Serological Screening of Immunoglobulin G against SARS-CoV-2 Nucleocapsid and Spike Protein before and after Two Vaccine Doses among Healthcare Workers in Japan.

This study sought to evaluate the effects of two vaccine doses and the extent of SARS-CoV-2 infection among healthcare workers. We measured immunoglobulin G antibody titers against SARS-CoV-2 nucleocapsid and spike protein among healthcare workers at Gunma University Hospital. In March 2021, prior to BNT-162b2 vaccination, two of 771 participants were seropositive for nucleocapsid and spike protein, whereas 768 were seronegative. The remaining one participant was seropositive for nucleocapsid protein but seronegative for spike protein. A total of 769 participants were seropositive for spike protein after two vaccination doses. The two seropositive participants prior to vaccination showed the highest antibody titers after the second vaccination. They were probably infected with SARS-CoV-2 without clinical symptoms before March 2021. Four weeks after the second vaccination, a younger age was associated with higher antibody titers against SARS-CoV-2 spike protein. Thirty-two weeks after the second vaccination, blood samples were collected from 342 of 769 participants. Antibody titers at 32 weeks after the second vaccination significantly decreased compared with those at 4 weeks after the second vaccination among all age groups. The rate of decrease in antibody titers between 4 and 32 weeks after the second vaccination was greater in the female participants. No sex differences were observed in the antibody titers within each age group. BNT-162b2 vaccination thus induced seroconversion in an age-dependent manner. Serological screening could further establish the likelihood of subclinical SARS-CoV-2 infection.

[Successful management of acquired factor V deficiency developing shortly after induction of hemodialysis].

Autoimmune factor V deficiency (AiF5D) is caused by autoantibodies to coagulation factor V (FV); its clinical manifestations range from asymptomatic to fatal hemorrhage. Herein, we report the case of a 68-year-old man who was diagnosed with end-stage renal disease at the time of a femoral fracture and developed AiF5D after initiating hemodialysis. A wound infection that occurred after joint replacement was treated with antibiotics; however, it was poorly controlled. One month after the procedure, his coagulation time prolonged. The infection was improved by debridement and antibiotics; however, the coagulation time was not decreased and poor hemostasis at the shunt was still persistent. Because ELISA detected anti-FV-binding IgG with FV activity of <2.8% and FV inhibitor levels were 11.8 BU/ml, AiF5D was diagnosed. Oral prednisolone (PSL) was started. Dialysis was initially performed without anticoagulants, but blood clots were not found in the circuit. Anticoagulants were resumed when the coagulation time decreased. After achieving complete remission, PSL dose was tapered and finally discontinued. Few reports have described the management of AiF5D via dialysis. We consider that our report would be useful for the management of patients with similar manifestations.

[Thyroid storm and exacerbation of autoimmune hemolytic anemia following childbirth].

A 32-year-old woman was diagnosed with autoimmune hemolytic anemia (AIHA) at 12 weeks of a pregnancy examination and followed up closely without treatment. At 40 weeks of gestation, she underwent emergency caesarean section because of premature rupture. On postoperative day one, the patient exhibited worsening hemolysis and tachycardia and developed high-output heart failure; she was diagnosed with Basedow disease based on the tachycardia pattern and thyroid storm based on the presence of hyperthyroidism, fever, tachycardia, and heart failure. She was administered thiamazole and potassium iodide, which improved her thyroid function, hemolytic anemia, and heart failure. AIHA is rarely associated with Basedow disease, and hemolytic anemia can be aggravated by hyperthyroidism. In pregnant women with AIHA, management of hyperthyroidism is crucial as delivery can lead to thyroid storm.

[Recurrence of acquired factor V inhibitor after four years of remission].

Acquired factor V inhibitor (AFV-I) is a rare bleeding disorder wherein autoantibodies are developed against coagulation factor V (FV). The clinical symptoms are variable, from laboratory abnormalities without bleeding to life-threatening hemorrhage. We report herein the case of a patient with AFV-I with two relapses 4 years after the first remission. A 66-year-old male was diagnosed with AFV-I in March 20XX-4. He was treated with prednisolone (PSL) at 50 mg/day and achieved remission within 1 month. PSL dose was tapered to oral administration of 2.5 mg every other day, and long-term remission was maintained. He had been treated with dual antiplatelet therapy (DAPT) for old myocardial infarction. FV activity was markedly reduced to 3.4%, and FV inhibitor was detected (1.0 BU/ml) in May 20XX. We followed the patient without increasing the treatment dose for 2 months, but no spontaneous improvement was seen. Because DAPT was ongoing, we judged that the bleeding risk was high, although only minor bleeding symptoms appeared. PSL was therefore increased to 40 mg/day in June. FV inhibitor rapidly disappeared. When PSL dose was gradually decreased, FV activity decreased, and subcutaneous bleeding occurred in February 20XX＋1. PSL dose was increased again for the second relapse, and the patient achieved remission. Few reports have described recurrent AFV-I, and no cases of two relapses have been reported. We believe that this case report is useful for examining the long-term management of AFV-I.

A novel CYCS mutation in the α-helix of the CYCS C-terminal domain causes non-syndromic thrombocytopenia.

We report a patient with thrombocytopenia from a Japanese family with hemophilia A spanning four generations. Various etiologies of thrombocytopenia, including genetic, immunological, and hematopoietic abnormalities, determine the prognosis for this disease. In this study, we identified a novel heterozygous mutation in a gene encoding cytochrome c, somatic (CYCS, MIM123970) using whole exome sequencing. This variant (c.301_303del:p.Lys101del) is located in the α-helix of the cytochrome c (CYCS) C-terminal domain. In silico structural analysis suggested that this mutation results in protein folding instability. CYCS is one of the key factors regulating the intrinsic apoptotic pathway and the mitochondrial respiratory chain. Using the yeast model system, we clearly demonstrated that this one amino acid deletion (in-frame) resulted in significantly reduced cytochrome c protein expression and functional defects in the mitochondrial respiratory chain, indicating that the loss of function of cytochrome c underlies thrombocytopenia. The clinical features of known CYCS variants have been reported to be confined to mild or asymptomatic thrombocytopenia, as was observed for the patient in our study. This study clearly demonstrates that thrombocytopenia can result from CYCS loss-of-function variants.

Darunavir concentration in PBMCs may be a better indicator of drug exposure in HIV patients.

PURPOSE: The clinical efficacies of some antiretroviral drugs are known to not depend on its concentration in blood. To establish a method of dosage adjustment for darunavir (DRV) based on pharmacokinetic theory, we analyzed the correlation between DRV levels in peripheral blood mononuclear cells (PBMCs) and plasma. METHODS: The concentrations of DRV and ritonavir (RTV) in plasma and PBMCs of 31 samples obtained from 19 patients were analyzed. An in vitro kinetic study using MOLT-4 cells was performed to assess the contribution of RTV to the intracellular accumulation of DRV. RESULTS: DRV levels in PBMCs varied between 7.91 and 29.36 ng/106 cells (CV 37.5%), while those in plasma were greater. No significant correlation was found between the trough level of DRV in plasma and that in PBMCs (p = 0.575). The inter-day difference in DRV levels in PBMCs seemed smaller than that in plasma (- 41.6-23.0% vs - 83.3-109.1%). In the in vitro study, the elimination half-life of cellular efflux of DRV was 15.7 h in the absence of RTV and extended to 47.6 h in the presence of RTV. CONCLUSIONS: We found a poor correlation between intracellular DRV and plasma DRV levels in patients receiving highly active antiretroviral therapy. The efflux rate of DRV from cells was slow; therefore, the concentration of DRV in PBMCs may reflect average exposure to the drug and clinical efficacy.

[Giant hemophilic pseudotumors in brothers with non-severe hemophilia B].

Hemophilic pseudotumors can occur in patients with hemophilia because of recurrent bleeding and poor hemostasis. A man in his 30s with hemophilia B and human immunodeficiency virus/hepatitis C virus co-infection complicated by liver cirrhosis presented with a large pseudotumor in the left iliopsoas muscle. However, resting to stop bleeding was difficult with his daily work. Osteolytic changes in the left ilium progressed over 8 years. A large osteolytic pseudotumor in the pelvis was also incidentally identified in his younger brother during his 30s. The same mutations in F9 (p. Arg294Gln, hemizygous mutation) associated with a non-severe phenotype were detected in both brothers. The clinical courses of the brothers suggested that large pseudotumors can occur in patients with non-severe hemophilia and underline the importance of patient education.

[Congenital factor V and factor VIII deficiency discovered in an elderly patient with abnormal bleeding after trauma].

Congenital combined deficiency of coagulation factor V (FV) and factor VIII (FVIII) (F5F8D) is a rare autosomal recessive bleeding disorder caused by mutations in lectin mannose-binding type 1 (LMAN1) or multiple coagulation factor deficiency 2 (MCFD2) encoding chaperone molecules involved in the intracellular transport of FV and FVIII. Here, we report a case of F5F8D in an elderly patient diagnosed with hematoma after a right thigh injury. A 71-year-old male had a history of abnormal bleeding after tooth extraction and cholecystectomy. The patient injured his right thigh with a kitchen knife; he was urgently hospitalized to a referral hospital 8 days later due to the occurrence of hematoma at the same site. Owing to prolongation of the coagulation time (PT 16.1 s, 1.72; APTT, 66.1 s), he received hemostatic treatment with fresh-frozen plasma. He was then referred to our hospital for examination of PT and APTT prolongation. FV and FVIII activities were moderately decreased to about 15%, and no inhibitor was detected. Whole-exome sequencing identified a previously reported homozygous nonsense mutation in LMAN1, revealing F5F8D in the proband. In this case, FFP infusion alone was not sufficient for increasing coagulation factor activities. Definitive diagnosis of F5F8D provides him with the treatment option with FVIII concentrates.

Successful Management of a Patient with Autoimmune Hemorrhaphilia due to Anti-Factor XIII/13 Antibodies Complicated by Pulmonary Thromboembolism.

Autoimmune hemophilia-like disease (hemorrhaphilia) due to anti-factor XIII (FXIII) antibodies (AH13) is a very rare, life-threatening bleeding disorder. A 77-year-old woman developed macrohematuria and a right renal pelvic hematoma. The coagulation times were not prolonged, but FXIII activity and antigen levels were severely and moderately reduced to 9 and 29% of normal values, respectively. Accordingly, the FXIII-specific activity turned out to be low. FXIII inhibitor and anti-FXIII-A subunit autoantibodies were detected by a 1:1 crossmixing test and immunoblot and immunochromatographic assays. She was therefore diagnosed with "definite AH13" and treated with plasma-derived FXIII concentrates to arrest the hemorrhage. In addition to a highly compressed inferior vena cava by a huge renal pelvic hematoma, deep vein thrombosis (DVT) and pulmonary thromboembolism (PE) were identified by systemic computed tomography. The patient was immediately started on anticoagulation therapy with low-dose heparin. Emboli disappeared quickly, probably because under-crosslinked thrombi caused by severe FXIII deficiency are vulnerable to fibrinolysis. After about 1.5 years, anti-FXIII-A subunit autoantibodies still remained despite the use of rituximab, steroid pulse therapy, oral prednisolone, and oral cyclophosphamide treatments. In conclusion, an extremely rare AH13 case complicated by DVT and PE was successfully managed by balancing anticoagulation therapy with hemostatic therapy.

Cytomegalovirus retinitis followed by immune recovery uveitis in an elderly patient with rheumatoid arthritis undergoing administration of methotrexate and tofacitinib combination therapy.

Cytomegalovirus (CMV) retinitis is an opportunistic ocular infection most commonly observed in patients infected with human immunodeficiency virus (HIV). We present a rare case of CMV retinitis that developed in a non-HIV patient with rheumatoid arthritis (RA). Over the preceding 5 months, a family doctor had been treating the 78-year-old male patient with a combination therapy of methotrexate (MTX) and tofacitinib (TOF). CMV retinitis occurred when the patient's CD4+ T cells were low (196 cells/µl), and preceded the onset of Pneumocystis pneumonia. MTX and TOF were stopped after the diagnosis of CMV retinitis. While intravenous and intravitreal ganciclovir administration significantly improved the CMV retinitis, uveitis developed 3 months later during the maintenance therapy with oral valganciclovir, concomitantly with the recovery of the CD4+ T cell counts. As we believed this uveitis was caused by the immune reconstitution mechanism, we treated the patient with a retrobulbar injection of corticosteroids. During the 6 months following the cessation of MTX and TOF, there was no flare-up of the RA. Cases of CMV retinitis and immune recovery uveitis in RA patients have been rarely reported in the literature. In the current case, the intensive immunosuppressive therapy in this elderly patient might have been the cause of this unusual opportunistic complication of RA.

[Successful Treatment of Immune Reconstitution Inflammatory Syndrome with Corticosteroid for Patient with Kaposi's Sarcoma Extending into the Respiratory Tract and Both Lungs].

An HIV-infected man in his 30s was transferred to our hospital after the discontinuation of antiretroviral therapy (ART) for 4 years. An intraoral tumor was identified, and a biopsy was performed. The diagnosis was Kaposi's sarcoma (KS) and disseminated lesions were detected in his respiratory tract and both lungs on computed tomography (CT) and 2-[18F] fluoro-2-deoxy-D-glucose positron-emission tomography (FDG/PET) imaging with increasing standardized uptake volume (SUV: max 7.4). On the 7th day, he was intubated to maintain his airway, then antiretroviral therapy and chemotherapy with pegylated liposomal doxorubicin were immediately initiated. After a period of remission, pulmonary lesions were detected again. We regarded them as a manifestation of immune reconstitution inflammatory syndrome (IRIS) and gave him short term corticosteroids. The lesions were then successfully controlled without additional chemotherapy. This case suggests that early induction of ART and intensive care can result in the survival of patients with KS having serious stenosis of the respiratory tract. Furthermore, this presenting case suggested that the use of corticosteroids could be a candidate to control IRIS even in patients with Kaposi's sarcoma.

Late-onset neutropenia after rituximab therapy in patients with autoimmune thrombotic and hemostatic disorders: a single center analysis.

Autoimmune thrombotic and hemostatic disorders, caused by autoantibodies against various factors regulating thrombosis and hemostasis, are rare. Rituximab (RTX) is on occasion used for treating these disorders. Late-onset neutropenia (LON) has been described as a side effect of RTX treatment for patients with hemato-oncological and/or rheumatological diseases but not for those with autoimmune thrombotic and hemostatic disorders. Eleven patients with autoimmune thrombotic and hemostatic disorders received RTX in our institution. Four of these 11 cases (36.4%) developed LON after a median 72.6 days of RTX administration (range 43-122). Three cases required G-CSF, but no severe infections developed.

Acquired immune-mediated von Willebrand syndrome accompanied by antiphospholipid syndrome.

Acquired von Willebrand syndrome (AvWS) is a rare bleeding disorder with laboratory findings resembling those of congenital von Willebrand disease. AvWS usually occurs in association with a variety of underlying disorders, such as lymphoproliferative disease or cardiovascular disease, but autoimmune AvWS is very rare. We now describe the case of a 42-year-old woman with autoimmune AvWS with concurrent antiphospholipid syndrome (APS). The patient was suffering from epistaxis and menorrhagia from few years prior to referral. She was referred to our hospital because of Activated Partial Thromboplastin Time (APTT) prolongation. Autoimmune AvWS was diagnosed as hemostatic and immunological analyses showed very low (<6%) levels of vWF ristocetin cofactor activity, low (6%) levels of vWF antigen and the presence of anti-vWF antibodies (IgG1 and IgG2) as well as antiphospholipid antibodies. Steroid therapy led to prompt AvWS remission, but deep vein thrombosis occurred in the left leg, on day 36 and APS was diagnosed. A combination of steroid and anti-coagulant was given for approximately 3 years. Thrombosis has not recurred; but AvWS re-exacerbated with steroid tapering. This is the first case report of autoimmune AvWS concurrent with APS, and the long-term disease course described here can be beneficial to clinicians.

Acquired thrombotic thrombocytopenic purpura with a smoldering clinical course.

Acquired thrombotic thrombocytopenic purpura (aTTP) is caused by a deficiency of ADAMTS13 activity due to neutralizing auto-autoantibodies (inhibitors) against ADAMTS13. Patients with aTTP show a rapid and fatal clinical course, unless an effective therapeutic intervention such as plasma exchange therapy (PEX) is performed. There is, however, a small population of patients who show a smoldering clinical course, for which no effective treatment strategy has yet been established. We herein report a 77-year-old man, who had repeated episodes of cerebral infarction and persistent thrombocytopenia over several months, but was not correctly diagnosed as having aTTP at a local hospital. However, sudden progression to an unconsciousness state together with thrombocytopenia prompted his physician to make a clinical diagnosis of aTTP, and the patient was then referred to our hospital, where the diagnosis of aTTP was confirmed by analyzing ADAMTS13. An improvement of his clinical signs was achieved with PEX and steroid therapy, but the latter had to be discontinued due to the development of grade 3 liver dysfunction. Despite discontinuation of steroid therapy, his clinical condition remained stable, but with a persistently low level of ADAMTS13 activity associated with the presence of low-titer inhibitors. Our experience may raise awareness of the diagnosis and treatment of aTTP with a smoldering clinical course.

Refractory thrombotic thrombocytopenic purpura achieving complete remission with rituximab treatment.

Some patients with thrombotic thrombocytopenic purpura (TTP) are refractory to standard treatment regimens comprised of plasma exchange (PEX) and steroids. This report describes a 40-year-old woman with refractory TTP who achieved complete remission (CR) in response to rituximab. She was referred to our institution from a rural hospital with purpura of the extremities, severe thrombocytopenia, anemia, and rapidly progressive disturbance of consciousness. TTP was diagnosed based on the clinical symptoms of TTP, low ADAMTS13 activity (<0.5%), and high ADAMTS13 inhibitor (4.4 BU/ml) titers. High-dose prednisolone was immediately administered and PEX was started. This approach was initially effective, but the thrombocytopenia and disturbance of consciousness worsened on the sixth day of treatment. We considered this patient to have refractory TTP and administered weekly rituximab. CR was achieved on day 20, and the disease status of this patient has remained stable over the long term. Our experience with this patient and five others who were similarly treated at our hospital over the past eight years indicates that rituximab is effective for refractory TTP.

[Acquired thrombotic thrombocytopenic purpura after vascular prosthesis implantation for impending rupture of an abdominal aortic aneurysm].

Acquired thrombotic thrombocytopenic purpura (TTP) is caused by autoantibodies against ADAMTS13. TTP patients run a rapidly fatal course unless immediate plasma exchange (PEX) is initiated upon diagnosis. Herein, we report a 72-year-old man with TTP, which developed after he underwent artificial blood vessel replacement surgery for an abdominal aneurysm with impending rupture. In the perioperative period, the patient received several platelet transfusions for severe thrombocytopenia (minimum platelet count: 0.6×10(4)/µl). Thereafter, he was admitted to our department for rapidly progressing coma with multiple cerebral infarctions, and was transferred to the ICU. Based on the tentative diagnosis of TTP, we immediately began PEX and steroid pulse therapy. The diagnosis was confirmed thereafter by markedly reduced ADAMTS13 activity (<0.5%) and his being positive for the ADAMTS13 inhibitor. We performed PEX for five consecutive days and administered high-dose prednisolone (PSL). On the second hospital day (HD), his platelet count rose along with improvement of his consciousness level. The ADAMTS13 inhibitor was not detected on the 10th HD. TTP did not relapse and his general condition improved despite tapering of PSL. In this case, by closely monitoring ADAMTS13-related parameters and minimizing the number of plasma exchanges, the patient was able to achieve a remission without the use of boosting inhibitors.

[Development of acquired hemophilia A during maintenance therapy for immune thrombocytopenia].

Acquired hemophilia A (AHA) is a rare coagulation disorder caused by autoantibodies against coagulation factor VIII (FVIII). We report herein a very rare case of AHA complicated by immune thrombocytopenia (ITP). A 30-year-old woman was hospitalized with severe thrombocytopenia. Her platelet count was 5,000/µl on admission, at which time APTT was normal. ITP was diagnosed and she was treated with γ-globulin, platelet transfusion, and prednisolone at 1 mg/kg/day. She was discharged after platelet count normalization and prednisolone was tapered to 5 mg/day. During the prednisolone tapering, purpura appeared on both thighs and in the left inguinal region, and APTT was found to be prolonged. She was referred to our hospital for examination of APTT prolongation. FVIII activity was markedly decreased to 7.7% and the FVIII inhibitor was positive (1.5 BU/ml), based on which AHA was diagnosed. We carefully followed this patient without intensification of immunosuppressive therapy for 7 weeks, but her platelet count decreased from 150,000/µl to 70,000/µl and the FVIII inhibitor increased to 4 BU/ml. We therefore increased prednisolone to 30 mg/day, after which her platelet count increased and complete remission of AHA was achieved by day 42. In addition, we examined the relationship of the FVIII inhibitor and FVIII binding antibody in this case.

[Remission of acquired hemophilia A following radiation therapy for esophageal cancer].

Although acquired hemophilia A (AHA) often develops in patients with neoplasms, there are few reports on the efficacy of radiation therapy during the bleeding phase of AHA in the prior literature. We herein present a case of AHA experiencing remission soon after radiation therapy for esophageal cancer. A man in his seventies, who had a history of radical nephrectomy for left renal cell carcinoma, received a diagnosis of esophageal cancer. Three months later, he noticed a right thigh hematoma, and was transferred to our hospital. Laboratory data revealed a marked reduction of coagulation factor VIII (FVIII) activity at 0.9% and the inhibitor to FVIII was detected in his serum at 21.8 BU/ml. Under a diagnosis of AHA, the patient received high-dose oral prednisolone, which failed to achieve disease remission. He then underwent radiation therapy to eradicate the underlying esophageal cancer. Despite tapering of the prednisolone dosage, FVIII inhibitor declined to undetectable levels. In this case, radiation therapy for the underlying cancer was associated with achieving complete remission of AHA.

Gene polymorphisms of mannose-binding lectin confer susceptibility to Pneumocystis pneumonia in HIV-infected patients.

BACKGROUND: Mannose-binding lectin (MBL) plays an important role in innate immunity. The aim of this study was to determine whether genetic variants of MBL confer susceptibility to Pneumocystis pneumonia (PCP) in patients with advanced human immunodeficiency virus (HIV) infections. OBJECTIVE: HIV patients (n = 53) having CD4 counts <200/µL who were admitted to our hospital were analyzed. Of these 53 patients, 30 had PCP at admission, and 23 did not. Genotypes at six single nucleotide polymorphisms (SNP) in MBL2 gene and serum MBL levels were determined for each patient, and compared between patients with or without PCP. We also examined whether MBL enhances phagocytosis of macrophages against rat-type Pneumocystis organism in vitro. RESULTS: Genotypes associated with low production of MBL were significantly more common in the PCP group than in the non-PCP group (P = 0.049, odds ratio 2.17, 95% CI 1.02-4.63). Serum MBL levels were significantly higher in the non-PCP group (P = 0.039). Findings from in vitro experiments indicated that MBL act as a direct opsonin enhancing macrophage-mediated phagocytosis of Pneumocystis organisms. CONCLUSION: Genetic variation of MBL production influences susceptibility to PCP in patients with advanced HIV infection, and can be regarded as a risk factor for PCP.

Retrospectively diagnosed autoimmune VWF deficiency in a patient with repeated hemorrhagic events after two common colds.

Autoimmune von Willebrand factor (VWF) deficiency (AiVWFD) caused by anti-VWF autoantibodies is a rare bleeding disorder, whereas "non-immune" acquired von Willebrand syndrome (AVWS) caused by other etiologies is more common. Therefore, only 40 patients with AiVWFD have been identified in Japan through an ongoing nationwide survey on autoimmune coagulation factor deficiencies. This may be due to the inability to efficiently detect anti-VWF antibodies, as anti-VWF antibody testing is not routine. An 80-year-old Japanese woman developed AVWS and experienced bleeding after two separate common colds. She took the same cold medicine each time and recovered spontaneously after discontinuation of the medicine. Severe VWF deficiency normalized each time. Initial immunological tests did not detect anti-VWF autoantibodies, and thus a diagnosis of "non-immune" AVWS of unknown origin was made. However, after 6 years, new ELISA assays using purified VWF proteins detected free anti-VWF autoantibodies, which led to a retrospective diagnosis of AiVWFD. It is probable that the cold medicine (and/or cold virus infection) induced the autoantibodies, as the recurrence and normalization of the same coagulation abnormality and the clinical course (including drug administration and discontinuation) were completely synchronized. If AiVWFD is suspected, highly sensitive autoantibody tests should be performed.