Biobanking for COVID-19 research.

BACKGROUND: Biobanks are imperative infrastructures, particularly during outbreaks, when there is an obligation to acquire and share knowledge as quick as possible to allow for implementation of science-based preventive, diagnostic, prognostic, and therapeutic strategies. METHODS: We established a COVID-19 biobank with the aim of collecting high-quality and well-annotated human biospecimens, in the effort to understand the pathogenic mechanisms underlying COVID-19 and identify therapeutic targets (COVID-BioB, NCT04318366). Here we describe our experience and briefly review the characteristics of the biobanks for COVID-19 that have been so far established. RESULTS: A total of 46,677 samples have been collected from 913 participants (63.3% males, median [IQR] age 62.2 [51.2-74.0] years) since the beginning of the program. Most patients (66.9%) had been admitted to hospital for COVID-19, with a median length of stay of 15.0 (9.0-27.0) days. A minority of patients (13.3% of the total) had been admitted for other reasons and subsequently tested positive for SARS-CoV-2. The remainder were managed at home after being seen at the Emergency Department. CONCLUSIONS: Having a solid research infrastructure already in place, along with flexibility and adaptability to new requirements, allowed for the quick building of a COVID-19 biobank that will help expand and share the knowledge of SARS-CoV-2.

Testosterone in males with COVID-19: a 12-month cohort study.

BACKGROUND: Male patients with COVID-19 have been found with reduced serum total testosterone (tT) levels and with more severe clinical outcomes. OBJECTIVES: To assess total testosterone (tT) levels and the probability of recovering eugonadal tT levels during a minimum 12-month timespan in a cohort of men who have been followed over time after the recovery from laboratory-confirmed COVID-19. MATERIALS AND METHODS: Demographic, clinical and hormonal values were collected for the overall cohort. Hypogonadism was defined as tT ≤9.2 nmol/l. The Charlson Comorbidity Index was used to score health-significant comorbidities. Descriptive statistics was used to compare hormonal levels at baseline versus 7-month (FU1) versus 12-month (FU2) follow-up, respectively. Multivariate cox proportional hazards regression model was used to identify the potential predictors of eugonadism recovery over time among patients with hypogonadism at the time of infection. RESULTS: Of the original cohort of 286 patients, follow-up data were available for 121 (42.3%) at FU1 and 63 (22%) patients at FU2, respectively. Higher median interquartile range (IQR) tT levels were detected at FU2 (13.8 (12.3-15.3) nmol/L) versus FU1 (10.2 [9.3-10.9] nmol/L) and versus baseline (3.6 [3.02-4.02] nmol/L) (all p < 0.0001), whilst both LH and E2 levels significantly decreased over the same time frame (all p ≤ 0.01). Circulating IL-6 levels further decreased at FU2 compared to FU1 levels (19.3 vs. 72.8 pg/ml) (p = 0.02). At multivariable cox regression analyses, baseline tT level (HR 1.19; p = 0.03 [1.02-1.4]) was independently associated with the probability of tT level normalization over time, after adjusting for potential confounders. CONCLUSIONS: Circulating tT levels keep increasing over time in men after COVID-19. Still, almost 30% of men who recovered from COVID-19 had low circulating T levels suggestive for a condition of hypogonadism at a minimum 12-month follow-up.

Combined plasma levels of IL-10 and testosterone, but not soluble HLA-G5, predict the risk of death in COVID-19 patients.

BACKGROUND: The identification of biomarkers correlated with coronavirus disease 2019 (COVID-19) outcomes is a relevant need for clinical management. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is characterized by elevated interleukin (IL)-6, IL-10, HLA-G, and impaired testosterone production. OBJECTIVES: We aimed at defining the combined impact of sex hormones, interleukin-10, and HLA-G on COVID-19 pathophysiology and their relationship in male patients. MATERIALS AND METHODS: We measured by chemiluminescence immunoassay, electrochemiluminescent assays, and enzyme-linked immunosorbent assay circulating total testosterone, 17ß-estradiol (E2 ), IL-10, and -HLAG5 as well as SARS-CoV-2 S1/S2 Immunoglobulin G from 292 healthy controls and 111 COVID-19 patients with different disease severity at hospital admission, and in 53 COVID-19 patients at 7-month follow-up. RESULTS AND DISCUSSION: We found significantly higher levels of IL-10, HLA-G, and E2 in COVID-19 patients compared to healthy controls and an inverse correlation between IL-10 and testosterone, with IL-10, progressively increasing and testosterone progressively decreasing with disease severity. This correlation was lost at the 7-month follow-up. The risk of death in COVID-19 patients with low testosterone increased in the presence of high IL-10. A negative correlation between SARS-CoV-2 Immunoglobulin G and HLA-G or IL-10 at hospitalization was observed. At the 7-month follow-up, IL-10 and testosterone normalized, and  HLA-G decreased. CONCLUSION: Our findings indicate that combined evaluation of IL-10 and testosterone predicts the risk of death in men with COVID-19 and support the hypothesis that IL-10 fails to suppress excessive inflammation by promoting viral spreading.

Chitinase-3-like protein-1 at hospital admission predicts COVID-19 outcome: a prospective cohort study.

Infectious and inflammatory stimuli elicit the generation of chitinase-3-like protein-1 (CHI3L1), involved in tissue damage, repair and remodeling. We evaluated whether plasma CHI3L1 at disease onset predicts clinical outcome of patients with Coronavirus 2019 (COVID-19) disease. Blood from 191 prospectively followed COVID-19 patients were collected at hospital admission between March 18th and May 5th, 2020. Plasma from 80 survivors was collected one month post-discharge. Forty age- and sex-matched healthy volunteers served as controls. Primary outcome was transfer to intensive care unit (ICU) or death. CHI3L1 was higher in COVID-19 patients than controls (p < 0.0001). Patients with unfavorable outcome (41 patients admitted to ICU, 47 died) had significantly higher CHI3L1 levels than non-ICU survivors (p < 0.0001). CHI3L1 levels abated in survivors one month post-discharge, regardless of initial disease severity (p < 0.0001), although remaining higher than controls (p < 0.05). Cox regression analysis revealed that CHI3L1 levels predict primary outcome independently of age, sex, comorbidities, degree of respiratory insufficiency and systemic inflammation or time from symptom onset to sampling (p < 0.0001). Kaplan-Meier curve analysis confirmed that patients with CHI3L1 levels above the median (361 ng/mL) had a poorer prognosis (log rank test, p < 0.0001). Plasma CHI3L1 is increased in COVID-19 patients and predicts adverse outcome.

Chromogranin A plasma levels predict mortality in COVID-19.

BACKGROUND: Chromogranin A (CgA) and its fragment vasostatin I (VS-I) are secreted in the blood by endocrine/neuroendocrine cells and regulate stress responses. Their involvement in Coronavirus 2019 disease (COVID-19) has not been investigated. METHODS: CgA and VS-I plasma concentrations were measured at hospital admission from March to May 2020 in 190 patients. 40 age- and sex-matched healthy volunteers served as controls. CgA and VS-I levels relationship with demographics, comorbidities and disease severity was assessed through Mann Whitney U test or Spearman correlation test. Cox regression analysis and Kaplan Meier survival curves were performed to investigate the impact of the CgA and VS-I levels on in-hospital mortality. RESULTS: Median CgA and VS-I levels were higher in patients than in healthy controls (CgA: 0.558 nM [interquartile range, IQR 0.358-1.046] vs 0.368 nM [IQR 0.288-0.490] respectively, p = 0.0017; VS-I: 0.357 nM [IQR 0.196-0.465] vs 0.144 nM [0.144-0.156] respectively, p<0.0001). Concentration of CgA, but not of VS-I, significantly increased in patients who died (n = 47) than in survivors (n = 143) (median 0.948 nM [IQR 0.514-1.754] vs 0.507 nM [IQR 0.343-0.785], p = 0.00026). Levels of CgA were independent predictors of in-hospital mortality (hazard ratio 1.28 [95% confidence interval 1.077-1.522], p = 0.005) when adjusted for age, number of comorbidities, respiratory insufficiency degree, C-reactive protein levels and time from symptom onset to sampling. Kaplan Meier curves revealed a significantly increased mortality rate in patients with CgA levels above 0.558 nM (median value, log rank test, p = 0.001). CONCLUSION: Plasma CgA levels increase in COVID-19 patients and represent an early independent predictor of mortality.

No Evidence of Long-Term Disruption of Glycometabolic Control After SARS-CoV-2 Infection.

PURPOSE: To assess whether dysglycemia diagnosed during severe acute respiratory syndrome coronavirus 2 pneumonia may become a potential public health problem after resolution of the infection. In an adult cohort with suspected coronavirus disease 2019 (COVID-19) pneumonia, we integrated glucose data upon hospital admission with fasting blood glucose (FBG) in the year prior to COVID-19 and during postdischarge follow-up. METHODS: From February 25 to May 15, 2020, 660 adults with suspected COVID-19 pneumonia were admitted to the San Raffaele Hospital (Milan, Italy). Through structured interviews/ medical record reviews, we collected demographics, clinical features, and laboratory tests upon admission and additional data during hospitalization or after discharge and in the previous year. Upon admission, we classified participants according to American Diabetes Association criteria as having (1) preexisting diabetes, (2) newly diagnosed diabetes, (3) hyperglycemia not in the diabetes range, or (4) normoglycemia. FBG prior to admission and during follow-up were classified as normal or impaired fasting glucose and fasting glucose in the diabetes range. RESULTS: In patients with confirmed COVID (n = 589), the proportion with preexisting or newly diagnosed diabetes, hyperglycemia not in the diabetes range and normoglycemia was 19.6%, 6.7%, 43.7%, and 30.0%, respectively. Patients with dysglycemia associated to COVID-19 had increased markers of inflammation and organs' injury and poorer clinical outcome compared to those with normoglycemia. After the infection resolved, the prevalence of dysglycemia reverted to preadmission frequency. CONCLUSIONS: COVID-19-associated dysglycemia is unlikely to become a lasting public health problem. Alarmist claims on the diabetes risk after COVID-19 pneumonia should be interpreted with caution.

Unconventional CD147-dependent platelet activation elicited by SARS-CoV-2 in COVID-19.

BACKGROUND: Platelet activation and thrombotic events characterizes COVID-19. OBJECTIVES: To characterize platelet activation and determine if SARS-CoV-2 induces platelet activation. PATIENTS/METHODS: We investigated platelet activation in 119 COVID-19 patients at admission in a university hospital in Milan, Italy, between March 18 and May 5, 2020. Sixty-nine subjects (36 healthy donors, 26 patients with coronary artery disease, coronary artery disease, and seven patients with sepsis) served as controls. RESULTS: COVID-19 patients had activated platelets, as assessed by the expression and distribution of HMGB1 and von Willebrand factor, and by the accumulation of platelet-derived (plt) extracellular vesicles (EVs) and HMGB1+ plt-EVs in the plasma. P-selectin upregulation was not detectable on the platelet surface in a fraction of patients (55%) and the concentration of soluble P-selectin in the plasma was conversely increased. The plasma concentration of HMGB1+ plt-EVs of patients at hospital admission remained in a multivariate analysis an independent predictor of the clinical outcome, as assessed using a 6-point ordinal scale (from 1 = discharged to 6 = death). Platelets interacting in vitro with SARS-CoV-2 underwent activation, which was replicated using SARS-CoV-2 pseudo-viral particles and purified recombinant SARS-CoV-2 spike protein S1 subunits. Human platelets express CD147, a putative coreceptor for SARS-CoV-2, and Spike-dependent platelet activation, aggregation and granule release, release of soluble P-selectin and HMGB1+ plt-EVs abated in the presence of anti-CD147 antibodies. CONCLUSIONS: Hence, an early and intense platelet activation, which is reproduced by stimulating platelets in vitro with SARS-CoV-2, characterizes COVID-19 and could contribute to the inflammatory and hemostatic manifestations of the disease.

CXCL10 levels at hospital admission predict COVID-19 outcome: hierarchical assessment of 53 putative inflammatory biomarkers in an observational study.

BACKGROUND: Host inflammation contributes to determine whether SARS-CoV-2 infection causes mild or life-threatening disease. Tools are needed for early risk assessment. METHODS: We studied in 111 COVID-19 patients prospectively followed at a single reference Hospital fifty-three potential biomarkers including alarmins, cytokines, adipocytokines and growth factors, humoral innate immune and neuroendocrine molecules and regulators of iron metabolism. Biomarkers at hospital admission together with age, degree of hypoxia, neutrophil to lymphocyte ratio (NLR), lactate dehydrogenase (LDH), C-reactive protein (CRP) and creatinine were analysed within a data-driven approach to classify patients with respect to survival and ICU outcomes. Classification and regression tree (CART) models were used to identify prognostic biomarkers. RESULTS: Among the fifty-three potential biomarkers, the classification tree analysis selected CXCL10 at hospital admission, in combination with NLR and time from onset, as the best predictor of ICU transfer (AUC [95% CI] = 0.8374 [0.6233-0.8435]), while it was selected alone to predict death (AUC [95% CI] = 0.7334 [0.7547-0.9201]). CXCL10 concentration abated in COVID-19 survivors after healing and discharge from the hospital. CONCLUSIONS: CXCL10 results from a data-driven analysis, that accounts for presence of confounding factors, as the most robust predictive biomarker of patient outcome in COVID-19.

X-linked recessive TLR7 deficiency in ~1% of men under 60 years old with life-threatening COVID-19.

Autosomal inborn errors of type I IFN immunity and autoantibodies against these cytokines underlie at least 10% of critical COVID-19 pneumonia cases. We report very rare, biochemically deleterious X-linked TLR7 variants in 16 unrelated male individuals aged 7 to 71 years (mean: 36.7 years) from a cohort of 1,202 male patients aged 0.5 to 99 years (mean: 52.9 years) with unexplained critical COVID-19 pneumonia. None of the 331 asymptomatically or mildly infected male individuals aged 1.3 to 102 years (mean: 38.7 years) tested carry such TLR7 variants (p = 3.5 × 10-5). The phenotypes of five hemizygous relatives of index cases infected with SARS-CoV-2 include asymptomatic or mild infection (n=2, 5 and 38 years), or moderate (n=1, 5 years), severe (n=1, 27 years), or critical (n=1, 29 years) pneumonia. Two boys (aged 7 and 12 years) from a cohort of 262 male patients with severe COVID-19 pneumonia (mean: 51.0 years) are hemizygous for a deleterious TLR7 variant. The cumulative allele frequency for deleterious TLR7 variants in the male general population is < 6.5x10-4 We also show that blood B cell lines and myeloid cell subsets from the patients do not respond to TLR7 stimulation, a phenotype rescued by wild-type TLR7 The patients' blood plasmacytoid dendritic cells (pDCs) produce low levels of type I IFNs in response to SARS-CoV-2. Overall, X-linked recessive TLR7 deficiency is a highly penetrant genetic etiology of critical COVID-19 pneumonia, in about 1.8% of male patients below the age of 60 years. Human TLR7 and pDCs are essential for protective type I IFN immunity against SARS-CoV-2 in the respiratory tract.

Testosterone in males with COVID-19: A 7-month cohort study.

BACKGROUND: Circulating testosterone levels have been found to be reduced in men with severe acute respiratory syndrome coronavirus 2 infection, COVID-19, with lower levels being associated with more severe clinical outcomes. OBJECTIVES: We aimed to assess total testosterone levels and the prevalence of total testosterone still suggesting for hypogonadism at 7-month follow-up in a cohort of 121 men who recovered from laboratory-confirmed COVID-19. MATERIALS AND METHODS: Demographic, clinical, and hormonal values were collected for all patients. Hypogonadism was defined as total testosterone ≤9.2 nmol/L. The Charlson Comorbidity Index was used to score health-significant comorbidities. Descriptive statistics and multivariable linear and logistic regression models tested the association between clinical and laboratory variables and total testosterone levels at follow-up assessment. RESULTS: Circulating total testosterone levels increased at 7-month follow-up compared to hospital admittance (p < 0.0001), while luteinizing hormone and 17ß-estradiol levels significantly decreased (all p ≤ 0.02). Overall, total testosterone levels increased in 106 (87.6%) patients, but further decreased in 12 (9.9%) patients at follow-up, where a total testosterone level suggestive for hypogonadism was still observed in 66 (55%) patients. Baseline Charlson Comorbidity Index score (OR 0.36; p = 0.03 [0.14, 0.89]) was independently associated with total testosterone levels at 7-month follow-up, after adjusting for age, BMI, and IL-6 at hospital admittance. CONCLUSIONS: Although total testosterone levels increased over time after COVID-19, more than 50% of men who recovered from the disease still had circulating testosterone levels suggestive for a condition of hypogonadism at 7-month follow-up. In as many as 10% of cases, testosterone levels even further decreased. Of clinical relevance, the higher the burden of comorbid conditions at presentation, the lower the probability of testosterone levels recovery over time.

Pre-Existing Diabetes and COVID-Associated Hyperglycaemia in Patients with COVID-19 Pneumonia.

AIM: The aim of the current study was to compare clinical characteristics, laboratory findings, and major outcomes of patients hospitalized for COVID-19 pneumonia with COVID-associated hyperglycaemia or pre-existing diabetes. METHODS: A cohort of 176 adult patients with a diagnosis of pre-existing diabetes (n = 112) or COVID-associated hyperglycaemia (n = 55) was studied. RESULTS: Patients with COVID-associated hyperglycaemia had lower BMI, significantly less comorbidities, and higher levels of inflammatory markers and indicators of multi-organ injury than those with pre-existing diabetes. No differences between pre-existing diabetes and COVID-associated hyperglycaemia were evident for symptoms at admission, the humoral response against SARS-CoV-2, or autoantibodies to glutamic acid decarboxylase or interferon alpha-4. COVID-associated hyperglycaemia was independently associated with the risk of adverse clinical outcome, which was defined as ICU admission or death (HR 2.11, 95% CI 1.34-3.31; p = 0.001), even after adjustment for age, sex, and other selected variables associated with COVID-19 severity. Furthermore, at the same time, we documented a negative association (HR 0.661, 95% CI 0.43-1.02; p = 0.063) between COVID-associated hyperglycaemia to swab negativization. CONCLUSIONS: Recognizing hyperglycaemia as a specific clinical entity associated with COVID-19 pneumonia is relevant for early and appropriate patient management and close monitoring for the progression of disease severity.

Neutralizing antibody responses to SARS-CoV-2 in symptomatic COVID-19 is persistent and critical for survival.

Understanding how antibody responses to SARS-CoV-2 evolve during infection may provide important insight into therapeutic approaches and vaccination for COVID-19. Here we profile the antibody responses of 162 COVID-19 symptomatic patients in the COVID-BioB cohort followed longitudinally for up to eight months from symptom onset to find SARS-CoV-2 neutralization, as well as antibodies either recognizing SARS-CoV-2 spike antigens and nucleoprotein, or specific for S2 antigen of seasonal beta-coronaviruses and hemagglutinin of the H1N1 flu virus. The presence of neutralizing antibodies within the first weeks from symptoms onset correlates with time to a negative swab result (p = 0.002), while the lack of neutralizing capacity correlates with an increased risk of a fatal outcome (p = 0.008). Neutralizing antibody titers progressively drop after 5-8 weeks but are still detectable up to 8 months in the majority of recovered patients regardless of age or co-morbidities, with IgG to spike antigens providing the best correlate of neutralization. Antibody responses to seasonal coronaviruses are temporarily boosted, and parallel those to SARS-CoV-2 without dampening the specific response or worsening disease progression. Our results thus suggest compromised immune responses to the SARS-CoV-2 spike to be a major trait of COVID-19 patients with critical conditions, and thereby inform on the planning of COVID-19 patient care and therapy prioritization.

Blood neurofilament light chain and total tau levels at admission predict death in COVID-19 patients.

BACKGROUND AND AIMS: Patients infected with SARS-CoV-2 range from asymptomatic, to mild, moderate or severe disease evolution including fatal outcome. Thus, early predictors of clinical outcome are highly needed. We investigated markers of neural tissue damage as a possible early sign of multisystem involvement to assess their clinical prognostic value on survival or transfer to intensive care unit (ICU). METHODS: We collected blood from 104 patients infected with SARS-CoV-2 the day of admission to the emergency room and measured blood neurofilament light chair (NfL), glial fibrillary acidic protein (GFAP), ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), and total tau protein levels. RESULTS: We found that NfL, GFAP, and tau were significantly increased in patients with fatal outcome, while NfL and UCH-L1 in those needing ICU transfer. ROC and Kaplan-Meier curves indicated that total tau levels at admission accurately predict mortality. CONCLUSIONS: Blood neural markers may provide additional prognostic value to conventional biomarkers used to predict COVID-19 outcome.

Higher-order connections between stereotyped subsets: implications for improved patient classification in CLL.

Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B-cell receptor (BcR) immunoglobulins. Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR immunoglobulin stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR immunoglobulin stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. To address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29 856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed "satellites," were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL.

COVID-19 survival associates with the immunoglobulin response to the SARS-CoV-2 spike receptor binding domain.

BACKGROUNDSerological assays are of critical importance to investigate correlates of response and protection in coronavirus disease 2019 (COVID-19), to define previous exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in populations, and to verify the development of an adaptive immune response in infected individuals.METHODSWe studied 509 patients confirmed to have COVID-19 from the San Raffaele Hospital of Milan and 480 samples of prepandemic organ donor sera collected in 2010-2012. Using fluid-phase luciferase immune precipitation (LIPS) assays, we characterized IgG, IgM, and IgA antibodies to the spike receptor binding domain (RBD), S1+S2, nucleocapsid, and ORF6 to ORF10 of SARS-CoV-2, to the HCoV-OC43 and HCoV-HKU1 betacoronaviruses spike S2, and the H1N1Ca2009 flu virus hemagglutinin. Sequential samples at 1 and 3 months after hospital discharge were also tested for SARS-CoV-2 RBD antibodies in 95 patients.RESULTSAntibodies developed rapidly against multiple SARS-CoV-2 antigens in 95% of patients by 4 weeks after symptom onset and IgG to the RBD increased until the third month of follow-up. We observed a major synchronous expansion of antibodies to the HCoV-OC43 and HCoV-HKU1 spike S2. A likely coinfection with influenza was neither linked to a more severe presentation of the disease nor to a worse outcome. Of the measured antibody responses, positivity for IgG against the SARS-CoV-2 spike RBD was predictive of survival.CONCLUSIONThe measurement of antibodies to selected epitopes of SARS-CoV-2 antigens can offer a more accurate assessment of the humoral response in patients and its impact on survival. The presence of partially cross-reactive antibodies with other betacoronaviruses is likely to impact on serological assay specificity and interpretation.TRIAL REGISTRATIONCOVID-19 Patients Characterization, Biobank, Treatment Response and Outcome Predictor (COVID-BioB). ClinicalTrials.gov identifier: NCT04318366.FUNDINGIRCCS Ospedale San Raffaele and Università Vita Salute San Raffaele.

Severely low testosterone in males with COVID-19: A case-control study.

BACKGROUND: Circulating androgens could have a relevant pathobiological role in clinical outcomes in men with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (COVID-19). OBJECTIVES: We aimed to assess: (a) circulating sex steroids levels in a cohort of 286 symptomatic men with laboratory-confirmed COVID-19 at hospital admission compared to a cohort of 281 healthy men; and (b) the association between serum testosterone levels (tT), COVID-19, and clinical outcomes. MATERIALS AND METHODS: Demographic, clinical, and hormonal values were collected for all patients. Hypogonadism was defined as tT ≤9.2 nmol/l. The Charlson Comorbidity Index (CCI) was used to score health-significant comorbidities. Severe clinical outcomes were defined as patients either transferred to intensive care unit (ICU) or death. Descriptive statistics and multivariable linear and logistic regression models tested the association between clinical and laboratory variables and tT levels. Univariable and multivariable logistic regression models tested the association between tT and severe clinical outcomes. RESULTS: Overall, a significantly lower levels of LH and tT were found in patients with COVID-19 compared to healthy controls (all p < 0.0001); conversely, healthy controls depicted lower values of circulating E2 (p < 0.001). Testosterone levels suggestive for hypogonadism were observed in 257 (89.8%) patients at hospital admission. In as many as 243 (85%) cases, hypogonadism was secondary. SARS-CoV-2 infection status was independently associated with lower tT levels (p < 0.0001) and greater risk of hypogonadism (p < 0.0001), after accounting for age, BMI, CCI, and IL-6 values. Lower tT levels were associated with higher risk of ICU admission and death outcomes (all p ≤ 0.05), after accounting for clinical and laboratory parameters. CONCLUSIONS: We unveil an independent association between SARS-CoV-2 infection status and secondary hypogonadism already at hospital admission, with lower testosterone levels predicting the most severe clinical outcomes.

Robust Neutralizing Antibodies to SARS-CoV-2 Develop and Persist in Subjects with Diabetes and COVID-19 Pneumonia.

CONTEXT: Demonstrating the ability to mount a neutralizing antibody response to SARS-CoV-2 in the presence of diabetes is crucial to understand COVID-19 pathogenesis, reinfection potential, and vaccine development. OBJECTIVE: The aim of this study was to characterize the kinetics and durability of neutralizing antibody (Nab) response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the presence of hyperglycemia. METHODS: Using a lentiviral vector-based SARS-CoV-2 neutralization assay to measure Nabs, we characterized 150 patients randomly selected from a cohort of 509 patients with confirmed COVID-19 pneumonia. We analyzed Nab response according to the presence of diabetes or hyperglycemia, at the time of hospitalization and during the postdischarge follow-up: 1-, 3-, and 6-month outpatient visits. RESULTS: Among 150 randomly selected patients 40 (26.6%) had diabetes. Diabetes (hazard ratio [HR] 8.9, P < .001), glucose levels (HR 1.25 × 1.1 mmol/L, P < .001), and glucose variability (HR 1.17 × 0.6 mmol/L, P < .001) were independently associated with an increased risk of mortality. The neutralizing activity of SARS-CoV-2 antibodies in patients with diabetes was superimposable, as for kinetics and extent, to that of patients without diabetes. It was similar across glucose levels and correlated with the humoral response against the SARS-CoV-2 spike protein. Positivity for Nabs at the time of hospital admission conferred protection on mortality, both in the presence (HR 0.28, P = .046) or absence of diabetes (HR 0.26, P = .030). The longevity of the Nab response was not affected by diabetes. CONCLUSION: Diabetes and hyperglycemia do not affect the kinetics and durability of the neutralizing antibody response to SARS-CoV-2. These findings provide the rational to include patients with diabetes in the early phase of the vaccination campaign against SARS-CoV-2.

Antibody response to multiple antigens of SARS-CoV-2 in patients with diabetes: an observational cohort study.

AIMS/HYPOTHESIS: The aim of the study was to characterise the humoral response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in patients with diabetes. Demonstrating the ability to mount an appropriate antibody response in the presence of hyperglycaemia is relevant for the comprehension of mechanisms related to the observed worse clinical outcome of coronavirus disease 2019 (COVID-19) pneumonia in patients with diabetes and for the development of any future vaccination campaign to prevent SARS-CoV-2 infection. METHODS: Using a highly specific and sensitive measurement of antibodies by fluid-phase luciferase immunoprecipitation assays, we characterised the IgG, IgM and IgA response against multiple antigens of SARS-CoV-2 in a cohort of 509 patients with documented diagnosis of COVID-19, prospectively followed at our institution. We analysed clinical outcomes and antibody titres according to the presence of hyperglycaemia, i.e., either diagnosed or undiagnosed diabetes, at the time of, or during, hospitalisation. RESULTS: Among patients with confirmed COVID-19, 139 (27.3%) had diabetes: 90 (17.7%) had diabetes diagnosed prior to the hospital admission (comorbid diabetes) while 49 (9.6%) had diabetes diagnosed at the time of admission (newly diagnosed). Diabetes was associated with increased levels of inflammatory biomarkers and hypercoagulopathy, as well as leucocytosis and neutrophilia. Diabetes was independently associated with risk of death (HR 2.32 [95% CI 1.44, 3.75], p = 0.001), even after adjustment for age, sex and other relevant comorbidities. Moreover, a strong association between higher glucose levels and risk of death was documented irrespective of diabetes diagnosis (HR 1.14 × 1.1 mmol/l [95% CI 1.08, 1.21], p < 0.001). The humoral response against SARS-CoV-2 in patients with diabetes was present and superimposable, as for timing and antibody titres, to that of non-diabetic patients, with marginal differences, and was not influenced by glucose levels. Of the measured antibody responses, positivity for IgG against the SARS-CoV-2 spike receptor-binding domain (RBD) was predictive of survival rate, both in the presence or absence of diabetes. CONCLUSIONS/INTERPRETATION: The observed increased severity and mortality risk of COVID-19 pneumonia in patients with hyperglycaemia was not the result of an impaired humoral response against SARS-CoV-2. RBD IgG positivity was associated with a remarkable protective effect, allowing for a cautious optimism about the efficacy of future vaccines against SARs-COV-2 in people with diabetes. Graphical abstract.