Association Between Sex Hormones Levels and Clinical Outcomes in Patients With COVID-19 Admitted to Hospital: An Observational, Retrospective, Cohort Study (preprint)

Background: Understanding the cause of sex disparities in COVID-19 outcomes is a major challenge. We investigate sex hormone levels and their association with outcomes in COVID-19 patients, stratified by sex and age.Methods: This observational, retrospective, cohort study included 138 patients aged 18 years or older with COVID-19, hospitalized in Italy between February 1 and May 30, 2020. The association between sex hormones (testosterone, estradiol, progesterone, dehydroepiandrosterone) and outcomes (ARDS, severe COVID-19, in-hospital mortality) was explored in 120 patients aged 50 years and over. STROBE checklist was followed.Findings: The median age was 73·5 years [IQR 61, 82]; 55·8% were male. In older males, testosterone was lower if ARDS and severe COVID-19 were reported than if not (3·6 vs. 5·3 nmol/L, p =0·0378 and 3·7 vs. 8·5 nmol/L, p =0·0011, respectively). Deceased males had lower testosterone (2·4 vs. 4·8 nmol/L, p =0·0536) and higher estradiol than survivors (40 vs. 24 pg/mL, p = 0·0006). Testosterone was negatively associated with ARDS (OR 0·849 [95% CI 0·734, 0·982]), severe COVID-19 (OR 0·691 [95% CI 0·546, 0·874]), and in-hospital mortality (OR 0·742 [95% CI 0·566, 0·972]), regardless of potential confounders, though confirmed only in the regression model on males. Higher estradiol was associated with a higher probability of death (OR 1·051 [95% CI 1·018, 1.084]), confirmed in both sex models. Interpretation: In males, higher testosterone seems to be protective against any considered outcome. Higher estradiol was associated with a higher probability of death in both sexes.Funding Information: The research was funded by Italian Ministry of Health “Fondi Ricerca Corrente, Project L1P5” for IRCCS Sacro Cuore Don Calabria Hospital.Declaration of Interests: We declare there is no conflict of interest.Ethics Approval Statement: Ethical approval for data collection was obtained in accordance with local regulations at each participating site. The study protocol received ethical approval and consent from the competent Ethics Committee (Comitato Etico per la sperimentazione Clinica delle Province di Verona e Rovigo) on September 1st, 2020 (protocol #46555).

High Dose Ivermectin for the Early Treatment of COVID-19 (COVER Study): A Randomised, Double-Blind, Multicentre, Phase II, Dose-Finding, Proof of Concept Clinical Trial (preprint)

Background: High concentrations of ivermectin demonstrated antiviral activity against SARS-CoV-2 in vitro. Aim of this study was to assess safety and efficacy of high-dose ivermectin in reducing viral load in individuals with initial SARS-CoV-2 infection. Methods: Randomised, double-blind, multicentre, phase II, dose-finding, proof-of-concept clinical trial performed in outpatients in Italy. Participants: adults recently diagnosed with asymptomatic/oligosymptomatic SARS-CoV-2 infection, providing informed consent. Exclusion criteria: pregnant or lactating women; CNS diseases; participants under dialysis; severe medical condition with prognosis < 6 months; warfarin treatment; antiviral/chloroquine phosphate/hydroxychloroquine treatment. Participants were assigned according to a randomized permuted block procedure to one of the following arms with allocation ratio 1:1:1: placebo (arm A); single dose ivermectin 600 μg/kg plus placebo for 5 days (arm B); single dose ivermectin 1200 μg/kg for 5 days (arm C). The pharmacist prepared the treatment according to the randomization list and on the basis of the participant’s weight. Primary outcomes: serious adverse drug reactions (SADR) and change of viral load at Day 7. The protocol was registered with ClinicalTrials.gov , NCT04438850. Findings. From 31th July, 2020 to 26th May, 2021, 32 participants were randomized to arm A, 29 to arm B and 32 to arm C. The recruitment was stopped on 10th June, because of a dramatic drop of cases. Eighty-nine participants were included in the safety analysis set, the change in viral load was calculated on 87 participants. No SADR were registered. The mean log10 viral load reduction was 2.9 in arm C (SD 1.6), 2.5 (2.2) in arm B and 2.0 (2.1) in arm A, with no significant differences (p=0.099 and 0.122 for C versus A and B versus A, respectively). Interpretation: High- dose ivermectin demonstrated safe, but did not prove efficacy to reduce viral load.Trial Registration: The protocol was registered with ClinicalTrials.gov , NCT04438850. Funding: The trial was partly funded by the Italian Ministry of Health.Declaration of Interest: None to declare. Ethical Approval: This study was approved by the national Ethics Committee of INMI – Spallanzani in Rome that is competent for all COVID-19 trials in Italy (resolution 139/2020 of 28th May, 2020), and by the Italian drug agency AIFA (resolution 136BIS/2020 of 18th May, 2020).

Direct Quantitation of SARS-CoV-2 Using Droplet Digital PCR in Suspected Samples With Very Low Viral Load (preprint)

Background: The reference test for SARS-CoV-2 detection is the reverse transcriptase real time PCR (real time RT-PCR). However, evidences reported that real time RT-PCR has a lower sensitivity compared with the droplet digital PCR (ddPCR) leading to possible false negative in low viral load cases. Methods: We used ddPCR for viral genes N1 and N2 on 20 negative (no detection) samples from symptomatic hospitalized COVID-patients presenting fluctuating real time RT-PCR results and 10 suspected samples (Ct value>35) from asymptomatic not hospitalized subjects. Results: ddPCR performed on RNA revealed 65% of positivity for at least one viral target in the hospitalized patients group of samples (35% for N1 and N2, 10% only for N1 and 20% only for N2) and 50% in the suspected cases (30% for N1 and N2, while 20% only for N2). On hospitalized patients’ samples, we applied also a direct ddPCR approach on the swab material, achieving an overall positivity of 83%. Conclusion: ddPCR, in particular the direct quantitation on swabs, shows a sensitivity advantage for the SARS-CoV-2 identification and may be useful to reduce the false negative diagnosis, especially for low viral load suspected samples.

ACoRE: Accurate SARS-CoV-2 genome reconstruction for the characterization of intra-host and inter-host viral diversity in clinical samples and for the evaluation of re-infections (preprint)

We report Accurate SARS-CoV-2 genome Reconstruction (ACoRE), an amplicon-based viral genome sequencing workflow for the complete and accurate reconstruction of SARS-CoV-2 sequences from clinical samples, including suboptimal ones that would usually be excluded even if unique and irreplaceable. We demonstrated the utility of the approach by achieving complete genome reconstruction and the identification of false-positive variants in >170 clinical samples, thus avoiding the generation of inaccurate and/or incomplete sequences. Most importantly, ACoRE was crucial to identify the correct viral strain responsible of a relapse case, that would be otherwise mis-classified as a re-infection due to missing or incorrect variant identification by a standard workflow.

Sensitivity, specificity and predictive values of molecular and serological tests for COVID-19. A longitudinal study in emergency room. (preprint)

Accuracy of diagnostic tests is essential for suspected cases of Coronavirus Disease 2019 (COVID-19). This study aimed to assess the sensitivity, specificity and positive and negative predictive value (PPV and NPV) of molecular and serological tests for the diagnosis of SARS-CoV-2 infection. A total of 346 consenting, adult patients were enrolled at the emergency room of IRCCS Sacro Cuore Don Calabria Hospital, Negrar, Italy. We evaluated three RT-PCR methods including six different gene targets; five serologic rapid diagnostic tests (RDT); one ELISA test. The final classification of infected/not infected patients was performed using Latent Class Analysis in combination with clinical re-assessment of incongruous cases and was the basis for the main analysis of accuracy. Of 346 patients consecutively enrolled, 85 (24.6%) were classified as infected. The molecular test with the highest sensitivity, specificity, PPV and NPV was RQ-SARS-nCoV-2 with 91.8% (C.I. 83.8-96.6), 100% (C.I. 98.6-100.0), 100.0% (C.I. 95.4-100.0) and 97.4% (C.I. 94.7-98.9) respectively, followed by CDC 2019-nCoV with 76.2% (C.I. 65.7-84.8), 99.6% (C.I. 97.9-100.0), 98.5% (C.I. 91.7-100.0) and 92.9% (C.I. 89.2-95.6) and by in-house test targeting E-RdRp with 61.2% (C.I. 50.0-71.6), 99.6% (C.I. 97.9-100.0), 98.1% (C.I. 89.9-100.0) and 88.7% (C.I. 84.6-92.1). The analyses on single gene targets found the highest sensitivity for S and RdRp of the RQ-SARS-nCoV-2 (both with sensitivity 94.1%, C.I. 86.8-98.1). The in-house RdRp had the lowest sensitivity (62.4%, C.I. 51.2-72.6). The specificity ranged from 99.2% (C.I. 97.3-99.9) for in-house RdRp and N2 to 95.0% (C.I. 91.6-97.3) for E. The PPV ranged from 97.1% (C.I. 89.8-99.6) of N2 to 85.4% (C.I. 76.3-92.00) of E, and the NPV from 98.1% (C.I. 95.5-99.4) of gene S to 89.0% (C.I. 84.8-92.4) of in-house RdRp. All serological tests had <50% sensitivity and low PPV and NPV. One RDT (VivaDiag IgM) had high specificity (98.5%, with PPV 84.0%), but poor sensitivity (24.7%). Molecular tests for SARS-CoV-2 infection showed excellent specificity, but significant differences in sensitivity. As expected, serological tests have limited utility in a clinical context.

Assessment of the direct quantitation of SARS-CoV-2 by droplet digital PCR (preprint)

Droplet digital PCR (ddPCR) is a sensitive and reproducible technology widely used for quantitation of several different viruses. The aim of this study was to compare the 2019-nCoV CDC ddPCR Triplex Probe Assay (BioRad) performance on the direct quantitation of SARS-CoV-2 on nasopharyngeal swab respect to the procedure applied to the extracted RNA. Moreover, the two widely used swab types were compared (UTM 3mL and ESwab 1mL, COPAN). A total of 50 nasopharyngeal swabs (n=25 UTM 3mL and n=25 ESwab 1mL) from SARS-CoV-2 patients collected during the pandemic from IRCCS Sacro Cuore Don Calabria Hospital (Veneto Region, North-East Italy) were used for our purpose. After heat inactivation, an aliquot of swab medium was used in order to perform the direct quantitation. Then, we compared the direct method with the quantitation performed on the RNA purified from nasopharyngeal swab by automated extraction. We observed that the direct approach achieved generally equal RNA copies compared to the RNA extracted. The results with the direct quantitation were more accurate on ESwab with a sensitivity of 93.33% [95% CI, 68.05 to 99.83] and specificity of 100.00% for both N1 and N2. On the other hand, on UTM we observed a higher rate of discordant results for N1 and N2. The human internal amplification control (RPP30) showed 100% of both sensitivity and specificity independent of swabs and approaches.In conclusion, we described a simple and fast approach for the quantitation of SARS-CoV-2 in nasopharyngeal swab. Our approach resulted in an efficient quantitation, without automated RNA extraction and purification. However, special care needs to be taken on the potential bias due to the conservation of samples as we used thawed material and to the heating treatment. Further studies on a larger cohort of samples are warranted to evaluate the clinical value of this direct approach.