pH-EVD: A pH-Paper-Based Extraction and Visual Detection System for Instrument-Free SARS-CoV-2 Diagnostics.

The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of deaths worldwide. However, most SARS-CoV-2 detection methods depend on time-consuming sample preparation and large detection instruments. Herein, a method employing nonbleeding pH paper to achieve both RNA extraction and visual isothermal amplification is proposed, enabling rapid, instrument-free SARS-CoV-2 detection. By taking advantage of capillary forces, pH-paper-based RNA extraction can be accomplished within 1 min without need for any equipment. Further, the pH paper can mediate dye-free visual isothermal amplification detection. In less than a 46-min sample-to-answer time, pH-paper-based extraction and visual detection (termed pH-EVD) can consistently detect 1200 genome equivalents per microliter of SARS-CoV-2 in saliva, which is comparable to TaqMan probe-based quantitative reverse transcription PCR (RT-qPCR). Through coupling with a chemically heated incubator called a smart cup, the instrument-free, pH-EVD-based SARS-CoV-2 detection method on 30 nasopharyngeal swab samples and 33 contrived saliva samples is clinically validated. Thus, the pH-EVD method provides simple, rapid, reliable, low-cost, and instrument-free SARS-CoV-2 detection and has the potential to streamline onsite COVID-19 diagnostics.

Concurrent Lymphoma and COVID-19: Diagnostic and Therapeutic Challenges of High-Grade Lymphoma and COVID-19.

The coronavirus disease 2019 (COVID-19) global pandemic has put an unprecedented strain on cancer care. The initial months were marred by fears of immunocompromised patients becoming opportunistic hosts to this deadly virus. We present a case of newly diagnosed high-grade B-cell lymphoma in a patient with COVID-19 and discuss the diagnostic and therapeutic challenges posed. A 76-year-old female presented with one month of progressive malaise, poor appetite, weight loss, and night sweats. A surveillance COVID-19 polymerase chain reaction (PCR) resulted positive. With strict isolation precautions, the daily focused physical examination masked several key findings including multifocal adenopathy. She developed hypoxic respiratory failure and progressive transaminitis and cytopenias. Image-guided, rather than excisional, biopsy revealed high-grade B-cell lymphoma. Superimposed COVID-19 infection presented multiple challenges, but she completed treatment and achieved remission. Suspicion for underlying malignancy was high. Institutional concerns included obtaining imaging studies and the gold standard excisional tissue biopsy while maintaining acceptable staff exposure. Fortunately, a lymph node core biopsy confirmed the histopathological diagnosis of high-grade B-cell lymphoma. The administration of chemoimmunotherapy (rituximab, cyclophosphamide, doxorubicin, dose-reduced vincristine, and prednisone (R-CHOP)) posed inherent risks, notably, worsening cytopenias and hepatotoxicity. The approach to treatment was further complicated as the interaction of high-grade lymphoma and COVID-19 remained unclear. Medical teams have faced delays executing formerly routine diagnostic studies and formulating timely and appropriate treatment strategies. Careful consideration of risks and benefits must be weighed. A multidisciplinary approach is crucial to successfully treat patients. The relationship between COVID-19 and cancer treatment is yet to be established, and large sample-size studies are required.

Instrument-free, CRISPR-based diagnostics of SARS-CoV-2 using self-contained microfluidic system.

Rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for early diagnostics and timely medical treatment of coronavirus disease 2019 (COVID-19). However, current detection methods typically rely on expensive and bulky instrumentation. Here, we developed a simple, sensitive, instrument-free, CRISPR-based diagnostics of SARS-CoV-2 using a self-contained microfluidic system. The microfluidic chip integrates isothermal amplification, CRISPR cleavage, and lateral flow detection in a single, closed microfluidic platform, enabling contamination-free, visual detection. To simplify the operation and transportation of the device, we lyophilized the CRISPR reagents in the reaction chamber and pre-stored the liquid solutions in blisters. We employed a low-cost, portable hand warmer to incubate the microfluidic chip without the need for electricity. The self-contained microfluidic system can detect down to 100 copies of SARS-CoV-2 RNA. Further, we clinically validated our method by detecting 24 COVID-19 clinical nasopharyngeal swab samples, achieving excellent sensitivity (94.1%), specificity (100%), and accuracy (95.8%). This simple, sensitive, and affordable microfluidic system represents a promising tool for point-of-care diagnostics of COVID-19 and other infectious diseases.

Autonomous lab-on-paper for multiplexed, CRISPR-based diagnostics of SARS-CoV-2.

The COVID-19 pandemic, caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), has become a public health emergency and widely spread around the world. Rapid, accurate and early diagnosis of COVID-19 infection plays a crucial role in breaking this pandemic. However, the detection accuracy is limited for current single-gene diagnosis of SARS-CoV-2. Herein, we develop an autonomous lab-on-paper platform for multiplex gene diagnosis of SARS-CoV-2 by combining reverse transcription recombinase polymerase amplification (RT-RPA) and CRISPR-Cas12a detection. The autonomous lab-on-paper is capable of simultaneously detecting nucleoprotein (N) gene and spike (S) gene of SARS-CoV-2 virus as well as human housekeeping RNAse P gene (an internal control) in a single clinical sample. With the developed platform, 102 copies viral RNA per test can be detected within one hour. Also, the lab-on-paper platform has been used to detect 21 swab clinical samples and obtains a comparable performance to the conventional RT-PCR method. Thus, the developed lab-on-paper platform holds great potential for rapid, sensitive, reliable, multiple molecular diagnostics of COVID-19 and other infectious diseases in resource-limited settings.

Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay.

The recent outbreak of novel coronavirus (SARS-CoV-2) causing COVID-19 disease spreads rapidly in the world. Rapid and early detection of SARS-CoV-2 facilitates early intervention and prevents the disease spread. Here, we present an All-In-One Dual CRISPR-Cas12a (AIOD-CRISPR) assay for one-pot, ultrasensitive, and visual SARS-CoV-2 detection. By targeting SARS-CoV-2's nucleoprotein gene, two CRISPR RNAs without protospacer adjacent motif (PAM) site limitation are introduced to develop the AIOD-CRISPR assay and detect the nucleic acids with a sensitivity of few copies. We validate the assay by using COVID-19 clinical swab samples and obtain consistent results with RT-PCR assay. Furthermore, a low-cost hand warmer (~$0.3) is used as an incubator of the AIOD-CRISPR assay to detect clinical samples within 20 min, enabling an instrument-free, visual SARS-CoV-2 detection at the point of care. Thus, our method has the significant potential to provide a rapid, sensitive, one-pot point-of-care assay for SARS-CoV-2.

Mild COVID-19 infection despite chronic B cell depletion in a patient with aquaporin-4-positive neuromyelitis optica spectrum disorder.

Coronavirus disease 2019 (COVID-19) is caused by the novel coronavirus SARS-CoV-2, which affects the lung and other organs. After an incubation period of 3-14 days, the infection presents with symptoms of variable severity, from mild flu-like disease to severe pneumonia and cytokine storm with increased mortality. Immunosuppressed patients may have higher risk of adverse outcomes; hence, there is an urgent need to evaluate the immune response and clinical outcomes of SARS-CoV-2 infection in these patients. Here, we report a 59-year-old woman with aquaporin-4-positive (AQPR4+) neuromyelitis Optica treated with rituximab who developed mild respiratory symptoms with COVID-19, despite B cell depletion at the time of infection.

GRK2 enforces androgen receptor dependence in the prostate and prostate tumors.

Metastatic tumors that have become resistant to androgen deprivation therapy represent the major challenge in treating prostate cancer. Although these recurrent tumors typically remain dependent on the androgen receptor (AR), non-AR-driven tumors that also emerge are particularly deadly and becoming more prevalent. Here, we present a new genetically engineered mouse model for non-AR-driven prostate cancer that centers on a negative regulator of G protein-coupled receptors that is downregulated in aggressive human prostate tumors. Thus, prostate-specific expression of a dominant-negative G protein-coupled receptor kinase 2 (GRK2-DN) transgene diminishes AR and AR target gene expression in the prostate, and confers resistance to castration-induced involution. Further, the GRK2-DN transgene dramatically accelerates oncogene-initiated prostate tumorigenesis by increasing primary tumor size, potentiating visceral organ metastasis, suppressing AR, and inducing neuroendocrine marker mRNAs. In summary, GRK2 enforces AR-dependence in the prostate, and the loss of GRK2 function in prostate tumors accelerates disease progression toward the deadliest stage.

Chylous Ascites: A Review of Pathogenesis, Diagnosis and Treatment.

Chylous ascites (CA) is a rare form of ascites that results from the leakage of lipid-rich lymph into the peritoneal cavity. This usually occurs due to trauma and rupture of the lymphatics or increased peritoneal lymphatic pressure secondary to obstruction. The underlying etiologies for CA have been classified as traumatic, congenital, infectious, neoplastic, postoperative, cirrhotic or cardiogenic. Since malignancy and cirrhosis account for about two-thirds of all the cases of CA in Western countries, in this article we have attempted to reclassify CA based on portal and non-portal etiologies. The diagnosis of CA is based on the distinct characteristic of the ascitic fluid which includes a milky appearance and a triglyceride level of >200 mg/dL. The management consists of identifying and treating the underlying disease process, dietary modification, and diuretics. Some studies have also supported the use of agents such as orlistat, somatostatin, octreotide and etilefrine. Paracentesis and surgical interventions in the form of transjugular intrahepatic portosystemic shunt (commonly known as TIPS), peritoneal shunt, angiography with embolization of a leaking vessel, and laparotomy remain as treatment options for cases refractory to medical management.

Primary Follicular Lymphoma of the Gastrointestinal Tract: Case Report and Review.

PURPOSE: Primary gastrointestinal follicular lymphoma (GI-FL) is considered a rare disease with fewer than 400 cases reported in the literature. It accounts for roughly 1-3 % of GI non-Hodgkins lymphomas (NHL). It originates in the GI tract and typically affects small bowel. The disease has an indolent course, and a prolonged survival can be expected in most cases. Due to its rarity, an optimal diagnostic work up and treatment plan has not been well established. METHODS: Endoscopic evaluation of the entire gastrointestinal (GI) tract using esophagogastroduodenoscopy (EGD), wireless capsule endoscopy, double-balloon enteroscopy, colonoscopy, and whole body examination with fluorodeoxyglucose-positron emission tomography (FDG-PET) has been suggested to more accurately stage the disease and guide the treatment plan. RESULTS: Treatment options for GI follicular lymphoma include watch and wait strategy, surgery, chemotherapy, radiation, immuno-radiotherapy, or a combination of these modalities. CONCLUSION: In this article, we have summarized the existing information regarding clinical presentation, diagnostic evaluation, and treatment options for this rare entity after presenting a case of GI-FL who was diagnosed during an EGD for evaluation of belching, heartburn, and weight loss.

Splenic morphological changes are accompanied by altered baseline immunity in a mouse model of sickle-cell disease.

Although functional asplenia from infarctions may be a major contributor to increased infectious mortality in sickle-cell disease (SCD), this relationship has not been fully defined. We used the transgenic Berkeley SCD mouse to define blood and splenic immunophenotypic differences in this model compared with C57BL/6 and hemizygous controls. In the serum of SCD mice, we found increased IgG2a and suppressed IgM, IgG2b, and IgA levels. Serum IL-6 levels in SCD mice were elevated, whereas IL-1α, CXCL10, and CCL5 levels were decreased. The blood of SCD mice had higher white blood cell counts, with an increased percentage of lymphocytes and decreases in other leukocytes. Immunophenotyping of lymphocytes revealed higher percentages of CD8(+) and T-regulatory cells and lower percentages of B cells. SCD mouse spleens exhibited histological disorganization, with reduction of defined lymphoid follicles and expansion of red pulp, a greater than fourfold increase in splenic mononuclear cells, marked expansion of the nucleated red blood cell fraction, and B-cell and CD8(+) T-cell lymphopenia. Within the splenic B-cell population, there was a significant decrease in B-1a B cells, with a corresponding decrease in IgA secreting plasma cells in the gut. Confocal microscopy of spleens demonstrated complete disruption of the normal lymphofollicular structure in the white pulp of SCD mice without distinct B, T, and marginal zones. Our findings suggest that altered SCD splenic morphological characteristics result in an impaired systemic immune response.

An 18-year-old woman with Kabuki syndrome, immunoglobulin deficiency and granulomatous lymphocytic interstitial lung disease.

Granulomatous lymphocytic interstitial lung disease, or GLILD, is an uncommon condition associated with common variable immunodeficiency (CVID). We present an interesting case of an 18-year-old woman with Kabuki syndrome and CVID who was seen in our clinic for an abnormal chest CT scan. She was subsequently diagnosed with GLILD. There are no established guidelines for the treatment of GLILD in CVID. Immune globulin replacement therapy is the main treatment for CVID and higher doses of intravenous immunoglobulin (IVIG) may prevent the progression of chronic lung disease. Patients with CVID and GLILD are at increased risk for malignancy and their prognosis is worse compared to patients with CVID without GLILD.