The operational and financial impact of adding anaerobic screening of platelets.

BACKGROUND AND OBJECTIVES: We evaluated the operational and safety impact of implementing anaerobic culture screening of apheresis and pooled platelets at the American Red Cross on the already established use of the aerobic culture screening of each donation performed no sooner than 24 h following collection. MATERIALS AND METHODS: Platelets were screened for bacterial contamination with the BACT/ALERT 3D® (bioMérieux, Durham, NC) microbial detection testing system. The addition of anaerobic culture to the already existing aerobic culture resulted in sampling an additional 8-10 mL from each donation. RESULTS: Implementation of anaerobic testing resulted in an approximate 3.5-fold increased rate of False Positive BACT/ALERT alarms. There was a modest increase in the rate of True Positive alarms of 1.4-fold with increased detection of Klebsiella and Propionibacterium species, including Cutibacterium acnes. In addition, there was an approximate 3.5-fold increase rate of False Positives and a 13.5-fold increase rate of Indeterminates, the majority (~57%) were due to Cutibacterium acnes. The combined costs and lost revenue associated with adding anaerobic screening increased by ~$1,000,000/year due to testing cost and product discards. CONCLUSION: The addition of anaerobic culture to aerobic culture to the original donation (without the introduction of sampling delay) resulted in a significant increase in the rate of alerts. The 40% increased rate of True Positive alarms may have modestly improved platelet safety. However, there was a disproportionate increase in the rate of False Positive and Indeterminate bacterial culture alarms, which added substantial cost and overall loss of platelet products.

How do we use the National Healthcare Safety Network for Hemovigilance in Massachusetts?

BACKGROUND: Use of the National Healthcare Safety Network (NHSN) has been essential to the success of the Massachusetts Hemovigilance Program and has allowed for the timely identification of signals and trends over a defined population that correlate with national and international hemovigilance (HV) data. Here, we outline how the NHSN system is used for monitoring HV data in Massachusetts and encourage adoption of NHSN for nationwide HV surveillance. STUDY DESIGN AND METHODS: A collaboration that grew over time between local HV stakeholders and the Massachusetts Department of Public Health (MDPH) resulted in the change from a paper-based method of reporting adverse reactions and monthly transfusion activity for compliance with state requirements to replacement with statewide adoption of reporting via NHSN. RESULTS: Over 1.5 million blood products were transfused in Massachusetts between 2017 and 2021, with 3000 adverse reactions among 10 defined types reported. Using NHSN, MDPH has been able to produce numerous reports, publications, and presentations that have made previously non-obtainable HV and blood utilization data available. DISCUSSION: Although limitations to these self-reported data exist, such as lack of external validation, successful statewide implementation of NHSN for hospital blood bank reporting is possible and has benefits beyond those for regulatory oversight. It results in standardized, actionable data at both the hospital and state level, enabling inter-facility comparisons, benchmarking, and opportunities for practice improvement.

A Comprehensive Approach to Parkinson's Disease: Addressing Its Molecular, Clinical, and Therapeutic Aspects.

Parkinson's disease (PD) is a gradually worsening neurodegenerative disorder affecting the nervous system, marked by a slow progression and varied symptoms. It is the second most common neurodegenerative disease, affecting over six million people in the world. Its multifactorial etiology includes environmental, genomic, and epigenetic factors. Clinical symptoms consist of non-motor and motor symptoms, with motor symptoms being the classic presentation. Therapeutic approaches encompass pharmacological, non-pharmacological, and surgical interventions. Traditional pharmacological treatment consists of administering drugs (MAOIs, DA, and levodopa), while emerging evidence explores the potential of antidiabetic agents for neuroprotection and gene therapy for attenuating parkinsonian symptoms. Non-pharmacological treatments, such as exercise, a calcium-rich diet, and adequate vitamin D supplementation, aim to slow disease progression and prevent complications. For those patients who have medically induced side effects and/or refractory symptoms, surgery is a therapeutic option. Deep brain stimulation is the primary surgical option, associated with motor symptom improvement. Levodopa/carbidopa intestinal gel infusion through percutaneous endoscopic gastrojejunostomy and a portable infusion pump succeeded in reducing "off" time, where non-motor and motor symptoms occur, and increasing "on" time. This article aims to address the general aspects of PD and to provide a comparative comprehensive review of the conventional and the latest therapeutic advancements and emerging treatments for PD. Nevertheless, further studies are required to optimize treatment and provide suitable alternatives.

[Approach to refractory gastroesophageal reflux disease]. / Abordaje de la enfermedad por reflujo gastroesofágico refractaria.

An adequate approach to refractory gastroesophageal reflux disease (rGERD) is essential for achieving therapeutic success. From the precise definition of rGERD to the detailed characterization of its phenotypes, it will pave the way for the customization of optimal therapy for each patient. In this narrative literature review, the aim is to provide an updated synthesis of the utility of various diagnostic tools and explore the wide range of therapeutic options, both medical and invasive, available for this condition.

Irritable bowel syndrome in medical students from Lima, Peru, during the COVID-19 pandemic, using virtual questionnaires according to the Rome IV criteria: prevalence and associated factors.

Irritable bowel syndrome (IBS) is a functional gastrointestinal (GI) disorder, characterized by changes of the intestinal habit associated with abdominal pain. This study analyzed factors associated with this pathology during the COVID-19 pandemic, and it was seen that the impact of IBS was higher in young women who had ongoing studies in the medical field. OBJECTIVES: Determine the prevalence of IBS in medical students and explore the associated factors with the increase of its symptoms, through the use of digital tools. MATERIALS AND METHODS: Descriptive observational study with exploration of associations, with non-probabilistic sampling, until reaching the minimum sample of 110 participants with a confidence interval of 95%, finally having a total sample of 131 students, distributed in 3 proportional and representative subgroups of the last 3 years of study of the medical career from a university from Lima, Peru. The inclusion criteria were students of both sexes and legal age, who gave their consent to participate and did not have risk factors for GI disease. Once the study was approved, the link of a validated virtual questionnaire was shared through the institutional email. To explore the associated factors, the Chi-square test was used with a statistical significance of p < 0.05. RESULTS: Responses were obtained from 195 students; 64 (32.82%) of them had at least one exclusion criteria, finally having a sample size of 131; 64 (48.85%) were women, and 52 (39.69%) were from 7th year. Using the Rome IV criteria, 23 participants were classified as having IBS, which indicates a prevalence of 17.56%, 14 (60.87%) of them were women and 10 (43.48%) were from the 7th year (last year of study of the medical career in Peru). It was observed that 1 in 3 students (32.06%) had chronic abdominal pain, and 1 in 2 (53.44%) reported having a history of Major Depression or Generalized Anxiety Disorder. Of the total, 51 (38.93%) had a history of having a positive COVID-19 test result. Regarding the associated factors, a significant association was only found between IBS and the diarrhea type, classified according to the Bristol Scale. CONCLUSIONS: The prevalence of IBS in medical students was 17.56%, the highest compared with studies in the general population and in medical students using Rome IV criteria. Of the associated factors, the clinical presentation of diarrhea, according to the Bristol scale, was associated with IBS.

Pigmentation abnormalities in Coffin-Siris syndrome.

Pigmentary abnormalities in Coffin-Siris Syndrome should be considered as part of the wide phenotypical spectrum of this patients.

How do I manage a blood product shortage?

BACKGROUND: The demand for blood products sometimes exceeds the available inventory. Blood product inventories are dependent upon the availability of donors, supplies and reagents, and collection staff. During prolonged extreme shortages, blood centers and transfusion services must alter practices to meet the needs of patients. STUDY DESIGN AND METHODS: The Association for the Advancement of Blood and Biotherapies Donor and Blood Component Management Subsection compiled some strategies from its blood center and hospital transfusion service members that could be implemented during blood product shortages. RESULTS: Some strategies that blood centers could use to increase their available inventories include increasing donor recruitment efforts, using alternate types of collection kits, manufacturing low-yield apheresis-derived platelets and/or whole blood-derived platelets, using cold-stored platelets, transferring inventory internally among centers of the same enterprise, using frozen inventory, decreasing standing order quantities, prioritizing allocation to certain patient populations, filling partial orders, and educating customers and blood center staff. Transfusion service strategies that could be implemented to maximize the use of the limited available inventory include increasing patient blood management efforts, using split units, finding alternate blood suppliers, trading blood products with other hospital transfusion services, developing a patient priority list, assembling a hospital committee to decide on triaging priorities, using expired products in extreme situations, and accepting nonconforming products after performing safety checks. DISCUSSION: Blood centers and transfusion services must choose the appropriate strategies to implement based on their needs.

Health utility in community-dwelling adults with dysvascular lower limb loss.

PURPOSE: Adults with dysvascular lower extremity amputation (LEA) experience a large number of secondary health conditions yet there is a gap in the literature on health utility scores for this population. A health utility score relates to a person's state of well-being, and is a single metric anchored at 0 (death) and 1 (perfect health). This study aimed to provide a descriptive account of health utility scores in community-dwelling adults with dysvascular LEA. METHODS: Participants were adults with dysvascular LEA who were 3 months post-amputation. Data collected included socio-demographic characteristics, the Special Interest Group in Amputee Medicine (SIGAM) grades, the dysvascular conditions scale (DCS), which is a scale developed for this study, and the Short Form-36 (SF-36). SF-6D health utility scores were derived from the SF-36 using a software algorithm. Participants were grouped into low-impact and high-impact groups based on self-reported severity of symptoms using the DCS. Health utility scores were compared between the low-impact and high-impact groups using independent t-tests. RESULTS: A total of 231 participants were enrolled in the study. The mean SF-6D health utility score was 0.689 (0.127). A significant association was found between health utility score and SIGAM grade (p < 0.001, η2 = .09). Health utility was positively associated with age (r = 0.137, p = 0.037) and months post-amputation (r = 0.141, p = 0.032), and negatively associated with DCS severity (r = -0.526, p < 0.001). Health utility scores were lower for participants in the DCS high-impact groups for conditions such as diabetes mellitus, phantom limb pain, musculoskeletal pain, back pain, psychological distress, depression, vision problems, and other pain. CONCLUSION: Cost-utility analyses rely on health utility estimates and our findings provide data for future economic evaluations that may assist policy makers in evidence informed allocation of healthcare resources for this population.

[Generation of a predictive test for the diagnosis of the etiology of variceal upper gastrointestinal bleeding]. / Generación de un test predictivo para el diagnóstico de la etiología de la hemorragia digestiva alta variceal.

Our objective was to develop a diagnostic test to predict the etiology of Variceal Upper Gastrointestinal Bleeding (VUGIB). We conducted a retrospective cohort study. Medical records of patients over 18 years of age with Upper Gastrointestinal Bleeding (UGIB) who attended the emergency service of Hospital Cayetano Heredia (HCH) in Lima-Peru between 2019 and 2022 were reviewed; demographic, laboratory and clinical data were collected. Subsequently, predictive variables of variceal upper gastrointestinal bleeding (VUGIB) were identified using multiple logistic regression. Each variable with predictive capacity was assigned a score with a cut-off point and served to build a predictive scale for VUGIB. 197 medical records of patients with UGIB were included, of which 127 (64%) had non-variceal bleeding, and 70 (36%), variceal. Four independent predictors were identified: hematemesis (red vomit) (OR: 4,192, 95% CI: 1.586-11.082), platelet count (OR: 3.786, 95% CI: 1.324-10.826), history of UGIB (OR: 2.634, 95% CI: 1.017-6.820), signs of chronic liver disease (OR: 11.244, 95% CI: 3.067-35.047), with which a predictive scale was constructed, with a cut-off point >7 and ≤7; which showed a sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, and negative of 58.6%, 90.6%, 77.4%, 79.9%, 6.20, and 0.46 respectively. In conclusion, the predictive scale with a cut-off point >7 is useful for predicting the presence of VUGIB in patients who attend the emergency room for UGIB.

Frequency of functional esophageal disorders in patients with refractory reflux symptoms in Lima, Peru.

Gastroesophageal reflux disease (GERD) is a clinical condition in which gastric reflux causes symptoms or damage to the esophageal mucosa. It is managed with proton pump inhibitors, however, up to 45% of patients with suspected GERD are refractory to treatment. It is necessary to establish a true GERD diagnosis by means of a digestive endoscopy, which does not show lesions in approximately 70% of patients. In this scenario, it is necessary to perform an esophageal pH-impedance measurement, a procedure that allows to determine whether exposure to gastric acid is pathological. Of this group, patients with pathological acid exposure are diagnosed as true non-erosive reflux disease (NERD). If, in addition to not presenting esophageal lesions, they have a physiological exposure to gastric acid, they suffer from esophageal hypersensitivity or functional heartburn, which are functional disorders. These require a different approach from that of GERD or NERD, as the symptoms are not due to pathological exposure to gastric acid. The aim was to calculate the frequency of esophageal hypersensitivity and functional heartburn in patients with suspected NERD. This was a cross-sectional study. Data was collected by reviewing pH-impedance and manometry reports, 166 patients were selected. The frequency for functional disorders was 86.15%, being 46.9% for functional heartburn and 39.2% for esophageal hypersensitivity. The frequency of functional disorders was higher than that reported in previous studies. In conclusion, age, psychological conditions, dietary, cultural, ethnic or lifestyle factors inherent to our environment might play important roles in the development of functional disorders.

Glioblastoma Treatment: State-of-the-Art and Future Perspectives.

(1) Background: Glioblastoma is the most frequent and lethal primary tumor of the central nervous system. Through many years, research has brought various advances in glioblastoma treatment. At this time, glioblastoma management is based on maximal safe surgical resection, radiotherapy, and chemotherapy with temozolomide. Recently, bevacizumab has been added to the treatment arsenal for the recurrent scenario. Nevertheless, patients with glioblastoma still have a poor prognosis. Therefore, many efforts are being made in different clinical research areas to find a new alternative to improve overall survival, free-progression survival, and life quality in glioblastoma patients. (2) Methods: Our objective is to recap the actual state-of-the-art in glioblastoma treatment, resume the actual research and future perspectives on immunotherapy, as well as the new synthetic molecules and natural compounds that represent potential future therapies at preclinical stages. (3) Conclusions: Despite the great efforts in therapeutic research, glioblastoma management has suffered minimal changes, and the prognosis remains poor. Combined therapeutic strategies and delivery methods, including immunotherapy, synthetic molecules, natural compounds, and glioblastoma stem cell inhibition, may potentiate the standard of care therapy and represent the next step in glioblastoma management research.

[Antibiotic resistance of Helicobacter pylori in the Peruvian population: a systematic review and meta-analysis of its prevalence in the general population]. / Resistencia antibiótica de Helicobacter pylori en la población peruana: una revisión sistemática y metaanálisis sobre su prevalencia en la población general.

Helicobacter pylori infection is quite common worldwide and is associated with gastric adenocarcinoma. The high rates of resistance found in Latin American countries justify the investigation of local resistance rates, which could improve the therapeutic approach and eradication rates. The objective is to evaluate the prevalence of resistance in Peru of Helicobacter pylori to commonly used antibiotics. All studies in the Peruvian population that revealed rates of antibiotic resistance of Helicobacter pylori were included. A systematic literature search was conducted up to January 2021, using PubMed and other databases. For the group of patient studies with the EUCAST cut-off point, the resistance was as follows: Amoxicillin 14% (95% CI: 6-25), Clarithromycin 43% (95% CI: 30-57), Metronidazole 58% (95% CI: 22-90) and 51% Quinolones (95% CI: 38-64). For the EUCAST cut-off group of sample studies, the following: Amoxicillin 57% (95% CI: 51-63), Clarithromycin 35% (95% CI 30-41), Metronidazole 67% (95% CI: 62-72) and 4% Tetracycline (95% CI: 2-7). A high antibiotic resistance of Helicobacter pylori was found in most of the groups studied, although with heterogeneity between the studies. The rate of resistance to tetracyclines was low and the studies were shown to be homogeneous.

SPI-1 is a missing host-range factor required for replication of the attenuated modified vaccinia Ankara (MVA) vaccine vector in human cells.

Modified vaccinia virus Ankara (MVA) is the leading poxvirus vector for development of vaccines against diverse infectious diseases. This distinction is based on high expression of proteins and good immunogenicity despite an inability to assemble infectious progeny in human cells, which together promote efficacy and safety. Nevertheless, the basis for the host-range restriction is unknown despite past systematic attempts to identify the relevant missing viral gene(s). The search for host-range factors is exacerbated by the large number of deletions, truncations and mutations that occurred during the long passage history of MVA in chicken embryo fibroblasts. By whole genome sequencing of a panel of recombinant host-range extended (HRE) MVAs generated by marker rescue with 40 kbp segments of vaccinia virus DNA, we identified serine protease inhibitor 1 (SPI-1) as one of several candidate host-range factors present in those viruses that gained the ability to replicate in human cells. Electron microscopy revealed that the interruption of morphogenesis in human cells infected with MVA occurred at a similar stage as that of a vaccinia virus strain WR SPI-1 deletion mutant. Moreover, the introduction of the SPI-1 gene into the MVA genome led to more than a 2-log enhancement of virus spread in human diploid MRC-5 cells, whereas deletion of the gene diminished the spread of HRE viruses by similar extents. Furthermore, MRC-5 cells stably expressing SPI-1 also enhanced replication of MVA. A role for additional host range genes was suggested by the restoration of MVA replication to a lower level relative to HRE viruses, particularly in other human cell lines. Although multiple sequence alignments revealed genetic changes in addition to SPI-1 common to the HRE MVAs, no evidence for their host-range function was found by analysis thus far. Our finding that SPI-1 is host range factor for MVA should simplify use of high throughput RNAi or CRISPR/Cas single gene methods to identify additional viral and human restriction elements.

Structure and dynamics of water plastic crystals from computer simulations.

Water has a rich phase diagram with several crystals, as confirmed by experiments. High-pressure and high-temperature water is of interest for Earth's mantle and exoplanetary investigations. It is in this region of the phase diagram of water that new plastic crystal phases of water have been revealed via computer simulations by both classical forcefields and ab initio calculations. However, these plastic phases still remain elusive in experiments. Here, we present a complete characterization of the structure, dynamics, and thermodynamics of the computational plastic crystal phases of water using molecular dynamics and the two-phase thermodynamic method and uncover the interplay between them. The relaxation times of different reorientational correlation functions are obtained for the hypothetical body-centered-cubic and face-centered-cubic plastic crystal phases of water at T = 440 K and P = 8 GPa. Results are compared to a high pressure liquid and ice VII phases to improve the understanding of the plastic crystal phases. Entropy results indicate that the fcc crystal is more stable compared to the bcc structure under the studied conditions.

Endoscopic removal of a toothbrush in a young adult with psychiatricdisorder.

Ingestion of foreign bodies is a relative common situation in the emergency department; however, ingestion of toothbrush is rarely reported in the literature. We present the case of a 27-year-old man with a previous diagnosis of obsessive-compulsive disorder, who presented to the emergency department 17 hours after an ingestion of a toothbrush. We performed an endoscopic removal using a polypectomy snare in the Gastroenterology Department under moderate sedation. No complications were reported in the procedure and the patient was ischarged few hours later. Ingestion of toothbrush is rare in the literature and some authors described techniques using overtube and retractable snares and forceps. Endoscopic removal of a toothbrush under moderate sedation can be a safe and successful procedure. However, if endoscopic removal fails, surgery should be performed.

Adoption trends of point of issue Verax PGD rapid test for bacterial screening of platelets between 2013 and 2018 among hospitals supplied by the American Red Cross and impact on platelet availability.

BACKGROUND: Point-of-issue tests, such as the Verax Pan Genera Detection (PGD) test, can be used to mitigate the occurrence of septic reactions. Little is known about how widespread the adoption of the PGD test is across US hospitals. STUDY DESIGN/METHODS: The Red Cross hemovigilance database was used to determine the numbers of PGD-positive test results between 2013 and 2018. An examination of the impact of a reactive PGD test on product availability was performed for 2018. RESULTS: The number of reported cases doubled, rising from approximately 50 to 100 per year between 2013 and 2018. A total of 475 initially reactive PGD tests during the entire study period were reported, and 7 (1.5%) of these were determined to be true positives. Hospitals surveyed reported testing platelet units once, with no repeat testing if the unit was PGD reactive. Evaluation of the reported PGD reactive cases for apheresis platelets for 2018 (n = 93) resulted in an estimated cost to the blood center of $87,000, which included the labor and material costs of working up the cases and the estimated value of the lost 64 units and co-components. In addition, there was a loss of the opportunity to collect over 85 apheresis platelets during the time the implicated donor was temporarily deferred. CONCLUSIONS: The number of hospital reports of reactive PGD tests has shown modest growth in the past 5 years. The number of initially reactive PGD tests that were confirmed was minimal. Blood centers can incur cost and platelet inventory loss from initially false-positive test results.

Minimal Clinically Important Differences in Functional Independence After a Knowledge Translation Intervention in Stroke Rehabilitation.

OBJECTIVE: To examine the odds of persons with stroke achieving minimal clinically important difference (MCID) in functional independence during inpatient rehabilitation relative to cognitive impairment (CI) severity, site, and rehabilitation teams' exposure to a Cognitive Orientation to daily Occupational Performance (CO-OP) knowledge translation (KT) intervention. DESIGN: A pre-post observational study was conducted using data from a centralized referral system. Our research team implemented a CO-OP KT intervention as part of a larger study aimed at training teams to use the CO-OP approach. SETTING: Five inpatient rehabilitation units. PARTICIPANTS: Cases extracted from a centralized referral system from the 5 participating units. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: The FIM instrument data from 12 months preintervention and 6 months postintervention were analyzed. A logistic regression was performed to determine the odds ratios (ORs) for achieving MCID based on sample cohort (historical control not exposed to CO-OP KT vs post-CO-OP KT intervention), controlling for site and severity of CI. RESULTS: A model that considered the intervention, admission score, CI severity, and site was the best fit for the cases analyzed. Those with severe CI were less likely to achieve FIM total MCID compared to those with no CI (P=<.001; OR=.18; 95% confidence interval, .09-.39). Taking site and CI into account, cases post CO-OP KT intervention were significantly more likely to achieve MCID on FIM motor (P=.048; OR=1.4; 95% confidence interval, 1.00-1.98) than historical controls. CONCLUSION: The CO-OP KT intervention is associated with increased odds of achieving MCID in the FIM motor subscale in inpatient stroke rehabilitation.

Hepatitis A and hepatitis E virus co-infection with right pleural effusion, ascites and acute acalculous cholecystitis. A case report.

Hepatitis A and hepatitis E are the leading causes of acute viral hepatitis in developing countries due to our poor sanitary conditions, both spread by fecal-oral route or through contaminated water and food. Being both self-limiting diseases, they are usually benign but may present with atypical clinical findings. A 32 year-old female with right pleural effusion, ascites and acalculous cholecystitis during the course of HAV and HEV co-infection is reported. Clinical improvement was observed with conservative management. As far as we know, this is the first case described of a patient with these three complications in the background of a hepatitis A virus and hepatitis E virus co-infection.

Surveillance of adverse events in the treatment of drug-resistant tuberculosis: first global report.

The World Health Organization (WHO) recommends that countries implement pharmacovigilance and collect information on active drug safety monitoring (aDSM) and management of adverse events.The aim of this prospective study was to evaluate the frequency and severity of adverse events to anti-tuberculosis (TB) drugs in a cohort of consecutive TB patients treated with new (i.e. bedaquiline, delamanid) and repurposed (i.e. clofazimine, linezolid) drugs, based on the WHO aDSM project. Adverse events were collected prospectively after attribution to a specific drug together with demographic, bacteriological, radiological and clinical information at diagnosis and during therapy. This interim analysis included patients who completed or were still on treatment at time of data collection.Globally, 45 centres from 26 countries/regions reported 658 patients (68.7% male, 4.4% HIV co-infected) treated as follows: 87.7% with bedaquiline, 18.4% with delamanid (6.1% with both), 81.5% with linezolid and 32.4% with clofazimine. Overall, 504 adverse event episodes were reported: 447 (88.7%) were classified as minor (grade 1-2) and 57 (11.3%) as serious (grade 3-5). The majority of the 57 serious adverse events reported by 55 patients (51 out of 57, 89.5%) ultimately resolved. Among patients reporting serious adverse events, some drugs held responsible were discontinued: bedaquiline in 0.35% (two out of 577), delamanid in 0.8% (one out of 121), linezolid in 1.9% (10 out of 536) and clofazimine in 1.4% (three out of 213) of patients. Serious adverse events were reported in 6.9% (nine out of 131) of patients treated with amikacin, 0.4% (one out of 221) with ethionamide/prothionamide, 2.8% (15 out of 536) with linezolid and 1.8% (eight out of 498) with cycloserine/terizidone.The aDSM study provided valuable information, but implementation needs scaling-up to support patient-centred care.

Identification of Poxvirus Genome Uncoating and DNA Replication Factors with Mutually Redundant Roles.

Genome uncoating is essential for replication of most viruses. For poxviruses, the process is divided into two stages: removal of the envelope, allowing early gene expression, and breaching of the core wall, allowing DNA release, replication, and late gene expression. Subsequent studies showed that the host proteasome and the viral D5 protein, which has an essential role in DNA replication, are required for vaccinia virus (VACV) genome uncoating. In a search for additional VACV uncoating proteins, we noted a report that described a defect in DNA replication and late expression when the gene encoding a 68-kDa ankyrin repeat/F-box protein (68k-ank), associated with the cellular SCF (Skp1, cullin1, F-box-containing complex) ubiquitin ligase complex, was deleted from the attenuated modified vaccinia virus Ankara (MVA). Here we showed that the 68k-ank deletion mutant exhibited diminished genome uncoating, formation of DNA prereplication sites, and degradation of viral cores as well as an additional, independent defect in DNA synthesis. Deletion of the 68k-ank homolog of VACV strain WR, however, was without effect, suggesting the existence of compensating genes. By inserting VACV genes into an MVA 68k-ank deletion mutant, we discovered that M2, a member of the poxvirus immune evasion (PIE) domain superfamily and a regulator of NF-κB, and C5, a member of the BTB/Kelch superfamily associated with cullin-3-based ligase complexes, independently rescued the 68k-ank deletion phenotype. Thus, poxvirus uncoating and DNA replication are intertwined processes involving at least three viral proteins with mutually redundant functions in addition to D5.IMPORTANCE Poxviruses comprise a family of large DNA viruses that infect vertebrates and invertebrates and cause diseases of medical and zoological importance. Poxviruses, unlike most other DNA viruses, replicate in the cytoplasm, and their large genomes usually encode 200 or more proteins with diverse functions. About 90 genes may be essential for chordopoxvirus replication based either on their conservation or individual gene deletion studies. However, this number may underestimate the true number of essential functions because of redundancy. Here we show that any one of three seemingly unrelated and individually nonessential proteins is required for the incompletely understood processes of genome uncoating and DNA replication, an example of synthetic lethality. Thus, poxviruses appear to have a complex genetic interaction network that has not been fully appreciated and which will require multifactor deletion screens to assess.