Survival of thermophilic spore-forming bacteria in a 90+ year old milk powder from Ernest Shackelton's Cape Royds Hut in Antarctica

Milk powder taken to Antarctica on Shackelton's British Antarctic Expedition in 1907 was produced in New Zealand by a roller drying process in the first factory in the world dedicated to this process. Thermophilic bacilli are the dominant contaminants of modern spray-dried milk powders and the 1907 milk powder allows a comparison to be made of contaminating strains in roller-dried and spray-dried powders. Samples of milk powder obtained from Shackelton's Hut at Cape Royds had low levels of thermophilic contamination (<500 cfu ml−1) but the two dominant strains (Bacillus licheniformis strain F and Bacillus subtilis) were typical of those found in spray-dried powders. Soil samples from the floor of the hut also contained these strains, whereas soils distant from the hut did not. Differences in the RAPD profiles of isolates from the milk powder and the soils suggest that contamination of the milk from the soil was unlikely. It is significant that the most commonly encountered contaminant strain in modern spray-dried milk (Anoxybacillus flavithermus strain C) was not detected in the 1907 sample

Snakebite envenoming.

Snakebite envenoming is a neglected tropical disease that kills >100,000 people and maims >400,000 people every year. Impoverished populations living in the rural tropics are particularly vulnerable; snakebite envenoming perpetuates the cycle of poverty. Snake venoms are complex mixtures of proteins that exert a wide range of toxic actions. The high variability in snake venom composition is responsible for the various clinical manifestations in envenomings, ranging from local tissue damage to potentially life-threatening systemic effects. Intravenous administration of antivenom is the only specific treatment to counteract envenoming. Analgesics, ventilator support, fluid therapy, haemodialysis and antibiotic therapy are also used. Novel therapeutic alternatives based on recombinant antibody technologies and new toxin inhibitors are being explored. Confronting snakebite envenoming at a global level demands the implementation of an integrated intervention strategy involving the WHO, the research community, antivenom manufacturers, regulatory agencies, national and regional health authorities, professional health organizations, international funding agencies, advocacy groups and civil society institutions

Production, characterization, and antigen specificity of recombinant 62-71-3, a candidate monoclonal antibody for rabies prophylaxis in humans

Rabies kills many people throughout the developing world every year. The murine monoclonal antibody (mAb) 62-71-3 was recently identified for its potential application in rabies postexposure prophylaxis (PEP). The purpose here was to establish a plant-based production system for a chimeric mouse-human version of mAb 62-71-3, to characterize the recombinant antibody and investigate at a molecular level its interaction with rabies virus glycoprotein. Chimeric 62-71-3 was successfully expressed in Nicotiana benthamiana. Glycosylation was analyzed by mass spectroscopy; functionality was confirmed by antigen ELISA, as well as rabies and pseudotype virus neutralization. Epitope characterization was performed using pseudotype virus expressing mutagenized rabies glycoproteins. Purified mAb demonstrated potent viral neutralization at 500 IU/mg. A critical role for antigenic site I of the glycoprotein, as well as for two specific amino acid residues (K226 and G229) within site I, was identified with regard to mAb 62-71-3 neutralization. Pseudotype viruses expressing glycoprotein from lyssaviruses known not to be neutralized by this antibody were the controls. The results provide the molecular rationale for developing 62-71-3 mAb for rabies PEP; they also establish the basis for developing an inexpensive plant-based antibody product to benefit low-income families in developing countries.—Both, L., van Dolleweerd, C., Wright, E., Banyard, A. C., Bulmer-Thomas, B., Selden, D., Altmann, F., Fooks, A. R., Ma, J. K.-C. Production, characterization, and antigen specificity of recombinant 62-71-3, a candidate monoclonal antibody for rabies prophylaxis in humans

Tracking antigen-specific T-cells during clinical tolerance induction in humans.

Allergen immunotherapy presents an opportunity to define mechanisms of induction of clinical tolerance in humans. Significant progress has been made in our understanding of changes in T cell responses during immunotherapy, but existing work has largely been based on functional T cell assays. HLA-peptide-tetrameric complexes allow the tracking of antigen-specific T-cell populations based on the presence of specific T-cell receptors and when combined with functional assays allow a closer assessment of the potential roles of T-cell anergy and clonotype evolution. We sought to develop tools to facilitate tracking of antigen-specific T-cell populations during wasp-venom immunotherapy in people with wasp-venom allergy. We first defined dominant immunogenic regions within Ves v 5, a constituent of wasp venom that is known to represent a target antigen for T-cells. We next identified HLA-DRB1*1501 restricted epitopes and used HLA class II tetrameric complexes alongside cytokine responses to Ves v 5 to track T-cell responses during immunotherapy. In contrast to previous reports, we show that there was a significant initial induction of IL-4 producing antigen-specific T-cells within the first 3-5 weeks of immunotherapy which was followed by reduction of circulating effector antigen-specific T-cells despite escalation of wasp-venom dosage. However, there was sustained induction of IL-10-producing and FOXP3 positive antigen-specific T cells. We observed that these IL-10 producing cells could share a common precursor with IL-4-producing T cells specific for the same epitope. Clinical tolerance induction in humans is associated with dynamic changes in frequencies of antigen-specific T-cells, with a marked loss of IL-4-producing T-cells and the acquisition of IL-10-producing and FOXP3-positive antigen-specific CD4+ T-cells that can derive from a common shared precursor to pre-treatment effector T-cells. The development of new approaches to track antigen specific T-cell responses during immunotherapy can provide novel insights into mechanisms of tolerance induction in humans and identify new potential treatment targets

Bites by exotic snakes reported to the UK National Poisons Information Service 2009-2020

Introduction: Snakebite is recognised as a neglected tropical disease and a cause of substantial morbidity and mortality. Whilst the most medically important snakes are typically native of Asia, Africa, Latin America and Oceania, the possibility of encountering these snakes is no longer limited by geography due to an increasing number of exotic (non-native) snakes being held in captivity. Methods: A retrospective review of snakebite enquiries to the UK National Poisons Information Service (NPIS) between 2009 and 2020. Enquiries about the European adder (Vipera berus) or where the identity of the snake was unknown were excluded. Results: There were 321 exotic snakebites in 300 patients involving 68 different species during this period. Ten patients were bitten on more than one occasion. The majority of patients (64.5%) were male. Most bites were inflicted by snakes of the family Colubridae (184/321, 57.3%); seventeen bites resulted in moderate symptoms (predominantly swelling of the bitten limb). There were 30 (9.3%) bites by Viperidae and 14 (4.3%) bites by Elapidae. All severe cases (n = 15) resulted from bites by either Viperidae (n = 10) or Elapidae (n = 5). Antivenom was given in 17 cases. One fatality was recorded. Conclusions: Despite their low incidence, exotic snakebites present a substantial challenge for UK healthcare professionals. Although rare, these bites typically occur in individuals (usually male) who keep snakes as part of their occupation or hobby and are therefore at risk of multiple bites. Bites can result in venom hypersensitisation and the risk of venom-induced anaphylaxis. Rapid access to expert clinical advice is available in the UK on a 24-hour basis through the National Poisons Information Service and is strongly recommended in all cases of exotic snakebite

BHPR research: qualitative1. Complex reasoning determines patients' perception of outcome following foot surgery in rheumatoid arhtritis

Background: Foot surgery is common in patients with RA but research into surgical outcomes is limited and conceptually flawed as current outcome measures lack face validity: to date no one has asked patients what is important to them. This study aimed to determine which factors are important to patients when evaluating the success of foot surgery in RA Methods: Semi structured interviews of RA patients who had undergone foot surgery were conducted and transcribed verbatim. Thematic analysis of interviews was conducted to explore issues that were important to patients. Results: 11 RA patients (9 ♂, mean age 59, dis dur = 22yrs, mean of 3 yrs post op) with mixed experiences of foot surgery were interviewed. Patients interpreted outcome in respect to a multitude of factors, frequently positive change in one aspect contrasted with negative opinions about another. Overall, four major themes emerged. Function: Functional ability & participation in valued activities were very important to patients. Walking ability was a key concern but patients interpreted levels of activity in light of other aspects of their disease, reflecting on change in functional ability more than overall level. Positive feelings of improved mobility were often moderated by negative self perception ("I mean, I still walk like a waddling duck”). Appearance: Appearance was important to almost all patients but perhaps the most complex theme of all. Physical appearance, foot shape, and footwear were closely interlinked, yet patients saw these as distinct separate concepts. Patients need to legitimize these feelings was clear and they frequently entered into a defensive repertoire ("it's not cosmetic surgery; it's something that's more important than that, you know?”). Clinician opinion: Surgeons' post operative evaluation of the procedure was very influential. The impact of this appraisal continued to affect patients' lasting impression irrespective of how the outcome compared to their initial goals ("when he'd done it ... he said that hasn't worked as good as he'd wanted to ... but the pain has gone”). Pain: Whilst pain was important to almost all patients, it appeared to be less important than the other themes. Pain was predominately raised when it influenced other themes, such as function; many still felt the need to legitimize their foot pain in order for health professionals to take it seriously ("in the end I went to my GP because it had happened a few times and I went to an orthopaedic surgeon who was quite dismissive of it, it was like what are you complaining about”). Conclusions: Patients interpret the outcome of foot surgery using a multitude of interrelated factors, particularly functional ability, appearance and surgeons' appraisal of the procedure. While pain was often noted, this appeared less important than other factors in the overall outcome of the surgery. Future research into foot surgery should incorporate the complexity of how patients determine their outcome Disclosure statement: All authors have declared no conflicts of interes

Oral abstracts 3: RA Treatment and outcomesO13. Validation of jadas in all subtypes of juvenile idiopathic arthritis in a clinical setting

Background: Juvenile Arthritis Disease Activity Score (JADAS) is a 4 variable composite disease activity (DA) score for JIA (including active 10, 27 or 71 joint count (AJC), physician global (PGA), parent/child global (PGE) and ESR). The validity of JADAS for all ILAR subtypes in the routine clinical setting is unknown. We investigated the construct validity of JADAS in the clinical setting in all subtypes of JIA through application to a prospective inception cohort of UK children presenting with new onset inflammatory arthritis. Methods: JADAS 10, 27 and 71 were determined for all children in the Childhood Arthritis Prospective Study (CAPS) with complete data available at baseline. Correlation of JADAS 10, 27 and 71 with single DA markers was determined for all subtypes. All correlations were calculated using Spearman's rank statistic. Results: 262/1238 visits had sufficient data for calculation of JADAS (1028 (83%) AJC, 744 (60%) PGA, 843 (68%) PGE and 459 (37%) ESR). Median age at disease onset was 6.0 years (IQR 2.6-10.4) and 64% were female. Correlation between JADAS 10, 27 and 71 approached 1 for all subtypes. Median JADAS 71 was 5.3 (IQR 2.2-10.1) with a significant difference between median JADAS scores between subtypes (p < 0.01). Correlation of JADAS 71 with each single marker of DA was moderate to high in the total cohort (see Table 1). Overall, correlation with AJC, PGA and PGE was moderate to high and correlation with ESR, limited JC, parental pain and CHAQ was low to moderate in the individual subtypes. Correlation coefficients in the extended oligoarticular, rheumatoid factor negative and enthesitis related subtypes were interpreted with caution in view of low numbers. Conclusions: This study adds to the body of evidence supporting the construct validity of JADAS. JADAS correlates with other measures of DA in all ILAR subtypes in the routine clinical setting. Given the high frequency of missing ESR data, it would be useful to assess the validity of JADAS without inclusion of the ESR. Disclosure statement: All authors have declared no conflicts of interest. Table 1Spearman's correlation between JADAS 71 and single markers DA by ILAR subtype ILAR Subtype Systemic onset JIA Persistent oligo JIA Extended oligo JIA Rheumatoid factor neg JIA Rheumatoid factor pos JIA Enthesitis related JIA Psoriatic JIA Undifferentiated JIA Unknown subtype Total cohort Number of children 23 111 12 57 7 9 19 7 17 262 AJC 0.54 0.67 0.53 0.75 0.53 0.34 0.59 0.81 0.37 0.59 PGA 0.63 0.69 0.25 0.73 0.14 0.05 0.50 0.83 0.56 0.64 PGE 0.51 0.68 0.83 0.61 0.41 0.69 0.71 0.9 0.48 0.61 ESR 0.28 0.31 0.35 0.4 0.6 0.85 0.43 0.7 0.5 0.53 Limited 71 JC 0.29 0.51 0.23 0.37 0.14 -0.12 0.4 0.81 0.45 0.41 Parental pain 0.23 0.62 0.03 0.57 0.41 0.69 0.7 0.79 0.42 0.53 Childhood health assessment questionnaire 0.25 0.57 -0.07 0.36 -0.47 0.84 0.37 0.8 0.66 0.4

Snakebite mortality in India: a nationally representative mortality survey

Background India has long been thought to have more snakebites than any other country. However, inadequate hospital-based reporting has resulted in estimates of total annual snakebite mortality ranging widely from about 1,300 to 50,000. We calculated direct estimates of snakebite mortality from a national mortality survey. Methods and Findings We conducted a nationally representative study of 123,000 deaths from 6,671 randomly selected areas in 2001–03. Full-time, non-medical field workers interviewed living respondents about all deaths. The underlying causes were independently coded by two of 130 trained physicians. Discrepancies were resolved by anonymous reconciliation or, failing that, by adjudication. A total of 562 deaths (0.47% of total deaths) were assigned to snakebites. Snakebite deaths occurred mostly in rural areas (97%), were more common in males (59%) than females (41%), and peaked at ages 15–29 years (25%) and during the monsoon months of June to September. This proportion represents about 45,900 annual snakebite deaths nationally (99% CI 40,900 to 50,900) or an annual age-standardised rate of 4.1/100,000 (99% CI 3.6–4.5), with higher rates in rural areas (5.4/100,000; 99% CI 4.8–6.0), and with the highest state rate in Andhra Pradesh (6.2). Annual snakebite deaths were greatest in the states of Uttar Pradesh (8,700), Andhra Pradesh (5,200), and Bihar (4,500). Conclusions Snakebite remains an underestimated cause of accidental death in modern India. Because a large proportion of global totals of snakebites arise from India, global snakebite totals might also be underestimated. Community education, appropriate training of medical staff and better distribution of antivenom, especially to the 13 states with the highest prevalence, could reduce snakebite deaths in India

Randomised controlled double-blind non-inferiority trial of two antivenoms for saw-scaled or carpet viper (Echis ocellatus) envenoming in Nigeria.

BACKGROUND: In West Africa, envenoming by saw-scaled or carpet vipers (Echis ocellatus) causes great morbidity and mortality, but there is a crisis in supply of effective and affordable antivenom (ISRCTN01257358). METHODS: In a randomised, double-blind, controlled, non-inferiority trial, "EchiTAb Plus-ICP" (ET-Plus) equine antivenom made by Instituto Clodomiro Picado was compared to "EchiTAb G" (ET-G) ovine antivenom made by MicroPharm, which is the standard of care in Nigeria and was developed from the original EchiTAb-Fab introduced in 1998. Both are caprylic acid purified whole IgG antivenoms. ET-G is monospecific for Echis ocellatus antivenom (initial dose 1 vial) and ET-Plus is polyspecific for E. ocellatus, Naja nigricollis and Bitis arietans (initial dose 3 vials). Both had been screened by pre-clinical and preliminary clinical dose-finding and safety studies. Patients who presented with incoagulable blood, indicative of systemic envenoming by E. ocellatus, were recruited in Kaltungo, north-eastern Nigeria. Those eligible and consenting were randomly allocated with equal probability to receive ET-Plus or ET-G. The primary outcome was permanent restoration of blood coagulability 6 hours after the start of treatment, assessed by a simple whole blood clotting test repeated 6, 12, 18, 24 and 48 hr after treatment. Secondary (safety) outcomes were the incidences of anaphylactic, pyrogenic and late serum sickness-type antivenom reactions. FINDINGS: Initial doses permanently restored blood coagulability at 6 hours in 161/194 (83.0%) of ET-Plus and 156/206 (75.7%) of ET-G treated patients (Relative Risk [RR] 1.10 one-sided 95% CI lower limit 1.01; P = 0.05). ET-Plus caused early reactions on more occasions than did ET-G [50/194 (25.8%) and 39/206 (18.9%) respectively RR (1.36 one-sided 95% CI 1.86 upper limit; P = 0.06). These reactions were classified as severe in 21 (10.8%) and 11 (5.3%) of patients, respectively. CONCLUSION: At these doses, ET-Plus was slightly more effective but ET-G was slightly safer. Both are recommended for treating E. ocellatus envenoming in Nigeria. TRIAL REGISTRATION: Current Controlled Trials ISRCTN01257358