SARS-CoV-2 replicates efficiently in the upper airways of humans and produces high loads of virus RNA and, at least in the initial phase after infection, many infectious virus particles. Studying virus ultrastructure, such as particle integrity or presence of spike proteins, and effects on their host cells in patient samples is important to understand the pathogenicity of SARS-CoV-2.Suspensions from swab samples with a high load of virus RNA (Ct < 20) were sedimented by desktop ultracentrifugation and prepared for thin section electron microscopy using a novel method which is described in detail. Embedding was performed in Epon or in LR White resin using standard or rapid protocols. Thin sections were examined using transmission electron microscopy.Virus particles could be regularly detected in the extracellular space, embedded in a background of heterogenous material , and within ciliated cells. Morphology  of virus particles in the swab samples was very similar to particle morphology in cell culture. However, in some of the samples the virus particles hardly revealed spikes. Infected ciliated cells occasionally showed replication organelles, such as double-membrane vesicles. The most common cells in all samples were keratinocytes from the mucosa and bacteria.The new method allows the ultrastructural visualization and analysis of coronavirus particles and of infected host cells from easy to collect naso/oropharyngeal patient swab samples.The online version contains supplementary material available at 10.1186/s12985-023-01981-9. The pandemic Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) primarily infects the respiratory tract . In mostThe upper airways are the best target for an analysis of the virus and the histopathology of the earlier phases of the COVID-19 disease because of the high virus RNA loads that can be measured. Biopsies would be suitable samples for studying such infections (e.g. ). HoweveNaso- and/or oropharyngeal swabs were taken from individuals by different operators using various standard swab collection systems (with and without transport medium). Dry swab brushes were transferred into 1 ml of phosphate-buffered saline (PBS). RNA extraction and qRT-PCR were conducted as described previously . Determi7 RNA copies/ml [g (20 psi) using the fixed-angle A-100/18 rotor . After centrifugation, the supernatant was removed and the airfuge vial was filled with 100 µl of warm (approx. 40 °C) 3% low-melting point agarose, which was solidified on ice. The hardened agarose bloc was removed from the vial with a small spatula and the red pellet was covered by a drop of 3% low-melting point agarose to avoid dispersal during further preparation steps , and twVaccinia virus at defined concentration (106 and 107 virus particles/ml). Virus propagation in cell culture and determination of particle concentration was done as described before [Determination of the detection limit of the entire procedure was done using suspensions of d before . Airfuged before .Transmission EM of the sections was performed at 120 kV  and images were recorded with a side-mounted CMOS camera . Electron tomography was performed at 120 kV using a bottom mounted CCD camera  operated at 2048 by 2048 pixels and the Tecnai tomography software . Reconstruction was done with the Inspect3D software  using the “Simultaneous Iterative Reconstruction Technique “ (SIRT) algorithm  and embedded them in LR White resin  the polA. And th0 min andVaccinia virus suspension at two defined concentrations , while with a suspension of 106 particles/ml only part of the pellets showed virus in the first Section (6 of 8 samples).We selected swab samples which most likely contained a high concentration of virus particles according to the RT-PCR measurements of the virus RNA load. To get an idea about the detection limit of our airfuge sedimentation method, we sedimented /ml; see  for a deConcentration of particles from naso-/oropharyngeal swabs by airfuge ultracentrifugation allowed the visualization of SARS-CoV-2 particles by thin section EM. Airfuge centrifugation in electron microscopy was mainly used in conjunction with negative staining of particles to increase particle number adsorbed at the sample carriers , 28. SamVirus particles could be detected in the extracellular space and within ciliated cells, which is the main target cell of the virus in the upper airways , 10. RemThe swab samples contained other cells and material besides virus particles and ciliated cells, mainly keratinocytes from the upper layers of the epidermis and, mostly coccoid, bacteria. Vesicular and needle-like crystal aggregates formed the background matrix, which most probably derived from cells or the swab material, respectively. The preservation of the keratinocytes, bacteria, viruses and background material was similar in frozen and non-frozen samples, while other cells, in particular ciliated cells, appeared extracted in the swab samples that were frozen. Thus, for optimal preservation of cells, especially if the cell biology of the virus is of interest, fresh swab samples should be considered. For diagnostic purposes, even frozen material is suitable.The coronavirus morphology seemed to be independent from the storage conditions (i.e. frozen or non-frozen) but was sample-dependent. Some samples showed many deformed particles and/or only virus particles without spikes. Deformation of virus particles and spike visibility in infected cell culture are most probably due to false or incomplete assembly at later stages of infection or virus production in cell culture . It can The sedimentation method for swab suspensions presented here is compatible with embedding in epoxy and acrylic resin, including rapid infiltration and polymerization. This allows using the method for rapid diagnostics in cases of emergencies, for instance, the emergence of unknown or unconventional pathogens. However, spike visibility was reduced after osmium tetroxide post-fixation and rapid embedding in the acrylic resin LR White. Block-contrasting with tannic acid and/or uranyl acetate, as performed in the epoxy embedding protocol, could probably help to increase spike visibility, but would increase processing time and limiting the benefit of using LR White. In case of SARS-CoV-2, the reduced spike visibility was not relevant for diagnostics because the viruses could be identified by finding them in intracellular membrane-bound compartments in high numbers which is a typical feature of coronaviruses .Vaccinia virus suspensions of defined concentration, a particle concentration of 107 particles/ml is necessary to allow an immediate detection of viruses without performing an extensive search of several sections at the microscope. At 106 particles/ml not all of the samples revealed viruses in the first section through the pellet, which would require taking more sections and extended time for microscopy. The SARS-CoV-2-positive swab samples used in this study contained RNA loads far above approx. 107 copies/ml . It wasIn summary, we presented a method for thin section EM of naso/oropharyngeal swab samples, which allows the detection and analysis of SARS-CoV-2 particles in the extra- and intracellular context of the acute phase of the infection, thereby complementing studies using autopsies, biopsies and cell cultures. The method could be relevant for the study of further virus variants of concern or of other emerging infectious pathogens affecting the upper respiratory tract, because it offers direct and easy access to the pathogens produced by the natural host including infected or affected cells for the study of their pathogenesis. Moreover, it is likely that the method could be extended to other sample types, such as broncho-alveolar lavage or urine.Additional file 1: Video S1. Recovery of the sediment after ultracentrifugationAdditional file 2: Table S1. Step-by-step protocol of sediment embedding in EponAdditional file 3: Table S2. Step-by-step protocol of rapid sediment embedding in LR White resinAdditional file 4: Fig. S1. Transmission EM of a thin section through the sediment of swab sample 2 (Epon embedding). A A group of SARS-CoV-2 particles (rectangle) is localized within mainly vesicular material and bacteria (ba) of various profile shape. At the lower edge of the image a keratinocyte (kc) is visible. B Enlarged view of the coronavirus particle group marked with a rectangle in frame A. Only few virus particles reveal spikes (arrowheads) and some particles appear deformed (arrows). Scale bar in A = 2 µm, in B = 100 nm.Additional file 5: Fig. S2. Transmission EM of thin sections through the sediment of swab sample 1 (Epon embedding). Single SARS-CoV-2 particles are shown in the vicinity of keratinocytes. A A SARS-CoV-2 particle (rectangle) is localized between two keratinocytes (kc) and a bacterial cell (ba). Inset: Magnified view of the virus particle which reveals the typical features of coronaviruses: i.e. an oval shape and a profile size within the 60–140 nm range, a limiting bio-membrane, club-shaped surface spikes and a granular particle interior. B The rim of another keratinocyte, which reveals characteristic tonofilaments (tf) and a SARS-CoV-2 particle (arrow) close to the cell surface. Scale bar in A = 1 µm, Inset = 100 nm, in B = 200 nm.Additional file 6: Fig. S3 Transmission EM of thin sections through the sediment of the PCR-negative swab sample 10 (Epon embedding). A Overview image of an observation field which shows a keratinocyte (kc) and, in the extracellular space, crystalline (arrow) and vesicular (arrowhead) structures (the image file is available in data set 6). Examples for smaller vesicular profiles, indicated by the three boxes, are magnified in B-D. For reference, virus particle profiles (arrowheads) from PCR-positive swab samples are shown in E-G. All particle profiles indicated by numbers in B-D are in the correct size range of virus particle profiles, but lack other typical features of virus particles: profile 1 lacks the spike proteins and the interior granules are smaller in size and less dense; profile2 lacks a clear membrane envelop, spikes and a granular interior; profiles 3 and 4 lack spikes and a granular interior; profiles 5 lack the typical electron-dense granular interior. Scale bar in A = 2 µm and in B-G = 100 nm.Additional file 7: Fig. S4. Electron tomography of thin sections through the sediment of swab sample 7. A A computed slice (approx. 10 nm thick) from a tomogram of four SARS-CoV-2 particles (1-4) demonstrates the spike morphology (arrowheads) and interior granular substructure (arrows) much clearer than by conventional transmission EM of sections at standard section thickness (i.e. 60–70 nm). B A computed slice (approx. 6 nm thick) from a tomogram of double-membrane vesicles (*) in a ciliated cell. Note that the inner membrane is not entirely continuous (arrows) which indicates instability. The loose filamentous content of the vesicles is typical and most likely represent double-stranded RNA [nded RNA . Scale bAdditional file 8: Fig. S5. Transmission EM of thin sections through the sediment of swab sample 6 (Epon embedding). Images show comparatively well-preserved non-infected cells. A Part of ciliated cell with cross- and longitudinal sections through cilia (arrows) and their basal bodies (arrowheads). Mitochondria (mi) reveal well-preserved membranes but a somewhat extracted matrix. B Part of an un-identified cell with dense cytoplasm, short microvilli (arrowheads) and well-preserved membranes, including the envelop (arrows) of the nucleus (nu). Mitochondria (mi) and several other membrane-bound compartments, such as the rough endoplasmic reticulum (er) are visible. Scale bars = 1 µm.Additional file 9: Fig. S6. Transmission EM of thin sections through the sediment of frozen and non-frozen swab samples (Epon embedding). A, B Ciliated cells in non-frozen samples A show more structural detail than ciliated cells in frozen samples (B). In non-frozen cells A membranes are at least partially preserved (arrowheads) and the cytoplasm reveals various organelles, such as mitochondria (mi). Membrane preservation in frozen samples B is poor  and the cytoplasm (*) appears extracted. C, D Keratinocytes in frozen (C) and non-frozen D samples reveal no obvious differences regarding their structural appearance. The cytoplasm is filled with numerous tonofilaments (tf). Scale bar in A, B = 1µm, in C, D = 0.5 µm.Additional file 10: Fig. S7.Transmission EM of a thin section through the sediment of a Vaccinia virus suspension (LR White embedding). A few virus particle profiles (*) are embedded in the filamentous matrix of the agarose (arrowheads). The inset shows a virus particle profile which reveals the characteristic dumb-bell-shaped inner core of poxviruses. Scale bars = 200 nm.


Human monkeypox is a rare viral zoonosis that was first identified in 1970; since then, this infectious disease has been marked as endemic in central and western Africa. The disease has always been considered rare and self-limiting; however, recent worldwide reports of several cases suggest otherwise. Especially with monkeypox being recognized as the most important orthopoxvirus infection in humans in the smallpox post-eradication era, its spread across the globe marks a new epidemic. Currently, there is no proven treatment for human monkeypox, and questions about the necessity of developing a vaccine persist. Notably, if we are to take lessons from the COVID-19 pandemic, developing a nanomedicine-based preventative strategy might be prudent, particularly with the rapid growth of the use of nanotechnology and nanomaterials in medical research. Unfortunately, the collected data in this area is limited, dispersed, and often incomplete. Therefore, this review aims to trace all reported nanomedicine approaches made in the monkeypox area and to suggest possible directions that could be further investigated to develop a counteractive strategy against emerging and existing viruses that could diminish this epidemic and prevent it from becoming a potential pandemic, especially with the world still recovering from the COVID-19 pandemic. Monkeypox is an infectious disease caused by the monkeypox virus (MPXV), a double-stranded DNA virus belonging to the Orthopoxvirus genus  ,2. In adCynomolgus macaques while conducting research on polio vaccines in 1958 [The MPXV was first isolated from  in 1958 . In 1970The genetic classification of the MPXV categorizes the virus into two clades: the central African clade and the west African clade. The former is endemic in Congo Basin countries, while the latter is endemic in West African countries. In addition to the distinct geographical distribution, the two clades differ in disease severity and transmissibility. The Congo Basin clade has been associated with more severe symptoms, higher transmissibility, and an increased case fatality rate of 1–10%.−5 to 10−6 mutations per replication site). Surprisingly, though, viruses isolated during the current outbreak exhibited more modifications than expected. On average, 50 single-nucleotide mutations were identified compared to the 2018–2019 virus [Phylogenetic analysis of the currently circulating MPXV suggests that the outbreak virus belongs to the less severe West African clade . Moreove19 virus ,16. Yet,19 virus .The MPXV is an enveloped, oval-shaped virus measuring 200–250 nm in size. The enveloped virus encloses a linear, double-stranded DNA genome and enzymes required for virus replication . The MPXThe main internalization route occurs through macropinocytosis for both MVs and EVs, although direct fusion with the plasma membrane is also documented for EVs . MacropiA study on an immune-deficient mouse model demonstrated diverse tissue tropisms for the MPXV, including in the brain, heart, kidney, ovary, liver, pancreas, and lung . HoweverThe MPXV is primarily considered to be a zoonotic viral infection. Transmission has occurred almost exclusively from animals to humans through bites, eating undercooked meat, or direct contact with biological fluids from infected animals . Before The MPX infection has an incubation period of 1–2 weeks, followed by the prodromal phase lasting 0–2 days, and the rash stage persisting for 1–3 weeks. The disease begins with general symptoms, such as fever, fatigue, headache, muscular ache, and lymphadenopathy . Then, aSeveral methods have been developed for detecting the MPXV nucleic acid, among which real-time PCR (RT-PCR) is the preferred routine method . TypicalAn enzyme-linked immunosorbent assay (ELISA) is another detection method that could be used to detect MPX infection. This technique is cheaper than RT-PCR and genome sequencing, costing around $6–8 per sample. An ELISA can detect the presence of specific IgM and IgG antibodies in the serum of the MPX patients at days 5–8 after the infection. Collected data showed a 4-fold increase in patients’ serum antibodies during the acute and convalescent stages of the MPX infection. However, due to the cross-reaction between the MPX viral antigens and the other poxviruses, the specificity of this technique is insufficient, making it inaccurate in identifying the MPXV . To assiThe MPXV is a re-emerging threat to humanity that has gained increasing global attention in recent years. As of January 2023, 84,916 confirmed human cases had been reported globally . Even thSimilarly, the effectiveness of vaccinia immunoglobulin, which is given intravenously, in human subjects still needs to be better established . FurtherTo face the outbreak, new MPX vaccines are being developed. Meanwhile, the USA has approved vaccines that were proven effective in eradicating smallpox. Observational studies demonstrated that smallpox vaccination prevents MPX 89% of the time ,47,50,51ACAM200, based on a live replication-competent virus, replicates and produces a mild infection once in the host, inducing an immune response similar to the natural infection. Nevertheless, in immunocompromised patients, infants, pregnant or breastfeeding women, and individuals with a weak immune system, there is a residual risk of a severe or even fatal infection, and the vaccine is contraindicated. Moreover, an increased risk of adverse effects may occur in individuals presenting cardiac disease (risk for myopericardititis) or dermatologic conditions  ,53. JYNNACAM2000 is supplied as a lyophilized powder, whereas JYNNEOS™ is in a frozen liquid form. However, both vaccines require storage at −20 °C before utilization, requiring cold chain monitoring to maintain their characteristics . The gloMoreover, the containment and eradication efforts will be challenging due to the spread of the virus globally and the circulation of the virus in both human and non-human reservoirs. Lessons learned from the ongoing COVID-19 pandemic have emphasized the importance of developing preventative and therapeutic measures to avoid any catastrophic outcomes of the current MPX outbreak . SeveralDespite the progress in developing conventional vaccines for the orthopoxvirus, improvements are still required, especially with the low immunogenicity, toxicity, instability, and multiple dose concerns associated with the conventionally available vaccines that currently employ a live attenuated vaccinia virus and not the MPXV itself or derived antigens. Furthermore, viral infections pose significant global health challenges, especially with the emergence of more resistant, rapidly evolving viruses causing new pandemics and epidemics, thus emphasizing the potential of nanomedicine applications in the field of antiviral therapies. In addition to providing delivery tools, nanomedicine can increase cellular and humoral immune responses. The involvement of nanoparticles in vaccine development can enhance the immunogenicity and stability of antigens and modulate the immune response. Therefore, over the past decades, nanoparticles such as polymers, liposomes, virus-like particles, immunostimulant complexes, inorganic nanoparticles, and emulsions have been designed and studied to stabilize vaccine antigens and act as adjuvants .Despite having many different nanoformulation-based vaccines, such as the virus-like particle, viral vector vaccines, and nucleic acid vaccines that are encapsulated in lipid-based nanoparticles, which are used in clinics for other viral diseases, as well as the others, including organic and inorganic nanomaterials, so far there are no nano-based vaccines in clinical use for the MPXV ,58,59,60In the present scenario, nanoparticles  and their unique chemical and physical properties are emerging as novel antiviral agents . This isPossible metal nanoparticles employed for this purpose include copper, zinc, titanium, magnesium, gold, alginate, and silver nanoparticles against the hepatitis B virus, respiratory syncytial virus, influenza virus, HIV-1, herpes simplex virus type 1, MPXV, and Tacaribe virus . InteresIn terms of organic nanoparticles , a research group developed lipid nanoparticle (LNP)-formulated mRNA vaccines and investigated their effect on enhancing the immune response both in vitro and in vivo for use in MPXV prevention. The results showed the mRNA constructs’ translation, secretion, and biological activation, as evidenced by the increase in the target monoclonal antibody levels . These data demonstrated the feasibility of inducing multiple antibodies through mRNA constructs using nanotechnology . AnotherDespite the great potential of the nanomedicine field, published work is limited to in vitro and in vivo research, while their applicability in clinical practice has yet to be achieved. However, the rapid spread of the MPXV in non-endemic countries will lay the foundation for accelerating next-generation MPX vaccines, antivirals, and diagnostics to monitor and control the MPXV .Innate immune cells typically act as the first line of defense following MPX infection, with numerous in vitro and in vivo studies demonstrating that monocytes and neutrophils are the initial targets and the predictors of MPXV lethality . FollowiFurther analysis revealed that the MPXV does not interfere with expression or intracellular transportation. Instead, it prevents T cell receptor-mediated T cell activation in trans, triggering a state of non-responsiveness by blocking antiviral T cell activation and inflammatory cytokine production, contributing to viral dissemination in the infected host . NotablyImmunotherapy is emerging as an effective preventative and treatment strategy for many diseases, including infectious diseases. Therefore, developing and studying immunomodulatory nanosystems can rapidly overcome many obstacles that are faced in the field of infectious diseases . To dateSimilarly, decorating nanoparticles with CD44, lectins, dextran, and folate makes them more recognizable by the corresponding receptors overexpressed on macrophages, with dextran having intrinsic targeting properties for macrophages . AdditioMoreover, not using the nanoparticle anti-infective vaccines often requires the assistance of adjuvants to effectively activate the immune systems. Virosome- and liposome-based nanovaccines were tested in the face of infectious diseases and showed promising efficacy in the human body. Until now, two nanoparticle-based vaccines  have been approved by the FDA for the prevention of malaria, influenza, and hepatitis A . NumerouAs with the development of other vaccines, using materials to develop nanovaccines is accompanied with many challenges and limitations, which vary depending on the type of nanomaterial used, with inorganic nanoparticles having more challenges than organic ones; and the type of vaccine carried . Indeed, there are many studies in the literature discussing the challenges associated with the type of nanomaterial used, thereby determining their potential drawbacks, such as ensuring their biodegradability and biocompatibility, tailoring the elected immune response, and the ability to minimize their toxicity . In termIn summary, despite the great international efforts, MPX cases continue to increase, and it is now detected in 110 countries, probably because it is underestimated since many cases were and still are unreported or undiagnosed. Because the MPXV was previously confined to Central and West African countries, with minimal studies and research conducted in this area, more effort is required to develop better preventative and treatment strategies, especially with only four returns found for the nanomedicine, nanoparticles, and MPXV keywords in PubMed (as of 20 January 2023), and even fewer returns in other research websites. This indicates how limited research was in this field until recently, when the focus shifted to the MPXV when the MPX disease spread to the rest of the world. Now the US has the world’s largest MPX outbreak, with more than 6600 cases across 48 states . For the


Background: Access to surgical care is poor in Tanzania. The country is at the implementation stage of its first National Surgical, Obstetric, and Anesthesia Plan  aiming to scale up surgery. This study aimed to calculate the costs of providing surgical care at the district and regional hospitals. Methods: Two district hospitals (DHs) and the regional referral hospital (RH) in Arusha region were selected. All the staff, buildings, equipment, and medical and non-medical supplies deployed in running the hospitals over a 12 month period were identified and quantified from interviews and hospital records. Using a combination of step-down costing (SDC) and activity-based costing (ABC), all costs attributed to surgeries were established and then distributed over the individual types of surgeries. These costs were delineated into pre-operative, intra-operative, and post-operative components.Results: The total annual costs of running the clinical cost centres ranged from $567k at Oltrumet DH to $3453k at Mt Meru RH. The total costs of surgeries ranged from $79k to $813k; amounting to 12%-22% of the total costs of running the hospitals. At least 70% of the costs were salaries. Unit costs and relative shares of capital costs were generally higher at the DHs. Two-thirds of all the procedures incurred at least 60% of their costs in the theatre. Open reduction and internal fixation (ORIF) performed at the regional hospital was cheaper ($618) than surgical debridement (plus conservative treatment) due to prolonged post-operative inpatient care associated with the latter ($1177), but was performed infrequently due mostly to unavailability of implants. Conclusion: Lower unit costs and shares of capital costs at the RH reflect an advantage of economies of scale and scope at the RH, and a possible underutilization of capacity at the DHs. Greater efficiencies make a case for concentration and scale-up of surgical services at the RHs, but there is a stronger case for scaling up district-level surgeries, not only for equitable access to services, but also to drive down unit costs there, and free up RH resources for more complex cases such as ORIF. Implications for policy makersGreater efficiencies and economies of scale and scope make the case for greater concentration and scaling up of the volume of surgical services at the referral hospital (RH). However, there is a stronger case for scaling up basic surgery at district hospitals (DHs), to increase equitable access to surgery and produce lower unit costs at the DH, also freeing up resources at the RH for more complex cases. While implants are prohibitively expensive for regional hospitals which have the technical capacity to perform open reduction and internal fixation (ORIF) of fractures, it does make economic sense to purchase them as the alternative management options incur high post-operative costs. Further interventions to scale up surgery at the DHs should prioritise less tangible rate limiting factors, such as staff skills, anaesthesia capacity, and supplies, over investments in physical infrastructure. To derive full benefits from the manpower available at DHs, disincentives for self-referrals to RHs , and a mechanism of continuing surgical training and supervision at the DHs  are needed.  Implications for the public With the launch of a National Surgical, Obstetric, and Anesthesia Plan (NSOAP) in 2018, Tanzania embarked on an ambitious journey to make safe and timely surgery available to everybody by 2025. The findings in this paper show that surgery carried out at a referral hospital (RH) may be more efficient but that investments are needed to scale up surgery at district hospitals (DHs), to promote equitable access to surgery and ensure that resources at RHs are freed up to manage more complex cases.1 Only about 6% of all the surgeries performed globally are performed in LMICs,2 despite the fact that these populations bear the greatest needs for surgical care: 38 disability-adjusted life years (DALYs) per 1 million people are lost to conditions amenable to surgery annually in sub-Saharan Africa alone.3 These translate to a projected productivity loss of about US$12 trillion between 2015 and 2030.2 An estimated 7.2 million DALYs could be averted annually in LMICs by investing in essential surgical, obstetric and anaesthesia (SOA) care.4 There is a need for empirical evidence to guide such investments. Access to surgery remains poor in low- and middle-income countries (LMICs), where as much as 95% of the people do not have access to safe and timely surgical care.5 Noting that Verguet and colleagues’ study omitted the cost requirements for relevant human resource development, Jumbam et al reported the costs of implementation of National Surgical, Obstetric, and Anesthesia Plans (NSOAPs) in 4 African countries – Rwanda, Zambia, Tanzania and Nigeria – to be US$5.6, US$18.1, US$10.57, and US$85.6 per capita, respectively.6 Differences in NSOAP priorities, local costs of interventions and costing methodologies account for the wide variations in costs,6 making cross-country comparisons difficult. Much is known about the required system-level financial investments for the scale-up of surgery in LMICs. Verguet et al estimated that achieving the target proposed by the Lancet Commission on Global Surgery (LCGS) of 5000 major surgical procedures per 100 000 persons per year would require between US$300 and US$420 billion.7 This makes costing studies particularly relevant. In SSA, only few such studies have been reported: on caesarean sections (CSs)8 and laparotomies9 in Rwanda; open heart surgeries in Nigeria10; neurosurgical management of infant hydrocephalus in Africa11; burn care in sub-Saharan Africa12; and common surgeries in Uganda.13 In Tanzania, to the best of our knowledge, only one study has investigated the cost of a major type of surgery: Open reduction and internal fixation (ORIF).14 That study took a tertiary hospital perspective. Little or nothing is known about the costs of surgeries at the district hospitals (DHs) and the financial burden of surgery on these facilities on which the majority of rural populations rely. The resource requirements and costs of individual types of surgeries in LMICs are less well-explored however. Surgeries are technically complex, and some of their costs are shared with other health services. Much of the associated costs may therefore be masked.15 The DHs  provide first-level major surgical services for patients referred from lower-level facilities . Some of the latter  have been upgraded to provide basic emergency SOA care.15 Surgeries at DHs are performed by medical officers (MOs) and assistant medical officers (AMOs), who are non-physician clinicians (NPCs) with advanced diplomas in medicine and surgery. Both cadres have undergone limited, non-specialist training in SOA care. DHs refer patients that they cannot manage primarily to regional referral hospitals (RHs). A total of 367 facilities provide major surgery services in Tanzania.15 Even though over 70% of the population lives in a rural area, most DHs do not have permanent SOA specialists, with lack of physician anaesthesiologists reported even up to the level of regional hospitals.16 In 2018, the country adopted its first NSOAP15 in line with the recommendation of the LCGS. It provides a systems-based pivot for all stakeholders towards a nationwide scale-up of safe surgery. The NSOAP clearly defines what surgeries are expected to be performed at every level of the health system. For instance, while it recommends non-operative fracture treatments at the DHs, the regional hospitals are expected to offer comprehensive elective and emergency orthopaedic services including ORIF.15 Tanzania lags behind in the LCGS indices, with staff shortages identified as the single most pressing deficit.17 This study in particular aimed to establish and compare the costs of performing common basic surgeries at DHs and a regional RH, taking into account both direct and indirect costs to the provider. SURG-Africa, which is a European Union (EU)-funded research project, has been implementing and evaluating a surgical mentorship model whereby specialists from higher-level hospitals provide mentorship and supervision support to surgical teams at DHs. The study was descriptive and cross-sectional, and took the perspective of the healthcare system. The period of interest was the 2017/2018 financial year . Retrospective data collection took place from July to September 2018. Two DHs in Arusha region of Tanzania were selected based on the volume of surgery performed  and considerations of convenience: Meru DH and Oltrumet DH. In addition, we included their main RH: Mount Meru Regional Hospital (Mt Meru RH) in Arusha town.18 This is an Excel-based tool for step-down accounting developed by the WHO-CHOICE  initiative to help countries set their healthcare priorities. It is particularly recommended for costing of health services in LMICs. CostIt consists of a series of linked template worksheets that allow recording and analysis of cost data. We developed a data collection approach to mirror the data requirements of CostIt. CostIt tool was used for data aggregation and analysis.19 Data on surgeries performed and other services were obtained from registers (theatre and ward) and aggregated Health Management Information System reports, using predesigned templates. Types of major surgeries of which at least 10 were performed at the RH during the 12-month study period and at least 5 at the DHs were selected for investigation. All other major and minor surgeries were grouped. At the RH, the other major surgeries were broken down by their respective subspecialties. Minor surgeries performed outside the main operating theatres (OT), ie, in minor theatres, out-patient department (OPD) or the casualty department, were excluded in the analysis. Classification of cases into “major” and “minor” was based on local practices as evidenced in the OT registers. It is noteworthy that the classifications were uniform across all the hospitals and essentially agreed with the common literature definition of a ‘major’ type of surgery: Any procedure occurring in a hospital OT, involving incision, excision, manipulation, or suturing of tissues, and usually under general or regional anaesthesia or sedation. Both economic and financial cost data were collected but only economic costs are reported in this paper. Hospital departments were divided into direct/clinical cost centres that deal with patients  and indirect/overhead cost centres that do not directly deal with patients but whose services are used by clinical cost centres. The sets of cost centres used in the analyses of the hospitals differed depending on their contexts. Details of these are presented in Tables S1-S3 See . The cos20 At Mt Meru RH, the full list of all the assets and their replacement costs, ages, assumed useful life (in years), and estimated remaining life were obtained from the hospital asset register. The asset valuation exercise had been conducted in 2016 by a professional valuing firm. At the DHs, there were no hospital asset registers, and therefore inventories of all available assets were established during data collection. In both cases, replacement costs of the items (equipment and furniture) were obtained from a combination of sources: Mt Meru RH asset register , procurement records, Medical Stores Department (MSD) annual price list, expert opinions , and online price quotations by local vendors. Economic capital costs were based on the replacement (current) costs, while financial costs were based on the historical (purchasing) costs.2. The costs of other buildings across the DHs were estimated by measuring their roof surface areas with Map Developers online tool – Area Calculator21 - and multiplying them by the average cost per m2. Historical costs of some of the buildings at Oltrumet DH were obtained from the managers. For these we calculated the average cost per m Conversions between historical and replacement costs were done by applying the appropriate World Bank gross domestic product (GDP) price deflators. In the absence of actual age data, the average ages from the Mt Meru RH asset register were used. Useful life was set at 66 years, 5 years and 10 years for buildings, vehicles and other assets (equipment/furniture), respectively. These numbers had been applied in the Mt Meru RH asset register.20 Calculation of the economic and financial capital costs is an automated process on the CostIt tool. Once the historical and replacement costs and the useful lives are entered, CostIt calculates the financial capital costs using a simple linear method and the economic capital costs using macro-generated annualization factors.22 Due to privacy concerns we were given the de-identified payroll records across the 3 hospitals. From this we calculated the average salary per level, per cadre. As the payroll records were not regularly updated with staff turnover, we obtained additional staff distribution and composition data from the heads of various departments and staff duty rosters. We multiplied the average salaries per cadre/level by the actual staff composition to arrive at a salary cost for each department. Distribution of salaries of clinicians over clinic (consultation), ward, OT and administrative (where applicable) duties were based on interviews of the clinicians. Costs of supplies to the various departments were estimated by multiplying the volumes used by the corresponding unit prices. Volumes of medical and non-medical supplies were obtained from the procurement office and pharmacy, respectively, using the supplies (requisition) records and ordering books. Unit prices for supplies were obtained from the procurement records and MSD official price lists. Other recurrent expenditures were obtained from the hospital annual financial reports. For recurrent costs, we assumed that the economic costs are equal to the financial costs. An overview of the cost items and their sources and calculations are presented in 20 After collecting the direct costs for each overhead and clinical cost centre, a combination of (top-down) step-down costing (SDC) and (bottom-up) activity-based costing (ABC) approaches was used to estimate the unit cost per surgical procedure. SDC was used to allocate costs from overhead cost centres to clinical cost centres. ABC was used to allocate costs from clinical cost centres to individual surgical procedures. A discount rate of 3% was used. Allocation criteria were determined together with staff members. Criteria were chosen based on relevance  and the availability of data. Commonly used allocation criteria were direct personnel cost, number of nurses, and economic costs of buildings. More details about the allocation criteria used can be found in Tables S1-S3 See . Not all clinical departments contribute substantially to surgery. In the absence of sufficiently discriminatory records, the share of the total cost of a department pertaining to surgeries was estimated by the surgical proportion of all the patients it served ie, based on simple patient counts . This ranged from 0% for departments that have little or no contact with surgery patients  to 100% for departments that provide surgical services exclusively . This proportion was multiplied by its total cost  to arrive at the total cost of surgery for the department. Only patients who were treated operatively were considered in this allocation formula. For instance, the fraction of total maternity ward cost allocated to surgery was equal to the ratio of all major obstetrics and gynaecological surgeries recorded in the theatres to the total number of patients admitted in the maternity wards during the study period. This approach assumes that both surgical and non-surgical patients consume resources equally. ABC was employed to establish the unit costs of the different types of surgery from the total costs of the different departments while reflecting the heterogeneity in the resource consumptions of the different surgeries. ‘Activities’ represent those areas in which different surgeries can realistically be expected to differ with regard to resource use and costs. They correspond to the domains in the surgical care pathway. The identified activities were: number of preoperative consultations, investigative activities , pre-operative ward days, time spent in OT, post-operative ward days, and number of follow-up consultations. Post-operative investigations were considered exceptions and not norms, and therefore excluded. We assumed that resource utilization per unit of a given activity  is constant, irrespective of the type of surgery. While intra-operative resource use would expectedly differ across different surgeries, OT duration was used as a proxy, assuming that longer surgeries would logically consume more resources .23 Each clinician was interviewed on specific procedures he/she performed most commonly. For a procedure performed by more than one clinician in a hospital, we obtained multiple activity estimates and used the arithmetic means in the analyses. As more types of surgery were done than the common basic ones under our consideration we had to make assumptions regarding the activities of the other types of surgery, as they also consumed resources meant for surgery. For these we took the average activity indices across other surgeries in the respective sub-specialties. For each type of surgery under our consideration, we estimated its “activity indices” by interviewing surgery clinicians  on their ‘standard practices’ . Such an approach had been applied in previous studies. Each activity constituted a discrete cost pool. The costs of surgery per clinical department were distributed over the corresponding activity cost pools. For instance, the cost of OT was assigned wholly to the intra-operative activity cost pool while ward costs were split between pre-operative and post-operative care cost pools. Each activity cost pool was subsequently allocated to individual types of surgery based on the estimated activity indices. By summing up all costs allocated to a type of surgery in this way we obtained a total cost per type of surgery. We arrived at a unit cost for a particular procedure by dividing this total cost by the number of surgeries of that type performed. Costs are reported in December 2017 United States Dollars, ie, at the mid-point of the study period. In order to understand how the uncertainties around our assumptions and input variables affect the results, we performed the following secondary analyses: 1. Univariate Deterministic Sensitivity Analysis:We changed our assumptions and input variables one at a time (keeping other factors constant). The following variables were adjusted:Step-down allocation factors: Five selected allocation factors were substituted in each hospital. The affected factors are presented in Tables S1-S3 See . Assumptions about allocation of departmental costs to surgery pool: The secondary model assumed that surgical patients consume twice as much resources as the non-surgical patients. i) Assets , (ii) Salaries, and (iii) Supplies. Assuming the input costs were increased or decreased by 20%: Input costs adjusted were; . Details of the surgeries and their volumes are presented in Table S4 See . The total direct costs of overhead and clinical departments of Mt Meru RH were $907k and $2616k, respectively. At Meru DH, the overhead and clinical centres incurred total direct costs of $294k and $1247k respectively. Overhead and clinical departments of Oltrumet costs were $139k and $443k respectively. Although the absolute figures differed among the hospitals as a function of their size, several patterns could be seen. Salaries took up the largest share of the costs. Of the overhead departments, the general administration department was the most costly, owing in part to the fact that certain overhead costs could not be allocated to particular direct cost centres. At Mt Meru, the pharmacy department was also relatively costly due to the large size of the building it occupies and the relatively large number of academically trained personnel in the department. The direct costs of the clinical departments were much higher than those of most overhead departments, owing mostly to their higher personnel counts. Across all the hospitals, the OPD and wards were generally the most costly departments, followed by diagnostic departments and the OT. Details of these costs are shown in Tables S5-S10 See . Following the step-down of overhead costs, the total economic costs of the clinical departments for the 12 month period were: $3453k for Mt Meru RH, $1543k for Meru DH, and $576k for Oltrumet DH. The relative size of the capital costs at Mt Meru RH was lower than that of the DHs. The details are presented in Tables S11-S13 See . Disaggregating the total costs of surgery into types of costs , salaries constituted at least 70% of the total cost of surgery care at the 3 hospitals (not shown on the plot). Again, the relative share of capital costs was lowest (2%) at Mt Meru , 6% at Meru DH and highest (11%) at Oltrumet DH . Comparing the total costs of surgical services to the total hospital costs, Mt Meru RH spent over a fifth (22%) of its resources on surgical care while Meru DH and Oltrumet DH spent 12% and 14%, respectively. Tables S14-S16 See  show the With regards to costs in different stages of care, about 64% of the procedures incurred at least 60% of their costs intraoperatively. At Mt Meru RH, surgical debridement (plus conservative treatment) stood out with high post-operative costs due to prolonged inward care of at least 4 weeks, but with low pre-operative cost, being an emergency operation. On the other hand, ORIF (mostly an elective procedure in this setting), with extended pre-operative consultation visits (up to 4), had a higher pre-operative cost. Ear-nose-throat procedures (tonsillectomies/adenectomies/adenotonsillectomies) were unique in that large percentages (78% on average) of their costs were incurred pre-operatively. This was due to their short OT times of 20–45 minutes and post-operative care of less than 24 hours. The results of the sensitivity analyses are presented in  Applying a combination of SDC and ABC, this study quantified the costs of performing common surgeries in a regional hospital and 2 DHs in Arusha region of Tanzania. It also gives insights into the costs of running the hospitals. As the Government of Tanzania implements its first NSOAP, this study therefore provides vital evidence for planning and budgeting of surgery scale-up at the regional and district facility levels. Unit costs and the relative shares of capital costs were generally lower at the RH. This reflects advantages of economies of scale and scope of surgery at the RH, and conversely, a possible underutilisation of capacities at the DHs. The total cost of running the clinical departments of Mt Meru RH (after overhead step-down) was 2 times and 6 times those of Meru DH and Oltrumet DH, respectively. However, the volume of surgery output was about 4 times and 9 times those of Meru DH and Oltrumet DH, respectively. Similarly, with 3 major operating rooms, Mt Meru RH performed nearly 3500 major surgeries over the one-year study period, giving an average of almost 1200 per theatre. In contrast, Meru and Oltrumet DHs, with 2 major operating rooms each, performed an average of about 400 and less than 200 per theatre, respectively.24 While this study did not evaluate demand side costs, there is ample evidence that patients incur substantial costs in seeking care at the higher hospitals which are often far from their homes; driven by transport, feeding, lodging and informal care.27 Moreover, judging by the NSOAP stipulations,15 the bulk of surgeries at the RH  are ideally provided at the DHs, and these could crowd out the more advanced RH-appropriate cases. A scale-up of the DH-level appropriate surgeries will not only drive down unit costs at this level but also allow the RHs to concentrate on and scale up the more complex and resource intensive cases,28 thus allowing even a more judicious application of the RH resources. From a provider perspective, it makes economic sense, especially in resource poor settings, to concentrate resources and surgery services at RHs for reasons of efficiency and reduced health system costs. However, such policies often come with consequences of increased access distance and costs of services on the patients, or outright lack thereof – potentially compromising health system equity objectives.29 While this is relevant, we contend that in many instances, as is seen in this study and elsewhere,7 there are trapped potentials at the DH that require only minimal input to be enabled. Efforts must be made to first identify and tackle such rather intangible, rate-limiting elements inhibiting full exploitation of the already installed  capacities at the DHs. These include the supplies, instruments, and staff mix. The absence of one anaesthetist for instance could render a battalion of doctors redundant.16 Our findings do not only support scale-up of DH-appropriate surgery, but also provide insights into how this could be achieved. Firstly, in discussions about surgery scale-up in LMICs, there is a tendency to prioritise extra investments in physical infrastructure expansion.30 Among those formally referred from the lower centres, lack of expertise alongside equipment was again the most commonly cited reason for referrals.30 To derive full benefits from the manpower available at the DHs, disincentives for self-referrals to RHs , and a mechanism of continuing surgical training and supervision at the DHs  will be needed.17 Secondly, having large numbers of clinicians at the DHs without the requisite skills induces inefficiencies. While the DHs employed several AMOs/MOs , only very few of them performed surgeries other than emergency CS cases encountered during on-call hours; such that less than 10% of all the surgeries at the DHs were non-CS. An earlier study in Tanzania reported that over 70% of the patients seen at the national hospital were self-referred, bypassing the DHs; nearly 70% were surgery cases; and as much as 96% cited lack of expertise at DH as the reason for self-referrals.23 a study done in Malawi found the share of salaries to be significantly lower (about 31% in the theatre).7 Malawi public service salaries are generally lower than Tanzania salaries,31 and surgeries at DHSs are provided mostly by clinical officers (COs)32 contrary to the practice in Tanzania, where surgeries at the DHs are performed by MOs and AMOs. As human resources constitute the bulk of costs of surgery, our findings suggest that from an economic point of view, task shifting is a wise and pragmatic approach (at least in the medium term) to providing surgery to rural populations in the face of severe manpower shortages. While there have been safety concerns about surgeries performed by NPCs,33 and there have been no randomized controlled trials comparing surgeries performed by medical doctors and NPCs,34 it is interesting to note that several controlled studies in Tanzania36 and elsewhere38 have reported absence of statistically significant differences in health outcomes. Thirdly, salaries accounted for at least 70% of the entire costs of surgical care across the 3 hospitals. Although similar findings have been reported in the past ,40 These entailed several weeks of post-operative inpatient care , compared to few days for ORIF patients. Granted that increased risk of infection with use of internal fixation as a first line treatment in open fractures may be one reason for this practice, the other major reason is unavailability of implants which are prohibitively expensive40; even closed fractures requiring surgical fixation are commonly managed conservatively for this reason.41 This study reveals that surgical debridement plus conservative treatment (US$1177) is in fact more costly than ORIF (US$618) as a result of this prolonged ward stay. Although the ORIF cost here excludes the cost of implants (about US$134 14), since they were not captured in the routine hospital cost records, it includes the costs of prolonged pre-operative clinic visits. Indeed, cost-effectiveness studies in Kenya42 and the United States43 had demonstrated that internal fixation offers better clinical outcomes at lower costs compared to non-operative fixation of fractures. This illustrates the need for scaling up basic surgery at the DH-level, as this can free up resources at the RH that could then be channelled to such critical inputs as orthopaedic implants. Lastly, this study makes an economic case for the provision of implants at the RH. Most open fractures at the RH were managed by surgical debridement and conservative treatments such as casting, slabbing, and traction .9 This approach gives a better insight into resource flows and utilisation thereby revealing the potentials for efficiency gains. This can guide hospital managers and clinicians in making more rational resource allocations as well as identifying necessary changes in clinical practices. Our study draws strength from the use of (primary) hospital-level data, thus making the results more policy relevant. Further, the study joins the few published surgery costing studies in sub-Saharan Africa that delineate pre-, intra-, and post-operative costs. The main limitation of our study is that data on “standard practice” applied in the ABC were obtained through interviews of clinicians rather than observation of actual practice, leaving room for respondent biases in the estimates. Moreover, these estimates of activities for ‘typical’ cases do not capture atypical situations or scenarios with complications. Interviewing of multiple clinicians about the same type of surgery was an attempt to reduce the possible bias however. Further studies and complementary approaches, for example time and motion studies, are encouraged to obtain finer insights into the cost dynamics of providing surgeries in LMICs. While there are economic reasons to concentrate surgeries at the bigger specialist hospitals, overconcentration of relatively simple surgeries at the RH leads to congestion at this level at the expense of specialist services. Evidence of underutilised capacities at the DHs provides justification and potentials for scaling up surgery at the DH-level. Further initiatives would need to prioritise the rate limiting factors at DHs, such as staff skills, anaesthesia capacity and supplies, over physical infrastructure extension. We wish to acknowledge the support of the Ministry of Health, Community Development, Gender, Elderly and Children (MOHCDGEC) of Tanzania; the President’s Office, Regional Administration and Local Government (PORALG) of Tanzania; Tanzania Surgical Society (TSA); East, Central and Sothern African Health Community (ECSA-HC); and Arusha Regional Medical Office. We thank specially the management teams and staff members of the hospitals involved in the study for their assistance and cooperation. This study was approved by the Kilimanjaro Christian Medical College Research Ethics and Review Committee , and the National Institute for Medical Research of Tanzania . The costing studies required collection of data that may be considered sensitive; especially regarding financial records, staff grades, salary levels and staff time allocation. Verbal informed consents were obtained at each of the institutions involved from the management team members, relevant clinicians and all other respondents. Authors declare that they have no competing interests. Conception and Design: MI, HB, LB. Acquisition of data: MI, AJ, HB, LB. Analysis and interpretation of data: MI, HB, LB. Drafting of Manuscript: MI, HB, LB. Critical revision of the manuscript for important intellectual content: All co-authors. Funding acquisition: JK, KC, EK, RB, LB. Administrative, technical, or material support: AJ, EK. Supervision: LB. This study was part of the Scaling up Safe Surgery for District and Rural Populations in Africa (SURG-Africa) project, which is funded by the European Union’s Horizon 2020 Programme for Research and Innovation, under grant agreement no: 733391.Supplementary file 1 contains Tables S1-S16.Click here for additional data file.


The current monkeypox virus global spread and lack of data regarding clinical specimens’ infectivity call for examining virus infectivity, and whether this correlates with results from PCR, the available diagnostic tool. We show strong correlation between viral DNA amount in clinical specimens and virus infectivity toward BSC-1 cell line. Moreover, we define a PCR threshold value , corresponding to negative viral cultures, which may assist risk-assessment and decision-making regarding protective-measures and guidelines for patients with monkeypox. To date, the current outbreak is mostly affecting men who have sex with men (MSM), and the disease caused by MPXV is manifested mainly with skin/mucosal lesions at multiple sites including genital, oropharyngeal or perianal areas [In early May 2022, monkeypox virus (MPXV) human infections were reported in the United Kingdom (UK). Since, increasing numbers of cases, also attributed to the MPXV Clade II, have been observed in other non-endemic countries for the virus, amounting, up to 5 August 2022, to over 28,220 cases worldwide . To datal areas . In thisSupplemental Table S1). Among the 32 patients, 10 had paired oropharynx and dermal-lesion exudate swabs and one patient had both oropharyngeal and rectal swabs . Swabs were stored in viral-transport-medium-(VTM) containing tubes up to 48 hours at 4°C before being subjected to viral DNA quantification (via the quantitation cycle (Cq) value in PCR) or infectious virus titre measurement (pfu/mL in plaque assay).A total of 43 clinical specimens from 32 male patients from six medical centres in Israel were included in this study. The specimens consisted of 21 oropharyngeal swabs, 20 dermal-lesion exudate swabs, and two rectal swabs . Cq values, one for each of these paired PCRs conducted on the respective 43 specimens were determined, the lowest of which was retained as the specimen Cq.To determine the Cq value for each specimen, MPXV DNA was extracted with the QIAamp DNA Mini Kit (Qiagen) using a protocol for blood and body fluids available from the manufacturer, in a QIAcube robot, and multiplex real-time PCR assays were performed using MPXV generic assay primers, as well as primers specific for MPXV Clade II  were derived from the Cq values using a standard curve based on calibrated MPXV DNA standard.Supplemental Material). Plates were fixed and stained 72 hours later, plaques were counted, and plaque forming units/mL (pfu/mL) were determined.To determine the MPXV infectivity of the same 43 samples, a plaque assay was employed. The VTM solutions, where swabs had respectively been stored, each underwent 10-fold serial dilutions, and the latter were used to infect BSC-1 cells  of all samples irrespective of swab type, a significant negative correlation was found  and oropharynx swabs (r = ‒ 0.8869) was found . Thus, COf the 43 specimens, 20 were paired swabs of oropharynx and dermal lesion from the same patient, thus enabling examination of the Cqs  or pfu/mCq values for 10 of the tested oropharynx swabs were relatively high > 33, . For mosThis suggests that in clinical swabs, Cq values ≥ 35 predict no or very low infectivity. In addition, we calculated a ratio of 172 DNA copies for 1 pfu to provide a comparable value across different laboratories. Altogether, based on our limit of detection (25 pfu/mL), the minimum amount of DNA required for a clinical sample to be infectious is 4,300 DNA copies/mL.MPXV belongs to the Orthopoxvirus genus within the Poxviridae family and is a double-stranded-DNA zoonotic virus ,5. AccorMPXV was originally reported in humans in central Africa in 1970, and thereafter sporadic outbreaks were observed mostly in Central and West Africa, where the virus is endemic. Since 2003, sporadic MPXV outbreaks have also occurred in non-African countries .Since early May 2022, when a set of cases of MPXV were diagnosed in the UK, a monkeypox outbreak is spreading rapidly, currently spanning several countries, where the virus is not endemic. MPXV cases are being diagnosed by PCR testing that determines viral DNA presence in patients’ specimens. However, data on infectivity relative to viral DNA content are lacking, thus impairing risk assessment and determination of the appropriate protective measures . In thisWe also tested paired swabs of oropharynx and dermal lesion from the same patients. Most tested patients appeared to present higher viral loads  in dermal lesion specimens than in oropharynx specimens, suggesting a higher infectivity risk from dermal lesions.Based on our data, a Cq value of ≥ 35 predicts no or very low infectivity. This concurs with results of Lapa et. al., who reported the successful recovery of MPXV from semen, in a sample with a Cq value of 29 .Supplementary Figure S2), poxvirus infectivity seems to be maintained following 24 hours of incubation at 4 °C and marginally affected upon incubation for 48 hours at the same temperature . Recent preliminary results with clinical specimens from the current MPXV outbreak additionally suggest that the virus infectivity is not affected after up to 48 hours of incubation at 4 °C (data not shown). This is in line with previous studies showing that poxviruses maintain their infection capacity for a relatively long time and across different conditions [We further calculated a ratio of 172 DNA copies for 1 pfu. Previous reports suggest values of 10–100 DNA copies for 1 pfu for orthopox viruses originating from cultured cells ,12. Thisnditions . Furthernditions .Overall, we suggest that a Cq value of ≥ 35  corresponds with non or marginal infectivity. Altogether, infectiousness should also be evaluated in context of the overall clinical manifestation including the course of disease, lesion location and stage, etc.There are several limitations to this study. First, data on virus infectivity from other poorly available types of clinical specimens, such as semen, scabs, and rectal swabs still require further evaluation. Second, due to the nature of the disease that manifests mostly with dermal lesions and less with oropharyngeal ones , tested This work highlights the strong correlation of MPXV Cq values to virus infectivity and defines a threshold  that predicts poorly- or non-infectious specimens. These data, together with the corresponding clinical manifestation, may provide valuable support for decision-making regarding protective measures and guidelines for monkeypox patients and their close contacts.


Vitis hosts, Silene latifolia and Chenopodium album. We investigated the presence of GPGV in grapevine and other plant species growing as weeds in the vineyard. Using RT-PCR, we identified GPGV in seven non-Vitis hosts: Ailanthus, Asclepias, Crataegus, Fraxinus, Rosa, Rubus, and Sambucus. In the case of Rosa and Rubus, this finding was supported by Northern blot detection of the virus. GPGV strains in non-Vitis hosts belong to the asymptomatic clade, and are clustered according to their original geographic locations. The presence of GPGV in species other than grapevine shows that besides well-known vector and propagating material-based infections, other possible entry sites for the virus can exist, which have to be taken into consideration when developing reliable regulation strategies.Grapevine Pinot gris virus (GPGV) was described in Italy using a metagenomic approach: next-generation sequencing of the virus-derived small RNAs. Since that time, it has been reported all over the world. The presence of GPGV is associated with grapevine disease, but most of the time, the disease is asymptomatic. Although the host range of this virus has not been investigated, it has been found in the non- Colomerus vitis, and was transmitted to healthy vines through mite infestation [Silene latifolia subsp. Alba (Mill) and Chenopodium album L. [Grapevine Pinot gris virus (GPGV), a member of the trichovirus (Betaflexiviridae) genus, was first identified in vineyards in Trentino, Italy, by small RNA (sRNA) high-throughput sequencing (HTS)  as a cauestation , revealialbum L. .GPGV variants show low genetic diversity (a sequence identity of higher than 97%) , but cleRubus, Rosa, and even in Fraxinus [Vitis hosts in more detail than in our initial observation, to gain information regarding the possible wider host range of GPGV.In a survey of endemic plants and invasive weeds surrounding the vineyards, GPGV could be detected in common milkweed, Fraxinus . In the Surveying vineyards and grapevine rootstock plantations, we detected GPGV infection in distinct parts of the country ,5. For tChenopodium album . The only exception was when we compared the movement protein-coding capacity of grapevine isolates to the reference genome. The phylogenetic relationship of the GPGV isolates was investigated to find out their evolutionary relationship . From thC. album, we could only amplify the 411 bp region of the virus. The phylogenetic tree, including this isolate, shows that it does not cluster with the grapevine isolate (SZ_Vitis) from the same geographical origin  clustered together with the symptomatic reference strain of the virus.An investigation of the presence of symptomatic and asymptomatic clade-specific SNPs revealed that although the above-mentioned E_Vitis and E_Rosa variants clustered together with the symptomatic variants, they did not have either the early stop codon at 6670 or the symptomatic variant-specific SNPs at 6400 and 6593 in the movement protein . It is iVitis hosts: Ailanthus, Asclepias, Crataegus, Fraxinus, Rosa, Rubus, and Sambucus. The presence of the virus was not accompanied by typical GPGV-specific symptoms, either on grapevine or on weeds. There are different theories to explain the symptomatology of GPGV. One explanation is that there are symptomatic and asymptomatic variants, which we will discuss later. Investigating virus variants together with virus concentrations led to another hypothesis: that the virus titer can influence the development of symptoms [Rosa and Rubus, we could support the RT-PCR result, and could detect the presence of GPGV using a less sensitive Northern blot assay, suggesting a relatively higher virus concentration in these two samples. In C. album we could only amplify a 411 bp product from the MP/CP region, while at Jaszszentlaszlo, we could only amplify GPGV if we used oligodT primers for cDNA synthesis. We could not amplify the 5′ part of the viral genome either in J_Fraxinus and J_Vitis, or in M_Ailanthus, M_Crataegus, or M_Sambucus, which are woody hosts at Mogyorod. Such failure in these cases suggests that, in both C. album and the three woody hosts, the virus concentration was low, below the sensitivity threshold of the method. It could also be possible that in these hosts, a variant with a mutation in the primer coding region was present, leading to an inability to detect them.GPGV was detected in all of the examined vineyards, and besides grapevine, its presence was detected in seven non-symptoms . In thatsymptoms . In the The other hypothesis for the symptomatic and asymptomatic behavior of GPGV is the existence of symptomatic and asymptomatic variants of the virus. The most striking difference between the two variants is the polymorphism at the end of the MP (6670 in the reference genome). The symptomatic variants encode an early stop codon, resulting in a protein that is shorter by six amino acids . In addiA comparison of the sequences of GPGV variants in different isolates showed very high identity, higher than 97% in all but one case. When we compared the sequences of the grapevine-originated GPGV isolates to the reference genome, the identity on the amino acid level was only 95.39–96.21%. The reason for this is the presence of the longer MP coding region, characteristic of asymptomatic variants, in the investigated grapevine isolates.Phylogenetic analysis of the 5′ part and the MP/CP region of the GPGV strains showed that the GPGV variants clustered according to the vineyard and not the host, suggesting an on-site infection. The shorter MP variant associated with the presence of symptoms was not found either in grapevine or in weeds, suggesting a widespread presence of the latent variant.GPGV variants at the old, neglected vineyard clustered distantly and not together. It is possible that GPGV infection at this place happened a longer time ago, allowing for a longer evolution and spread of GPGV within these perennial hosts, allowing the virus to incorporate several mutations and diverge.Colomerus vitis, the proven vector of GPGV, is monophagous and known to feed only on grape [S. latifolia and C. album [The host range of a virus is determined by several factors, but it is highly dependent on the virus vector. on grape . In lineC. album , we showThe presence of GPGV in a vineyard’s neighboring woody or perennial hosts suggests that GPGV may in fact be endemic in Eastern Europe. Although its probable center of origin center is located in Asia, it is suggested that an intercontinental jump to Europe in the middle of the 20th century occurred . Since tLeaf samples were collected at 5 regions in Hungary  from graRNA extracts of plants were used as templates for cDNA synthesis using random or oligodT with the RevertAid First Strand cDNA Synthesis Kit . The presence of GPGV was checked using diagnostic primers  amplifyiFor Northern blot analysis, 2–4 µg of total RNA was separated on formaldehyde/1.2% agarose gels and blotted to Nytran NX membrane  via the capillary method using 20× SSC. Hybridization was performed at 65 °C in Church buffer  overnight with the appropriate radioactively labeled probe, washed for 5 min in 2× SSC and 0.1% SDS, and incubated for 15 min in 0.5× SSC and 0.1% SDS at the hybridization temperature and exposed to X-ray film. Virus-specific P32-labeled DNA probes were prepared using the Thermo Scientific Decalabel DNA labeling kit.https://www.geneious.com) and MEGA11 [For the SNP and phylogenetic analysis of viral sequences, we used Geneious Prime 2022.0.2 (d MEGA11 . The vird MEGA11  was taked MEGA11 . InitialIn our work, we show that GPGV, a widespread virus infecting grapevine, can also be present in other species near vineyards. GPGV was proved to be transmitted via grafting and via a monophagous vector. Its presence in alternative hosts suggests the possibility of transmission via a polyphagous vector, which remains to be found and characterized.The presence of GPGV in alternative hosts has importance in virus epidemiology. There is no official regulation dealing with GPGV, but this fact has to be kept in mind when making decisions regarding its regulation. This could prevent it from spreading by transport and exchange between these possibly infected alternative hosts.


In 2021, the world marked 10 years free from rinderpest. The United Nations Food and Agriculture Organization and World Organisation for Animal Health have since made great strides in consolidating, sequencing, and destroying stocks of rinderpest virus–containing material, currently kept by only 14 known institutions. This progress must continue.  In 2011, ten years after the last confirmed outbreak, the Food and Agriculture Organization of the United Nations (FAO) and the World Organisation for Animal Health  jointly declared global freedom from rinderpest. Rinderpest, also known as cattle plague, is only the second infectious disease eradicated from the world, smallpox being the first. Over the 10 years since eradication, the main goal of the Rinderpest Post-Eradication Programme  containing unique rinderpest virus nucleic acid or amino acid sequences; and full-length genomic material, including from virus RNA and cDNA copies of virus RNA. Subgenomic fragments of morbillivirus nucleic acid not capable of incorporation into a replicating morbillivirus or morbillivirus-like viruses are not considered RVCM. Accounting for remaining RVCM is critical to limit the risk for reintroducing the pathogen by intentional or inadvertent release from a laboratory (https://www.oie.int/en/disease/rinderpest/#ui-id-3). Category A RHFs are designated for storing RVCM, excluding vaccine stocks; category B RHFs are approved for storing only manufactured vaccines and materials for their production. In 2011, a total of 150 countries were surveyed regarding their RVCM stocks . In addiTo confirm that no relevant findings unknown to WOAH had been published by research groups or laboratories, we reviewed the scientific literature to identify any publications about rinderpest virus research undertaken since 2011. A search of 21 databases identified 623 unique publications of which we evaluated 17 at the full-text level  Table 1.Members of the study team contacted a representative from each institute known by WOAH to keep RVCM as of August 2021 to arrange an interview to discuss current and historic RVCM stocks and laboratory biosecurity. Interviews were conducted remotely and accompanied by completion of a structured questionnaire. All institutes keeping RVCM, except for 1 located in the Middle East, responded to the request for an interview. Because of logistical difficulties and COVID-19–related challenges, interviews were not conducted with institutes in 2 countries in Europe. Therefore, during August 8–September 17, 2021, interviews proceeded with 11 of the 14 institutes known to keep RVCM. Several of the institute directors contacted were not familiar with the specific content of their RVCM stocks and indicated that these materials were simply in storage, which is concerning because of the critical nature of these materials. At present, Africa is the only region actively attempting to consolidate its RVCM into a single facility. According to Resolution 18, passed in 2011 during the 79th general session of the World Assembly of WOAH Delegates: “Rinderpest virus-containing material that is not in an approved BSL3 [Biosecurity Level 3] facility shall be destroyed by a validated process or transferred to an approved BSL3 facility.” Biosecurity levels for institutes keeping RVCM during 2011–2021 ranged from BSL2 to BSL4  meaning During recent genomic analysis of PPRV isolates held at an FAO/WOAH–designated RHF, 1 sample was found to contain a sequence that aligned with RPV in addition to PPRV sequences. A traceback investigation found that this sample, obtained from the field by another institute in the early 1970s, appears to have been destroyed. All materials derived from the original stock before the contaminated sample was identified were uncontaminated, but those derived from the contaminated stock were RPV-contaminated, so contamination appears to have occurred inside the institute, from an unknown source, but likely during a period in the late 1990s when both RPV and PPRV were being manipulated concurrently at the institute. All contaminated samples were destroyed. After examining all other samples being manipulated during the same period, the institute concluded that no others were contaminated. Records indicated that the institute had not shared this sample with other facilities and that it would thereafter screen all PPRV samples by PCR for RPV before sharing them. No contaminated samples had escaped containment and all processes to secure stocks appeared to be working well. Risks associated with remaining global stocks are being evaluated and will be presented in a future publication.We document discovery of RPV-contaminated PPRV samples; our findings suggest that because of risk for cross-contamination, other laboratories should take precautions with samples manipulated alongside RPV, especially PPRV. Although progress is being made in consolidating RVCM stocks, 2 of 6 nonapproved institutes known to keep RVCM stockpiles have indicated no plans to destroy or transfer them to an FAO/WOAH RHF. Therefore, in spite of the progress, much work remains. Current FAO/WOAH strategy is to continue removing RVCM from nonapproved laboratories and advocating for reduced RVCM stocks in FAO/WOAH-designated RHFs. Ultimately, the only remaining RCVM materials should be manufactured vaccines and materials for vaccine production and diagnostics. Additional information from a study of sequestration and destruction of rinderpest virus–containing material


As a result, RDV was available for a longer period of time to interact with RNA polymerase and inhibit.NV-CoV-2 is a nanoviricide that is covalently bonded with polyethylene glycol (PEG) and alkyl pendants. This molecular design is used to attack many strains of coronaviruses in a broad-spectrum manner. The ligand works by competitive inhibition and binds to the same site on the S-protein of SARS-CoV that attaches to the cognate cellular receptor, ACE2. This prevents SARS-CoV from binding and infecting the cell. NV-CoV-2 is designed to bind to the free virion particles at multiple points encapsulate the virus and disable its ability to infect the cells. The multi-point binding interaction, like a nano-velcro-tape, may lead to lipid-lipid fusion of the alkyl chains in the nanoviricide micelle with the lipid envelope of the virus. The virus becomes dismantled to a capsid form before the host immune system becomes involved. This putative mechanism is orthogonal to many other anti-coronavirus agents in development. Thus, it maybe possible to produce a stronger antiviral effect when combining NV-CoV-2 therapy with other anti-coronavirus therapies such as Remdesivir (RDV). NV-CoV-2 can encapsulate other antiviral compounds as well. In this study, RDV was encapsulated and protected from serum-mediated degradation  A nanoviricide wraps around the virus particle and encapsulates it, preventing the virus's ability to enter a cell. The viral resistance to the nanoviricide drug is unlikely because it still will bind to the same cell surface receptors, even if the virus mutates.https://www.genengnews.com/covid-19-candidates/nanoviricides/).NV-CoV-2 is a nanoviricide that is recently being developed to treat the SARS-CoV-2 infection. NV-CoV-2 is an off-white, odorless material. At room temperature the material is a waxy, non-crystalline semi-solid that tastes slightly sour in vivo and in vitro studies. The human coronavirus, CoV-NL63 was used in the virus-infected cell culture and rats in vitro and in vivoProof-of-Concept studies were completed through both 2.2.1.A nanoviricide is composed of a number of antiviral small chemical ligands that are covalently attached to a micelle-forming polymeric chain. The polymeric chain is a homopolymer of a monomer that is covalently bonded to polyethylene glycol (PEG), a connecting group and alkyl pendants. Self-assembly of the polymeric chain will occur in an aqueous solution where the PEG shell surrounds the hydrophobic alkyl core. This particular ligand is designed through molecular modeling to bind to the S-protein of SARS-CoV-1 and prevent the virus from binding to the cognate cellular receptor, ACE2. Therefore, the nanoviricide, NV-CoV-2 was designed to prevent coronaviruses such as SARS-CoV-1, SARS-CoV-2 and NL63 from binding to the ACE2 receptor. In addition, NV-CoV-2 has similar activity against the human coronavirus 229E (hCoV-229E), which uses Aminopeptidase N (APN) as its cellular receptor instead of ACE2 2.2.NV-CoV-2 is designed to bind to the free virion particles at different places through receptor-ligand interaction and encapsulate the virus. Encapsulation of the virus disables its ability to infect the cells. Similarly to “nano-velcro-tape”, this multiple binding interaction may lead to a lipid-lipid fusion of the alkyl chains in the nanoviricide micelle with the lipid envelope of the virus. This dismantles the virus to a capsid form, which does not require any involvement from the host immune system. This putative mechanism is orthogonal to many other anti-coronavirus agents that are in development The support of this mechanism is shown in the electron photomicrographs of 2.3.It's possible that NV-CoV-2 can only deliver encapsulated cargo into infected cells and not healthy cells. In fact, it's only the infected cells that display the viral antigen S-protein (S1 and S2 proteins) on either the budding virion or on the exposed cell membrane structures were NV-CoV-2 binds. In addition, NV-CoV-2 can encapsulate to protect other antiviral drugs and act as a double arm effector in humans.in vivo, which could result in rapid recoveries of the patients. The proposed combination is NV-CoV-2 encapsulating remdesivir (RDV) during the standard RDV treatment. RDV is the only antiviral drug approved and being used against the SARS-CoV-2 virus. RDV is the only anti viral that has recently been approved for using against SARS-CoV-2 virus. However, the efficacy of RDV in vivo studies is very low compared to in vitro cell culture studies. RDV is very unstable in the presence of plasma in vivo animal study infected with the CoV-NL63 virus.It is hypothesized that NV-CoV-2 may provide substantial antiviral effects 3.®” that is designed to attack most strains of coronaviruses in a broad-spectrum manner. It is composed of a number of antiviral small chemical ligands that are covalently bound to a micelle-forming polymeric chain. This particular ligand is designed through molecular modeling to bind to the S-protein of SARS-CoV-1 and prevent the virus from binding to the cognate cellular receptor, ACE2. Therefore, NV-CoV-2 was designed to prevent coronaviruses such as SARS-CoV-1, SARS-CoV-2 and NL63 from binding to the ACE2 receptor. NV-CoV-2 also has activity against the human coronavirus 229E (hCoV-229E), which binds to the Aminopeptidase N (APN). This appears to be because of the certain conserved elements in the quaternary structures of ACE2 and APN that are both peptidases with different specificities. The broad-spectrum activity of NV-CoV-2 against both h-CoV-NL63 and h-CoV-229E indicates that it may continue to be active against a number of coronaviruses, even after it mutates NV-CoV-2 is a “nanoviricideThe mechanism of action of NV-CoV-2 is not well defined yet, but is hypothesized to bind to free virion particles at multiple points and encapsulate the virus. Once the virus is encapsulated, it becomes neutralized and its ability to infect cells is disabled. This putative mechanism is orthogonal to many other anti-coronavirus agents that are being developed. Furthermore, it may be beneficial and possible to achieve a stronger antiviral effect by combining NV-CoV-2 nanoviricide with other anti-coronavirus therapies in the future.We have experienced that NV-CoV-2 works in three different ways to destroy the SARS-CoV-2 virus. which causes COVID-19. First, a nanoviricide micelle will bind to a virus particle because of the specific interaction between a ligand on the nanoviricide with the glycoproteins on the virus surface. The flexible nanoviricide can get close to the virus and cause additional ligands to bind to additional viral coat proteins, in a mode called “co-operative binding”. Co-operative binding is a well-known natural process that forms the basis of biological recognition such as antibody-antigen binding complexes, DNA hybridization, and protein assembly. The idea that multiple ligands are attached to a single polymer chain and multiple polymer chains make up a single micelle may lead to a very high avidity for the micelle to bind to the virus. This is analogous to the “Velcro” effect that results from cooperative interactions. This property enables the use of ligands that may individually have low affinity, but the polyvalency can result in very high avidity. This concept has been shown quite convincingly with polymeric vs. monomeric inhibitors in the scientific literature An analogy may be drawn between nanoviricides and neutralizing anti-viral antibodies. Antibodies are an integral part of the natural immune process to bring infections under control. Antibodies can ameliorate or prevent many viral infections and may aid in the resolution of viral infections. Some antibodies do this through a process called neutralization. Antibodies can neutralize viral infectivity by causing aggregation of the virus particles, inhibiting the virus from binding to its host cell surface receptor(s), blocking endocytic uptake into cells and/or preventing virus uncoating in endosomes. When anti-viral antibodies bind to the viruses, the resulting complexes can be lysed upon subsequent binding by complement factors. In addition, virus-antibody complexes can be taken up by phagocytes and destroyed. In contrast, a nanoviricide micelle binds to a virus particle through van der Waals interactions and promotes encapsulation of the virus particle. Re-arrangement of the polymeric chains takes place where the lipid tails of the nanoviricide micelle integrate with the lipid membrane coating the virus (for “enveloped” viruses). Encapsulation of the virus particle may lead to the viral envelope becoming dismantled, viral surface lipids getting stripped off and the removal of glycoproteins that are necessary for viral adsorption and cellular entry.Viruses bind to specific cell surface ligands in order to attach to cells and internalize. Nanoviricides become very useful by neutralizing these crucial steps of the viral particles. A nanoviricide is designed to act like a human cell decoy. When the virus sees the appropriate ligand displayed on a nanoviricide micelle, the virus is believed to bind to it. The nanoviricide is very flexible and can bind all over the viral particle surface, which could potentially lead to fusion with the lipid-coated virus surface by a phase-inversion. Basically, the fatty core of the nanoviricide could merge with the viral lipid coat and the hydrophilic shell of the nanoviricide could become the exterior of the particle, completely engulfing the virus. Once encapsulation occurs the coat proteins that are necessary for the virus to bind to other cells becomes unavailable. This highly targeted attack may also lead to a loss of viral coat proteins and the nanoviricide may further dismantle the engulfed virus capsid. The loss of virus particle integrity could render the virus non-infectious. This proposed mechanism of action  supportsThe antiviral efficacy, potency and the cytotoxicity of the antiviral compound NV-CoV-2 was studied in cell cultures Treatment with NV-CoV-2 and NV-CoV-2-R extended the survival of the rats infected intra-tracheally with a lethal dose of human CoV-NL63 virus. In fact, NV-CoV-2-R showed a better effect than NV-CoV-2, and both treatments were better than RDV treatment alone. Thus, NV-CoV-2-R clearly demonstrates antiviral efficacy in this lethal infection model with CoV-NL63 virus.