Single-Cell Transcriptomic Analysis of Epstein-Barr Virus-Associated Hemophagocytic Lymphohistiocytosis.

Epstein-Barr virus (EBV) infection can lead to infectious mononucleosis (EBV-IM) and, more rarely, EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH), which is characterized by a life-threatening hyperinflammatory cytokine storm with immune dysregulation. Interferon-gamma (IFNγ) has been identified as a critical mediator for primary HLH; however, the detailed role of IFNγ and other cytokines in EBV-HLH is not fully understood. In this study, we used single-cell RNA sequencing to characterize the immune landscape of EBV-HLH and compared it with EBV-IM. Three pediatric patients with EBV-HLH with different backgrounds, one with X-linked lymphoproliferative syndrome type 1 (XLP1), two with chronic active EBV disease (CAEBV), and two patients with EBV-IM were enrolled. The TUBA1B + STMN1 + CD8 + T cell cluster, a responsive proliferating cluster with rich mRNA detection, was explicitly observed in EBV-IM, and the upregulation of SH2D1A-the gene responsible for XLP1-was localized in this cluster. This proliferative cluster was scarcely observed in EBV-HLH cases. In EBV-HLH cases with CAEBV, upregulation of LAG3 was observed in EBV-infected cells, which may be associated with an impaired response by CD8 + T cells. Additionally, genes involved in type I interferon (IFN) signaling were commonly upregulated in each cell fraction of EBV-HLH, and activation of type II IFN signaling was observed in CD4 + T cells, natural killer cells, and monocytes but not in CD8 + T cells in EBV-HLH. In conclusion, impaired responsive proliferation of CD8 + T cells and upregulation of type I IFN signaling were commonly observed in EBV-HLH cases, regardless of the patients' background, indicating the key features of EBV-HLH.

Comparison of plasma proteomic profiles of patients with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis and infectious mononucleosis.

Primary Epstein-Barr virus (EBV) infection occasionally causes EBV-infectious mononucleosis (EBV-IM) and EBV-hemophagocytic lymphohistiocytosis (EBV-HLH). Although EBV-IM is mostly mild and self-limiting, EBV-HLH is a life-threatening disease characterized by excessive immune activation. However, the pathogenesis of EBV-HLH is yet to be fully elucidated. A diagnostic biomarker for EBV-HLH is desirable because early diagnosis and treatment are critical for the effective management of patients. In this study, the proteomic profiling of plasma was performed using liquid chromatography-mass spectrometry to identify proteins specific to EBV-IM and EBV-HLH. Furthermore, pathway analysis was performed for the proteins upregulated in patients with EBV-IM and EBV-HLH. Compared to healthy controls, 63 and 18 proteins were upregulated in patients with EBV-IM and EBV-HLH, respectively. Pathway and process enrichment analyses revealed that the complement system was the most enriched category of upregulated proteins in EBV-IM, whereas proteins related to immune effector processes were the most enriched in EBV-HLH. Among the 18 proteins upregulated in EBV-HLH, seven were exclusive to EBV-HLH. These specific proteins were associated with three pathways, and apolipoprotein E was commonly found in all the pathways. Proteomic analysis may provide new insights into the host response to EBV infection and the pathogenesis of EBV-related diseases.

Serum Proteomic Profiles of Patients with Chronic Recurrent Multifocal Osteomyelitis.

OBJECTIVES: Chronic recurrent multifocal osteomyelitis (CRMO) is an autoinflammatory disease characterized by sterile bone inflammation; however, its pathophysiology is poorly understood. Thus, this study aimed to characterize the serum proteomic profiles of patients with CRMO to better understand the molecular mechanisms underpinning CRMO pathogenesis. METHODS: Proteomic profiling of the sera collected from 11 patients with CRMO (five patients were in active phase, six were in inactive phase) was conducted using liquid chromatography-mass spectrometry. Sera from four children without inflammatory diseases were used as controls. Pathway analysis was performed to identify the upregulated and downregulated proteins in patients with active CRMO. RESULTS: Compared with the control group, 19 and 41 proteins were upregulated and downregulated, respectively, in patients with active CRMO. Pathway and process enrichment analyses revealed that axon guidance was the most enriched category of upregulated proteins in patients with active CRMO, followed by neutrophil degranulation and mitogen-activated protein kinase cascade regulation. In comparison to patients with inactive CRMO, 36 proteins, including 11 keratin proteins, were upregulated and highly enriched in the intermediate filament organization category. Rho GTPase pathway-related proteins were downregulated in ibuprofen-treated patients. CONCLUSION: Proteomic analysis identified upregulated proteins in the sera of patients with acute CRMO. These proteins can be used as biomarkers for disease diagnosis and activity. Furthermore, we anticipate that this study will contribute to a deeper understanding of the pathophysiology of CRMO, which, in turn, will contribute to the discovery of potential novel therapeutic targets.

Association between adeno-associated virus 2 and severe acute hepatitis of unknown etiology in Japanese children.

INTRODUCTION: Outbreaks of acute hepatitis of unknown etiology (AHUE) in children were reported in Western countries in 2022. Previous studies found that adeno-associated virus 2 (AAV2) and its helper viruses, such as human adenovirus (HAdV) and human herpesvirus-6 (HHV-6), are frequently detected in patients with AHUE. However, the existence of hepatitis associated with AAV2 prior to AHUE outbreaks in 2022 had not yet been investigated. We aimed to investigate the association between AAV2 and pediatric acute hepatitis in Japanese children, as well as the incidence of AAV2-related hepatitis prior to 2022. METHODS: Preserved blood samples obtained from 49 pediatric patients with acute hepatitis between 2017 and 2023 were retrospectively analyzed. Blood samples from 50 children with acute illnesses and 50 children with chronic conditions were used as controls. Viral DNA loads were quantitated using real-time PCR. RESULTS: AAV2 DNA was detected in 12 % (6/49) of acute hepatitis cases but in only one acute illness and none of the chronic-condition control cases. The concentration of AAV2 DNA in the six acute hepatitis cases was higher than that in the acute-illness control case. Co-infection with one or more helper viruses, including HAdV, HHV-6, cytomegalovirus, and Epstein-Barr virus, was observed in five AAV2-positive cases. CONCLUSIONS: Our results indicated the sporadic occurrence of pediatric severe hepatitis associated with AAV2 infection in Japan prior to the AHUE outbreaks in 2022. Our findings suggest that co-infection with AAV2 and helper viruses plays a role in developing severe hepatitis.

Comparison of Whole Blood and Plasma for Monitoring Cytomegalovirus and Epstein-Barr Virus.

Monitoring Epstein-Barr virus (EBV) and cytomegalovirus (CMV) infection after transplantation is recommended to enable preemptive therapy. However, the most suitable sample type remains unclear. Patients who underwent hematopoietic stem cell or liver transplantation were included in this study. Viral loads in sequential whole-blood and plasma samples were retrospectively analyzed. EBV DNA was detected more frequently in whole blood (55%) than in plasma (18%). The detection rate of CMV DNA was similar between the two sample types. The correlation of viral loads between the two sample types were 0.515 and 0.688 for EBV and CMV, respectively. Among paired samples in which EBV DNA was detected in whole blood, the plasma EBV detection rate was significantly higher in patients who underwent hematopoietic stem cell transplantation than in those who underwent liver transplantation. The viral DNA load in whole blood and plasma showed similar trends. The EBV detection rate was higher in whole blood, and a high correlation was observed between CMV DNA loads and whole blood and plasma. These results indicate that whole blood is more sensitive for monitoring both EBV and CMV, whereas plasma is a potential alternative sample for monitoring CMV.

Quantitative assessment of viral load in the blood and urine of patients with congenital cytomegalovirus infection using droplet digital PCR.

Congenital cytomegalovirus infection (cCMV) is a common cause of congenital infections, leading to neurodevelopmental sequelae. Real-time quantitative polymerase chain reaction (qPCR) has been widely used for the diagnosis and assessment of cCMV; however, the correlation between CMV DNA load and the severity of cCMV symptoms has been inconclusive. Droplet digital PCR (ddPCR) offers an improvement over the current qPCR methods through the absolute quantification of viral loads. We compared ddPCR and qPCR results for the quantification of CMV DNA in blood and urine specimens from 39 neonates with cCMV (21 symptomatic and 18 asymptomatic). There was no significant difference in blood CMV DNA loads measured by ddPCR and qPCR, with or without any clinical findings. However, developmental delays at 36 months were significantly more frequently observed in patients with high CMV DNA loads (≥2950 copies/ml), as measured by ddPCR at diagnosis, than in those with lower CMV DNA loads. The association of urine CMV DNA load with symptoms and developmental delay was not observed. CMV DNA loads in the blood might be used as a predictor of developmental outcomes in cCMV patients, and absolute quantitation of viral loads by ddPCR assay could contribute to the standardization of CMV load measurement.

MicroRNA expression profiling of cerebrospinal fluid/serum exosomes in children with human herpesvirus 6-associated encephalitis/encephalopathy by high-throughput sequencing.

Primary human herpesvirus 6 (HHV-6) infection is sometimes accompanied by acute encephalopathy with reduced subcortical diffusion (AED) in immunocompetent children. We investigated exosomal microRNA (miRNA) expression profiles in cerebrospinal fluid (CSF) and sera of patients with HHV-6-associated AED (n = 5) and febrile seizure (FS) (n = 5) using high-throughput sequencing. A total of 176 and 663 miRNAs were identified in CSF and serum exosomes, respectively. Comparative analysis determined that some miRNAs (miR-381-3p, miR-155) were exclusively expressed in the CSF exosomes of AED but not of FS patients, suggesting their potential application as novel diagnostic biomarkers for AED.

Detection of antiviral drug resistance in patients with congenital cytomegalovirus infection using long-read sequencing: a retrospective observational study.

BACKGROUND: Congenital human cytomegalovirus (cCMV) infection can cause sensorineural hearing loss and neurodevelopmental disabilities in children. Ganciclovir and valganciclovir (GCV/VGCV) improve long-term audiologic and neurodevelopmental outcomes for patients with cCMV infection; however, antiviral drug resistance has been documented in some cases. Long-read sequencing can be used for the detection of drug resistance mutations. The objective of this study was to develop full-length analysis of UL97 and UL54, target genes with mutations that confer GCV/VGCV resistance using long-read sequencing, and investigate drug resistance mutation in patients with cCMV infection. METHODS: Drug resistance mutation analysis was retrospectively performed in 11 patients with cCMV infection treated with GCV/VGCV. UL97 and UL54 genes were amplified using blood DNA. The amplicons were sequenced using a long-read sequencer and aligned with the reference gene. Single nucleotide variants were detected and replaced with the reference sequence. The replaced sequence was submitted to a mutation resistance analyzer, which is an open platform for drug resistance mutations. RESULTS: Two drug resistance mutations (UL54 V823A and UL97 A594V) were found in one patient. Both mutations emerged after 6 months of therapy, where viral load increased. Mutation rates subsided after cessation of GCV/VGCV treatment. CONCLUSIONS: Antiviral drug resistance can emerge in patients with cCMV receiving long-term therapy. Full-length analysis of UL97 and UL54 via long-read sequencing enabled the rapid and comprehensive detection of drug resistance mutations.

A STING inhibitor suppresses EBV-induced B cell transformation and lymphomagenesis.

Epstein-Barr virus-associated lymphoproliferative disease (EBV-LPD) is frequently fatal. Innate immunity plays a key role in protecting against pathogens and cancers. The stimulator of interferon genes (STING) is regarded as a key adaptor protein allowing DNA sensors recognizing exogenous cytosolic DNA to activate the type I interferon signaling cascade. In terms of EBV tumorigenicity, the role of STING remains elusive. Here we showed that treatment with the STING inhibitor, C-176, suppressed EBV-induced transformation in peripheral blood mononuclear cells. In an EBV-LPD mouse model, C-176 treatment also inhibited tumor formation and prolonged survival. Treatment with B cells alone did not affect EBV transformation, but suppression of EBV-induced transformation was observed in the presence of T cells. Even without direct B cell-T cell contact in a transwell system, the inhibitor reduced the transformation activity, indicating that intercellular communication by humoral factors was critical to prevent EBV-induced transformation. These findings suggest that inhibition of STING signaling pathway with C-176 could be a new therapeutic target of EBV-LPD.

Temporal dynamics of the plasma microbiome in recipients at early post-liver transplantation: a retrospective study.

BACKGROUND: Immunosuppression during liver transplantation (LT) enables the prevention and treatment of organ rejection but poses a risk for severe infectious diseases. Immune modulation and antimicrobials affect the plasma microbiome. Thus, determining the impact of immunosuppression on the microbiome may be important to understand immunocompetence, elucidate the source of infection, and predict the risk of infection in LT recipients. We characterized the plasma microbiome of LT recipients at early post-LT and assessed the association between the microbiome and clinical events. RESULTS: In this study, 51 patients who received LT at Nagoya University Hospital from 2016 to 2018 were enrolled. Plasma samples were retrospectively collected at the following time points: 1) within a week after LT; 2) 4 ± 1 weeks after LT; 3) 8 ± 1 weeks after LT; and 4) within 2 days after a positive blood culture. A total of 111 plasma samples were analyzed using shotgun next-generation sequencing (NGS) with the PATHDET pipeline. Relative abundance of Anelloviridae, Nocardiaceae, Microbacteriaceae, and Enterobacteriaceae significantly changed during the postoperative period. Microbiome diversity was higher within a week after LT than that at 8 weeks after LT. Antimicrobials were significantly associated with the microbiome of LT recipients. In addition, the proportion of Enterobacteriaceae was significantly increased and the plasma microbiome diversity was significantly lower in patients with acute cellular rejection (ACR) than non-ACR patients. Sequencing reads of bacteria isolated from blood cultures were predominantly identified by NGS in 8 of 16 samples, and human herpesvirus 6 was detected as a causative pathogen in one recipient with severe clinical condition. CONCLUSIONS: The metagenomic NGS technique has great potential in revealing the plasma microbiome and is useful as a comprehensive diagnostic procedure in clinical settings. Temporal dynamics of specific microorganisms may be used as indirect markers for the determination of immunocompetence and ACR in LT recipients.

Pediatric sepsis cases diagnosed with group B streptococcal meningitis using next-generation sequencing: a report of two cases.

BACKGROUND: Group B Streptococcus (GBS) is an important cause of invasive infection in neonates and infants. Cerebrospinal fluid (CSF) findings and culture may not show evidence of infection early in GBS meningitis. Next-generation sequencing (NGS) has the potential to detect microbial genetic material in patients with infectious diseases. We report two cases of infantile sepsis of GBS meningitis with negative results for CSF culture tests, but positive results for NGS analysis. CASE PRESENTATION: Patient 1 was a 22-day-old male infant diagnosed with sepsis and meningitis. His CSF findings showed pleocytosis, decreased glucose, and increased protein levels. However, CSF and blood culture results at admission were negative. He received a total of 3 weeks of treatment with ampicillin and cefotaxime, and showed clinical improvement. GBS was detected through NGS analysis of CSF collected at admission. Patient 2 was a 51-day-old male infant with sepsis. CSF findings on admission were normal, and blood and CSF cultures were also negative. Intravenous ampicillin and cefotaxime treatment were initiated. Treatment was de-escalated to ampicillin alone because Enterococcus faecalis was cultured from urine. He was discharged after a total of 1 week of antibiotic treatment. Six days after discharge, he was re-hospitalized for sepsis. Blood and CSF cultures were negative, and E. faecalis was again cultured from urine. He received a total of 3 weeks of ampicillin treatment for enterococcal-induced nephritis and did not relapse thereafter. NGS pathogen searches were retrospectively performed on both blood and CSF collected at the first and second admission. GBS was detected in the CSF collected at the first admission, but no significant pathogen was detected in the other samples. Inadequate treatment for GBS meningitis at the first admission may have caused the recurrence of the disease. CONCLUSION: Infantile sepsis may present bacterial meningitis that is not diagnosed by either culture testing or CSF findings. NGS analysis for CSF may be useful for confirming the diagnosis of bacterial meningitis.

Comprehensive Detection of Candidate Pathogens in the Lower Respiratory Tract of Pediatric Patients With Unexpected Cardiopulmonary Deterioration Using Next-Generation Sequencing.

OBJECTIVES: Next-generation sequencing has been applied to the investigation of microorganisms in several clinical settings. We investigated the infectious etiologies in respiratory specimens from pediatric patients with unexpected cardiopulmonary deterioration using next-generation sequencing. DESIGN: Retrospective, single-center, observational study. SETTING: Tertiary care, a children's hospital. SUBJECTS: The study enrolled a total of 16 pediatric patients with unexpected cardiopulmonary deterioration who were admitted to the PICU. Ten bronchoalveolar lavage fluid and six transtracheal aspirate samples were analyzed. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: RNA libraries were prepared from specimens and analyzed using next-generation sequencing. One or more bacterial/viral pathogens were detected in the bronchoalveolar lavage fluid or transtracheal aspirate specimens from 10 patients. Bacterial and viral coinfection was considered in four cases. Compared with the conventional culture and viral antigen test results, an additional six bacterial and four viral pathogens were identified by next-generation sequencing. Conversely, among 18 pathogens identified by the conventional methods, nine pathogens were detected by next-generation sequencing. Candidate pathogens (e.g., coxsackievirus A6 and Chlamydia trachomatis) were detected by next-generation sequencing in four of 10 patients in whom no causative pathogen had been identified by conventional methods. CONCLUSIONS: Our results suggest that viral and bacterial infections are common triggers in unexpected cardiopulmonary deterioration in pediatric patients. Next-generation sequencing has the potential to contribute to clarification of the etiology of pediatric critical illness.

Comprehensive pathogen detection in sera of Kawasaki disease patients by high-throughput sequencing: a retrospective exploratory study.

BACKGROUND: Kawasaki disease (KD) is an idiopathic systemic vasculitis that predominantly damages coronary arteries in children. Various pathogens have been investigated as triggers for KD, but no definitive causative pathogen has been determined. As KD is diagnosed by symptoms, several days are needed for diagnosis. Therefore, at the time of diagnosis of KD, the pathogen of the trigger may already be diminished. The aim of this study was to explore comprehensive pathogens in the sera at the acute stage of KD using high-throughput sequencing (HTS). METHODS: Sera of 12 patients at an extremely early stage of KD and 12 controls were investigated. DNA and RNA sequences were read separately using HTS. Sequence data were imported into the home-brew meta-genomic analysis pipeline, PATHDET, to identify the pathogen sequences. RESULTS: No RNA virus reads were detected in any KD case except for that of equine infectious anemia, which is known as a contaminant of commercial reverse transcriptase. Concerning DNA viruses, human herpesvirus 6B (HHV-6B, two cases) and Anelloviridae (eight cases) were detected among KD cases as well as controls. Multiple bacterial reads were obtained from KD and controls. Bacteria of the genera Acinetobacter, Pseudomonas, Delfita, Roseomonas, and Rhodocyclaceae appeared to be more common in KD sera than in the controls. CONCLUSION: No single pathogen was identified in serum samples of patients at the acute phase of KD. With multiple bacteria detected in the serum samples, it is difficult to exclude the possibility of contamination; however, it is possible that these bacteria might stimulate the immune system and induce KD.

Viral DNA Loads in Various Blood Components of Patients With Epstein-Barr Virus-Positive T-Cell/Natural Killer Cell Lymphoproliferative Diseases.

To evaluate diagnostic values for Epstein-Barr virus (EBV) DNA loads in different blood components of patients with EBV-positive T-cell/natural killer cell lymphoproliferative diseases, EBV DNA loads were compared among disease categories in each blood component from 59 patients. Plasma viral loads were significantly higher in "active" disease in chronic active EBV infection. EBV DNA was not detected in the plasma from 7 patients in whom EBV DNA was detected in peripheral blood mononuclear cells and whole blood. Diagnostic cutoff values for whole blood EBV DNA loads of patients with chronic active EBV infection compared with those of infectious mononucleosis was 104.2 (15 800) IU/mL.

Serological screening of immunoglobulin M and immunoglobulin G during pregnancy for predicting congenital cytomegalovirus infection.

BACKGROUND: Cytomegalovirus (CMV) is one of the most frequent pathogens for congenital infections. Most cases of congenital CMV infection (cCMV) are asymptomatic at birth, but sensorineural hearing loss (SNHL) or neurodevelopmental delay can appear later in childhood. This prospective study examined the practicability of serological screening for anti-CMV immunoglobulin (Ig) G and anti-CMV IgM in pregnant women. METHODS: A total of 11,753 pregnant women were examined for CMV IgG and CMV IgM during the first or second trimester. When IgM was positive, IgG was reevaluated more than two weeks later. When IgG was negative, IgG was reevaluated in the second or third trimester. All neonates from mothers with positive/borderline IgM or IgG seroconversion underwent polymerase chain reaction assay for CMV using urine samples to diagnose cCMV. Levels of IgG and IgM were compared between mothers with and without cCMV. Receiver operating characteristic (ROC) curves for IgM titers were analyzed. RESULTS: Eight of 500 neonates (1.6%) born from mothers with positive IgG and positive IgM, and 3 of 13 neonates (23.1%) born from mothers with IgG seroconversion were diagnosed with cCMV. Neither IgM titers nor IgG titers differed significantly between cCMV and non-cCMV groups. The area under the ROC curve was 0.716 and the optimal cut-off for IgM was 7.28 index (sensitivity = 0.625, specificity = 0.965, positive predictive value = 0.238, negative predictive value = 0.993). Titers of IgG were not frequently elevated in pregnant women with positive IgM during the observation period, including in those with cCMV. All 11 cCMV cases were asymptomatic at birth and none had shown SNHL or developmental delay as of the last regular visit (mean age, 40 months). CONCLUSIONS: Seroconversion of CMV IgG and high-titer IgM during early pregnancy are predictors of cCMV. High IgM titer (> 7.28 index) is a predictor despite relatively low sensitivity. Levels of IgG had already plateaued at first evaluation in mothers with cCMV. Maternal screening offered insufficient positive predictive value for diagnosing cCMV, but allowed identifying asymptomatic cCMV cases in an early stage.

Comparison of high-dose and low-dose corticosteroid therapy for refractory Mycoplasma pneumoniae pneumonia in children.

BACKGROUND: Mycoplasma pneumoniae pneumonia (MPP) is generally a self-limiting disease, but it may become refractory. It is thought that refractory MPP is linked to the excessive immunologic responses of the host. Consequently, the use of adjunctive systemic corticosteroids may have beneficial effects. In this study, we compared the effects of high- and low-dose corticosteroid therapy in a pediatric population with refractory MPP. METHODS: We retrospectively collected data from 91 pediatric MPP patients treated with adjunctive systemic corticosteroids between April 2014 and October 2016. The patients were divided into the following two groups: high-dose corticosteroid group (2 mg/kg/day or more of prednisolone equivalents; n = 38) and low-dose corticosteroid group (<2 mg/kg/day; n = 53). Additionally, we compared the number of febrile days post-corticosteroid administration. We used 25 paired patients in a propensity score matching analysis to correct for confounding factors both by age and by days (from onset till corticosteroid therapy initiation). RESULTS: We observed that in the high-dose corticosteroid group defervescence following corticosteroid therapy initiation was achieved significantly earlier and length of hospitalization was significantly shorter (0.8 ± 1.0 vs. 1.5 ± 1.4 days and 8.2 ± 2.4 vs. 10.7 ± 2.7 days, respectively). In the propensity score matching, we observed that significant differences in the length of fever following corticosteroid therapy initiation and hospitalization were still present. Further, neither of the groups developed corticosteroid-related adverse events. CONCLUSION: Our results suggest that patients with refractory MPP treated with high-dose corticosteroid could achieve defervescence earlier and have a shorter hospitalization.

Reactivation of human herpesviruses 6 and 7 in Kawasaki disease.

Objectives: Kawasaki disease (KD) is one of the most common childhood vasculitides. Some serological studies have suggested an etiological relationship between KD and human herpesvirus (HHV)-6 or HHV-7. However, primary or reactivated HHV-6 and -7 has not been fully investigated in patients with KD. Methods: Twenty-three patients with KD were prospectively enrolled in this study. Peripheral blood was collected in the acute and convalescence phases, and HHV-6 and -7 viral loads were measured by real-time PCR. Results: In the acute phase, HHV-6 and -7 DNA was detected in 7 (30%) patients each, compared to 13 (57%) and 9 (39%) patients in the convalescence phase, respectively. HHV-6 and -7 DNA loads were significantly higher in the convalescence phase than in the acute phase. Significant increases in HHV-6 and -7 DNA loads were not observed in disease control patients. Taking into account HHV-6 and -7 serostatus, reactivation of HHV-6 and -7 was observed in 7 and 9 patients, respectively. KD patients with HHV-6 reactivation showed higher C-reactive protein levels and more frequently required steroid therapies than patients without reactivation. Conclusion: HHV-6 and -7 reactivation is frequent in KD patients. HHV-6 reactivation might exacerbate the severity of KD.

Identification of potential pathogenic viruses in patients with acute myocarditis using next-generation sequencing.

Myocarditis is an inflammatory disease of the myocardium and leads to cardiac dysfunction and heart failure. Although viral infections are considered to be the most common etiology of myocarditis, the identification of the causative virus is still challenging. Recently, next-generation sequencing (NGS) has been applied in the diagnosis of infectious diseases. The aim of the current study was to comprehensively analyze potential pathogenic microorganisms using NGS in the sera of patients with myocarditis. Twelve pediatric and five adult patients hospitalized for acute myocarditis were included. Serum samples in the acute phase were obtained and analyzed using NGS to detect pathogen-derived DNA and RNA. Viral sequence reads were detected in 7 (41%) of the 17 myocarditis patients by NGS. Among these patients, detection of Epstein-Barr virus, human parvovirus B19, torque teno virus, and respiratory syncytial virus reads by NGS was consistent with polymerase chain reaction or antigen test results in one patient each. A large number of human pegivirus reads were detected from one patient by RNA sequencing; however, its pathogenicity to human is unknown. Conversely, the number of detected virus-derived reads was small in most cases, and the pathophysiological role of these viruses remains to be clarified. No significant bacterial or fungal reads other than normal bacterial flora was detected. These data indicate that comprehensive detection of virus-derived DNA and RNA using NGS can be useful for the identification of potential pathogenic viruses in myocarditis.

A case of refractory cytomegalovirus-related thrombocytopenia that achieved complete remission without antiviral therapy.

Cytomegalovirus (CMV) is one of the major infectious etiologies that induce thrombocytopenia. Although immune thrombocytopenic purpura (ITP) in children is often preceded by viral infections, thrombocytopenia associated with active CMV infection is considered CMV-related thrombocytopenia (CMV-thrombocytopenia), which can be distinguished from ITP. CMV-thrombocytopenia is reported to be less responsive to standard therapies for ITP and may require antiviral therapies. We herein report a case of refractory CMV-thrombocytopenia that achieved complete remission without antiviral therapy. A 20-month-old boy presented with a 2-day history of fever and systemic petechiae. There were no abnormal findings except for an extremely low platelet count (8000/µl) on blood examinations. He was clinically diagnosed with ITP, and intravenous immunoglobulin was administered twice, but his platelet count did not increase. CMV infection was suspected serologically, and a high CMV DNA load was detected in serum by real-time quantitative polymerase chain reaction (PCR). Without antiviral treatment, the CMV DNA load decreased below the detection limit on the 11th day of admission, followed by complete remission of the thrombocytopenia. The present case suggests that spontaneous recovery of thrombocytopenia can be expected in immunocompetent patients with CMV-thrombocytopenia in whom decreased CMV DNA load is observed.

Neurological Disorders Associated with Human Alphaherpesviruses.

Herpes simplex virus (HSV) encephalitis is the most common cause of sporadic fatal encephalitis worldwide, and central nervous system (CNS) involvement is observed in approximately one-third of neonatal HSV infections . In recent years, single-gene inborn errors of innate immunity have been shown to be associated with susceptibility to HSV encephalitis . Temporal lobe abnormalities revealed by magnetic resonance imaging-the most sensitive imaging method for HSV encephalitis-are considered strong evidence for the disease. Detection of HSV DNA in the cerebrospinal fluid by polymerase chain reaction (PCR) is the gold standard for the diagnosis of HSV encephalitis and neonatal meningoencephalitis. Intravenous acyclovir for 14-21 days is the standard treatment in HSV encephalitis. Neurological outcomes in neonates are improved by intravenous high-dose acyclovir for 21 days followed by oral acyclovir suppressive therapy for 6 months. Varicella-zoster virus (VZV) causes a wide range of CNS manifestations. VZV encephalitis typically occurs after primary infection, and reactivation of VZV may cause encephalitis. On the other hand, VZV infection of cerebral arteries produces vasculopathy, which can manifest as ischemic stroke. Vasculopathy can occur after primary infection or reactivation of VZV. PCR detection of VZV DNA in the cerebrospinal fluid can be used for the diagnosis of encephalitis or vasculopathy. Although there are no controlled treatment trials to assess VZV treatments of encephalitis or vasculopathy, intravenous acyclovir is a common treatment.