N-antigenemia detection by a rapid lateral flow test predicts 90-day mortality in COVID-19: A prospective cohort study.

OBJECTIVES: To evaluate if the detection of N antigen of SARS-CoV-2 in plasma by a rapid lateral flow test predicts 90-day mortality in COVID-19 patients hospitalized at the wards. METHODS: The presence of N-antigenemia was evaluated in the first 36 hours after hospitalization in 600 unvaccinated COVID-19 patients, by using the Panbio COVID-19 Ag Rapid Test Device from Abbott (Abbott Laboratories Inc., Chicago, IL, USA). The impact of N-antigenemia on 90-day mortality was assessed by multivariable Cox regression analysis. RESULTS: Prevalence of N-antigenemia at hospitalization was higher in nonsurvivors (69% (82/118) vs. 52% (250/482); p < 0.001). The patients with N-antigenemia showed more frequently RNAemia (45.7% (148/324) vs. 19.8% (51/257); p < 0.001), absence of anti-SARS-CoV-2 N antibodies (80.7% (264/327) vs. 26.6% (69/259); p < 0.001) and absence of S1 antibodies (73.4% (240/327) vs. 23.6% (61/259); p < 0.001). The patients with antigenemia showed more frequently acute respiratory distress syndrome (30.1% (100/332) vs. 18.7% (50/268); p = 0.001) and nosocomial infections (13.6% (45/331) vs. 7.9% (21/267); p = 0.026). N-antigenemia was a risk factor for increased 90-day mortality in the multivariable analysis (HR, 1.99 (95% CI,1.09-3.61), whereas the presence of anti-SARS-CoV-2 N-antibodies represented a protective factor (HR, 0.47 (95% CI, 0.26-0.85). DISCUSSION: The presence of N-antigenemia or the absence of anti-SARS-CoV-2 N-antibodies after hospitalization is associated to increased 90-day mortality in unvaccinated COVID-19 patients. Detection of N-antigenemia by using lateral flow tests is a quick, widely available tool that could contribute to early identify those COVID-19 patients at risk of deterioration.

Digital PCR applications for the diagnosis and management of infection in critical care medicine.

Infection (either community acquired or nosocomial) is a major cause of morbidity and mortality in critical care medicine. Sepsis is present in up to 30% of all ICU patients. A large fraction of sepsis cases is driven by severe community acquired pneumonia (sCAP), which incidence has dramatically increased during COVID-19 pandemics. A frequent complication of ICU patients is ventilator associated pneumonia (VAP), which affects 10-25% of all ventilated patients, and bloodstream infections (BSIs), affecting about 10% of patients. Management of these severe infections poses several challenges, including early diagnosis, severity stratification, prognosis assessment or treatment guidance. Digital PCR (dPCR) is a next-generation PCR method that offers a number of technical advantages to face these challenges: it is less affected than real time PCR by the presence of PCR inhibitors leading to higher sensitivity. In addition, dPCR offers high reproducibility, and provides absolute quantification without the need for a standard curve. In this article we reviewed the existing evidence on the applications of dPCR to the management of infection in critical care medicine. We included thirty-two articles involving critically ill patients. Twenty-three articles focused on the amplification of microbial genes: (1) four articles approached bacterial identification in blood or plasma; (2) one article used dPCR for fungal identification in blood; (3) another article focused on bacterial and fungal identification in other clinical samples; (4) three articles used dPCR for viral identification; (5) twelve articles quantified microbial burden by dPCR to assess severity, prognosis and treatment guidance; (6) two articles used dPCR to determine microbial ecology in ICU patients. The remaining nine articles used dPCR to profile host responses to infection, two of them for severity stratification in sepsis, four focused to improve diagnosis of this disease, one for detecting sCAP, one for detecting VAP, and finally one aimed to predict progression of COVID-19. This review evidences the potential of dPCR as a useful tool that could contribute to improve the detection and clinical management of infection in critical care medicine.

Low anti-SARS-CoV-2 S antibody levels predict increased mortality and dissemination of viral components in the blood of critical COVID-19 patients.

BACKGROUND: Anti-SARS-CoV-2 S antibodies prevent viral replication. Critically ill COVID-19 patients show viral material in plasma, associated with a dysregulated host response. If these antibodies influence survival and viral dissemination in ICU-COVID patients is unknown. PATIENTS/METHODS: We studied the impact of anti-SARS-CoV-2 S antibodies levels on survival, viral RNA-load in plasma, and N-antigenaemia in 92 COVID-19 patients over ICU admission. RESULTS: Frequency of N-antigenaemia was >2.5-fold higher in absence of antibodies. Antibodies correlated inversely with viral RNA-load in plasma, representing a protective factor against mortality (adjusted HR [CI 95%], p): (S IgM [AUC ≥ 60]: 0.44 [0.22; 0.88], 0.020); (S IgG [AUC ≥ 237]: 0.31 [0.16; 0.61], <0.001). Viral RNA-load in plasma and N-antigenaemia predicted increased mortality: (N1-viral load [≥2.156 copies/ml]: 2.25 [1.16; 4.36], 0.016); (N-antigenaemia: 2.45 [1.27; 4.69], 0.007). CONCLUSIONS: Low anti-SARS-CoV-2 S antibody levels predict mortality in critical COVID-19. Our findings support that these antibodies contribute to prevent systemic dissemination of SARS-CoV-2.

A Simple, Affordable, Rapid, Stabilized, Colorimetric, Versatile RT-LAMP Assay to Detect SARS-CoV-2.

The SARS-CoV-2 pandemic has forced all countries worldwide to rapidly develop and implement widespread testing to control and manage the Coronavirus Disease 2019 (COVID-19). reverse-transcription (RT)-qPCR is the gold standard molecular diagnostic method for COVID-19, mostly in automated testing platforms. These systems are accurate and effective, but also costly, time-consuming, high-technological, infrastructure-dependent, and currently suffer from commercial reagent supply shortages. The reverse-transcription loop-mediated isothermal amplification (RT-LAMP) can be used as an alternative testing method. Here, we present a novel versatile (real-time and colorimetric) RT-LAMP for the simple (one-step), affordable (~1.7 €/sample), and rapid detection of SARS-CoV-2 targeting both ORF1ab and N genes of the novel virus genome. We demonstrate the assay on RT-qPCR-positive clinical samples, obtaining most positive results under 25 min. In addition, a novel 30-min one-step drying protocol has been developed to stabilize the RT-LAMP reaction mixtures, allowing them to be stored at room temperature functionally for up to two months, as predicted by the Q10. This Dry-RT-LAMP methodology is suitable for potentially ready-to-use COVID-19 diagnosis. After further testing and validation, it could be easily applied both in developed and in low-income countries yielding rapid and reliable results.

Comparison of real-time and droplet digital PCR to detect and quantify SARS-CoV-2 RNA in plasma.

BACKGROUND: The presence of SARS-CoV-2 RNA in plasma has been linked to disease severity and mortality. We compared RT-qPCR to droplet digital PCR (ddPCR) to detect SARS-CoV-2 RNA in plasma from COVID-19 patients (mild, moderate, and critical disease). METHODS: The presence/concentration of SARS-CoV-2 RNA in plasma was compared in three groups of COVID-19 patients (30 outpatients, 30 ward patients and 30 ICU patients) using both RT-qPCR and ddPCR. Plasma was obtained in the first 24h following admission, and RNA was extracted using eMAG. ddPCR was performed using Bio-Rad SARS-CoV-2 detection kit, and RT-qPCR was performed using GeneFinder™ COVID-19 Plus RealAmp Kit. Statistical analysis was performed using Statistical Package for the Social Science. RESULTS: SARS-CoV-2 RNA was detected, using ddPCR and RT-qPCR, in 91% and 87% of ICU patients, 27% and 23% of ward patients and 3% and 3% of outpatients. The concordance of the results obtained by both methods was excellent (Cohen's kappa index = 0.953). RT-qPCR was able to detect 34/36 (94.4%) patients positive for viral RNA in plasma by ddPCR. Viral RNA load was higher in ICU patients compared with the other groups (P < .001), by both ddPCR and RT-qPCR. AUC analysis revealed Ct values (RT-qPCR) and viral RNA load values (ddPCR) can similarly differentiate between patients admitted to wards and to the ICU (AUC of 0.90 and 0.89, respectively). CONCLUSION: Both methods yielded similar prevalence of RNAemia between groups, with ICU patients showing the highest (>85%). RT-qPCR was as useful as ddPCR to detect and quantify SARS-CoV-2 RNAemia in plasma.

Viral RNA load in plasma is associated with critical illness and a dysregulated host response in COVID-19.

BACKGROUND: COVID-19 can course with respiratory and extrapulmonary disease. SARS-CoV-2 RNA is detected in respiratory samples but also in blood, stool and urine. Severe COVID-19 is characterized by a dysregulated host response to this virus. We studied whether viral RNAemia or viral RNA load in plasma is associated with severe COVID-19 and also to this dysregulated response. METHODS: A total of 250 patients with COVID-19 were recruited (50 outpatients, 100 hospitalized ward patients and 100 critically ill). Viral RNA detection and quantification in plasma was performed using droplet digital PCR, targeting the N1 and N2 regions of the SARS-CoV-2 nucleoprotein gene. The association between SARS-CoV-2 RNAemia and viral RNA load in plasma with severity was evaluated by multivariate logistic regression. Correlations between viral RNA load and biomarkers evidencing dysregulation of host response were evaluated by calculating the Spearman correlation coefficients. RESULTS: The frequency of viral RNAemia was higher in the critically ill patients (78%) compared to ward patients (27%) and outpatients (2%) (p < 0.001). Critical patients had higher viral RNA loads in plasma than non-critically ill patients, with non-survivors showing the highest values. When outpatients and ward patients were compared, viral RNAemia did not show significant associations in the multivariate analysis. In contrast, when ward patients were compared with ICU patients, both viral RNAemia and viral RNA load in plasma were associated with critical illness (OR [CI 95%], p): RNAemia (3.92 [1.183-12.968], 0.025), viral RNA load (N1) (1.962 [1.244-3.096], 0.004); viral RNA load (N2) (2.229 [1.382-3.595], 0.001). Viral RNA load in plasma correlated with higher levels of chemokines (CXCL10, CCL2), biomarkers indicative of a systemic inflammatory response (IL-6, CRP, ferritin), activation of NK cells (IL-15), endothelial dysfunction (VCAM-1, angiopoietin-2, ICAM-1), coagulation activation (D-Dimer and INR), tissue damage (LDH, GPT), neutrophil response (neutrophils counts, myeloperoxidase, GM-CSF) and immunodepression (PD-L1, IL-10, lymphopenia and monocytopenia). CONCLUSIONS: SARS-CoV-2 RNAemia and viral RNA load in plasma are associated with critical illness in COVID-19. Viral RNA load in plasma correlates with key signatures of dysregulated host responses, suggesting a major role of uncontrolled viral replication in the pathogenesis of this disease.