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.

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.

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 (CeO2, TiO2 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 CeO2 and TiO2, 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.

Background The FLU-PRO Plus is a patient-reported outcome data collection instrument assessing symptoms of viral respiratory tract infections across eight body systems. This study evaluated the measurement properties of FLU-PRO Plus in a study enrolling individuals with COVID-19. Methods Data from a prospective cohort study (EPICC) in US Military Health System (MHS) beneficiaries evaluated for COVID-19 was utilized. Adults with symptomatic SARS-CoV-2 infection with FLU-PRO Plus survey information within one week of symptom onset were included. Reliability of FLU-PRO Plus was estimated using intraclass correlation coefficients (ICC; 2 days reproducibility). Known-groups validity was assessed using patient global assessments (PGA) of disease severity. Patient report of return to usual health was used to assess responsiveness (day 1-6/7). Results 226 SARS-CoV-2 positive participants were included in the analysis. Reliability among those who reported no change in their symptoms from one day to the next was high for most domains (ICC range 0.68-0.94 for day 1 to day 2). Construct validity was demonstrated by moderate to high correlation between the PGA rating of disease severity and domain and total scores (e.g., total scores correlation: 0.69 (influenza-like illness severity), 0.69 (interference in daily activities), and -0.58 (physical health)). In addition, FLU-PRO Plus demonstrated good known-groups validity, with increasing domain and total scores observed with increasing severity ratings. Conclusions FLU-PRO Plus performs well in measuring signs and symptoms in SARS-CoV-2 infection with excellent construct validity, known-groups validity, and responsiveness to change. Standardized data collection instruments facilitate meta-analyses, vaccine effectiveness studies, and other COVID-19 research activities.

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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.

The first case of non-travel related SARS-COV-2 was detected late February in California, however initially stringent testing criteria and subsequent delay of diagnostic testing made it difficult to identify those who could have acquired it through community spread. Early speculation was that the virus may have been circulating from at least January [1], and environmental sampling suggests that versions of this virus were found months before the first human samples were identified [2]. Here we performed a retrospective study from residual samples collected from a global respiratory surveillance effort to establish a tentative timeline by which this virus was circulating in the US military population. We performed RT-PCR for SARS-COV-2 and compared the dates of these cases to the first cases identified in respective states and counties using the Johns Hopkins COVID tracker website. Twenty-four positive samples were identified out of approximately 7,000 tested. Although we found some early cases in certain regions, we did not see circulation before late February in samples collected both in the US and outside the USA.

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.