virus. A high score (8–12 points) indicates the participant correctly answered 8–12 questions and suggests the respondent had a good understanding of microcephaly and Guillain-Barré syndrome.Of the 262 survey participants, 149 (56.9%) were women and 113 (43.1%) were men; age range was 18–94  years. More than half (56.9%) of participants were foreign born, 185 (70.6%) considered themselves Hispanic or Latino, and 138 (52.7%) were married. More women (36.9%) than men (31.0%) scored high (8–12 points) for Zika virus knowledge  Table.A total of 53.0% of women and 49.6% of men felt somewhat confident they could protect their households from contracting Zika . PersonaTaking action to protect oneself against Zika virus infection  and knowing someone pregnant (cue to action)  were associated with a higher knowledge of Zika virus . This hiAmong women, Zika virus knowledge was higher among those who had taken action to prevent Zika virus infection  and those with bachelor’s degrees . However, among men, Zika virus knowledge was higher among those who knew someone at risk for Zika  and those who took action to prevent Zika virus infection .Our analysis indicates that women were more concerned about Zika than were men in Miami-Dade County and that those with bachelor’s degrees were more knowledgeable than were those without. Therefore, targeting prevention and treatment interventions by sex and education level should be considered to maximize positive outcomes in high-risk areas during outbreaks (Characteristics of Miami-Dade County residents, by sex, Florida, USA, 2016.


Xiphinema. GFLV and ArMV move from cell to cell as virions through tubules formed into plasmodesmata by the self-assembly of the viral movement protein. Five surface-exposed regions in the coat protein called R1 to R5, which differ between the two viruses, were previously defined and exchanged to test their involvement in virus transmission, leading to the identification of region R2 as a transmission determinant. Region R4 (amino acids 258 to 264) could not be tested in transmission due to its requirement for plant systemic infection. Here, we present a fine-tuning mutagenesis of the GFLV coat protein in and around region R4 that restored the virus movement and allowed its evaluation in transmission. We show that residues T258, M260, D261, and R301 play a crucial role in virus transmission, thus representing a new viral determinant of nematode transmission.Grapevine fanleaf virus (GFLV) and arabis mosaic virus (ArMV) are nepoviruses responsible for grapevine degeneration. They are specifically transmitted from grapevine to grapevine by two distinct ectoparasitic dagger nematodes of the genus  Capsid proteins (CPs) not only protect the genome of plant viruses, but are often crucial in a variety of other functions including vector transmission, replication, translation, cell-to-cell and long distance movements, pathogenicity, inactivation of plant antiviral defenses, or host range determination . CPs of Nepovirus in the family Secoviridae .,22.C. quCP coding sequence in the GFLV-EGFP infectious clone to give the G4-EGFP construct  when compared to the high values obtained with the wild-type GFLV-EGFP control  visualized by ISEM and detected by DAS-ELISA  was shown to be compatible with the production of VLPs [N. benthamiana leaves, the fusion protein G4 2CCP:TR was detected by anti-TagRFP antibodies in western blot  or in an ArMV R4 context (recombinant G4-EGFP). This result further indicates that the nature of region R4 is of importance for capsid formation and in planta movement.The point mutant E264A-EGFP exhibited a phenotype similar to that of recombinant virus G4-EGFP since it induced the formation of small sized foci extending over several cell layers, was undetectable in DAS-ELISA, only allowed amplification of the F2 fragment in IC-RT-PCR, and did not spread systemically . AdditioAS-ELISA . In contAS-ELISA . HoweverAS-ELISA . WhetherCP subunit  and the spatially very close residue R301 belonging to region R5 . First, CP  as efficient as GFLV-EGFP . FurtherCP region R4 and residue R301, we succeeded in generating an infectious recombinant virus containing three residues of region R4 (and one in region R5) of ArMV origin, for which it was now possible to evaluate the impact on the virus transmission by its nematode vector Xiphinema index.Thanks to our site-directed mutagenesis on the GFLV 2CX. index was evaluated in controlled conditions in the greenhouse and compared to the transmission of both the wild type GFLV deriving from the infectious clones (noted GFLV) and a natural ArMV isolate (ArMV-Co) as positive and negative controls, respectively. Two independent transmission experiments were performed with slightly different acquisition procedures . Transmission was assessed by DAS-ELISA on a total of 26 to 28 bait plants (X. index.The transmissibility of the recombinant virus TMDR_ArMV by t plants . Like fot plants . In bothOf the five peptidic regions (R1 to R5) previously predicted to be exposed at the surface of the GFLV virions and potentially accessible for interactions, either with the nematode for specific transmission or with the plant for cell-to-cell and long-distance movement, region R4 was investigated in detail in this work. Through the use of a recombinant virus encoding EGFP, we could visualize the cell-to-cell spread of the infection and discriminate between a local site of infection produced by the wild type virus and a subliminal infection caused by a mutant impaired in capsid formation or stability.CP subunit of GFLV contains region R4 of ArMV origin, is able to protect its genomic RNAs in an RNase protection assay, the recombinant virus G4-EGFP appeared to be restricted to small sites in inoculated leaves. We could show that this defective cell-to-cell movement was due to improper capsid formation rather than to the loss of interaction between the mutated capsid and the tubules, as previously hypothesized. EGFP visualization-assisted point mutagenesis was then performed to recover a mutant affected in region R4 of the 2CCP protein, which is still capable of producing a systemic infection. To this end, we produced mutants where four amino acids , were either changed into alanines or into their V258, N260, N261, and S301 counterparts in polyprotein P2-U of ArMV-S. The latter, named TMDR_ArMV, was fully infectious and was thus tested for transmission by X. index. The loss of transmission of this chimeric virus identifies these four residues as an additional important determinant of GFLV transmission.Although the chimeric virus G4, where the 2CCP residues involved in GFLV transmission by X. index . Similarly, the limited but indubitable cell-to-cell movement of G3-EGFP, despite its poor capsid formation, suggests that its residues are likely not involved in the specific transport of GFLV by its cognate tubules. Furthermore, the clear correlation between the low number of capsids observed in fixed tissues infected by the G3- and G4-EGFP recombinants and the inefficiency of these viruses to move over several cell layers, argues against the existence of an alternative, like a ribonucleoprotein complex, to the movement of the virus as virions in tubules, as proposed for other tubule-forming viruses like brome mosaic virus ,41,42.CP for cell-to-cell and long-distance movements differ, even for viruses with a tubule guided movement strategy, as previously reported for viruses using other movement strategies like tobacco etch virus [CP protein. However, such a function has not been established for GFLV or ArMV 2CCP proteins. Additionally, although this hypothesis cannot be ruled out, it is expected that such an impairment of silencing suppression would also have a drastic effect locally on the size of the infection foci [The mutant TMDR_Ala-EGFP, where the four amino acids T258, M260, D261, and R301 were replaced by four alanines, is also of very high interest. This mutant produces easily detectable capsids, encapsidates its genomic RNAs as deduced from IC-RT-PCR analyses, and moves cell-to-cell as well as the wild type GFLV-EGFP control. However, this locally effective infection is only systemic in 10% of the inoculated plants, highlighting that the requirements of the 2Cch virus , cucumbech virus , or olivch virus . These dch virus  and one ion foci . A more In this manuscript, we showed that a limited GFLV cell-to-cell movement is possible even when capsids are improperly formed, although, efficient tubule-guided cell-to-cell movement depends on the quality of the assembled particles. We also showed that cell-to-cell and long-distance virus movement can be uncoupled, as the requirements for these two movements differ, but both movements are critical for high virus accumulation in the roots and uptake by the nematode vector. Importantly, we identified a new viral determinant involved in the transmission of GFLV by its nematode vector, consisting of four residues in the 2CCP protein: T258, M260, D261 and R301. This new viral determinant is in close proximity to a positively charged cavity of the capsid, which strengthens the hypothesis that this cavity could constitute the ligand-binding pocket of GFLV in the nematode’s mouthparts. In the future, this work could be highly valuable to look for the GFLV receptor within the nematode.


Gremmeniella abietina (Lagerberg) Morelet includes causal agents of shoot blight and stem canker of several conifers in Europe and North America, which are known to host a diverse virome. GaRV6 is the latest and sixth mycovirus species reported within G. abietina. Before its description, one victorivirus and one gammapartitivirus species were described in biotype A, two mitoviruses in both biotypes A and B and a betaendornavirus in biotype B. Possible phenotypic changes produced by mycoviruses on G. abietina mycelial growth have been reported in Spanish mitovirus-free and GaRV6-hosting G. abietina isolates, which had higher growth rates at the optimal temperature of 15 °C, but no other major differences have been observed between partitivirus-like dsRNA and dsRNA-free isolates. In this review, we reappraise the diversity of viruses found in G. abietina so far, and their relevance in clarifying the taxonomy of G. abietina. We also provide evidence for the presence of two new viruses belonging to the families Fusariviridae and Endornaviridae in Spanish isolates.The European race of the ascomycetous species  Gremmeniella abietina is a virulent haploid ascomycete responsible for shoot dieback and Scleroderris canker on conifers including spruces, firs, larches, pines, and junipers in North, Central, and South Europe, northeastern North America, and East Asia . It. ItG. aba B type E, and be3 (DdV3)  and seveFusarium graminearum . I. I36]. Iminearum , Rhizocta solani , C. para annosum , and thetuberata . The clo5 (FgV5)  and Fusa5 (FgV5) . The FgVe family ,37,50. Me family  have notG. abietina, where its genetic diversity was minimal, despite its relatively high abundance (46% of 50 isolates screened) [GaRV6 primarily appeared in the Spanish population of creened) . GaRV6 hcreened) .G. abietina commonly hosts more than one virus in a single isolate [Gremmeniella viruses not reported yet. Nothing is known about the interactions of multiple viruses co-inhabiting the same mycelia, nor of their effects on the host phenotype. isolate ,31,32,36 isolate , in B ty isolate  and, in G. abietina noted by Botella et al. [G. abietina have been included in the previously mentioned studies about GaRV-MS1, GMV1 and GaRV6 [Here, we provide further information about the exceptional richness of the virus community hosted by Spanish isolates of a et al. . Two dsRnd GaRV6 ,36,38.−3, belonging to a hypothetical ATP-dependent RNA helicase HrpB of Haemophilus influenzae (Accession number PRK11664).A partial 2309 bp sequence of the ca. 6 kb dsRNA fragment  and 5 reverse transcription (RT) PCR products. Specific primers  were desG. abietina [G. abietina populations has not been determined, with more specific methods such as reverse transcription (RT) PCR.A similar banding pattern has also been found in other isolates of abietina , such asVictorivirus. The BLASTX comparison of the partial RdRp sequence had the highest similarity (78%) with Sclerotinia nivalis victorivirus 1, followed by Aspergillus foetidus slow virus 1 (52%)  were determined from a 43-clone contig from a ca. 5 kb dsRNA fragment of Spanish isolate 06P . The BLA 1 (52%) , but wasVictorivirus, this virus should be considered a strain of Sclerotinia nivalis victorivirus 1 because it shows 78% RdRp and 81% CP similarity to Sclerotinia nivalis victorivirus 1. Therefore, we designate the virus as Sclerotinia nivalis victorivirus 1—strain 1 from Gremmeniella abietina (SnVV1-Ga1)In the case of the partial CP fragment, the consensus sequence derived from a seven-clone contig of 1261 bp also showed the highest similarity (81%) with Sclerotinia nivalis victorivirus 1, followed by Tolypocladium cylindrosporum virus 1 (68%) . AccordiG. abietina [G. abietina isolates has not been determined with specific methods.A similar dsRNA banding pattern has also been found in other isolates of Spanish abietina , such asG. abietina into different biotypes, and possible species [One of the fundamental questions in evolutionary biology is the degree to which the diversification of parasites and/or symbionts is associated with the diversification of their hosts . Mycovir species , is mostG. abietina is known to host only one species of gammapartitivirus (GaRV-MS1) [G. abietina to Turkey or, alternatively, in Turkey with an unrecognised biotype A population. In the first scenario, biotype B isolates arrived in Turkey with GaRV-MS1 viruses and, in the second scenario, they would have received the viruses in Turkey. A third possibility is that GaRV-MS1 viruses occurred in both biotypes during their evolutionary history, but were later lost in B-type with the exception of the Turkish population. The presence of GaRV-MS1 in the European race (biotype A) in North America, introduced from Europe in 1977 [The European race of aRV-MS1) . In nortaRV-MS1) ,34. On t in 1977 , indicatG. abietina population supports its origin as biotype A [The virus community inhabiting the Spanish iotype A  because Cryphonectria sp. and Cryphonectria parasitica [Sclerotinia homoeocarpa and Ophiostoma ulmi [O. ulmi and Ophiostoma novo-ulmi [Heterobasidion [Sclerotinia [Rhizoctonia [G. abietina has never been shown to exist in Spain and, therefore, the timing and place of this possible host jump remains open. It could have occurred in the past, or even today, in the mountains of northern Spain, where the snow cover in wintertime is enough to fulfil its need [The occurrence of GMV2 in both biotype B and the Spanish population, together with its extremely low genetic variability, would be in accordance with a recent host switch and subsequent adaptation to a new host . Naturalrasitica , Sclerotoma ulmi , O. ulmiovo-ulmi , and witbasidion ,25,26, Serotinia ,28, and zoctonia . Howeverits need ,13,62. FG. abietina is the only one hosting the two different mitoviruses, which underlines its unique position within the taxonomy of G. abietina var. abietina, extending also to its mycovirus community.No evidence is available for the presence of mitoviruses in the Swiss (Alpine biotype), Turkish (biotype B), and North American isolates (biotype A). Therefore, the Spanish population of G. abietina biotypes (or species) cohabit. In Finland, viruses occurring in biotype A and biotype B diverge  have been reported [A certain level of specialisation is detected in Finland ,32,35, w diverge . The FinG. abietina, has not been tested directly, but the lack of corresponding dsRNA banding patterns in Finnish populations suggests its unlikeliness. Therefore, it can also be hypothesised that these viruses represent transmission from some distantly related fungi, such as Sclerotinia nivalis, to the Spanish population of G. abietina. This possibility is supported by the emerging evidence of virus transmissions over wide taxonomic distances known from other fungi [Interestingly, we report, here, that the Spanish population hosts a fusarivirus, GFV1, and also an unrelated victorivirus, SnVV1, the presence of which, in other populations of er fungi ,63,64,65G. abietina in North America, Spain, and Turkey.Altogether, phylogenetic and prevalence studies of the distinct viruses provide evidence that support separation of biotypes A and B from each other, as well as on the origin of separate populations of G. abietina biotype A isolates harbouring GaRV-L and GaRV-MS dsRNA patterns were performed, but no firm conclusion could be drawn on the effect of dsRNA viruses on G. abietina pathogenicity towards Pinus sylvestris [Two pathogenicity tests with lvestris . All isolvestris .G. abietina at its optimum growing temperature, 15 °C. Whether this increase in mycelial growth is connected to the virulence of their hosts has not been tested. In contrast, GaRV6 occurrence had a negative impact on the growth rate of G. abietina on artificial media [Romeralo et al.  noted thal media .G. abietina. However, conclusions about the real and specific role of each type of these viruses cannot be fully determined, since multiple virus infections are common in this conifer pathogen [Fusarivirus and Victorivirus reported in this review, and had the lowest growth rate in the experiment by Botella et al. [G. abietina isolates; unfortunately, up to now, virus removal trials with higher temperatures or cycloheximide have failed with this pathogen [G. abietina in dual culture experiments used for C. parasitica since the 1960s  and, therefore, artificial transmission methods should also be developed for G. abietina viruses.Based on the results described above, at least some viruses may have effects on the phenotype of pathogen ,36,55,56G. abietina hosts a distinct and abundant community of viruses belonging to different genera and families of mycoviruses. Its diversification and spread in different countries has also affected the divergence of its virus community. Based on a single study, it appears that multiple virus infections may affect the phenotype of the host, but the effects of single viruses remain obscure, and should be determined by in vivo and in vitro experiments using isogenic pairs of single-infected and virus-free isolates.


Gynecology and dermatology share the diagnosis and the management of some disorders of the female external genital area. In the last decade, clinical diagnosis in dermatology has dramatically improved, thanks to the introduction of dermatoscopy.  Dermatoscopy is a noninvasive, rapid, and simple technique performed with an affordable handheld instrument called dermatoscope, endowed with a light source and a high-quality lens achieving 10 times magnification.  The use of dermatoscopy for the diagnosis of some common external genital disorders, i.e., genital warts (GW), vestibular papillomatosis (VP), molluscum contagiosum (MC), angiokeratoma (AK), and pediculosis pubis (PP), is presented and discussed.  The use of dermatoscopy in a gynecologic office may considerably help the specialist to enhance in selected cases the clinical diagnosis and to avoid unnecessary and cumbersome investigations which may be time and money consuming. Gynecology and dermatology share the diagnosis and the management of some disorders of the female genital area as it represents an anatomical region of common interest. In the last decade, clinical diagnosis in dermatology has dramatically improved, thanks to the introduction of dermatoscopy, a new noninvasive technique . This arDermatoscopy is a noninvasive, rapid, and simple technique performed with an affordable (from ∼USD 600) handheld instrument called dermatoscope, endowed with a light source and a high-quality lens achieving 10 times magnification . It alloGW  are common communicable benign growths of the genital and perigenital areas, more often observed in young and sexually active individuals, caused by infection from human papilloma viruses (HPVs), most commonly HPV 6/11. They represent one of the most frequent sexually transmissible diseases. Clinically, GW appear as raised, variably sized and colored, soft, and fleshy papules, occurring as single elements or in clusters , often sDermatoscopy of GW varies depending on the clinical presentation. Papular lesions show the so-called “mosaic pattern,” consisting of a white reticular network circumscribing areas centered by dotted vessels Figures ; finger-VP  is the female equivalent of pearly penile papules in men. It is a common and benign condition present in about 1% of women seeking gynecological advice. Clinically, VP appear as soft, flesh-colored, symmetric, and asymptomatic papules lining both sides of the vulva that generally remain unchanged with time . The maiDermatoscopy is a useful tool to differentiate these two conditions, as it allows optimal identification of some of these criteria. In VP, and not in GW, it clearly shows pinkish projections, regularly distributed and linearly arranged in a symmetric distribution, displaying a separated base; linear vessels may sometimes be observed  5]. His. His5]. MC is a common skin infection caused by a Poxvirus. It mostly affects children but may also be observed on the genital areas or in its close proximity (lower abdomen or upper thigh), in sexually active individuals (15–29 years). MC appears as a small, pink to skin-colored, dome-shaped papule, with a central umbilication; the lesions are often multiple and grouped . The diaAt dermatoscopy, MC shows a typical pattern consisting of a central yellowish-white, lobulated, amorphous structure with a peripheral crown of linear, fine, and sometimes blurred vessels that do not cross the center of the lesion  1, 6]. , 6. 1, 6AK of the vulva, unlike its male equivalent , is considered a rare condition in women, more often reported between 20 and 40 years of age. It is a benign tumor characterized by numerous dilated dermal vessels with overlapping epidermal hyperplasia, which may occur as a single or multiple and disseminated lesions. AKs clinically present as keratotic papules of color ranging from red, reddish-blue to black ; they arDermatoscopy can help to rule out these conditions showing the presence of typical red to dark lacunae, which are likely the result of dilated vessels with or without thrombosis, and of a whitish veil, that histologically corresponds to acanthosis or hyperkeratosis . In addiPhthirus pubis that generally occurs as a sexually transmitted infection. It peaks between 15 and 40 years, when sexual activity is higher, and has a male predilection probably because of a greater amount of body hairs that favours parasite transmission. The most frequently involved areas are the pubic and inguinal regions, but axillae and limbs may also be involved. Clinically, pruritus and scratching marks are major complaints. When present, maculae caeruleae, asymptomatic bluish-gray macules caused by crab bites, are a characteristic finding. Nits or lice grasped to the pubic hairs may be detected by simple naked eye inspection. Clinically, disorders that can mimic lice infestation are folliculitis, dermatophytosis, scabies, seborrheic scales, contact dermatitis, etc.Pediculosis pubis  is an ectoparasitosis caused by Dermatoscopy allows a clear and unequivocal visualization of both parasites  and nitsThe diagnosis of common external disorders of the female genital area is generally made through an accurate visual inspection. The use of dermatoscopy, initially for the diagnosis of pigmented skin lesions including those appearing in the vulvar area , has conGiven the high incidence of some external genital disorders as GW, which have a worldwide prevalence up to 44% in some studies, with an economic burden, in terms of diagnosis and treatment, of about 4 billion dollars a year only in the US , the posDermatoscopy may therefore considerably help the office gynecologist to reach, in selected cases, the final diagnosis more easily so to avoid unnecessary and cumbersome investigations which may be time and money consuming. An extra advantage is that, in case of infective disorders, early diagnosis followed by a prompt eradication may considerably improve public health, timely suppressing potential sources of contamination/diffusion. Finally, a multidisciplinary approach with a strict collaboration between gynecologists and dermatologists should be encouraged for an optimal diagnostic approach to some genital disorders.


There are few published data on the acute care or long-term costs after acute/critical limb or visceral ischaemia (ACLVI) events. Using data from patients with acute events in a population based incidence study (Oxford Vascular Study), the present study aimed to determine the long-term costs after an ACLVI event.All patients with first ever incident ACLVI from 2002 to 2012 were included. Analysis was based on follow up until January 2017, with all patients having full 5 year follow up. Multivariate regressions were used to assess baseline and subsequent predictors of total 5 year hospital care costs. Overall costs after an ACLVI event were benchmarked against those after stroke in the same population, during the same period.p < 0.001). Results of the multivariate analyses showed that patients with diabetes and those undergoing above knee amputations incurred additional costs of €11,804 (p = 0.014) and €25,692 (p < 0.001), respectively. Five year hospital care costs after an ACLVI event were significantly higher than after stroke , but similar after including long-term costs of institutionalisation .Among 351 patients with an ACLVI event, mean 5 year total care costs were €35,211 , of which €6443 (18%) were due to long-term institutionalisation. Costs differed by type of event (acute visceral ischaemia €16,476; acute limb ischaemia €24,437; critical limb ischaemia €46,281; Long-term care costs after an ACLVI event are considerable, especially after critical limb ischaemia. Hospital care costs were significantly higher than for stroke over the long term, and were similar after inclusion of costs of institutionalisation. This is the first prospective, population based study of acute/critical limb or visceral ischaemia (ACLVI) events in a population of 92,728 in Oxfordshire, UK. The study shows that the long-term costs after such events are substantial, and that costs vary considerably across type of event. When compared with stroke, hospital care costs are significantly higher after an acute visceral and peripheral arterial event, and similar after inclusion of institutionalisation costs. The present data will be useful to other researchers to perform comparisons in attempts to better understand the likely economic consequences of ACLVI events in their own setting.Cardiovascular disease is the leading cause of death and disability, costing the US and European Union healthcare systems $196 billion and €111 billion, respectively.However, although peripheral arterial disease (PAD) has a poor prognosis,Using data from patients with an acute/critical limb or visceral ischaemia (ACLVI) event in a population based incidence study (Oxford Vascular Study [OXVASC]) ascertained between 2002 and 2012, the present study aimed to reliably determine the absolute long-term costs and their baseline and subsequent predictors. In addition, given that the costs of other vascular disease, in this case stroke, have been estimated in the same population and using the same methodology,The Oxford Vascular Study (OXVASC) population comprises all individuals  registered in nine general practices across Oxfordshire, UK. The study methods have been described elsewhere,All diagnoses were reviewed by a vascular surgeon. Cardiovascular examination included assessment of the peripheral pulses, Buerger's test,All patients with a first ever incident ACLVI event from April 1, 2002 to March 31, 2012 from the study registered practices were included and followed-up. ALI was defined as an arterial event of sudden onset and <2 weeks in duration resulting in symptomatic limb ischaemia. AVI was defined as acute arterial events of sudden onset and <2 weeks in duration resulting in symptomatic visceral ischaemia . CLI was defined as an event with symptoms present for >2 weeks with ischaemic rest pain or tissue loss of sufficient severity to warrant urgent hospital admission and thought to be secondary to large or small vessel arterial disease. History of stable PAD was defined as symptomatic PAD without prior ACLVI history or emergency intervention. These patients typically have intermittent claudication  with any pain at rest or ulceration.All surviving patients were followed up by the following methods: 1) 6 month face to face follow up; 2) primary care records; 3) ongoing study ascertainment of hospital contacts of study participants and the wider study population; 4) routine review of administrative hospital and centralised care records; 5) mortality records; and 6) review of Coroner's office death reports for deaths outside hospital or during surgical procedures. If a vascular event was suspected, the patient was reassessed by a study physician. All patients had mortality follow up.The following healthcare costs were included: accident and emergency visits, emergency transport by ambulance, inpatient care stays, day cases, and outpatient care visits. In terms of social care, costs associated with stays in a nursing or residential care home were included. Hospital care resource use was obtained from computerised patient hospital records from the date of first ever incident ACLVI event until death or 5 years after the event, whichever was first.Patients' centralised Hospital Episodes Statistics (HES) records, as well as those from the Oxford University Hospitals NHS Foundation Trust  and nine Oxfordshire community hospitals, were reviewed. Information on any accident and emergency visit, emergency transport, outpatient care visit, day case, or hospitalisation was obtained. For each hospital admission, information was recorded on the date of admission and discharge, including the dates of transfers between different specialty wards. Hospitalisations during which patients were admitted and discharged on the same day were classified as day cases. Hospital admissions resulting from the incident ACLVI event were defined as the first admission occurring within 7 days of the event. Subsequent admissions resulting from an ACLVI event were defined as: 1) admissions within 7 days of any subsequent ACLVI event; 2) admissions within 7 days of any subsequent intervention or investigation because of ACLVI events; and 3) hospital admissions  immediately after incident or subsequent ACLVI events, or because of interventions or investigations. For the purposes of this study, long-term institutionalisation was defined as admission into a nursing or residential care home. Date of institutionalisation was obtained from patients' study, primary, and hospital care records.Regardless of when hospital resources were consumed by patients, all resource use was priced using 2015/16 unit costs. All healthcare unit costs were derived from the schedule of National Health Service reference costs.In the main analysis, costs incurred by patients after the first year have not been discounted to present value terms. However, sensitivity analyses are presented in which costs incurred after the first year were discounted using a 3.5% annual rate to reflect current UK recommendations .As part of the analysis, the 5 year care costs after an ACLVI event were compared with those after stroke,All costs were converted from UK pounds sterling (£) to Euros (€), using the rate of purchasing power parities in 2016 .Time to institutionalisation was defined as the difference between the date on which the incident ACLVI event occurred and the date of admission into a nursing or residential care home. The 5 year risk of institutionalisation in a long-term care home was evaluated using Kaplan–Meier techniques, adjusted for mortality. Statistically significant differences in risk between subgroups were assessed using Cox's proportional hazards model.•All cases, in which mean annual costs for all patients, including patients who died, are presented;•Surviving available cases, in which mean annual costs for surviving patients with complete data for that year are presented; and•Only 5 year surviving cases, in which mean annual costs for 5 year survivors only are presented.All 5 year costs are presented as means together with SD and 95% CI under the Central Limit Theorem hypothesis. Given the high 5 year mortality after an ACLVI event,t test or analysis of variance to examine the differences between/among stratified groups. Using the same baseline variables, a generalised linear gamma model with log identity was used to assess the independent baseline predictors of total costs after the event.The main predictors of hospital care costs at 5 years were assessed. By means of a univariate analysis, the importance was assessed of each baseline variable at determining hospital costs after the incident ACLVI event. The baseline variables included were: age; gender; history of each of smoking, diabetes mellitus, hypertension, hyperlipidaemia, atrial fibrillation, heart failure, coronary artery disease, previous stroke or TIA, and stable PAD; type of ACLVI event ; severity of ischaemia on admission; and deprivation (as measured using the index of multiple deprivation by postcode of residence at event onset). For all continuous variables (age and index of multiple deprivation), appropriate cut off points were used to stratify patients. All comparisons between groups were performed using two tailed Student In addition, using both uni- and multivariate analysis, the impact of subsequent ACLVI events and surgical and endovascular interventions was assessed on hospital costs. However, as many of these interventions are not performed in patients with AVI, for a more meaningful interpretation of these costs, only patients with limb ischaemia were included (i.e. ALI and CLI).t test was used to compare differences in hospital, long-term institutionalisation and overall mean costs between patients with an ACLVI event and patients with stroke. Statistical significance was set at p < 0.050. Model specification was tested using the Ramsey reset test, a general specification test for the linear regression model used to test whether non-linear combinations of the fitted values helped explain costs.A two tailed Student n = 174). Half the sample  had a confirmed history of stable PAD, and a significant proportion of patients had other previous symptomatic vascular disease  were ALI, 71 (20%) AVI, and 199 (57%) CLI. The mean age was 76 (SD 12) years, with males accounting for half the sample , of which 18% (€6443) were due to long-term institutionalisation admission and 72%  were hospital care costs . As showp < 0.0001). Although patients with AVI had the lowest overall 5 year costs, they had the highest long-term institutionalisation costs, with half of all costs  incurred from institutionalisation, with AVI patients facing a higher risk of institutionalisation in the first 6 months after an incident ACLVI event  for AVI; €24,437  for ALI; and €46,281  for CLI  as opposed to 33% (n = 27) for ALI and 21% (n = 42) for CLI (p < 0.001), who had the highest overall costs. Overall, 5 year life expectancy was 1.25 years after AVI; 2.62 years after ALI; and 2.76 after CLI , as did patients with diabetes mellitus  and hyperlipidaemia  when compared with ALI, whereas AVI patients incurred significantly lower costs . Age and previous history of heart failure were also found to be significant predictors of reduced overall costs over 5 years, which could be explained, as well as the lower costs incurred by AVI patients, by high case fatality rates (p = 0.014) than those without the condition, partly reflecting the association with CLI.As with the univariate analysis, ACLVI event type was a significant independent predictor of total 5 year costs . After aty rates . Patientp < 0.0001, p = 0.026) and AVI .Average 5 year hospital care costs for the 89 patients who experienced one or more subsequent ACLVI event types after the incident event were €52,649, compared with €20,655 for those who did not . Patients undergoing a surgical or endovascular intervention incurred significantly higher costs at 5 years than those who did not  had at least one endovascular or surgical intervention. A considerable proportion of patients underwent angioplasty or stenting ( did not .p < 0.001) and €25,692 (p < 0.001), respectively, over 5 years.For patients with limb ischaemia, after adjusting for the type of limb ischaemia event (i.e. ALI or CLI) and subsequent surgical interventions, subsequent ACLVI events did not significantly increase 5 year hospital care costs . Endovasp = 0.004, p = 0.010 and p = 0.0004, respectively).Between April 2002 and March 2007, there was complete 5 year follow up for 793 patients with a first-in-study-period stroke. The mean 5 year total hospital care costs after stroke were €22,623 , €6145 lower than those after an ACLVI event . Overall, when including hospital and long-term institutionalisation care costs, 5 year total care costs after an ACLVI event were similar to those after stroke .Five year costs of long-term institutionalisation after stroke were €12,769 , which was significantly higher than the 5 year institutionalisation costs after an ACLVI event , and highest costs after CLI . With very high mortality rates shortly after the event, patients with AVI, and to a lesser degree ALI, were less likely to incur costs over the long-term. As the present study was undertaken using a long-term time perspective, it was possible to compare the costs incurred at each follow up year. This showed that over half of total costs (54%) were incurred over the first year following the incident ACLVI event.p = 0.004). Despite higher mortality rates than for stroke, the increased costs after ACLVI events are, in part, explained by the high rates of procedures in these patients. In OXVASC, 82% of patients with limb ischaemia underwent either endovascular or surgical procedures. As shown in the multivariate analyses, these interventions significantly increased costs over 5 years, with every above knee amputation increasing care costs by €25,692 in patients with limb ischaemia. By contrast, 5 year long-term institutionalisation costs were significantly lower after an ACLVI event than stroke . Therefore, it was found that, overall, 5 year total care costs after an ACLVI event were comparable with those after stroke . Given that the costs of stroke have been widely assessed in a number of countries and settings,Given that 5 year hospital care and long-term institutionalisation costs after stroke have previously been evaluated in the same patient population using the same methodology and unit costs to value resource use,Although the present study did not compare the care costs following stroke and an ACLVI event with those after myocardial infarction (MI) in OXVASC, previous studies have shown that the care costs of stroke are higher in the long term than those of MI.In addition to previously noted limitations of the study,Current European guidelines on peripheral arterial disease summarise the evidence base for diagnosis and management of acute and critical limb ischaemia and acute visceral ischaemia, and identify gaps in evidence, pointing out that contemporary data on the epidemiology and “real world” outcomes of peripheral arterial disease are lacking.The present results also highlight the considerable cost differences observed across ACLVI events, but also show that when taken together the hospital care costs after these events are greater than those after stroke and remained comparable even after long-term institutionalisation costs were taken into account.None.Wellcome Trust (WT095626), Wolfson Foundation, Dunhill Medical Trust (OSRP2/1006), and the National Institute for Health Research (NIHR)Biomedical Research Centre, Oxford. PMR has a NIHR and Wellcome Trust Senior Investigator Award. The funding sources had no involvement with the studyThe Oxford Vascular Study has been funded by the


Moreover, we find out that under the slow switching PJ, increasing ρ may either enhance or degrade the BER.This paper considers the DCSK system under jamming environment. We consider both fast and slow switching pulse jamming (PJ). We derive approximations of the system bit error rates (BERs) in analytic expressions containing only well-known special functions, from which we show that for both jamming cases increasing the spreading factor can enhance the BER. In addition, we reveal that under the fast switching PJ, the BER does not depend on the jamming duty cycle  Chaotic communications (CC) has recently been considered as a promising alternative to the conventional direct sequence spread-spectrum (SS) systems. The basic idea of CC is to replace pseudo-noise sequences by chaotic sequences, which can be directly generated by chaotic maps. Among various chaotic maps, logistic has been extensively exploited due to its simplicity and good performance . ChaoticThe fundamental coherent and non-coherent modulation schemes are chaotic shift keying (CSK) and differential CSK (DCSK), respectively. The performances of chaotic systems with CSK and DCSK modulation schemes, referred as CSK and DCSK systems, over the additive white Gaussian noise (AWGN) channel are extensively investigated in –6]. It . It 6]. Although the noise performances of coherent and non-coherent systems have been well studied –6], ver, ver6], E [xk] = 0, β, called spreading factor. In the second time slot, either the reference sequence or its inverted version is transmitted depending upon the information bit. As a result the transmitted signal of the lth bit, bl, can be expressed asThe considered system consists of a pair of source-destination and a jammer. The DCSK scheme is employed for modulating and demodulating legitimate signals. In addition, the logistic map is exploited for generating chaotic sequences. A mathematical representation of the map is given by xk+1=1-2sk is affected by pulse jamming and additive white Gaussian noise (AWGN). The channel with jamming and AWGN is considered here so that we can solely focus on the effect of jamming on the system performance. This assumption has also been adopted in several related works, i.e. . Th. Thsk isPj/ρ in the equivalent baseband discrete-time model, where Pj is the average power of the jammer , and var[⋅] denote the probability, expectation, and the variance, respectively. Let’s first consider the FSPJ case, in which the mean and variance of the output of the correlator yl can be derived by considering the following two cases: λ ≤ β and λ > β.Although the F-distribution based approach for deriving an exact BER of a DCSK system is presented in , its resmated as BERl=Pr = 0, E [Cjl] = 0, andi = {1, 2, 3}, j = {1, ⋯, 5}, andFrom xk, jk, and the AWGN are all zero, we can readily prove that the covariance between any pairs of {Al, Bl1, Bl2, Bl3, Cl1, Cl2, Cl3, Cl4, Cl5} is zero. Consequently, we obtainIn addition, by exploiting the fact that the means of lth bit BERl. In addition, from the fact that BERl does not depend on l, the whole system error probability BER will be the same as BERl and is given as followsFinally, replacing the results given in ρ ≤ 0.5, the system BER is independent of ρ.When ρ > 0.5, increasing its value degrades the system BER. The reason is that ρ enlarges in β and 2lβ − χ. χ is used here to make sure that every symbol is jammed. Depending upon the value of Tstart we can have the following cases: only chaotic sequences are jammed, only information bearing sequences are jammed, two sequences of every symbol are jammed, and chaotic and information bearing sequences belonging to any two consecutive symbols are jammed. In the figure, S and T denote simulation and theory, respectively. It is shown that regardless of the value of Tstart, our findings about the effect of the FSPJ on the system performance still hold and our analytic results still follow the simulated ones tightly. In addition, we observe that increasing Pj/Ps degrades the system performance, as expected.In Pj/Ps under the STJ, FSPJ, and the SSPJ environments in Pj/Ps. However, when Pj/Ps goes large, the FSPJ causes a much larger BER than that provided by the slow switching counterpart. The reason is that in the low region of Pj/Ps, the effect of jamming is dominated by that of the AWGN. On the contrary, in the high region of Pj/Ps, the effect of jamming becomes superior, and since under the FSPJ scenario it is most likely that every symbol is jammed, the corresponding error probability is larger than that caused by the SSPJ. Finally, the figure shows that as ρ tends to 1, the two BERs under the two PJs converge to each other and they approach the BER under the STJ in the high region of Pj/Ps.We illustrate the system BER versus β = 100. We observe that the DCSK scheme provides the worst performance, the NR-DCSK scheme provides the best performance, and the I-DCSK and the SR-DCSK give similar performance. In addition, it is noticed that the four scheme follow the same trend as the ratio Pj/Ps increases.Finally, we present a comparison of the performance of the DCSK system and its derivatives under the FSPJ environment in ρ when ρ ≤ 0.5, however when ρ > 0.5, the system BER is degraded when ρ increases. Moreover, we pointed out that under the slow switching pulse jamming scenario, increasing ρ may either improve or decrease the system BER. Furthermore, our simulation pointed out that the single-tone jamming is a more effective jamming type than the two pulse jamming counterparts. For our future works, we may consider the application of chaotic communications in physical layer security to enhance the secrecy performance of wireless communication systems ..ρ when ρ


Hepatic glycogen storage diseases (GSDs) are a group of rare genetic disorders in which glycogen cannot be metabolized to glucose in the liver because of enzyme deficiencies along the glycogenolytic pathway. GSDs are well‐recognized diseases that can occur without the full spectrum, and with overlapping in symptoms.We analyzed a cohort of 125 patients with suspected hepatic GSD through a next‐generation sequencing (NGS) gene panel in Ion Torrent platform. New variants were analyzed by pathogenicity prediction tools.n = 53), followed by Ib (n = 23). The most frequent variants were p.Arg83Cys  and p.Gln347*  in G6PC gene, and p.Leu348Valfs  in SLC37A4 gene.Twenty‐seven new variants predicted as pathogenic were found between 63 variants identified. The most frequent GSD was type Ia  for diagnosis. The molecular diagnosis of hepatic GSDs enables the characterization of diseases with similar clinical symptoms, avoiding hepatic biopsy and having faster results. These defects are caused by pathogenic variants that result in enzymatic deficiencies for glycogen breakdown or synthesis, or problems in proteins that regulate glycogen metabolism. The consequence is accumulation of glycogen in tissues, especially in liver , and all patients and guardians provided written informed consent for participation.Were analyzed 125 patients with clinical symptoms of hepatic GSD. Blood samples were collected in EDTA vacuum container. DNA was extracted with Easy‐DNA Purification kit (Thermo Fisher). DNA samples were quantificated in NanoDrop 1000 (Thermo Fisher) and through Qubit dsDNA HS Assay Kit (Thermo Fisher).The gene panel amplicon was designed with Ion Ampliseq Designer software (Thermo Fisher), and included the exons and flanking 40 bp into introns of 11 genes involved in hepatic GSD (Table ® (Varstation) were used to detect and classify variants. To determine the variants causing disease, the following were considered: ACMG guideline , Variant Effect Predictor (Ensembl), Ion Reporter (Thermo Fisher) and VarstationValidations of NGS results were realized by Sanger sequencing in patients and in parents when the sample was available. The unbiased capture and deep coverage of each coding exon and adjacent intronic region of all genes in this panel ensure accuracy of variant detection.3We analyzed 125 patients with clinical suspicion of hepatic GSD. All samples were successfully sequenced. We found 63 different variants in 110 families, and 27 of those were new variants Tables .n = 48, RJ n = 2, MG n = 10, ES n = 3), 50 from the south , eight from the northeast , two from the Midwest  and two from the north (PA n = 2) . In five patients, no variant was identified Appendix . All ideEight families included in this study had multiple affected individuals. For patients 5, 11, and 12 (11 and 12 are sisters), their parents reported consanguinity Appendix . These iSixty‐three alleles were identified, in which 26 are missense variants (41.2%), 16 are nonsense variants (25.3%), six are splice site variants (9.5%), 11 deletions (17.4%), three insertions (4.7%) and one duplication (1.5%) , 23 with GSD Ib (18%), 14 with GSD III (11%), two with GSD VI (1.6%), 16 GSD IXa (12%), six with GSD IXb (4.8%), six with GSD IXc (4.8%), and five were not diagnosed (4%) Appendix .G6PC gene, causing GSD Ia. The other frequent variant, p.Leu348Valfs in SLC37A4 gene, was observed in 10% of alleles causing GSD Ib.The most frequent variants in patients were p.Arg83Cys, observed in 39 alleles (18.5%), and p.Gln347* present in 14 alleles (6.6%), both in Variants not described in the literature were evaluated for protein impact by nine in silico pathogenicity prediction algorithms. All new missense variants were predicted as pathogenic. In the bioinformatics analyses of new splice site variants, all were confirmed to modify the exon–intron structures in different forms importantly, showing sufficient entropy forces to perform the incorrect splicing.4This is one of the largest screening of variants causing the different forms of GSDs in patients, including 125 patients and describing 63 different variants, of which 27 are novel.The GSD Ia and GSD Ib represent 60% of the analyzed patients. In other analyzed cohorts, Vega et al.  reportedG6PC is in the active center of the enzyme G6Pase and presented no detectable activity in transient expression assays  in GSD Ib. This variant was present in 39% of Serbians patients , since GSD type IX had never been described in Brazilian patients.AGL. This gene has the highest number of gaps in coverage of NGS and is one of the biggest genes in panel, with 36 exons. However, the gaps were analyzed by Sanger sequencing and no variant was found. The results obtained from these patients are contradictory, since the variants found in both cases are described for GSD type III; however both patients presented inconsistent clinical findings. One of them has Down syndrome and liver histology similar to GSD III, but no biochemical results are compatible with the disease and the patient does not present any clinical symptoms. The other patient presented hypoglycemia from birth, however, currently associated with hyponatremia and metabolic acidosis. The liver biopsy was inconclusive and not suggestive of GSD. Therefore, it is possible that variants or technical artifacts are eliminating the amplification of the mutated allele or the variants are in regulatory regions, not covered by the panel, but the absence of disease is a possibility  instead of two. Both patients presented variants in In five patients with no identified variants, the clinical suspicions are mild or inconclusive, once they did not have clear clinical indications or laboratory findings supporting the diagnosis of GSD, besides hypoglycemia and/or hepatomegaly. The NGS was a diagnostic exclusion test; therefore, it was an expected result. These patients probably do not have GSD, once hepatomegaly and hypoglycemia are difficult to distinguish from other metabolic storage disorders without more clinical findings. Another possibility is the presence of variants in nontargeted deep intronic and regulatory regions , which does not justify the disease.The patient 120 is possibly a GSD patient because he presented clinical symptoms such as hypoglycemia and ketonuria but no other clinical signs, however only synonymous variants were found in This variety of results reflects the profile of an extremely large country with an interesting and important mix of people from all over the world. The presence of immigrants from the most diverse origins, such as Africans, Asians, and Amerindians justifies the variability of alleles found in a highly mixed population. Genetic analyses indicate that Latin Americans trace their ancestry mainly in the intermixing of Native Americans, Europeans, and Sub‐Saharan Africans. Historically, Latin America has a continuous, differential, and diverse intra‐ and intercontinental migration events, and presents higher prevalence of metabolic diseases (Adhikari, Chacón‐Duque, Mendoza‐Revilla, Fuentes‐Guajardo, & Ruiz‐Linares, Among the advantages of NGS diagnosis, patients undiagnosed by traditional means were investigated and correctly diagnosed in the present study. This method is especially promising for mixed populations with high level of heterogeneity. This method also allows the identification of unexpected diagnoses in the supposed typical phenotypes. Rare genetic diseases can be a diagnostic challenge, sometimes an odyssey. The NGS technologies can provide a fast diagnosis, advantages for treatment management, in reproductive choices, genetic counseling, and fertility services (Schofield et al., This diagnosis is an important advancement for patients with nontypical forms of disease, especially for those who need agile actions, since it evaluates 11 genes at the same time.This was an important step to increase the knowledge about the genetics of the different types of hepatic GSDs in Brazilian patients, since they have a genetically heterogeneous origin, and it is reflected in the variability of types and variants (Vega et al., G6PC in case of GSD Ia or p.Leu348Valfs in SLC37A4 for GSD Ib. Sanger sequencing approach is the most cost‐effective to solve up to 40% of the cases. However, for the cases without prevalent mutations or without suspected type of GSD, NGS is the most effective solution.Differentiated therapeutic management among GSD justifies the population characterization of patients. If NGS analyses are not available or expensive, the molecular diagnosis should be conducted first through the search for the pathogenic variants p.Arg83Cys and p.Gln347* in This study emphasizes that molecular genetic analysis is a reliable and convenient alternative to the assay of enzymatic activity in a fresh liver biopsy specimen for the diagnosis of GSDs. This type of study is an important tool for the estimation of disease progression, since different types of GSDs present variations in their clinical course and treatment, besides serving as a basis for genetic counseling and prenatal diagnosis.The discovery of a significant number of new mutations reinforces the allelic variability of different GSDs and proves that the diagnosis of GSDs in Brazil can be challenging, showing the validity of NGS gene panel use for diagnosis.The authors declare no conflict of interest.  Click here for additional data file.


Mops condylurus) trapped in the city of N’Zerekore and in a nearby village.In 2018, a previously unknown Ebola virus, Bombali virus, was discovered in Sierra Leone. We describe detection of Bombali virus in Guinea. We found viral RNA in internal organs of 3 Angolan free-tailed bats ( Ebolavirus (family Filoviridae), Bombali virus (BOMV), was discovered in Sierra Leone (Chaerephon pumilus (little free-tailed bat) and Mops condylurus (Angolan free-tailed bat). Both bat species are widespread in Africa, and their ranges include countries where human Ebola virus disease (EVD) outbreaks have occurred. Forbes et al.  and in a pool of liver and spleen tissues (Ct 28.2) of an M. condylurus bat from a school in the city of N’Zerekore in March 2019  in China, and another study showed that 3 distinct groups of unclassified filoviruses are circulating in Eonycteris spelaea and Rousettus spp. fruit bats in China (Marí Saéz et al. (EBOV IgG was detected in the human population of Sierra Leone in 2006, 8 years before the EVD outbreak began in that country (M. condylurus bats provides additional confirmation that BOMV could amplify in these bats and that this species is a reservoir host of this virus.Although BOMV had been detected in the northern part of Sierra Leone (Mops condylurus bats, Guinea.Additional information on Bombali virus in


On July 24, 2017, the Ohio Department of Health (ODH) was notified of a positive rabies test result from a domestic cat in Summit County, a county considered free from terrestrial rabies. Oral rabies vaccination (ORV) of raccoons, in the form of consumable bait, is conducted each year along the Ohio-Pennsylvania border to prevent the westward expansion of the raccoon rabies virus variant (RVV). In the United States, several distinct rabies virus variants exist; raccoon RVV is enzootic along the eastern parts of the United States (from Florida to Maine), including several counties in northeast Ohio . ODH iniIn early February 2017, two adults traveled from North Carolina to Arkansas with two dogs and 13 cats. The stray cats (including cat A) were taken in as personal pets while the couple lived in North Carolina. They departed Arkansas on February 17, and arrived in Summit County, Ohio, for temporary residence with family members on February 21. The cats were co-housed in a garage until May 3. During that time, the cats were not permitted out of the garage, and no known exposures to other mammals occurred. While housed in the garage, cat A and another cat gave birth to an unknown number of kittens, several of which died of unknown causes.During April 28–May 3, because of inability to care for the animals, the owner surrendered 15 cats to the Humane Society of Summit County , including cat A and her four kittens. Upon intake, the animals received a physical exam, routine vaccines, an antihelminthic, flea and tick control, and feline immunodeficiency virus and feline leukemia virus testing. Because the rabies vaccine is administered at time of spay/neuter at this facility, cat A did not receive a rabies vaccine. Cat A and her four kittens were placed in a temporary home during May 5–July 11 to allow the kittens to be weaned. Cat A was examined by a veterinarian on May 24, June 1, and June 9 for an upper respiratory tract infection. On July 8, cat A was reported to be “making abnormal vocalizations,” which were attributed to estrus. Upon return to the shelter on July 11, no abnormalities were noted in either cat A or the kittens. On July 18, almost 5 months after arriving in Ohio, cat A developed abnormal behavior including excessive panting, agitation, open-mouthed breathing, and hind-limb ataxia. Despite treatment, symptoms progressed rapidly and cat A was euthanized on July 20. Although no one was bitten and there was no evidence that cat A had been exposed to a rabies vector species, the cat’s head was submitted for rabies testing. The ODH laboratory reported a positive rabies result on July 24, the same day the sample was received. After consultation with CDC, a brain tissue sample was submitted for further characterization to determine the likely origin of the virus. Antigenic typing confirmed that the virus was raccoon RVV, and molecular characterization of the rabies virus N-gene determined that it was most similar to a clade found in central North Carolina, suggesting that cat A had been infected before being translocated to Ohio.ODH developed a risk assessment tool to identify persons and animals potentially exposed to cat A during the viral shedding period (July 8–20), defined as 10 days before development of clinical signs (July 18) until the time of the cat’s death (July 20) , Animal and Plant Health Inspection Service Wildlife Services commits over $20 million annually to oral rabies vaccine (ORV) programs in the eastern United States. In Ohio, the ORV zone covers several counties in the northeastern part of the state. Vaccine-laden baits are dropped in these areas to create a barrier of immunized animals that aids in reducing the number of animal rabies cases and prevents the westward spread of raccoon RVV. Enhanced rabies surveillance is conducted along the ORV zone, which includes Summit County, to ensure that ORV breaches are quickly recognized and confirmed; risk assessments are conducted to guide the necessity and scope for emergency management action. When breaches occur, emergency vaccination operations are typically conducted by USDA in collaboration with federal, state, and local partners.Phylogenetic and epidemiologic evidence was used to rule out a breach by natural expansion; in this case, human intervention appears to have been responsible for the translocation of raccoon RVV from North Carolina (an enzootic state) into Summit County, Ohio, a county free from the viral variant. Molecular characterization of the virus variant was essential in determining the source of the virus, which guided public health intervention efforts on local, state, and federal levels. Had the evidence suggested a natural expansion event, an emergency ORV baiting operation would likely have been initiated. However, given that the translocation event was likely human-mediated, and the event was quickly contained, additional ORV baiting was deemed unnecessary.Human-mediated translocation  of rabid animals has occurred in the past. These events can have profound public health implications, as seen by the translocation of a rabid raccoon from Florida to Virginia in the 1970s and the translocation of a rabid raccoon from Vermont to Hamilton, Canada, in 2015  up to the time of surrender in May. Therefore, cat A likely was infected with the raccoon RVV as a stray followed by a minimum 5-month incubation period, which is longer than the typical incubation period of 3 weeks to 3 months (None of the 13 translocated cats had a known history of rabies vaccination. Because of the public health and agriculture risks associated with translocation of unvaccinated rabies-susceptible animals, numerous federal and state animal movement laws have been enacted. These include requirements for rabies vaccination before interstate travel and procurement of a health certificate from a veterinarian (Translocation of wildlife is responsible for spreading rabies in the United States, and has led to the introduction of new rabies variants and establishment of terrestrial rabies in areas where it was previously undetected.While incubating the rabies virus, a cat potentially traveled through eight states, three of which are terrestrial rabies free. If the cat had become infectious during travel, a rabies epizootic could have occurred.Local rabies vaccination laws vary greatly and animals frequently cross state lines without proper veterinary care and medical documentation. Animal rabies testing, prompt investigation, interagency collaboration, and the utilization of molecular epidemiology are important in determining proper public health interventions.