Transjugular intrahepatic portosystemic shunt (TIPS) is a well-validated decompressive therapy option to manage ascites and variceal bleeding secondary to portal hypertension. Complications following TIPS procedures include hepatic encephalopathy, liver failure, and TIPS dysfunction. TIPS dysfunction is due to occlusion or stenosis of the TIPS shunt and can be caused by acute or chronic thrombosis. TIPS thrombosis is often treated with mechanical thrombectomy or catheter-directed thrombolytic therapy. Most cases of in-stent occlusion can be treated via a transjugular approach with recanalization or placement of additional stents. We present a case of a 72-year-old female who presented with worsening ascites 17 months after initial TIPS procedure; she was found to have a large thrombus completely occluding the TIPS stent. In our case, a combined transhepatic and transjugular approach was required for TIPS revision given the extent of well-organized clot located near the hepatic venous end of the stent, resulting from prolonged stent occlusion. This was an extremely challenging scenario with two overlapping covered stents and a bare metal stent at the hepatic venous end in the setting of chronic thrombosis and a well-organized fibrous cap. The case highlights the need for optimal initial placement of the primary TIPS shunt to avoid the need for subsequent complex interventions to maintain TIPS shunt patency. Transjugular intrahepatic portosystemic shunt (TIPS) is a well-validated decompressive therapy option to manage ascites and variceal bleeding secondary to portal hypertension The incidence of TIPS thrombosis has decreased significantly with the widespread use of polytetrafluroethylene stents instead of bare metal stents ,5. HowevWe present a case of a 72-year-old-female who presented for a TIPS revision and was found to have a large thrombus completely occluding the TIPS stent. She was treated via a combined transjugular and transhepatic approach to cannulate the TIPS stent, with subsequent thrombectomy performed due to the large clot burden.A 72-year-old female with a past medical history of cirrhosis secondary to non-alcoholic steatohepatitis, esophageal varices status-post multiple band ligations, bilateral carotid endarterectomy, cerebrovascular accident, and coronary artery bypass grafting presented to our tertiary medical center for TIPS revision 17 months after initial placement. She underwent initial TIPS shunt placement for management of persistent ascites requiring large volume paracentesis twice per week. After this, her paracentesis burden was reduced to once per week, however, at 13 months following initial TIPS, she once again began to require large volume paracentesis twice per week. TIPS revision was then performed by means of TIPS stent angioplasty and placement of an extension stent at the portal venous end, and two additional stents at the hepatic venous end, one covered and one uncovered. However, the patient experienced only minimal improvement in symptoms prompting further evaluation at our institution. Both the initial TIPS procedure and first revision were performed at an outside hospital. Doppler ultrasound 3 months prior to the revision described in this report showed an occluded TIPS without significant flow and thrombus extending into the superior mesenteric vein. . The patContrast-enhanced CT abdomen performed at our hospital showed a thrombus within the TIPS stent and our interventional radiology team was consulted for TIPS revision . The rigA 5-Fr MPB catheter was advanced into the TIPS stent over the .035-inch “flossing” Glidewire and after multiple attempts, the access to the portal vein through the stent was established after 3mm balloon dilatation. Given the extreme clot burden within the TIPS stent and the difficulty in securing and increasing the size of the access, the decision was made to proceed with mechanical thrombectomy. Thrombectomy was performed using the Indigo System Lightning 12 , and a well-formed clot measuring 31 cm in length, and 2-3 cm in thickness was removed. A well-formed fibrous cap was seen correlating with the stent's hepatic venous end .Fig. 5LaThe previously placed portal venous end extension stent was detached from the main TIPS stent and was completely occluded. Multiple attempts were made to re cannulate the occluded detached stent but were unsuccessful, as the stent did not appear to be in the expected location of the portal vein as seen in On follow-up one month later, the patient expressed marked symptomatic improvement and stated she felt significantly better. At this time, she was requiring one large-volume paracentesis per week, down from two per week prior to the procedure. Additionally, the removed volume decreased from 10 liters pre-TIPS revision to 3 liters post-TIPS revision. She was planned for follow-up portal venography and additional stent extension of the hepatic venous end given residual stenosis. Patient was then lost to follow-up.Shunt stenosis or occlusion is a common complication after TIPS procedures with meta-analysis showing that reintervention to maintain shunt patency is required in 70%-90% of patients within 2 years of creation Given the widespread use of covered stents, most cases of TIPS thrombosis are commonly caused by structural obstruction of flow related to suboptimal shunt position or configuration, which can occur due to initial malpositioning or migration of the shunt following the initial procedure. Malpositioned TIPS stents at risk for thrombosis are more commonly related to the hepatic venous end, with the highest risk related to the superior margin terminating within the hepatic parenchymal tract abutting the hepatic vein or IVC wall. Although initial portal venography after initial shunt insertion may show appropriate stent position and flow, removal of the guidewire or catheter can cause changes in stent positioning resulting in compromised flow through the TIPS. In our patient, thrombosis was attributed to the need for placement of several hepatic venous end extension stents and a non-anatomical non-overlapping portal venous end extension stent.Treatment options for TIPS thrombosis include thrombectomy, balloon angioplasty, catheter-directed pharmacologic thrombolysis, systemic anticoagulation, or definitive treatment with liver transplantation. In most cases, a combined approach with thrombectomy and catheter-directed pharmacologic thrombolysis is used. Thrombectomy performed for management of TIPS thrombosis can be performed through multiple approaches – mechanical, hydrodynamic, and rheolytic methods have all been described Most cases of in-stent occlusion in TIPS can be managed via the transjugular approach with recanalization or placement of additional stents. In our patient, this approach could not be used to cannulate the occluded stent given the extent of well-organized clot located near the hepatic venous end after prolonged duration of stent occlusion of 13 months. As a result, a combined transjugular and transhepatic approach was used, which was first reported by Haskal and Cope in a series of 4 patients  In summary, stent thrombosis and occlusion are complications of TIPS procedures that present with recurrent symptoms of portal hypertension, which in our case was an increase in paracentesis burden. TIPS thrombosis can occur due to structural obstruction of flow related to suboptimal TIPS shunt initial placement or configuration, which can occur due to initial malpositioning or migration of the shunt following the initial procedure. Treatment options for TIPS occlusion involve recanalization or placement of additional stents, which in our case was performed via a combined transjugular and transhepatic approach. For patients with extensive thrombosis of the shunt, combined treatment with mechanical thrombectomy and catheter-directed thrombolysis may be needed.Written, informed consent was obtained from the patient for all procedures performed in this study.


A 28-year-old male with a recent diagnosis of human immunodeficiency virus presented to the emergency department with odynophagia and dysphagia for a month. Physical exam revealed Kaposi sarcoma partially occluding the airway. Point-of-care ultrasound was used to assist with the diagnosis of reactive lymphadenopathy, and computed tomography revealed systemic disease. Otolaryngology was urgently consulted, and the patient was admitted for prompt tracheostomy the following day.Kaposi sarcoma is a violaceous vascular neoplasm that is an acquired immuno-deficiency syndrome (AIDS)-defining illness. Mucocutaneous membranes should be thoroughly evaluated with patients suspected of AIDS. This case demonstrates the vital evaluation of the patient’s airway to assess patency. Highly active antiretroviral therapy should be initiated promptly, as well as chemotherapy in severe systemic cases. A 28-year-old male presented to the emergency department (ED) with progressively worsening odynophagia and dysphagia. He had been diagnosed with human immunodeficiency virus five months prior after initially presenting to an outside hospital with odynophagia and weight loss. Given difficulty affording his antiretrovirals, he reported only intermittently taking emtricitabine-tenofovir and was lost to follow-up. He was unaware of his oral lesions and overall grim clinical status. Seven days prior to presentation he developed mild dyspnea as well as worsening dysphagia. Physical examination of the oral cavity revealed a 3 × 3 centimeter (cm) violaceous, pedunculated, midline posterior mass partially occluding the airway, as well as a 2 × 2 cm anterior hard palate, violaceous mass .The exam was also notable for extensive cervical and inguinal lymphadenopathy, global wasting, and a muffled voice. He was able to speak in full sentences, was tolerating his secretions, and did not have evidence of respiratory distress. Point-of-care ultrasound (POCUS) revealed reactive cervical lymphadenopathy .Computed tomography soft tissue neck  and chesPneumocystis jirovecii pneumonia prophylaxis. Otolaryngology was consulted in the ED and performed a bedside laryngoscopy.. He was admitted for tracheostomy the following day. Excisional biopsy confirmed the diagnosis of disseminated Kaposi sarcoma. Given his extensive disease burden, he was started on systemic chemotherapy.He was started on broad spectrum antibiotics including trimethoprim/sulfamethoxazole for Kaposi sarcoma traditionally occurs in patients with immunosuppression, such as those with acquired immunodeficiency syndrome (AIDS), or those who are immunosuppressed secondary to organ transplant. Kaposi sarcoma related to AIDS occurs in patients with cluster of differentiation four (CD4) counts less than 200 cells per cubic millimeter and is an AIDS-defining illness.3In our case, we used POCUS to confirm the diagnosis of extensive reactive lymphadenopathy. Other differentials included lymphoma, metastasis, abscess, and tuberculosis. Gray scale sonography can evaluate nodal morphology by noting size, shape, and architecture. Metastatic lymph nodes have loss of hilar architecture and the presence of intranodal calcification and necrosis.5What do we already know about this clinical entity?Kaposi sarcoma is an acquired immunodeficiency syndrome-defining illness.What is the major impact of the image(s)?Few images of Kaposi sarcoma of the oral cavity exist. This multimodality image approach illustrates the pathology and severity of the illness beyond cutaneous findings.How might this improve emergency medicine practice?Thorough physical examination and multimodal imaging could aid the emergency physician in making this life-altering diagnosis.


Supplemental Digital Content is available in the text. Coronavirus Disease-2019 presents risk to both patients and medical teams. Staff-intensive, complex procedures such as extracorporeal membrane oxygenation (ECMO) or extracorporeal cardiopulmonary resuscitation (eCPR) may increase chances of exposure and spread. This investigation aimed to rapidly deploy an in situ Simulation-based Clinical Systems Testing (SbCST) framework to identify Latent Safety Threats (LSTs) related to ECMO/eCPR initiation during a pandemic.The adapted SbCST framework tested systems related to ECMO/eCPR initiation in the Neonatal and Pediatric Intensive Care Units. Systems were evaluated in six domains . We conducted three high-fidelity simulations with members from the Neonatal Intensive Care Unit General Surgery, Pediatric Intensive Care Unit Cardiovascular Surgery (CV), and Pediatric Intensive Care Unit General Surgery teams. Content experts evaluated systems issues during simulation, and LSTs were identified during debriefing. Data were analyzed for frequency of LSTs and trends in process gaps.Sixty-six LSTs were identified across three scenarios. Resource issues comprised the largest category (26%), followed by Process/System issues (24%), Infection Control issues (24%), Communication issues (17%), and Facility and Clinical Performance issues (5% each). LSTs informed new team strategies such as the use of a “door/PPE monitor” and “inside/outside” team configuration.The adapted SbCST framework identified multiple LSTs related to ECMO/eCPR cannulation and infection control guidelines in the setting of Coronavirus Disease-2019. Through SbCSTs, we developed guidelines to conserve PPE and develop optimal workflows to reduce patient/staff exposure in a high-risk procedure. This project may guide other hospitals to adapt SbCSTs strategies to test/adjust rapidly changing guidelines. The number of essential team members and equipment crowded the patient’s isolation room, and communication was difficult due to the use of personal protective equipment (PPE). The operating room staff wasted supplies due to isolation practices. PPE, already in short supply, was not conserved safely and responsibly. These events demonstrated a need to improve and streamline our internal systems for both patient and staff safety.Over 5100 patients with COVID-19 have been supported with Extracorporeal Membrane Oxygenation (ECMO) as of early 2021.7 This methodology identified Latent Safety Threats (LSTs) during ECMO/eCPR cannulation and facilitated recommendations for process improvements.The purpose of this safety report is to describe the rapid deployment of the Colman et al’s Simulation-based Clinical Systems Testing (SbCST) framework to develop COVID-19 infection control and emergency preparedness institutional guidelines.A request for assistance to rapidly test our process was made to the Center for Pediatric Simulation (CPS). The Center for Pediatric Simulation (CPS) at our institution is composed of multidisciplinary healthcare providers trained in simulation methodology to provide simulation-based training, systems testing, and research for the entire organization. The CPS convened a multidisciplinary key stakeholder group to develop and implement in situ ECMO/eCPR simulations in light of COVID-19 clinical experiences. This group included Pediatric Intensive Care Unit (PICU), Cardiac Intensive Care Unit (CICU) and Neonatal Intensive Care Unit physicians, nursing, respiratory therapists, pharmacy staff, ECMO team, General Surgery (GS) and Cardiovascular Surgery (CVS), Operating Room staff, radiology, and echocardiogram technicians, and Infection Prevention and Control staff.7 The SbCST approach incorporates peer-review into three phases  to make design decisions in newly built healthcare facilities in the months preceding patient exposure. It utilizes evidence-based safe design principles and usually takes place over months to a year.7 This process utilizes the following standardized tools: scenario facilitator guides, observation tools to document LSTs, and Failure Modes and Effects Analysis reports that synthesize LST findings recorded during scenarios.We utilized the SbCST framework adapted from Colman et al to provide a consistent approach to test clinical processes and human and environmental factors through simulation scenarios. This process is used to detect and report LSTs.7This approach was condensed over several weeks to facilitate the need for urgent testing during the COVID-19 pandemic. The developmental phase was significantly abbreviated, and implementation was rapid. All components of the developmental phase, including stakeholder engagement, needs assessment, process mapping and scenario development were addressed virtually. We adapted the SbCST facilitator guide to provide a standard template during simulation. It includes the scenario overview, timeline, participant roster and roles, equipment and supplies, and testing objectives. The adapted facilitator guide provided a template for planning, documenting, and debriefing. A COVID-19 ECMO/eCPR comprehensive summary, similar to the SbCST Failure Modes and Effects Analysis, was generated after each simulation scenario to address our institutional needs in the domains of Design/Facility, Resource accessibly/workflow efficiency, COVID process/systems, Clinical Performance, Infection Control, and Communication.The high-fidelity simulation was executed three times; twice in the PICU (once with the CVS team and once with the GS team) and once in the NICU with the GS team. A standardized pre-brief described the goals of the SbCST and emphasized process evaluation of each scenario. Two authors (RS and AS) with a combined simulation experience of over 15 years co-facilitated and debriefed the simulation. Although scenario content was consistent, each subsequent simulation was modified based on mitigation strategies learned from the observations and debriefing of the previous scenarios.Seven observers representing key stakeholders and infection prevention were positioned both inside and outside the patient room. They utilized the adapted facilitator guide during the simulation to guide the debriefing process and discuss challenges, LSTs, and possible mitigation strategies. We used Promoting Excellence and Reflective Learning in Simulation and Advocacy Inquiry debriefing frameworks as adjuncts to chronological debriefing.A COVID-19 comprehensive summary report for each simulation categorized the LSTs as high or low priority, as determined by key stakeholders based on the frequency of observation and level of safety threat to staff. Simulation staff revised the pre-briefing for each subsequent simulation to include the revised mitigation steps for the high-priority LSTs. Final mitigation strategies were incorporated into our institutional recommendations for the facility’s standard COVID-19 guidelines for ECMO/eCPR cannulations.8Frequency and percentages of each LST domain type were calculated for each scenario to uncover the most prevalent system issues. Analysis was performed in R, version 3.5.Three simulation scenarios and debriefings were completed in April 2020, integrating approximately 60 participants and seven observers. Twenty-three participants completed optional evaluations. The application of the SbCST framework identified 66 LSTs during these scenarios Table . ResourcThe first scenario identified Resource LSTs with repeated errors in donning and doffing PPE. The lowest Likert score received on the evaluation by participants was related to their ability to use the proper PPE. Confusion surrounding the availability of proper PPE was addressed through the employment of centralized PPE via a bedside cart. PPE super-users (trained nurse monitors donning and doffing of PPE) were integrated during high-risk events. However, contacting the PPE super-users in a timely fashion was later identified as an LST.We tested and recommended optimal team configuration. Creating the “inside” and “outside” team is a mitigation strategy that crossed multiple LSTs and domains. The design further emphasized the “inside team”, particularly the recording nurse who assumed the communicator role. Subsequent simulations tested the safety and feasibility of this new process and successfully reduced required personnel from 15–17 to 10–12 people. An additional member was added to the “outside team” after the first scenario to mitigate inefficiencies in transferring supplies; decrease multiple, prolonged door openings; and facilitate communication. This “door monitor” is in airborne PPE precautions and decreased door openings by 38% (26–16 occurrences) by the third simulation.Communication LSTs were prevalent despite various modes of communication. A communicator role for both “inside” and “outside” teams was necessary to facilitate effective communication. Multiple modalities were tested in isolation, but two modalities were ultimately recommended. Another communication LST led to an additional field in the ECMO/eCPR activation page to denote the COVID-19 status of the patient allowing for participants in subsequent scenarios to arrive with proper PPE. The additional information in the ECMO/eCPR paging system decreased confusion for the cannulation teams responding to pages. This system was used 37 times, and 25 patients were ultimately cannulated.See document, Supplemental Digital Content 1, which shows…http://links.lww.com/PQ9/A346)Facility LSTs were difficult to augment; however, recommendations were made to cannulate in the most isolated bed spaces for optimal COVID-19 isolation during ECMO/eCPR cannulation. A specific CVS COVID-19 surgical cart was tested to prevent unnecessary wasting of supplies. Through the SbCST process, we learned this cart could be shared between the surgical services. A COVID-19 eCPR surgical checklist and detailed informational email was provided to all appropriate surgical staff.  delineates staff configurations and PPE protocols.We developed multiple job aides to support the clinical staff. These included infographics, PowerPoint presentations, and surgical checklists. An infographic specific to COVID-19 ECMO/eCPR cannulation (9 Similarly, our institution utilized SbCST framework to assess preparedness and improve safety for ECMO/eCPR cannulations during the COVID-19 pandemic. This framework’s ability to be utilized in rapid deployment has also been demonstrated in testing alternative care sites capacity and safety during the COVID-19 pandemic.10During the 2014 Ebola virus disease epidemic, multiple health care institutions and the Society for Simulation in Healthcare recognized and leveraged simulation education as a key component in ongoing and future infectious disease outbreaks.9 Within our already established ECMO-simulation program, we could focus on patient and staff safety by utilizing infection prevention experts. Limiting team size was a primary concern, and our work identified strategies to improve this process. The creation of the “inside” and “outside” teams proved feasible and limited the exposure risk to staff.Through various highly contagious infectious diseases outbreaks including the Ebola virus disease epidemic, it remains evident that rapid deployment of training education is necessary. Furthermore, the protection of healthcare workers remains a top priority.For frontline team members, these simulations promoted confidence in the system amidst uncertainty while performing high-level clinical tasks during emergent procedures. A specific COVID-19 ECMO/eCPR cannulation infographic available on the organization’s internal website promotes sustainability of the mitigation strategies and accessibility of the process for staff to easily review.The SbCST simulations demonstrated 66 LSTs that we could quantify and develop mitigation strategies for in a multidisciplinary team during a time of high anxiety and intensity for staff. Resource, Process/System, and Infection Control domains were identified at the highest frequency suggesting the application of the SbCST framework is suitable for rapid testing of emerging hospital guidelines. With this project, we provide a new application of SbCST to rapidly assess and allow frontline providers to test evolving guidelines.The communication LSTs proved to be the most challenging to address. The communicator role for both “inside” and “outside” teams is recommended to facilitate effective communication. No single model of communication was sufficiently reliable; thus, employment of at least two modes of communication is recommended.These systems test developed strategies that have been implemented in other high-risk scenarios, particularly the use of the PPE super-users, the “door monitor,” and the various communication strategies with the modified team configuration. Additionally, the COVID-19 status embedded within the ECMO/eCPR cannulation notification page remains active to assure that all staff members responding to the ECMO/eCPR cannulation have the appropriate PPE. Through the SbCST process, the team focused on how to preserve the safety of team members responding to emergent surgical procedures for patients in extremis.Our team adapted the SbCST framework to perform a complex system test, identify LSTs and develop solutions to improve patient and staff safety during ECMO utilization during the COVID-19 pandemic. This project demonstrates the value of utilizing an adapted framework to rapidly improve team systems for complex clinical events such as ECMO/eCPR cannulation.The authors have no financial interest to declare in relation to the content of this article.The authors acknowledge those who made this endeavor possible, including Marita Thompson, MD, Medical Director of Pediatric ECMO and Jeremy Affolter, MD, for their ECMO and resuscitation expertise. Jessica Brunkhorst, MD, Neonatal Simulation Director and Kasey Davis, MD Associate Medical Director of Simulation, Texas Children’s Hospital – Woodlands for their medial simulation expertise. Jessica Rindels, RN infection prevention liaison, for her infection prevention and control insight. The Children’s Mercy Hospital, Kansas City, Missouri, Pediatric Institutional Review Board reviewed this project and determined it did not meet the definition of research involving human subjects.


Bacteriophages (phages) were discovered in the early part of the 20th century, and their ability to eliminate bacterial infections as bacterial viruses gathered interest almost immediately. Bacteriophage therapy was halted in the Western world due to inconclusive results in early experiments and the concurrent discovery of antibiotics. The spread of antibiotic-resistant bacteria has elicited renewed interest in bacteriophages as a natural alternative to conventional antibiotic therapy. Interest in the application of bacteriophages has also expanded to include the environment, such as wastewater treatment, agriculture and aquaculture. Although the complete phage is important in bacteriophage therapy, the focus is shifting to purified phage enzymes. These enzymes are an attractive option for pharmaceutical companies with their patent potential. They can be bio-engineered for enhanced adjuvant properties, such as a broadened spectrum of activity or binding capability. Enzymes also eliminate the concern that the prophage might integrate resistance genes into the bacterial genome. From a clinical perspective, the first randomised clinical controlled phage therapy trial was conducted with more pioneering phase I/II clinical studies on the horizon. In this opinion paper, the authors outline bacteriophages as naturally occurring bactericidal entities, their therapeutic potential against antibiotic-resistant bacteria and compare them to antibiotics. Their potential multipurpose application in the medical field is also addressed, including the use of bacteriophages for vaccination, and utilisation of the antimicrobial enzymes that they produce. Staphylococci isolated from patients in a London hospital demonstrated resistance to penicillin by 1946.1 Antibiotic resistance has subsequently proliferated at an alarming rate to the level that by 2050, 10 million deaths due to antibiotic-resistant infections may occur.2 The emergence and spread of pan-drug resistant bacteria render the need for new treatment approaches a priority.As anticipated by Alexander Fleming, soon after discovering penicillin in the 1920s, 14% of 3 Alternative approaches include using non-lytic bacteriophages as a delivery vehicle to transport deoxyribonucleic acid (DNA) vaccines or using purified phage components such as cell wall hydrolases as novel modalities to treat antibiotic-resistant infections. Bacteriophage therapy does have potential limitations that include but are not limited to toxic shock following bacterial lysis or the generation of neutralising bacteriophage antibodies after repeated treatment.4Bacteriophages are viruses that kill bacteria following amplification and bacterial lysis to release virion progeny into the environment (lytic-lifecycle). Bacteriophage therapy exploits this lifecycle  without 5Lytic bacteriophages offer several potential advantages over antibiotic therapy . Most im6 As auto-replication occurs after one dose, less phage is required to achieve the optimum therapeutic effect.Another major advantage of using therapeutic phages is their self-limiting amplification capability, as they only persist as long as the bacteria are present. Notably, the use of localised phage therapy leads to deep penetration as long as the infection is present, as opposed to antibiotics, where from a pharmacokinetic point of view specific targets need to be maintained with repetitive doses to maintain concentrations.7 It is unclear if this response is because of the phage itself or the lysed bacterial components. Zimecki et al. demonstrated that prophylactic phage administration in an immunosuppressed mouse model could overcome neutrophil deficit in the clearance of Staphylococcus aureus infection and mobilise cells from both lymphocytic as well as myelocytic lineages. It was postulated in this study that phages could promote the recruitment of immature neutrophil cell types from bone marrow precursors and trigger a rapid output of functional mature neutrophils into the peripheral circulation to clear the bacterial infection.8Most bacteriophages promote a strong antibody response and are highly immunogenic.in vivo may lead to potential health concerns in patients. A particularly useful strategy may be to use lysis deficient bacteriophages, which decreases the release of pro-inflammatory cytokines.9 However, considering the minor side effects reported for bacteriophage therapy, such as occasional serum-like illness after intravenous administration, multiple side effects have been observed with antibiotics. Anaphylaxis following bacteriophage therapy has never been described in the literature. The initial reporting of ‘shock’ was most likely because of a low level of purification and contaminants in phage preparations.10 In this regard, protein toxins produced by pathogenic species and gram-negative bacteria endotoxins are impurities in phage lysates that are harmful to humans. Therefore, similar to antibiotic purification, procedures should be rapid, scalable and efficient in removing impurities to ensure patient safety.11Endotoxin released from bacteria lysed 12Bacteriophage therapy is a promising alternative to antibiotics in vulnerable population groups such as pregnant population and neonates, where numerous antibiotic contraindications pertain. Minimal disruption of the host microbiota during the antenatal and perinatal periods is beneficial. In addition, the transplacental transfer of phages has been observed in guinea pigs following intravenous maternal phage administration. Further studies are required to explore in utero bacteriophage treatment strategies.13 This is especially true when the phage component is applied at high titres topically and therefore not easily removed by the immune system. Furthermore, phages encapsulated in hydrogels can result in a long-term release of active particles, whilst liposome preparations applied topically protect against immune system components and enzymatic hydrolysis.15 It is unlikely; however that bacteriophage therapy will replace antibiotics but combining antibiotics with phage therapy should reduce bacterial resistance because the pathogen must acquire two separate mechanisms to survive. Altering binding targets for both antibiotics and phages may come at a fitness cost for the bacteria because mutations can influence essential biological functions. The concept of a trade-off of antibiotic resistance mechanisms and phage resistance is referred to as ‘phage steering’.17 Further research is required to explore the evolutionary response of bacterial strains to the phage-antibiotic combinations. The argument supports the idea that a phage can be selected against every antibiotic or phage-resistant bacterium from an evolutionary standpoint. The ongoing process of natural selection achieves this.18There is a clear potential to use phage therapy concurrently with antibiotics, especially with increasingly antibiotic-resistant pathogens.19 Alternatively, the non-lytic bacteriophage discussed in the following sections may be used to deliver DNA encoding phage-proteins that affect bacterial cell division, DNA replication, transcription and translation.20A strategy to prevent phage resistance would be to use complex mixtures of bacteriophages or ‘phage cocktails’ to circumvent phage resistance.21 In targeted drug delivery, a targeting moiety is displayed on the filamentous phage coat with phage display. The cytotoxic drug is then chemically linked with an ester bond to the bacteriophage. The drug is a prodrug with no cytotoxic activity in the conjugated state. Once the prodrug is dissociated from the phage at the target site in a controlled manner, it is activated. Targeted drug delivery has been shown in vitro between S. aureus and chloramphenicol.22 Adapted bacteriophage vectors have been used to restore antibiotic sensitivity by reversing antibiotic resistance in pathogens. For example, a proof-of-concept model exploited the temperate phage to introduce the rpsL and gyrA genes and confer sensitivity to streptomycin and nalidixic acid, respectively, where these antibiotics were resistant.23 Unlike conventional bacteriophage therapy, the authors proposed a scenario where these phages can deliver genetic constructs and render bacteria sensitive to previously resistant antibiotics. Rather than administering phages to the patient, they may also be applied to hospital surfaces and, in the process, potentially reduce environmental contamination by antibiotic-resistant pathogens.Many novel antibiotic agents are excluded from clinical use because of their inability to be selected explicitly for bacterial versus human cells. This may result in toxicity. During ‘phage display’, an antigen gene is fused with a gene coding for a coat-surface protein and, when expressed, it is displayed as a fusion protein on the capsule of the bacteriophage.24Various methods have been employed to increase phage uptake and stability to target intracellular pathogens. In this Trojan horse approach, pages are loaded onto a carrier vehicle during receptor-mediated uptake. These novel technologies include nanocrystal, liposome, polymer encapsulation and delivery, genetically engineered phage, nanofibre entrapment, nanoparticle absorbed and hydrogel embedded phages.26 This is a bio-engineered structure that entails a DNA vaccine contained within a phage capsule under the expression of a eukaryotic promoter.21 Once the phage transporting the DNA vaccine is engulfed into a eukaryotic antigen-presenting cell, the vaccine gene is expressed. Following this, major histocompatibility complexes (MHC) present the processed antigens to the immune system. Antigens presented to MHC II generate an antibody response, while a cellular response is activated through cross-priming with the MHC I pathway. Phage display vaccines can also directly activate B cells to produce antibodies.26 Haq et al. reported on studies proposing a hybrid phage with phage display and DNA vaccine capability.27 The advantage of an engineered DNA phage vaccine is to treat and prevent infection. Bacteriophage vaccines are also suitable to immunise large communities because they are relatively easy to apply and inexpensive to produce. Jepson and March, in 2004 postulated the concept of bacteriophage DNA vaccines administered orally via drinking water.28 Notably, during such a proposed vaccination programme, a high phage titre would be required as it appears that gut transit and phage penetration after oral administration are dose-dependent.29Bacteriophages vaccination is an innovative and novel vaccine system.30 Their synergistic mechanism of action with a range of other antimicrobials or fellow peptidoglycan hydrolases is of interest. Endolysins are less likely to invoke resistance because of the coevolution of bacteriophages and bacteria.31 The purified lysins can also be applied directly to sensitive bacteria. Enzyme preparations include aerosols for inhalation, formulations for systematic injection, topical ointments and creams.32 The lytic activity spectrum of lysins is usually narrow because lysin receptors are distributed in a strain-specific manner. Furthermore, the binding affinity is equivalent to mature antibodies, making them an irreversible inhibitor.33 This alone promises that lysins could be highly efficacious when compared to antibiotics. An anti-staphylococcal lysin  demonstrated a 42.8% higher clinical response rate when given alongside the standard of care antibiotic compared to the antibiotic alone when treating methicillin-resistant Staphylococcus aureus (MRSA) bacteremia, including infective endocarditis. As a result, this is the first lysin to have entered a phase III human trial to treat S. aureus.36Purified lysins are promising antibacterial phage components currently under development. They consist of a class of cell wall hydrolases that translocate into the peptidoglycan wall of bacteria and cleave the bonds, which results in the release of viral progeny during a viral infection cycle. Endolysins have a characteristic structure with multiple cell wall binding domains. They degrade the peptidoglycan wall with lytic transglycosylase, amidase glycosidase or endopeptidase activities.37 An enzybiotic is a bacteriophage enzyme with cell wall degrading capability against bacteria or fungi. It is a hybrid term derived from the words enzyme and antibiotic. The cell wall hydrolysis and receptor binding domains of the structure can be separated while maintaining their activity. These domains can be fused in a complementary fashion to other lysins to alter catalytic function, redirect binding or both. These bioengineered lysins are the so-called next-generation lysins.38 Artilysins are a group of next-generation lysins with an additional lipopolysaccharide disrupter to increase outer membrane penetration of gram-negative bacteria. A limited number of lysins can be recombined to generate a highly variable number to avoid bacterial resistance. So far, no neutralising antibodies or lysine resistance has been detected under the same conditions that favour antibiotic resistance and similarly no toxic side effects have been demonstrated.39 Lysins have shown promising results to clear pathogens on the mucous membranes in animal models and in vitro. The chimeric ectolysin P128 was manufactured to clear nasal MRSA infections. In situ efficacy was seen with a P128 hydrogel formulation against Staphylococci recovered from the nares of 31 healthy individuals, including mupirocin-resistant isolates. In addition, an MRSA nasal decolonisation rat model compared P128 formulated as a hydrogel to 2% mupirocin ointment. P128 cleared colonisation twofold, whereas the mupirocin alone was ineffective in the rat nares.41 A randomised, double-blind placebo-controlled study to determine the efficacy and safety of P128 applied intra-nasally to healthy volunteers has been completed (results not publicly available) to evaluate S. aureus clearance and re-colonisation rates.42An interesting feature of these ‘enzybiotics’ is their structural versatility.43 LysGH15 inhibits biofilm formation in vitro, but at higher doses, it distorts the integrity of the biofilm by dislodging the bacteria instead of destroying the biofilm matrix.44 The bacteriophage-derived lysin PlySs2 reduced colony-forming units by 99% and biofilm by 75% relative to vancomycin in vitro.45 Lastly, killing slow or non-growing organisms associated with bacterial infections of a chronic nature that may be naturally resistant to phage treatment or antibiotics could also be susceptible to lysins.46Although further attention is needed in preclinical testing with lysins, applications could include the treatment of antibiotic-resistant bacterial biofilms on medical devices such as catheters.Pseudomonas aeruginosa in ventilated patients and Klebsiella pneumoniae lung infections are promising.48 Furthermore, phage therapy co-administered with antibiotics has been successfully utilised in a refractory case of a complicated intra-abdominal infection because of MDR Acinetobacter baumannii.49 Even though phage resistance emerged in this patient with a pancreatic pseudocyst infection, it was rapidly circumvented with the selection and purification of a salvage phage that was subsequently added to the treatment regime.49 Notably, a case series in critically ill patients with prosthetic or native valve endocarditis because of S. aureus treated with a bacteriophage displayed reduced bacterial counts, clinical improvement and corresponding decreases in inflammatory markers.50 Other applications include gut decolonisation strategies, such as eradicating MDR, carbapenemase-producing K. pneumoniae isolates, following intra-rectal and oral therapy with a lytic bacteriophage preparation.51Several case reports highlight the potential use of phages in various clinical syndromes. Case studies illustrating the successful eradication of multidrug-resistant (MDR) 52 only two have progressed to phase III to our knowledge. The first is an active open-label, single-group assigned intervention of a nebulised liquid pyobacteriophage complex that entails the irrigation of mucous membranes in children with confirmed acute tonsillitis.53 A phase III randomised, placebo-controlled, double-blind trial was recently conducted in patients undergoing transurethral resection of the prostate with urinary tract infections. Treatment with intravesicular phage administration was safe and non-inferior in outcome compared to standard antibiotic treatment.55Although several phase I/II clinical studies on bacteriophage therapy have been published with many case reports that include different routes of phage administration, sources of infection and targeted pathogens,56 Possible factors for phage failure include sub-optimal titres and quality of phage preparations, the production of serum anti-phage antibodies, insufficient phage coverage of targeted pathogens, phage-eukaryotic cell interactions that might modify the effect of phage therapy and external factors such as medication  as well as food and drinks .58 A better focus and understanding of these confounding factors during trial design might lead to more favourable outcomes in the future.The poor clinical efficacy of phage therapy in many previous phase I/II trials could be related to design and technical issues.59Although not the focus of this overview, mention should be made to bacteriophage’s unique capability to detect specific bacteria for pathogen diagnostic purposes. Fluorescently stained phages that bind to targeted bacteria can be visualised under a fluorescence microscope as halo structures. Alternatively, genetically engineered phages express reporter genes during phage-bacteria binding. Quantum dots  bioconjugated to the capsid surface can be visualised using a microscope during phage-bacteria interaction. In optical and electrochemical transducer systems, phage heads are immobilised onto electrodes exposing their tails to capture specific bacteria that can identify microorganisms.in vivo studies, phage safety, efficacy and phage biobanking in the African context remain a priority.60 Although no single phage manufacturing centre has been established in Africa yet, research groups have been initiated to foster collaboration with Phages for Global Health.60 This initiative is focused on initially teaching scientists in African countries to isolate and characterise phages.Phage research is gaining interest on the African continent, but few groups have entered phage product design and development stages. As a result, 60 Current regulatory frameworks should support phage research and academic institutions to promote development, be flexible in compassionate use cases and be open to possible alternative pathways for the approval of phage therapy.It remains unclear if phages should be classified as biological or chemical agents (lysins), food additives or pharmaceutical drugs. This uncertainty complicates legislation, regulatory processes for bacteriophage therapy and investment.62 The possibility for an individual phage to be patented is unlikely; however, firms have adopted strategies to commercialise phage therapy. This includes patenting specific phage sequences or phage components mentioned above. Establishing regional phage banks across Africa that have already isolated, characterised, sequenced and purified phages could contribute to sustainable and cost-effective phage therapy.63 In addition, inaugurating networks of phage banks across the globe between reference phage laboratories and low-income regions could further help transfer knowledge and lower the cost of bacteriophage therapy.In this regard, a pertinent question for the future of phage therapy is whether manufacturers are willing to invest in the development of a product with limited patent protection. Intellectual property status is one of the major contributing factors to the general reluctance to develop phage therapy in developed countries. Pharmaceutical companies will not undergo expensive and rigorous clinical trials for drug approval and registration without patent protection.66 Until bacteriophage bio-control becomes the next frontier against pathogens, focusing on research that could provide regulatory bodies with the evidence to justify phage commercialisation and clinical use remains essential. This includes research on their complimentary bactericidal activity against antibiotic-resistant infections and their ability to reduce the gastro-intestinal carriage of MDR pathogens, from an infection prevention control point of view. In addition, designing phage formulations specifically in the African context that are easy to mass administer, have a long shelf life, are robust for transportation at different temperatures, and obtaining large-scale production skills should not be ignored.67 Finally, clinical data on bacteriophage and phage-derived products remain limited, with more rigorous clinical trials and reference standards needed to substantiate phage efficacy, determine appropriate treatment regimens and confirm its safety profile.In the future, treatment of antibiotic-resistant bacterial infections with phage therapy might be preceded by bacteriophage control of pathogens from an ecological perspective. To prevent infection with phage rather than to treat it is the focus for future research in agriculture, water treatment and food-related industries.


IgA nephropathy (IgAN) is the most common primary glomerulonephritis in the world. It was first described in 1968 by Jean Berger and Nicole Hinglais as the presence of intercapillary deposits of IgA. Despite this simple description, patients with IgAN may present very broad clinical features ranging from the isolated presence of IgA in the mesangium without clinical or biological manifestations to rapidly progressive kidney failure. These features are associated with a variety of histological lesions, from the discrete thickening of the mesangial matrix to diffuse cell proliferation. Immunofluorescence on IgAN kidney specimens shows the isolated presence of IgA or its inconsistent association with IgG and complement components. This clinical heterogeneity of IgAN clearly echoes its complex and multifactorial pathophysiology in humans, inviting further analyses of its various aspects through the use of experimental models. Small-animal models of IgAN provide the most pertinent strategies for studying the multifactorial aspects of IgAN pathogenesis and progression. Although only primates have the IgA1 subclass, several murine models have been developed in which various aspects of immune responses are deregulated and which are useful in the understanding of IgAN physiopathology as well as in the assessment of IgAN therapeutic approaches. In this manuscript, we review all murine IgAN models developed since 1968 and discuss their remarkable contribution to understanding the disease. The true prevalence of IgAN cannot be exactly determined. IgA deposits on the kidney are indeed frequent in asymptomatic patients and were reported in 16.1% of a population of kidney donors in Japan : IgAN isThere is significant heterogeneity in the clinical manifestations of IgAN since even healthy subjects with no clinical signs of IgAN may have mesangial deposits. By contrast, for those patients developing glomerulopathy with proteinuria and/or hematuria, one-third of the cases will progress to end stage renal disease (ESRD) . High blIgA antibodies (Ab) are exceptionally abundant at mucosal surfaces; more than 80% of mammalian Ab secreting plasma cells reside in the gut and secrete IgA. Notably, with the exception of germ-free animals or germ-free mucosal tissues, these antibodies arise prominently during homeostasis in the absence of overt inflammation and immunization. Consistently, the influence of super antigens expressed by bacteria present early in the intestinal flora has recently been demonstrated . At the These IgA share an interspecies reactivity because they are often polyreactive and partly produced by B-lymphocytes in a T-independent context. Second, during the local immune response induced by pathogens, protective high affinity IgA are produced in the mucosa-associated lymphoid tissue (MALT) germinal centers. This T-dependent response is similar to that of a general response establishing specific immunity as effective as a response generated after a systemic infection. The immune response to the presence of various commensal microorganisms constitute a homeostatic immune repertoire that echoes the variability of the microbiota which evolves according to age, environment, and diet and which is accompanied by an increase in the IgA affinity, whether T-dependent or not. This repertoire is not limited to controlling the commensal microbiota, it is also the source of protective immunity against pathogens ,10. AbnoIgAN is a mysterious disease. The heterogeneity of the clinical features and prognosis variability between patients explain why IgAN pathophysiology remains poorly understood despite the remarkable achieved advances. It is known that IgAN is not a primary (intrinsic) renal defect. The recurrence of IgA deposits on a normal kidney transplanted into a patient with IgAN or, on the contrary, the disappearance of deposits on kidneys from donors with IgA deposits accidentally transplanted into individuals without IgAN are two arguments supporting the extra-renal origin of the disease . CurrentOver time, the idea of a genetic component in IgAN has been raised. This is especially evident with the wide disparity of IgAN incidence by ethnicity and geographic region . Most IgIgA glycosylation plays important roles in protein conformation, stability, transport, and clearance from the circulation . The hinBactéroïdes and Escherichia-Shigella is higher in patients with IgAN compared to healthy subjects unlike other bacteria such as Bifidobacterium and Prevotella 7 that are present in much lower levels [Environmental factors play a potential role in the pathogenesis of IgAN from IgA production to its deposition at the mesangial level. Specific pathogens such as streptococcus or staphylococcus could be implicated in IgAN pathophysiology. Mesangial deposition of IgA binding streptococcal M protein has been detected in patients with IgA nephropathy . Macroscr levels .Experimental studies have confirmed the implication of environmental factors. Notably, in the α1KI mouse model expressing the heavy chain of human IgA1 (hIgA1) instead of the mouse IgM, environmental antigenic challenges accelerated IgA mesangial deposition. Mice transfer from pathogen-free zone to a conventional immune stimulation zone yielded an increase in IgA polymerization levels as well as changes in the IgA glycosylation profile ,32. MoreThe first described IgAN experimental model was published in 1979 . Rifai’sA few years later, in 1985, the spontaneous ddY mouse model was described. ddY animals were characterized by glomerulonephritis associated with spontaneous mesangial IgA deposition co-localized with IgM, IgG, and C3 deposits. This model was also characterized by the extreme variability of disease onset and severity. Based on histologic grading in serial biopsies, ddY mice were classified in early onset, late-onset, and quiescent groups . In 1997The presence of circulating fibronectin-containing IgA complexes and their deposition in the mesangium has been reported in patients with IgAN. In 1999, Zheng et al. developed a uteroglobin-deficient mouse model. Uteroglobin, a protein known for its anti-inflammatory and immunomodulatory properties, has a strong affinity for fibronectin. In wild type mice, uteroglobin normally binds to the fibronectin heterodimer preventing its polymerization and thus the formation of IgA-fibronectin complexes. Uteroglobin-invalidated animals developed a kidney disease with microscopic hematuria associated with IgA and C3 deposits in the mesangium but neither proteinuria nor alteration of kidney function were mentioned. Circulating IgA-fibronectin complexes were also detected in uteroglobin-deficient mice. Interestingly, uteroglobin injection prevented the formation and deposition of these complexes. This study defined an essential role for uteroglobin in preventing mouse IgA nephropathy . This mo+/+CD89+/+ mice by breeding this model to hIgA1-producing mice. Mouse follow-up did not show any exacerbation of glomerular lesions despite the presence of circulating IgA-CD89 immune complexes [The presence of circulating soluble Fcα receptor (CD89)-IgA complexes was detected in patients with IgAN. Based on this observation, researchers have focused on the implication of IgA receptors in IgAN development. In 1999, Renato Monteiro’s team developed a transgenic mouse model expressing human CD89 (no CD89 homologue exist in rodents) on macrophages and monocytes. CD89 expression was under the control of the CD11b gene promoter. Interestingly, CD89 transgenic mice having circulating CD89-IgA complexes spontaneously developed massive mesangial IgA deposition, mesangial matrix expansion, hematuria, and proteinuria . In 2012omplexes . Marked The human Bcl-2 transgenic mouse (NZW x C57BL/6) F1-hbcl-2 model was described in 2004 . Bcl-2 pIn parallel, Wang and his collaborators tested the role of T-cell dysregulation in IgAN development by focusing on LIGHT secreted by T cells. LIGHT is the ligand of the lymphotoxin β receptor (LTβR) expressed by stromal cells whose interaction creates a local environment activating IgA-producing B cells in the intestine. LIGHT transgenic mice developed T-cell-mediated intestinal inflammation with dysregulated polymeric IgA production, transportation, and clearance. Mice produced elevated levels of polymeric IgA, anti-DNA IgG and IgA antibodies, in addition to IgA and C3 mesangial deposition accompanied by proteinuria and hematuria. Importantly, this model highlighted a direct contribution of T-cell-mediated mucosal immunity to IgAN pathogenesis .In 2006, another mouse model overexpressing the B-cell-activating factor (BAFF) was developed. BAFF ensures the survival of B-lymphocytes by promoting their differentiation into mature B cells and Ig class switching. Overexpression of BAFF in mice resulted in increased circulating IgA levels and mesangial deposition with glomerulonephritis. These glomerular lesions might have been most likely due to an autoimmune disorder in these mice . FurtherAs mouse IgA lacks the hinge region, the study of aberrant glycosylation in IgAN development seems to be complicated. In 2007, a mouse model deficient in β-1,4 galactosyltransferase -I was developed. Knockout of the β4GalT-I coding gene implied the complete absence of galactosylation and sialylation in murine IgA molecules. Circulating IgA levels were significantly increased with the polymeric form predominating and mesangial deposition associated with mesangial matrix expansion . In paraAnother mouse model developed in 2014 suggested that the dysregulation of IgA production at the germinal center (GC) level led to the formation of IgA molecules prompt to deposition. Animals lacking the follicular dendritic cell secreted protein (FDC-SP) expression, a suppressive protein for IgA production secreted by FDCs, showed an increase in IgA levels in both intestinal wash and serum. This resulted from the accumulation of IgA B cells in the blood, bone marrow, Peyer’s patches, and lymph nodes. IgA deposits with proteinuria were identified in 6-month-deficient mice . This stMore recently, the hypothesis of altered affinity maturation on IgA mesangial deposition was raised. The α1KI mouse model expressing the heavy chain of human IgA1 (hIgA1) in an AID-deficient background was set up . In thisGiven the wide heterogeneity of IgAN clinical features added to the multifactorial aspect of the pathology, the development of experimental models constitutes a huge challenge for researchers. Since 1979, several animal models have been developed for a better understanding of IgAN pathogenesis. Although multiple models were able to reproduce some of the IgAN characteristics, they could not cover the full spectrum of pathological manifestations observed in patients. It is evident that animal models can constitute useful tools, taking into consideration marked differences between human and mouse systems especially in IgA biosynthesis, dominant circulating forms, molecules half-life and clearance mechanisms . Most imThe heterogeneity of human disease and animal models likely reflects the varying influence of genetic and environmental factors on a multitude of complex pathogenic mechanisms modulating the disease phenotype in different individuals and populations. Another explanation is that IgAN may not be a “single disease” but rather a group of distinct diseases showing a common path of mesangial IgA deposition.


Human amniotic membrane (HAM) has been used as a biologic dressing for burn wounds since 1955 but limited due to availability, size, and processing costs. In 2021 a new porcine placental product was FDA-approved overcoming challenges with human-sourced products. Our study is the first case series to report outcomes using porcine placental extracellular matrix (PPECM) in the use of adult burn patients.Adults with thermal burns resulting in partial-thickness burn wounds (PTBW) were consented and included in the study from 03/2021 to 09/2021. Patients with full-thickness injures, concomitant trauma, or adverse beliefs to porcine products were not included in the study. Serial still images and initial wound measurements were obtained intraoperatively and post-operatively. PPECM trial product processed with a proprietary decellularization method to produce single sheets up to 15x20cm was approved by the facility value assessment committee. Adverse events were defined a priori as infection, increased pain or itching relative to adjacent autografts, or failure to heal. Infection was defined as a PPECM treatment site requiring any change from standard of care or initiation of local or systemic antibiotics. Pain was assessed using a visual analogue scale. Itching was assessed at discharge and follow-up. Healing was assessed using the FDA guidance for wound closure with 2 consecutive visits 2 weeks apart demonstrating 100% epithelialization without drainage or dressing requirements.Four patients were treated during the study period with wounds involving the torso and major joints such as the hands/wrists and knees. None of the PPECM wounds demonstrated failure to heal or required revision excision, or autograft. None of the PPECM wounds had evidence of infection. PPECM wounds had decreased pain/itching relative to adjacent burn wounds which were treated with split-thickness autograft, autologous skin cell suspension, or allogeneic cultured skin substitute (VAS mean 1 vs 3.1). Healing was noted in all wounds at 1-week primary dressing removal with confirmation at 2-week interval follow-up.PPECM treatment of PTBW was not associated with adverse events and resulted in favorable outcomes clinically. The large size, ease of use, and lower costs relative to HAM is an intriguing alternative for PTBW. Comparative studies are needed in the field to determine best practices and overall value.


If microorganisms are isolated from certain plants, there is undoubtedly a strong possibility of obtaining beneficial endophytes strains producing host-specific secondary metabolites for their potential applications in sustainable agriculture, pharmaceuticals and other industrial sectors. Few products derived from endophytes are being used for cultivating resilient crops and developing non-toxic feeds for livestock. Our better understanding of the complex relationship between endophytes and their host will immensely improve the possibility to explore their unlimited functionalities. Successful production of host-secondary metabolites by endophytes at commercial scale might progressively eliminate our direct dependence on high-valued vulnerable plants, thus paving a viable way for utilizing plant resources in a sustainable way.Endophytes represent microorganisms residing within plant tissues without typically causing any adverse effect to the plants for considerable part of their life cycle and are primarily known for their beneficial role to their host-plant. These microorganisms can  Tolerance to drought conditions and enhancement of growth are common non-habitat adapted benefits, irrespective of origin of habitat, whereas benefits of habitat adapted are specific to the habitat with selective pressures that include salinity, pH and temperatures The microorganisms such as fungi, bacteria including actinomycetes and viruses that reside within plant tissues are known as endophytes Considerable attention in the extensive investigation of beneficial microorganisms from the plant tissues fully demonstrate their unique abilities to produce secondary metabolites of the host plant and collection of functionalities  with theIdentification of fungal endophytes has been carried out by studying the morphological characteristics after sporulation. However, classification of non-sporulating fungi is problematic and it is carried out through phylogenetic analyses of rDNA-ITS sequences after the amplification of DNA extracted from the fungal mycelia 3.Microbial endophytes are well-known for their ability to produce a wide range of pharmacologically important compounds with enormous therapeutic potentials; which have been identified as antiviral, antifungal, antibacterial, antitumor and anticancer agents. A number of endophytes are prospective source of plant growth promoting factors, and plant hormones. They can synthesize compounds of applications in the field of agriculture, iron chelating agents, compounds with nematocidal, insecticidal activities and abiotic stress tolerant properties. Some endophytes have shown their ability to secrete wide range of extracellular enzymes, such as phosphatase enzyme to convert insoluble phosphates to soluble form for its easy assimilation by plants. Endophytes produce molecules suitable for the production of bio-fuels and degrade complex organic and inorganic substances with suitable use in industrial sectors. The useful properties of endophytes are listed below with their potential significance in respective sectors.3.1.Published work state that endophytes are definitely an excellent source of metabolites and desired functions that could prove to be beneficial in organic farming system. Some of the endophytes could be used as bio-pesticides against phytopathogens due to their antimicrobial, nematocidal and insecticidal qualities.3.1.1.Phleum pratense, demonstrated myco-toxic properties, which were secreted by a systemic grass symbiont fungal endophyte Epichloe typhina. The antifungal properties of the extracts were detected against Cladosporium herbarumLarix laricina displayed insecticidal property against larvae of spruce budworm. Chitinase, known to degrade chitin polymers that are essential part of a fungal cell wall, was produced by bacteria, an endophyte of Sinapis arvensis. The bacterial endophyte was identified as Bacillus cereus strainRhizoctonia solaniThe extracts from a perennial grass native to most of Europe Neotyphodium sp. (AR601) producing large quantities of alkaloids such as loline and ovaline inoculated into a cultivar ‘Jackal’ of turf tall fescue have shown birds deterring ability Strain of 3.1.2.Hymenoscyphus ericae, that produced several enzymes along with phosphate solubilization properties et al isolated Colletotrichum sp. B501 from the healthy stems of Artemisia annua L, secreted IAA and 3β-hydroxy-ergosta-5-ene, these compounds that showed properties for plant growth In vitro investigation of endophyte-plant interaction in Echinacea purpurea demonstrated that colonization potential of bacterial strains belonging to Pseudomonas and Arthrobacter genus were tissue specific in host plants from which they were originally obtained but did not show similar specificity in non-host plants Bacillus sp. isolated from Thymus vulgaris demonstrated plant growth promoting traits in Solanum lycopersicum L under salt stress along with showing antagonistic activity against Fusarium oxysporum and reduced the antioxidant stress on plants viz. roots, stem, leaf and flower of Origanum vulgare L Pseudomonas and Bacillus were the most represented genera of endophytes in Lavandula dentata that demonstrated multiple PGP traits Endophytes have been identified to solubilize phosphates, produce siderophores, secrete plant growth promoting factors and increase soil nutrition by degrading complex organic molecules. It was observed that ericoid plants were able to thrive in extreme conditions due to the presence of an endophyte, Some of the bacterial and fungal endophytes and their potential applications in agriculture are listed in 3.2.Fusarium sp. PN8 isolated from Panax notogensingProducts derived from natural sources are a major area of research for discovering the range of their functions, that could be used in pharmaceutical industries 3.2.1.11) an antimicrobial agent was extracted from Phomopsis sp., isolate no. MF6031 obtained from the twigs of Salix gracilostyla var. melanostachys. The compound 11 exhibited antibacterial activity against Bacillus subtilis, Salmonella gallinarium and Staphylococcus aureus with some amount of antagonism towards Candida tropicaliset al isolated an endophytic fungus from the needles of Larix laricina (Du Roi) K. Koch 12) showing antibacterial activity against Vibrio salmonicida, S. aureus and Pseudomonas aeruginosa.Some species of endophytes are known to produce antimicrobial compounds. Phomopsichalasin , 3β,5α-dihydroxy-6β-phenylacetyloxy-ergosta-7,22-diene (21), 3β-hydroxy-ergosta-5-ene (15), 3-oxo-ergosta-4,6,8(14),22-tetraene (16) and 3β,5α-dihydroxy-6β-acetoxy-ergosta-7,22-diene (20) showed antibacterial activity against Gram-positive and Gram-negative bacteria. Compounds 15, 20 and 21 had antifungal properties, compound 15, 19 and 20 demonstrated fungistatic property In another study, a 3.2.2.Taxus brevifolia Nutt., Taxomyces andreanae, was able to produce Taxol, the host secondary metabolite, in a broth culture medium Hypericum perforatum L. Phomopsis vexans isolated from Solanum virginianum L. Few endophytes show the medicinal properties as anticancer and antitumor in their metabolites. An endophyte isolated from The secondary metabolites and other functions from endophytes could have potential applications in therapeutics without causing damage to the respective plant species. The bacterial and fungal endophytes suitable for therapeutic purposes are listed in 3.3.Nocardiopsis sp., isolated from Pachyrhizus erosus L., was found to secrete a thermostable α-amylase, which is useful for starch degradation Candida guillermondii from Ricinus communis L. produce lipase and helps in the synthesis of methyl oleate Microorganisms and their derivatives play a significant role in processing of substrate into several products for use in industrial sectors. There are many reports of enzymes being produced by endophytes isolated from different plant species. Enzymes like amylase, pectinase and lipases obtained from different endophytes have been known to hydrolyze starch, pectin and oils, respectively. Other enzymes include cellulases, xylanases, amylase, laccase, and proteases, which have application in various industrial sectors Hypoxylon sp. from Persea indica (L.) Spreng. was found to secrete 1,4-Cyclohexadi-ene (94). The compound 94 readily oxidizes to benzene, which is a main component of crude oil Gliocladium roseum (NRRL 50072) isolated from Eucryphia cordifolia Cav. produced a bio-fuel known as myco-diesel Some endophytes are also known to produce bio-fuels, as alternate source of conventional fuels. A fungal isolate, While some of the endophytes are known to degrade polyurethane which are of great value to the industrial sector 3.4.in vitro conditions. The genome mining approach could reduce the time taken to identify the putative genes required for the synthesis of secondary metabolites Burkholderia species. Poplar trees augmented with a modified strain of endophyte, Pseudomonas putida W619-TCE, showed increased reduction (90%) of trichloroethylene evapotranspiration under field tests Some microorganisms are known to synthesize only a limited number of secondary metabolites (SMs) as compared to the ones estimated through genome mining 4.in vitro conditions. Some of the strains have found their place in modern agricultural practices, such as perennial ryegrass (PRG) due to its endophyte, Neotyphodium lolii, was able to protect the host plant from Argentine stem weevil infection without producing any toxic compounds harmful to livestock. A product of Rye grass, AR1, infected with the endophyte has been beneficial for livestock production in places with lesser number of black beetles. Another strain of PRG, Endosafe, has shown better survival response in places dominated with black beetles but with decreased biomass production compared to AR1 In an effort to meet the increasing demand of food and feeds, chemical fertilizers and pesticides have been commonly used in agricultural system for improving soil fertility and controlling pests, respectively. The adverse effect of use of toxic chemicals in agriculture has resulted in increasing interest in sustainable farming practices Muscodor albus isolated from cinnamon tree, has shown properties related to bio-fumigation and it may replace the use of methyl bromide for fumigation of soils Adaptive Symbiotic Technologies in Seattle, USA, have developed several products under the brand name BioEnsure® using a combination of beneficial endophytes. The products are able to induce tolerance in crops to drought, high salt concentration and temperature; it improves water utilization by plants and is fairly stable in different climate and soil types. The microbial formulations can easily be applied to fields along with other agriculture inputs and are non-competitive against other normal microbial flora of the soil. The products have a viability of more than two years at 4 °C. BioEnsure® products not genetically modified are classified as organic products by Organic Material Review Institute, Eugene, USA 5.There have been numerous reports on the production of plant secondary metabolites by endophytes outside its host but there are no products as such that have successfully been produced in mass scale and commercialized. Production of Taxol by endophytic fungus in the early 90s was thought to develop the process of obtaining metabolites from endophytes with eventual decrease in over-use of plants. However, apart from the use of few endophytes in agricultural system, not a single product from endophytes has made it to the market with a significant advance in secondary metabolite industry et al their evolutionary significance The reason for the production of host metabolites by the endophytes could be hidden in their genes that must have undergone genetic recombination during the time of their evolution in vitro system.Some of the constraints involved in the production of secondary metabolites under laboratory conditions include: the low-yield of secondary metabolites, optimization of growth-conditions involving variety of abiotic factors and silent gene clusters, synthesis of metabolites with unidentified functions, unclear understanding of pathways involved in the production of metabolites, role of secondary metabolites in different pathways and lack of a complete knowledge on secondary metabolites 6.in vivo trials could satisfactorily establish the practical possibility of endophytes commercialization. Specific mechanisms involved in the complex interactions, types of selection pressures that properly govern the crosstalk between endophytes and their suitable host, efficient production of host secondary metabolites, and possible ways to effectively manipulate the biochemical-pathways, would undoubtedly require comprehensive understanding before the successful commercialization of bioactive metabolites from endophytes.Endophytic microorganisms have convincingly demonstrated their remarkable ability to typically produce an abundance of pharmacological metabolites with possible usage in drug manufacturing. Extensive search for newer metabolites is important to deal with multi-drug resistant microorganisms and to find alternative therapeutic drugs for several diseases. Secretion of plant growth-promoting factors and antagonistic agents against phytopathogens could easily substitute chemical inputs in sustainable agriculture practices with suitable endophytes. Novel enzymes with better specific-activity obtained from endophytes could be valuable in fermentation industries. However, most of the published findings are from controlled experiments and similar results from 7.in vitro systems. These properties of endophytes not only make them suitable candidates for exploring their ability to produce various bioactive compounds, enzymes, and biopigments, etc., but it may also reduce the dependency of humans on endangered plant species for their secondary metabolites, thus resulting in sustainable use of plant-based bio-resources. The necessary factors controlling growth of endophytes for biosynthesis of host secondary metabolites in vitro, are required to be optimised for commercial-scale production of plant-derived natural compounds employing these endophytes.It is estimated that there are more than quarter million species of plants in this planet with a possibility of obtaining more than one million endophytes from these plants. Very few of these microorganisms from their diverse group have been isolated and studied so far. Apart from producing array of metabolites and functions advantageous to its host plant, these microbial resources have proven to secrete similar secondary metabolites even outside its host using Click here for additional data file.


Recent clinical trials have demonstrated the capability of safely delivering prostate radiotherapy with a simultaneous focal boost. These studies have also indicated limitations to achieving focal boosts whilst trying to limit normal tissue toxicity. More guidance in the location and level of the required boost could alleviate such limitations. Tumour hypoxia is one of the causes of clinically observed radioresistance and hypoxic volumes represent prime candidates for a focal boost. Multiparametric magnetic resonance imaging (mpMRI) has the potential to quantitatively describe the extent and spatial distribution of hypoxia in prostate cancer. In this article we demonstrate a biologically targeted radiotherapy approach that can utilise this information to target hypoxia for favourable treatment outcomes.Purpose: Hypoxia has been linked to radioresistance. Strategies to safely dose escalate dominant intraprostatic lesions have shown promising results, but further dose escalation to overcome the effects of hypoxia require a novel approach to constrain the dose in normal tissue.to safe levels. In this study, we demonstrate a biologically targeted radiotherapy (BiRT) approach that can utilise multiparametric magnetic resonance imaging (mpMRI) to target hypoxia for favourable treatment outcomes. Methods: mpMRI-derived tumour biology maps, developed via a radiogenomics study, were used to generate individualised, hypoxia-targeting prostate IMRT plans using an ultra- hypofractionation schedule. The spatial distribution of mpMRI textural features associated with hypoxia-related genetic profiles was used as a surrogate of tumour hypoxia. The effectiveness of the proposed approach was assessed by quantifying the potential benefit of a general focal boost approach on tumour control probability, and also by comparing the dose to organs at risk (OARs) with hypoxia-guided focal dose escalation (DE) plans generated for five patients. Results: Applying an appropriately guided focal boost can greatly mitigate the impact of hypoxia. Statistically significant reductions in rectal and bladder dose were observed for hypoxia-targeting, biologically optimised plans compared to isoeffective focal DE plans. Conclusion: Results of this study suggest the use of mpMRI for voxel-level targeting of hypoxia, along with biological optimisation, can provide a mechanism for guiding focal DE that is considerably more efficient than application of a general, dose-based optimisation, focal boost. Hypoxia has been widely reported to exist in prostate cancer (PCa) and has been shown to be linked to local treatment failure, increased risk of metastases and radioresistance . To overConventional dose-based planning approaches aim to deliver a prescribed dose to the prostate (and a boost dose to the tumour if applicable). In contrast, biologically targeted radiation therapy (RT) considers the specific characteristics, on a voxel-wise basis, of the tumour in the treatment planning process, and aims to maximise the tumour control probability of each voxel whilst minimising normal tissue complication probability. Biologically targeted RT has, up until recent times, not been widely practised due to the inability to spatially map tumour characteristics. Quantitative imaging, however, has demonstrated an ability to define pathological and physiological features of tissue at the voxel-level and hence the potential to provide patient-level input parameters for a biological approach to treatment planning . In the In this article, we present the first in silico study to investigate the benefits of a biologically targeted RT approach (which we define as “BiRT”) to overcome the effects of hypoxic tumour sub-volumes. We compare this approach with dose-based optimisation methods using the hypo-FLAME dose fractionation schedule, and include whole-gland treatments, whole-gland treatments with a tumour-defined boost volume, and whole-gland treatments with a boost volume and hypoxic sub-volume.7 for the five patients  . Patientpatients , which iPatient-specific hypoxia score maps were derived from mpMRI using the top eight performing candidate radiomics features identified by Sun et al. . BrieflyThe voxel resolution of the tumour biology maps was resampled to 2 mm × 2 mm × 2.5 mm from the original resolution of 0.22 mm × 0.22 mm × 2.5 mm for improved computation time.As patient data did not include CT imaging, a single CT image set  was selected from an established clinical trial for dose calculation purposes. The selected CT data set contained a prostate volume that was similar in shape and size to the MRI defined prostate volumes of the five patients, but slightly larger such that the prostate contours delineated on the T2w MRI were wholly contained within the CT-defined prostate contour. The organs at risk (OARs) including the rectum, bladder, and head of femurs (HOFs) were delineated on the CT images.th voxel of the clinical target volume (CTV) with n fractions was calculated using Equation [th voxel is given by Equation [All model equations are presented in Equation . In thisEquation . Most moEquation . Tumour quations  and 4]..ith voxequations . Assuminquations .Treatment planning was performed using a MATLAB based open-source program, matRad  uniform-dose planning, (2) focal DE to the tumour, (3) focal DE to the tumour and hypoxic sub-volumes and 4) robust biological optimisation. Each of these methods are presented in schematic format in  robust bThe main objective of the uniform-dose planning method was to provide a homogeneous dose prescription of 35 Gy to the planning target volume (PTV) while satisfying OAR DV constraints. The hypo-FLAME trial recommended a planning margin of 4-5 mm, however, for consistency across all 4 planning approaches we accounted for geometric uncertainties using a modelling approach to produce the robust biologically optimised treatment plans (Method 4). These uncertainties were considered by noting that random uncertainties increasingly behave like systematic errors with hypofractionation ; the geoThe overall distribution and the resultant margin for the three principal directions used for uniform-dose planning are summarised in The OAR dose constraints were identical to those used for the hypo-FLAME trial. The PTV and CTV treatment planning objectives were V33.25Gy  ≥ 99% and V35Gy  ≥ 99%, respectively .The tumour voxels were segmented and defined as the gross tumour volume (GTV). Based on the hypo-FLAME trial objectives, the focal DE plans were designed to deliver a dose of 35 Gy to the PTV and up to 50 Gy to the GTV while satisfying the OAR dose constraints.The voxels classified as hypoxic were segmented as hypoxic target volumes (HTVs) for each HF. A previous Monte Carlo study estimated that an increase of 20–50% of tumour dose is required to increase the TCP if a significant portion of chronic hypoxia is identified [Uncertainties in the delivery of highly modulated treatment fields can result in significant differences between planned and delivered dose distributions. Hence, incorporating robust treatment planning approaches is necessary for biologically targeted radiotherapy approaches. In this study we utilise the term “expectation value” to indicate the reported values of TCP and normal tissue complication probability (NTCP) account for treatment delivery uncertainties and are termed <TCP> and <NTCP> respectively. In addition to the uncertainties included in the planning margin for the uniform-dose planning approach, uncertainties associated with each of the image-registration steps were included for biological optimisation. These were MR-to-histology registration  and MR-tBiological optimisation aimed to maximise <TCP>, while simultaneously minimising <NTCP>, of the rectum and bladder. The methods described in Witte et al.  were use2Gy>, was computed using Equation [NTCP is based on the generalised equivalent uniform dose (EUD) . The expEquation , and <NTEquation .To investigate the effect of hypoxia on <TCP> of dose-based planning methods, <TCP> of the plans generated with uniform-dose (Method 1), focal tumour DE (Method 2) and focal tumour+hypoxia DE (Method 3) approaches were calculated for all oxygenation status considered: HF of 0% (normoxic), 20%, 40%, 60% and 80% see .The overall dose distributions of the biologically optimised plans (Method 4) were linearly scaled to attain the same <TCP> as the corresponding focal tumour + hypoxia DE plans (Method 3). Biologically optimised plans were assumed to be invariant with dose scaling. Dose to the rectum and bladder was compared by calculating the <EUD>. Mean doses were evaluated for the HOFs.t-test was performed to compare focal tumour + hypoxia DE plans and biologically optimised plans of the same HF.A paired To investigate the sensitivity of robust biological optimisation to different OER values, hypoxia-targeting biological plans were generated for patients with the lowest and the highest total number of clonogens (Patient 2 and 4) and modelling of the HF at 20% and 80% . Includip-value) less than 0.05 was considered significant.All statistical analyses were performed with the R statistical language . A test statistic . Differences in mean dose to HOFs were not statistically significant.The results so far applied an OER value of 1.4 as our radiogenomics study was not designed to provide a quantitative measure of OER. As shown by 2Gy doses reported in the FLAME and hypo-FLAME studies (106.3 Gy and 133.3 Gy respectively) [Dose escalation (DE) to the GTV using the hypo-FLAME protocol Method 2) demonstrated a substantial improvement in <TCP> compared to uniform-dose planning Method 1) planned without consideration of oxygenation and is consistent with higher EQDctively) . In this planned ctively) , further demonstrIn this in silico study we applied the dose fractionation schedule of the hypo-FLAME study based on the promising results of this clinical trial. These clinical findings also support the conclusions of our previous work where we demonstrated the normal tissue sparing benefits of biologically targeted, hypofractionated schedules compared with conventional fractionation schedules .3 representing typical prostate volumes. Whilst large prostate volumes may impact on the ability to limit the dose to the OARs, we would expect that tumour volume would be the predominant factor in challenging the biologically targeted RT approach we have described. We would propose future studies consider a larger sample size with a range of factors, such as prostate and tumour volume that may determine the upper limit for our proposed approach to be feasible. Furthermore the urethra was not included as an OAR as it could not be easily delineated on the MR datasets. Future studies should include a urethra-sparing approach using contrast or modelling from ground-truth histology for urethra position. As the urethra runs through the centre of the prostate, it is however anticipated that hypoxia-targeting, biologically optimised plans will suffer significantly from “streakiness” in the effort to provide a doughnut-shaped dose distribution. As such, hypoxia-targeting biologically targeted radiotherapy approaches may be better realised with brachytherapy. Biological optimisation of radioactive seed placement for low-dose-rate brachytherapy has previously demonstrated improved tumour control whilst simultaneously minimising dose to OARs, including the urethra when population-based tumour biology information was employed [In this study we applied the CT scan of a single patient for the purpose of dose calculation. We would expect that the conclusions of our study would be unchanged if we had used a synthetic CT derived from the MR data for each patient due to the relative homogeneity of tissue densities within the prostate and surrounding tissue. Our small sample size of five patients included prostates with a range of cell density distributions and tumour sizes to demonstrate their effects on biological optimisation. The volumes of these prostates ranged from 27.6–57.0 cmemployed . The resemployed ,38. WithLimitations in the modelling of hypoxia in this work are acknowledged. Although it is well-known that hypoxia exists in a spatially and temporally heterogeneous distribution, tumour hypoxia is a complex process that is still not well understood. In the absence of ground-truth information, a predictive model of tumour hypoxia could not be developed. Instead, surrogates based on genetic profiling and immunohistochemistry staining of histology were used to derive the candidate features for defining the spatial distribution of hypoxia . AdditioIn addition to hypoxia modelling, the TCP model used in this study has further limitations in describing tumour radiation response. In this study we have chosen to use our previously validated TCP model , a modelThis work explored the focal DE and biologically targeted radiotherapy approaches for targeting hypoxia in an ultra-hypofractionated prostate radiotherapy schedule. By using patient-specific, mpMRI-derived cell density and hypoxia maps, improved rectal and bladder sparing was achievable for biological optimisation when compared to isoeffective focal DE plans. The results of this study support further investigation of biologically targeted approaches using intensity modulated radiotherapy, brachytherapy and potentially particle therapy to take advantage of the knowledge of the spatial distribution of tumour heterogeneity.


Emerging cross-sectional evidence comparing cognitive profiles in treatment resistant schizophrenia to treatment-responsive schizophrenia has indicated that verbal memory and language functions may be more impaired in treatment resistance. We sought to confirm this finding by comparing cognitive performance between antipsychotic non-responders (NR) and responders (R) using a brief cognitive battery for schizophrenia, with a primary focus on verbal tasks compared against other measures of cognition.Cross-sectional.This cross-sectional study recruited antipsychotic treatment R and antipsychotic NR across four UK sites. Cognitive performance was assessed using the Brief Assessment of Cognition in Schizophrenia (BACS).One hundred and six participants aged 18–65 years with a diagnosis of schizophrenia or schizophreniform disorder were recruited according to their treatment response, with 52 NR and 54 R cases.Composite and subscale scores of cognitive performance on the BACS. Group (R vs NR) differences in cognitive scores were investigated using univariable and multivariable linear regressions adjusted for age, gender and illness duration.Univariable regression models observed no significant differences between R and NR groups on any measure of the BACS, including verbal memory  and verbal fluency . This pattern of findings was consistent in multivariable models.The lack of group difference in cognition in our sample is likely due to a lack of clinical distinction between our groups. Future investigations should aim to use machine learning methods using longitudinal first episode samples to identify responder subtypes within schizophrenia, and how cognitive factors may interact within this.REC: 15/LO/0038. The study examined cognitive performance in a relatively large and multicentre sample of antipsychotic responders and non-responders.Cognition was assessed with the Brief Assessment of Cognition in Schizophrenia, a reliable and brief test battery specifically designed for schizophrenia.The lack of significant group differences in cognition between antipsychotic responders and non-responders may reflect limited clinical separation between these groups.Up to one-third of patients with a schizophrenia diagnosis have inadequate symptomatic improvement despite having at least two antipsychotic drugs, one being a second-generation antipsychotic excluding clozapine, at adequate doses and duration  guidelines)Cognitive impairment in schizophrenia may provide some insight into antipsychotic treatment response. Performance on tasks of verbal memory has often been reported to be impaired in schizophrenia samples,dl=−0.59, p<0.001) and language functions , with smaller but still statistically significant impairments in tasks across other cognitive domains, relative to their responder counterparts.In a recent meta-analysis comparing mostly cross-sectional studies of treatment resistant cases and responders, TRS cases exhibited greatest cognitive impairments on tasks of verbal memory and learning , a consortium which included King’s College London , University of Manchester , Cardiff University  and University of Edinburgh . The aim of the STRATA consortium is to identify neurobiological, cognitive and genetic biomarkers of antipsychotic treatment resistance and NR within schizophrenia and other related psychotic disorders.During the early development of the study the views and recommendations of service users and carers regarding the use of stratified medicine research were assessed. Consultations were undertaken with the Institute of Psychiatry, Psychology and Neuroscience’s Service User Advisory Group. Service user researchers in London, Manchester and Edinburgh (18 people) carried out focus groups, and one carer focus group in London (8 people). Focus groups were digitally recorded, the transcripts analysed in NVivo V.10 using a simple thematic analysis, and quotations deidentified to protect participants. The results of this research are published in BioMed Central.et al.One hundred and six participants were recruited following a screening of patients across four sites: King’s College London N=38), University of Manchester (N=32), Cardiff University (N=16) and University of Edinburgh (N=18). Inclusion criteria were as follows: aged 18–65 years, with a schizophrenia or schizophreniform disorder diagnosis as per Diagnostic and Statistical Manual of Mental Disorders, fifth edition, Univers’). Fifty-four treatment responders were recruited into the study.Participants were defined as antipsychotic treatment R if they had been treated with only one antipsychotic drug since illness onset or if their antipsychotic drug had been changed only for reasons of adverse effects as opposed to NR. In addition to this, responders needed to have a Clinical Global Impression-Schizophrenia scale (CGI-SCH)Antipsychotic treatment NR was defined as having documented treatment with at least two antipsychotics each above the minimum therapeutic dose as defined by the British National Formulary for >4 weeks each, a CGI-SCH severity score of >3, a PANSS total severity rating of at least 70, and a CRS adherence score of >3. Fifty-two participants met criteria for antipsychotic NR.Previous and existing drug use were measured using the Alcohol, Drug and Tobacco Inventory. Participants’ disorder severity was measured using the Mini-International Neuropsychiatric Interview ,Cognitive data were collected using the BACS2 tests were used to compare cognitive performance across sites in case of site differences. Univariable regressions were used to compare cognitive performance between groups. Multivariable regression analyses were used to adjust univariable results for age, gender and illness duration, due to the reported relationship of age,All analyses were conducted using STATA V.15/SE.10.1136/bmjopen-2021-054160.supp1Supplementary data10.1136/bmjopen-2021-054160.supp2Supplementary dataDescriptive statistics of demographic and clinical variables between responder groups are reported in Mean scores for each group on all BACS tasks and standardised composite scores are displayed in Univariable linear regression analyses  observedThe present investigation sought to compare specific cognitive deficits in antipsychotic R and antipsychotic NR using the BACS,Previous cross sectional research investigating differences in cognitive performance between antipsychotic treatment R and treatment resistant cases have identified poorer performance in verbal, executive function, full-scale IQ cognitive measures,et al).The lack of significant differences in cognitive performance observed between R and NR groups in our study may be partly explained by the criteria used to define these groups. Unlike earlier investigations, our study did not include clozapine-treated patients, and there may have been less clinical separation between the R and NR groups than in some previous studies  have been attributed to D2 dopamine receptors and functioning in the striatum, as evidenced by animal models.Another consideration is that our study focused on younger patients early in their treatment trajectories to reduce the potential effects of chronicity and previous medication, with a mean length of treatment of 3–4 years. Most previous cross-sectional investigations also include older samples with a longer duration of illness,Likewise, this more restricted focus may explain why there was smaller sampling of females in comparison to previous investigations. A recent nationwide cohort study found that on average females are more likely to be first diagnosed with a mood disorder prior to a psychotic diagnosis.et alDespite not detecting significant differences between antipsychotic R groups, it is worth mentioning the importance of conducting research using clinically transferable measures of cognitive impairment. It may be possible for future researchers to use machines learning algorithms to identify subgroups of schizophrenia from cognitive outcomes. Bak Within this cross-sectional investigation, we observed no differences in cognitive performance between antipsychotic R and NR. This may be because there was less clinical separation between these groups in our sample in comparison to previous investigations. Future investigations should consider the role of machine learning techniques to investigate the role of cognitive functions in identifying subgroups of schizophrenia using first episode cohorts and how this may differ in future stages of treatment resistance. Such research using antipsychotic-naïve patients vs healthy controls has observed strong group discrimination using cognitive measures in comparison to electrophysiology and MRI methods,74