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Introduction Known as the “golden hour,” survival of most critically injured patients is highly dependent on providing the required treatment within the first hour of injury. Recent technological advances in additive manufacturing (also known as three-dimensional [3D] printing) allow for austere deployment and point-of-care rapid fabrication of a variety of medical supplies, including human tissues and bioactive bandages, in prolonged field care scenarios. In this pilot project, our aim was to investigate the ability to 3D print a range of potential biomedical supplies and solutions in an austere field environment. Materials and Methods We specifically designed and fabricated novel surgical tools, bioactive bandages, objects (screw and anatomic models), and human meniscal tissue in an austere African desert environment. A total of seven packages were sent using a commercial carrier directly to the end destination. A multi-tool ruggedized 3D printer was used as the manufacturing platform for all objects fabricated downrange. Human mesenchymal stem cells were shipped for 3D bioprinting of human menisci and bioactive bandages. Design and fabrication for all 3D-printed products utilized computer-aided design (CAD) tools. Results Initial shipment from a single U.S. site to the sub-Saharan Africa location was relatively prompt, taking an average of 4.7 days to deliver three test packages. However, the actual delivery of the seven packages from Orlando, FL, to the same sub-Saharan Africa site took an average of 16 days (range 7-23 days). The ruggedized printer successfully fabricated relevant medical supplies using biocompatible filament, bioink hydrogels, and stem cell–loaded bioinks. This prototype did not, however, have the capacity to provide a sterile environment. A multi-material complete bandage was 3D printed using polyamide polyolefin and cellulose, live cells, neomycin salve, and adhesive. The bandage, wound covering backing, and adhesive backing print took under 2 min to 3D print. Surgical instrument CAD files were based on commercially available medical-grade stainless-steel instruments. The screw CAD file was downloaded from the NIH 3D Print Exchange website. The prints of the two surgical tools and screw using thermoplastic material were successful. Menisci, relatively complex forms of the cartilage, were 3D bioprinted with a gel that held their form well after printing and were then solidified slightly using a cross-linking solution. After 2 min of solidification, it was possible to remove and handle the menisci. Conclusion The current and future challenges of prolonged field care need to be addressed with new techniques, training, and technology. Ruggedized, deployable 3D printers allow for the direct fabrication of medical tools, supplies, and biological solutions for austere use. Delivery of packages can vary, and attention to routes and location is key, especially for transit of time-sensitive perishable supplies such as live cells. The significance of this study provides the real possibility to 3D print “just-in-time” medical solutions tailored to the need of an individual service member in any environment. This is a potentially exciting opportunity to bring critical products to the war front.

Context.— Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin G (IgG) testing is used for serosurveillance and will be important to evaluate vaccination status. Given the urgency to release coronavirus disease 2019 (COVID-19) serology tests, most manufacturers have developed qualitative tests. Objective.— To evaluate clinical performance of 6 different SARS-CoV-2 IgG assays and their quantitative results to better elucidate the clinical role of serology testing in COVID-19. Design.— Six SARS-CoV-2 IgG assays were tested using remnant specimens from 190 patients. Sensitivity and specificity were evaluated for each assay with the current manufacturer's cutoff and a lower cutoff. A numeric result analysis and discrepancy analysis were performed. Results.— Specificity was higher than 93% for all assays, and sensitivity was higher than 80% for all assays (≥7 days post–polymerase chain reaction testing). Inpatients with more severe disease had higher numeric values compared with health care workers with mild or moderate disease. Several discrepant serology results were those just below the manufacturers' cutoff. Conclusions.— Severe acute respiratory syndrome coronavirus 2 IgG antibody testing can aid in the diagnosis of COVID-19, especially with negative polymerase chain reaction. Quantitative COVID-19 IgG results are important to better understand the immunologic response and disease course of this novel virus and to assess immunity as part of future vaccination programs.

Md A Nahid,1 Jaclyn M Griffin,2 Michael B Lustik,3 Jordan J Hayes,3 Keith SK Fong,3 Timothy S Horseman,3 Massimo Menguito,4 Erik C Snesrud5 ,† Jason C Barnhill,4,6 Michael A Washington4 1Department of Dental and Craniomaxillofacial Trauma Research, United States Army Institute of Surgical Research, JBSA Fort Sam, Houston, TX 78234, USA; 2Department of Vascular Surgery, Tripler Army Medical Center, Honolulu, HI, 96859, USA; 3Department of Clinical Investigation, Tripler Army Medical Center, Honolulu, HI 96859, USA; 4Department of Chemistry and Life Science, United States Military Academy, West Point, NY 10996, USA; 5Multidrug-Resistant Organism Repository and Surveillance Network (MRSN), Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA; 6Department of Radiology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA†Dr Erik C Snesrud passed away on February 10, 2020Correspondence: Md A NahidUnited States Army Institute of Surgical Research, JBSA Fort Sam, Houston, TX 78234, USATel +1 210-539-6419Fax +1 210-539-3877Email md.a.nahid.mil@mail.milPurpose: The biology of chronic wounds is complex and many factors act concurrently to impede healing progress. In this study, the dynamics of microflora changes and their antibiotic susceptibility patterns were evaluated longitudinally over 30 days using data from 28 patients with a total of 47 chronic lower extremity wounds.Materials and Methods: In this study, colonized wound isolates were characterized using cultural, biochemical, and VITEK 2 methods. Antibiotic susceptibility patterns of the wound isolates were analyzed using various phenotypic assays. Furthermore, antimicrobial resistance patterns and the presence of mutations were evaluated by a genotypic assay, whole-genome sequencing (WGS).Results: Staphylococcus aureus and Pseudomonas aeruginosa were found to be the most common strains at early time points, while members of Enterobacteriaceae were prevalent at later stages of infection. Antimicrobial resistance testing and whole-genome sequencing revealed that the molecular and phenotypic characteristics of the identified wound pathogens remained relatively stable throughout the study period. It was also noted that Enterobacter and Klebsiella species may serve as reservoirs for quinolone resistance in the Pacific region.Conclusion: Our observations showed that wounds were colonized with diverse bacteria and interestingly their numbers and/or types were changed over the course of infection. The rapid genetic changes that accompanied the first 4 weeks after presentation did not directly contribute to the development of antibiotic resistance. In addition, standard wound care procedures did not appear to select for resistant bacterial strains. Future efforts should focus on defining those genetic changes associated with the wound colonizing microorganisms that occur beyond 4 weeks.Keywords: anti-microbial resistance, antimicrobial susceptibility profiling, non-healing wound, wound microflora, wound healing

Most known SARS-CoV-2 neutralizing antibodies (nAbs), including those approved by the FDA for emergency use, inhibit viral infection by targeting the receptor-binding domain (RBD) of the spike (S) protein. Variants of concern (VOC) carrying mutations in the RBD or other regions of S reduce the effectiveness of many nAbs and vaccines by evading neutralization. Therefore, therapies that are less susceptible to resistance are urgently needed. Here, we characterized the memory B-cell repertoire of COVID-19 convalescent donors and analyzed their RBD and non-RBD nAbs. We found that many of the non-RBD-targeting nAbs were specific to the N-terminal domain (NTD). Using neutralization assays with authentic SARS-CoV-2 and a recombinant vesicular stomatitis virus carrying SARS-CoV-2 S protein (rVSV-SARS2), we defined a panel of potent RBD and NTD nAbs. Next, we used a combination of neutralization-escape rVSV-SARS2 mutants and a yeast display library of RBD mutants to map their epitopes. The most potent RBD nAb competed with hACE2 binding and targeted an epitope that includes residue F490. The most potent NTD nAb epitope included Y145, K150, and W152. As seen with some of the natural VOC, the neutralization potencies of COVID-19 convalescent-phase sera were reduced by 4- to 16-fold against rVSV-SARS2 bearing Y145D, K150E, or W152R spike mutations. Moreover, we found that combining RBD and NTD nAbs did not enhance their neutralization potential. Notably, the same combination of RBD and NTD nAbs limited the development of neutralization-escape mutants in vitro, suggesting such a strategy may have higher efficacy and utility for mitigating the emergence of VOC. IMPORTANCE The U.S. FDA has issued emergency use authorizations (EUAs) for multiple investigational monoclonal antibody (MAb) therapies for the treatment of mild to moderate COVID-19. These MAb therapeutics are solely targeting the receptor-binding domain of the SARS-CoV-2 spike protein. However, the N-terminal domain of the spike protein also carries crucial neutralizing epitopes. Here, we show that key mutations in the N-terminal domain can reduce the neutralizing capacity of convalescent-phase COVID-19 sera. We report that a combination of two neutralizing antibodies targeting the receptor-binding and N-terminal domains may be beneficial to combat the emergence of virus variants.

Most known SARS-CoV-2 neutralizing antibodies (nAbs), including those approved by the FDA for emergency use, inhibit viral infection by targeting the receptor-binding domain (RBD) of the spike (S) protein. Variants of concern (VOC) carrying mutations in the RBD or other regions of S reduce the effectiveness of many nAbs and vaccines by evading neutralization. Therefore, therapies that are less susceptible to resistance are urgently needed. Here, we characterized the memory B-cell repertoire of COVID-19 convalescent donors and analyzed their RBD and non-RBD nAbs. We found that many of the non-RBD-targeting nAbs were specific to the N-terminal domain (NTD). Using neutralization assays with authentic SARS-CoV-2 and a recombinant vesicular stomatitis virus carrying SARS-CoV-2 S protein (rVSV-SARS2), we defined a panel of potent RBD and NTD nAbs. Next, we used a combination of neutralization-escape rVSV-SARS2 mutants and a yeast display library of RBD mutants to map their epitopes. The most potent RBD nAb competed with hACE2 binding and targeted an epitope that includes residue F490. The most potent NTD nAb epitope included Y145, K150 and W152. As seen with some of the natural VOC, the neutralization potencies of COVID-19 convalescent sera were reduced by 4-16-fold against rVSV-SARS2 bearing Y145D, K150E or W152R spike mutations. Moreover, we found that combining RBD and NTD nAbs modestly enhanced their neutralization potential. Notably, the same combination of RBD and NTD nAbs limited the development of neutralization-escape mutantsin vitro, suggesting such a strategy may have higher efficacy and utility for mitigating the emergence of VOC.ImportanceThe US FDA has issued emergency use authorizations (EUAs) for multiple investigational monoclonal antibody (mAb) therapies for the treatment of mild to moderate COVID-19. These mAb therapeutics are solely targeting the receptor binding domain of the SARS-CoV-2 spike protein. However, the N-terminal domain of the spike protein also carries crucial neutralizing epitopes. Here, we show that key mutations in the N-terminal domain can reduce the neutralizing capacity of convalescent COVID-19 sera. We report that a combination of two neutralizing antibodies targeting the receptor binding and N-terminal domains may have higher efficacy and is beneficial to combat the emergence of virus variants.

The coronavirus disease 2019 (COVID-19) global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to place an immense burden on societies and health care systems. A key component of COVID-19 control efforts is serological testing to determine the community prevalence of SARS-CoV-2 exposure and quantify individual immune responses to prior SARS-CoV-2 infection or vaccination. Here, we describe a laboratory-developed antibody test that uses readily available research-grade reagents to detect SARS-CoV-2 exposure in patient blood samples with high sensitivity and specificity. We further show that this sensitive test affords the estimation of viral spike-specific IgG titers from a single sample measurement, thereby providing a simple and scalable method to measure the strength of an individual's immune response. The accuracy, adaptability, and cost-effectiveness of this test make it an excellent option for clinical deployment in the ongoing COVID-19 pandemic.IMPORTANCE Serological surveillance has become an important public health tool during the COVID-19 pandemic. Detection of protective antibodies and seroconversion after SARS-CoV-2 infection or vaccination can help guide patient care plans and public health policies. Serology tests can detect antibodies against past infections; consequently, they can help overcome the shortcomings of molecular tests, which can detect only active infections. This is important, especially when considering that many COVID-19 patients are asymptomatic. In this study, we describe an enzyme-linked immunosorbent assay (ELISA)-based qualitative and quantitative serology test developed to measure IgG and IgA antibodies against the SARS-CoV-2 spike glycoprotein. The test can be deployed using commonly available laboratory reagents and equipment and displays high specificity and sensitivity. Furthermore, we demonstrate that IgG titers in patient samples can be estimated from a single measurement, enabling the assay's use in high-throughput clinical environments.

The Bronx was an early epicenter of the COVID-19 pandemic in the USA. We conducted temporal genomic surveillance of SARS-CoV-2 genomes across the Bronx from March-October 2020. Although the local structure of SARS-CoV-2 lineages mirrored those of New York City and New York State, temporal sampling revealed a dynamic and changing landscape of SARS-CoV-2 genomic diversity. Mapping the trajectories of variants, we found that while some became ‘endemic’ to the Bronx, other, novel variants rose in prevalence in the late summer/early fall. Geographically resolved genomes enabled us to distinguish between cases of reinfection and persistent infection in two pediatric patients. We propose that limited, targeted, temporal genomic surveillance has clinical and epidemiological utility in managing the ongoing COVID pandemic.One sentence summaryTemporally and geographically resolved sequencing of SARS-CoV-2 genotypes enabled surveillance of novel genotypes, identification of endemic viral variants, and clinical inferences, in the first wave of the COVID-19 pandemic in the Bronx.

The coronavirus disease 2019 (COVID-19) global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to place an immense burden on societies and health care systems. A key component of COVID-19 control efforts is serological testing to determine the community prevalence of SARS-CoV-2 exposure and quantify individual immune responses to prior SARS-CoV-2 infection or vaccination. Here, we describe a laboratory-developed antibody test that uses readily available research-grade reagents to detect SARS-CoV-2 exposure in patient blood samples with high sensitivity and specificity. We further show that this sensitive test affords the estimation of viral spike-specific IgG titers from a single sample measurement, thereby providing a simple and scalable method to measure the strength of an individual’s immune response. The accuracy, adaptability, and cost-effectiveness of this test make it an excellent option for clinical deployment in the ongoing COVID-19 pandemic. IMPORTANCE Serological surveillance has become an important public health tool during the COVID-19 pandemic. Detection of protective antibodies and seroconversion after SARS-CoV-2 infection or vaccination can help guide patient care plans and public health policies. Serology tests can detect antibodies against past infections; consequently, they can help overcome the shortcomings of molecular tests, which can detect only active infections. This is important, especially when considering that many COVID-19 patients are asymptomatic. In this study, we describe an enzyme-linked immunosorbent assay (ELISA)-based qualitative and quantitative serology test developed to measure IgG and IgA antibodies against the SARS-CoV-2 spike glycoprotein. The test can be deployed using commonly available laboratory reagents and equipment and displays high specificity and sensitivity. Furthermore, we demonstrate that IgG titers in patient samples can be estimated from a single measurement, enabling the assay’s use in high-throughput clinical environments.

Summary There is an urgent need for vaccines and therapeutics to prevent and treat COVID-19. Rapid SARS-CoV-2 countermeasure development is contingent on the availability of robust, scalable, and readily deployable surrogate viral assays to screen antiviral humoral responses, define correlates of immune protection, and down-select candidate antivirals. Here, we generate a highly infectious recombinant vesicular stomatitis virus (VSV) bearing the SARS-CoV-2 spike glycoprotein S as its sole entry glycoprotein and show that this recombinant virus, rVSV-SARS-CoV-2 S, closely resembles SARS-CoV-2 in its entry-related properties. The neutralizing activities of a large panel of COVID-19 convalescent sera can be assessed in a high-throughput fluorescent reporter assay with rVSV-SARS-CoV-2 S, and neutralization of rVSV-SARS-CoV-2 S and authentic SARS-CoV-2 by spike-specific antibodies in these antisera is highly correlated. Our findings underscore the utility of rVSV-SARS-CoV-2 S for the development of spike-specific therapeutics and for mechanistic studies of viral entry and its inhibition., Graphical Abstract, Highlights • Highly infectious recombinant VSV expressing SARS-CoV-2 spike (S) was generated • rVSV-SARS-CoV-2 S resembles SARS-CoV-2 in entry and inhibitor/antibody sensitivity • rVSV-SARS-CoV-2 S affords rapid screens and forward-genetic analyses of antivirals, Surrogate systems are needed to evaluate COVID-19 vaccines and therapeutics rapidly and at scale. Dieterle & Haslwanter et al. describe a highly infectious recombinant vesicular stomatitis virus encoding the SARS-CoV-2 spike protein that is suitable for screening and mechanistic studies of small molecule inhibitors, recombinant biologics, and convalescent plasma.

Lyme disease is a continuing threat to military personnel operating in arboriferous and mountainous environments. Here we present the case of a 24-year-old Second Lieutenant, a recent graduate from the United States Military Academy, with a history of Lyme disease who developed recurrent knee effusions following surgery to correct a hip impingement. Although gonococcal arthritis was initially suspected from preliminary laboratory results, a comprehensive evaluation contradicted this diagnosis. Despite antibiotic therapy, aspiration of the effusions, and steroid treatment to control inflammation, the condition of the patient deteriorated to the point where he was found to be unfit for duty and subsequently discharged from active military service. This case illustrates the profound effect that latent Lyme disease can have on the quality of life and the career of an active duty military member. It highlights the need for increased surveillance for Borrelia burgdorferi (B. burgdorferi) in military training areas and for the early and aggressive diagnosis and treatment of military personnel who present with the symptoms of acute Lyme disease.

Whole genome sequencing (WGS) is increasingly employed in clinical settings, though few assessments of turnaround times (TAT) have been performed in real-time. In this study, WGS was used to investigate an unfolding outbreak of vancomycin resistant Enterococcus faecium (VRE) among 3 patients in the ICU of a tertiary care hospital. Including overnight culturing, a TAT of just 48.5 h for a comprehensive report was achievable using an Illumina Miseq benchtop sequencer. WGS revealed that isolates from patient 2 and 3 differed from that of patient 1 by a single nucleotide polymorphism (SNP), indicating nosocomial transmission. However, the unparalleled resolution provided by WGS suggested that nosocomial transmission involved two separate events from patient 1 to patient 2 and 3, and not a linear transmission suspected by the time line. Rapid TAT’s are achievable using WGS in the clinical setting and can provide an unprecedented level of resolution for outbreak investigations.

Virulence is the relative capacity of a pathogenic microorganism to cause damage in susceptible host cells such as those found in airway passages and the gut. In this study, the effect of clinical bacterial isolates on the monolayer integrity of cultured human alveolar basal epithelial cells (A549) was evaluated using the Electric Cell-Substrate Impedance Sensing (ECIS) system. ECIS is a morphological biosensor which records electrical properties of cell-covered microelectrodes in an AC circuit including impedance (ohm), resistance (ohm), and capacitance (μFarad). In the current study, fluctuations in the electrical properties of cell-covered microelectrodes reflect dynamic changes in cell morphology resulting from disrupted cell monolayers following exposure to bacteria. Using the ECIS system, real-time changes of cell morphology and disruption of monolayer integrity of cell-cultures in vitro were revealed for A549 cells infected with either Pseudomonas aeruginosa , ESBL Escherichia coli, Staphylococcus aureus (MRSA), or Enterococcus (VRE). We determined empirically that the optimal signal response was obtained for resistance (ohm) measurements at 4000 hertz. Following infection of A549 cells, the data revealed that Pseudomonas aeruginosa resulted in little change in microelectrode resistance (ohm @4 kHz) as compared to pathogen-free controls within the first 12 h. In contrast, E. coli , MRSA , and VRE caused significant changes in electrode resistance (ohm @4 kHz) values in the infected cells compared to controls over the first 5 h. Resistance (ohm @4 kHz) changes were also observed in cell monolayers infected with different bacterial concentrations for all isolates over 24 h. The highest concentration of bacteria caused the measured resistance (ohm @4 kHz) to drop faster than its' immediate lower concentration, suggesting a dose-dependent effect. Compared to live bacteria, cells exposed to heat-killed bacteria did not show significant changes in resistance (ohm @4 kHz) over 48 h post-exposure. Functionally, cytokine responses were different between cells treated with live and heat-killed bacteria. Of note, live bacteria induced IFNγ, IL-13, and IL-1β production in A549 cells, whereas heat-killed bacteria induced IL-8 production suggesting a differential interaction with cells that could reveal the underlying causes of resistance (ohm @4 kHz) changes. Our findings indicate that ECIS provides a means to quantify, automate, and measure bacterial virulence , which may have broader implications governing the course of treatment compared to traditional methods alone.

Hawaii has one of the highest incidences of Campylobacteriosis in the United States, but there remains little published data on circulating strains or antimicrobial resistance. We characterized 110 clinical Campylobacter isolates (106 C. jejuni, 4 C. coli) processed at Tripler Army Medical Center in Honolulu, HI from 2012–2016. Twenty-five percent of C. jejuni isolates exhibited fluoroquinolone (FQ) resistance, compared with 16% for tetracycline (TET), and 0% for macrolides. Two of the four C. coli isolates were resistant to FQ, TET, and macrolides. C. jejuni isolates further underwent multilocus sequence typing, pulsed-field gel electrophoresis, and molecular capsular typing. Nineteen capsule types were observed, with two capsule types (HS2 and HS9) being associated with FQ resistance (p

Staphylococcus sciuri is an emerging gram-positive bacterial pathogen that is infrequently isolated from cases of human disease. This organism is capable of rapid conversion from a state of methicillin sensitivity to a state of methicillin resistance and has been shown to express a set of highly effective virulence factors. The antibiotic-resistance breakpoints of S. sciuri differ significantly from the more common Staphylococcus species. Therefore, the rapid identification of S. sciuri in clinical material is a prerequisite for the proper determination of the antibiotic-resistance profile and the rapid initiation of antimicrobial therapy. Here, we present a brief literature review of S. sciuri and an entomological case study in which we describe the colonization of an American cockroach with this agent. In addition, we discuss potential implications for the distribution and evolution of antibiotic-resistant members of the genus Staphylococcus.