Human movement is accomplished through muscle contraction, yet there does not exist a portable system capable of monitoring muscle length changes in real time. To address this limitation, we previously introduced magnetomicrometry, a minimally-invasive tracking technique comprising two implanted magnetic beads in muscle and a magnetic field sensor array positioned on the body’s surface adjacent the implanted beads. The implant system comprises a pair of spherical magnetic beads, each with a first coating of nickel-copper-nickel and an outer coating of Parylene C. In parallel work, we demonstrate submillimeter accuracy of magnetic bead tracking for muscle contractions in an untethered freely-roaming avian model. Here, we address the clinical viability of magnetomicrometry. Using a specialized device to insert magnetic beads into muscle in avian and lagomorph models, we collect data to assess gait metrics, bead migration, and bead biocompatibility. For these animal models, we find no gait differences post-versus pre-implantation, and bead migration towards one another within muscle does not occur for initial bead separation distances greater than 3 cm. Further, using extensive biocompatibility testing, the implants are shown to be non-irritant, non-cytotoxic, non-allergenic, and non-irritating. Our cumulative results lend support for the viability of these magnetic bead implants for implantation in human muscle. We thus anticipate their imminent use in human-machine interfaces, such as in control of prostheses and exoskeletons and in closed-loop neuroprosthetics to aid recovery from neurological disorders. This technique senses myoelectric signals riations . While svia sEMG , even myvia sEMG  cannot fvia sEMG . Recent via sEMG . HoweverTo address this need, we recently developed a strategy for tracking muscle tissue lengths. This technique, known as magnetomicrometry (MM), uses implanted pairs of magnetic beads to wirelessly track muscle tissue lengths in real time see  Taylor . In paraTowards translating MM for human use, we here develop an improved coating strategy for 3-mm-diameter spherical magnetic bead implants and a translatable surgical protocol for implantation. With this new strategy, we apply a first coat of gold and a top coat of Parylene to the magnetic beads, following stringent medical device standards for coating, cleaning, and sterilization. We then address three features that are essential for the clinical use of magnetic bead implants: 1) lack of implant discomfort 2) lack of implant migration, and 3) implant biocompatibility.These implants are intended for permanent implantation like the tantalum beads used in radiostereographic analysis (RSA). Since the standardization of RSA in 1974 , tens ofin vivo turkey model to implant magnetic bead pairs in the right gastrocnemius muscle, and we analyze the percentage of stride time in stance for walking and running before and after implantation.Changes in movement patterns have been routinely analyzed to capture the secondary musculoskeletal effects of discomfort or pain, commonly referred to as antalgic gait . Followiin vivo turkey model to implant magnetic bead pairs in the lateral gastrocnemius and tibialis cranialis muscles, and we monitor long-term magnetic bead positions for migration.To provide a consistent measurement of tissue length and to ensure patient health, the magnetic implants must 1) not move relative to the surrounding tissue, 2) not move relative to one another, and 3) not migrate out of the muscle. Spherical steel beads and cylindrical titanium-encapsulated magnetic beads implanted individually into the tongue muscle in animal models proved to be stable against migration in short-term studies (6–24 days) . In prevThe biocompatibility of magnetic bead implants must also be verified before clinical use. In previous work, as a proof of concept, we used non-clinical-grade Parylene-coated magnets (7 μm coating thickness) with a non-clinical-grade insertion process, and we found only minor inflammation . Recent Meleagris gallopavo, adult, seven male [7.7–11.32 kg], three female ) from local breeders and cared for them in the Animal Care Facility at Brown University on an ad libitum water and poultry feed diet.We implanted magnetic beads in ten wild turkeys over an eight-month period. All turkey experiments were approved by the Institutional Animal Care and Use Committees (IACUC) at Brown University and the Massachusetts Institute of Technology. We obtained the wild turkeys  and albino guinea pigs  were obtained from Charles River Laboratories and maintained in the WuXi AppTec animal facility according to NIH and AAALAC guidelines on an ad libitum water and certified commercial feed diet.All biocompatibility testing protocols were reviewed and approved by the WuXi AppTec IACUC prior to the initiation of testing. Twelve rabbits and seventeen guinea pigs were used in the biocompatibility testing portion of this work. Albino rabbits  and plated with nickel-copper-nickel see . This baTo ensure the implants were free from debris, SCS shipped the Parylene-coated beads in double vacuum packaging to KKS Ultraschall AG, who prepared the implants for magnetization by cleaning and packaging them in polyvinyl chloride tubes and caps received from SM Magnetics. Specifically, in their ISO-8 clean room, KKS ultrasonically cleaned these tubes and caps, inserted 32 beads into each tube, placed the caps on each end, and individually double-packaged each tube into two sealed, well-fitting bags. SM Magnetics then placed each double-packaged tube into a magnetizing coil and used a first magnetizing pulse to align the beads, followed by a second magnetizing pulse to magnetize the beads.via pressing and shearing, then autoclaved the bead set. For outside laboratory biocompatibility testing (WuXi AppTec), HBI inserted the beads into these cartridges  directly from the tubes via pressing and shearing, triple-packaged each cartridge into a clamshell container, and sterilized each bead set via gamma sterilization before providing them to the biocompatibility testing facility.To prepare the beads for surgical insertion, Halifax Biomedical, Inc. (HBI) manufactured cartridges to hold the biocompatibly-coated magnetic beads see . For theWe contracted HBI to manufacture a customized magnetic bead insertion device see  using thvia surface anatomic landmarks and further confirmed its location via ultrasound. We then identified specific insertion sites within the muscle, ensuring an intermagnet distance greater than 3 cm, and marked these planned insertion sites with a sterile operative pen. At each insertion site, we made a 1 cm length incision through all layers of skin using a No. 11 surgical scalpel. We then dissected through the underlying subcutaneous fat layer down to the underlying fascia, then incised the fascia with the No. 11 surgical scalpel to expose the underlying target muscle. Finally, we made a 1 cm length incision through the epimysium to expose the underlying muscle fibers.We performed all implantation procedures under sterile conditions in spaces approved for veterinary interventions. For each procedure, we placed the turkey under general anesthesia and draped the turkey in typical sterile fashion, as described in previous work . We idenAfter loading a magnetic bead set into the Halifax Insertion Device, we marked the planned implantation depth for each insertion site  on the shaft of the insertion device and on a pair of fine surgical scissors (using a sterile operative pen and sterile ruler). With the aid of the fine surgical scissors, we pretunneled the intramuscular insertion path to the marked depth. We then placed the shaft of the Halifax magnetic bead inserter into the pretunneled path, using the depth marking as a guide. Keeping the insertion device in position, we deployed each magnetic bead implant by firmly grasping the insertion device and inserting the pushrod into the device until the knob of the pushrod contacted the cap, then withdrew the insertion device with the pushrod still in contact with the cap. Additional surgical instruments were utilized as necessary due to animal-specific anatomy .via MM and ultrasound, approximated the skin edges at each implantation site using 4-0 polypropylene interrupted percutaneous stitches, then confirmed the magnetic bead positions once again.After insertion, we reapproximated the epimysial edges at each site using 4-0 chromic interrupted stitches. While suturing, we took care to not bring ferromagnetic surgical instruments closer than necessary to the implants to avoid inadvertent displacement of the magnetic beads. Finally, we confirmed the implantation depth and distance between the magnetic beads We defined comfort as the absence of change in the percentage of stride time in the stance phase for the walking and running animals following magnetic bead implantation. Following the same techniques listed above, we implanted a magnetic bead pair in the mid-belly of the right gastrocnemius of the three female turkeys.To minimize variability in movement patterns, we trained each turkey to walk and run on an enclosed treadmill at 1.5, 2.0, 2.5, 3.0, and 3.5 m/s. In total, there were 15 training sessions: nine times prior to implantation and six times following implantation. During training, we randomized treadmill speed every 2 min for a total of 10 min. We collected pre-implantation biomechanics at session 9 and post-implantation biomechanics at session 15 (3 weeks post-implantation) on a high-speed camera  at 120 Hz. For each speed, we visually confirmed that the turkey’s movement pattern had stabilized before collecting data.We also manually identified toe strikes and toe offs for at least 20 consecutive gait cycles at each speed, during selected periods of gait that visually minimized irregular movements . Post process, we confirmed consistent movement patterns by excluding strides that were greater than three standard deviations from the mean percentage of right leg stride time in stance, then subsequently replaced inconsistent strides with the next stride recorded.post-hoc Tukey tests identified pairwise differences between individual speeds. We report effect sizes as ηp2 and Cohen’s d for main effects and pairwise comparisons, respectively. Values of d > 0.2, 0.5, and 0.8 indicate small, moderate, and large effects, respectively . A linear mixed model tested for the fixed main effect of speed  and fixed main effect of unilateral implants  on the percentage of right leg stride time in stance. Using the theory of generalized estimating equations , the linectively . All staIn the seven male turkeys, we intentionally placed the magnetic beads at a range of distances and in multiple muscles to explore different circumstances in which the magnetic beads might migrate toward one another. The distance between magnets was measured as described in previous work , except To analyze tissue response to the clinical-grade implants inserted using the Halifax magnetic bead inserter, in-house observations were made first, with a total of eight histological samples taken from four of the male turkeys, each at 8 months post-implantation. Histology was performed as in previous work , with thTo test the device biocompatibility under good laboratory practice (GLP) compliance , we submitted fully manufactured magnetic bead sets and insertion devices to WuXi AppTec for intramuscular implantation, cytotoxicity, intracutaneous irritation, and sensitization testing. All magnetic beads used in the testing were deployed from the magnetic bead cartridges using the insertion device, and all tests were conducted in compliance with international standard ISO 10993-12:2012, Biological Evaluation of Medical Devices, Part 12: Sample Preparation and Reference Materials.To test for local effects of the magnetic beads contacting skeletal muscle, three magnetic beads and three negative control articles  were implanted into the left and right paravertebral muscles, respectively, in nine rabbits. Four rabbits were used for the two-week implantation test, and the remaining five rabbits were reserved for the twenty-six-week implantation test. At the test endpoints, the muscles were explanted, fixed in 10% neutral buffered formalin, and sent to the study pathologist for analysis.via clinical, gross, and histopathologic information. Each site was examined microscopically and scored for inflammation and tissue response on a scale from 0 (no inflammation/response) to 4 (severe inflammation/response). The average of the magnetic bead site scores minus the control article site scores was used to determine an irritant ranking score, with a negative value considered 0.0. As directed in the ISO standard, a score from 0.0 up to 2.9 was considered to indicate that the magnetic bead implants were non-irritant. The intramuscular implantation tests were conducted in accordance with the ISO Test Method for Implantation in Muscle, ISO 10993-6: 2016, Biological Evaluation of Medical Devices - Part 6: Tests For Local Effects After Implantation.Each implantation site was analyzed 2/1 ml (surface area per volume) in Eagle’s minimal essential medium (E-MEM) supplemented with 5% volume-per-volume fetal bovine serum (FBS) for 72 h at 37°C, then shaken well. A negative control (HDPE) extraction, positive control  extraction, and cell control were also prepared in parallel to and under equivalent conditions as the magnetic bead extraction.To test for toxicity of the implants to mammalian cells, 54 magnetic beads were extracted at a ratio of 3 cmCompleted extractions were added to fully-formed cell culture wells containing L-929 mouse fibroblast cells  after removal of the culture maintenance medium. Specifically, 1 ml of each extraction was inserted into each of three cell culture wells, and the cell culture wells were incubated in a humidified atmosphere for 72 h.In Vitro Cytotoxicity.The cell cultures were examined microscopically and scored for cytopathic effects  at 24, 48, and 72 h on a scale from 0 (no reactivity) to 4 (severe reactivity). As directed in the ISO standard, a score of 0, 1, or 2 was considered to indicate that the magnetic bead implants were non-cytotoxic. This cytotoxicity testing was conducted in compliance with ISO 10993-5:2009, Biological Evaluation of Medical Devices, Part 5: Tests for 2/1 ml (surface area per volume) into each of 5.1, 8.1, and 10.1 ml of 0.9% normal saline  and 5.1, 8.1, and 10.1 ml of sesame oil . These extractions were freshly prepared for corresponding phases of the test and were performed over 72 h at 50°C, with agitation during the course of the extraction. Vehicle controls (containers of liquid without magnetic beads) were prepared in parallel to and under equivalent conditions as the magnetic bead extractions.To test for the induction of allergic reactions, magnetic beads were extracted at a ratio of 3 cmOne-tenth of 1 ml of each extraction was injected intracutaneously into the dorsal dermis of eleven guinea pigs (one on each side of the midline). Adjacent these first two injections, two additional 0.1 ml injections of an immunostimulant  were injected subcutaneously. An additional 0.05 ml of each extraction was then mixed with 0.05 ml of the immunostimulant, and each was injected subcutaneously adjacent the first injections. All six injections were repeated using negative (vehicle) controls in place of the magnetic bead extractions in each of six additional guinea pigs. Six days post-injection, the injection sites were treated with 10% sodium lauryl sulfate in mineral oil. After 24 h, the sites were then cleaned, and a 2 cm × 4 cm filter paper saturated with magnetic bead extract  was applied dermally to the site for 48 h, then removed.After an additional 15 days, two 2 cm × 2 cm filter papers saturated with magnetic bead extract and vehicle control, respectively, were applied dermally to the right and left flanks, respectively, of each animal for 24 h, then removed. At 24–48 h after patch removal, these 2 cm × 2 cm exposure sites were observed and scored for irritation and sensitization reaction on a scale from 0 (no erythema/edema) to 3 (intense erythema/edema). As directed in the ISO standard, a score of 0  was considered to indicate that the magnetic bead implants did not elicit a sensitization response. This sensitization testing was conducted in compliance with ISO 10993-10: 2010, Standard, Biological Evaluation of Medical Devices, Part 10: Tests for Irritation and Skin Sensitization.2/1 ml (surface area per volume) into each of 6 ml of 0.9% normal saline (64 magnetic beads) and 6 ml of sesame oil (64 magnetic beads). These extractions were performed over 72 h at 50°C, with agitation during the course of the extraction, and vehicle controls (containers of liquid without magnetic beads) were prepared in parallel to and under equivalent conditions as the magnetic bead extractions.To test for local irritation of dermal tissue due to potential extractables or leachables, magnetic beads were extracted at a ratio of 3 cmOne milliliter of each extraction and of each vehicle control was then injected intracutaneously into the dorsal dermis in each of three rabbits. Specifically, the magnetic bead extractions were injected to the right of the midline, and the vehicle controls were injected to the left of the midline. Each extraction was delivered as five 0.2 ml injections at locations spatially distributed along the cranial-caudal axis, with the normal saline extractions being delivered medial to the sesame oil extractions.The injection sites were observed and scored for gross evidence of erythema and edema at 24, 48, and 72 h on a scale from 0 (no erythema/edema) to 4 (severe erythema/edema). As directed in the ISO standard, a difference in score of less than 1.0 between the magnetic bead extraction and vehicle control extraction was considered to indicate that the magnetic bead implants met the requirements of the intracutaneous reactivity test. This intracutaneous irritation testing was conducted in compliance with ISO 10993-10: 2010, Standard, Biological Evaluation of Medical Devices, Part 10: Tests for Irritation and Skin Sensitization.p = 0.234, ηp2 = 0.002) or a significant speed-surgery interaction effect  on the percentage of right leg stride time in stance , five strides were replaced due to irregular movement . Linear mixed model analysis revealed a significant main effect of speed  on the percentage of right leg stride time in stance. The percentage of right leg stride time in stance significantly decreased with increasing speed .The percent of the total stride time spent in stance during treadmill running was evaluated before and after surgery for the leg that received implants (the right leg) in three turkeys. We did not observe a significant surgery main effect  for the magnetic bead implants, indicating that the fully manufactured magnetic beads in this study are non-irritants as compared to the control articles. No abnormal clinical signs were noted, and no difference was noted macroscopically between the test and control sites in any of the rabbits. All rabbits survived to the scheduled study endpoint.The cytotoxicity test resulted in a score of 0 for the magnetic bead extraction at 24, 48, and 72 h, indicating that the magnetic bead implants of this study are non-cytotoxic under the conditions of the cytotoxicity test. The negative and cell controls also received scores of 0 , while the positive control received a score of 4 (severe reactivity and complete or nearly-complete destruction of the cell layers), indicating that the test was functioning normally.The sensitization test resulted in a score of 0 for dermal observations of all exposure sites for both the normal saline and sesame oil extractions, indicating that the magnetic bead implants did not elicit a sensitization response, and thus that the implants are non-allergenic under the conditions of the test. No abnormal clinical signs were noted, and all animals survived to the scheduled study endpoint.The intracutaneous irritation test resulted in a comparative score of 0.0 for erythema and edema against the vehicle control for both the normal saline and sesame oil extractions, indicating that the magnetic beads met the requirements of the intracutaneous reactivity test, and thus that the implants are non-irritating under the conditions of the test. No abnormal clinical signs were noted, and no dermal reactions were observed at the test or control sites in any of the rabbits at any of the observation points. All animals survived to the scheduled study endpoint.In this study, we investigate the clinical viability of magnetic bead implants in muscle. Using a specialized device to insert magnetic beads into muscle in avian and lagomorph models, we collect data to assess bead migration, gait metrics, and bead biocompatibility. We find that implanted-leg stance time is preserved from pre- to post-implantation of the magnetic beads in muscle, migration does not occur when the magnets are implanted a sufficient distance from one another, and no inflammation occurs in response to the magnetic bead implantation. In addition, GLP-compliant testing confirms that the implants are non-irritant, non-cytotoxic, non-allergenic, and non-irritating. These results suggest that MM is a viable approach to muscle tissue length tracking in humans.In support of our hypothesis, we found no evidence of magnetic bead implant discomfort from measures of pre- and post-implant gait. Following a series of training sessions pre- and post-implantation, we did not observe an effect of surgery on the percentage of right leg stride time in stance across a range of walking and running speeds. The absence of any sign of antalgic gait in a relatively small animal 3 weeks following implantation supports the idea that the implant would not cause significant discomfort in humans, where the implant would represent a much smaller fraction of muscle volume.In previous work, no magnets migrated toward one another from an initial separation distance of 2.15 cm or above . Due to The migration model used here to predict the migration cutoffs may help in understanding one aspect of migration but is insufficient on its own to fully capture what is happening within the body with magnetic bead implant pairs. While in this study we employed the same material type for coating as in previous work, our updated coating, cleaning, and insertion processes resulted in no observed inflammation, which may have reduced the likelihood of migration. Variations in the magnetization strength of each implant due to magnetic bead size tolerances and magnetic material alignment may also be a cause for both in-study and cross-study variations, as could small differences in the surgical procedure . Finally, our model holds as a fundamental assumption that the maximum contraction ratio is equivalent between any two beads, regardless of implant location, suggesting that the minimum distance between the beads occurs during full muscle contraction and can be predicted from the distance between the beads at rest. However, muscle contraction is not spatially uniform , which weakens this assumption when the beads are not placed in precisely the same locations within the muscle. Further, this assumption is also weakened by the possibility of subject-to-subject variability in contraction ratios. Importantly, a highly conservative estimate of the migration threshold should be used to account for the many biological factors of the implants and implantation process. As such, we maintain the importance of an initial 3 cm separation distance at the time of implantation for these 3-mm-diameter spherical magnetic beads to ensure stability against migration.all other implants for all possible joint configurations, including distinct muscle contraction states resulting in all possible joint configurations. We wish to also underscore the importance of implanting at, or beyond, the minimum distance threshold even when only a single magnetic bead is implanted per muscle, as has been proposed in previous work , operation of local machines , and control of oneself or one’s tools in an augmented or virtual reality environment. Muscle length control could also enable improved navigation in a weightless environment, such as in control of underwater thrusters or air thrust rotors. Specifically, muscle tissue length tracking ubmerged . In all Each pair of magnetic beads should ideally be implanted along a muscle fascicle to enable biomimetic control. In addition, to maximize signal-to-noise ratio for any MM-based control strategy, the magnetic beads should ideally be implanted as maximally separated as possible within the muscle fascicle. via muscle tissue vibration has been well tested via vibramotors on the skin surface, and works by activating muscle spindles to deliver the sensation of illusory joint positions and movements (Magnetic bead implants may also be used for delivering proprioceptive feedback. Illusory kinesthetic feedback ovements . Howeverovements . For traWhen implanted next to cutaneous receptors, such as in fingertips, magnetic implants can confer cutaneous sensing of low-frequency magnetic fields . SeekingIn this work, we manufacture clinical-grade magnetic bead implants and develop a clinical-grade implantation strategy, and we verify implant comfort, stability against migration, and biocompatibility. Our results demonstrate that when implanted as discussed here, these magnetic beads are viable for use in human muscle.


Little evidence is available on the epidemiological and economical dimensions of certain foodborne diseases such as wild mushroom poisoning. This study aimed to investigate the epidemiology and estimate the costs of poisoning with cyclopeptide-containing mushrooms in Kermanshah province in 2018. In this study, poisoning was investigated in different subgroups. The cost of illness method with a bottom-up approach was used to estimate the poisoning costs. Both direct and indirect costs of the poisoning were included in the analysis. The perspective of the study was society. Required data were obtained from the medical records of Imam Khomeini hospital and completed through a line survey with the patients. Two hundred eighty-three patients were poisoned in Kermanshah due to poisoning with cyclopeptide-containing mushrooms. Of 283 patients, 143 (50.53%) were men and 59.01% of patients were rural residents. About 43% of admissions were out-patient cases and ~40% of patients were hospitalized within 1–3 days. Also, eight patients were pronounced dead in the area. The total cost of poisoning with cyclopeptide-containing mushrooms in Kermanshah province was $ 1,259,349.26. Of that, $ 69,281.65 was related to direct medical costs and $ 10,727.23 was direct non-medical costs. The indirect costs of death were estimated to be $ 1,125,829.7. The current study revealed that there is a significant financial burden due to cyclopeptide-containing mushrooms on patients, the health system, and society as a whole. Further studies are recommended to clarify the epidemiological and economic burden of foodborne illnesses related to wild mushroom poisonings. Sharing the outputs with health authorities, and informing the general public are warranted to reduce the burden of such diseases. Foodborne diseases (FBDs) are a main public health threat estimated to be the consequence of more than 600 million morbidities and 420,000 mortalities in the world annually, accounting for about 33 million Disability Adjusted Life Years (DALYs) . The estMushroom poisoning outbreaks occur frequently following the consumption of wild mushrooms that have been grown due to seasonal climate changes . Most caAmanita  ] , 10.The cyclopeptide-containing mushrooms are often mistakenly eaten instead of edible mushrooms. Since the toxins are heat stable, consuming in either raw or cooked way cannot abrogate the nature of toxicity . Importapoison and remedy.One might speculate that preventing the consumption of wild mushrooms is the best strategy to avoid morbidity and mortality of poisonous ones. However, the behavior of harvesting and consuming the wild mushrooms may be normal in some parts of the world to the extent that they may be categorized as mycophilic  or mycophobic . People of mycophilic regions are extremely eager in consuming wild mushrooms that originated in their long history and culture while the mycophobic inhabitants scarcely harvested and consumed wild mushrooms . UnfortuLepiota brunneioncarnata was determined by the Department of Botany, Iranian Research Institute of Plant Protection, Agricultural Research, Education and Extension Organization, Tehran, Iran). Nevertheless, the epidemiological and economical dimensions of these cases have not been addressed in the past. Therefore, this study aimed to determine the epidemiology and estimate the economic burden of these poisonings. The findings of the present study will help health policymakers to establish a better preventive program and take more robust measures.In the spring of 2018, an unprecedented foodborne outbreak of mushroom poisoning occurred in Iran, with more than 800 poisoned cases of which more than 300 cases occurred in Kermanshah province, in the west of Iran. This epidemic phenomenon was the result of climate changes and exceptional precipitations accelerating the growth of cyclopeptide-containing mushrooms in Kermanshah  method based on the human capital method was used .Costs were calculated using a bottom-up and prevalence approach. It should be noted that in cases such as food poisoning due to the short period of the disease, incidence, and prevalence are almost the same. The bottom-up approach usually starts from a specific population group with the disease in question and records all costs associated with that disease. Then, the costs of this subgroup are used to estimate the total costs of the disease in the population. In this study, we calculated the total cost of the disease from the perspective of society. The total economic burden of illnesses consists of two parts, one being the direct costs and the other the indirect costs :(A) Direct costs: These included both medical and non-medical costs. Direct medical costs measure the economic resources used to treat poisoning, namely, out-patient, in-patient, medicine, and other medical costs. Direct non-medical costs included transportation costs, travel costs to medical centers, and accommodation costs in other cities for treatment.(B) Indirect costs: Human capital approach (HCA) was used to calculate the indirect economic burden of poisoning. In HCA, the value of human capital depends on the future income of patients. In this method, the period that the patient loses due to absence from work is used as a basis for the calculation of indirect cost. Therefore, the higher the income and the longer the absence from work, the higher the indirect cost of the disease. In this study, indirect costs were divided into indirect costs caused by lost days (due to disability and receiving treatment) for patients and caregivers and GDP lost due to premature death. Indirect costs for each family depended on daily income and sick leave, average daily income for each caregiver, and the length of time absent from work for nursing and patient care. The gross domestic product was used to calculate the potential lost production due to premature death. GDP per capita in Iran was obtained from World Bank data .1. Production lost due to premature death: for measuring the production lost the following formula was used:W is GDP per capita, g is the average rate of economic growth in Iran (3.7%), i is a year lost due to premature death, and r is the discount rate (5%). GDP per capita was considered 6,000 $ according to Iran's GDP in 2017.Where 2. Production lost due to disability or seeking treatment: This cost is calculated by multiplying the number of hospitalization days or days lost (due to disability or/and seeking treatment) for patients and caregivers by the average daily wage for them.3. Other indirect costs: Includes some costs such as burial expenses that were calculated for the deceased. Finally, by summing up all types of production lost costs, the total production lost due to poisoning with mushrooms was calculated. For all types of direct and indirect costs, average costs were calculated by different subgroups of patients. Each US dollar is equal to 32,000 Iranian Rials in this study.According to data extracted from hospital records, 283 patients were referred to Imam Khomeini hospital in Kermanshah due to poisoning with cyclopeptide-containing mushrooms. Of these, 143 (50.53%) were men. The highest incidence of poisoning was in the age group of 20–39 years, and the lowest was in persons over 60 years old. Out of the total referrals, 43.5% were out-patient and ~40% of patients were hospitalized within 1–3 days. A total of 59.0% of patients were rural residents. Eight patients died due to poisoning. A total of 52.43% of the 185 patients who were interviewed by telephone were men. The characteristics of patients are shown in According to hospital records, $ 67,385.10 was spent in the hospital for out-patient and in-patient treatment, of which $ 6,573.52 was franchised. The average cost per patient was $ 223.13 shown in Per capita, direct medical costs were US$ 236.18 for men and US$ 253.63 for women. The direct non-medical costs for men and women were US$ 33.40 and US$ 42.87, respectively. By age, the highest per capita, direct medical costs (US$ 677.99) and direct non-medical costs (US$ 56.97) were in the age group of 60 years and older. Per capita, direct medical and non-medical costs were positively correlated with a prolonged hospital stay, with per capita, direct medical costs being only US$ 53.93 for out-patient cases, compared with US$ 744.49 for patients hospitalized for 7–14 days. Per capita, direct medical costs were $ 189.34 for the rural residents and US$ 324.67 for the urban population. Per capita, non-medical direct costs were higher for rural than urban (US$ 71.85 vs. US$ 19.52). Direct medical and non-medical costs for the deceased were significantly different from other patients. Per capita, direct costs were $ 268.80.Regarding the indirect costs associated with disability, women (US$ 91.12) 13- to 19-year-old patients (US$ 115.20), patients with hospitalization duration of 7–14 days (US$ 355.65), and rural patients (US$ 92.77) had higher costs than their counterparts. Per capita, the indirect cost of disability for patients and caregivers was US$ 85.95 and US$ 26.94, respectively. Other indirect costs  were US$ 1,757. Per capita, direct medical and non-medical costs and indirect costs are shown in Four men and four women died due to poisoning. Production lost due to premature deaths was US$ 1,125,829.70. The highest cost of death was in the age group of 59–40 years shown in The total cost of poisoning with cyclopeptide-containing mushrooms in Kermanshah province was US$ 1,259,349.26. Of that, US$ 69,281.65 was related to direct medical costs , and US$ 10,727.23 was direct non-medical costs. The indirect costs of death were estimated to be US$ 1,125,829.7. The total direct and indirect costs are shown in Altogether, premature death imposed 90% of the costs of mushroom poisoning, followed by direct medical costs .Food poisoning through the consumption of wild mushrooms is considered a public health concern worldwide. However, epidemiological data related to poisonous wild mushrooms vary depending on social factors such as food culture, eating habits, and tendency to be mycophilic, and environmental conditions such as favorable weather and rainfall in different parts of the world. Countries, namely, South Korea, China, Thailand, Russia, Switzerland, Turkey, Nepal, and Italy reported different morbidity and mortality associated with the use of poisonous mushrooms , 16–22.The pattern of wild mushroom poisonings in Iran, as an Asian country, reported different rates of morbidity and mortality in different provinces in previous studies. Pajoumand et al. by reviewing 72,421 poisoned cases of all causes admitted in Loghman-Hakim Hospital Poison Center from 1992 to 2002 found that 0.05% (37 patients) of the population study was due to wild mushroom poisoning .Another study conducted in Mashhad, Iran, from February 2005 to 2011 shows that mushroom poisoning caused a high mortality rate (22%), despite low frequency  . A recenn = 8) died due to severe consequences of the disease.Our study indicated that of the total of 283 poisoned patients admitted to Kermanshah referral hospital due to poisoning with cyclopeptide-containing mushrooms in spring 2018. Of them, 143 (50.53%) were men and 59.01% of patients came from rural areas. About 43% of admissions were out-patient patients and ~40% of cases were hospitalized within 1–3 days. Also, an amount of 2.83% of the population study  and occurred mostly in remote, less developed areas in which people believed that consuming wild mushrooms picked from nature is good for their health . ResearcAltuntas et al. in their study of the records of patients who were admitted to an emergency department of a University hospital in Turkey between January 2008 and December 2012 reported a total of 420 poisoned cases with wild mushrooms. The ratio of men to women was 1/1.5. The patients had a mean age of 46 years. Wild mushroom poisoning of composed of 13.3% of other posing cases. Of the patients, 47.6% were rural residents whereas 38.6% were urban and 13.8% live in city centers. The majority of admissions occurred in autumn (57.6%). Eighty-six percent of poisoning cases were due to the consumption of wild mushrooms picked from nature. Even though most of the patients show mild gastrointestinal symptoms and were discharged after supportive therapy, two cases died and ten cases were referred for liver transplantation. They concluded that wild mushroom poisoning can bring about the critical clinical outcome and thus the public needs to be educated regarding the potential adverse consequences of taking wild mushrooms that are often paper in nature . AnotherA. phalloides and related species) had occurred. In sum, 14 poisoned patients reported being admitted for medical treatments. Even though 11 patients recovered after receiving medical treatment, three patients needed liver transplantation as a result of fulminant hepatic failure and one patient succumbed to permanent neurological damage despite receiving liver transplantation. Therefore, the average length of stay in the hospital was as high as almost 9.36 days indicating that this wild mushroom poisoning imposed a relatively long stay in the hospital   .To our knowledge, this is the first COI study on the economic impact of wild mushroom poisonings in our country and also worldwide. We adopted the COI method to estimate the economic burden of the mentioned disease because it is one of the most popularly accepted methods employed to estimate the total economic costs of a disease . We estiSut et al. estimated the average ICU costs for acute poisonings per day as $344 while the cost per ICU stay was valued as high as $821 that they concluded that was lower in comparison with the US costs (the US $925) in Turkey during 2002–2006. Their study shows that admitting acute poisoning cases imposed a substantial cost on the healthcare system in particular ICU wards in Turkey .In our study, the total costs of the mushroom poisoning outbreak were estimated to amount of $ 1,259,349.26. We found that the amount of $ 69,281.65 was allocated to direct medical costs. The amount of $ 10,727.23 was related to direct non-medical costs. The indirect costs of death were estimated to be $ 1,125,829.7.Descamps et al. calculated the direct costs of poisoned cases admitted to a referral university hospital in Ghent, Belgium, for both the government and the patients in 2017. They found that a total of 1,214 cases were admitted due to acute poisoning, of which 96.9%  were under the cover of insurance . Of all admitted patients, 54.2% received out-patient care, 24.7% were forced to stay for 24 h in the emergency department for close observations, 17.8% were hospitalized, and 3.3% were referred to the intensive care unit. The total direct cost for the treatment of all cases of poisoning  was $1,830,870, of which 13.1% were allocated to the emergency department costs and 86.9% went to hospitalization costs. They show that the cost of poisoning is considerable for the government and patients and that the length of hospital stay and accommodation caused a significant share of all costs. However, they did not include the indirect costs of poisoned patients and thus they may underestimate the economic burden of poisoning on their society .Krajewski et al. by investigating an analysis of poisoned cases admitted in Illinois hospitals in 2010, found that there was a total of 425,491 poisoned cases that 222,339 of them were in-patients (52.3%) with an average length of stay of 5.5 days in the hospital. The average total hospital costs was $ 3,398 ± 4,081 in out-patients  and $ 32,391 ± 60,115 for in-patients . The total hospital cost for all out-patient cases was $ 690,400,129 and $ 7,201,628,216 for in-patient patients which translated to an estimate of about $7.9 billion in the total cost .Our study shows that the two major shares of costs of mushroom poisoning outbreaks are attributed to the premature deaths resulting in the loss of productivity, and then medical costs. Premature death imposed 90% of the costs of mushroom poisoning, followed by direct medical costs . These findings are in agreement with previous economic studies on foodborne outbreaks –40. Of nOur study shows that there is a significant financial burden due to cyclopeptide-containing mushrooms on patients, the health system, and society as a whole. The present study results will be a useful tool for health policymakers to establish suitable frameworks for the prevention and control of seasonal food poisonings related to the ingestion of cyclopeptide-containing mushrooms in the future. Further studies are needed to elucidate the epidemiological and economic burden of foodborne illnesses related to wild mushroom poisonings. Providing gratuitous consultation from mycology and toxicology expects for educating people to be able to differentiate between poisonous and edible mushrooms before harvesting and consumption, can reduce the burden of wild mushroom poisonings.The raw data supporting the conclusions of this article will be made available by the corresponding authors with reasonable requests.The studies involving human participants were reviewed and approved by the Ethics Committee of Kermanshah University of Medical Sciences (IR.KUMS.REC.1397.427). The patients/participants provided their written informed consent to participate in this study.BK: conceptualization, methodology, funding acquisition, and writing—review and editing. MA-S: investigation, methodology, and writing—review and editing. SR: formal analysis, software, and validation. AH: visualization and data curation. AK-K: supervision, project administration, software, and funding acquisition. All authors contributed to the article and approved the submitted version.This study was extracted from the Kermanshah University of Medical Sciences approved project and was funded and supported by the Research Deputy of Kermanshah University of Medical Sciences (Grant No. 97459).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Patients with fibromyalgia report persistent widespread pain, fatigue, and substantial functional limitations, which may lead to high health resource use (HRU) and lost productivity. Previous analyses of the U.S. population have not examined the direct and indirect costs of fibromyalgia by severity level.To assess (a) HRU, direct and indirect costs associated with fibromyalgia in routine clinical practice in the United States using a patient-centric approach, and (b) the relationship of fibromyalgia severity level to HRU and costs.This study recruited a nonprobability convenience sample of 203 subjects aged 18 through 65 years between August 2008 and February 2009 from 20 U.S. community-based physician offices. Subjects had a prior diagnosis of fibromyalgia by a rheumatologist, neurologist, or pain specialist; received treatment at the enrolling physician’s practice for at least 3 months; experienced widespread pain for at least 3 months; and experienced pain in the previous 24 hours. Subjects completed a 106-item patient questionnaire that included 5 validated health-related quality-of-life instruments and study-specific questions about demographics; clinical history; overall health; treatment satisfaction; and impact of fibromyalgia on cognitive function, daily activities, and employment status. Subjects also self-reported hours of unpaid informal caregiver time because of inability to perform daily activities , out-of-pocket expenses for medical and nonmedical services, and lost productivity related to fibromyalgia for the previous 4 weeks. The 20-item Fibromyalgia Impact Questionnaire total score was used to stratify subjects into fibromyalgia severity groups . Staff at each site recorded clinical characteristics, HRU, and medication use attributable to fibromyalgia on a paper clinical case report form (CRF) based on a 3-month retrospective medical chart review. Unit costs for 2009 were assigned to the 3-month HRU data reported on the CRF and 4-week subject-reported lost productivity. Costs were then annualized and reported in the following categories: direct medical, direct nonmedical, and indirect. Differences across severity levels were evaluated using the Kruskal-Wallis test (continuous measures) and Pearson chi-square or Fisher’s exact tests  at the 0.05 alpha level.Of the 203 subjects, 21 (10.3%) had mild, 49 (24.1%) had moderate, and 133 (65.5%) had severe fibromyalgia. For subjects with mild, moderate, and severe fibromyalgia, respectively, the number of fibromyalgia-related medications  and office visits to health care providers  significantly differed across severity levels. Across severity levels, total medical and nonmedical out-of-pocket costs also differed (P = 0.025). Mean [median] 3-month total direct costs  were $1,213 , $1,415 , and $2,329  for subjects with mild, moderate, and severe fibromyalgia, respectively (P = 0.002); and mean [median] 3-month indirect costs  were $1,341 [$0], $5,139 , and $8,285  (P  less than  0.001). Mean total indirect costs accounted for 52.5%, 78.4%, and 78.1% of mean total costs for subjects with mild, moderate, and severe fibromyalgia, respectively.Direct and indirect costs related to fibromyalgia are higher among subjects with worse fibromyalgia severity. Indirect costs account for a majority of fibromyalgia-related costs at all fibromyalgia severity levels.


This study estimates neonatal costs to hospital discharge for this group of very preterm babies in England.Babies born between 27Retrospective analysis of resource use data recorded within the National Neonatal Research Database.Neonatal units in England.+0 and 31+6 weeks of gestation in England and discharged from a neonatal unit between 2014 and 2018.Babies born between 27Days receiving different levels of neonatal care were costed, along with other specialised clinical activities. Mean resource use and costs per baby are presented by gestational age at birth, along with total costs for the cohort.Based on data for 28 154 very preterm babies, the annual total costs of neonatal care were estimated to be £262 million, with 96% of costs attributable to routine daily care provided by units. The mean (SD) total cost per baby of this routine care varied by gestational age at birth; £75 594 (£34 874) at 27 weeks as compared with £27 401 (£14 947) at 31 weeks.Neonatal healthcare costs for very preterm babies vary substantially by gestational age at birth. The findings presented here are a useful resource to stakeholders including NHS managers, clinicians, researchers and policymakers. Existing cost estimates for very preterm care in England are now over a decade old and may not reflect modern care practices.This study has generated current cost estimates at the level of the individual baby and for the cohort as a whole in England and confirmed the previously reported inverse relationship between healthcare costs and gestational age at birth.The outputs from this work provide a valuable resource for research assessing the economic implications of interventions to prevent preterm birth, the provision of care for preterm babies, as well as helping inform National Health Service resource allocation decisions.+0 and 31+6 weeks of gestation  represent the largest group of very preterm babies requiring National Health Service (NHS) care.Babies born between 27Previous work in the UK and elsewhere has attempted to estimate the healthcare and societal cost of preterm birth.https://www.imperial.ac.uk/neonatal-data-analysis-unit/). All NHS neonatal units in England, Scotland, Wales and the Isle of Man use their Electronic Patient Record (EPR) supplier systems to routinely submit detailed information to the NNRD on the clinical care they provide to babies. Submitted data are quality assured and curated to a research standard. Data comprise demographics, diagnoses, health outcomes and daily interventions  and treatments administered during the inpatient episode.For this retrospective analysis, we included all admissions to neonatal units in England for babies born at 27–31 weeks who were discharged or died between January 2014 and December 2018. Admissions were identifiable through the NNRD, which was created in 2007 to support activities including audit, evaluations and clinical, health services and policy research and is maintained at the Neonatal Data Analysis Unit at Imperial College London  was available for each day of the admission episode. Babies missing daily intervention records, or for whom daily records were available, but level of care data had not been recorded, were excluded from the primary analysis.The cost analysis was conducted from the perspective of the NHS in England. To calculate the costs associated with the routine daily neonatal care received by each baby, we multiplied the number of days spent receiving each level of care, by level-specific national average bed day costs sourced from the 2018/2019 National Schedule of NHS Costs.10.1136/bmjpo-2022-001818.supp1Supplementary dataper diem unit costs. Surfactant replacement and palivizumab were costed when given using unit costs specific to a baby’s weight. Types of neonatal surgery were identified from their corresponding Operating Procedure Code Standard (OPCS) and the International Statistical Classification of Diseases and Health Related Problems (ICD-10) codes for costing purposes and unit costs obtained from the 2018/2019 National Schedule of NHS Costs. Unit costs for these non-routine procedures are detailed in Not all major clinical activities are included in these neonatal critical care HRGs. After in-depth discussions with clinical experts, we identified five high-cost non-routine procedures captured within the NNRD, that were considered to be important in this particular population. These were nitric oxide, surfactant replacement, total parental nutrition (TPN) (over 14 days of use), palivizumab use and surgical care. Days receiving nitric oxide and TPN (beyond 14 days) were costed using When describing neonatal and maternal characteristics, counts and proportions and means and SD were used for categorical and continuous variables, respectively. Analyses of resource use data and costs were performed by gestational age at birth. Means (SD) were used to summarise days provided at each level of care with associated 95% CI following a Poisson distribution. Counts were made of the numbers of babies for whom non-routine procedures were recorded, along with the number of cases  or days  for which such treatment was given. Total costs were reported for the cohort as a whole and again by gestational age at birth. A secondary analysis used multiple imputation to impute routine care costs at each level  for babies with missing daily record and level of care data  and those with missing data on the level of daily care provided (n=377), a total of 28 173 babies  remained and were included in the cost analysis (full details of the record matching process and data cleaning is found in The mean (SD) cost per baby for the various levels of care and non-routine procedures are shown by gestational age at birth in The overall total cost estimated for the complete case cohort (n=28 154) over the 5 years from 2014 to 2018  revealed the Canadian cost estimates to be consistently lower across all gestational age groups.et al reported consistently lower mean lengths of stay for all gestational ages compared with our estimates due to their focus on intensive care units and the exclusion of the costs of care in lower dependency neonatal units. Thus, the Rios et al estimates do not fully capture the true costs of healthcare provision for this group of babies.A number of studies have previously estimated neonatal care costs for preterm babies across a range of gestational ages.In 2009, Mangham and colleagues used a decision analytic model to estimate neonatal costs for a hypothetical cohort of preterm babies born in England and Wales.This study makes a number of contributions to the published literature. First, existing UK cost estimates for neonatal care following very preterm birth are over a decade old and so are unlikely to reflect current standards of practice.A number of limitations must also be acknowledged. This study considered only healthcare costs associated with the initial period of hospitalisation, though the economic consequences of preterm birth extend over prolonged periods of time, in some cases over the lifetime of the individual.This study has generated up-to-date estimates of the costs of providing neonatal care to very preterm babies in England. Resource use and costs increase as gestational age decreases. The outputs from this work can be used to inform clinical and budgetary service planning and ensure the efficient allocation of healthcare resources. The estimates will also be of interest to countries with neonatal care provision similar to England.


International health agencies, working in concert, bridge the gaps in health care for populations at risk for RG-4 viral pathogenexposure. RG-4 virus research incorporates Biodefense Program and Biosafety Laboratory (BSL)-4 technologies. RG-4 viruses include Arena- Economic, social, and ecological instability, human migration, global warming, and vector and reservoir spread, all contribute to increased numbers of emergent and re-emergent viruses, including RG-4 viruses. Very few laboratories can work withRG-4 viruses since such research is restricted to Biosafety Level 4 (BSL4) laboratories.  The NIH viridae. [A few examples of emerging and re-emerging potential pandemic viruses include human immunodeficiency virus (HIV) in the1980s, severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV-2) in 2003, and re-emerging influenza virus pandemicsin 1890, 1918, 1957, 1968, and 2010. Further, ZIKA virus in 2017, COVID-19 in 2019 and Monkey pox virus in 2022 were observed. The most dangerous viruses involve all the WHO RG-4 virus pathogens. These virus families include Arena-, Filo-, Flavi-, Herpes-,Nairo-, Paramyxo-, and Pox-Arena-viridae includes Junin, Lassa Fever, Lujo Hemorrhagic Fever, Chapare, Guanarito, Machupo, Sabia, Argentinian Hemorrhagic Fever, Bolivian Hemorrhagic Fever, Venezuelan Hemorrhagic Fever, and Lymphocytic Choriomeningitis viruses.The host range and reservoirs of Arena viruses have expanded to include humans, rodents, bats, ixodid lone star ticks, fish, and reptiles (snakes). Lassa fever virus is endemic in West sub-Saharan Africa with occasional detection in the United States, Israel,the United Kingdom, Canada, Netherlands, Japan, and Germany. In endemic areas, the case fatality rate (CFR) is approximately 1-2% with an annual prevalence of 300,000 estimated infections. However, in hospitals in these areas, the CFR is closer to 69%, whereinfections are confirmed. Moreover, the high degree of virus genome sequence variability may contribute to variations in symptomatology. [Filo-viridae includes Ebola, Marburg, Sudan, Bundibugyo, Ravn, and Tai forest viruses. Of these viruses, Ebola is well-known and there was a large Ebola epidemic in West Africa, 2013-2016. The Ebola CFR ranges from 25-90%. In additionto human virus reservoirs, bats are included as well. In humans, persistent virus infections occur. Subsequent sexual transmission is observed as well, and has been seen up to 500 days after initial transmission. The virus genome undergoes significantsequence variation; thus, quantitative real-time polymerase-chain-reaction assay (qRT-PCR) is limited. To increase the reliability of qRT-PCR and minimize false negatives, two separate genome target sequences are used. Clinical diagnostic methods requireimprovement and vaccine improvements are also in the pharmaceutical pipeline. [ipeline. , 8Flavi-viridae: include Kyasanur Forest disease, Tick Borne Encephalitis, Omsk, Alkhurma, Yellow fever, Zika, Spondweni, West Nile, Yellow fever, St. Louis Encephalitis, Dengue, Japanese Encephalitis, Powassan viruses. These viruseshave a wide range prevalence range, totaling 499 million, with Dengue topping the list at 390 million. Virus reservoirs include humans, mosquitoes, non-human primates, birds, pigs, rodents, lagomorphs , and deer. The members ofthis virus family demonstrate a propensity for mutations in their genomes that are involved with spread among various vector mosquitoes and their hosts which likely increases their ability to infect humans. Global biomedical organizations attempt rapidtracking and containment of any viruses with the potential of becoming the next pandemic. [Herpes-viridae (Herpes B virus) recently caused deaths in China. Herpes B virus, although a benign herpes virus for macaque monkeys, is very pathogenic in humans with a CFR of 70-80%. At least 50 people were infected with Herpes B virusas of 2022. Herpes B virus is also known as Herpesvirus simiae, Macacine herpesvirus 1, Macacine Alphaherpesvirus 1, and Cercopithecine herpesvirus 1 (CeHV-1). In 1932, the first case of human Herpes B virus was reported. The individual diedof progressive encephalomyelitis 15 days after a normal monkey bit him. The virus was named Herpes B virus by Sabin in 1934. It is observed that people who work with macaque monkeys or have them as pets are at risk for Herpes B virus infection.[fection., 11Nairo-viridae genus in the family of Bunyaviruses includes Crimea-Congo hemorrhagic fever, Ganjam, Finch creek, Nairobi sheep disease, Kupe, and Dugbe viruses. Additional sub-groups of Nairo viruses include Hughes, Der Ghazi Kahn,Qalyub, Sakhalin, and Thiafora. The geographic distribution of this virus genus ranges from sub-Saharan Africa to China and is entirely tick-borne. Crimea-Congo hemorrhagic fever, although uneventful in animal's infections, has a human CFR up to30%. This virus is reported in the former Soviet Union, sub-Saharan Africa, Turkey, Bulgaria, Pakistan, India, China, the Arabian Peninsula, Iran, Iraq, and northern Greece. [ Greece. ,13Paramyxo-viridae genus includes Henipa-, Respiro-, Rubula-, and Morbilli-virus groups. Hendra and Nipah viruses (Henipa group) were the first dangerous paramyxoviruses that emerged and were identified in the last century: Hendrain August of 1995 in Australia and Nipah in September 1998 in Malaysia. The related Cedar virus is not as dangerous to humans. This virus genus is zoonotic, and Hendra and Nipah human infections stem from the Pteropus bat species. However,these and other members of the Paramyxo-viruses are spreading to other mammals, and their phylogenetically defined clades are diverging. They are considered at risk of becoming hazardous emergent viruses. Mammals in addition to humans that are being infectedinclude goats, cats, horses, pigs, dogs, and cows. [The nd cows. , 15, 16Pox-viridae family includes the Orthopox genus, which includes Variola, cowpox, Vaccinia, and mpoxviruses. There are additional novel Orthopox viruses under investigation. Variola, as a humanpathogen, emerged from East Africa approximately 3,000 - 4,000 years ago. Smallpox was eradicated by 1980; however, the global prevalence had reached 400 million people prior to its eradication. Mpox was first detected in primates in Denmark in 1959 and isendemic in the Democratic Republic of the Congo, Sudan, Cameroon, Central African Republic, Guinea, and Gabon. Mpox virus related disease recently emerged in clusters in non-endemic areas in what appears to be human to human transmission via close contact.Mpox virus shows neurological involvement and interacts with host immunologic response; in a way it may affect host circadian rhythms. Overall, caveats are manifest, since emergent pox viruses are most likely to continue, despite the eradication of smallpox.[The mallpox., 21Emergent and re-emergent of RG-4 viruses are highly pathogenic and pose profound risks for increased spread among humans, vectors, and animals. Healthcare organizations, international databases, artificial intelligence, and improved Hazmat interventionsare included in the arsenals used by healthcare workers in zones where these viruses emerge. Global health and security also require monitoring RG-4 pathogen potential weaponization threats as well as corresponding countermeasures. To produce molecularmechanisms that inhibit RG-4 viruses, specific gene pathogenic mechanisms need further development. The mechanisms of inhibitions, as well as vaccines and therapeutics, could then be used as countermeasures. Further risk-benefit analyses are needed toappraise laboratories as well as open public debate about RG-4 pathogens.


Extracts of double-stranded RNA from three fig cultivars from an Apulian  germplasm collection were used for high-throughput sequencing and revealed the presence of two distinct, recently described closteroviruses. Sequences obtained from these Apulian isolates belong to fig virus A and fig virus B and cover 38 and 25% of their RNA genome, respectively. Primer sets designed on selected contigs confirmed the presence of each virus in infected plants. A close phylogenetic relationship, investigated in a fragment of HSP70h protein, occurs among these isolates and the reference genomes. A nucleotide divergence  was observed among our isolates and the reference genomes. This is the first finding of these virus species in autochthonous fig accessions in Europe. Ficus carica L.) is considered one of the oldest agricultural practices in the Mediterranean basin [Closteroviridae [Domestication of fig trees . The combination of different co-infecting virus species brings forth an extraordinary range of symptoms  of the condition commonly as known ‘mosaic disease’ . The keyaravirus ).The degraded sanitary status of currently used fig varieties often implies yield losses of an unacceptable level for sustainable cultivation. To avoid the spread of regulated viruses (Regulations EU 2019/2072 and 2018/2019), a generic diagnostic tool to screen the propagation material is needed. High-throughput sequencing (HTS) demonstrates enough sensitivity for the detection of known and the de novo discovery of unknown systemic pathogens in certification programs of vegetatively propagated crops —namely, Closterovirus with a variable identity degree, albeit below the species demarcation threshold [For the Illumina-indexed libraries, an average of 28 million reads was achieved, with more than a million passed reads  per barcode inside each library. Several independent, small-size contigs, obtained from Illumina libraries in 2018, were sorted into two distinct groups; these groups were selectively mapped to members of the genus hreshold . The eviTotal RNA extracted by VdN, VCC and Ric plants was then analyzed by RT–PCR amplification. Some of the DNA fragments amplified from primer sets of two Illumina contigs per each accession, and from one read of the VCC nanopore barcoded library, were cloned in a plasmid vector and conventionally sequenced see . Since tA phylogenetic relationship among selected sequences produced in this study and similar regions of other closteroviruses from the GenBank repository was evaluated. An overlapping portion of 72 amino acids in the partial HSP70h translated proteins of the two related viruses was found in VdN contig 59184 and VCC contig 62228. Sequences of the related viruses, spanning the same borders of the VCC/VdN overlapping fragments, were aligned using the MUSCLE algorithm  and the In the case of Illumina libraries, due to the enriched virus-derived dsRNA fraction that was used as a template for sequencing, a proportion of the assembled contigs belonging to already known fig viruses  were identified.When nanopore-derived reads became available from our samples in 2021, these sequences were used to conduct a new BlastN database interrogation. We gained evidence that the closterovirus-like sequences previously found in those accessions should be attributed to a couple of recently described closterovirus species. These were fig virus A  and fig virus B . This finding was confirmed by the re-analysis of the Illumina contigs.A similar homology between the virus sequences analyzed in our experiment and the fig viruses A and B   from a JA selected array of Illumina-derived contigs from VCC and VdN barcoded libraries, having a significant length  and showing homology with the closer related viruses through Blast search, was listed in 6 passed reads (with a median read length of 315 nt) for the eight different barcodes mixed in the single run, the percentage of total reads from the three fig barcodes gave a similar representation . However, a few reads in the single fig–derived barcoded sequences could be identified by mapping against the investigated virus reference genomes. No assembly could be produced because of the scattered position of the reads along the genomes; however, these reads were large enough to be univocally annotated by BlastN/X. The error rate of the nanopore method, higher than Illumina sequencing, had the major drawback of hampering the precise reconstruction of the coding frame inside the few obtained reads.A different picture is drawn by nanopore sequencing. In the bulk of 4.3 × 10For the Ric accession, three independent virus reads were identified when annotated by BlastN: two were overlapping at the very 5′ portion of the genome (Ric b1f6bb93 and Ric d08096f4), while a third one was located almost in the middle (Ric e739c51c). Conversely, only one read was observed for VCC (VCC read 12eb-c33) and two for VdN  . All theAll the primers designed on VdN Illumina contigs and nanopore reads correctly amplified both VdN and Ric cDNAs, demonstrating that isolates of the same virus were present in both accessions. VdN cloned sequences mapped with the original VdN contigs, with an identity ranging from 95.3 to 98.8%. While no PCR cross-reactivity was detected with specific primers between the two groups (VCC vs. VdN/Ric and viceversa), the cloned sequences generally recognized—when blasted—the heterologous virus at a lower extent than the homologous virus; this was due to the high similarity at the whole genome level (56.7% nt identity) between the two viruses .Closterovirus. The derived bootstrapped dendrogram (Ampelovirus) and blueberry virus A  behave as outgroups. VCC and VdN isolates, each one closer to the homologous virus in a specific node, belong to a phylum formed by two fig-infecting closteroviruses (a predictive single lineage including fig mild mottling-associated virus (FMMaV): fig leaf mottle-associated virus 1 and 3 (FLMaV 1 and 3). This feature may support the hypothesis that an ancestral closterovirus was at the origin of the currently observed species in fig. This cluster thus shares minimal distance and a common ancestor node with those viruses (raspberry leaf mottle virus (RLMV) and blackcurrant closterovirus 1 (BCCV-1)) that occur with major identity scores when many independent contigs, along the whole genome of VCC or VdN, are BlastN annotated.To investigate the phylogenetic relationship of the Apulian FiVA and FiVB isolates, a small stretch at 5′ of hsp70 protein could be used since only two contigs (VCC_62228 and VdN_59184) overlapped in this region and could be compared with the aligned proteins of other members in the genus ndrogram  is in agThe discovery of these isolates of FiVA and FiVB in local fig germplasm  indicates a potentially widespread distribution of these viruses. A genetic divergence of these isolates from those described in South America and Asia, and their relative genetic closeness, is also remarkable. The identified viruses share a co-infection condition with other viral pathogens in affected fig trees and, consequently, no association with specific symptoms could be attributed to these two viruses. Therefore, it was worthwhile to develop molecular tools for their detection in the aim of research and containment purposes.A single accession of each Apulian autochthonous fig cultivar , Vito Carlo Casamassima (VCC) and Ricotta (Ric)) showing mosaic disease symptom, was collected in 2014—in the frame of a Regional Project on the preservation of crop biodiversity—in different locations in Apulia and kept self-rooted in a collection plot at CRSFA . In 2018, to check the global sanitary status of the germplasm collection, leaf tissues (up to 500 mg) were collected from each tree, and total nucleic acid was extracted with guanidine isothiocyanate buffer . The firIn 2021, a new dsRNA extraction from the same two fig accessions, and an additional one from the Ric source, were performed using the protocol described above. Every single dsRNA was separately heat denatured and reverse transcribed for the synthesis of cDNA-PCR barcoded libraries, according to the SQK-PCB109 cDNA-PCR sequencing kit  and using a nonamer anchored primer (5′-ACTTGCCTGTCGCTCTATCTTCNNNNNNNNN-3′)  and the http://hannonlab.cshl.edu/fastx_toolkit/ (accessed on 27 February 2023)), keeping only reads with a Phred score higher than 20 and adapter trimmed. Such reads were then de novo assembled in contigs using Spades v.3.15.3 [−6 and 10−4, respectively, for BLASTn and BLASTx outputs. Bowtie software v.2.3.5.1 [Illumina libraries were checked for their quality using the fastxtoolkit (v.3.15.3  with k-m.2.3.5.1  and Mini.2.3.5.1  were use