Fucus evanescens (FeF) and its enzymatically prepared high-molecular-weight (FeHMP) and low-molecular-weight (FeLMP) fractions to inhibit different stages of AMRV infection in Vero cells was studied. The structures of derivatives obtained were determined using nuclear magnetic resonance (NMR) spectroscopy. We found that fucoidan and its derivatives exhibited significant antiviral activity by affecting the early stages of the AMRV lifecycle, notably virus attachment and penetration. The FeHMP and FeLMP fractions showed the highest anti-adsorption activity by inhibiting AMRV focus formation, with a selective index (SI) > 110; FeF had an SI of ~70. The FeLMP fraction showed a greater virucidal effect compared with FeF and the FeHMP fraction. It was shown by molecular docking that 2O-sulphated fucotetrasaccharide, a main component of the FeLMP fraction, is able to bind with the AMRV envelope glycoproteins Gn/Gc and with integrin β3 to prevent virus–cell interactions. The relatively small size of these sites of interactions explains the higher anti-AMRV activity of the FeLMP fraction.The Hantaan orthohantavirus (genovariant Amur–AMRV) is a rodent-borne zoonotic virus; it is the causative agent of haemorrhagic fever with renal syndrome in humans. The currently limited therapeutic options require the development of effective anti-orthohantavirus drugs. The ability of native fucoidan from  Hantaviridae, order Orthohantavirus) are enveloped, negative-stranded RNA viruses that belong to the most widely distributed zoonotic rodent-borne viruses . Accumulation of the FeHMP fraction with a molecular weight of about 71 kDa indicates the presence in FeF of extended regions with the regular  and [→4)Fucp2S-(1→3)-Fucp2S-Fucp2S residues. Presumably, minor 4O-sulphation is part of a transition zone between regions with regular 2O-sulphation and regular alternating 2O-sulphation and 2,4diO-sulphation.In this work, we have demonstrated the prospects for the use of enzymes in tandem with NMR spectroscopy to establish the structural features of fucoidans. The study of the structure of the FeLMP and FeHMP fractions of FeF, from the brown alga Obtaining standardised fucoidan preparations with defined structures can be the basis of a strategy to develop promising anti-orthohantavirus agents. We have demonstrated for the first time the anti-orthohantavirus activity of FeF and its enzymatically prepared derivatives, the FeHMP and FeLMP fractions, which have defined structures. We showed that FeF and the FeHMP and FeLMP fractions exhibit significant anti-AMRV activity, mainly due to their ability to block the AMRV envelop glycoproteins Gc and Gn and integrin β3 of the host cells. The sites of interaction of between the glycoprotein Gn/Gc and integrin β3 with the fucoidans are relatively small; hence, the FeLMP fraction has relatively higher antiviral activity that FeF and the FeHMP fraction. The data obtained indicate that 2O-sulphated fucooligosaccharides are promising anti-orthohantavirus agents.


Scientific Reports 10.1038/s41598-019-53807-w, published online 29 November 2019Retraction of: The Authors have retracted this article because they are no longer confident that all of the adipose tissue used in this study was obtained under the protocol approved by the Ethics Committee.All Authors agree with the retraction and its wording.


The complex immunological mechanisms of vaccines bring about an inevitable risk of immune-mediated adverse reactions. Of special interest in this time of epidemic is the safety of COVID-19 vaccines and, in particular, the emerging evidence that the ChAdOx1 nCoV-19 adenoviral vector vaccine from AstraZeneca can cause vaccine-induced immune thrombotic thrombocytopenia (VITT). We present the case of a patient who developed an acute paracentral scotoma after having received this vaccine.A 27-year-old female was referred for ophthalmology evaluation because of visual disturbances in her left eye. Her past medical history was unremarkable. Her only medication was an oral contraceptive (combined desogestrel and ethinylestradiol). As she worked in a hospital, she had been prioritised for COVID-19 vaccination and had recently received the first dose of the AstraZeneca vaccine. The same day she developed flu-like symptoms. These resolved 2 days later. However, a left paracentral scotoma appeared at this point.On clinical examination the best-corrected visual acuity was 20/20 in both eyes. Threshold perimetry of the left eye showed a modest paracentral scotoma in the upper temporal quadrant. The intraocular pressure was normal in both eyes, and there were no signs of intraocular inflammation. Fundoscopy of the left eye revealed a delicate teardrop-shaped macular lesion nasally to the fovea Fig. , which wThe signs and symptoms of our patient were consistent with acute macular neuroretinopathy (AMN). The exact pathophysiology of AMN is unknown, but several risk factors have been identified; the most common associations are non-specific flu-like illness or fever and use of oral contraceptives . The pre


Background and objectiveHigh-frequency alternating current (HFAC) can yield a rapid-acting and reversible nerve conduction block. The present study aimed to demonstrate the successful implementation of HFAC block delivery via regenerative macro-sieve electrodes (MSEs).MethodsDual-electrode assemblies in two configurations  were fabricated from pairs of MSEs and implanted in the transected and subsequently repaired sciatic nerves of two male Lewis rats. After four months of postoperative nerve regeneration through the MSEs' transit zones, the efficacy of acute HFAC block was tested for both configurations. Frequencies ranging from 10 kHz to 42 kHz, and stimulus amplitudes with peak-to-peak voltages ranging from 2 V to 20 V were tested. Evoked muscle force measurement was used to quantify the nerve conduction block.ResultsHFAC stimulation delivered via DMSE assemblies obtained a complete block at frequencies of 14 to 26 kHz and stimulus amplitudes of 12 to 20 V p-p. The threshold voltage for the complete block showed an approximately linear dependence on frequency. The threshold voltage for the partial conduction block was also approximately linear. For those frequencies that displayed both partial and complete block, the partial block thresholds were consistently lower.ConclusionThis study provides a proof of concept that regenerative MSEs can achieve complete and reversible conduction block via HFAC stimulation of regenerated nerve tissue. A chronically interfaced DMSE assembly may thereby facilitate the inactivation of targeted nerves in cases wherein pathologic neuronal hyperactivity is involved. Various neurological diseases are characterized by hyperactivity of sensory or motor neurons leading to chronic neuropathic pain or dystonia. These conditions are usually treated via blockade of nerve conduction, thereby alleviating symptoms . ChemicaNumerous extraneural and intraneural electrodes have been used to exploit the benefits of HFAC block in animal models. A conduction block must be achieved at a suitably low stimulation threshold to maintain the nerve’s health and integrity with chronic use. The threshold frequency depends on the blocking electrode’s design and distance to axons ,12. PrevOne alternative to extraneural electrodes is regenerative sieve electrodes. This class of electrodes relies on peripheral nerves’ innate capacity to spontaneously regenerate after transection, as observed following a nerve injury or amputation. Implantation of a regenerative electrode requires placement of the electrode between the proximal and distal stumps of a transected peripheral nerve. Regenerating axons pass through pores  perforating the electrode’s surface. Interspersed among these pores is an array of electrodes that allows for highly selective and specific neural interfacing . The macThe aim of this study was to leverage the MSE as a unique vehicle for the delivery and achievement of HFAC nerve blockade in vivo. We hypothesized that an intimate neuron-electrode interface would require lower stimulus thresholds to achieve nerve block and increase the longevity of this modality. We employed MSEs to specifically block the fascicles under the corresponding electrode at high frequency and low amplitude. Moreover, a highly localized block would be less disruptive to surrounding axons. We believe that this pilot study will lay the groundwork for future studies that will examine the ability of MSEs to deliver the fascicle- and fiber-specific blockade necessary for targeting specific neural pathways.DMSE constructionThe MSE fabrication was performed as described in a prior report . Since tDMSE-I and DMSE-II differed in the separation of their two MSEs. For DMSE-I, the MSEs were separated by a thin layer of medical-grade adhesive  to create an inter-sieve gap of 0.3 mm. For DMSE-II, an inter-sieve gap of 1 mm was created using a segment of silicone guidance conduit . Subsequent assembly proceeded along similar lines. Sixteen microwire leads from a male Omnetics connector  were soldered to the MSEs’ contacts; 2-mm sections of the silicone guidance conduit were attached to either side of the DMSE using the same medical-grade adhesive. The solder points were insulated with medical-grade epoxy . Before surgery, the Omnetics connector terminal was wrapped in Parafilm (Parafilm PM996 Wrap) to prevent tissue deposition on its leads post-implantation.Surgical implantationDMSE-I and DMSE-II were implanted surgically in two adult male Lewis rats weighing 225-250 g Figure . All surExperimental setupFour months postoperatively, implanted rats underwent a non-survival surgery that exposed the sciatic nerve and distal musculature in order to assess the efficacy of the HFAC nerve block. A bipolar cuff electrode placed proximally to the DMSE was connected to an MS-16 stimulus isolator and RX7 base station . This electrode delivered excitatory stimuli to the sciatic nerve. The base station was programmed to generate monophasic, rectangular current pulses at 1-Hz frequency. The pulses had an amplitude of 1 mA and a width of 0.2 ms (muscle force measurements peaked at this width).After injecting the rat subcutaneously with 3 ml of saline, the Omnetics connector was retrieved from its dorsal subcutaneous pocket and connected to a function generator (BK Precision 4003A). This delivered the HFAC blocking signal in the form of a charge-balanced sinusoidal waveform that passed from all eight of the proximal MSE’s channels to all eight of the distal MSE’s channels. All combinations of nine frequencies and nine peak-to-peak voltages were tested for a total of 81 trials. Frequencies tested were 10 kHz, 14 kHz, 18 kHz, 22 kHz, 26 kHz, 30 kHz, 34 kHz, 38 kHz, and 42 kHz. Peak-to-peak voltages tested were 4 V, 6 V, 8 V, 10 V, 12 V, 14 V, 16 V, 18 V, and 20 V. Figure The evoked muscle force was measured in the tibialis anterior (TA) muscle by the following procedure. The sciatic nerve was exposed from the sciatic notch to the popliteal fossa. Next, the TA muscle was exposed by an incision along the anterior portion of the right leg. The distal tendons of the TA were exposed, isolated, and severed distally at the site of insertion into the bone. Animals were immobilized with a C-clamp, and their femoral condyles and distal tendons were anchored to thin-film load cells , which transduced muscle forces evoked by electrical stimulation of the sciatic nerve.Trial structureEach trial had three 10-second phases. In the first phase, the stimulation of the 1-Hz current was initiated and maintained through the remaining two phases. In the second phase, HFAC stimulation was initiated. Finally, in the third phase, HFAC stimulation was terminated. Evoked muscle force measurement continued through all phases to quantify the conduction block Figure .All implanted DMSEs and silicone nerve guidance conduits survived the study duration without causing significant host tissue reactions. MSEs maintained proper placement and orientation perpendicular to the sciatic nerve. Gross observation of DMSEs confirmed the devices’ chronic stability and successful regeneration of nerve tissue through the MSEs’ transit zones. We further confirmed successful nerve regeneration through the implanted device via gross examination upon explantation at the terminal time point.The DMSE-I assembly (0.3-mm inter-sieve distance) failed to deliver HFAC block at any given voltage and frequency. In contrast, the DMSE-II assembly (1-mm inter-sieve distance) successfully delivered a complete and quickly reversible block for frequencies of 14-26 kHz and peak-to-peak voltages of 12-20 V (Figure This study demonstrated the successful initiation of a quickly reversible HFAC nerve block using sinusoidal waveforms passed between the proximal and distal units of a DMSE assembly. The peak-to-peak voltage threshold necessary to achieve complete block increased with frequency, and the block persisted for all amplitudes beyond the threshold. Threshold voltages for partial block also increased with frequency in an approximately linear fashion. For frequencies that exhibited both partial block and complete block, thresholds for the partial block were consistently lower than thresholds for the complete block. Evoked muscle force measurements demonstrated the DMSE’s ability to deliver a complete and reversible conduction block of regenerated nerve fibers using HFAC. Our findings reveal specific parameters of the DMSE system to achieve a successful HFAC block in a mammalian model.An effective HFAC conduction block depends on multiple factors, including frequency, voltage, electrode geometry, the distance between bipolar blocking electrodes, and the distance from the electrodes to their target axonal population . RegardiThe present study examined two DMSE configurations to determine the appropriate minimum inter-sieve distance required to achieve an effective block. DMSE-I’s inter-sieve distance was 0.3 mm; DMSE-II’s was 1 mm. While DMSE-I failed to demonstrate complete or partial nerve block, DMSE-II attained complete block for multiple combinations of frequency and voltage. Our findings are consistent with Ackermann et al.'s study, which showed that the smallest block thresholds for bipolar cuffs were associated with an inter-electrode distance of 1-2 mm . They foLimitationsOur aim in this study was to establish a proof of concept that regenerative MSEs are capable of achieving rapidly reversible HFAC blocks. Our results are limited by a number of factors. Firstly, the sample size was small as it included only two rats. This was chiefly due to the complexity of device construction. Secondly, the validation of the HFAC block on sensory nerve fibers via the measurement of CNAP conduction was not performed. However, the use of evoked muscle measurement and demonstration of muscle activation immediately before and after the HFAC block confirmed that the block of motor fibers was achieved and not due to muscle fatigue. Finally, despite the intimacy of the electrode-axon interface afforded by the MSEs, relatively higher frequencies and voltages were required to achieve blocks compared with prior studies’ cuff electrodes. As mentioned previously, these unique variations are most likely attributable to the morphological parameters and average diameter of axons crossing the MSE, compared with healthy nerve tissue.Future directionsThe MSE shows significant potential as a highly selective, long-lasting interface to peripheral nerve tissue. This study demonstrated the ability of a novel dual macro-sieve assembly to achieve the HFAC block and highlighted the optimum parameters for achieving this block. Our protocol delivered the sinusoidal blocking signal using all 16 DMSE channels and therefore did not leverage the MSE’s inherent selectivity. Future studies will explore the potential of selective blockade of discrete micro-fascicles using cross-sectionally aligned electrode pairs from the proximal and distal MSEs. A successful outcome would have significant clinical implications as it would enable a selective block of nerve fibers associated with specific motor or sensory targets. Moreover, a selective block of pain fibers at higher frequencies without affecting large motor or sensory fibers may be useful in chronic pain management. The DMSE-mediated signal block and fiber selectivity may offer a unique advancement in integrated neural interfacing technology for neural prostheses and non-pharmacologic pain therapies.The DMSE assembly system is capable of delivering a complete and reversible HFAC block after nerve transection and regeneration. We established appropriate inter-sieve distance and block threshold configurations, suggesting that the efficacy of the HFAC block with DMSE depends on the exact in vivo configuration parameters. Maintenance of DMSE over the time period of the study demonstrated longevity of the stable interface for chronic neuromodulation. The intimate electrode-axon contact and the capacity for selective axonal recruitment have set the stage for exploring selective blocking of nerve fibers in the future.


Infected non-unions of the femur can be difficult to treat with a high risk of reinfection and persisting nonunion. Here we demonstrate a case of a 66-year-old female with a chronic infected nonunion of the left femur. The patient fell during temporary external fixation of the infected nonunion and sustained an additional proximal femur shaft fracture. The case was successfully managed with a low-amount silver coating of a long proximal femur nail and an additional augmentation locking plate along with local and systemic antibiotics. After a follow-up of 26 months, both the fracture and also the infected nonunion healed completely without recurrence of infection and without any signs of adverse events in response to the silver coating. In conclusion, silver coated fracture fixation devices can be helpful in difficult to treat infection cases without adverse events. The treatment of infected non-unions of long bones can be difficult. The overall goal for this entity is infection control with a subsequent successful bone healing without recurrence of infection. Antimicrobial coatings of implants has been shown to the successful and without any severe complications in the management of complex infections in trauma, periprosthetic joint infections and orthopaedic oncology To our best knowledge, we here report the first case on the use of a silver-coated intramedullary nail. In this case, an infected non-union of the femur with an additional fresh fracture of the proximal shaft area was managed with a gamma nail with a low-amount silver coating.A 66-year-old female sustained a polytrauma accident with a Gustilo-Anderson type I open distal femur shaft fracture. The open femur fracture was initially treated with external fixation that was then changed to an intramedullary nail a few days later. The patient developed an acute fracture-related infection of the nail, which was then removed with a debridement of the infected fracture site and a hybrid fixateur with monolateral half pin at the proximal and ring fixator construct at the distal fragment was placed. Four months after initial trauma, the patient presented a persisting draining sinus at the lateral aspect of the fracture site. X-rays showed no signs of callus formation and the case was considered as chronically infected non-union of the distal femur . A 2-staStaphylococcus aureus and Staphylococcus epidermidis were found along with E. coli from intraoperative tissue specimens. Systemic antibiotic therapy with meropenem (3x1g i.v.) and vancomicin (1 g i.v.) was started.For stage 1, the hybrid fixateur with all indwelling pins was removed and pin tracks were debrided with a curette and cleaned. The infected non-union site was accessed via a lateral approach and thoroughly debrided resulting in a 3 cm long semi-circumferential defect of the dorsal femur around the non-union site. The intramedullary canal was also debrided by intramedullary reaming and gentamicin-loaded antibiotic beads  were placed intro the intramedullary canal. The 3 cm semi-circumferential defect of the dorsal femur was filled with a vancomycin and gentamicin-loaded PMMA spacer. The femur was stabilized by a monolateral fixateur and the primary wound closure was performed . MethiciThe patient fell two weeks after the first revision on her left site and sustained an additional fresh fracture of the proximal femur shaft. The patient was taken to the OR on the same day and close reduction of the fracture was performed together with extension of the monolateral fixateur to the proximal part of the femur .Fig. 3X-Preoperative software-based planning revealed a 370 mm long and 11 mm thick gamma nail  to be suitable for re-osteosynthesis both for stabilization of the fresh fracture of the proximal and of the infected non-union site at the distal part of the femoral shaft . A gammaThe patient showed successful fracture healing of the proximal fracture site within six weeks E + F andAspergillus fumimatusTo our best knowledge, this is the first case to report about low-amount silver coating of an intramedullary nail for the treatment of an infected nonunion. It was previously reported for successful management with silver-coated knee arthrodesis implants for a recurrent periprosthetic knee infection S. aureus and S. epidermidis, as well against gram-negative strains, such as E. coliThe infected non-union in this case was complicated by an additional fresh fracture due to a fall of the patient during the 2-stage treatment of the infected non-union that was finally treated with a silver-coated gamma nail. This silver coating technology uses only a relatively low amount of silver, which makes silver specific adverse events, such as argyria with dyscolorization of the skin around the indwelling silver-coated implant as reported for high-amount silver coating technologies The released silver ions did not influence successful bone healing neither at the fracture site nor at the former infected nonunion site. This suggests that the low-amount silver coating technology does not negatively interfere with fracture or non-union healing.1.individual low-amount silver coating of intramedullary nails is possible and can be used in complex bone infections surgery.2.Antimicrobial aspects of the released silver ions showed to be beneficial in the presented case to avoid recurrence of infection in the presented case3.Low-amount silver coating technology did not interfere with fracture or non-union bone healing in this case.This case illustrates the following learning points:Volker Alt is consultant for Bio-Gate AG, Nürnberg, Germany.


Pediatric distal forearm fractures are very commonly seen in orthopedic and trauma services. The majority of such fractures involve either distal radius alone or concomitant with distal ulna physis. Isolated injuries of the distal ulna physis are exceedingly rare. Moreover, only one case of isolated Salter-Harris type III distal ulnar physeal injury has been reported in English literature. We report the second such case of isolated distal ulnar physeal injury with Salter-Harris type III pattern in a 15-year-old male child. The injury was treated conservatively in a short-arm splint for four weeks with a good outcome. At the last follow-up of one year, there was no physeal abnormality or growth disturbance detected. Such injuries need to be ruled out in all pediatric distal forearm fractures and can be managed conservatively, if undisplaced, with good outcomes. These patients also need regular monitoring till the closure of the physis to look for the development of any ulnar shortening or deformity due to physeal growth arrest. Injuries of the distal forearm are quite common in childhood and adolescence . Most ofA 15-year-old boy presented to the orthopedic outpatient department of our institute after falling from six feet height on his left hand (non-dominant hand) while playing. He complained of pain in the ulnar aspect of the left wrist and difficulty in holding objects. There was no history of injury to any other part of the body. On examination, he was found to have tenderness and swelling over the left distal ulna, and the wrist range of movement was painful. There was no tenderness over the distal radius or the distal radio-ulnar joint. There were no signs of distal radio-ulnar joint instability. There were no clinical signs suggestive of triangular fibrocartilage complex (TFCC) injury. Plain radiograph of the left wrist with forearm showed negative ulnar variance and a linear vertical undisplaced fracture line in the epiphysis of distal ulna without any physeal abnormality, categorizing it as a type III Salter-Harris injury of distal ulnar physis  score of 7.5 and a Patient-Rated Wrist Evaluation (PRWE) score of 6 ,5. The oPediatric orthopedic injuries commonly involve the distal forearm . The majPhyseal injuries involving the distal radius are often associated with distal radio-ulnar joint injury as well. In patients where the physes are still open, such disruption of the distal radio-ulnar joint is often seen as distal ulnar physeal separation with associated epiphysiolysis of the distal ulna. Such lesions have been described as Galeazzi-equivalent fractures .Isolated distal ulnar physeal injuries are extremely rare to find in English literature ,9. SuthaThere has been only one case of isolated type III Salter-Harris injury of distal ulnar physis described in English literature. Yukata et al. described a rare case of isolated type III Salter-Harris injury of distal ulna in a 13-year-old patient, treated conservatively with good outcome and without any physeal arrest or other complications . Our patDistal forearm injuries, being one of the common trauma cases in the pediatric age group, need to be diagnosed and managed promptly and effectively. We reported a very rare variety of such injuries - an isolated type III Salter-Harris physeal injury involving distal ulna. This is the only second such case reported in English literature. Identification and appropriate management of these fractures hold paramount significance as their mismanagement can lead to physeal arrest/deformity. Such injuries should not be missed upon presentation and can have good outcomes with conservative management. Also, these patients need to be followed up till skeletal maturity to identify any evolving growth-related complications.


To analyze the factors associated with mortality in fracture patients with concomitant COVID-19 infection based on the available published data.Keywords such as “fracture” and “COVID or COVID-19” were searched through three major databases includes PubMed, EMBASE, and Google Scholar. Selection criteria were all published reports providing the mortality related information of COVID-19 positive fracture patients. Published papers containing mortality data of COVID-19 positive fracture patients were considered for qualitative review. For meta-analysis, the presenting individual’s data were considered to study the different parameters association with mortality.The rate of mean mortality in COVID-19 positive fracture patients was 34%, and 91.7% of patients had hip fractures. Older age and hip fractures had a significant association with higher mortality rates in COVID-19 positive fracture patients.The mortality rates are considerably higher in COVID-19 positive patients with fractures compared to COVID-19 positive patients without fractures and to the COVID-19 negative fracture patients. Early surgical intervention should be preferred in hip fractures among COVID-19 positive patients for general stabilization and improved respiratory function. Older age and hip fractures are the main predictors of mortality in these patients. The COVID-19 pandemic has become a significant healthcare issue worldwide. The negative impact has created different domains in patient’s management by considering the high mortality rates  and otheAdditionally, the active phase duration and progression of disease is difficult to predict in fractured patients because the fracture severity and extent can itself bring a change to the level of the inflammatory markers and their levels may further change due to fracture-related complications. The currently available evidence suggests a role of cardiometabolic disease and end-organ failures in non-fractured COVID-19 affected patients to be the chief predictors of mortality . Howeverth June 2020 by Complete abbreviation (PRISMA) guidelines. We used the key terms such as “Fracture” and “COVID or COVID-19”. These two authors were screened the titles and abstracts of the search results for their relevance to the mortality in COVID-19 affected patients presenting with fractures. The full texts of the included studies were analyzed by the two authors. Those articles were included in the study which were presented the mortality-related information of COVID-19 affected fracture patients. All of the included articles were considered for the qualitative review. Those articles were examined for additional quantitative analysis and they were provided the mortality details of individual patients. A secondary manual search was performed for additional articles by scrutinizing bibliographies of publications identified. We excluded those studies with no description of COVID-19 affected fracture patients and those merely discussing fracture management during the COVID-19 pandemic. In accordance with the preferred reporting items for systematic reviews and meta-analyses, two of the present study authors independently searched through Pubmed, Google Scholar and Embase on 27Statistical Analysis 2) of less than 50% were aimed for, and studies lying grossly outside the funnel plot were excluded for individual analysis. A p-value of <0.05 indicated statistical significance.The quantifiable parameters related to individual patients were recorded and the data were pooled in an Excel sheet for statistical analysis. The parameters were broadly classified among the following categories: patient’s characteristics and comorbidities, fracture characteristics, inflammatory markers and surgery-related parameters. Review Manager  was used to perform a meta-analysis of the studies providing quantitative data related to mortality in COVID-19 infected fracture patients. The dichotomous data were analyzed using the combined estimates of the odds ratios (OR) correlating the variation of the above stated parameters to the mortality in COVID-19 infected fracture patients. The continuous data were analyzed using weighted mean differences (WMD) and 95% confidence intervals (95% CI) of the above stated parameters divided into mortality and survival groups. For each of the analyses, the fixed-effect model and heterogeneity  with maximum deaths reported in patients with hip or proximal femoral fractures. In COVID-19 positive patients with hip fractures, the mortality was 56%. Interestingly, 91.7% of the fractures among the reviewed studies belonged to the proximal femoral fractures in elderly patients suggesting a low energy trauma profile of the presenting fractures. The mean age was 77.32 years suggesting an overall elderly cohort of patients. The detailed observations from the reviewed studies have been provided in et al., [et al., [et al., [The elderly patients with lower limb fractures carry a risk of chest deterioration if not mobilized early. The accompanying COVID-19 illness can pose a major challenge in the operative management of these patients due to either preoperative COVID-19 pneumonia or postoperative respiratory deterioration. Catellani [et al.,  advocate[et al.,  advocateet al., [et al., [et al., [et al., [et al., [Cheung et al.,  observed[et al.,  observed[et al.,  observed[et al.,  observed[et al.,  comparedQuantitative Analysis  Eight articles were considered for the quantitative synthesis as they provided information of individual COVID-19 positive fracture patients , 17, 18.Patient’s Characteristics p=0.0007, Among the patient’s characteristics that considered patients’ age, gender, individual comorbidities and the total number of comorbidities, we found that the older age of the patients had a significant influence on increased mortality , D-dimer values, Procalcitonin, Lactate Dehydrogenase levels, and CRP levels. While no statistically significant associations were observed with TLC, D-dimer values and CRP levels, the associations of Procalcitonin and Lactate dehydrogenase levels could not be analysed due to insufficient comparative data for meta-analysis .Surgery Related Parameters Among the surgery-related parameters, we analyzed the mortality associated with operative management vs. non-operative management, ASA grades, delay in operative management, and hemiarthroplasty vs. cephalomedullary nailing for hip fractures. However, no statistically significant association was observed  and 3.What is known about mortality in COVID-19 patients? The mortality rates with COVID-19 infection are high. The earlier predicted overall death rate with COVID-19 infection was approximately 4% . The disHow Mortality Differs in COVID-19 Patients with Fractures? The current evidence concerning fracture management is limited in COVID-19 positive patients. The restrictions around the world with lockdown measures have markedly brought down the overall fracture incidence . The patShould Surgery Be Delayed in Fracture Patients? While there had been a general predilection towards delaying surgery, the lower limb fractures especially hip fractures should be prioritized considering the complications related to delayed fracture management. However, the COVID-19 management should be prioritized and the fracture management can be delayed in patients with symptomatic COVID-19 related illness . SurgeryCurrent Consensus in Operative Management of Fractures in COVID-19 Patients The families of the COVID-19 positive fracture patients should be counseled regarding the significantly higher mortality and complications especially with hip fractures. COVID-19 infection causes an increase in perioperative mortality in proximal femoral fractures. All trauma patients who require surgery should undergo screening for COVID-19 to avoid unnecessary risk to the patient if the surgery can be delayed by a couple of weeks with minimal impact on outcome . All effSuggestions from the Current Review With very limited evidence available regarding the mortality in COVID-19 fractures affected patients, it is difficult to determine the prognosis of the patients with operative and non-operative management. No consensus has been established whether to delay the fracture surgery or not and what could be the possible outcomes. A knowledge about the factors influencing the mortality related outcomes in fracture management can help surgeons in choosing the appropriate management according to the patient characteristics and fracture patterns. Our review suggests significantly higher early mortality with increasing age and hip fractures in COVID-19 infected patients. However, more evidence is required to establish valid recommendations due to limited number of studies related to fracture management in such patients with wide confidence intervals in the meta-analysis . Limitations There were several limitations of this review. The review is based on a small number of observational studies performed over a short period of time. The quality of evidence belongs to level III and level IV studies. The samples sizes of the included studies were small. Not all the parameters influencing the mortality in COVID-19 positive fracture patients were studied in every reviewed study. Most of the fractures studied belonged to the geriatric hip fracture group, as such patients frequently require early inpatient care. The outcomes with upper limb fractures may vary, and can potentially change the overall results as well. Lastly, the mortality rates accounted for the inpatient mortality while the patients were still under observation. A further change in mortality rates can occur with post-discharge follow-ups. Nevertheless, the current review fulfills the purpose of a comprehensive analysis which is lacking in the current scenario. Additionally, further evidence with a longer time frame would be needed for a detailed review especially meta-analysis that could establish a stronger inference. The mortality rates in COVID-19 positive patients with fractures are considerably higher compared to COVID-19 positive patients without fractures and to the COVID-19 negative fracture patients. The available evidence suggests that older age and hip fractures are the chief parameters associated with higher mortality rates in COVID-19 positive fracture patients. Early surgical intervention should be preferred in hip fractures among COVID-19 positive patients for general stabilization and improvement of respiratory function.


Metabolism is a common cellular feature. Cancer creates a suppressive microenvironment resulting in inactivation of antigen-specific T cells by metabolic reprogramming. Development of approaches that enhance and sustain physiologic properties of T cell metabolism to prevent T cell inactivation and promote effector function in the tumor microenvironment is an urgent need for improvement of cell-based cancer immunotherapies. Due to these well-established anti-tumor properties, T cell-based immunotherapies, particularly chimeric antigen receptor (CAR) T cells, have extended our armories in the battle against cancer and, together with checkpoint inhibitor blockade, have revolutionized cancer immunotherapy [T cells are central regulators of anti-tumor immunity . Cytolytotherapy . Althougotherapy ,4, the sotherapy –7. Variootherapy ,9.Metabolism, is a key mechanism that shapes the properties of the TME. Cancer creates a hostile metabolic microenvironment by various mechanisms including nutrient deprivation, metabolic competition, hypoxia, lactate production and oxidative stress ,11. ThesA recent study attempted to target this specific step of T cell activation by employing a novel approach to enhance initiation of T cell signaling in order to overcome the detrimental challenges of the TME . BecauseThis study provides evidence for the first time that this cell-surface anchor-engineering approach provides the opportunity to use high concentrations of metabolism-modulating drugs to target specifically and directly antigen-specific T cells prior to adoptive transfer. This approach minimizes the non-specific effects that might arise from systemic administration of a drug with the purpose and hope to target T cells of the TME. This approach provides a sustained metabolism-modifying impact that overcomes the detrimental implications of the TME on the specific module of T cell metabolism that governs TCR signal transduction. Importantly, this seems to have a long-lasting impact on T cell function.The recent success in anchoring an active drug at the cell plasma membrane opens new avenues in therapeutic intervention at a cell-specific manner. This approach is promising particularly in the context of cell-based therapies, where ex vivo or in vitro modification prior to adoptive transfer is feasible. Of note, although in vitro metabolic modulation of T cells before adaptive transfer has been previously proposed and tested, this approach is limited by the metabolically hostile TME that is capable of alternating the intrinsic metabolic state of infiltrating T cells. Anchoring a metabolism-modulating drug in tumor-specific T cells prior to adoptive transfer might overcome this limitation and allow therapeutic exploitation of metabolism for improvement of T cell function in cancer therapy.Several challenges and questions remain to be answered. For example: how stable is the maintenance of a membrane-anchored engineered compound? Do engineered T cells undergo unimpeded divisions after antigen encounter despite the modification? Do daughter cells preserve the engineered compound on their cell surface, and at what levels? Can engineered T cells undergo differentiation to T memory cells after exposure to tumor-associated antigens in the TME and survive beyond effector phase to provide immune surveillance that prevents cancer relapse? Can such modifications be implemented for therapeutic alteration of intracellular molecules such as enzymes or transcription factors that imprint distinct metabolic and functional fates in T cells? Despite the many unanswered questions, the new technology provides hope for a new era in the generation of engineered antigen-specific T cells for improvement of cell-based immunotherapies in cancer.


Plants have evolved a plethora of pathways at the thylakoid membrane but also in the chloroplast stroma to avoid acceptor side limitation. For instance, reduced ferredoxin can be recycled in cyclic electron flow or reducing equivalents can be indirectly exported from the organelle via the malate valve, a coordinated effort of stromal malate dehydrogenases and envelope membrane transporters. For a long time, the NADP(H) was assumed to be the only nicotinamide adenine dinucleotide coenzyme to participate in diurnal chloroplast metabolism and the export of reductants via this route. However, over the last years several independent studies have indicated an underappreciated role for NAD(H) in illuminated leaf plastids. In part, it explains the existence of the light-independent NAD-specific malate dehydrogenase in the stroma. We review the history of the malate valve and discuss the potential role of stromal NAD(H) for the plant survival under adverse growth conditions as well as the option to utilize the stromal NAD(H) pool to mitigate PSI damage.Plant productivity greatly relies on a flawless concerted function of the two photosystems (PS) in the chloroplast thylakoid membrane. While damage to PSII can be rapidly resolved, PSI repair is complex and time-consuming. A major threat to PSI integrity is acceptor side limitation e.g., through a lack of stromal NADP ready to accept electrons from PSI. This situation can occur when oscillations in growth light and temperature result in a drop of CO This enabled oxygen-requiring metabolic and biosynthetic pathways which form the basis for higher multicellular life on earth  at the level of the thylakoid membrane  across the chloroplast thylakoid membrane in response to light intensity shifts  on to the first e− acceptor of the electron transport chain (ETC). The e− gap in the oxidized P680+ molecule is filled through the splitting of water molecules which results in the release of e− and molecular O2  to plastoquinol (PHQ) reduction through the lipid matrix of the thylakoid membrane to the cytochrome b6f complex (Cyt b6f). Cyt b6f transfers the e− over to the luminal e− carrier protein plastocyanin (PC). Reduced PC travels the longest distance in the ETC by diffusion to the PSI donor side where it re-reduces P700+  reaction steps, the stromal enzyme ferredoxin-NADP+ oxidoreductase (FNR) reduces NADP+ to NADPH  production and concomitant PSI damage  causes charge separation in the chlorophyll a (Chl a) molecules located at the P680 reaction center of PSII . SubsequPH Mulo, . In summPH Mulo, . During Scheibe, . NADPH cScheibe, . A short− rushing toward PSI via LEF. In addition, high ΔpH slows down PQH2 oxidation at Cyt b6f and therefore LEF e− transport. This process is called photosynthetic control  . PTOX oxidizes PQH2 by reducing O2 to H2O  around PSI. CEF diverts energy into the ATP pool without NADPH production , STN7 becomes active resulting in LHCII phosphorylation and migration to PSI (State 2). This situation supports CEF and overall protects PSI . However, if this were correct it seems hard to comprehend why C3 plant species possess NAD-dependent MDHs with confirmed enzyme activity in light and dark-incubated chloroplasts  in the inner envelope . Conversely, the physiologically assigned role for NAD-MDH has been participation in the chloroplast's dark metabolism, i.e., the incomplete glycolysis  may also play a role in daytime chloroplast metabolism.While all these assumptions have merit, several discoveries from the C3 plants relied heavily on the NADP dependent malate valve, shouldn't we expect more dramatic effects especially on photosynthesis and PSI function in NADP-mdh loss-of-function lines? To our knowledge, PSI acceptor side limitation in NADP-mdh mutants has not been reported. Moreover, expression of an NADP-MDH variant that is no longer controlled by the redox poise and thus active during the night did not affect the mutants' growth under long-day conditions. This indicates that the enzyme activity did not significantly affect the OPPP as originally assumed. Interestingly, the mutants were more susceptible to fluctuating light and extended night periods  Loss of the sole NADP-MDH gene does not result in a noticeable phenotype  If a lack of NADP-MDH could be backed up by NAD-MDH, as increases in Jackson, .3) By now we know a handful of NAD(H)-dependent stromal reactions. Is it really feasible that these strictly occur in the dark when according to the original hypothesis flux through plastid glycolysis would be sufficient to build up a decently sized stromal NADH pool? Disputing this idea, chloroplast fatty acid synthesis, which includes NAD(H)-dependent steps, is also active during the day  What are the consequences of losing stromal NAD-MDH activity and the export of reducing equivalents via this route? How does it affect photosynthesis and PSI function? These questions have not been investigated but deserve research attention.NAD-MDH gene loss via T-DNA insertions is lethal  background. Several studies on different Arabidopsis accessions have shown that small genetic variations play a big part in the phenotypic response to light regimes  background.Recent findings on NAD-MDH mutants are highly intriguing but complicate future studies. Independent groups have shown that a homozygous NAD-mdh point mutant som410 (suppressor of mosaic death 1). This non-functional NAD-MDH mutant resembles the wild type  but more biomass than wild-type plants if grown under constant light  which in Arabidopsis are expressed in heterotrophic tissues  over NADP(H): (1) NAD-specific glyceraldehyde-3-phosphate (Gap) dehydrogenases , pgdh3 mutants revealed an increased transient NPQ level under non-saturating actinic light compared to wild-type plants  when fluctuating growth light was applied. In contrast, pgdh3 plants were capable to adjust to constant highlight stress which suggests other mechanisms provide protection under such conditions. Altogether, the data indicate that the NADH-dependent pathway provides a well-needed alternative route to export reducing equivalents from the stroma -dependent pathway in illuminated chloroplasts could inspire new strategies to secure plant productivity during light stress e.g., by protecting PSI through avoiding acceptor side limitations and concomitant damage to PSI. To reach this goal we suggest a few research directions and potential challenges.pgdh3 mutants . However, only PGDH1 seemed to be directly regulated by TRX according to It will be critical to further exploit assays that allow activity monitoring of either MDH type simultaneously. This could be realized through employing genetically encoded sensors. A number of studies from Arabidopsis report significant progress toward this goal  and NADP(H). When we started our research, we were surprised how little information was available in the newer literature. Over 30 years ago, a dual coenzyme specificity for NAD(H) and NADP(H) was shown for the chloroplast GapA/B isoforms. Other NAD(H)-dependent reactions or enzymes with dual coenzyme specificity may have been overlooked. Regardless if additional NAD(H) or dual coenzyme specific dehydrogenases will emerge, the data will greatly improve our perspective of the stromal redox poise.Finally, we propose that characterization of  NAD(P) dehydrogenases should generally determine V3 photosynthesis and the constraints their flux has on photosynthetic efficiency. Once this has been achieved, engineering these pathways could improve plant yields in the field, for instance through avoidance of PSI acceptor side limitations and irreversible PSI damage. Even decades after their first description, the chloroplast malate valve and its two MDH enzymes remain therefore highly interesting. Thorough future research employing a combination of new and more traditional tools such as in vivo imaging of genetically encoded sensors and chlorophyll fluorescence spectroscopy of plants exposed to standard and dynamic growth conditions may yield insights to unlock the full potential of the malate valve and its linked pathways for biotechnological applications.Understanding the relative light-dependent activity of NADP(H) vs. NAD(H)-coupled reactions in the stroma and their contribution to malate export from the chloroplast will yield a better understanding of CH-HK wrote the manuscript. MK edited text, references, and designed the figure. Both authors contributed to the article and approved the submitted version.H-HK was funded by the 3rd call ERA-CAPS grant (NSF IOS-1847382) and a National Science Foundation Career Award (IOS-1553506).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.


This suggests that the fitness cost of nAb-evading mutations is low. We then used evolutionary modeling to predict the frequency of immune escape before and after the widespread presence of nAbs due to vaccines, passive immunization or natural immunity. Our modeling suggests that SARS-CoV-2 mutants with one or two mildly deleterious mutations are expected to exist in high numbers due to neutral genetic variation, and consequently resistance to vaccines or other prophylactics that rely on one or two antibodies for protection can develop quickly -and repeatedly- under positive selection. Predicted resistance timelines are comparable to those of the decay kinetics of nAbs raised against vaccinal or natural antigens, raising a second potential mechanism for loss of immunity in the population. Strategies for viral elimination should therefore be diversified across molecular targets and therapeutic modalities.The spike protein receptor-binding domain (RBD) of SARS-CoV-2 is the molecular target for many vaccines and antibody-based prophylactics aimed at bringing COVID-19 under control. Such a narrow molecular focus raises the specter of viral immune evasion as a potential failure mode for these biomedical interventions. With the emergence of new strains of SARS-CoV-2 with altered transmissibility and immune evasion potential, a critical question is this: how easily can the virus escape neutralizing antibodies (nAbs) targeting the spike RBD? To answer this question, we combined an analysis of the RBD structure-function with an evolutionary modeling framework. Our structure-function analysis revealed that epitopes for RBD-targeting nAbs overlap one another substantially and can be evaded by escape mutants with ACE2 affinities comparable to the wild type, that are observed in sequence surveillance data and infect cells  The deployment of vaccines against SARS-CoV-2 brings the question of mutational escape from antibody prophylaxis to the forefront. Rapid evolutionary evasion of neutralizing antibodies (nAbs) poses a number of threats to biomedical interventions aimed at bringing the virus under control, namely the risk of reduced vaccinal efficacy over time as resistant variants continue to emerge , the risk of waning effectiveness of natural immunity as a result of evasion of common nAbs, and the risk of antibody-dependent enhancement (ADE).-3 substitutions per base per year [SARS-CoV-2 is commonly considered to acquire mutations more slowly than other RNA viruses ,2. Howevper year  peak neaper year . Thus, tper year . Hence, −6 sub/nuc/yr) [−2 sub/nuc/yr)  , others /nuc/yr) . There i/nuc/yr) . StudiesTo better understand the potential for immune evasion mediated by SARS-CoV-2 RBD mutations in the presence of nAbs, singly or in combination, we focused on three questions. First, what is the evolutionary cost of harboring nAb-evading RBD mutations? Second, given this evolutionary cost, how likely is it that SARS-CoV-2 patients will harbor viruses with pre-existing nAb-evading RBD mutations as their dominant viral sequence? Third, how rapidly will such nAb-evading RBD mutants become fixed in the population once nAb vaccines and therapies are deployed widely?To explore the diversity of the B-cell response against the RBD, we catalogued the reported spike RBD epitopes recognized by the natural human immune response. Consistent with prior work , we founin vitro resistance to SARS-CoV-2 nAbs . We determined how well changes in RBD binding to ACE2 or RBD expression caused by a mutation  predicteTaken together, the narrow focus of the immune response on a specific region of the RBD , the immin vitro, which could lead to reduced host-host transmission [de novo mutation which allows them to exist at nonzero frequencies. However, once nAbs are common in the population, these mutants will have a selective advantage. If they already exist at sufficient frequency in the population, the escape mutants will expand deterministically and lead to widespread SARS-CoV-2 resistance to nAbs.Based on this assessment, we used evolutionary theory to predict the frequency of immune escape mutants in the population both before and after the widespread presence of nAbs due to vaccines, passive immunization or natural immunity. Before immunity or antibody prophylaxis is widely established in the population, there is no transmission advantage for viruses with immune escape mutations since most people are equally susceptible to infection from wild-type and mutant SARS-CoV-2. Instead, these mutations may have a small evolutionary fitness cost due to negative effects on ACE2 binding affinity or other factors, similar to the observed fitness cost of drug resistance mutations in HIV  and conssmission . Althoug-5 to 1x10-4 (Methods) [Using mathematical modeling methods developed to study intrahost evolutionary dynamics during HIV infection, we calculated the expected number of infected individuals whose dominant viral sequence harbors one or more mildly deleterious immune-evading mutations under drift conditions  . Most reMethods) . Many nAMethods) . We inveThe expected number of SARS-CoV-2-infected individuals whose dominant viral sequence harbors one or two immune escape point mutations is high enough to eventually lead to widespread resistance to nAbs. Over a range of fitness costs and assuming a population of at least 5 million active infections (which we note is vastly lower than estimates as of 12/28/2020), we predicted that over 10,000 SARS-CoV-2-infected individuals worldwide would harbor a dominant viral sequence capable of evading one antibody . This nuIf more than two mutations are required for a virus to escape nAbs, it is much less likely that population-level resistance will arise immediately. Each triple mutant is expected to be at appreciable frequencies only when the fitness cost of immune-evading mutations is lower than 0.04 . SpecifiEven if a specific combination of mutations that confers resistance to an antibody combination is not present before the intervention is released, spontaneous mutation and positive selection will eventually lead to expansion of an escape mutant. To estimate how quickly population-level resistance to SARS-CoV-2 antibodies will emerge under natural selection, we modeled the acquisition of multiple mutations over time as a fitness valley-crossing problem (Methods). To acquire a specific combination of mutations that confers therapeutic resistance, the wild-type virus must transit through a valley of intermediate lower-fitness genotypes that have some, but not all, of the mutations required for immune escape. Previous theoretical expressions describing the time required to cross a fitness valley  were use-4 (Methods), differences between antibody epitope sizes or changes in the mutation rate of the virus population over time [-5 (-3 per transmission), resistance could emerge against even combinations of nAbs that require the acquisition of 4 mutations  suggests that SARS-CoV-2 has extensive capacity to evolve to evade nAbs targeting a small number of antigenic regions. This capacity will negatively impact SARS-CoV-2 immunity in humans, whether active  or passive (nAb prophylactics).The work described in this paper points to the mutation tolerance of SARS-CoV-2 spike protein, placing this property in context with the mutation rates and pandemic sizes (number of active infections) to estimate the ease with which the virus will mutate to defeat combinations of neutralizing antibodies. Numerous COVID-19 antibody prophylactics and vaccines target the spike protein ,26, and in vitro replication fitness to wild-type virus [in vitro infectivity  or that do not exert a specific selective pressure on the virus  are key to reducing the risk of immune escape. In this context, vaccines that do not provide sterilizing immunity (and therefore continue to permit transmission) will lead to the buildup of large standing populations of virus , greatlyThe evolvability of SARS-CoV-2 in response to selection pressure will determine the ultimate tractability of our efforts at disease control. Our work suggests that the capacity of SARS-CoV-2 to evade the immune system may be greater than originally anticipated and raises the specter of a process of ongoing and continuous evolution in response to antibody-based prophylaxis, occurring on a timescale that may not be convenient or tractable for the design of novel biomedical interventions. Thus, our findings speak to the need for both public health and biomedical intervention strategies targeting SARS-CoV-2 to be designed to account for the risk of rapid evolutionary response to biomedical interventions.st, 2020 which matched the criteria “Source Organism Taxonomy Name equals SARS-2 AND Source Organism Taxonomy Name equals Homo sapiens”. Structures were included if there were patient-derived nAbs present and the authors reported the binding residues. Structures were excluded if they were not patient-derived, if they were not nAbs, or if the authors did not report the binding epitope because the resolution was too low to identify it precisely. Additionally, other epitopes that met the inclusion criteria but were not found in the PDB were included on an ad hoc basis. The structures used are listed in The authors performed a comprehensive search of all entries in the Protein Data Bank (PDB)  as of SeAfter the search was completed, it was determined that there were too few epitopes reported outside of the RBD to attempt clustering in those residues. Thus, the clustering analysis was limited to the RBD. The RBD was defined as in .The antibodies were clustered using the Density-Based Spatial Clustering of Applications with Noise (DBSCAN) algorithm . The disin vitro infectivity of SARS-CoV-2 pseudovirus, we examined the predictivity of changes in RBD binding to ACE2 or expression according to [in vitro infectivity was evaluated based on the ability to predict a 90% or greater infectivity relative to the wildtype (WT) strain.Tolerability of SARS-CoV-2 to changes in ACE2 binding affinity or RBD expression were examined based on a receiver operating characteristic (ROC) analysis. Based on publications measuring the impact of 44 spike RBD mutations on the rding to  for grearding to . These mrding to  or experrding to ,51. PredS), individuals infected with WT virus (I0), infected with mutant virus (I1), and recovered or resistant to infection from WT virus (R). The number of individuals in each compartment obey the following differential equations:β is the transmission rate of WT virus, μ is the per transmission probability of mutation at a specific site, s is the fitness cost to transmission of the mutation, and δ is the recovery rate from the infection. The infected compartments of this SIR model have the same mathematical description as the virus-infected cells in the intrahost virus dynamics model presented by Ribeiro and co-workers [S and R can be treated as constant. Therefore, for the frequency of virus mutants present in the population at steady state before establishment of widespread immunity or vaccination, we used the frequency of individuals infected with a virus with a single mutation f1 = μ/s, which agrees with the expected frequency of single mutants under mutation-selection equilibrium [k mutations isTo investigate the emergence of SARS-CoV-2 resistance to nAbs, we modeled virus transmission dynamics using a modified deterministic susceptible-infectious-recovered (SIR) model with mutation. The simplest version of this model includes two viral genotypes, WT and mutant viruses with a specific single nucleotide change. At the population level there are four different compartments: susceptible individuals , viruses with antibody escape mutations will have a fitness advantage over the WT virus. This advantage comes from the ability of the mutant virus to infect individuals who are immune to the WT virus and depends on the fraction of the population who are immune. If a single mutant has the ability to infect recovered/resistant individuals, the SIR model equations for infected individuals change toN is constant, if a single mutation is sufficient to lead to immune escape, the time needed to establish an immune escape mutant is exponentially distributed with expected time 1/Nμw generations [In order for a virus with one or more mutations that confers immune escape to expand deterministically due to positive selection and establish in the population, the variant must first be created through mutation of a single virion. Then, the mutant virus must infect enough individuals so that it is unlikely to go extinct due to stochastic drift. Assuming the total number of infected individuals erations .k mutations are required for immune escape and all intermediates with less than k mutations have fitness cost s, the time τk to establishment of the k-mutant was approximated asγ is Euler’s constant, pi of an i-mutant to be successful was approximated asTo estimate the time needed for establishment of double-, triple-, or higher-order mutants that confer immune escape, we adapted previously reported work on the dynamics of asexual populations crossing fitness valleys . If k muNμ < 1). Following the argument for large populations in [Nμ > 1 we treated mutants with few mutations deterministically. We again used the results in [i-mutants at steady state under mutation-selection equilibrium fi for i < = k. The minimum value of i for which the estimated i-mutant population size Nfi was < 1/μ was taken as the mutant with the most mutations that could be treated deterministically as a constant-sized population. Denoting this number of mutations as j, the modified expression for the expected time to escape for large population sizes isThis approximation holds for intermediate population sizes .(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 FigTime required to establish a resistant viral single (A), double (B), triple (C), or quadruple (D) mutant with different fitness costs for intermediate mutants. In our model, viral variants with some, but not all, mutations required for resistance to an antibody intervention have a fitness cost (ranging from 1–9% less infectious). Increasing the fitness cost of these intermediates prolongs the time required for a resistant variant with a specific combination of 2–4 mutations (B-D) to establish in the population.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


There is an urgent unmet clinical need for the identification of novel therapeutics for the treatment of COVID-19. A number of COVID-19 late phase trial platforms have been developed to investigate (often repurposed) drugs both in the UK and globally (e.g. RECOVERY led by the University of Oxford and SOLIDARITY led by WHO). There is a pressing need to investigate novel candidates within early phase trial platforms, from which promising candidates can feed into established later phase platforms. AGILE grew from a UK-wide collaboration to undertake early stage clinical evaluation of candidates for SARS-CoV-2 infection to accelerate national and global healthcare interventions.AGILE is a seamless phase I/IIa platform study to establish the optimum dose, determine the activity and safety of each candidate and recommend whether it should be evaluated further. Each candidate is evaluated in its own trial, either as an open label single arm healthy volunteer study or in patients, randomising between candidate and control usually in a 2:1 allocation in favour of the candidate. Each dose is assessed sequentially for safety usually in cohorts of 6 patients. Once a phase II dose has been identified, efficacy is assessed by seamlessly expanding into a larger cohort. AGILE is completely flexible in that the core design in the master protocol can be adapted for each candidate based on prior knowledge of the candidate . This information is detailed in each candidate specific trial protocol of the master protocol.Few approved treatments for COVID-19 are available such as dexamethasone, remdesivir and tocilizumab in hospitalised patients. The AGILE platform aims to rapidly identify new efficacious and safe treatments to help end the current global COVID-19 pandemic. We currently have three candidate specific trials within this platform study that are open to recruitment.2020-001860-27 14 March 2020EudraCT Number: ClinicalTrials.gov Identifier: NCT04746183 19 February 2021ISRCTN reference: 27106947The online version contains supplementary material available at 10.1186/s13063-021-05458-4. In 2019, a novel coronavirus disease (COVID-19) emerged in Wuhan, China, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) . The proGiven the rapid and global spread of COVID-19 and the emergence of new variants in the UK, South Africa, India and Brazil, a robust but rapid assessment of potential treatments is needed . The AGIPhase I—To determine the optimal dose of each candidate (or combination of candidates) entered into the platform. Phase II—To determine the efficacy and safety of each candidate entered into the platform, compared to the current Standard of Care (SoC), and recommend whether it should be evaluated further in later phase II and III platforms.AGILE is a multicentre, multi-candidate, multi-dose, multi-stage, randomised phase I/IIa Bayesian adaptive platform trial to determine the safety and efficacy of multiple candidate agents for the treatment of COVID-19. The multi-candidate design allows many candidates to be tested simultaneously , in order to increase efficiency compared to multiple single-candidate studies. The multi-dose feature allows progression beyond the licensed dose dependent on adequate safety and tolerability data and promising efficacy. The multi-stage feature allows for pre-specified analyses that can be used to determine dropping of ineffective doses or candidates or recommending doses or candidates for further phase II/III testing, further increasing efficiency of the study. The adaptive platform design allows for the removal of unpromising candidates, promising candidates to be recommended for further testing in external phase II/III studies, and the addition of new potential treatments to be added during the trial. Candidates will be added into the AGILE platform study via candidate specific trial (CST) protocols to the master protocol.2)/fractional inspired oxygen (FiO2) ≥ 150 or peripheral capillary oxygen saturation (SpO2)/FiO2 ≥ 200], 8 [hospitalised mechanical ventilation PaO2/FiO2 < 150 (SpO2/FiO2 < 200) or vasopressors] or 9 . Group B (mild-moderate disease)—ambulant or hospitalised patients with SpO2 > 94% room air. If any CSTs include participants from the community setting, the CST protocol will clarify whether patients with suspected SARS-CoV-2 infection (in addition to laboratory-confirmed infection) are also eligible. Group C —to be defined in each CST protocol. Participants are currently being recruited from the UK, but we intend to open up recruitment at international sites later in 2021.Patient populations can vary between CSTs, but the main eligibility criteria include adult patients (≥ 18 years) who have laboratory-confirmed infection with SARS-CoV-2. We will include both severe and mild-moderate patients as well as healthy volunteers defined as follows: group A (severe disease)—patients with WHO Clinical Progression Scale  of gradeThe comparator is the current SoC, and in some CSTs, placebo is added to the current SoC. Candidates that prevent uncontrolled cytokine release, prevention of viral replication, and other anti-viral treatment strategies are at various stages of development for inclusion into AGILE. Other CSTs will be added over time. There is not a set limit on the number of CSTs we can include within the AGILE master protocol, and we will upload each CST into this publication as each opens to recruitment.2 < 92%) or hospitalisation or death, or change in time-weighted viral load [measured up to 29 days from randomisation].Phase I—Dose limiting toxicities using Common Terminology Criteria for Adverse Events (CTCAE) Version 5 Grade ≥ 3 adverse events. Phase II—Agreed on a CST basis depending on mechanism of action of the candidate and patient population, but may include time to clinical improvement of at least 2 points on the WHO Clinical Progression Scale [measured up to 29 days from randomisation], progression of disease  and between 32 and 40 participants are required across the dose-finding and efficacy evaluation when capping the maximum number of participants contributing to the evaluation of a treatment at 40. Each CST protocol will give details on the sample size needed for that candidate and a justification for any CST needing more than 40 participants.www.agiletrial.net  will evaluate and prioritise candidate agents for inclusion in the study. The design of AGILE allows for the assessment of multiple candidates at different doses, with the ability to add candidates as they are identified or drop them as their evaluation is completed. Promising candidates will move to an external trial for further evaluation in the phase II/III setting. AGILE is completely flexible and each CST design is catered to the requirement of the candidate whereby population, primary endpoint and sample size can be amended. AGILE has full regulatory approval  and has three CSTs currently open to recruitment with a number of others at various stages of development. AGILE has the potential to rewrite the treatment landscape for COVID-19 and shape our response to future global pandemics.The AGILE master protocol Version 9 (17 February 2021) has full regulatory approval and utilises several digital technology solutions, including Medidata’s Rave Electronic Data Capture (EDC), Randomization and Trial Supply Management (RTSM) for randomisation and drug distribution, patient eConsent on iPads and electronic Clinical Outcome Assessment (eCOA) to collect patient reported outcome measures via an App on the participants smart phones.CST-2 has full regulatory approval, has completed phase I and has successfully determined a dose for phase II evaluation. Phase II recruitment is ongoing following a planned interim analysis by an Independent Data Monitoring Committee. Molnupiravir is being evaluated in adult ambulant patients with laboratory confirmed COVID-19, who are within 5 days of symptom onset in the UK. Molnupiravir (EIDD-2801) is the 5′-isopropyl ester prodrug of the broadly active, direct-acting antiviral ribonucleoside analogue EIDD-1931. Molnupiravir is being developed for the treatment of infections caused by RNA viruses, specifically for COVID-19 and other coronavirus infections, influenza and Venezuelan equine encephalitis virus , 11.CST-3A has full regulatory approval and has recently completed recruitment. Nitazoxanide is being evaluated in healthy adults in the UK. Nitazoxanide is a thiazolide FDA-approved antiparasitic medicine used for the treatment of cryptosporidiosis and giardiasis diarrhoea but has been studied previously for viruses including influenza . HoweverCST-5 has full regulatory approval and phase I is currently recruiting. VIR-7832 and VIR-7831 are being evaluated in adult ambulant patients with laboratory-confirmed COVID-19, who are within 5 days of symptom onset in the UK. VIR-7832 and VIR-7831 are both human monoclonal antibodies that neutralise COVID-19 by targeting the receptor-binding domain of the SARS-CoV-2 Spike (S) glycoprotein . The binAdditional file 1.. AGILE Master Protocol Version 9 17-Feb-2021


An international HIV pharmacology specialty laboratory (PSL) was established at the University of Zimbabwe to increase bioanalytical and investigator capacities. Quantitation of plasma nevirapine in samples from the AIDS Clinical Trials Group protocol 5279 was compared between the University of Nebraska Medical Center PSL and the University of Zimbabwe PSL. Both PSLs employed internally developed methods utilising reverse-phase high-performance liquid chromatography with ultraviolet detection. Eighty-seven percent of the cross-validation results exhibited ± 20% difference. The UNMC-PSL served as the AIDS Clinical Trials Group 5279 (A5279) protocol designated laboratory. The UNMC-PSL, during the time of the A5279 study, participated in the Clinical Pharmacology Quality Assurance proficiency testing (PT) programme to assure nevirapine assay performance and accuracy. Although each PSL validated the nevirapine assay separately, the lack of data comparing the bioanalytical capacity of pharmacology laboratories in low-middle income countries using clinical samples (rather than spiked plasma samples) was the justification for this project.The Clinical Pharmacology Quality Assurance programme, which monitors all PSLs within the AIDS Clinical Trials Group, provided technical guidance to and developed a comparative nevirapine validation study for the UZ-IPSL to analyse replicate HIV clinical samples. Validation of the nevirapine bioanalytics methodTherefore, implementation of the project was the last stage in determining UZ-IPSL’s readiness to begin assaying human samples for clinical trials.Data files used in this comparison study were de-identified and Clinical Pharmacology Quality Assurance was blinded to any data associated with samples. This article followed all ethical standards for research without direct contact with human or animal subjects.n = 95) that were available at the UZ College of Health Sciences Clinical Trial Research Center (UZCHS-CTRC) for cross-validation of nevirapine assays between the UNMC-PSL and the UZ-IPSL. A5279 was a multi-centre, phase III clinical trial investigating the use of short-course rifapentine or isoniazid for the prevention of active tuberculosis in HIV-positive adults.6 In A5279, nevirapine pharmacokinetics when co-administered with rifapentine and isoniazid was one of the exploratory objectives. All A5279 primary aliquots collected from the UZ AIDS Clinical Trials Group CTRC were analysed for nevirapine by the protocol designated PSL at the UNMC-PSL.The UZ-IPSL utilised the A5279 replicate samples . The plasma aliquots were frozen (–70 °C) and shipped to the UNMC-PSL. The UZCHS-CTRC retained secondary aliquots for the UZ-IPSL cross-validation assay.7A5279 primary plasma aliquots were shipped from UZCHS-CTRC on dry ice to the UNMC-PSL for determination of the nevirapine. The UNMC-PSL nevirapine determination assay had a quantitation range of 25 ng/mL to 10 000 ng/mL. Samples measuring above the quantitation range were diluted and reanalysed using a validated dilution protocol. Solid-phase extraction was utilised to prepare samples for analysis. The UNMC-PSL utilised a Waters e2695 HPLC coupled to a Waters 2489 ultraviolet  detector, which was controlled with Empower 2  software as the analytical platform. The performance of the assay was compliant with the United States Food and Drug Administration bioanalytical guidelines for method validation.n = 95) from A5279 available at the UZCHS-CTRC were shipped to the UZ-IPSL for determination of the nevirapine concentration for cross-validation. The UZ-IPSL measured nevirapine using a validated HPLC-ultraviolet assay detailed previously.2 The UZ-IPSL chromatographic system consisted of a Shimadzu LC20A HPLC using ultraviolet photodiode array detection (model SPD-M20A) and LabSolutions Software . The assay quantitation range was 500 ng/mL to 15 000 ng/mL. Samples that were above the quantitation range were diluted and reanalysed. The performance of the assay was compliant with United States Food and Drug Administration bioanalytical guidelines. Peak purity for nevirapine was assessed to confirm the absence of interferences from potential impurities also extracted from the patient’s sample. A5279 samples were assayed over five runs; the two last runs included samples that were above the upper limit of quantification and were diluted (1:8) according to the validated dilution protocol.2Replicate plasma aliquots  software.The UZ-IPSL nevirapine results were submitted to the AIDS Clinical Trials Group Data Management Center portal using the Data Submission Utility . The A5279 study team approved the release of the UNMC A5279 nevirapine concentration results for comparison with the UZ-IPSL results. The original nevirapine concentration data from the UNMC-PSL were used in the specified A5279 protocol pharmacokinetics analysis; no UZ-IPSL derived pharmacokinetics data were used in the per-protocol study analysis. The Data Management Center compiled the data and provided the de-identified data files to the Clinical Pharmacology Quality Assurance PT unit to perform blinded, comparative statistical analyses of the nevirapine results. After examination (Omnibus 2) and finalisation of the data, the analyses of the paired n = 92) ranged between 2000 ng/mL and 20 000 ng/mL and the remaining 3% (n = 3) were above 20 000 ng/mL and not contiguous .5 Of the data, 93% (n = 88) were 15 000 ng/mL or less (9 while 7% (n = 7) were above this value. Therefore, the seven sample pairs were excluded from the method comparison. After exclusion, an additional sample pair was identified as a significant outlier using the Grubbs test and was also excluded.Ninety-five replicate plasma aliquots were bioanalysed. The nevirapine concentration range in the analysed samples was ~2000 ng/mL – 34 000 ng/mL (~17-fold). Of this, 97%   9 showed analysis 9 and thean plots 9 furtheranalysis 9, the co8. Both nevirapine assays from the two laboratories can detect concentrations that might be below the target concentration for virologic suppression or above the upper limit of the therapeutic range. Although the UZ-IPSL nevirapine concentrations were higher than those of UNMC-PSL and this difference was statistically significant, it is unlikely to have clinical significance. The United States Food and Drug Administration does not provide a window of acceptance for cross-validation studies but allows laboratories to determine a priori the acceptance criteria. Using the established acceptance window for Clinical Pharmacology Quality Assurance PT samples, about 20% of the final target value, and expected concentrations of nevirapine, this difference was acceptable.Drug concentrations for nevirapine reported in the literature for HIV-positive individuals indicate a steady state maximum of 5740 ng/mL (5000–7440) and minimum of 3730 ng/mL (3200–5080)The outcomes of the cross-validation also presented an opportunity to perform a root-cause analysis. The UZ-IPSL’s trend of PT outcomes before and during the A5279 sample analysis period was explored. While all PT results were satisfactory, agreement of initial PT sample analyses showed unbiased accuracy, while during subsequent analyses which coincided with A5279 sample analysis, the UZ-IPSL values exhibited a biased, high trend (4% – 19%) as compared to the final target values of that PT round. This shift in bias and accuracy was provided within the PT report and the UZ-IPSL was able to complete a root-cause analysis and remediate as needed.One limitation to consider is that the calibration ranges for the two equipment sets used for the nevirapine analysis were not identical. Future comparisons of this nature may benefit from evaluations where the calibration range is the same for both laboratories. In addition, limited data points above 20 000 ng/mL entailed difficulties in quantitation of proportional error at that level.The cross-validation study provided evidence that the UZ-IPSL performance using a nevirapine assay that was validated in their laboratory was acceptable and correlated with the results of an experienced PSL. This study afforded the UZ-IPSL a valuable opportunity to implement operations using its validated nevirapine assay for the analysis of samples obtained from a clinical research protocol and adopt procedures for handling of protocol specimens based on experience. Furthermore, the outcomes of the cross-validation emphasised the value of PT and provided an occasion to perform root-cause analysis.


Men who have sex with men (MSM) are at increased risk for extra-genital sexually transmitted infections (STIs). Without extra-genital screening, many chlamydia and gonorrhea infections would be missed among MSM. Yet, many barriers exist to extra-genital testing, and, in particular, to rectal collection. Self-collection increases screening and detection of asymptomatic chlamydia and gonorrhea among at-risk MSM and transgender women. This feasibility study assessed use of rectal self-collection and its acceptance among patients and primary care providers (PCPs) at a large, general practice community health center. The primary objective of this project was to assess the feasibility of including rectal self-collection as part of an implementation study looking to embed an STI care program in a safety-net primary care setting that would shift routine screening tasks to non-provider clinical team members such as medical assistants and nurses.Three PCPs identified and offered rectal self-collection to their MSM and transgender female patients who were due for routine or risk-based STI screening. For those patients who elected to participate in the study, the PCP’s medical assistant (MA) reviewed the self-collection instructions with them as part of their routine preventive care duties, and patients collected their own sample. Patients and PCPs completed brief cross-sectional surveys assessing the self-collection process.Of 1191 patients with sexual orientation and gender identity (SOGI) data on file who were seen for a medical visit by one of the three PCPs, 87 (7.3%) identified as MSM or transgender female. Seventy-five were due for rectal screening, of whom 33 (44%) were offered and completed rectal self-collection. Survey results indicated that self-collection was acceptable to and preferred over clinician-collection by both PCPs and patients.This study demonstrated that rectal self-collection is feasible as part of STI screening in a high-volume primary care setting, and can be administered as part of the clinical tasks that MAs routinely conduct. The overall acceptance by both PCPs and patients will allow the inclusion of rectal self-collection in an implementation study looking to increase STI screening at a large community health center by facilitating MA-led collection during medical provider visits and by establishing standalone nurse-led STI visits. Chlamydia trachomatis (chlamydia) and 616,000 cases of Neisseria gonorrhoeae (gonorrhea) reported in 2019 [Sexually transmitted infection (STI) rates continue to rise in the USA, with 1.8 million cases of  in 2019 . A 2018  in 2019 . Safety- in 2019 , presentThe United States STI National Strategic Plan 2021‑2025 identifies MSM as a priority population for STI risk reduction and care improvement  and are Barriers to chlamydia and gonorrhea rectal screening include increased visit complexity and time compared to urogenital testing; lack of provider awareness regarding need for extra-genital screening; stigma experienced or perceived by patients and/or providers related to sexual behavior; and discomfort with taking or providing a detailed sexual risk assessment and with the collection of rectal samples . Self-coTo-date, the use of rectal self-collection as part of routine STI screening for MSM and transgender women has not been studied at a large, high-volume general population community health center. Challenges to feasibility in this setting include identifying patients at increased risk who would benefit from routine screening; patient discomfort and acceptability of rectal collection given the sensitive and invasive nature of the test; and PCP time, effort, and uneasiness with sexual health-related assessments.This study was conducted among asymptomatic adult MSM and transgender female patients of three primary care providers (PCPs) at Community Health Center, Inc. (CHCI), a large, multi-site FQHC in Connecticut. The primary objective of this project was to assess the feasibility of using rectal self-collection for STI screening in MSM and transgender women during a primary care medical visit in a busy community health center setting. If the primary feasibility outcomes are met, rectal self-collection will be incorporated as an essential component of an implementation study that would examine shifting routine STI screening tasks to non-provider clinical team members such as medical assistants (MAs) and nurses. The secondary objective was to develop an understanding of whether having sexual orientation and gender identity (SOGI) information in the chart helped PCPs offer rectal self-collection to those MSM and transgender women who required screening.We hypothesized that we would be able to develop a process to identify patients at increased risk who would benefit from routine screening, and that self-collection would be easy to adopt and would be accepted and preferred by the majority of patients and PCPs.Cross-sectional, observational feasibility study.Our study enrolled MSM and transgender female patients identified by the PCP as requiring rectal chlamydia and gonorrhea screening during a primary care medical visit. Those offered participation had a visit during the study period, were 18+, English-speaking, reported history of receptive anal sex, and were due for routine or risk-based rectal STI testing. Patients were recruited from the panels of three PCPs practicing at three sites of a multi-site FQHC. This study followed the Consolidated Standards of Reporting Trials (CONSORT) statement extension to pilot trials reporting guideline .PCPs identified eligible patients due for rectal STI screening during medical visits based on their review of SOGI data from the electronic health record (EHR), sexual risk assessments, and recent STI testing. Patients agreeing to rectal STI screening were offered participation in the rectal self-collection study or standard of care which would require the patient to undress, be draped, and have a chaperone present with the PCP for the intimate examination and specimen collection. Patients who opted to participate in the study were consented by the PCP or MA toward the end of the medical visit, verbally instructed on how to self-collect, given the opportunity to ask questions, and provided with a set of printed instructions. MAs provided the instruction and received the self-collected specimen, which allowed the PCP to wrap up the medical visit as per their typical workflow and move on to the next scheduled patient. Patients were then left to self-collect their specimen in private in the exam room or bathroom. Patients and PCPs completed a short survey after self-collection or at the end of the study, respectively.For our primary objective, the feasibility of implementing rectal self-collection was contingent on PCPs’ and patients’ acceptability of the rectal self-collection process. We used cross-sectional self-report surveys to measure acceptability based on the following primary outcomes: PCPs’ time and effort; PCPs’ likelihood of rectal screening if self-collection was offered; and PCPs’ preference for self-collection vs. clinician-collection; patients’ ability to self-collect a specimen; patients’ physical comfort with self-collection; and patients’ preference for self-collection versus clinician-collection. We also captured qualitative data on the types of questions patients asked regarding self-collection, and operational data on number of self-collected specimens that were acceptable for laboratory analyses.For our secondary objective, we wanted to understand whether having SOGI information in the chart helped the PCPs offer rectal self-collection to those MSM and transgender women who required screening. We used the following measures to assess our secondary outcomes: total number of patients seen by the PCPs for medical visits; total number of those patients who had SOGI information in the chart; of those, the number of people who identified as MSM or transgender female; the proportion of those patients who were due for rectal screening; and the proportion of those patients who were offered rectal screening.Our sample consisted of patients with a medical visit to the three participating PCPs during the observation period who self-identified as MSM or transgender female, were due for rectal screening, and opted for self-collection versus clinician-collection.Descriptive statistics were analyzed using SPSS version 27 . Qualitative data on patient questions were captured and thematic content analysis was conducted using NVivo version 12 . The study was approved by the Institutional Review Board at CHCI.Figure n = 33) represented 37.9% of MSM and transgender women who were seen by the participating PCPs for a medical visit (n = 87). The sample of patients who self-collected were similar to the general population of MSM and transgender women seen during the study period with respect to age, race, ethnicity, and self-reported sexual orientation and gender identity, and thus deemed sufficient to draw conclusions on the potential feasibility of rectal self-collection adoption.Recruitment was initiated on March 1, 2018, following implementation of a Health Resources and Services Administration (HRSA)-mandated process to routinely collect and report SOGI data at FQHCs  and CHCIn = 87) of the 1345 patients who presented for care during the study period identified as MSM (n = 67) or transgender female (n = 20) based on SOGI information from EHR data, and were potentially eligible for inclusion. Of the 87 potentially eligible patients, twelve (13.8%) were identified as having had a rectal STI screen within the past year, and were not due for screening on the visit date. Thirty-nine patients had screening deferred on the visit date . Thirty-six patients who required and agreed to rectal screening were offered participation in the self-collection study. Three of these patients (8.3%) opted for clinician-collection due to current or prior history of rectal symptoms. All other patients  chose self-collection  with 26 identifying their gender as male (78.8%), five as transgender female (15.2%), and two chose not to disclose (6.0%). Participants were 39.4% White, 18.2% Black, 39.4% Hispanic/Latinx, and 3.0% other race/ethnicity, with mean age 40.0 . Most identified their sexual orientation as gay  (Table Table PCPs’ acceptability of self-collectionTime and effort. All PCPs (n = 3) agreed that self-collection was time-efficient and not disruptive to the clinical workflow.Likelihood of rectal screening. Two PCPs agreed and one somewhat agreed that self-collection increased their likelihood to obtain a rectal specimen from a patient.Preference. Two of the three PCPs preferred patient self-collection to clinician-collection, and the third expressed no preference.Patients’ acceptability of self-collectionAbility to self-collect. All 33 study participants endorsed comfort with self-collection and the majority agreed self-collection was easy . The most common self-collection questions patients asked were as follows: (1) how to seal the swab into the tube after collection; (2) how far into the rectum to insert the swab; and (3) how much to move the swab once inserted rectally. Nearly all self-collected specimens  were suitable for laboratory analysis.Physical comfort. The majority of patients  were neutral, agreed, or strongly agreed that they did not feel pain during self-collection.Preference. Two-thirds of participants preferred self-collection over clinician-collection , and the remaining third expressed no preference .Please refer to Table Ability to identify patients for rectal self-collectionn = 1191) had SOGI data documented in the EHR, and 87 identified as MSM or transgender female. Twelve of these patients would have qualified for routine rectal screening based on SOGI and sexual risk, but had already been screened within the prior year. Over half of the remaining 75 MSM and transgender female patients (n = 39) were not offered screening  and will examine whether participants perceive any stigma when asked to self-collect their specimen.We are aware that there may be a power dynamic that might influence a patient’s response to survey questions administered by members of the clinical team. Future studies should attempt to use non-clinical team members, preferably with lived experience, to administer surveys on perception of self-collection to all patients undergoing STI screening.Our feasibility study was conducted in a U.S. state and at a medical practice that provided above-average acceptance, support, and affirmation of sexual and gender minority people, which may limit generalizability of these findings to other settings where patients may feel less accepted and potentially uncomfortable disclosing sexual behaviors with their primary care team. The study was also concentrated at a primary care FQHC that collected SOGI data routinely. Such collection may help identify MSM and transgender women for potential screening more readily than at a practice that did not ask about SOGI information.This study assessed provider and patient acceptability of rectal self-collection and feasibility of offering it during a medical visit. We found that incorporating self-collection for rectal chlamydia and gonorrhea screening among MSM and transgender women was efficient, highly acceptable, and preferred by patients and clinicians. Our findings are in line with other studies indicating that patients prefer self-collection , 18 and Neglecting extra-genital testing in MSM and transgender women leads to missed diagnoses and can contribute to persistent disparities in STIs among sexual and gender minorities. Rectal mucosa is vulnerable to STIs and symptomatic or asymptomatic extra-genital chlamydia and gonorrhea infections are associated with increased risk of HIV transmission among MSM , 13, 28.The Centers for Disease Control and Prevention’s Recommendations for Providing Quality Sexually Transmitted Disease Clinical Services (2020) note that STIs are increasingly treated in primary care  and recon = 1191, 88.6%) had SOGI data on file. We found that 7.3% (n = 87) of these patients self-identified as MSM or transgender female, which exceeds the estimated percentage of U.S. adults who self-report any sexual and gender minority identity (4.5%) [Nevertheless, continued emphasis on SOGI data collection in primary care health centers is critical. SOGI information must be recorded in the EHR in a systematic manner that allows for effective patient-level use by clinical teams and organization-level use for population health. The majority of our sample (y (4.5%) . This ilDespite the availability of SOGI information in the EHR, 39 patients potentially eligible for rectal screening did not receive it. Clinical decision support tools such as dashboards using SOGI data can thus identify patients with potential risks for STIs and consequently trigger any member of the clinical team to offer rectal self-collection, standardize and normalize STI screening, and provide opportunities to offer PrEP and maximize HIV prevention efforts. As this study demonstrated, MAs were able to facilitate patient rectal self-collection. Since nurses and MAs are already able to collect pharyngeal and urine specimens, rectal self-collection can shift the burden of STI testing from PCPs to clinical support staff by extending capability for MAs to assist with screening and for nurses to conduct comprehensive assessment and screening under standing orders from a PCP.Our future study will implement STI services at this large FQHC that will leverage SOGI data and standardized sexual risk assessments, utilize a clinical decision support tool for MAs and nurses (the organization’s planned care dashboard), and set up templates and standardized protocols for nurse-led STI visits. Care team members will receive training on comprehensive STI screening that will permit them to work collaboratively to assess and respond to patients’ needs based on SOGI and sexual risk. Improved documentation of sexual risk in structured data fields in the patient’s chart is needed in order to accurately track screening rates among patients for whom it is medically indicated . We anticipate the ability to make this change in our practice as part of implementing a larger study.STI rates continue to rise in the USA, disproportionately affecting certain populations like MSM and transgender women, particularly those of racial and ethnic minorities. As access to STI clinics becomes more limited, primary care centers, especially those catering to the medically underserved, need to ensure they offer comprehensive STI services. Rectal self-collection can and should be considered as part of any strategy to increase STI screening rates given that rectal specimen collection is the most invasive, discomforting, and time-consuming of all STI testing required for MSM and transgender women. Our study indicated that rectal self-collection was highly accepted and preferred by both PCPs and patients and was easily implemented in a busy safety-net primary care practice. This is a positive indication supporting use of rectal self-collection as a core component of our future study that will assess implementation of comprehensive STI services, leveraging the whole primary care clinical team including MAs and nurses, in a large FQHC setting.