Recent research suggests that SARS-CoV-2-infected individuals can be highly infectious while asymptomatic or pre-symptomatic, and that an infected person may infect 5.6 other individuals on average. This situation highlights the need for rapid, sensitive SARS-CoV-2 diagnostic assays capable of high-throughput operation that can preferably utilize existing equipment to facilitate broad, large-scale screening efforts. We have developed a CRISPR-based assay that can meet all these criteria. This assay utilizes a custom CRISPR Cas12a/gRNA complex and a fluorescent probe to detect target amplicons produced by standard RT-PCR or isothermal recombinase polymerase amplification (RPA), to allow sensitive detection at sites not equipped with real-time PCR systems required for qPCR diagnostics. We found this approach allowed sensitive and robust detection of SARS-CoV-2 positive samples, with a sample-to-answer time of ~50 min, and a limit of detection of 2 copies per sample. CRISPR assay diagnostic results obtained nasal swab samples of individuals with suspected COVID-19 cases were comparable to paired results from a CDC-approved quantitative RT-PCR (RT-qPCR) assay performed in a state testing lab, and superior to those produced by same assay in a clinical lab, where the RT-qPCR assay exhibited multiple invalid or inconclusive results. Our assay also demonstrated greater analytical sensitivity and more robust diagnostic performance than other recently reported CRISPR-based assays. Based on these findings, we believe that a CRISPR-based fluorescent application has potential to improve current COVID-19 screening efforts.

Platelets can bind and phagocytose infectious microorganisms and so enable their transport for a prolonged time. To investigate the subcellular events of these interactions, platelets were incubated either with Staphylococcus aureus or with HIV and analyzed by electron microscopy (EM) and immuno-EM. HIV and bacteria internalization occurred exclusively within platelets showing morphological evidence of activation. Platelet activation enhanced the degree of bacterial internalization. Immunolabeling revealed that the engulfing vacuoles and the open canalicular system (OCS) were composed of distinct antigens. The engulfing vacuoles eventually became the site of prominent alpha-granule release. In platelets incubated with HIV, characteristic endocytic vacuoles were identified close to the plasma membrane, tightly surrounding 1 or 2 HIV particles. Virus particles were also located within the OCS. Immunogold labeling for the viral core protein p24 confirmed the presence of HIV within platelets. Finally, examination of platelets from a patient with acquired immunodeficiency syndrome and high viremia suggested that HIV endocytosis may also occur in vivo.

Idiopathic myelofibrosis (MF) is a myeloproliferative syndrome characterized by an increase in bone marrow collagen. Megakaryocytes (Mks), which store growth factors in their α granules, are known to be involved in the pathogenesis of MF. Previously, mice given bone marrow grafts infected with a retrovirus carrying murine thrombopoietin (TPO) complementary DNA developed a disease resembling human idiopathic MF. In this study, we used this murine model (TPO mice) to determine whether release of α granules is responsible for fibroblast activation and development of fibrosis. The intracellular trafficking of several α-granule proteins (von Willebrand factor, fibrinogen, and transforming growth factor β (TGFβ), which are stored in the granule matrix; and αIIbβ3 integrin and P-selectin (CD62p), which are located in the α-granule membrane) was studied with immune electron microscopy in bone marrow Mks from TPO mice. P-selectin immunolabeling increased consistently and was occasionally found lining the demarcation membrane system. Evidence of extensive emperipolesis was also found in TPO mouse Mks, involving almost exclusively neutrophil and eosinophil polymorphonuclear (PMN) cells with altered morphologic features. In parallel, the host Mks had myeloperoxidase-positive granules scattered in their cytoplasm, associated with marked ultrastructural cytoplasmic alterations and ruptured α-granule membranes. Similar observations were made in bone marrow biopsy specimens from 12 patients with idiopathic MF; indeed, there was an increased rate of emperipolesis involving mostly PMN cells, abnormal P-selectin expression, and mutual subcellular PMN and Mk alterations. This study indicates that in idiopathic MF, abnormal P-selectin distribution in Mks induces selective sequestration of PMN cells. This results in a release of α-granular proteins and growth factors, which in turn induces fibroblast activation and fibrosis deposition.