Prophylactic vaccination inducing anti-Env antibodies can result in protection against HTLV-1 challenge in macaques.

HTLV-1 infection occurs by cell-to-cell transmission and can induce fatal adult T-cell leukemia (ATL). Vaccine development is critical for the control of HTLV-1 transmission. However, determining whether vaccine-induced anti-Env antibodies can prevent cell-to-cell HTLV-1 transmission is challenging. Here, we examined the protective efficacy of a vaccine inducing anti-Env antibodies against HTLV-1 challenge in cynomolgus macaques. Eight of ten vaccinated macaques produced anti-HTLV-1 neutralizing antibodies (NAbs) and were protected from an intravenous challenge with 108 HTLV-1-producing cells. In contrast, the two vaccinated macaques without NAb induction and ten unvaccinated controls showed HTLV-1 infection with detectable proviral load after challenge. Five of the eight protected macaques were administered with an anti-CD8 monoclonal antibody, but proviruses remained undetectable and no increase in anti-HTLV-1 antibodies was observed even after CD8+ cell depletion in three of them. Analysis of Env-specific T cell responses did not suggest involvement of vaccine-induced Env-specific T cell responses in the protection. These results indicate that anti-Env antibody induction by vaccination can result in functionally sterile HTLV-1 protection, implying the rationale for strategies aimed at anti-Env antibody induction in prophylactic HTLV-1 vaccine development.

Generation of JC Polyoma Pseudovirus for High-Throughput Measurement of Neutralizing Antibodies.

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system (CNS) caused by reactivation of dormant JC polyomavirus (JCPyV). PML was mainly observed in immunocompromised individuals, such as HIV-positive patients, autoimmune disease patients, and cancer patients. Given that the presence of anti-JCPyV antibodies in serum is a risk indicator for PML development, it is essential to monitor anti-JCPyV antibody levels. In the present study, we established reporter-based single-infection neutralization assays for JCPyV and the genetically similar BK polyoma virus (BKPyV). We then confirmed the lack of cross-reactivity between the two viruses using test sera obtained from mice immunized with plasmids encoding the JCPyV or BKPyV capsid. Next, we compared neutralization antibody titers in sera from healthy donors, patients with multiple sclerosis (MS), and HIV-positive patients using an in-house enzyme-linked immunosorbent assay (ELISA) with JCPyV-like particles (virus-like particles; VLPs). A positive correlation was demonstrated between the neutralization titer (75% infectious concentration; IC75) against JCPyV and the antibody titer obtained by VLP-based JCPyV ELISA. This assay system may be applied to detect antibodies against other PyVs by generation of pseudoviruses using the respective capsid expression plasmids, and is expected to contribute to the surveillance of PyV as well as basic research on these viruses.

Acute acalculous cholecystitis following extended administration of nirmatrelvir/ritonavir for persistent SARS-CoV-2 infection.

Immunocompromised patients with hematologic malignancies, particularly those treated with anti-CD20 antibodies such as rituximab and obinutuzumab, are known to be at risk of prolonged infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Prolonged administration or combination therapy with antiviral medications reportedly yields favorable outcomes in these patients. However, knowledge regarding the adverse events associated with such therapeutic approaches is limited. Herein, we report a case of acute acalculous cholecystitis (AAC) following extended administration of nirmatrelvir/ritonavir (NMV/r) in a 68-year-old Japanese man with persistent SARS-CoV-2 infection. The patient had received obinutuzumab and bendamustine for follicular lymphoma and was diagnosed with coronavirus disease 2019 (COVID-19) approximately one year after treatment initiation with these drugs. Subsequently, he was admitted to a different hospital, where he received antiviral drugs, monoclonal antibodies, and steroids. Despite these interventions, the patient relapsed and was subsequently transferred to our hospital due to persistent SARS-CoV-2 infection. Remdesivir administration was ineffective, leading to the initiation of extended NMV/r therapy. One week later, he exhibited elevated gamma-glutamyl transpeptidase (GGT) levels, and one month later, he developed AAC. Cholecystitis was successfully resolved via percutaneous transhepatic gallbladder drainage and administration of antibiotics. We speculate that extended NMV/r administration, in addition to COVID-19, may have contributed to the elevated GGT and AAC. During treatment of persistent SARS-CoV-2 infection with extended NMV/r therapy, patients should be carefully monitored for the appearance of findings suggestive of biliary stasis and the development of AAC.

18F-THK5351 Positron Emission Tomography Clearly Depicted Progressive Multifocal Leukoencephalopathy After Mantle Cell Lymphoma Treatment.

An 84-year-old Japanese woman presented with left hemiplegia 8 months after completing chemotherapy for mantle cell lymphoma. Brain magnetic resonance imaging (MRI) revealed a hyperintense lesion extending from the right parietal lobe to the left parietal lobe. Compared with these MRI results, 18F-THK5351 PET revealed more extensive accumulation. A brain biopsy showed progressive multifocal leukoencephalopathy (PML). Immunohistochemistry and John Cunningham virus (JCV) DNA-polymerase chain reaction indicated JCV infection. Therefore, a diagnosis of PML was made. 18F-THK5351 PET, indicative of activated astrocytes, clearly depicted PML lesions composed of reactive and atypical astrocytes. 18F-THK5351 PET may capture fresh progressive PML lesions better than MRI.

Dengue virus infection induces selective expansion of Vγ4 and Vγ6TCR γδ T cells in the small intestine and a cytokine storm driving vascular leakage in mice.

Dengue is a major health problem in tropical and subtropical regions. Some patients develop a severe form of dengue, called dengue hemorrhagic fever, which can be fatal. Severe dengue is associated with a transient increase in vascular permeability. A cytokine storm is thought to be the cause of the vascular leakage. Although there are various research reports on the pathogenic mechanism, the complete pathological process remains poorly understood. We previously reported that dengue virus (DENV) type 3 P12/08 strain caused a lethal systemic infection and severe vascular leakage in interferon (IFN)-α/ß and γ receptor knockout mice (IFN-α/ß/γRKO mice), and that blockade of TNF-α signaling protected mice. Here, we performed transcriptome analysis of liver and small intestine samples collected chronologically from P12/08-infected IFN-α/ß/γRKO mice in the presence/absence of blockade of TNF-α signaling and evaluated the cytokine and effector-level events. Blockade of TNF-α signaling mainly protected the small intestine but not the liver. Infection induced the selective expansion of IL-17A-producing Vγ4 and Vγ6 T cell receptor (TCR) γδ T cells in the small intestine, and IL-17A, together with TNF-α, played a critical role in the transition to severe disease via the induction of inflammatory cytokines such as TNF-α, IL-1ß, and particularly the excess production of IL-6. Infection also induced the infiltration of neutrophils, as well as neutrophil collagenase/matrix metalloprotease 8 production. Blockade of IL-17A signaling reduced mortality and suppressed the expression of most of these cytokines, including TNF-α, indicating that IL-17A and TNF-α synergistically enhance cytokine expression. Blockade of IL-17A prevented nuclear translocation of NF-κB p65 in stroma-like cells and epithelial cells in the small intestine but only partially prevented recruitment of immune cells to the small intestine. This study provides an overall picture of the pathogenesis of infection in individual mice at the cytokine and effector levels.

A nationwide cross-sectional study using a web-based questionnaire survey of the duration of isolation of COVID-19 inpatients with cancer at Japanese cancer centers.

There is no clear consensus regarding the optimal isolation duration for immunocompromised patients with coronavirus disease 2019 (COVID-19). Therefore, we conducted a questionnaire survey at eight Japanese cancer centers to investigate the practices of infectious disease specialists regarding the duration of isolation for COVID-19 inpatients with cancer. For asymptomatic to severely ill COVID-19 inpatients without severe immunodeficiency, four centers reported at least 10 days of isolation without testing, and two reported at least 20 days. Two centers incorporated polymerase chain reaction (PCR) as a criterion for terminating the isolation of inpatients without severe immunodeficiency. For severely immunocompromised COVID-19 inpatients, at least 20 days of isolation were required in seven facilities, regardless of illness severity. Additionally, seven centers had implemented Ct or antigen quantification test values as criteria for de-isolating severely immunocompromised inpatients. No cases caused nosocomial outbreaks after isolation was terminated based on each facility's criteria for isolation termination. Thus, cancer patients required longer isolation periods than the general population in most facilities, and for those with severe immunodeficiency, the isolation periods were longer and more tightly controlled with tests.

BK Polyomavirus-Associated Urothelial Carcinoma of the Bladder with a Background of BK Polyomavirus Nephropathy in a Kidney Transplant Recipient.

Renal transplant recipients are at increased risk for the development of a malignant neoplasm. Polyomavirus-associated urothelial carcinoma is a rare tumor that occurs in renal transplant recipients, with approximately 41 cases reported since 2002. It accounts for 27-31% of all post-transplant urothelial carcinomas and develops at an average of 8.5 years after transplantation. Histologically, it shows high-grade urothelial carcinoma (95.1%) with a high frequency of glandular differentiation and micropapillary structures (58.5%) and positive immunohistochemistry for polyomavirus large T antigen, p53 (92.9%), and p16 (100%). We encountered a case of BK polyomavirus (BKPyV)-associated urothelial carcinoma of the bladder diagnosed 54 months after kidney transplantation. Histologically, it was a high-grade urothelial carcinoma with micropapillary features, and immunohistochemically, it was diffusely positive for polyomavirus large T antigen, p16, and p53. BKPyV DNA and mRNA for BKPyV large T antigen have been identified in tissues using real-time polymerase chain reaction. The same sequence of the BKPyV VP1 genome hypervariable region was detected in both transplanted kidney tissue with polyomavirus nephropathy and urothelial carcinoma tissue, suggesting that polyomavirus-associated urothelial carcinoma developed in a background of persistent polyomavirus nephropathy. This case showed typical histological features and was detected and treated at an earlier stage than has been reported. It is important to keep in mind that polyomavirus-associated urothelial carcinoma can develop early after transplantation and might be associated with polyomavirus nephropathy. Because of its rapidly progressive nature, careful follow-up with urine cytology and cystoscopy is necessary. We report this case with a literature review.

COVID-19 Analysis in Tissue Samples Acquired by Minimally Invasive Autopsy in Out-of-Hospital Deaths with Postmortem Degeneration.

Minimally invasive autopsy (MIA) is an alternative to a full autopsy for the collection of tissue samples from patients' bodies using instruments such as a biopsy needle. MIA has been conducted in many cases of coronavirus disease 2019 (COVID-19) and has contributed to the elucidation of the disease pathogenesis. However, most cases analyzed are hospital deaths, and there are few reports on the application of MIA in out-of-hospital deaths with varying extents of post-mortem changes. In this study, MIA and autopsies were performed in 15 patients with COVID-19 2-30 days after death, including 11 out-of-hospital deaths. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome detection by reverse transcriptase quantitative polymerase chain reaction using MIA samples was mostly consistent with autopsy samples, particularly lung tissue, even in out-of-hospital cases. MIA had high sensitivity and specificity (> 0.80). Histological examination of lung tissue obtained by MIA showed characteristics of COVID-19 pneumonia, with 91% agreement with autopsy samples, whereas localization of SARS-CoV-2 protein in lung tissue was indicated by immunohistochemistry, with 75% agreement. In conclusion, these results suggest that MIA is applicable to out-of-hospital deaths due to COVID-19 with various postmortem changes, especially when autopsies are not available.

Apple-shaped obesity: A risky soil for cytokine-accelerated severity in COVID-19.

Obesity has been recognized as one of the most significant risk factors for the deterioration and mortality associated with COVID-19, but the significance of obesity itself differs among ethnicity. Multifactored analysis of our single institute-based retrospective cohort revealed that high visceral adipose tissue (VAT) burden, but not other obesity-associated markers, was related to accelerated inflammatory responses and the mortality of Japanese COVID-19 patients. To elucidate the mechanisms how VAT-dominant obesity induces severe inflammation after severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection, we infected two different strains of obese mice, C57BL/6JHamSlc-ob/ob (ob/ob), C57BLKS/J-db/db (db/db), genetically impaired in the leptin ligand and receptor, respectively, and control C57BL/6 mice with mouse-adapted SARS-CoV-2. Here, we revealed that VAT-dominant ob/ob mice were extremely more vulnerable to SARS-CoV-2 due to excessive inflammatory responses when compared to SAT-dominant db/db mice. In fact, SARS-CoV-2 genome and proteins were more abundant in the lungs of ob/ob mice, engulfed in macrophages, resulting in increased cytokine production including interleukin (IL)-6. Both an anti-IL-6 receptor antibody treatment and the prevention of obesity by leptin replenishment improved the survival of SARS-CoV-2-infected ob/ob mice by reducing the viral protein burden and excessive immune responses. Our results have proposed unique insights and clues on how obesity increases the risk of cytokine storm and death in patients with COVID-19. Moreover, earlier administration of antiinflammatory therapeutics including anti-IL-6R antibody to VAT-dominant patients might improve clinical outcome and stratification of the treatment for COVID-19, at least in Japanese patients.

Chimeric flavivirus causes vascular leakage and bone marrow suppression in a mouse model.

Previously, we demonstrated the utility of a recombinant chimeric flavivirus (DV2ChimV), which carries the premembrane (prM) and envelope (E) genes of a type 2 DENV clinical (Thai) isolate on a backbone of Japanese encephalitis virus, for evaluating the protective efficacy of antidengue envelope antibodies both in vitro and in vivo. Here, to assess the potential use of this model for pathological studies, we aimed to characterize interferon-α/ß-γ-receptor double-knockout mice (IFN-α/ß/γR dKO mice) infected with DV2ChimV. Vascular leakage and bone marrow suppression are unique features of severe dengue. In the current model, DV2ChimV caused vascular leakage in the liver and intestine at the moribund stage. High levels of virus were detected in the bone marrow, and strong bone marrow suppression (i.e., disappearance of megakaryocytes and erythroblastic islets) was observed. These observations suggest that the DV2ChimV-infected mouse model mimics the vascular leakage and bone marrow suppression observed in human cases.

Nationwide Laboratory Surveillance of Progressive Multifocal Leukoencephalopathy in Japan: Fiscal Years 2011-2020.

Progressive multifocal leukoencephalopathy (PML) is a devastating demyelinating disease caused by JC virus (JCV), predominantly affecting patients with impaired cellular immunity. PML is a non-reportable disease with a few exceptions, making national surveillance difficult. In Japan, polymerase chain reaction (PCR) testing for JCV in the cerebrospinal fluid (CSF) is performed at the National Institute of Infectious Diseases to support PML diagnosis. To clarify the overall profile of PML in Japan, patient data provided at the time of CSF-JCV testing over 10 years (FY2011-2020) were analyzed. PCR testing for 1537 new suspected PML cases was conducted, and 288 (18.7%) patients tested positive for CSF-JCV. An analysis of the clinical information on all individuals tested revealed characteristics of PML cases, including the geographic distribution, age and sex patterns, and CSF-JCV-positivity rates among the study subjects for each type of underlying condition. During the last five years of the study period, a surveillance system utilizing ultrasensitive PCR testing and widespread clinical attention to PML led to the detection of CSF-JCV in the earlier stages of the disease. The results of this study will provide valuable information not only for PML diagnosis, but also for the treatment of PML-predisposing conditions.

A human T-lymphotropic virus-1 carrier who developed progressive multifocal leukoencephalopathy following immunotherapy for sarcoidosis: a case report.

BACKGROUND: Progressive multifocal leukoencephalopathy (PML) is a devastating demyelinating disorder of the central nervous system caused by opportunistic infection of the JC virus (JCV). CASE PRESENTATION: A 58-year-old Japanese woman was admitted to our hospital for aphasia. She had a 5-year history of untreated sarcoidosis and was a human T cell lymphotropic virus-1 (HTLV-1) carrier. Serum angiotensin-converting enzyme, soluble interleukin-2 receptor, lysozyme, and calcium levels were elevated. JCV-DNA was not detected in cerebrospinal fluid by PCR testing. Skin biopsy revealed noncaseating granuloma formation. Bilateral multiple nodular lesions were present on chest X-ray. Brain magnetic resonance imaging showed left frontal and temporal lesions without gadolinium enhancement. As we suspected that systemic sarcoidosis had developed into neurosarcoidosis, we started steroid and infliximab administration. After treatment, the chest X-ray and serum abnormalities ameliorated, but the neurological deficits remained. At 1 month after immunotherapy, she developed right hemiparesis. Cerebrospinal fluid was positive for prototype (PML-type) JCV on repeated PCR testing. Brain biopsy revealed demyelinating lesions with macrophage infiltration, atypical astrocytes, and JCV antigen-positive cells. We diagnosed her with PML and started mefloquine, leading to partial remission. CONCLUSIONS: Sarcoidosis and HTLV-1 infection both affect T cell function, especially CD4+ T cells, and may developped the patient's PML. The comorbidity of sarcoidosis, PML, and HTLV-1 infection has not been reported, and this is the world's first report of PML associated with HTLV-1 infection and sarcoidosis.

Successful treatment of proven coronavirus disease 2019-associated pulmonary aspergillosis with liposomal amphotericin B in a patient with bronchiolitis obliterans syndrome after allogeneic hematopoietic stem cell transplantation.

Coronavirus disease 2019 (COVID-19)-associated pulmonary aspergillosis (CAPA) is being increasingly recognized as a severe complication that contributes to poor prognoses among patients with COVID-19. However, little is known regarding the clinical course of CAPA with hematological malignancies, especially after allogeneic hematopoietic stem cell transplantation (HSCT). A 29-year-old woman was diagnosed with proven CAPA with an Aspergillus fumigatus identified by cultures of bronchoalveolar lavage and lung biopsy four years after haploidentical HSCT for acute myelogenous leukemia. She had been taking oral prednisolone for bronchiolitis obliterans syndrome that developed after HSCT. Although prolonged RT-PCR positivity for SARS-CoV-2 (133 days after the onset of COVID-19) without shedding of viable virus was observed, the COVID-19 was treated with favipiravir, remdesivir, dexamethasone, and enoxaparin. However, the CAPA did not respond to combination therapy, which included triazole (voriconazole, itraconazole, posaconazole) and echinocandin (caspofungin, micafungin), even though the Aspergillus fumigatus isolate was found to be susceptible to these agents in vitro. Nevertheless, a total of 16 weeks of liposomal amphotericin B (L-AMB) therapy led to a favorable response, and the patient was discharged from the hospital on day 213. This case provided essential experience of CAPA treated with L-AMB in a recipient with chronic respiratory disease after HSCT.

Anti-N-methyl-D-aspartate receptor encephalitis with concurrent human herpes virus-6A deoxyribonucleic acid detection: An autopsy case.

We report an autopsy case of anti-N-methyl-D-aspartate (NMDA) receptor (NMDAR) encephalitis with concurrent human herpes virus-6 (HHV-6) A deoxyribonucleic acid (DNA) detection in cerebrospinal fluid (CSF). A 38-year-old previously healthy Japanese man presented with a generalized seizure. Brain magnetic resonance imaging (MRI) findings were unremarkable, but CSF revealed pleocytosis. On Day 11, HHV-6 DNA was detected in CSF, and IgG antibodies against the NR1 subunit of the NMDAR (GluN1) were subsequently detected. Since HHV-6 encephalitis was initially suspected, the patient was treated with foscarnet and ganciclovir, but the HHV-6A copy number increased from 200 (Day 22) to 2000 copies/mL (Day 47), and the therapy was ineffective. As typical symptoms of anti-NMDAR encephalitis developed, we changed the patient's treatment to combat anti-NMDAR encephalitis. He was repeatedly treated with first-line immunotherapy, and GluN1 antibody titer decreased. He was not treated with second-line immunotherapy because of recurrent infections; he died on Day 310. Postmortem examinations did not show systemic tumors. Microscopic examination of the brain revealed only severe neuronal rarefaction in the hippocampal cornu ammonis (CA) 3-4 areas with gliosis. Early initiation of aggressive immunotherapy may be required in a refractory case of anti-NMDAR encephalitis, even with HHV-6A DNA detection, because the significance of this concurrent detection in autoimmune encephalitis remains unclear.

Performance evaluation of the Roche Elecsys® Anti-SARS-CoV-2 immunoassays by comparison with neutralizing antibodies and clinical assessment.

Quantitative measurement of SARS-CoV-2 neutralizing antibodies is highly expected to evaluate immune status, vaccine response, and antiviral therapy. The Elecsys® Anti-SARS-CoV-2 S (Elecsys® anti-S) was developed to measure anti-SARS-CoV-2 S proteins. We sought to investigate whether Elecsys® anti-S can be used to predict neutralizing activities in patients' serums using an authentic virus neutralization assay. One hundred forty-six serum samples were obtained from 59 patients with COVID-19 at multiple time points. Of the 59 patients, 44 cases were included in Group M (mild 23, moderate 21) and produced 84 samples (mild 35, moderate 49), while 15 cases were included in Group S (severe 11, critical 4) and produced 62 samples (severe 43, critical 19). The neutralization assay detected 73% positive cases, and Elecsys® anti-S and Elecsys® Anti-SARS-CoV-2 (Elecsys® anti-N) showed 72% and 66% positive cases, respectively. A linear correlation between the Elecsys® anti-S assay and the neutralization assay were highly correlated (r = 0.7253, r2 = 0.5261) than a linear correlation between the Elecsys® anti-N and neutralization assay (r = 0.5824, r2 = 0.3392). The levels of Elecsys® anti-S antibody and neutralizing activities were significantly higher in Group S than in Group M after 6 weeks from onset of symptoms (p < 0.05). Conversely, the levels of Elecsys® anti-N were comparable in both groups. Three immunosuppressed patients, including cancer patients, showed low levels of anti-S and anti-N antibodies and neutralizing activities throughout the measurement period, indicating the need for careful follow-up. Our data indicate that Elecsys® anti-S can predict the neutralization antibodies in COVID-19.

Neutralizing mAbs against SFTS Virus Gn Protein Show Strong Therapeutic Effects in an SFTS Animal Model.

Severe fever with thrombocytopenia syndrome (SFTS) is an infectious disease with a high case fatality rate caused by the SFTS virus, and currently there are no approved specific treatments. Neutralizing monoclonal antibodies (mAbs) against the virus could be a therapeutic agent in SFTS treatment, but their development has not sufficiently been carried out. In the present study, mouse and human mAbs exposed to the viral envelope proteins Gn and Gc (16 clones each) were characterized in vitro and in vivo by using recombinant proteins, cell culture with viruses, and an SFTS animal model with IFNAR-/- mice. Neutralization activities against the recombinant vesicular stomatitis virus bearing SFTS virus Gn/Gc as envelope proteins were observed with three anti-Gn and six anti-Gc mAbs. Therapeutic activities were observed among anti-Gn, but not anti-Gc mAbs with neutralizing activities. These results propose an effective strategy to obtain promising therapeutic mAb candidates for SFTS treatment, and a necessity to reveal precise roles of the SFTS virus Gn/Gc proteins.

Acute Myocarditis with Severe Fever and Thrombocytopenia Syndrome.

A 67-year-old man, hospitalized with fever and pancytopenia, experienced cardiogenic shock on the 3rd day of hospitalization. He complained of chest pain and exhibited cardiac dysfunction, upregulated serum troponin levels, and an ST elevation on electrocardiogram. Severe fever with thrombocytopenia syndrome (SFTS) was suspected based on the symptom course after a tick bite and was definitively diagnosed using the serum polymerase chain reaction (PCR) test. An endomyocardial biopsy performed in the convalescent phase revealed a sign of myocardial inflammation with increases in CD3- and CD68-positive cells. We herein report the first case of acute myocarditis complicated with SFTS.

A Fatal Breakthrough COVID-19 Case Following Bendamustine-Rituximab Therapy.

Although messenger ribonucleic acid vaccines are substantially effective toward SARS-CoV-2 infection, patients with hematologic malignancies are still prone to the virus. Herein, we report a fatal case of breakthrough SARS-CoV-2 Delta variant infection in a patient with mucosa-associated lymphoid tissue lymphoma with remission by bendamustine-rituximab (BR) therapy completed a year ago. The serologic study revealed impaired responsiveness toward vaccines and prolonged high viral load after infection. BR therapy seemingly induced an immune escape. Prevention and treatment strategies for such vulnerable patients should be clarified immediately.

Microthrombosis as a cause of fulminant myocarditis-like presentation with COVID-19 proven by endomyocardial biopsy.

Myocardial injury has been reported as a complication of COVID-19. Although several mechanisms have been proposed as its cause, they are mostly based on autopsy studies, We report a 49-year-old male with COVID-19-associated myocardial injury presented like fulminant myocarditis. We performed endomyocardial biopsy on day 2 and we confirmed the presence of microthrombosis histologically. He died on day 5 due to cardiogenic shock.