Cell-free DNA methylation analysis as a marker of malignancy in pleural fluid.

Diagnosis of malignant pleural effusion (MPE) is made by cytological examination of pleural fluid or histological examination of pleural tissue from biopsy. Unfortunately, detection of malignancy using cytology has an overall sensitivity of 50%, and is dependent upon tumor load, volume of fluid assessed, and cytopathologist experience. The diagnostic yield of pleural fluid cytology is also compromised by low abundance of tumor cells or when morphology is obscured by inflammation or reactive mesothelial cells. A reliable molecular marker that may complement fluid cytology for the diagnosis of malignant pleural effusion is needed. The purpose of this study was to establish a molecular diagnostic approach based on pleural effusion cell-free DNA methylation analysis for the differential diagnosis of malignant pleural effusion and benign pleural effusion. This was a blind, prospective case-control biomarker study. We recruited 104 patients with pleural effusion for the study. We collected pleural fluid from patients with: MPE (n = 48), indeterminate pleural effusion in subjects with known malignancy or IPE (n = 28), and benign PE (n = 28), and performed the Sentinel-MPE liquid biopsy assay. The methylation level of Sentinel-MPE was markedly higher in the MPE samples compared to BPE control samples (p < 0.0001) and the same tendency was observed relative to IPE (p = 0.004). We also noted that the methylation signal was significantly higher in IPE relative to BPE (p < 0.001). We also assessed the diagnostic efficiency of the Sentinel-MPE test by performing receiver operating characteristic analysis (ROC). For the ROC analysis we combined the malignant and indeterminate pleural effusion groups (n = 76) and compared against the benign group (n = 28). The detection sensitivity and specificity of the Sentinel-MPE test was high (AUC = 0.912). The Sentinel-MPE appears to have better performance characteristics than cytology analysis. However, combining Sentinel-MPE with cytology analysis could be an even more effective approach for the diagnosis of MPE. The Sentinel-MPE test can discriminate between BPE and MPE. The Sentinel-MPE liquid biopsy test can detect aberrant DNA in several different tumor types. The Sentinel-MPE test can be a complementary tool to cytology in the diagnosis of MPE.

Late Onset of Rivaroxaban-Associated Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis.

Although anti-thyroid drugs (ATDs) are the most common cause of drug-associated anti-neutrophil cytoplasmic antibody (ANCA) vasculitis (AAV), many other classes of drugs can lead to drug-associated AAV. We present a unique case of rivaroxaban-associated AAV. A 76-year-old female with a past medical history of atrial fibrillation on rivaroxaban presented with fatigue, bilateral lower extremity purpura, and hemoptysis to an outside hospital. Investigations revealed a positive cytoplasmic-ANCA (c-ANCA) titer of 1:320 and a positive anti-myeloperoxidase (anti-MPO), and negative perinuclear-ANCA (p-ANCA) and anti-proteinase 3 (anti-PR3). In addition, chest imaging demonstrated bilateral ground-glass opacities which raised suspicion for diffuse alveolar hemorrhage (DAH). A lung biopsy revealed acute and ongoing DAH with focally active capillaritis and characteristic pathological findings, which strongly suggested that was likely secondary to rivaroxaban. Rivaroxaban was discontinued, and the patient received pulses of intravenous glucocorticosteroids and rituximab. Her symptoms improved. She continued immunosuppressive therapy with rituximab for 2 years. She presented to our hospital for a second opinion regarding the discontinuation of rituximab, and we decided to discontinue rituximab. After discontinuation, the patient remained stable after 1.5 years of follow-up and did not have any relapses. This is a unique case of rivaroxaban-associated AAV. Clinicians should consider drug-associated AAV in all patients who present with an atypical clinical presentation and/or pathological findings of AAV. Given the broad and rapidly increasing use of novel anticoagulants, it is important to raise awareness of this potential complication. Prompt discontinuation of the drug and initiation of immunosuppressant treatment in severe cases may be lifesaving.

Cell-Free DNA Methylation Analysis as a Marker of Malignancy in Pleural Fluid.

Background: Diagnosis of malignant pleural effusion (MPE) is made by cytological examination of pleural fluid or histological examination of pleural tissue from biopsy. Unfortunately, detection of malignancy using cytology has an overall sensitivity of 50%, and is dependent upon tumor load, volume of fluid assessed, and cytopathologist experience. The diagnostic yield of pleural fluid cytology is also compromised by low abundance of tumor cells or when morphology is obscured by inflammation or reactive mesothelial cells. A reliable molecular marker that may complement fluid cytology malignant pleural effusion diagnosis is needed. The purpose of this study was to establish a molecular diagnostic approach based on pleural effusion cell-free DNA methylation analysis for the differential diagnosis of malignant pleural effusion and benign pleural effusion. Results: This was a blind, prospective case-control biomarker study. We recruited 104 patients with pleural effusion for the study. We collected pleural fluid from patients with: MPE (n = 48), PPE (n = 28), and benign PE (n = 28), and performed the Sentinel-MPE liquid biopsy assay. The methylation level of Sentinel-MPE was markedly higher in the MPE samples compared to BPE control samples (p < 0.0001) and the same tendency was observed relative to PPE (p = 0.004). We also noted that the methylation signal was significantly higher in PPE relative to BPE (p < 0.001). We also assessed the diagnostic efficiency of the Sentinel-MPE test by performing receiver operating characteristic analysis (ROC). For the ROC analysis we combined the malignant and paramalignant groups (n = 76) and compared against the benign group (n = 28). The detection sensitivity and specificity of the Sentinel-MPE test was high (AUC = 0.912). The Sentinel-MPE appears to have better performance characteristics than cytology analysis. However, combining Sentinel-MPE with cytology analysis could be an even more effective approach for the diagnosis of MPE. Conclusions: The Sentinel-MPE test can discriminate between BPE and MPE. The Sentinel-MPE liquid biopsy test can detect aberrant DNA in several different tumor types. The Sentinel-MPE test can be a complementary tool to cytology in the diagnosis of MPE.

SARS-CoV-2 Rapid Antigen Testing of Symptomatic and Asymptomatic Individuals on the University of Arizona Campus.

SARS-CoV-2, the cause of COVID19, has caused a pandemic that has infected more than 80 M and killed more than 1.6 M persons worldwide. In the US as of December 2020, it has infected more than 32 M people while causing more than 570,000 deaths. As the pandemic persists, there has been a public demand to reopen schools and university campuses. To consider these demands, it is necessary to rapidly identify those individuals infected with the virus and isolate them so that disease transmission can be stopped. In the present study, we examined the sensitivity of the Quidel Rapid Antigen test for use in screening both symptomatic and asymptomatic individuals at the University of Arizona from June to August 2020. A total of 885 symptomatic and 1551 asymptomatic subjects were assessed by antigen testing and real-time PCR testing. The sensitivity of the test for both symptomatic and asymptomatic persons was between 82 and 90%, with some caveats.

An Unusual Case of Chronic Hemoptysis.

CASE PRESENTATION: A 62-year-old African American man was admitted to the hospital with hemoptysis. He had a complicated medical history significant for active tobacco use (>50 pack-year history), coronary artery disease, and heart failure with reduced ejection fraction. He reported intermittent episodes of coughing up streaks of blood in the sputum for the past 3 years. For the past few days before this presentation, he had multiple episodes of coughing up over a tablespoon of only blood. He was not on any anticoagulant agents. There were no risk factors for TB, nor was there a history of fevers, chills, shortness of breath, leg swelling, changes in his urine color and frequency or urgency, or unintended weight loss. On admission, he was noted to be breathing comfortably. Vital signs revealed a temperature of 36.6ºC, BP of 138/70 mm Hg, heart rate of 66 beats/min, respiratory of rate of 18 breaths/min, and a blood oxygen saturation level of 98% on room air. Physical examination was significant for decreased bilateral breath sounds with no wheezing, crackles, or rhonchi. Cardiovascular examination revealed normal cardiac rhythm without murmur, rubs, or gallops. There was no clubbing or edema on his extremities.

Orthogonal SARS-CoV-2 Serological Assays Enable Surveillance of Low-Prevalence Communities and Reveal Durable Humoral Immunity.

We conducted a serological study to define correlates of immunity against SARS-CoV-2. Compared to those with mild coronavirus disease 2019 (COVID-19) cases, individuals with severe disease exhibited elevated virus-neutralizing titers and antibodies against the nucleocapsid (N) and the receptor binding domain (RBD) of the spike protein. Age and sex played lesser roles. All cases, including asymptomatic individuals, seroconverted by 2 weeks after PCR confirmation. Spike RBD and S2 and neutralizing antibodies remained detectable through 5-7 months after onset, whereas α-N titers diminished. Testing 5,882 members of the local community revealed only 1 sample with seroreactivity to both RBD and S2 that lacked neutralizing antibodies. This fidelity could not be achieved with either RBD or S2 alone. Thus, inclusion of multiple independent assays improved the accuracy of antibody tests in low-seroprevalence communities and revealed differences in antibody kinetics depending on the antigen. We conclude that neutralizing antibodies are stably produced for at least 5-7 months after SARS-CoV-2 infection.

Detection, prevalence, and duration of humoral responses to SARS-CoV-2 under conditions of limited population exposure.

We conducted an extensive serological study to quantify population-level exposure and define correlates of immunity against SARS-CoV-2. We found that relative to mild COVID-19 cases, individuals with severe disease exhibited elevated authentic virus-neutralizing titers and antibody levels against nucleocapsid (N) and the receptor binding domain (RBD) and the S2 region of spike protein. Unlike disease severity, age and sex played lesser roles in serological responses. All cases, including asymptomatic individuals, seroconverted by 2 weeks post-PCR confirmation. RBD- and S2-specific and neutralizing antibody titers remained elevated and stable for at least 2-3 months post-onset, whereas those against N were more variable with rapid declines in many samples. Testing of 5882 self-recruited members of the local community demonstrated that 1.24% of individuals showed antibody reactivity to RBD. However, 18% (13/73) of these putative seropositive samples failed to neutralize authentic SARS-CoV-2 virus. Each of the neutralizing, but only 1 of the non-neutralizing samples, also displayed potent reactivity to S2. Thus, inclusion of multiple independent assays markedly improved the accuracy of antibody tests in low seroprevalence communities and revealed differences in antibody kinetics depending on the viral antigen. In contrast to other reports, we conclude that immunity is durable for at least several months after SARS-CoV-2 infection.