Persistence of SARS-CoV-2 in saliva: Implications for late-stage diagnosis and infectious duration.

Saliva has been a COVID-19 diagnostic specimen of interest due to its simple collection, scalability, and yield. Yet COVID-19 testing and estimates of the infectious period remain largely based on nasopharyngeal and nasal swabs. We sought to evaluate whether saliva testing captured prolonged presence of SARS-CoV-2 and potential infectiousness later in the disease course. We conducted an observational study of symptomatic COVID-19 patients at University Hospital in Newark, NJ. Paired saliva and nasal specimens from 96 patients were analyzed, including longitudinal analysis of paired observations from 28 of these patients who had multiple time-points. Saliva detected significantly more cases of COVID-19 beyond 5 days (86.1% [99/115] saliva vs 48.7% [56/115] nasal, p-value < 0.001), 9 days (79.4% [50/63] saliva vs 36.5% [23/63] nasal, p-value < 0.001) and 14 days (71.4% [20/28] saliva vs 32.1% [9/28] nasal, p-value = 0.010) of symptoms. Additionally, saliva yielded lower cycle thresholds across all time periods, indicative of higher viral loads in saliva. In the longitudinal analysis, a log-rank analysis indicated that the survival curve for saliva was significantly different from the curve for nasal swabs (p<0.001) with a median survival time for saliva of 18 days compared to 13 days for nasal swabs. We additionally performed saliva viral cultures among a similar COVID-19 patient cohort and noted patients with positive saliva viral cultures between 7 to 28 days of symptoms. Findings from this study suggest that SARS-CoV-2 RNA persists longer and in higher abundance in saliva compared to nasal swabs, with potential of prolonged propagating virus. Testing saliva may thus increase yield for detecting potentially infectious virus even beyond the first five days of symptomatic COVID-19.

A randomized controlled trial to evaluate outcomes with Aggrenox in patients with SARS-CoV-2 infection.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is an immunoinflammatory and hypercoagulable state that contributes to respiratory distress, multi-organ dysfunction, and mortality. Dipyridamole, by increasing extracellular adenosine, has been postulated to be protective for COVID-19 patients through its immunosuppressive, anti-inflammatory, anti-coagulant, vasodilatory, and anti-viral actions. Likewise, low-dose aspirin has also demonstrated protective effects for COVID-19 patients. This study evaluated the effect of these two drugs formulated together as Aggrenox in hospitalized COVID-19 patients. METHODS: In an open-label, single site randomized controlled trial (RCT), hospitalized COVID-19 patients were assigned to adjunctive Aggrenox (Dipyridamole ER 200mg/ Aspirin 25mg orally/enterally) with standard of care treatment compared to standard of care treatment alone. Primary endpoint was illness severity according to changes on the eight-point COVID ordinal scale, with levels of 1 to 8 where higher scores represent worse illness. Secondary endpoints included all-cause mortality and respiratory failure. Outcomes were measured through days 14, 28, and/or hospital discharge. RESULTS: From October 1, 2020 to April 30, 2021, a total of 98 patients, who had a median [IQR] age of 57 [47, 62] years and were 53.1% (n = 52) female, were randomized equally between study groups (n = 49 Aggrenox plus standard of care versus n = 49 standard of care alone). No clinically significant differences were found between those who received adjunctive Aggrenox and the control group in terms of illness severity (COVID ordinal scale) at days 14 and 28. The overall mortality through day 28 was 6.1% (3 patients, n = 49) in the Aggrenox group and 10.2% (5 patients, n = 49) in the control group (OR [95% CI]: 0.40 [0.04, 4.01], p = 0.44). Respiratory failure through day 28 occurred in 4 (8.3%, n = 48) patients in the Aggrenox group and 7 (14.6%, n = 48) patients in the standard of care group (OR [95% CI]: 0.21 [0.02, 2.56], p = 0.22). A larger decrease in the platelet count and blood glucose levels, and larger increase in creatinine and sodium levels within the first 7 days of hospital admission were each independent predictors of 28-day mortality (p < 0.05). CONCLUSION: In this study of hospitalized patients with COVID-19, while the outcomes of COVID illness severity, odds of mortality, and chance of respiratory failure were better in the Aggrenox group compared to standard of care alone, the data did not reach statistical significance to support the standard use of adjuvant Aggrenox in such patients.

RT-PCR negative COVID-19.

BACKGROUND: COVID-19 is a multi-system infection with emerging evidence-based antiviral and anti-inflammatory therapies to improve disease prognosis. However, a subset of patients with COVID-19 signs and symptoms have repeatedly negative RT-PCR tests, leading to treatment hesitancy. We used comparative serology early in the COVID-19 pandemic when background seroprevalence was low to estimate the likelihood of COVID-19 infection among RT-PCR negative patients with clinical signs and/or symptoms compatible with COVID-19. METHODS: Between April and October 2020, we conducted serologic testing of patients with (i) signs and symptoms of COVID-19 who were repeatedly negative by RT-PCR ('Probables'; N = 20), (ii) signs and symptoms of COVID-19 but with a potential alternative diagnosis ('Suspects'; N = 15), (iii) no signs and symptoms of COVID-19 ('Non-suspects'; N = 43), (iv) RT-PCR confirmed COVID-19 patients (N = 40), and (v) pre-pandemic samples (N = 55). RESULTS: Probables had similar seropositivity and levels of IgG and IgM antibodies as propensity-score matched RT-PCR confirmed COVID-19 patients (60.0% vs 80.0% for IgG, p-value = 0.13; 50.0% vs 72.5% for IgM, p-value = 0.10), but multi-fold higher seropositivity rates than Suspects and matched Non-suspects (60.0% vs 13.3% and 11.6% for IgG; 50.0% vs 0% and 4.7% for IgM respectively; p-values < 0.01). However, Probables were half as likely to receive COVID-19 treatment than the RT-PCR confirmed COVID-19 patients with similar disease severity. CONCLUSIONS: Findings from this study indicate a high likelihood of acute COVID-19 among RT-PCR negative with typical signs/symptoms, but a common omission of COVID-19 therapies among these patients. Clinically diagnosed COVID-19, independent of RT-PCR positivity, thus has a potential vital role in guiding treatment decisions.

Highly versatile antibody binding assay for the detection of SARS-CoV-2 infection and vaccination.

Monitoring the burden and spread of infection with the new coronavirus SARS-CoV-2, whether within small communities or in large geographical settings, is of paramount importance for public health purposes. Serology, which detects the host antibody response to the infection, is the most appropriate tool for this task, since virus-derived markers are most reliably detected during the acute phase of infection. Here we show that our ELISA protocol, which is based on antibody binding to the Receptor Binding Domain (RBD) of the S1 subunit of the viral Spike protein expressed as a novel fusion protein, detects antibody responses to SARS-CoV-2 infection and vaccination. We also show that our ELISA is accurate and versatile. It compares favorably with commercial assays widely used in clinical practice to determine exposure to SARS-CoV-2. Moreover, our protocol accommodates use of various blood- and non-blood-derived biospecimens, such as breast milk, as well as dried blood obtained with microsampling cartridges that are appropriate for remote collection. As a result, our RBD-based ELISA protocols are well suited for seroepidemiology and other large-scale studies requiring parsimonious sample collection outside of healthcare settings.

Sample collection and transport strategies to enhance yield, accessibility, and biosafety of COVID-19 RT-PCR testing.

Introduction. Non-invasive sample collection and viral sterilizing buffers have independently enabled workflows for more widespread COVID-19 testing by reverse-transcriptase polymerase chain reaction (RT-PCR).Gap statement. The combined use of sterilizing buffers across non-invasive sample types to optimize sensitive, accessible, and biosafe sampling methods has not been directly and systematically compared.Aim. We aimed to evaluate diagnostic yield across different non-invasive samples with standard viral transport media (VTM) versus a sterilizing buffer eNAT- (Copan diagnostics Murrieta, CA) in a point-of-care diagnostic assay system.Methods. We prospectively collected 84 sets of nasal swabs, oral swabs, and saliva, from 52 COVID-19 RT-PCR-confirmed patients, and nasopharyngeal (NP) swabs from 37 patients. Nasal swabs, oral swabs, and saliva were placed in either VTM or eNAT, prior to testing with the Xpert Xpress SARS-CoV-2 (Xpert). The sensitivity of each sampling strategy was compared using a composite positive standard.Results. Swab specimens collected in eNAT showed an overall superior sensitivity compared to swabs in VTM (70 % vs 57 %, P=0.0022). Direct saliva 90.5 %, (95 % CI: 82 %, 95 %), followed by NP swabs in VTM and saliva in eNAT, was significantly more sensitive than nasal swabs in VTM (50 %, P<0.001) or eNAT (67.8 %, P=0.0012) and oral swabs in VTM (50 %, P<0.0001) or eNAT (58 %, P<0.0001). Saliva and use of eNAT buffer each increased detection of SARS-CoV-2 with the Xpert; however, no single sample matrix identified all positive cases.Conclusion. Saliva and eNAT sterilizing buffer can enhance safe and sensitive detection of COVID-19 using point-of-care GeneXpert instruments.

Risk Factors and Outcomes of Hospitalized Patients With Severe Coronavirus Disease 2019 (COVID-19) and Secondary Bloodstream Infections: A Multicenter Case-Control Study.

BACKGROUND: Coronavirus disease 2019 (COVID-19) has become a global pandemic. Clinical characteristics regarding secondary infections in patients with COVID-19 have been reported, but detailed microbiology, risk factors, and outcomes of secondary bloodstream infections (sBSIs) in patients with severe COVID-19 have not been well described. METHODS: We performed a multicenter case-control study including all hospitalized patients diagnosed with severe COVID-19 and blood cultures drawn from 1 March 2020 to 7 May 2020 at 3 academic medical centers in New Jersey. Data collection included demographics, clinical and microbiologic variables, and patient outcomes. Risk factors and outcomes were compared between cases (sBSI) and controls (no sBSI). RESULTS: A total of 375 hospitalized patients were included. There were 128 sBSIs during the hospitalization. For the first set of positive blood cultures, 117 (91.4%) were bacterial and 7 (5.5%) were fungal. Those with sBSI were more likely to have altered mental status, lower mean percentage oxygen saturation on room air, have septic shock, and be admitted to the intensive care unit compared with controls. In-hospital mortality was higher in those with an sBSI versus controls (53.1% vs 32.8%, P = .0001). CONCLUSIONS: We observed that hospitalized adult patients with severe COVID-19 and sBSI had a more severe initial presentation, prolonged hospital course, and worse clinical outcomes. To maintain antimicrobial stewardship principles, further prospective studies are necessary to better characterize risk factors and prediction modeling to better understand when to suspect and empirically treat for sBSIs in severe COVID-19.