Patient characteristics, angiographic and echocardiographic procedures evaluations
This was a multicenter observational cohort study aimed to investigate the relationship between thrombus viral load, thrombus dimension, and in-hospital outcomes in ASAP STEMI patients. We examined patients with first STEMI treated with the primary percutaneous coronary intervention (PPCI) and thrombus aspiration (TA) without coronavirus disease 2019 (COVID-19) between February 2020 and November 2020. Patients with clinical evidence of COVID-19 were excluded from the study. All patients met the guideline definition of STEMI [9]. Routine analyses were obtained on admission before coronary angiography and before the initiation of full medical therapy. All patients with STEMI had baseline serological samples before cardiac catheterization for full blood count, renal and liver function tests, C-reactive protein, D-dimer, fibrinogen, lactate dehydrogenase (LDH), and high sensitivity (hs)-Troponin T. We considered eligible for the study all patients with: correspondence between ECG findings and suspected culprit artery; a minimum visual estimate of 50% stenosis in the culprit artery, and feasibility of performing TA, as judged by the treating physician; the age of 18 years or greater; presentation to the cardiac catheterization laboratory for PPCI in the setting of first STEMI. Patients with left ventricular ejection fraction less than 25%, with previous myocardial infarction or previous PPCI and/or coronary by-pass grafting, or who had received fibrinolytic therapy were instead excluded from the study.
Coronary angiography
Coronary angiography was performed either via the radial or femoral artery. The culprit lesion was identified and crossed with an angioplasty guidewire. During primary PCI, unfractionated heparin was administered in a loading dose of 70–100 U/kg with the activated clotting time (ACT) maintained > 250 s. ACTs were recorded at 10–15 min intervals after the initial dose of heparin. Glycoprotein (GP) IIb/IIIa inhibitors were used at the operator’s discretion.
Thrombus burden
The thrombus content was classified by a modified TIMI Thrombus Grade Classification [10]. This classification scores the thrombus in five grades: Grade 0 (G0): No angiographic characteristics of thrombus are present; Grade 1 (G1): Possible thrombus is present (reduced contrast density, haziness, or irregular lesion contour); Grade 2 (G2): There is definite thrombus, with greatest dimensions ≤ half the vessel diameter; Grade 3 (G3): Definite thrombus, with greatest linear dimension > half the vessel diameter but < 2 vessel diameters; Grade 4 (G4): Definite thrombus, with the largest dimension ≥ 2 but < 4 vessel diameters; Grade 5 (G5): Definite thrombus, with the largest dimension ≥ 4 vessel diameters [2]. To date, two experienced interventional cardiologists independently evaluated all angiographic parameters. Two independent pathologists, blinded to study protocol, evaluated the thrombus dimension. After TA, thrombus surface area was defined as the product of its length, height, and thickness. Therefore, the thrombus dimension was expressed as surface area in mm2.
Thrombus aspiration
2017 Guidelines of the European Society of Cardiology for the management of acute myocardial infarction in patients presenting with ST-segment elevation [11] and 2018 Guidelines on myocardial revascularization [12] do not recommend routine use of thrombus aspiration (class III, level A). Nevertheless, the same guidelines state that thrombus aspiration may be considered in large residual thrombus burden cases. According to these recommendations, with the support of the flowchart proposed by Junhua Ge et al. [13], manual TA was performed based on angiographic selection criteria (e.g., the presence of a visible thrombus on angiography, large vessel easy to pass with the catheter, localization of the thrombus at the proximal or middle segments of the target vessel, TIMI Thrombus Grade Classification Grade G3–G5), followed by conventional PCI to the culprit's vessel. The TA started before crossing the lesion, with a minimum of two syringes (40 mL) of aspirate recommended. Investigators were appropriately trained to ensure that the guide catheter was engaged with the coronary ostia when removing the thrombectomy catheter. Finally, the guide catheter was aspirated after thrombectomy to avoid embolization of either air or thrombus from the guide catheter.
Myocardial blush grade
The Myocardial Blush Grade (MBG) was defined according to the Zwolle criteria [14]. Angiographic assessment of myocardial reperfusion in patients treated with primary angioplasty for acute myocardial infarction: myocardial blush grade. However, we defined the blush grade into grades from 0 to 3. Thus, grade 0 indicated no myocardial blush or contrast density; or persistent “staining,” suggesting leakage of the contrast medium into the extravascular space. The grade 1 blush was defined as minimal myocardial blush or contrast density. Grade 2 was indicative of moderate blush or obtained on the contralateral/ipsilateral non-IRA, and grade 3 was indicative of normal blush. The angiography studies were evaluated at 2 independent centers by experienced interventional cardiologists in both cases. The observers were blinded to the remaining clinical information. Laboratory 1 (Lab 1) is the institution that carried out the study, the catheterization laboratory of a teaching hospital, and laboratory 2 (Lab 2), an independent catheterization core laboratory with extensive experience measuring the myocardial blush index. Grades 2 and 3 were considered normal perfusion. The interobserver variability was 10% for detecting grades 2 and 3 blush and 20% for grade 3 blush in Lab 1, 13% and 15%, respectively, in Lab 2.
Echocardiographic evaluation
In the current study, two experienced physicians in echocardiography performed a trans-thoracic two-dimensional echocardiogram with M-mode, conventional Doppler imaging (TDI) measurements in each patient admitted for acute ST-elevation myocardial infarction (STEMI). For the examinations, we used a Philips iE33 echocardiography (Eindhoven, The Netherlands). The echocardiography was performed at hospital admission and at hospital discharge. Then, physicians acquired the images of echocardiography in the parasternal long and short-axis views. Thus, we calculated left ventricle end-diastolic diameter (LVEDD), end-diastolic volume (LVEDV), end-systolic diameter (LVESD), end-systolic volume (LVESV), and we determined left ventricle ejection fraction (LVEF) with the Simpson biplane method (ref). However, the physicians systematically performed averaged measurements in five consecutive samples to have final calculation measures. The physicians involved in the echocardiographic evaluation performed and analyzed each examination independently and blinded to the study protocol. Finally, two observers blinded to measures performed previously reviewed all measurements. The observers were blinded to study protocol [15].
The study follows the principles outlined in 1976, the Declaration of Helsinki, and its later amendments for the use of human tissue or subjects. The Institutional Review Board of University of Campania “Luigi Vanvitelli,” Naples, Italy approved the protocol (Ethical Committee number 268 for study on SARS-COV-2, and number 151 for study TA).
Nasal/pharyngeal swab and thrombus SARS-COV-2 analysis
Post-catheterization, all patients underwent routine nasal/pharyngeal swab and thrombus analysis for the SARS-CoV-2 virus using real time-polymerase chain reaction (RT-PCR). The sample was dissolved by homogenization and then extracted by ripospinvrd kit (GeneAll). The RT-PCR was performed on CFX-96 Real-time PCR system (Bio-Rad) by Allplex 2019-nCoV assay, based on the analysis of four fluorophores: FAM for the revelation of E gene, Cal Red 610 for RdRP gene, Quasar 670 for N gene, and HEX to analyze the internal control (IC). The result was evaluated through Seegene Viewer. Respiratory and thrombus specimens were collected by the local CDC and then shipped to designated authoritative laboratories to detect SARS-CoV-2 presence and load.
The viral load has been detected with cycle threshold (CT) values [5, 16]. CT values are the number of cycles needed to detect each genetic marker identified by real-time reverse transcription-polymerase chain reaction testing. A lower CT value indicates a higher amount of viral RNA. Paired values for each resident are depicted using a different shape. Positivity was defined as a cycle threshold (CT) value ≤ 38.0. Based on the admission SARS-CoV-2 RNA nasopharyngeal swab, patients were clustered in two groups: ASAP STEMI patients (positive nasopharyngeal swab) and SANE STEMI patients (negative nasopharyngeal swab). Patients with laboratory-confirmed SARS-CoV-2 infection were defined as asymptomatic, who has no symptoms at the time of first clinical assessment and had no symptoms at the end of follow-up. The end of follow-up was defined as one or more negative respiratory specimen RT-PCR test results.
In-hospital outcomes
The primary endpoints were thrombus dimension + thrombus viral load effects on microvascular bed perfusion in the infarct area (Blush grade) after PPCI. The secondary endpoints during hospitalization were major adverse cardiovascular events (MACEs) defined as a composite of cardiovascular death, nonfatal acute AMI, and heart failure during hospitalization.
Statistical analysis
Descriptive statistical analyses were performed using SPSS Statistics version 23.0 (IBM). A 2-sided p value < 0.05 defined statistical significance. Variables are expressed accounts (percentages), mean ± standard deviation (SD), and median [lower quartile-upper quartile] as appropriate. Chi-squared analysis or Fisher’s-exact test was used to compare categorical data between groups. The independent samples Student t test or ANOVA test was used to compare normally distributed continuous data between groups, and the Mann-Whitney U test was used to compare the distribution of continuous skewed data between groups.
Correlation performed using Pearson’s correlation analysis and Spearman’s correlation analysis in the case of skewed variables. Event rates were derived as Kaplan–Meier estimates and compared by log-rank test.
Transforming the MBG as as a dichotomic variable, indicating with 0 the MBG < 2 (MBG 0, and 1), and indicating as 1 the cut off of blush grade in the case of MBG > 2 (MBG 2, and 3) we build a multiple logistic regression analysis to evaluate the independent association of SARS-CoV-2 infection and thrombus viral load with thrombus dimension independently of age, sex, metabolic risk factors, hypertension, and smoking. Thus, in a first model (A) we tested this analysis for all study population (n = 176). Then, we build a second model (B) and third model (C) including in the analysis also the SARS-CoV-2 infection for B, and excluding the thrombus dimension from the analysis in the model C. The models B and C were built to evaluate the multiple logistic regression for ASAP patients (n = 46). The odds ratio and 95% confidence intervals (CI) were also calculated.
To date, the composite score for MACE has been build summarizing the variables as cardiovascular death, nonfatal acute AMI, and heart failure during hospitalization. Therefore, the MACE as study endpoint was diagnosed by the evidence at least of one of the indicated conditions (cardiovascular death, nonfatal acute AMI, and heart failure during hospitalization).
The sample size, estimated according to a global effect size of 27% with type I error of 0.05 and a power of 90%, was 140 patients. To date, we calculated a post hoc sample size for the study primary endpoint as global effect that was about 30 participants as ASAP and 110 participants as SANE.