HomeCirculationVol. 144, No. 6In-Depth Evaluation of a Case of Presumed Myocarditis After the Second Dose of COVID-19 mRNA Vaccine Free AccessCase ReportPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissionsDownload Articles + Supplements ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toSupplemental MaterialFree AccessCase ReportPDF/EPUBIn-Depth Evaluation of a Case of Presumed Myocarditis After the Second Dose of COVID-19 mRNA Vaccine Alagarraju Muthukumar, Madhusudhanan Narasimhan, Quan-Zhen Li, Lenin Mahimainathan, Imran Hitto, Franklin Fuda, Kiran Batra, Xuan Jiang, Chengsong Zhu, John Schoggins, James B. Cutrell, Carol L. Croft, Amit Khera, Mark H. Drazner, Justin L. Grodin, Benjamin M. Greenberg, Pradeep P.A. Mammen, Sean J. Morrison and James A. de Lemos Alagarraju MuthukumarAlagarraju Muthukumar Department of Pathology (A.M., M.N., L.M., I.H., F.F.), University of Texas Southwestern Medical Center, Dallas. , Madhusudhanan NarasimhanMadhusudhanan Narasimhan Department of Pathology (A.M., M.N., L.M., I.H., F.F.), University of Texas Southwestern Medical Center, Dallas. , Quan-Zhen LiQuan-Zhen Li Department of Immunology (Q.-Z.L.), University of Texas Southwestern Medical Center, Dallas. Department of Internal Medicine (Q.-Z.L., X.J., C.Z., J.B.C., C.L.C., A.K., M.H.D., J.L.G., P.P.A.M., J.A.d.L.), University of Texas Southwestern Medical Center, Dallas. , Lenin MahimainathanLenin Mahimainathan Department of Pathology (A.M., M.N., L.M., I.H., F.F.), University of Texas Southwestern Medical Center, Dallas. , Imran HittoImran Hitto https://orcid.org/0000-0002-9928-4175 Department of Pathology (A.M., M.N., L.M., I.H., F.F.), University of Texas Southwestern Medical Center, Dallas. , Franklin FudaFranklin Fuda Department of Pathology (A.M., M.N., L.M., I.H., F.F.), University of Texas Southwestern Medical Center, Dallas. , Kiran BatraKiran Batra Department of Radiology (K.B.), University of Texas Southwestern Medical Center, Dallas. , Xuan JiangXuan Jiang Department of Internal Medicine (Q.-Z.L., X.J., C.Z., J.B.C., C.L.C., A.K., M.H.D., J.L.G., P.P.A.M., J.A.d.L.), University of Texas Southwestern Medical Center, Dallas. , Chengsong ZhuChengsong Zhu Department of Internal Medicine (Q.-Z.L., X.J., C.Z., J.B.C., C.L.C., A.K., M.H.D., J.L.G., P.P.A.M., J.A.d.L.), University of Texas Southwestern Medical Center, Dallas. , John SchogginsJohn Schoggins Department of Microbiology (J.S.), University of Texas Southwestern Medical Center, Dallas. , James B. CutrellJames B. Cutrell Department of Internal Medicine (Q.-Z.L., X.J., C.Z., J.B.C., C.L.C., A.K., M.H.D., J.L.G., P.P.A.M., J.A.d.L.), University of Texas Southwestern Medical Center, Dallas. , Carol L. CroftCarol L. Croft Department of Internal Medicine (Q.-Z.L., X.J., C.Z., J.B.C., C.L.C., A.K., M.H.D., J.L.G., P.P.A.M., J.A.d.L.), University of Texas Southwestern Medical Center, Dallas. , Amit KheraAmit Khera https://orcid.org/0000-0001-7255-6874 Department of Internal Medicine (Q.-Z.L., X.J., C.Z., J.B.C., C.L.C., A.K., M.H.D., J.L.G., P.P.A.M., J.A.d.L.), University of Texas Southwestern Medical Center, Dallas. , Mark H. DraznerMark H. Drazner https://orcid.org/0000-0003-3054-4757 Department of Internal Medicine (Q.-Z.L., X.J., C.Z., J.B.C., C.L.C., A.K., M.H.D., J.L.G., P.P.A.M., J.A.d.L.), University of Texas Southwestern Medical Center, Dallas. , Justin L. GrodinJustin L. Grodin https://orcid.org/0000-0003-2400-3196 Department of Internal Medicine (Q.-Z.L., X.J., C.Z., J.B.C., C.L.C., A.K., M.H.D., J.L.G., P.P.A.M., J.A.d.L.), University of Texas Southwestern Medical Center, Dallas. , Benjamin M. GreenbergBenjamin M. Greenberg https://orcid.org/0000-0002-2091-8201 Department of Neurology and Neurotherapeutics (B.M.G.), University of Texas Southwestern Medical Center, Dallas. Department of Pediatrics (B.M.G.), University of Texas Southwestern Medical Center, Dallas. , Pradeep P.A. MammenPradeep P.A. Mammen https://orcid.org/0000-0001-5688-7091 Department of Internal Medicine (Q.-Z.L., X.J., C.Z., J.B.C., C.L.C., A.K., M.H.D., J.L.G., P.P.A.M., J.A.d.L.), University of Texas Southwestern Medical Center, Dallas. , Sean J. MorrisonSean J. Morrison https://orcid.org/0000-0003-1587-8329 Howard Hughes Medical Institute (S.J.M.), University of Texas Southwestern Medical Center, Dallas. Children's Medical Center Research Institute (S.J.M.), University of Texas Southwestern Medical Center, Dallas. and James A. de LemosJames A. de Lemos Correspondence to James A. de Lemos, MD, Department of Internal Medicine, University of Texas Southwestern Medical Center, 5939 Harry Hines Boulevard, Dallas, TX 75390. Email E-mail Address: [email protected] https://orcid.org/0000-0003-2211-7261 Department of Internal Medicine (Q.-Z.L., X.J., C.Z., J.B.C., C.L.C., A.K., M.H.D., J.L.G., P.P.A.M., J.A.d.L.), University of Texas Southwestern Medical Center, Dallas. Originally published16 Jun 2021https://doi.org/10.1161/CIRCULATIONAHA.121.056038Circulation. 2021;144:487–498Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: June 16, 2021: Ahead of Print Initial PresentationA 52-year-old man presented to the emergency department ≈90 min after the onset of substernal chest pain. Three days before presentation, he received his second dose of mRNA-1273 (Moderna) vaccine for coronavirus disease 2019 (COVID-19), and the next day had a severe reaction that he described as being the "worst he had ever felt." He had subjective high fevers, shaking chills, myalgias, and a headache. These symptoms largely resolved by the third day after vaccination except for a positional headache that was unusual for him. On the morning of hospitalization, he walked 3 to 4 miles and felt fine. Later that day, while in a meeting, he developed persistent midsternal chest discomfort without radiation, prompting him to seek evaluation in a university hospital emergency department. The pain subsided spontaneously after approximately 3 hours. He had no associated dyspnea, palpitations, dizziness, fever, chills, or myalgia.The patient had a past medical history of hypertension, hypercholesterolemia, obstructive sleep apnea treated with an oral appliance, and minor elevations in liver function tests attributed to possible hepatic steatosis. A recent screening coronary artery calcium scan demonstrated coronary artery calcium at the 81st percentile for age and sex. The patient had no previous history of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. His medications included aspirin 81 mg, simvastatin 40 mg, ezetimibe 10 mg, and lisinopril 10 mg daily, and he took no supplements. He drank alcohol socially and denied use of tobacco and all recreational drugs.On physical examination, the following vital signs were recorded: oral temperature 36.8°C, pulse 73/min, blood pressure 124/76, and respiratory rate 18/min, and his oxygen saturation was 100% on room air. Pulmonary and cardiac examinations were normal without a pericardial friction rub. The remainder of his physical examination was normal.In the emergency department, his initial ECG showed sinus rhythm with left axis deviation and incomplete right bundle-branch block without ST or T wave changes (Figure 1A). His initial high-sensitivity cardiac troponin I was 2768 ng/L. Point-of-care echocardiogram showed normal left ventricular function and volumes, and no wall motion abnormalities. Urgent coronary angiography showed mild nonobstructive coronary artery disease with no stenoses or visible thrombus and no evidence of coronary embolism or dissection (Figure 1B and 1C).Download figureDownload PowerPointFigure 1. ECG and coronary angiogram.A, The ECG on presentation to the emergency department. B, A posterior anterior cranial projection of a dominant right coronary artery and with no severe angiographic stenoses or flow-limiting lesions in the main vessel or its branches. C, A right anterior oblique caudal projection of a bifurcating left coronary artery and no severe angiographic stenoses or flow-limiting lesions in the main vessel or its branches.His initial laboratory panel revealed normal white blood cells 6.3 × 109/L (76% polymorphonuclear leukocytes, 14% lymphocytes, 9% monocytes, 0.5% eosinophils, and 0.2% basophils), hemoglobin 14.9 g/L, and platelets 207 × 109/L. Chemistries were remarkable for glucose of 172 mg/dL, but creatinine 0.87 mg/dL and alanine aminotransferase 58 U/L were consistent with his baseline. High-sensitivity cardiac troponin I peaked at 6770 ng/L at 7 hours after admission and remained elevated (551 ng/L) even after 4 days. In contrast, high-sensitivity cardiac troponin T and creatine kinase-MB biomarkers showed modest elevation (Table 1). C-reactive protein, erythrocyte sedimentation rate, and D-dimer were elevated in the first sample taken at the time of admission but resolved to near normal levels within 1 to 2 days. Antinuclear antibodies were negative.Table 1. Relevant Biochemical Parameters in the Case of InterestDescriptionCIS1CIS2CIS3CIS4Reference rangecTnI HS (ng/L)677015961440551<26cTnT HS (V gen P) (ng/L)n/an/an/a138≤15.0BNP (pg/mL)50n/an/an/a<100CRP (mg/L)19.112.85.9n/a≤5.0ESR (mM/h)2542n/an/a0–15CK-MB Index4.83.2n/an/a0.0-3.0Ferritin (ng/mL)162119n/an/a22–275D-dimer (mg/L FEU)0.740.57n/an/a≤0.59IL-6 (pg/mL)<2.0<2.0n/an/a<2.0Glucose (mg/dL)17211310716670–139AST (U/L)49n/an/an/a10–50ALT (U/L)58n/an/an/a10–50Abnormal results are shown in bold. CIS1, CIS2, CIS3, and CIS4 indicate case of interest sample at day 1, day 2, day 3, and day 4 after symptom onset, respectively. ALT indicates alanine aminotransferase; AST, aspartate aminotransferase; BNP, B-type natriuretic peptide; CK-MB, creatine kinase-MB; CRP, c-reactive protein; cTnI HS, high-sensitivity cardiac troponin I; cTnT HS, high-sensitivity cardiac troponin T; ESR, erythrocyte sedimentation rate; IL-6, interleukin 6; and n/a, not tested or not available.Additional Clinical TestingA repeat echocardiogram performed on hospital day 2 revealed a normal ejection fraction without wall motion abnormalities and no valvular or pericardial abnormalities. Contrast-enhanced cardiac magnetic resonance imaging (MRI) with parametric mapping was performed on a 1.5T MRI scanner (Siemens Healthineers) on hospital day 3. Delayed contrast-enhanced phase-sensitive images showed midmyocardial and subepicardial linear and nodular late gadolinium enhancement in the inferoseptal, inferolateral, anterolateral, and apical walls. The left ventricle showed mild dilatation and "low normal" left ventricular ejection fraction at 54%. The right ventricular ejection fraction was normal at 58%. Parametric mapping showed elevated T1 relaxation time and relative inhomogeneity and focal elevation of the T2 relaxation values (Figure 2). In addition, wall motion abnormalities with mild hypokinesis of the lateral and inferior apical walls were noted. These findings were consistent with myocarditis on the basis of the modified Lake Louise criteria.1Download figureDownload PowerPointFigure 2. Phase-sensitive inversion-recovery cardiac magnetic resonance imaging.Right, Short-axis views demonstrating linear and curvilinear delayed enhancement in the subepicardial inferior basal and mesocardial midventricular region, compatible with nonischemic pattern of delayed enhancement. Middle, A native T1 map showing globally increased T1 values (1054 ms), Local native myocardial T1 (short axis [SA] and 4 chamber [4CH] midwall) (965 ± 35) and specifically higher values in the regions of delayed enhancement. The color map shows relaxation times with normal relaxation time in green and increased relaxation time in red and orange. Left, Native T2 map with heterogenous relative increased T2 values within the same segments (arrows) (maximum T2 value was 65 ms) (local normal T2 values for our institution, 45–64 ms). Color scale shows time in milliseconds.Hospital CourseThe chest discomfort in the patient had fully resolved within 3 hours after onset and did not recur. He reported feeling normal throughout the remainder of his 4-day hospital stay. Endomyocardial biopsy was not performed because of his resolution of symptoms, preserved left ventricular ejection fraction, and the absence of any hemodynamic or arrhythmic complications. The patient was treated with low-dose lisinopril and carvedilol, but no immunosuppressive or anti-inflammatory medications. At the time of discharge, the patient remained asymptomatic, and his high-sensitivity cardiac troponin T levels had fallen to 138 ng/L. His NT-proBNP (N-terminal pro-B-type natriuretic peptide) at discharge was <27 pg/mL.Postdischarge CourseThe patient has had no recurrent symptoms in >3 months since hospital discharge. Given the presumptive diagnosis of myocarditis, exercise has been restricted, and he has remained on β-blocker and angiotensin-converting enzyme inhibitor medications. Repeat high-sensitivity cardiac troponin I was 10 ng/L 4 days after discharge and undetectable (<5 ng/L) 2 weeks after discharge. Serial cardiac MRIs have been performed showing a gradual reduction in left ventricular volumes and reduction in the degree of late gadolinium enhancement abnormalities, with normalization of T1 relaxation time and decrease in the T2 relaxation time (Table 2).Table 2. Left Ventricular Volume and Late Gadolinium Enhancement Abnormalities in the Case of InterestVariableLeft ventricular volumeLate gadolinium enhancementT1 relaxation time (ms)T2 relaxation time (ms)Hospitalization196 mL+++105450–642 wk after discharge163 mL++101543–5212 wk after discharge138 mL++96942–47+++ indicates high; ++, moderate; normal native myocardial T1 (short axis [SA] and 4 chamber [4CH] midwall): 965±35; and normal T2 values: 40–64 ms.Exploratory Studies to Investigate Potential Pathological MechanismsMyopericarditis has been reported to the US national passive Vaccine Safety Surveillance System (VAERS) as a rare adverse event after vaccinations, with most reports associated with smallpox vaccination.2 However, at the time of the patient's presentation, there were no reported cases of myocarditis caused by COVID-19 vaccination.To explore potential mechanisms of myocardial injury in temporal association with vaccination in the present case, written informed consent was obtained for additional in-depth analysis of viral, cytokine, and autoimmune panels and subsequent research publication of the case. Samples from the patient of interest were compared with excess, remnant blood specimens that were available in the laboratory after routine clinical testing. Samples from the case of interest (CI) were collected on days 1 to 4 after symptom onset (CIS1–S4) and were compared with 4 groups: naive unvaccinated (NUV; n=8), unvaccinated patients hospitalized with COVID-19 (n=10), naive vaccinated (NV; n=10), and age-matched controls receiving Moderna vaccine (NM, n=2). NV and NM groups were tested ≈?2 weeks after receiving their second vaccine dose. The studies were performed as part of a biorepository protocol approved by the University of Texas Southwestern Institutional Review Board, and waiver of Institutional Review Board consent was obtained to use the remnant blood specimens. Detailed methods are provided in the Methods in the Data Supplement.Results of Exploratory StudiesAntibody response to viral antigens and SARS-CoV-2 nucleocapsid and spike proteins serum immunoglobulin (Ig) G antibodies against 18 different viral antigens and SARS-CoV-2 serology testing were measured using a custom developed proteome array and US Food and Drug Administration–approved standard assays, respectively, using the methods described in the Methods in the Data Supplement. These studies confirmed the absence of previous COVID-19 infection (negative reactivity for SARS-CoV-2 nucleocapsid IgG) (Figures 3 and 4). As expected, a clear immune response to the vaccine (SARS-CoV-2 spike as a component) was observed in the case of interest 5 and 6 days after the second dose of Moderna vaccine, which corresponds with the third and fourth day after symptom onset in the case of interest (CIS3 and CIS4) (Figure 3). Comparison of the strength of vaccine-induced immune responses in the case of interest at the measured sampling period CIS2 and CIS4 with either NV or NM did not reveal abnormally elevated SARS-CoV-2 spike IgG or SARS-CoV-2 spike IgM levels (Figure 4). Low IgG serology reactivity was noted in the case patient samples (CIS1–S4) for the other viral antigens, including cytomegalovirus, Epstein-Barr virus, influenza A, and respiratory syncytial virus compared with vaccinated control samples (Figure 3). It is interesting that, although anti-spike antibody levels were higher than the manufacturer-recommended positive threshold, the SARS spike protein antibody levels were either lower or just comparable in the case compared with NV (Figure 4). This may be partly explained by a difference in the timing of blood sampling after immunization among the vaccinated controls (2 weeks) versus case patient (5–6 days) for assessing antibody response. Concurrent clinical evaluation for known infectious causes of acute myocarditis, including multiple SARS-CoV-2 nasopharyngeal polymerase chain reaction tests and Food and Drug Administration–approved multiplex respiratory viral polymerase chain reaction panels and serologies, were all negative with 2 exceptions. An IgG antibody for Mycoplasma pneumoniae was positive but IgM antibody was negative, consistent with previous exposure and not acute infection. In addition, an IgG titer of 1:320 was reported for coxsackie B virus 4, but IgM antibody titers were negative. However, convalescent serum antibody testing 3 weeks later revealed a titer of 1:160, consistent with remote and not acute or recent infection.Download figureDownload PowerPointFigure 3. Antibody profile to viral antigens in the case of interest as compared with naive vaccinated, naive unvaccinated, and COVID-19 unvaccinated patients. The heatmap shows immunoglobulin G reactivity expressed in terms of row z-score for a respective antigen across different patient samples. Each antigen is organized into rows color-coded by virus, for serum specimens organized into columns classified as naive unvaccinated (NUV, 8 samples), (COVUV, 10 samples), naive vaccinated (NV, 10 samples), naive Moderna vaccinated controls (NM, 2 samples), and case of interest samples (CIS, collections at day 1, day 2, day 3, and day 4 after symptom onset: S1, S2, S3, and S4 in the respective order). Reactivity is represented by color (light blue=low, black=mid, ellow=high). The heatmap has normalized row z-score values, a typical scaling method that helps better visualization of analytes with varying trends in the expression/reactivity between samples. Although a normalized row z-score can better represent the nonrandomness of directionality within a dataset, a negative z-score does not indicate a complete absence of expression/reactivity. A negative z-score means comparatively a lower raw scores/absolute expression. CMV indicates cytomegalovirus; COVID-19, coronavirus disease 2019; EBV, Epstein-Barr virus; and RSV, respiratory syncytial virus.Download figureDownload PowerPointFigure 4. SARS-CoV-2–related antibody status in the case of interest as compared with naive vaccinated, naive unvaccinated, and COVID-19–unvaccinated patients. Comparison of SARS-CoV2–related antibody response in the case of interest with naive vaccinated, naive unvaccinated, and COVID unvaccinated patients. A, Evaluation of spike-specific IgG antibody response. B, Comparison of spike-specific IgM levels. C, Comparison of nucleocapsid-specific antibody response. For A through C, all the patient samples in the NV group were immunized with Pfizer vaccine. AU indicates arbitrary units; CIS2, case of interest sampled at day 2 after symptom onset; CIS4, case of interest sampled at day 4 after symptom onset; COVID-19, coronavirus disease 2019; COVUV, COVID-19 unvaccinated; Ig, immunoglobulin; NM, age- and vaccine (Moderna)–matched naive (positive controls for case of interest); NUV, naive unvaccinated; NV, naive vaccinated; SARS-CoV2, severe acute respiratory syndrome coronavirus 2; and SP, spike. Dashed brown line indicates the manufacturer-recommended positive threshold of the respective antibody assays used.Genetic TestingGiven that inherited cardiomyopathy may present clinically as myocarditis,3 a panel test for variants in 121 genes potentially linked to cardiomyopathy was performed (Invitae, San Francisco, CA). No pathogenic variants and 1 intronic variant of unknown significance (heterozygous, ACTN2, c2367+5G>A) were identified, suggesting that the known gene variants are not the cause of myocarditis in the case patient.Screening of Cytokine ResponseAlthough the vaccine-induced immune response is chiefly linked to protective immunity, an exaggerated and unwarranted immune reaction could potentially heighten inflammation and augment the risk of immunopathology. We measured a panel of 48 cytokines and chemokines in the case patient using fluorescent bead-based Bio-Plex Pro Human Cytokine Screening Panel, per the manufacturer's instructions (Bio-Rad, CA), as described in the Methods in the Data Supplement. Cytokine levels in the case patient were NV or NM (Figure 5). The trend of cytokine changes in the case of interest along with the control groups is shown in Table 3. To aid efficient interpretation of this data, we considered as abnormal only the analytes with ≥2.0-fold increase (bold) or a ≥2.0 decrease (bold and italics) in CIS1–S4 versus both NM and NUV groups. The NUV comparison provides a reference interval to interpret the case patient's cytokine results. Given the inclusion of 2 comparators NM and NUV, if the 2-fold change is in 1 direction versus 1 comparator and in the opposite direction for another comparator, then those cytokine changes are indicated in italics. This analysis revealed in the case patient elevated levels of 4 cytokines (IL-1ra, IL-5, IL-16, and MIG), diminished levels of 1 cytokine LIF (leukemia inhibitory factor), and 3 other cytokines (IL-10, MIF, and VEGF) with bidirectional pattern (increase or decrease) relative to the comparators, NM or NUV (Table 3). Although statistical inference is not possible because of the single case patient, and the clinical relevance of the magnitude of difference seen is not clear, some of the following changes are of potential interest. The level of IL-1ra (IL-1 receptor antagonist) in the first sample from the case patient after symptom onset (CIS1; 1174 pg/mL) was comparable with levels in patients with active COVID-19 infection (unvaccinated patients hospitalized with COVID-19; 1183 pg/mL). Generation of IL-1ra could be a compensatory counterattacking mechanism to limit excessive inflammation. In support of this notion, it has been documented that treatment with IL-1ra rescues myocarditis-associated end-stage heart failure.4 Around the time of symptom onset, the case patient also displayed elevated levels of other cytokines, IL-5, IL-16, and MIG (CXCL9), which play inflammatory roles in either myocarditis or related cardiac complications in humans or in experimental animal models.5–8 In contrast, relative to NM or NUV, the first sample of the case patient (CIS1) showed a decrease in the levels of cytokine LIF, which provides cellular stability and ensures survival of cardiomyocytes during stress.9 The other 3 cytokines, VEGF, IL-10, and MIF, did not reveal a unidirectional regulatory pattern with comparators (NM and NUV); however, each spiked above the NUV reference group and has been individually implicated in immune vasculitis.10–12 Additional clinical laboratory assessment of IL-1β, IL-2, and IL-6 cytokines revealed normal levels of these cytokines (data not shown).Table 3. Cytokine Profile in Naive Unvaccinated, COVID-19 Unvaccinated, Naive Vaccinated, and the Case of InterestCytokineNUVCOVUVNVNMCIS1CIS2CIS3CIS4CCL271168581621634542668610803CCL11761201071507211712040bFGF4667323736323128G-CSF12733113422212215012295GM-CSF1.15.73.13.92.34.23.72.1CXCL1662671641627647669535678HGF5212687363316396372331725IFN-α211207811879IFNγ1596304817252410IL-1α1533131519191315IL-1β1.63.01.21.21.91.31.21.5IL-1ra18311832952971174†308235181IL-25.714.26.66.07.57.55.74.1IL-2Rα471895710040636243IL-30.010.720.140.090.160.080.160.01IL-40.91.70.81.01.10.91.00.9IL-50.052.729.517.31.784.5†69.2†25.6IL-60.818.93.03.32.64.45.21.8IL-7141198171474IL-88631313815109IL-9311253221190284207112284IL102118134‡171510IL-12 (P-70)2.84.84.63.03.22.81.91.9IL-12 (P-40)1213899510314711281112IL-131.92.92.01.92.62.82.42.0IL-150.0356.6248.7289.20.0453.0370.6224.2IL-163389261193607†199169132IL-17101679121179IL-1842592012058475474IP109301759378302794607521718LIF215722292*323314MCP135194578525435023MCP30.0121.451.662.163.312.201.661.66M-CSF13.469.818.023.519.829.722.018.7MIF4605348421028092702527020531223‡MIG2802955409407941†1342†918†501MIP-1α2.26.31.62.12.02.72.02.1MIP-1β22117315514122915997228β-NGF1.45.64.24.63.05.74.04.1PDGF-BB45501168842387116822589375RANTES13 77670027027478414 8315225231120 040SCF67199801254811111581SCGFβ116 351188 15084 74770 20094 735109 16395 983107 049SDF1α94453578110778437597841493TNFα10211980711058456104TNFβ0.011.05.07.71.511.59.45.3TRAIL4445424537545257VEGF44490343447149‡622549345Presented are the median pg/mL values for the groups, NUV (n=8), NV (n=10), and COVUV (n=10), average pg/mL values for NM (n=2), and individual pg/mL values for the groups CIS1–S4 (n=1 in each group). To help efficiently interpret these data, we considered only the analytes with a fold change of ≥2.0 in CIS1–S4 versus both NM and naive unvaccinated healthy control (NUV). CIS1, CIS2, CIS3, and CIS4 indicate case of interest sample at day 1, day 2, day 3, and day 4, respectively, after symptom onset; COVID-19, coronavirus disease 2019; COVUV, COVID-19 unvaccinated; n/a, not tested or not available; NM, age- and vaccine (Moderna)–matched naive (positive controls for case of interest); and NV, naive vaccinated.* ≥2.0-fold decrease (bold and italics);† ≥2.0-fold increase (bold);‡ >2.0-fold change (italics) in either direction of comparison with NM or NUV.Download figureDownload PowerPointFigure 5. Cytokine profile in the case of interest as compared with naive vaccinated, naive unvaccinated, and COVID-19–unvaccinated patients. The heatmap shows reactivity expressed in terms of row z-score for a respective antigen across different patient samples. Each row in the graphics represent a cytokine for serum specimens organized into columns classified as naive unvaccinated (NUV, 8 samples), COVID-19 unvaccinated (COVUV, 10 samples), naive vaccinated (NV, 10 samples), naive Moderna vaccinated controls (NM, 2 samples), and case of interest samples (CIS, 4 different collection days at day 1, day 2, day 3, and day 4 after symptom onset: S1, S2, S3, and S4 in the respective order). The reactivity intensity ranges from turquoise (low) to black (moderate) or yellow (high). For the groups NM and CIS, each patient sample was run in duplicates that were averaged and represented. Some of the samples that displayed values below the least detection range were arbitrarily assigned a lowest value. COVID-19 indicates coronavirus disease 2019.It should be emphasized that these cytokine analyses are exploratory and limited by the absence of baseline measurements in the case patient before vaccination. Although this empirical evidence cannot identify a specific cytokine candidate or signature, this approach represents a first step of searching for such a cytokine signature in COVID-19 vaccine–associated myocarditis and may provide important insights for subsequent studies in larger numbers of patients.AutoantibodiesImmunizations with adverse effects typically induce disproportionate autoantibody generation.13,14 Thus, we next investigated whether the COVID-19 mRNA vaccine and the associated nonviral acute myocarditis seen in the patient of interest may be a consequence of an autoimmune response, using a proteome array printed with HuProtTM version 3.1 arrays (CDI Laboratories, Mayaguez, PR) comprised of ≈19,500 unique full-length human proteins (Methods in the Data Supplement).Analyses for potentially informative autoantibodies were clustered into 3 separate subpanels representing common, COVID-specific, and CIS-specific groups for both IgM and IgG classes of circulating autoantibodies (Figure 6A and 6B). In the common subpanel, the case patient was characterized by higher levels of 2 IgM autoantibodies (CRK and UNC45B) (Figure 6A) and 6 IgG autoantibodies (IL-10, KCNK5, PARP1, VCL, AKAP5, and IFNγ) compared with the patient with active COVID-19 and NUV controls (Figure 6B), suggesting potential specific associations with myocarditis. Autoantibodies against IL-10 and IFNγ have been detected in patients with life-threatening COVID-19, and previous reports indicate a cardioprotective effect for these cytokines in humans and rodents.15–17 IgM autoantibodies against several common antigens, including TNNC1 (troponin C1) and IL-1RN, were elevated in both the case patient and the patient with COVID-19, which is expected given the presence of cardiac injury and inflammation present in both disease scenarios.Download figureDownload PowerPointFigure 6. Antibody profiles to self-antigens in the case patient relative to unvaccinated naive and COVID-19 patient samples. The heatmap shows the Phenolyzer-prioritized candidate proteins involved in cardiac disease expressed in terms of mean of the individual signal intensities from the duplicate samples that were corrected for background intensity followed by variance stabilizing normalization (VSN). Each row in the graphics represent the analytes for serum specimens organized into column
Abstract The rapid spread of SARS-CoV-2 Variants of Concern (VOC) necessitates systematic efforts for epidemiological surveillance. The current method for identifying variants is viral whole genome sequencing (WGS). Broad clinical adoption of sequencing is limited by costly equipment, bioinformatics support, technical expertise, and time for implementation. Here we describe a scalable, multiplex, non-sequencing-based capillary electrophoresis assay to affordably screen for SARS-CoV-2 VOC.
Abstract Background The success of COVID-19 mRNA vaccines (Co-mV) has provided a renewed impetus in developing “onetime universal flu, COVID”, and other mRNA-based vaccines to offer broader and long-lasting protection. However, to bring this endeavor to fruition, it is of prime importance to address the mechanism(s) underlying poor vaccine response in some patients and close this gap of inequitable effectiveness through improvement strategies, which is the focus of our current study. To date, there is no ‘empirical’ evidence to link the perturbation of translation, a rate-limiting step for mRNA vaccine efficiency, to its dampened response. Methods This IRB approved study involved 1) an assessment of a total of 1009 immunocompromised (IC) patients and immunocompetent subjects who had received 2 or more doses of Co-mV, 2) in vitro cell-culture experiments, and 3) in vivo animal studies. Impact of immunosuppressants (ISs), tacrolimus (T), mycophenolate (M), rapamycin/sirolimus (S), and their combinations on Pfizer Co-mV translation were determined by the Spike (Sp) protein expression following Co-mV transfection in HEK293 cells. In vivo impact of ISs on SARS-CoV-2 spike specific antigen (SpAg) and associated antibody levels (IgGSp) in serum were longitudinally assessed (26 days) in Balb/c mice after two doses (2D) of the Pfizer vaccine. Spike Ag and IgGSp levels were assessed in 259 IC patients and 50 healthy controls (HC) who received 2D of Pfizer or Moderna Co-mV. In addition, 67 immunosuppressed solid organ transplant (SOT) patients and 843 non-transplanted (NT) subjects following three doses (3D) of Co-mV were assessed. Expression of Sp and p70S6K phosphorylation (translation surrogate) were evaluated following higher vaccine doses and transient drug holidays. Statistical analysis was performed using GraphPad software 9.3.1. p < 0.05 was considered as statistically significant. Results The 2D and 3D Co-mV received IC patients showed a significantly lower IgGSP response (p < 0.0001) relative to their matched controls. M or S profoundly dampened (p < 0.001) the IgGSP response following Co-mV in the IC patients relative to those that were not on these drugs. M and S, when used individually or in combination in HEK293 cells, significantly (p < 0.05) attenuated the Co-mV-induced Sp expression concurrently with translation surrogates. In contrast, T did not change these indices. Notably, the cellular uptake of Co-Mv was not altered by these drugs. In vivo sirolimus combo pretreatment significantly (p < 0.05) attenuated the Co-mV induced IgMSp and IgGSp production. This correlated with a decreasing trend in the early levels (after day 1) of Co-mV-induced Sp immunogen levels. Neither higher Co-mV concentrations (6μg) nor a 1-day break of S could overcome the repressed Sp protein levels. Interestingly, 3-days of S holiday or using T alone rescued Sp levels in vitro. Conclusion This is the first study to reveal that ISs, sirolimus and mycophenolate restrain Co-mV-induced Sp protein synthesis via translation suppression. Transient holiday of sirolimus or selective use of tacrolimus at the time of vaccination can be a potential option to rescue translation-dependent Sp protein production. These compelling findings lay a solid foundation for guiding future studies aimed at improving mRNA-based vaccine effectiveness in high-risk IC patients.
Two-month-old mice were placed in cages with (Ex) or without exercise running wheels with free access to the wheel 24 h/day for 10 mo. An equal amount of food for both groups was provided daily. Ex mice ran an average of 33.67 km/wk initially, and exercise decreased gradually with age. Ex mice had gained an average of 43.5% less body weight at the end of the experiment. Although serum lipid peroxides were not altered by exercise, superoxide dismutase and glutathione peroxidase activities in serum were significantly increased. Flow cytometric analysis of spleen cells revealed an increased percentage of CD8 + T cells and a decreased percentage of CD19 + B cells in Ex mice ( P< 0.05). Exercise decreased apoptosis in total splenocytes and CD4 + cells incubated with medium alone or with H 2 O 2 , dexamethasone, tumor necrosis factor-α (TNF-α), or anti-CD3 monoclonal antibody ( P < 0.05) and CD8 + cells with medium alone or with TNF-α ( P < 0.05). Even though exercise did not alter the intracellular cytokines (TNF-α and interleukin-2) or Fas ligand, it did significantly lower interferon-γ in CD4 + and CD8 + cells ( P < 0.05). In summary, voluntary wheel exercise appears to decrease H 2 O 2 -induced apoptosis in immune cells as well as decrease interferon-γ production.
Introduction: The Friedewald equation (F-LDL-C) and the Martin-Hopkins algorithm (MH-LDL-C) estimate direct LDL-C from a standard lipid panel. Discordant LDL-C estimates by the two methods may carry significant clinical implications. We evaluated the clinical variables associated with discordant LDL-C estimates and the association of discordance with risk of incident atherosclerotic cardiovascular disease (ASCVD) in the Dallas Heart Study (DHS), a multi-ethnic, population based prospective cohort. Methods: We estimated F-LDL-C and MH-LDL-C in 2824 DHS participants (42% male; mean age 43.5 years) with TG ≤ 400 mg/dL, who were not on baseline lipid lowering therapy and were free of prior ASCVD. We divided the cohort into quintiles of LDL-C discordance (MH-LDL-C minus F-LDL-C, in mg/dL) and assessed associations with ASCVD risk factors. We evaluated associations between discordance and incident ASCVD by sequentially adjusted Cox regression models, and we generated restricted cubic spline plots of discordance and hazard for ASCVD. Results: There were 228 ASCVD events over a median of 12.3 years. Clinical characteristics across discordance quintiles are shown in the Table . After adjustment for traditional ASCVD risk factors, there was a linear association between higher LDL-C discordance and increased risk of ASCVD events ( Figure ) with the highest hazard in Quintile 5 (HR 1.5, 95% CI 1.1 - 2.0). Conclusions: Discordant LDL-C estimates were largely associated with male sex, White and Hispanic races, and characteristics of the metabolic syndrome. Individuals in the highest quintile of discordant LDL-C estimates, with MH-LDL-C > F-LDL-C, had greater risk for incident ASCVD.
Emergency department (ED) and hospital overcrowding is a major issue impacting health care institutions all over the country. Approximately 6% of all ED visits and approximately 27% of all admissions are related to chest pain.1 According to some studies, utilization of the newer high-sensitivity troponin (hs-TnT) assays is an effective way to improve early identification of acute coronary syndrome (ACS).2 However, to our knowledge, there are no studies evaluating the impact of hs-TnT testing on hospital operations. We hypothesize that implementation of the new hs-TnT with accelerated diagnostic pathway (ADP) will result in decreased admission rates compared to the use of older fourth-generation troponin T (TnT). Secondary outcomes were ED length of stay (LOS) and laboratory use. University of Texas Southwestern's Clements University Hospital in Dallas, Texas, recently replaced a TnT and a point-of-care troponin I (TnI) assays with a new hs-TnT assay from Roche Diagnostics Elacsys. Prior to this change, patients evaluated for ACS were tested using TnT or TnI at the providers' discretion. On the day the hs-TnT assay was implemented, both TnT and TnI were simultaneously discontinued. This resulted in three separate, significant changes in evaluation of ACS: the near instantaneous point-of-care testing was discontinued requiring all assays to be processed by a central laboratory, new cutoff limits required a change in interpretation (normal limits changed from <0.01 ng/mL for TnT and TnI to <12 ng/L for hs-TnT),3 and evaluation was standardized to follow a specific ADP4 instead of relying on an individual provider's discretion. This ADP used both hs-TnT results and time of onset of symptoms to direct subsequent testing and disposition. The implementation of ADP was automated within a clinical decision support system using a deterministic finite state machine computational model that offered nursing and physician guidance on when next assay was required and ultimately the ADP had completed. To evaluate the impact of this transition, we examined a convenience sample of all patients who presented to the ED between June 1, 2018, and June 1, 2019 who underwent troponin testing as part of their ED evaluation. During the first part of the study between June 1, 2018, and October 22, 2018, patients requiring troponin testing were evaluated with a combination of TnT and TnI. Patients presenting after this time period were evaluated only with the new hs-TnT plus the ADP that resulted in each patient being classified as "ruled out" or "abnormal."4 Providers were then encouraged to use the HEART score for risk stratification5 to help make the final disposition. Standard ED operational metrics were used to assess the implementation of the hs-TnT in association with an ADP on the operational characteristics. The aims of this before-and-after study are to compare the impact of the new hs-TnT assay and ADP on the hospital admission rates. We also examined its impact on ED LOS and phlebotomy/laboratory utilization. ED LOS is defined as the time between patient arriving to an ED treatment room and the time of disposition order; this metric reduces the impact of confounders such as ED overcrowding and ED boarding on this analysis. Continuous variables if normally distributed were depicted with mean (±SD) and compared with Welch's t-test; otherwise, they were depicted with median (IQR) and compared with the Wilcoxon rank-sum test. Categorical data were depicted with total count and percentages and compared using two-sample test of proportions. The changes to ED operational characteristics are summarized in Table 1. During both study periods, approximately 27% of ED patients were evaluated with a troponin assay, and both groups had similar distributions of chief complaints. The admission rate for patients who did not undergo troponin testing was similar during TnT/TnI and hs-TnT periods (25.0% vs 28.3%, p = 0.08). On the other hand the admission rate for all patients evaluated with a troponin assay decreased from 54.3% for TnT/TnI to 51.4% for hs-TnT (p < 0.01), but resulted in a 7-minute increase in LOS (95% CI = 3 to 11 minutes, p < 0.01) and 0.76 increase in troponin assay utilization per patient encounter (p < 0.01). In the subgroup of patients with the chief complaint of chest pain, implementation of hs-TnT and ADP had even greater effect on admission rate and LOS—admissions decreased from 42.6% to 31.5% (p < 0.01) and the median LOS increased by 22 minutes (95% CI = 12 to 28 minutes, p < 0.01). A decrease in admission rates was not observed in patients who presented with chief complaints other than chest pain (Table 1). TnT/TnI (Jun 2018–Oct 2018) hs-TnT (Oct 2018–Jun 2019) Difference (95%CI) This study is one of the first to describe the impact of implementation of ADP using fifth-generation troponin on the ED throughput in the United States. The study demonstrates a significant reduction in patient admissions following hs-TnT and ADP implementation. This reduction is not only a reflection of improved sensitivity compared to prior fourth-generation troponin assays, but it also suggests that the ADP guides providers toward recommended management based on the final risk strata of their patients. Previously utilized risk stratification tools such as the HEART score5 or EDACS6 have been plagued by significant variation in testing strategies and disposition following troponin testing. However, in this study, the standardization of patient evaluation within the ADP helped minimize this variability and potentially contributed to the decrease in admission rates. The decrease in admission rates comes at a price of increased ED LOS and increased number of troponin assay tests. While the 22-minute increase in LOS and 0.7 increase in tests per patient were statistically significant, these numbers need to be placed in an appropriate perspective. In a subgroup with chest pain, for every nine patients tested, one less required an admission. This suggests that every obviated admission costs an additional 198 minutes in ED room occupancy and six extra tests. For some hospitals this might be a cheap price to free up an inpatient bed, while for others with small EDs it might be too high. This cost–benefit analysis needs to be made on a per system basis. Because multiple interventions occurred in parallel, change to troponin assay and initiation of the ADP, it is possible that the increased LOS is a result of an algorithm that encourages repeat testing. In addition, the new hs-TnT assay and ADP were implemented within a new electronic health records–based clinical decision support system that simplified interpretation of this diagnostic algorithm and offered the ability to alert providers when the ADP had completed and automatically discontinue any unnecessary troponin orders. This automation may directly impact LOS and limit the generalizability of our results. This study was conducted at a quaternary hospital with patients who likely demanded higher number of troponin tests within the ADP. Health systems with lesser acuity might require fewer troponin tests and therefore have shorter ED LOS and per patient testing while still benefiting from the decreased admission rate. Implementation of hs-TnT–based ACS evaluation utilizing a novel ADP was associated with decreased hospital admissions for chest pain patients, but at a cost of longer ED room times and more per patient testing. It also helped to standardize a highly variable chest pain evaluations and admission strategies for high-risk patients. With increased emphasis on decreasing costs and improving outcomes, implementation of the high-sensitivity troponin assay with ADP may offer improved risk stratification and resource utilization.
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