The ongoing COVID-19 pandemic has caused an unprecedented need for rapid diagnostic testing. The Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) recommend a standard assay that includes an RNA extraction step from a nasopharyngeal (NP) swab followed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect the purified SARS-CoV-2 RNA. The current global shortage of RNA extraction kits has caused a severe bottleneck to COVID-19 testing. We hypothesized that SARS-CoV-2 RNA could be detected from NP samples via a direct RT-qPCR assay that omits the RNA extraction step altogether, and tested this hypothesis on a series of blinded clinical samples. The direct RT-qPCR approach correctly identified 92% of NP samples (n = 155) demonstrated to be positive for SARS-CoV-2 RNA by traditional clinical diagnostic RT-qPCR that included an RNA extraction. Thus, direct RT-qPCR could be a front-line approach to identify the substantial majority of COVID-19 patients, reserving a repeat test with RNA extraction for those individuals with high suspicion of infection but an initial negative result. This strategy would drastically ease supply chokepoints of COVID-19 testing and should be applicable throughout the world. MAINThe ongoing COVID-19 pandemic has put exceptional strain on public health laboratories, hospital laboratories, and commercial laboratories as they attempt to keep up with demands for SARS-CoV-2 testing. The current diagnostic testing methods recommended by the Centers for Disease Control and Prevention (CDC) in the United States and the World Health Organization (WHO) are traditional RT-qPCR assays that require two steps: first, an RNA extraction from patient nasopharyngeal (NP) swab material, followed by RT-qPCR amplification of the extracted RNA to detect viral RNA 1-3 . The major bottleneck to widespread SARS-CoV-2 testing lies at the RNA extraction step. The simplest manual kit (the Qiagen Viral RNA Mini) is no longer available, and reagents and supplies for the larger automated instruments are extremely limited with uncertain supply chains. While substitution of other RNA extraction kits 4,5 is possible, they too are in limited supply. The current bottleneck is not simply the availability of RNA extraction kits, but also the cost of the extraction assay, the labor and time required to perform it, and the fact that it is rate limiting compared to the downstream RT-qPCR analysis. To address this issue, we tested the unconventional approach of skipping the RNA extraction step altogether and directly loading patient swab material into the RT-qPCR mix. Herein, we report that this approach (which we refer to hereafter as "direct RT-qPCR") correctly identified 92% of samples (n =155) previously shown to be positive for SARS-CoV-2 RNA by conventional RT-qPCR featuring an RNA extraction. Thus, our results suggest that this streamlined assay could greatly alleviate constraints to COVID-19 testing in many regions of the world.

The ongoing COVID-19 pandemic has created an unprecedented need for rapid diagnostic testing. The World Health Organization (WHO) recommends a standard assay that includes an RNA extraction step from a nasopharyngeal (NP) swab followed by reverse transcription–quantitative polymerase chain reaction (RT-qPCR) to detect the purified SARS-CoV-2 RNA. The current global shortage of RNA extraction kits has caused a severe bottleneck to COVID-19 testing. The goal of this study was to determine whether SARS-CoV-2 RNA could be detected from NP samples via a direct RT-qPCR assay that omits the RNA extraction step altogether. The direct RT-qPCR approach correctly identified 92% of a reference set of blinded NP samples ( n = 155) demonstrated to be positive for SARS-CoV-2 RNA by traditional clinical diagnostic RT-qPCR that included an RNA extraction. Importantly, the direct method had sufficient sensitivity to reliably detect those patients with viral loads that correlate with the presence of infectious virus. Thus, this strategy has the potential to ease supply choke points to substantially expand COVID-19 testing and screening capacity and should be applicable throughout the world.

Understanding the DNA elements that constitute and control the regulatory genome is critical for the appropriate therapeutic management of complex diseases. Here, using chromosome Y (ChrY) consomic mouse strains on the C57BL/6J (B6) background, we show that susceptibility to two diverse animal models of autoimmune disease, experimental allergic encephalomyelitis (EAE) and experimental myocarditis, correlates with the natural variation in copy number of Sly and Rbmy multicopy ChrY genes. On the B6 background, ChrY possesses gene regulatory properties that impact genome-wide gene expression in pathogenic CD4+ T cells. Using a ChrY consomic strain on the SJL background, we discovered a preference for ChrY-mediated gene regulation in macrophages, the immune cell subset underlying the EAE sexual dimorphism in SJL mice, rather than CD4+ T cells. Importantly, in both genetic backgrounds, an inverse correlation exists between the number of Sly and Rbmy ChrY gene copies and the number of significantly up-regulated genes in immune cells, thereby supporting a link between copy number variation of Sly and Rbmy with the ChrY genetic element exerting regulatory properties. Additionally, we show that ChrY polymorphism can determine the sexual dimorphism in EAE and myocarditis. In humans, an analysis of the CD4+ T cell transcriptome from male multiple sclerosis patients versus healthy controls provides further evidence for an evolutionarily conserved mechanism of gene regulation by ChrY. Thus, as in Drosophila, these data establish the mammalian ChrY as a member of the regulatory genome due to its ability to epigenetically regulate genome-wide gene expression in immune cells.

Objective-Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS), characterized by a global increasing incidence driven by relapsingremitting disease in females. p38 MAP kinase (MAPK) has been described as a key regulator of inflammatory responses in autoimmunity, but its role in the sexual dimorphism in MS or MS models remains unexplored.Methods-Toward this end, we used experimental autoimmune encephalomyelitis (EAE), the principal animal model of MS, combined with pharmacologic and genetic inhibition of p38 MAPK activity and transcriptomic analyses.Results-Pharmacologic inhibition of p38 MAPK selectively ameliorated EAE in female mice. Conditional deletion studies demonstrated that p38α signaling in macrophages/myeloid cells, but not T cells or dendritic cells, recapitulated this sexual dimorphism. Analysis of CNS inflammatory infiltrates showed that female, but not male mice lacking p38α in myeloid cells exhibited reduced immune cell activation compared with controls, while peripheral T cell priming was unaffected in both sexes. Transcriptomic analyses of myeloid cells revealed differences in p38α-controlled transcripts comprising female-and male-specific gene modules, with greater p38α dependence of pro-inflammatory gene expression in females.Interpretation-Our findings demonstrate a key role for p38α in myeloid cells in CNS autoimmunity and uncover important molecular mechanisms underlying sex differences in disease pathogenesis. Taken together, our results suggest that the p38 MAPK signaling pathway represents a novel target for much needed disease modifying therapies for MS.

BackgroundCoxsackievirus B3 (CVB3) induces myocarditis, an inflammatory heart disease, which affects men more than women. Toll-like receptor (TLR) signaling has been shown to determine the severity of CVB3-induced myocarditis. No direct role for signaling through TLR2 had been shown in myocarditis although published studies show that cardiac myosin is an endogenous TLR2 ligand and stimulates pro-inflammatory cytokine expression by dendritic cells in vitro. The goal of this study is to determine which TLRs show differential expression in CVB3 infected mice corresponding to male susceptibility and female resistance in this disease.MethodsMale and female C57Bl/6 mice were infected with 102 PFU CVB3 and killed on day 3 or 6 post infection. Hearts were evaluated for virus titer, myocardial inflammation, and TLR mRNA expression by PCR array and microarray analysis. Splenic lymphocytes only were evaluated by flow cytometry for the number of TLR+/CD3+, TLR+/CD4+, TLR+F4/80+ and TLR+/CD11c+ subpopulations and the mean fluorescence intensity to assess upregulation of TLR expression on these cells. Mice were additionally treated with PAM3CSK4 (TLR2 agonist) or ultrapure LPS (TLR4 agonist) on the same day as CVB3 infection or 3 days post infection to confirm their role in myocarditis susceptibility.ResultsDespite equivalent viral titers, male C57Bl/6 mice develop more severe myocarditis than females by day 6 after infection. Microarray analysis shows a differential expression of TLR2 at day 3 with female mice having higher levels of TLR2 gene expression compared to males. Disease severity correlates to greater TLR4 protein expression on splenic lymphocytes in male mice 3 days after infection while resistance in females correlates to preferential TLR2 expression, especially in spleen lymphocytes. Treating male mice with PAM reduced mortality from 55% in control CVB3 infected animals to 10%. Treating female mice with LPS increased mortality from 0% in control infected animals to 60%.ConclusionCVB3 infection causes an up-regulation of TLR2 in female and of TLR4 in male mice and this differential expression between the sexes contributes to disease resistance of females and susceptibility of males. While previous reports demonstrated a pathogenic role for TLR4 this is the first report that TLR2 is preferentially up-regulated in CVB3 infected female mice or that signaling through this TLR directly causes myocarditis resistance.