RESUMO
The COVID-19 pandemic has significantly impacted work, economy, and way of life. The SARS-CoV-2 virus displays unique features including widely varying symptoms and outcomes between infected individuals. Sensitive measurement of SARS-CoV-2 specific antibodies would provide new insight into virus transmission dynamics, pre-existing cross-reactive immunity, and the nuances of SARS-CoV-2 pathogenesis. To date, existing SARS-CoV-2 serology tests have limited utility due to insufficient detection of antibody levels lower than what is typically present after several days of symptoms. To measure lower quantities of SARS-CoV-2 IgM, IgG, and IgA with higher resolution than existing assays, we developed a new ELISA protocol with a distinct plate washing procedure and timed plate development via use of a standard curve. This BU ELISA method exhibits very low signal from plasma or serum samples added to uncoated wells at as low as a 1:5 dilution. Use of this method revealed circulating SARS-CoV-2 receptor binding domain (RBD) and nucleocapsid protein (NP) reactive antibodies from blood samples drawn prior to May 2019. Of our prepandemic cohort, no SARS-CoV-2 RBD-reactive IgG antibodies were detected in subjects over 70 years of age, and SARS-CoV-2 NP-reactive antibodies were present at similar levels to infected subjects in some individuals and very low in others. Also, samples drawn in May 2020 from two individuals with no symptoms or no known virus exposure contained SARS-CoV-2 RBD-reactive antibodies at intermediate amounts compared with other subject groups (higher than pre-pandemic and lower than confirmed SARS-CoV-2 infected). The one asymptomatic SARS-CoV-2 convalescent subject in our study possessed comparable amounts of SARS-CoV-2 NP-specific IgM and IgG but drastically lower IgA than the symptomatic counterparts. Also, our assay detected positive signal from samples that gave negative results in a commercially available Lateral Flow Device (LFD) and the EUA approved Abbott IgG chemiluminescent microparticle immunoassay for SARS-CoV-2 antibody detection. We propose that this improved ELISA protocol, which is straightforward to perform, low cost, and uses readily available commercial reagents, is a useful tool to elucidate new information about SARS-CoV-2 infection and has promising implications for improved detection of all analytes measurable by this platform.
RESUMO
BackgroundA subset of COVID-19 patients exhibit clinical features of cytokine storm. However, clinicopathologic features diagnostic of hemophagocytic lymphohistiocytosis (HLH) have not been reported. Pathologic studies to date have largely focused on the pulmonary finding of diffuse alveolar damage (DAD). To this aim, we study the reticuloendothelial organs of four consecutive patients dying of COVID-19 and correlate with clinical and laboratory parameters to detect HLH. MethodsAutopsies restricted to chest and abdomen were performed on four patients who succumbed to COVID-19. Spleen, liver, and multiple pulmonary hilar/mediastinal lymph nodes were sampled in all cases. Bone marrow was obtained by rib squeeze in a subset of cases. Routine H&E staining as well as immunohistochemical staining for CD163 was performed to detect hemophagocytosis. Clinical and laboratory results from pre-mortem blood samples were used to calculate H-scores. FindingsAll four cases demonstrated DAD within the lungs. Three of the four cases had histologic evidence of hemophagocytosis within pulmonary hilar/mediastinal lymph nodes. One case showed hemophagocytosis in the spleen but none showed hemophagocytosis in liver or bone marrow. Lymphophagocytosis was the predominant form of hemophagocytosis observed. One patient showed diagnostic features of HLH with an H-score of 217 while a second patient was likely HLH with a partial H-score of 145 due to missing triglyceride level. Both patients exhibited high fever and early onset rise in serum ferritin; however, neither bicytopenia, pancytopenia, nor hypofibrinogenemia were observed in either. The remaining two patients had H-scores of 131 and 96. InterpretationThis is the first report of SARS-CoV-2 associated HLH. Identification of HLH in a subset of patients with severe COVID-19 will inform clinical trials of therapeutic strategies.
