Establishment of SARS-CoV-2 genomic surveillance within the Military Health System during 1 March-31 December 2020.

This report describes SARS-CoV-2 genomic surveillance conducted by the Department of Defense (DoD) Global Emerging Infections Surveillance Branch and the Next-Generation Sequencing and Bioinformatics Consortium (NGSBC) in response to the COVID-19 pandemic. Samples and sequence data were from SARS-CoV-2 infections occurring among Military Health System (MHS) beneficiaries from 1 March to 31 December 2020. There were 1,366 MHS samples sequenced from 10 countries, 36 U.S states or territories, and 5 Geographic Combatant Commands, representing approximately 2% of DoD cases in 2020. Genomes from these samples were compared with other public sequences; observed trends were similar to those of Centers for Disease Control and Prevention national surveillance in the U.S. with B.1, B.1.2, and other sub-lineages comprising the dominant variants of SARS-CoV-2. Sequence data were used to monitor transmission dynamics on U.S. Navy ships and at military training centers and installations. As new variants emerge, DoD medical and public health practitioners should maximize the use of genomic surveillance resources within DoD to inform force health protection measures.

Mass COVID-19 patient screening using UvsX and UvsY mediated DNA recombination and high throughput parallel sequencing.

The Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), also known as 2019 novel coronavirus (2019-nCoV), is a highly infectious RNA virus. A percentage of patients develop coronavirus disease 2019 (COVID-19) after infection, whose symptoms include fever, cough, shortness of breath and fatigue. Acute and life-threatening respiratory symptoms are experienced by 10-20% of symptomatic patients, particularly those with underlying medical conditions. One of the main challenges in the containment of COVID-19 is the identification and isolation of asymptomatic/pre-symptomatic individuals. A number of molecular assays are currently used to detect SARS-CoV-2. Many of them can accurately test hundreds or even thousands of patients every day. However, there are presently no testing platforms that enable more than 10,000 tests per day. Here, we describe the foundation for the REcombinase Mediated BaRcoding and AmplificatioN Diagnostic Tool (REMBRANDT), a high-throughput Next Generation Sequencing-based approach for the simultaneous screening of over 100,000 samples per day. The REMBRANDT protocol includes direct two-barcoded amplification of SARS-CoV-2 and control amplicons using an isothermal reaction, and the downstream library preparation for Illumina sequencing and bioinformatics analysis. This protocol represents a potentially powerful approach for community screening of COVID-19 that may be modified for application to any infectious or non-infectious genome.

Early Identification of SARS-CoV-2 Emergence in the Department of Defense via Retrospective Analysis of 2019-2020 Upper Respiratory Illness Samples.

The first U.S. case of non-travel-related severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection was detected in late February 2020 in California, but the prevailing delay in diagnostic testing and initial stringent testing criteria made it difficult to identify those who could have acquired the virus through community spread. The emergence of the virus in the western Pacific region in late 2019 and the global distribution of Department of Defense (DoD) personnel present the risk that DoD members may have been exposed and contracted the virus earlier then U.S. detections. Here, a retrospective study from residual samples collected from a global DoD Respiratory Surveillance Program was conducted to establish a tentative timeline of when this virus began circulating in the DoD population. Quantitative real-time reverse-transcription polymerase chain reaction testing for SARS-CoV-2 was performed and the specimen collection dates of positive results were compared to the dates of the first infections previously identified in respective states and counties. Twenty-four positive samples were identified out of approximately 7,000 tested. Although this retrospective testing found early cases in 8 locations, there were no results indicative of circulation before late February.

Multisystem Inflammatory Syndrome in Children in February 2020 and Implications of Genomic Sequencing for SARS-CoV-2.

Multisystem inflammatory syndrome in children (MIS-C) is a newly recognized disease process that can complicate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We present what we believe to be the earliest case of MIS-C, occurring in February 2020. Our patient's SARS-CoV-2 infection was caused by an emerging lineage with the D614G variant in the spike protein. This lineage would subsequently become the predominant cause of SARS-CoV-2 outbreaks in Europe and the United States where MIS-C was first described.

Differentiation of Francisella tularensis Subspecies and Subtypes.

The highly infectious and zoonotic pathogen Francisella tularensis is the etiologic agent of tularemia, a potentially fatal disease if untreated. Despite the high average nucleotide identity, which is >99.2% for the virulent subspecies and >98% for all four subspecies, including the opportunistic microbe Francisella tularensis subsp. novicida, there are considerable differences in genetic organization. These chromosomal disparities contribute to the substantial differences in virulence observed between the various F. tularensis subspecies and subtypes. The methods currently available to genotype F. tularensis cannot conclusively identify the associated subpopulation without using time-consuming testing or complex scoring matrices. To address this need, we developed both single and multiplex quantitative real-time PCR (qPCR) assays that can accurately detect and identify the hypervirulent F. tularensis subsp. tularensis subtype A.I, the virulent F. tularensis subsp. tularensis subtype A.II, F. tularensis subsp. holarctica (also referred to as type B), and F. tularensis subsp. mediasiatica, as well as opportunistic F. tularensis subsp. novicida from each other and near neighbors, such as Francisella philomiragia, Francisella persica, and Francisella-like endosymbionts found in ticks. These fluorescence-based singleplex and non-matrix scoring multiplex qPCR assays utilize a hydrolysis probe, providing sensitive and specific F. tularensis subspecies and subtype identification in a rapid manner. Furthermore, sequencing of the amplified F. tularensis targets provides clade confirmation and informative strain-specific details. Application of these qPCR- and sequencing-based detection assays will provide an improved capability for molecular typing and clinical diagnostics, as well as facilitate the accurate identification and differentiation of F. tularensis subpopulations during epidemiological investigations of tularemia source outbreaks.

A plasmid containing the human metallothionein II gene can function as an antibody-assisted electrophoretic biosensor for heavy metals.

Different forms of heavy metals affect biochemical systems in characteristic ways that cannot be detected with typical metal analysis methods like atomic absorption spectrometry. Further, using living systems to analyze interaction of heavy metals with biochemical systems can be laborious and unreliable. To generate a reliable easy-to-use biologically-based biosensor system, the entire human metallothionein-II (MT-II) gene was incorporated into a plasmid (pUC57-MT) easily replicated in Escherichia coli. In this system, a commercial polyclonal antibody raised against human metal-responsive transcription factor-1 protein (MTF-1 protein) could modify the electrophoretic migration patterns (i.e. cause specific decreases in agarose gel electrophoretic mobility) of the plasmid in the presence or absence of heavy metals other than zinc (Zn). In the study here, heavy metals, MTF-1 protein, and polyclonal anti-MTF-1 antibody were used to assess pUC57-MT plasmid antibody-assisted electrophoretic mobility. Anti-MTF-1 antibody bound both MTF-1 protein and pUC57-MT plasmid in a non-competitive fashion such that it could be used to differentiate specific heavy metal binding. The results showed that antibody-inhibited plasmid migration was heavy metal level-dependent. Zinc caused a unique mobility shift pattern opposite to that of other metals tested, i.e. Zn blocked the antibody ability to inhibit plasmid migration, despite a greatly increased affinity for DNA by the antibody when Zn was present. The Zn effect was reversed/modified by adding MTF-1 protein. Additionally, antibody inhibition of plasmid mobility was resistant to heat pre-treatment and trypsinization, indicating absence of residual DNA extraction-resistant bacterial DNA binding proteins. DNA binding by anti-DNA antibodies may be commonly enhanced by xenobiotic heavy metals and elevated levels of Zn, thus making them potentially effective tools for assessment of heavy metal bioavailability in aqueous solutions and fluid obtained from metal implant sites.

Disseminated vaccine-strain varicella as initial presentation of the acquired immunodeficiency syndrome: a case report and review of the literature.

Varicella-zoster virus (VZV) infections have declined in many industrialized countries due to vaccination with the attenuated Oka strain virus. Rare cases of severe, disseminated vaccine-strain VZV infection have occurred in the immunocompromised, although rarely in HIV-infected persons. We describe a man with previously-undiagnosed human immunodeficiency virus (HIV) infection who received VZV vaccination and subsequently presented to a combat hospital in Afghanistan with disseminated varicella, respiratory failure, and sepsis. The patient recovered with ventilator and hemodynamic support, intravenous acyclovir, and empiric antibiotic therapy. DNA sequencing detected Oka strain virus from patient blood specimens. Although safe in most populations, the VZV vaccine may cause life-threatening disease in immunocompromised patients. Improved detection of HIV infection may be useful in preventing such cases.

Imaging interactions of metal oxide nanoparticles with macrophage cells by ultra-high resolution scanning electron microscopy techniques.

Use of engineered metal oxide nanoparticles in a plethora of biological applications and custom products has warned about some possible dose-dependent cytotoxic effects. Macrophages are key components of the innate immune system used to study possible toxic effects and internalization of different nanoparticulate materials. In this work, ultra-high resolution field emission scanning electron microscopy (FE-SEM) was used to offer new insights into the dynamical processes of interaction of nanomaterials with macrophage cells dosed with different concentrations of metal oxide nanoparticles (CeO(2), TiO(2) and ZnO). The versatility of FE-SEM has allowed obtaining a detailed characterization of processes of adsorption and endocytosis of nanoparticles, by using advanced analytical and imaging techniques on complete unstained uncoated cells, including secondary electron imaging, high-sensitive backscattered electron imaging, X-ray microanalysis and stereoimaging. Low voltage BF/DF-STEM confirmed nanoparticle adsorption and internalization into endosomes of CeO(2) and TiO(2), whereas ZnO develop apoptosis after 24 h of interaction caused by dissolution and invasion of cell nucleus. Ultra-high resolution scanning electron microscopy techniques provided new insights into interactions of inorganic nanoparticles with macrophage cells with high spatial resolution.

Role of hyaluronidase in subcutaneous spread and growth of group A streptococcus.

Group A streptococcus (GAS) depends on a hyaluronic acid (HA) capsule to evade phagocytosis and to interact with epithelial cells. Paradoxically, GAS also produces hyaluronidase (Hyl), an enzyme that cleaves HA. A common assumption is that Hyl digests structurally identical HA in human tissue to promote bacterial spread. We inactivated the gene encoding extracellular hyaluronidase, hylA, in a clinical Hyl(+) isolate. Hyl(+) and an isogenic Hyl(-) mutant were injected subcutaneously into mice with or without high-molecular-weight dextran blue. The Hyl(-) strain produced small lesions with dye concentrated in close proximity. The Hyl(+) strain produced identical lesions, but the dye diffused subcutaneously. However, Hyl(+) bacteria were not isolated from unaffected skin stained by dye diffusion. Thus, Hyl digests tissue HA and facilitates spread of large molecules but is not sufficient to cause subcutaneous diffusion of bacteria or to affect lesion size. GAS capsule expression was assayed periodically during broth culture and was reduced in Hyl(+) strains relative to Hyl(-) strains at the onset and the end of active capsule synthesis but not during peak synthesis in mid-exponential phase. Thus, Hyl is not sufficiently active to remove capsule during peak synthesis. To demonstrate a possible nutritional role for Hyl, GAS was shown to grow with N-acetylglucosamine but not d-glucuronic acid (both components of HA) as a sole carbon source. However, only Hyl(+) strains could grow utilizing HA as a sole carbon source, suggesting that Hyl may permit the organism to utilize host HA or its own capsule as an energy source.