Summary SARS-CoV-2 Spike protein is critical for virus infection via engagement of ACE2 1 , and is a major antibody target. Here we report chronic SARS-CoV-2 with reduced sensitivity to neutralising antibodies in an immune suppressed individual treated with convalescent plasma, generating whole genome ultradeep sequences over 23 time points spanning 101 days. Little change was observed in the overall viral population structure following two courses of remdesivir over the first 57 days. However, following convalescent plasma therapy we observed large, dynamic virus population shifts, with the emergence of a dominant viral strain bearing D796H in S2 and ΔH69/ΔV70 in the S1 N-terminal domain NTD of the Spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype diminished in frequency, before returning during a final, unsuccessful course of convalescent plasma. In vitro , the Spike escape double mutant bearing ΔH69/ΔV70 and D796H conferred modestly decreased sensitivity to convalescent plasma, whilst maintaining infectivity similar to wild type. D796H appeared to be the main contributor to decreased susceptibility but incurred an infectivity defect. The ΔH69/ΔV70 single mutant had two-fold higher infectivity compared to wild type, possibly compensating for the reduced infectivity of D796H. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy associated with emergence of viral variants with evidence of reduced susceptibility to neutralising antibodies.

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Objective To evaluate the effectiveness and costs of a multifaceted flexible educational programme aimed at reducing antibiotic dispensing at the practice level in primary care.Design Randomised controlled trial with general practices as the unit of randomisation and analysis. Clinicians and researchers were blinded to group allocation until after randomisation.Setting 68 general practices with about 480 000 patients in Wales, United Kingdom.Participants 34 practices were randomised to receive the educational programme and 34 practices to be controls. 139 clinicians from the intervention practices and 124 from control practices had agreed to participate before randomisation. Practice level data covering all the clinicians in the 68 practices were analysed.Interventions Intervention practices followed the Stemming the Tide of Antibiotic Resistance (STAR) educational programme, which included a practice based seminar reflecting on the practices’ own dispensing and resistance data, online educational elements, and practising consulting skills in routine care. Control practices provided usual care.Main outcome measures Total numbers of oral antibiotic items dispensed for all causes per 1000 practice patients in the year after the intervention, adjusted for the previous year’s dispensing. Secondary outcomes included reconsultations, admissions to hospital for selected causes, and costs.Results The rate of oral antibiotic dispensing (items per 1000 registered patients) decreased by 14.1 in the intervention group but increased by 12.1 in the control group, a net difference of 26.1. After adjustment for baseline dispensing rate, this amounted to a 4.2% (95% confidence interval 0.6% to 7.7%) reduction in total oral antibiotic dispensing for the year in the intervention group relative to the control group (P=0.02). Reductions were found for all classes of antibiotics other than penicillinase-resistant penicillins but were largest and significant individually for phenoxymethylpenicillins (penicillin V) (7.3%, 0.4% to 13.7%) and macrolides (7.7%, 1.1% to 13.8%). There were no significant differences between intervention and control practices in the number of admissions to hospital or in reconsultations for a respiratory tract infection within seven days of an index consultation. The mean cost of the programme was £2923 (€3491, $4572) per practice (SD £1187). There was a 5.5% reduction in the cost of dispensed antibiotics in the intervention group compared with the control group (−0.4% to 11.4%), equivalent to a reduction of about £830 a year for an average intervention practice.Conclusion The STAR educational programme led to reductions in all cause oral antibiotic dispensing over the subsequent year with no significant change in admissions to hospital, reconsultations, or costs.Trial registration ISRCT No 63355948.

One hundred methicillin-resistant Staphylococcus aureus (MRSA) strains, isolated between 1983 and 1999, were tested alongside the vancomycin hetero-resistant S. aureus (hVRSA) strain Mu 3, and vancomycin-resistant S. aureus (VRSA) strain Mu 50, for their resistance to vancomycin. This was achieved using the screening method described by Hiramatsu, gradient plates, agar incorporation, standard Etest, macrodilution Etest and a modified population analysis. Using Hiramatsu's screening method, 5% of the 100 MRSA were identified as VRSA and 5% identified as hVRSA, the gradient plates identified 7% hVRSA, and the standard and macrodilution Etests identified no hVRSA. Mu 3 appeared to be vancomycin-susceptible using both the agar incorporation and standard Etest methods, but was classified as hVRSA using the macrodilution Etest. The modified population analysis reliably detected vancomycin hetero-resistance in Mu 3 and identified no hVRSAs within the 100 MRSA sample.

The sensitivity and specificity of seven methods (agar dilution, broth microdilution, Etest at 0.5 and 2.0 McFarland (McF) inocula, two agar screening methods, and population studies [PS]) were evaluated in a double-blind study involving 284 methicillin-resistant Staphylococcus aureus (MRSA) strains and 45 Staphylococcus strains with reduced susceptibilities to vancomycin (SRSV). The results were compared to the population analysis profile-area under the curve ratio method (PAP-AUC ratio compared to that of Mu3) as described by Wootton et al. The agar screening method using brain heart infusion agar (6 g of vancomycin per ml) gave a sensitivity of 22% and a specificity of 97%. A similar method using Mueller-Hinton agar (5 g of vancomycin per ml) gave a sensitivity of 20% and a specificity of 99%. The PS method detected 34 false positives (12%) and gave a sensitivity of 71% and a specificity of 88%. Etest using 0.5 and 2.0 McF inocula gave sensitivities and specificities of 82 and 93% and of 96 and 97%, respectively. The best Etest interpretative criteria for the 2.0 McF inoculum was >8 mg of vancomycin per liter and >8 g teicoplanin per ml or >12 g of teicoplanin per ml. The direct colony suspension inoculum for this method was found to be equally accurate in detecting (hetero-) glycopeptide-intermediate S. aureus compared to the overnight broth inoculum preparation method. Agar dilution and broth microdilution using the NCCLS breakpoint criteria for vancomycin gave sensitivities and specificities of 20 and 100% and of 11 and 100%, respectively. Using the Etest with a 2.0 McF inoculum, six different media were assessed against a selection of SRSV (n ‫؍‬ 48) and MRSA (n ‫؍‬ 12). Brain heart infusion agar yielded the highest sensitivity and specificity values: 88 and 88%, respectively.

eThe uncontrolled, often inappropriate use of antibiotics has resulted in the increasing prevalence of antibiotic-resistant pathogens, with major cost implications for both United States and European health care systems. We describe the utilization of a lowmolecular-weight oligosaccharide nanomedicine (OligoG), based on the biopolymer alginate, which is able to perturb multidrug-resistant (MDR) bacteria by modulating biofilm formation and persistence and reducing resistance to antibiotic treatment, as evident using conventional and robotic MIC screening and microscopic analyses of biofilm structure. OligoG increased (up to 512-fold) the efficacy of conventional antibiotics against important MDR pathogens, including Pseudomonas, Acinetobacter, and Burkholderia spp., appearing to be effective with several classes of antibiotic (i.e., macrolides, ␤-lactams, and tetracyclines). Using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM), increasing concentrations (2%, 6%, and 10%) of alginate oligomer were shown to have a direct effect on the quality of the biofilms produced and on the health of the cells within that biofilm. Biofilm growth was visibly weakened in the presence of 10% OligoG, as seen by decreased biomass and increased intercellular spaces, with the bacterial cells themselves becoming distorted and uneven due to apparently damaged cell membranes. This report demonstrates the feasibility of reducing the tolerance of wound biofilms to antibiotics with the use of specific alginate preparations.

The emergence of Staphylococcus aureus resistant to vancomycin has caused considerable concern. Such strains are currently rare, although they have been isolated from many areas of the world. Considerable controversy surrounds strains of S. aureus displaying heterogeneous resistance to vancomycin regarding their definition and methods for detection. This has led to considerable variance in estimates of prevalence (0-1.3%-20% in Japan) and has hindered efforts to define the clinical relevance of these strains. The mechanism of resistance involves a complex reorganization of cell wall metabolism, leading to a grossly thickened cell wall with reduced peptidoglycan cross-linking. There may be many different ways in which strains achieve this endpoint. Current knowledge and theories are summarized.

Glycopeptide-intermediate Staphylococcus aureus (GISA) and heterogeneous GISA (hGISA) strains are notoriously difficult to detect in the diagnostic laboratory. The clinical importance of GISA, and particularly hGISA, will only be obvious when a definitive detection method is available. A few novel GISA and hGISA detection methods have been proposed; however, their validity has never been tested on a significant scale and in different laboratories. This study compares three screening methods for detecting GISA and hGISA strains in 12 laboratories, using a blind panel of 48 strains with known glycopeptide susceptibilities. The three screening methods used were brain heart infusion agar with 6 mg/liter vancomycin (BHIA6V) (CDC/CLSI), Mueller-Hinton agar with 5 mg/liter teicoplanin (MHA5T) (European Antimicrobial Resistance Surveillance System [EARSS]), and the macrodilution method Etest (MET) (EARSS), with population analysis profile-area under the curve analysis as the gold standard. Sensitivity and specificity were highest for MHA5T and MET, which identified 82.5% and 85.9% of strains, respectively. BHIA6V had poor sensitivity, particularly for hGISA (11.5% of strains were detected), and gave the largest interlaboratory variation in performance. MET exhibited the least interlaboratory variation. It is essential that laboratories use appropriate methods to detect GISA/ hGISA strains so that the prevalence and clinical importance of these strains can be assessed properly.