An assessment of the airborne longevity of group A Streptococcus.

Group A streptococcus (GAS) infections result in more than 500 000 deaths annually. Despite mounting evidence for airborne transmission of GAS, little is known about its stability in aerosol. Measurements of GAS airborne stability were carried out using the Controlled Electrodynamic Levitation and Extraction of Bioaerosols onto a Substrate (CELEBS) instrument. CELEBS measurements with two different isolates of GAS suggest that it is aerostable, with approximately 70 % of bacteria remaining viable after 20 min of levitation at 50 % relative humidity (RH), with lower survival as RH was reduced. GAS airborne viability loss was driven primarily by desiccation and efflorescence (i.e. salt crystallization), with high pH also potentially playing a role, given reduced survival in bicarbonate containing droplet compositions. At low enough RH for efflorescence to occur, a greater proportion of organic components in the droplet appeared to protect the bacteria from efflorescence. These first insights into the aerosol stability of GAS indicate that airborne transmission of these respiratory tract bacteria may occur, and that both the composition of the droplet containing the bacteria, and the RH of the air affect the duration of bacterial survival in this environment. Future studies will explore a broader range of droplet and air compositions and include a larger selection of GAS strains.

Use of saliva-based qPCR diagnostics for the accurate, rapid, and inexpensive detection of strep throat.

OBJECTIVES: Outpatient health care facilities are essential for quickly diagnosing common infectious diseases such as bacterial and viral pharyngitis. The only form of pharyngitis requiring antibiotics is strep throat (ST); however, antibiotic prescription rates are much higher than ST prevalence, suggesting antibiotics are being inappropriately prescribed. Current rapid ST diagnostics may be contributing to this problem due to the low sensitivity and variable specificity of these tests. It is best practice to verify a negative ST diagnosis with a group A Streptococcus (GAS) culture, but many clinics do not perform this test due to the additional cost and 24-72 h required to obtain results. This indicates there is great need for more accurate rapid diagnostic tools in outpatient facilities. We hypothesized that next generation qPCR technology could be adapted to detect GAS DNA from saliva samples (instead of the traditional throat swab) by creating a simple, fast, and inexpensive protocol. METHODS: Saliva specimens collected from patients at James Madison University Health Center were used to test the effectiveness of our Chelex 100-based rapid DNA extraction method, followed by a fast protocol developed for the Open qPCR machine to accurately detect ST. RESULTS: Our final saliva processing and qPCR protocol required no specialized training to perform and was able to detect ST with 100 % sensitivity and 100 % specificity (n=102) in 22-26 min, costing only $1.12 per sample. CONCLUSIONS: Saliva can be rapidly analyzed via qPCR for the accurate and inexpensive detection of ST.

Assessment of changes in Streptococcus pyogenes levels using N-acetylgalactosamine-6-sulfatase marker and pharyngeal airway space with appliance therapy in mouth breathers - An ELISA-based study.

Background: The frequency of adenotonsillar hypertrophy in mouth-breathing children when compared to the average found in the general population is considered to be higher. Mouth breathing is considered as one of the causative factors for tonsillitis in children. Through continuous irritation on tonsillar wall, tonsils swell up and inflammation develops. Purpose: The purpose of the study is to evaluate Streptococcus pyogenes count using colony-forming units (CFUs) and N-acetylgalactosamine-6-sulfatase side chain marker on ELISA (enzyme linked immunosorbent assay) in mouth breathers and to establish its correlation with pharyngeal airway space pre- and post-oral screen appliance therapy. Materials and Methods: A total number of 24 (n) mouth breathers aged between 5 and 12 years were included in the study and given oral screen appliance therapy. The subjects were evaluated for the various parameters before the delivery of a habit-breaking appliance and then reevaluated for the same parameters (presence of S. pyogenes and its counts, size of tonsils, and pharyngeal airway space dimensions) after 6 months of appliance usage. Results: A statistically significant difference was seen in levels of S. pyogenes using ELISA and CFUs. Furthermore, statistically significant difference was observed in Friedman tonsil scoring and pharyngeal airway space and pre- and post-oral screen appliance therapy. Conclusion: Oral screen appliance therapy reduced the frequency of occurrence of tonsillitis in mouth breathers by decreasing the counts of S. pyogenes bacteria. Upper and lower pharyngeal airway space dimensions were increased after 6 months of appliance therapy in mouth breathers.

A pilot study to develop assessment tools for Group A Streptococcus surveillance studies.

Introduction: Group A Streptococcus (GAS) causes pharyngitis (sore throat) and impetigo (skin sores) GAS pharyngitis triggers rheumatic fever (RF) with epidemiological evidence supporting that GAS impetigo may also trigger RF in Australian Aboriginal children. Understanding the concurrent burden of these superficial GAS infections is critical to RF prevention. This pilot study aimed to trial tools for concurrent surveillance of sore throats and skins sore for contemporary studies of RF pathogenesis including development of a sore throat checklist for Aboriginal families and pharynx photography. Methods: Yarning circle conversations and semi-structured interviews were performed with Aboriginal caregivers and used to develop the language and composition of a sore throat checklist. The sore throat story checklist was combined with established methods of GAS pharyngitis and impetigo surveillance (examination, bacteriological culture, rapid antigen detection and serological tests) and new technologies (photography) and used for a pilot cross-sectional surveillance study of Aboriginal children attending their health clinic for a routine appointment. Feasibility, acceptability, and study costs were compiled. Results: Ten Aboriginal caregivers participated in the sore-throat yarning circles; a checklist was derived from predominant symptoms and their common descriptors. Over two days, 21 Aboriginal children were approached for the pilot surveillance study, of whom 17 were recruited; median age was 9 years [IQR 5.5-13.5], 65% were female. One child declined throat swabbing and three declined finger pricks; all other surveillance elements were completed by each child indicating high acceptability of surveillance assessments. Mean time for screening assessment was 19 minutes per child. Transport of clinical specimens enabled gold standard microbiological and serological testing for GAS. Retrospective examination of sore throat photography concorded with assessments performed on the day. Conclusion: Yarning circle conversations were effective in deriving culturally appropriate sore throat questionnaires for GAS pharyngitis surveillance. New and established tools were feasible, practical and acceptable to participants and enable surveillance to determine the burden of superficial GAS infections in communities at high risk of RF. Surveillance of GAS pharyngitis and impetgio in remote Australia informs primary RF prevention with potential global translation.

A Systematic Framework for Prioritizing Burden of Disease Data Required for Vaccine Development and Implementation: The Case for Group A Streptococcal Diseases.

Vaccine development and implementation decisions need to be guided by accurate and robust burden of disease data. We developed an innovative systematic framework outlining the properties of such data that are needed to advance vaccine development and evaluation, and prioritize research and surveillance activities. We focus on 4 objectives-advocacy, regulatory oversight and licensure, policy and post-licensure evaluation, and post-licensure financing-and identify key stakeholders and specific requirements for burden of disease data aligned with each objective. We apply this framework to group A Streptococcus, a pathogen with an underrecognized global burden, and give specific examples pertinent to 8 clinical endpoints. This dynamic framework can be adapted for any disease with a vaccine in development and can be updated as vaccine candidates progress through clinical trials. This framework will also help with research and innovation priority setting of the Immunization Agenda 2030 (IA2030) and accelerate development of future vaccines.

Design, molecular docking study of synthesised N-heteroaryl substituted gallamide derivatives and their antibacterial assessment.

A series of N-heteroaryl substituted Gallamide derivatives 3a-3g were synthesised and the obtained structures were further confirmed by different spectral studies. For in-vitro antibacterial activity, the synthesised compounds were evaluated against three UTI (Urinary Tract Infection) bacterial strains including Staphylococcus aureus, Escherichia coli, and Streptococcus pyogenes. Furthermore, the designed compounds were docked with bacterial DNA gyrase and dihydropteroate synthase. All the compounds had shown good inhibition against S. aureus whereas compound 3e has produced significant inhibition at 28 and 26 mm against S.aureus and E.coli, respectively. The MIC value of the conjugate 3e and 3d was 3.12 and 6.25 µg/mL against S. aureus andE.coli, respectively. Compound 3,4,5-trihydroxy-N-(4-(N-(5-methyl isoxazol-3-yl) sulfamoyl) phenyl)benzamide 3d had shown the highest binding energy against both the targets along with good antibacterial action.

Health-Economic Value of Vaccination Against Group A Streptococcus in the United States.

BACKGROUND: Vaccines are needed to reduce the burden of group A Streptococcus (GAS). We assessed the potential health-economic value of GAS vaccines achievable through prevention of invasive disease and acute upper respiratory infections in the United States. METHODS: We estimated annual incidence of invasive GAS disease and associated costs incurred from hospitalization and management of long-term sequelae, as well as productivity losses resulting from acute illness, long-term disability, and mortality. We also estimated healthcare and productivity costs associated with GAS pharyngitis, sinusitis, and acute otitis media. We estimated costs averted by prevention of invasive disease and acute upper respiratory infections for vaccines with differing efficacy profiles; our base case considered vaccines meeting the World Health Organization Preferred Product Profile (WHO-PPP) with a 6-year average duration of protection. RESULTS: Costs of invasive GAS disease and acute upper respiratory infections totaled $6.08 (95% confidence interval [CI], $5.33-$6.86) billion annually. Direct effects of vaccines meeting WHO-PPP characteristics and administered at ages 12 and 18 months would avert $609 (95% CI, $558-$663) million in costs annually, primarily by preventing noninvasive disease; with an additional dose at age 5 years, averted costs would total $869 (95% CI, $798-$945) million annually. Adult vaccination at age 65 years would avert $326 (95% CI, $271-$387) million in annual costs associated with invasive GAS disease. Indirect effects of vaccination programs reducing incidence of GAS diseases across all ages by 20% would avert roughly $1 billion in costs each year. CONCLUSIONS: The economic burden of GAS is substantial. Our findings should inform prioritization of GAS vaccine development and evaluation.

Burden of Group A Streptococcal Pharyngitis, Rheumatic Fever, and Rheumatic Heart Disease in India: A Systematic Review and Meta-Analysis.

OBJECTIVE: To estimate the burden of group A streptococcal pharyngitis (GAS) pharyngitis, rheumatic fever (RF), and rheumatic heart disease (RHD) in India using existing data sources, as well as to recognize the most serious gaps in GAS disease burden data. METHODS: Four electronic databases-PubMed, Scopus, Embase, and Web of Science were searched using a comprehensive search strategy. Data were identified primarily from observational studies including school surveys, community-based and hospital-based studies. The standard methodological procedures as per Cochrane guidelines were used. Eligible studies were pooled for estimating prevalence, incidence, and case fatality rate using R software version 3.3.3. The protocol was registered with PROSPERO; registration number CRD42018075742. RESULTS: The pooled prevalence of GAS pharyngitis among asymptomatic children and pharyngitis cases aged 5 to 15 y was estimated as 2.79 percent [95% Confidence interval (CI): 1.58-4.89] and 13 percent (95% CI: 3.18-41.97), respectively. The prevalence rate of rheumatic fever was found to be 0.04% (95% CI: 0.01-0.17). The pooled prevalence rate of RHD among children aged 5-15 y using clinical auscultation and echocardiography was estimated as 0.36 percent (95% CI: 0.02-7.52) and 0.28 percent (95% CI: 0.08-1.03), respectively. CONCLUSION: The study emphasizes the importance of developing a population-based surveillance framework to track patterns, management strategies, and outcomes in order to develop informed recommendations for launching contextual measures to regulate RF and RHD.

Global patterns of necrotizing soft tissue infections: A systematic review and meta-analysis.

BACKGROUND: Frequency, microbiology, and outcomes of necrotizing soft tissue infections vary based on locoregional and environmental factors; however, there has been no global survey of these patterns. We performed a systematic review/meta-analysis on published reports of necrotizing soft tissue infections from across the globe. METHODS: Peer-reviewed empirical studies examining rates of polymicrobial and monomicrobial necrotizing soft tissue infections with microbial isolation and overall mortality rate were extracted along with geographic location using PubMed, Scopus, ProQuest, and Web of Science. Random-effects meta-analyses and sensitivity analyses were performed, adjusting for publication bias. Meta-regression analyses examined moderator effects of risk factors. RESULTS: One hundred and five studies (8,718 total patients) were included. Pooled prevalence of polymicrobial and monomicrobial infections were 53% and 37.9%, respectively. Truncal necrotizing soft tissue infections were commonly polymicrobial (P < .001), whereas monomicrobial infections prevailed in extremities (P = .008). Global prevalence of monomicrobial necrotizing soft tissue infections was observed to increase by 1.1% annually (P = .003). Staphylococcus aureus was the most common organism globally and in North America, Asia, the Middle East, and Africa, followed by Streptococcus pyogenes and Escherichia coli. Methicillin-resistant S. aureus accounted for 16% of necrotizing soft tissue infections globally. Overall mortality was 23.1%, observed to decline globally over the last decade (P = .020). No regional differences were noted for mortality. CONCLUSION: Although polymicrobial infections remain predominant worldwide, the incidence of monomicrobial infections is increasing. The observed decline in necrotizing soft tissue infection-related mortality is encouraging and may reflect advances in management, despite major variations in available healthcare resources globally.

In-depth assessment of the PAM compatibility and editing activities of Cas9 variants.

A series of Cas9 variants have been developed to improve the editing fidelity or targeting range of CRISPR-Cas9. Here, we employ a high-throughput sequencing approach primer-extension-mediated sequencing to analyze the editing efficiency, specificity and protospacer adjacent motif (PAM) compatibility of a dozen of SpCas9 variants at multiple target sites in depth, and our findings validate the high fidelity or broad editing range of these SpCas9 variants. With regard to the PAM-flexible SpCas9 variants, we detect significantly increased levels of off-target activity and propose a trade-off between targeting range and editing specificity for them, especially for the near-PAM-less SpRY. Moreover, we use a deep learning model to verify the consistency and predictability of SpRY off-target sites. Furthermore, we combine high-fidelity SpCas9 variants with SpRY to generate three new SpCas9 variants with both high fidelity and broad editing range. Finally, we also find that the existing SpCas9 variants are not effective in suppressing genome instability elicited by CRISPR-Cas9 editing, raising an urgent issue to be addressed.

Rheumatic heart disease in Indigenous young peoples.

Indigenous children and young peoples live with an inequitable burden of acute rheumatic fever and rheumatic heart disease. In this Review, we focus on the epidemiological burden and lived experience of these conditions for Indigenous young peoples in Australia, New Zealand, and Canada. We outline the direct and indirect drivers of rheumatic heart disease risk and their mitigation. Specifically, we identify the opportunities and limitations of predominantly biomedical approaches to the primary, secondary, and tertiary prevention of disease among Indigenous peoples. We explain why these biomedical approaches must be coupled with decolonising approaches to address the underlying cause of disease. Initiatives underway to reduce acute rheumatic fever and rheumatic heart disease in Australia, New Zealand, and Canada are reviewed to identify how an Indigenous rights-based approach could contribute to elimination of rheumatic heart disease and global disease control goals.

Immunogenicity Assessment of Cell Wall Carbohydrates of Group A Streptococcus via Self-Adjuvanted Glyco-lipopeptides.

Identifying the immunogenic moieties and their precise structure of carbohydrates plays an important role for developing effective carbohydrate-based subunit vaccines. This study assessed the structure-immunogenicity relationship of carbohydrate moieties of a single repeating unit of group A carbohydrate (GAC) present on the cell wall of group A Streptococcus (GAS) using a rationally designed self-adjuvanted lipid-core peptide, instead of a carrier protein. Immunological evaluation of fully synthetic glyco-lipopeptides (particle size: 300-500 nm) revealed that construct consisting of higher rhamnose moieties (trirhamnosyl-lipopeptide) was able to induce enhanced immunogenic activity in mice, and GlcNAc moiety was not found to be an essential component of immunogenic GAC mimicked epitope. Trirhamnosyl-lipopeptide also showed 75-97% opsonic activity against four different clinical isolates of GAS and was comparable to a subunit peptide vaccine (J8-lipopeptide) which illustrated 65-96% opsonic activity.

The economic and health burdens of diseases caused by group A Streptococcus in New Zealand.

OBJECTIVES: In preparation for the future arrival of a group A Streptococcus (GAS) vaccine, this study estimated the economic and health burdens of GAS diseases in New Zealand (NZ). METHODS: The annual incidence of GAS diseases was based on extrapolation of the average number of primary healthcare episodes managed each year in general practices (2014-2016) and on the average number of hospitalizations occurring each year (2005-2014). Disease incidence was multiplied by the average cost of diagnosing and managing an episode of disease at each level of care to estimate the annual economic burden. RESULTS: GAS affected 1.5% of the population each year, resulting in an economic burden of 29.2 million NZ dollars (2015 prices) and inflicting a health burden of 2373 disability-adjusted life years (DALYs). Children <5 years of age were the most likely age group to present for GAS-related healthcare. Presentations for superficial throat and skin infections (predominantly pharyngitis and impetigo) were more common than other GAS diseases. Cellulitis contributed the most to the total economic and health burdens. Invasive and immune-mediated diseases disproportionately contributed to the total economic and health burdens relative to their frequency of occurrence. CONCLUSION: Preventing GAS diseases would have substantial economic and health benefits in NZ and globally.

Evaluation of Homology-Independent CRISPR-Cas9 Off-Target Assessment Methods.

The ability to alter genomes specifically by CRISPR-Cas gene editing has revolutionized biological research, biotechnology, and medicine. Broad therapeutic application of this technology, however, will require thorough preclinical assessment of off-target editing by homology-based prediction coupled with reliable methods for detecting off-target editing. Several off-target site nomination assays exist, but careful comparison is needed to ascertain their relative strengths and weaknesses. In this study, HEK293T cells were treated with Streptococcus pyogenes Cas9 and eight guide RNAs with varying levels of predicted promiscuity in order to compare the performance of three homology-independent off-target nomination methods: the cell-based assay, GUIDE-seq, and the biochemical assays CIRCLE-seq and SITE-seq. The three methods were benchmarked by sequencing 75,000 homology-nominated sites using hybrid capture followed by high-throughput sequencing, providing the most comprehensive assessment of such methods to date. The three methods performed similarly in nominating sequence-confirmed off-target sites, but with large differences in the total number of sites nominated. When combined with homology-dependent nomination methods and confirmation by sequencing, all three off-target nomination methods provide a comprehensive assessment of off-target activity. GUIDE-seq's low false-positive rate and the high correlation of its signal with observed editing highlight its suitability for nominating off-target sites for ex vivo CRISPR-Cas therapies.

Antibacterial Assessment of Zinc Sulfide Nanoparticles against Streptococcus pyogenes and Acinetobacter baumannii.

BACKGROUND: Due to the appearance of resistant bacterial strains against the antimicrobial drugs and the reduced efficiency of these valuable resources, the health of a community and the economies of countries have been threatened. OBJECTIVE: In this study, the antibacterial assessment of zinc sulfide nanoparticles (ZnS NPs) against Streptococcus pyogenes and Acinetobacter baumannii has been performed. METHODS: ZnS NPs were synthesized through a co-precipitation method using polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA) and polyethylene glycol (PEG-4000). The size and morphology of the synthesized ZnS NPs were determined by a scanning electron microscope (SEM) and it was found that the average size of the applied NPs was about 70 nm. In order to evaluate the antibacterial effect of the synthesized ZnS NPs, various concentrations (50µg/mL, 100 µg/mL and 150 µg/mL) of ZnS NPs were prepared. Antibacterial assessments were performed through the disc diffusion method in Mueller Hinton Agar (MHA) culture medium and also the optical density (OD) method was performed by a UV-Vis spectrophotometer in Trypticase™ Soy Broth (TSB) medium. Then, in order to compare the antibacterial effects of the applied NPs, several commercial antibiotics including penicillin, amikacin, ceftazidime and primaxin were used. RESULTS: The achieved results indicated that the antibacterial effects of ZnS NPs had a direct relation along with the concentrations and the concentration of 150 µg/mL showed the highest antibacterial effect in comparison with others. In addition, the ZnS NPs were more effective on Acinetobacter baumannii. CONCLUSION: The findings of this research suggest a novel approach against antibiotic resistance.

Reconstruction of low-resolution molecular structures from simulated atomic force microscopy images.

BACKGROUND: Atomic Force Microscopy (AFM) is an experimental technique to study structure-function relationship of biomolecules. AFM provides images of biomolecules at nanometer resolution. High-speed AFM experiments produce a series of images following dynamics of biomolecules. To further understand biomolecular functions, information on three-dimensional (3D) structures is beneficial. METHOD: We aim to recover 3D information from an AFM image by computational modeling. The AFM image includes only low-resolution representation of a molecule; therefore we represent the structures by a coarse grained model (Gaussian mixture model). Using Monte-Carlo sampling, candidate models are generated to increase similarity between AFM images simulated from the models and target AFM image. RESULTS: The algorithm was tested on two proteins to model their conformational transitions. Using a simulated AFM image as reference, the algorithm can produce a low-resolution 3D model of the target molecule. Effect of molecular orientations captured in AFM images on the 3D modeling performance was also examined and it is shown that similar accuracy can be obtained for many orientations. CONCLUSIONS: The proposed algorithm can generate 3D low-resolution protein models, from which conformational transitions observed in AFM images can be interpreted in more detail. GENERAL SIGNIFICANCE: High-speed AFM experiments allow us to directly observe biomolecules in action, which provides insights on biomolecular function through dynamics. However, as only partial structural information can be obtained from AFM data, this new AFM based hybrid modeling method would be useful to retrieve 3D information of the entire biomolecule.

ENT involvement and orobuccal movements' disorders in Pandas patients: assessment and rehabilitations tools.

OBJECTIVE: PANDAS are known as the spectrum of autoimmune pathologies related to a previous or current infection by group A beta-hemolytic streptococcus (SBEGA), dealing with several neuropsychiatric manifestations that mainly affect pediatric age. The main features consist of behavioral disease or movement disease characterized by acute-onset, presenting especially through infant period or adolescence. Specific manifestations, occurring during the progression of the disease, are the presence of otorhinolaryngologic symptoms (ENT) and orofacial movement disorders associated with temporomandibular joint pain. PATIENTS AND METHODS: We enrolled 130 children (5-15 years) with a clinical diagnosis of PANDAS between 2012 and 2018. Participants were assessed using ENT specific parameters, PSG to examine respiratory disorders and conventional audiological evaluation. Descriptive and comparative statistical analyses were performed with a control group of 51 healthy patients. RESULTS: The prevalence of ENT symptoms associated was significantly detected in 88 patients of 130 in Group A (relative frequency (%) 67.6; p=0.041) and in 51 patients of 130 in the control Group B (relative frequency (%) 39.2; p=0.063). In relation to prevalence of SDB, 54 subjects have presented nocturnal respiratory obstructive symptoms from mild to severe (relative frequency (%) 61.3; p=0.033) vs. 20 patients of Group B (relative frequency (%) 39.2; p=0.055). The obstructive severity average type was correlated to the consensual adenotonsillar development (size 3-4), (relative frequency (%) 45.4; p=0.047). The audiological deficits found were mostly of transmissive type with OME correlated and linked to the presence of occasional episodes of AOM. The four PANDAS patients who presented orobuccal dystonia (relative frequency (%) 4.54; p=0.091) achieved an improvement of the algic symptoms through the exercises of self-rehabilitation. CONCLUSIONS: Findings from our study show that respiratory diseases, characterizing a group of patients with pandas, are the direct consequences of the malformed or hypertrophic condition and suggesting in these conditions surgical therapy as an approaching tool.

The Path to Group A Streptococcus Vaccines: World Health Organization Research and Development Technology Roadmap and Preferred Product Characteristics.

Group A Streptococcus (GAS) infections result in a considerable underappreciated burden of acute and chronic disease globally. A 2018 World Health Assembly resolution calls for better control and prevention. Providing guidance on global health research needs is an important World Health Organization (WHO) activity, influencing prioritization of investments. Here, the role, status, and directions in GAS vaccines research are discussed. WHO preferred product characteristics and a research and development technology roadmap, briefly presented, offer an actionable framework for vaccine development to regulatory and policy decision making, availability, and use. GAS vaccines should be considered for global prevention of the range of clinical manifestations and associated antibiotic use. Impediments related to antigen diversity, safety concerns, and the difficulty to establish vaccine efficacy against rheumatic heart disease are discussed. Demonstration of vaccine efficacy against pharyngitis and skin infections constitutes a key near-term strategic goal. Investments and collaborative partnerships to diversify and advance vaccine candidates are needed.