In this prospective study, we analyzed covid-19 hypertensive patients consecutively admitted to the Department of Infectious Disease at University of Campania “Luigi Vanvitelli”, Naples, Italy between February 10, 2020 and April 20, 2020. Covid-19 infection was categorized as follow: a) Mild, patients with fever and no pneumonia evidence in imaging; b) Moderate, patients with fever, respiratory tract symptoms, pneumonia confirmed at imaging without the need for invasive ventilation; c) Critical, occurrence of respiratory failure requiring mechanical ventilation, presence of shock, other organ failure requiring monitoring and treatment in intensive care unit (1).
patients with previous inflammatory disorders, malignancy, renal diseases; unavailability of a written informed consent; patients without cardiac biomarkers evaluation, including values of high-sensitivity troponin I (hs-TNI) and creatinine kinase–myocardial band (CK-MB). The diagnosis of hypertension was made following the international guidelines (10), and/or by known history of hypertension and current anti-hypertensive therapy.
All enrolled patients were treated with the same standard protocol: non-invasive oxygen therapy; hydroxychloroquine (400 mg/daily) and lopinavir/ritonavir (200/50 mg daily). According to AB0 blood group, patients were then categorized as “0 and non-0 blood group”, (6, 7). Established cardiac biomarkers, including hs-TNI, CK-MB, and myo-hemoglobin, were collected for every participant at hospital admission by 2 investigators (V.M. and C.S.).The investigation conforms to the principles outlined in the Declaration of Helsinki for the study of human subjects or tissues. The institutional ethics committee of the University of Campania “Luigi Vanvitelli” approved the study protocol. Written informed consent was obtained from all participating patients.
Study Outcomes. In this study, we investigated the inflammatory and coagulative status, and the cardiac injury and deaths in hypertensive patients with covid-19, with the aim to compare 0 vs. non-0 blood group. Cardiac injury and death were reported in a previous study for patients with covid-19 (11).Cardiac injury was defined as blood levels of cardiac biomarkers (hs-TNI) above the 99th -percentile upper reference limit (11). Data on cardiac injury and death were collected by two independent physicians (P.M; R.M) during clinical examination, laboratory and imaging tests in hospitalized patients, and by examination of hospital discharge schedules (11).
Laboratory and imaging evaluations.
-Real-time reverse transcription (RT-PCR assay for SARS-CoV-2. Respiratory specimens were collected from each patient and then shipped to specialized laboratories designated by the Italian government for confirming covid-19 infection. The presence of SARS-CoV-2 in respiratory specimens was detected by established RT-PCR methods. Laboratory analyses were obtained on admission before starting covid-19 medical therapy and during hospitalization.
-Clinical and laboratory parameters. We tested respiratory specimens, including nasal and pharyngeal swabs or sputum, to exclude evidence of other viral infections, including influenza, respiratory syncytial virus, avian influenza, para-influenza, and adenovirus. We also performed routine bacterial and fungal examinations. Laboratory assessments consisted of a complete blood count, blood chemical analysis, coagulation testing, evaluation of liver and renal function, and measures of electrolytes, C-reactive protein, procalcitonin, lactate dehydrogenase, and creatine kinase. Venous blood for IL-6 (Human ELISA Kit, RD System) and D-dimer (Human ELISA Kit, Invitrogen) levels was collected in EDTA-coated tubes immediately after patients arrived at the department and weekly during hospitalization.
The AB0 phenotypes were ascertained by genotyping for four single nucleotide polymorphisms of the AB0 gene: G261del, A297 G, G703A and C526G, as described (9). Briefly, we used single nucleotide polymorphisms of the C526G to decipher the O303 allele, which, unlike other O alleles, does not have a deletion at nucleotide position 261 (9). We determined genotyping by using the multiplexing capability of the MassARRAY homogenous MassEXTEND assay of the Sequenom system (San Diego, CA, USA). Therefore, the DNA fragments surrounding the single nucleotide polymorphisms sites were amplified by PCR, treated with shrimp alkaline phosphatase to dephosphorylate unincorporated dNTPs, followed by the extension primers that form allele-specific extension products. However, each extension product had a unique mass, measured using MALDI-TOF. Genotypes were automatically assigned to each sample using the Mass ARRAY RT software. The presence or absence of FV Leiden (A1691 G, R506Q) and the prothrombin G20210A polymorphism was assessed by standard methods (9). All patients underwent ECG at hospital admission, and in case of elevation of cardiac biomarkers during hospitalization; findings compatible with myocardial ischemia included T-wave depression and inversion, ST-segment depression, and Q waves. Two blinded physician (C.S, R.M) reviewed and analyzed ECG patterns. Radiologic assessments included chest radiography and/or computed tomography (CT) at admission and weekly during hospitalization, and all laboratory testing was performed according to the clinical care needs of each patient. We determined the presence of radiologic abnormalities on the basis of the documentation or description in medical charts; if imaging scans were available, they were reviewed by attending physicians in respiratory medicine who extracted the data. Two blinded physician experienced in lung imaging (G.G, V.C.) reviewed and analyzed chest radiography and CT patterns. Major disagreement between two reviewers was resolved by consultation with a third reviewer.
Statistical Analysis. Continuous variables were expressed as medians and interquartile ranges or simple ranges, as appropriate. Categorical variables were summarized as counts and percentages. We performed only descriptive statistics, because the cohort of patients in our study was not derived from random selection. We performed a risk adjusted Cox-regression analysis to assess survival from cardiac injury and deaths through days of hospitalization; Cox models were adjusted for; age, gender, body mass index, heart rate, cholesterol, high density lipoprotein-cholesterol, low density lipoprotein-cholesterol, triglycerides levels, heart diseases, dyslipidemia, diabetes, current smoking, beta-blockers, ace-inhibitors, calcium inhibitors, thiazide diuretics, aspirin. Only variables presenting a p value ≤ 0.25 at the univariate analysis were included in the model. We used a stepwise method with backward elimination, and we calculated odds ratios (OR) with 95% confidence intervals. The model was evaluated with a Hosmer and Lemeshow test. Kaplan-Meier survival analysis was performed for cardiac injury events and deaths in patients divided in: 0 vs. non-0 blood group. A p value < 0.05 was considered statistically significant. All calculations were performed using the software SPSS23.