Neutrophil Extracellular Traps (NETs): An Emerging Therapeutic Target to Improve Infectious Diseases Outcomes.

Neutrophils possess a diverse repertoire of pathogen clearance mechanisms, one of which is the formation of neutrophil extracellular traps (NETs). NETs are complexes of histone proteins and DNA coated with proteolytic enzymes that are released extracellularly to entrap pathogens and aid in their clearance, in a process known as NETosis. Intravascular NETosis may drive a massive inflammatory response that has been shown to contribute to morbidity and mortality in many infectious diseases, including malaria, dengue fever, influenza, bacterial sepsis, and SARS-CoV-2 infection. In this review we seek to: (1) summarize the current understanding of NETs; (2) discuss infectious diseases in which NET formation contributes to morbidity and mortality; and (3) explore potential adjunctive therapeutics that may be considered for future study in treating severe infections driven by NET pathophysiology. This includes drugs specifically targeting NET inhibition and FDA-approved drugs that may be repurposed as NET inhibitors.

Pediatric COVID-19 Health Disparities and Vaccine Equity.

While most children with coronavirus 2019 (COVID-19) experience mild illness, some are vulnerable to severe disease and develop long-term complications. Children with disabilities, those from lower-income homes, and those from racial and ethnic minority groups are more likely to be hospitalized and to have poor outcomes following an infection. For many of these same children, a wide range of social, economic, and environmental disadvantages have made it more difficult for them to access COVID-19 vaccines. Ensuring vaccine equity in children and decreasing health disparities promotes the common good and serves society as a whole. In this article, we discuss how the pandemic has exposed long-standing injustices in historically marginalized groups and provide a summary of the research describing the disparities associated with COVID-19 infection, severity, and vaccine uptake. Last, we outline several strategies for addressing some of the issues that can give rise to vaccine inequity in the pediatric population.

Potent Activity of Ertapenem Plus Cefazolin Within Staphylococcal Biofilms: A Contributing Factor in the Treatment of Methicillin-Susceptible Staphylococcus aureus Endocarditis.

Background: Besides antistaphylococcal beta-lactams and source control, there are limited validated antimicrobial salvage options in patients with prolonged methicillin-susceptible Staphylococcus aureus (MSSA) bacteremia, including infective endocarditis (IE). Methods: MSSA IE cases treated with ertapenem (ETP) plus cefazolin (CZ) were compared with matched IE cases treated with standard beta-lactam monotherapy. The bactericidal activity of ETP plus CZ was also compared with nafcillin (NAF), CZ, and ETP alone using an in vitro MSSA biofilm model. Results: The median duration of bacteremia experienced by patients (n = 12) while on CZ or NAF was 4 days (range 1-16 days) compared with 1 day (range 1-3 days) for patients (n = 5) treated with ETP + CZ (P = .01, Mann-Whitney U test). Cefazolin and NAF alone or in combination did not achieve biofilm eradication at clinically relevant concentrations. However, the addition of ETP to CZ led to bactericidal eradication within biofilms at standard dosing. Conclusions: Ertapenem reduces CZ concentrations required to eradicate MSSA biofilms to those achievable in vivo by standard dosing, translating into shorter bacteremia duration in patients with MSSA endocarditis. Larger studies are needed to investigate ETP plus CZ therapy in the treatment of biofilm-related MSSA infections such as endocarditis.

Azithromycin: An Underappreciated Quinolone-Sparing Oral Treatment for Pseudomonas aeruginosa Infections.

Outpatient treatment of Pseudomonas aeruginosa infections is challenged by increasing rates of resistance to fluoroquinolones, the only class of antibiotics which offers an established oral route of administration against this organism. Azithromycin does not demonstrate activity against P. aeruginosa when evaluated under standard methods of susceptibility testing with bacteriologic media. However, growing evidence shows that azithromycin is very active against P. aeruginosa when using physiologic media that recapitulate the in vivo milieu and is supported by animal models of infection and various clinical settings, including cystic fibrosis. We present three cases of outpatient management of P. aeruginosa otolaryngological infections successfully treated with oral azithromycin, 500 mg daily ranging from 3-8 weeks, where use of fluoroquinolones was not possible due to either resistance or patient intolerance. We review the previous data supporting this clinical approach, in the hope that this will alert clinicians to this treatment option and to inspire a more thorough clinical trial evaluation of azithromycin in this environment of growing medical need.

Impact of Clopidogrel on Clinical Outcomes in Patients with Staphylococcus aureus Bacteremia: a National Retrospective Cohort Study.

Activated platelets have known antimicrobial activity against Staphylococcus aureus. Accelerated clearance of platelets induced by S. aureus can result in thrombocytopenia and increased mortality in patients. Recent studies suggest that P2Y12 inhibition protects platelets from accelerated clearance. We therefore evaluated the effect of P2Y12 inhibition on clinical outcomes in patients with S. aureus bacteremia across a large national cohort. Our retrospective cohort (2010 to 2018) included patients admitted to Veterans Affairs (VA) hospitals with blood cultures positive for S. aureus and treated with standard-of-care antibiotics. Employing propensity score-matched Cox proportional hazards regression models, we compared clinical outcomes in patients treated with clopidogrel for at least the 30 days prior to admission and continuing for at least 5 days after admission to patients without any P2Y12 inhibitor use in the year preceding admission. Mortality was significantly lower among clopidogrel users than P2Y12 inhibitor nonusers (n = 147 propensity score-matched pairs): the inpatient mortality hazard ratio (HR) was 0.11 (95% confidence interval [CI], 0.01 to 0.86), and 30-day mortality HR was 0.43 (95% CI, 0.19 to 0.98). There were no differences in 30-day readmission, 30-day S. aureus reinfection, microbiological clearance, or thrombocytopenia. Clopidogrel use at the time of infection reduced in-hospital mortality by 89% and 30-day mortality by 57% among a cohort of patients with S. aureus bacteremia. These results support the need to further study the use of P2Y12 inhibitors as adjunctive therapy in S. aureus bloodstream infections.

SARS-CoV-2 acquisition and immune pathogenesis among school-aged learners in four diverse schools.

BACKGROUND: Understanding SARS-CoV-2 infection in children is necessary to reopen schools safely. METHODS: We measured SARS-CoV-2 infection in 320 learners [10.5 ± 2.1 (sd); 7-17 y.o.] at four diverse schools with either remote or on-site learning. Schools A and B served low-income Hispanic learners; school C served many special-needs learners, and all provided predominantly remote instruction. School D served middle- and upper-income learners, with predominantly on-site instruction. Testing occurred in the fall (2020), and 6-8 weeks later during the fall-winter surge (notable for a tenfold increase in COVID-19 cases). Immune responses and mitigation fidelity were also measured. RESULTS: We found SARS-CoV-2 infections in 17 learners only during the surge. School A (97% remote learners) had the highest infection (10/70, 14.3%, p < 0.01) and IgG positivity rates (13/66, 19.7%). School D (93% on-site learners) had the lowest infection and IgG positivity rates (1/63, 1.6%). Mitigation compliance [physical distancing (mean 87.4%) and face-covering (91.3%)] was remarkably high at all schools. Documented SARS-CoV-2-infected learners had neutralizing antibodies (94.7%), robust IFN-γ + T cell responses, and reduced monocytes. CONCLUSIONS: Schools can implement successful mitigation strategies across a wide range of student diversity. Despite asymptomatic to mild SARS-CoV-2 infection, children generate robust humoral and cellular immune responses. IMPACT: Successful COVID-19 mitigation was implemented across a diverse range of schools. School-associated SARS-CoV-2 infections reflect regional rates rather than remote or on-site learning. Seropositive school-aged children with asymptomatic to mild SARS-CoV-2 infections generate robust humoral and cellular immunity.

Ticagrelor Increases Platelet-Mediated Staphylococcus aureus Killing, Resulting in Clearance of Bacteremia.

Platelets are a critical immune defense against Staphylococcus aureus bloodstream infections. Staphylococcus aureus α-toxin is a virulence factor that decreases platelet viability and accelerates platelet clearance. It has been shown that ticagrelor blocks α-toxin-mediated platelet injury and resulting thrombocytopenia, protecting mice in a lethal S. aureus sepsis model. We now present the use of ticagrelor as adjunctive therapy in a patient with a S. aureus endovascular infection and thrombocytopenia, associated with restoration of platelet count and bacteremia clearance. Ticagrelor enhanced platelet killing of the S. aureus bloodstream isolate from the treated patient in vitro.

SARS-CoV-2 Acquisition and Immune Pathogenesis Among School-Aged Learners in Four Diverse Schools.

BACKGROUND: Understanding SARS-CoV-2 infection in children is necessary to reopen schools safely. METHODS: We measured SARS-CoV-2 infection in 320 learners [10.5 ± 2.1(sd); 7-17 y.o.] at four diverse schools with either remote or on-site learning. Schools A and B served low-income Hispanic learners; school C served many special-needs learners; and all provided predominantly remote instruction. School D served middle- and upper-income learners, with predominantly on-site instruction. Testing occurred in the fall (2020), and 6-8 weeks later during the fall-winter surge (notable for a tenfold increase in COVID-19 cases). Immune responses and mitigation fidelity were also measured. RESULTS: We found SARS-CoV-2 infections in 17 learners only during the surge. School A (97% remote learners) had the highest infection (10/70, 14.3%, p<0.01) and IgG positivity rates (13/66, 19.7%). School D (93% on-site learners) had the lowest infection and IgG positivity rates (1/63, 1.6%). Mitigation compliance [physical distancing (mean 87.4%) and face covering (91.3%)] was remarkably high at all schools. Documented SARS-CoV-2-infected learners had neutralizing antibodies (94.7%), robust IFN-γ+ T cell responses, and reduced monocytes. CONCLUSION: Schools can implement successful mitigation strategies across a wide range of student diversity. Despite asymptomatic to mild SARS-CoV-2 infection, children generate robust humoral and cellular immune responses. KEY POINTS: Successful COVID-19 mitigation was implemented across a diverse range of schools.School-associated SARS-CoV-2 infections reflect regional rates rather than remote or on-site learning.Seropositive school-aged children with asymptomatic to mild SARS-CoV-2 infections generate robust humoral and cellular immunity.

SARS-CoV-2 vaccine testing and trials in the pediatric population: biologic, ethical, research, and implementation challenges.

As the nation implements SARS-CoV-2 vaccination in adults at an unprecedented scale, it is now essential to focus on the prospect of SARS-CoV-2 vaccinations in pediatric populations. To date, no children younger than 12 years have been enrolled in clinical trials. Key challenges and knowledge gaps that must be addressed include (1) rationale for vaccines in children, (2) possible effects of immune maturation during childhood, (3) ethical concerns, (4) unique needs of children with developmental disorders and chronic conditions, (5) health inequities, and (6) vaccine hesitancy. Because COVID-19 is minimally symptomatic in the vast majority of children, a higher acceptable risk threshold is required when evaluating pediatric clinical trials. Profound differences in innate and adaptive immunity during childhood and adolescence are known to affect vaccine responsiveness for a variety of childhood diseases. COVID-19 and the accompanying social disruption, such as the school shutdowns, has been disproportionately damaging to minority and low-income children. In this commentary, we briefly address each of these key issues, specify research gaps, and suggest a broader learning health system approach to accelerate testing and clinical trial development for an ethical and effective strategy to implement a pediatric SARS-CoV-2 vaccine as rapidly and safely as possible. IMPACT: As the US begins an unprecedented implementation of SARS-CoV-2 vaccination, substantial knowledge gaps have yet to be addressed regarding vaccinations in the pediatric population. Maturational changes in the immune system during childhood have influenced the effectiveness of pediatric vaccines for other diseases and conditions, and could affect SARS-CoV-2 vaccine responsiveness in children. Given that COVID-19 disease is far milder in the majority of children than in adults, the risk-benefit of a pediatric SARS-CoV-2 vaccine must be carefully weighed. The needs of children with developmental disabilities and with chronic disease must be addressed. Minority and low-income children have been disproportionately adversely affected by the COVID-19 pandemic; care must be taken to address issues of health equity regarding pediatric SARS-CoV-2 vaccine trials and allocation. Research and strategies to address general vaccine hesitancy in communities must be addressed in the context of pediatric SARS-CoV-2 vaccines.

Granadaene Photobleaching Reduces the Virulence and Increases Antimicrobial Susceptibility of Streptococcus agalactiae.

Streptococcus agalactiae, also known as Group B Streptococcus (GBS), is increasingly recognized as a major cause of soft tissue and invasive diseases in the elderly and diabetic populations. Antibiotics like penicillin are used with great frequency to treat these infections, although antimicrobial resistance is increasing among GBS strains and underlines a need for alternative methods not reliant on traditional antibiotics. GBS granadaene pigment is related to the hemolysin/cytolysin of GBS, which is critical for the pathogenesis of GBS diseases. Here, we show that photobleaching granadaene dampens the hemolytic activity of GBS. Furthermore, photobleaching of this antioxidant was found to increase GBS susceptibility to killing by reactive oxygen species like hydrogen peroxide. Treatment with light was also shown to affect GBS membrane permeability and contribute to increased susceptibility to the cell membrane-targeting antibiotic daptomycin. Overall, our study demonstrates dual effects of photobleaching on the virulence and antimicrobial susceptibility of GBS and suggests a novel approach for the treatment of GBS infection.

Photo-Disassembly of Membrane Microdomains Revives Conventional Antibiotics against MRSA.

Confronted with the rapid evolution and dissemination of antibiotic resistance, there is an urgent need to develop alternative treatment strategies for drug-resistant pathogens. Here, an unconventional approach is presented to restore the susceptibility of methicillin-resistant S. aureus (MRSA) to a broad spectrum of conventional antibiotics via photo-disassembly of functional membrane microdomains. The photo-disassembly of microdomains is based on effective photolysis of staphyloxanthin, the golden carotenoid pigment that gives its name. Upon pulsed laser treatment, cell membranes are found severely disorganized and malfunctioned to defense antibiotics, as unveiled by membrane permeabilization, membrane fluidification, and detachment of membrane protein, PBP2a. Consequently, the photolysis approach increases susceptibility and inhibits development of resistance to a broad spectrum of antibiotics including penicillins, quinolones, tetracyclines, aminoglycosides, lipopeptides, and oxazolidinones. The synergistic therapy, without phototoxicity to the host, is effective in combating MRSA both in vitro and in vivo in a mice skin infection model. Collectively, this endogenous chromophore-targeted phototherapy concept paves a novel platform to revive conventional antibiotics to combat drug-resistant S. aureus infections as well as to screen new lead compounds.

Cefazolin and Ertapenem Salvage Therapy Rapidly Clears Persistent Methicillin-Susceptible Staphylococcus aureus Bacteremia.

Cefazolin and ertapenem combination therapy was used successfully to salvage 11 cases (6 endocarditis) of persistent methicillin-susceptible Staphylococcus aureus (MSSA) bacteremia, including immediate clearance (≤24 hours) in 8 cases. While in vitro synergy was modest, cefazolin plus ertapenem exhibited synergistic action in a rat model of MSSA endocarditis. The combination of cefazolin and ertapenem provides potent in vivo activity against MSSA beyond what is predicted in vitro and warrants further clinical study in the treatment of refractory MSSA bacteremia and endocarditis.

Azithromycin Exerts Bactericidal Activity and Enhances Innate Immune Mediated Killing of MDR Achromobacter xylosoxidans.

Azithromycin (AZM), the most commonly prescribed antibiotic in the United States, is thought to have no activity against multidrug-resistant Gram-negative pathogens such as Achromobacter xylosoxidans (AX) per standard minimum inhibitory concentration testing in cation-adjusted Mueller Hinton Broth. Here we provide the first report of AZM bactericidal activity against carbapenem-resistant isolates of AX, with a multifold decrease in minimum inhibitory concentration across 12 clinical isolates when examined under physiologic testing conditions that better recapitulate the in vivo human environment. This pharmaceutical activity, evident in eukaryotic tissue culture media, is associated with enhanced AZM intracellular penetration and synergistic killing with human whole blood, serum, and neutrophils. Additionally, AZM monotherapy inhibited preformed AX biofilm growth in a dose-dependent manner together with a reduction in viable bacteria. In an illustrative case, AZM in combination with piperacillin-tazobactam exerted clear therapeutic effects in a patient with carbapenem-resistant AX mediastinitis, sternal osteomyelitis, and aortic graft infection. Our study reinforces how current antimicrobial testing practices fail to recapitulate the host environment or host-pathogen interactions and may misleadingly declare complete resistance to useful agents, adversely affecting patient outcomes. We conclude that AZM merits further exploration in the treatment of drug-resistant AX infections. Novel approaches to antimicrobial susceptibility testing that better recapitulate the host environment should be considered, especially as infections caused by multidrug-resistant Gram-negative bacterial pathogens are expanding globally with high morbidity and mortality.

Association of Enterovirus D68 with Acute Flaccid Myelitis, Philadelphia, Pennsylvania, USA, 2009-2018.

Acute flaccid myelitis (AFM) is a polio-like disease that results in paralysis in previously healthy persons. Although the definitive cause of AFM remains unconfirmed, enterovirus D68 (EV-D68) is suspected based on 2014 data demonstrating an increase in AFM cases concomitant with an EV-D68 outbreak. We examined the prevalence in children and the molecular evolution of EV-D68 for 2009-2018 in Philadelphia, Pennsylvania, USA. We detected widespread EV-D68 circulation in 2009, rare detections in 2010 and 2011, and then biennial circulation, only in even years, during 2012-2018. Prevalence of EV-D68 significantly correlated with AFM cases during this period. Finally, whole-genome sequencing revealed early detection of the B1 clade in 2009 and continued evolution of the B3 clade from 2016 to 2018. These data reinforce the need to improve surveillance programs for nonpolio enterovirus to identify possible AFM triggers and predict disease prevalence to better prepare for future outbreaks.

Avibactam Sensitizes Carbapenem-Resistant NDM-1-Producing Klebsiella pneumoniae to Innate Immune Clearance.

Infections caused by New Delhi metallo-ß-lactamase (NDM)-producing strains of multidrug-resistant Klebsiella pneumoniae are a global public health threat lacking reliable therapies. NDM is impervious to all existing ß-lactamase inhibitor (BLI) drugs, including the non-ß-lactam BLI avibactam (AVI). Though lacking direct activity against NDMs, AVI can interact with penicillin-binding protein 2 in a manner that may influence cell wall dynamics. We found that exposure of NDM-1-producing K. pneumoniae to AVI led to striking bactericidal interactions with human cathelicidin antimicrobial peptide LL-37, a frontline component of host innate immunity. Moreover, AVI markedly sensitized NDM-1-producing K. pneumoniae to killing by freshly isolated human neutrophils, platelets, and serum when complement was active. Finally, AVI monotherapy reduced lung counts of NDM-1-producing K. pneumoniae in a murine pulmonary challenge model. AVI sensitizes NDM-1-producing K. pneumoniae to innate immune clearance in ways that are not appreciated by standard antibiotic testing and that merit further study.

The neural substrate of gesture recognition.

Previous studies have linked action recognition with a particular pool of neurons located in the ventral premotor cortex, the posterior parietal cortex and the superior temporal sulcus (the mirror neuron system). However, it is still unclear if transitive and intransitive gestures share the same neural substrates during action-recognition processes. In the present study, we used event-related functional magnetic resonance imaging (fMRI) to assess the cortical areas active during recognition of pantomimed transitive actions, intransitive gestures, and meaningless control actions. Perception of all types of gestures engaged the right pre-supplementary motor area (pre-SMA), and bilaterally in the posterior superior temporal cortex, the posterior parietal cortex, occipitotemporal regions and visual cortices. Activation of the posterior superior temporal sulcus/superior temporal gyrus region was found in both hemispheres during recognition of transitive and intransitive gestures, and in the right hemisphere during the control condition; the middle temporal gyrus showed activation in the left hemisphere when subjects recognized transitive and intransitive gestures; activation of the left inferior parietal lobe and intraparietal sulcus (IPS) was mainly observed in the left hemisphere during recognition of the three conditions. The most striking finding was the greater activation of the left inferior frontal gyrus (IFG) during recognition of intransitive actions. Results show that a similar neural substrate, albeit, with a distinct engagement underlies the cognitive processing of transitive and intransitive gestures recognition. These findings suggest that selective disruptions in these circuits may lead to distinct clinical deficits.

Recognition of point-light biological motion: mu rhythms and mirror neuron activity.

Changes in power in the mu frequency band (8-13Hz) of the electroencephalogram (EEG) is thought to indirectly reflect the activity of mirror neurons in premotor cortex. Activation of these neurons by self-performed, observed or imagined motor actions is assumed to produce asynchronous firing and a reduction in mu rhythm oscillation (referred to as mu suppression) in sensorimotor cortex. A recent fMRI study by Saygin et al. [Saygin AP, Wilson SM, Hagler Jr DJ, Bates E, Sereno MI. Point-light biological motion perception activates human premotor cortex. J Neurosci 2004;24:6181-8] revealed that the premotor brain regions containing mirror-neurons are also activated in response to point-light human motion. The perceived movement of these light cues are integrated into one percept of a complete human action (e.g. jumping jacks), rather than seen as individual moving lights. The present study examined whether recruitment of the mirror neuron system, as reflected in mu rhythm suppression, mediates recognition of point-light biological motion. Changes in mu power were recorded while subjects viewed point-light biological motion videos, matched scrambled versions of these animations, and visual white-noise (baseline). The results revealed that point-light biological animations produced mu suppression relative to baseline, while scrambled versions of these animations did not. This supports the hypothesis that the mirror neuron system is involved in inferring human actions by recovering object information from sparse input.