Novel STAT1 Variants in Japanese Patients with Isolated Mendelian Susceptibility to Mycobacterial Diseases.

PURPOSE: Heterozygous dominant-negative (DN) STAT1 variants are responsible for autosomal dominant (AD) Mendelian susceptibility to mycobacterial disease (MSMD). In this paper, we describe eight MSMD cases from four kindreds in Japan. METHODS: An inborn error of immunity-related gene panel sequencing was performed using genomic DNA extracted from whole blood samples. The identified variants were validated using Sanger sequencing. Functional analysis was evaluated with a luciferase reporter assay and co-transfection assay in STAT1-deficient cells. RESULTS: Patient 1.1 was a 20-month-old boy with multifocal osteomyelitis and paravertebral abscesses caused by Mycobacterium bovis bacillus Calmette-Guérin (BCG). Although the paravertebral abscess was refractory to antimycobacterial drugs, the addition of IFN-γ and drainage of the abscess were effective. Intriguingly, his mother (patient 1.2) showed an uneventful clinical course except for treatment-responsive tuberculous spondylitis during adulthood. Patient 2.1 was an 8-month-old boy with lymphadenopathy and lung nodules caused by BCG. He responded well to antimycobacterial drugs. His mother (patient 2.2) was healthy. Patient 3.1 was a 11-year-old girl with suspected skin tuberculosis. Her brother (patient 3.2) had BCG-osis, but their mother (patient 3.3) was healthy. Patient 4 was an 8-month-old girl with left axillary and supraclavicular lymphadenopathy associated with BCG vaccination. Kindreds 1, 2, and 3 were shown to have novel heterozygous variants (V642F, R588C, and R649G) in STAT1, respectively. Kindred 4 had previously reported heterozygous variants (Q463H). A luciferase reporter assay in STAT1-deficient cells followed by IFN-γ stimulation confirmed that these variants are loss-of-function. In addition, with co-transfection assay, we confirmed all of these variants had DN effect on WT STAT1. CONCLUSION: Four kindred MSMD subjects with 3 novel variants and 1 known variant in STAT1 were identified in this study. AD STAT1 deficiency might be prevalent in Japanese patients with BCG-associated MSMD.

Tetanus in a partially immunized child.

A 13-year-old boy developed tetanus, although he had protective antitoxin antibody raised by three doses of tetanus toxoid vaccine. Four days after injury, he presented with muscle rigidity of his posterior neck, excessive diaphoresis, and risus sardonicus and was subsequently diagnosed with tetanus. Tetanus is rare in developed countries, particularly during childhood, but must be promptly diagnosed based on clinical symptoms.

Predictive factors of response to IVIG in pediatric immune thrombocytopenic purpura.

BACKGROUND: Immune thrombocytopenic purpura (ITP) is commonly treated with i.v. immunoglobulin (IVIG). METHODS: We retrospectively evaluated whether pretreatment clinical and laboratory finding could predict the short- and long-term response to IVIG. RESULTS: Short-term response was estimated by platelet count 2 weeks after IVIG, and long-term response was assessed on thrombocytopenia-free survival (TFS). TFS was defined as the probability of survival without treatment failure after initial IVIG, such as relapse, requirement for additional therapeutic interventions, or progressing to chronic ITP. Seventy-six patients with newly diagnosed ITP who were initially treated with IVIG were evaluated. Fifty-three patients (69.7%) were determined as responders at 2 weeks after IVIG. On multivariate analysis, age ≥23 months (P = 0.020) and platelet count <9.0 × 109 /L (P = 0.018) were considered to be unfavorable factors for short-term response. Cumulative proportion of long-term (1 year) good prognosis was estimated at 53.0% (95%CI: 40.8-65.2). On multivariate analysis of unfavorable factors for long-term response, age ≥23 months (P = 0.020) was the only significant factor. CONCLUSIONS: For new-onset ITP in patients aged >2 years, corticosteroid therapy in addition to IVIG may be considered as the initial treatment.

Long-term prognosis of human herpesvirus 6 reactivation following allogeneic hematopoietic stem cell transplantation.

BACKGROUND: Patients undergoing hematopoietic stem cell transplantation (HSCT) frequently have HHV-6 reactivation typically during the early phase following HSCT. The long-term clinical complications and prognosis, however, remain unclear. METHODS: Between September 2010 and October 2012, whole blood samples from 105 patients collected weekly from prior to 6 weeks after HSCT underwent multiplex polymerase chain reaction (PCR) to screen for viral DNA, followed by real-time PCR for quantitative estimation. In 48 patients, only HHV-6 was detected in at least one sample. In 30 patients, no viral DNA was detected. Long-term clinical records were reviewed in March 2016. All 48 HHV-6-positive patients, and 24 patients in whom no viral DNA detected, were followed up. RESULTS: Median maximum HHV-6 DNA load in the blood of the HHV-6 reactivation group (n = 48) was 11 800 copies/µg peripheral blood leukocyte DNA (range, 52-310 000 000). Hemophagocytic syndrome (HPS) was diagnosed in two subjects with HHV-6 reactivation. Acute graft-versus-host disease (GVHD) developed more frequently in patients with HHV-6 reactivation than in patients without viral reactivation (P = 0.002), but there was no difference in incidence of chronic GVHD. There was no difference in engraftment of neutrophils and platelets between groups. There was also no difference in overall survival between groups. Onset of HPS, however, was associated with lower overall survival (P = 0.009). CONCLUSIONS: Human herpesvirus 6 reactivation was associated with acute GVHD, but not with chronic GVHD, engraftment or overall survival. Onset of HPS, however, predicts lower overall survival.

A case report of cutaneous polyarteritis nodosa in siblings.

Cutaneous polyarteritis nodosa (CPAN) is characterized by a necrotizing vasculitis of small and medium-sized arteries in the skin, which can be associated with fever, arthralgia, myalgia, and neuropathy, but, unlike polyarteritis nodosa (PAN), there is no visceral involvement. CPAN is rare in childhood. We report two siblings who developed CPAN during childhood. Interestingly, both had Mediterranean fever gene (MEFV) mutation, i.e. heterozygous E148Q. They also shared HLA-A24, -DR15 alleles. Simultaneous occurrence of MEFV mutation and HLA alleles with CPAN has never been reported in Japan. These cases could provide some hereditary clue for the development of CPAN.

Clinical and Molecular Characteristics of Human Rotavirus G8P[8] Outbreak Strain, Japan, 2014.

During March-July 2014, rotavirus G8P[8] emerged as the predominant cause of rotavirus gastroenteritis among children in Hokkaido Prefecture, Japan. Clinical characteristics were similar for infections caused by G8 and non-G8 strains. Sequence and phylogenetic analyses suggest the strains were generated by multiple reassortment events between DS-1-like P[8] strains and bovine strains from Asia.

Virus reactivations after autologous hematopoietic stem cell transplantation detected by multiplex PCR assay.

Several studies have indicated that viral reactivations following allogeneic hematopoietic stem cell transplantation (allo-HSCT) are frequent, but viral reactivations after autologous HSCT (auto-HSCT) have not been investigated in detail. We performed multiplex polymerase chain reaction (PCR) assay to examine multiple viral reactivations simultaneously in 24 patients undergoing auto-HSCT between September 2010 and December 2012. Weekly whole blood samples were collected from pre- to 42 days post-HSCT, and tested for the following 13 viruses; herpes simplex virus 1 (HSV-1), HSV-2, varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV-6), HHV-7, HHV-8, adeno virus (ADV), BK virus (BKV), JC virus (JCV), parvovirus B19 (B19V), and hepatitis B virus (HBV). Fifteen (63%) patients had at least one type of viral reactivation. HHV6 (n = 10; 41.7%) was most frequently detected followed by EBV (n = 7; 29.2%). HHV-6 peaked on day 21 after HSCT and promptly declined. In addition, HBV, CMV, HHV7, and B19V were each detected in one patient. HHV6 reactivation was detected in almost half the auto-HSCT patients, which was similar to the incidence in allo-HSCT patients. The incidence of EBV was unexpectedly high. Viral infections in patients undergoing auto-HSCT were higher than previously reported in other studies. Although there were no particular complications of viral infection, we should pay attention to possible viral reactivations in auto-HSCT patients. J. Med. Virol. 89:358-362, 2017. © 2016 Wiley Periodicals, Inc.

Pneumococcal vaccination reduces the risk of community-acquired pneumonia in children.

BACKGROUND: The seven-valent pneumococcal conjugate vaccine (PCV7) was introduced to Japan in 2009, after which there was a rapid decline in invasive pneumococcal disease. There are few data, however, on the effectiveness of PCV7 against community-acquired pneumonia (CAP). We conducted an ambispective cohort study among children aged 0-6 years old who attended day-care centers. METHODS: A total of 624 children at 10 day-care centers in Sapporo, Japan participated in the study. The parents reported whether their child had received PCV7 one or more times, as well as the exact dates of vaccination from records in maternal and child health handbooks marked by pediatricians. Each CAP event was reported by parents according to doctor diagnosis. A Cox proportional hazards regression model was used to calculate the hazard ratio (HR) and 95%CI of CAP incidence reduced by PCV7 inoculation. RESULTS: During the observational period, 94 subjects contracted CAP. After adjusting for potentially confounding variables, inoculation with PCV7 was significantly associated with a reduced risk of CAP (HR, 0.22; 95%CI: 0.13-0.34). On stratified analysis by age, PCV7 was significantly associated with a reduced risk of CAP in both children aged <3 years (HR, 0.31; 95%CI: 0.14-0.71), and those ≥3 years (HR, 0.20; 95%CI: 0.09-0.43). CONCLUSION: PCV7 is highly effective in reducing the risk of CAP in children attending day-care centers.

Immunochromatography test for rapid diagnosis of Mycoplasma pneumoniae infection.

The sensitivity and specificity of a new rapid Mycoplasma pneumoniae antigen immunochromatography (IC) test, DK-MP-001, were determined using particle agglutination (PA) antibody response and loop-mediated isothermal amplification (LAMP) gene detection as the gold standard. Of 165 patients, 59 were diagnosed with M. pneumoniae infection based on a ≥fourfold rise of serum PA antibody during the course of the illness. Of the first visit swabs, 60 were positive for M. pneumoniae on LAMP, and 49 were positive for M. pneumoniae antigen on IC test. Compared with PA antibody and LAMP, the sensitivity/specificity of the IC test were 81.4% (48/59) and 99.1% (105/106); and 81.7% (49/60) and 100% (105/105), respectively. IC test detected antigen in pharyngeal swabs more sensitively than in nasal swabs for the same subjects (P < 0.05). The IC test performs well enough to be used with pharyngeal swabs at the first examination.

HHV-6 encephalitis may complicate the early phase after allogeneic hematopoietic stem cell transplantation: Detection by qualitative multiplex PCR and subsequent quantitative real-time PCR.

Viral reactivation following hematopoietic stem cell transplantation (HSCT) can cause various complications especially viral encephalitis. In this prospective study, we investigated the correlation of post-HSCT viral reactivation in blood with CNS dysfunction. We employed a multiplex PCR that detects 13 kinds of viruses as a first-line screening test and real-time PCR for subsequent quantitative evaluation. Five hundred ninety-one whole blood samples were collected from 105 patients from before until 42 days after HSCT. Seven patients developed CNS dysfunction such as altered consciousness. In six of the seven, the multiplex PCR test detected HHV-6 DNA in at least one sample. In contrast, DNA from other viruses, such as CMV, EBV, HHV-7, adenovirus, and HBV was never detected in any of the seven patients throughout the study period. Quantitative measurement of whole blood HHV-6 DNA levels demonstrated four of the six HHV-6 DNA loads were elevated at successive time points during the CNS dysfunction. In addition, the virus DNA peaks were temporally associated with the development of CNS dysfunction. CSF was tested in two of the four patients and high HHV-6 DNA levels comparable to those in whole blood were confirmed in both. These four patients were, thus, suspected to have developed HHV-6 encephalitis, a rate of 3.8% in the study population. Our results suggest that early diagnosis of probable HHV-6 encephalitis can be improved by confirming high HHV-6 DNA load in blood.

Quantitative analysis of human parvovirus B19 DNA in maternal and fetal serum, and amniotic fluid during an early stage of pregnancy.

Simple and accurate diagnosis of vertical transmission of human parvovirus B19 (B19V) infection remains an important issue in pregnancy. There are few reports on quantitative analysis of B19V in amniotic fluids. Quantitative estimation of B19V DNA in amniotic fluids was comparerd with those in maternal or fetal serum obtained at an early stage of pregnancy with possible mother-to-fetus transmission. All pregnant women contracted B19V infection between 13 to 14 weeks gestation. The B19V DNA amount in 3 maternal serum and amniotic fluid sample pairs collected between 16 to 27 weeks gestation was quantified by a real-time polymerase chain reaction assay. Serum from 2 fetuses was included. The B19V DNA concentrations in maternal sera and amniotic fluids ranged from 10(4) to 10(5) copies/ml and from 10(7) to 10(8) copies/ml, respectively. The B19V DNA in the amniotic fluids concentration coincided with those of each fetal serum. The concentrations in amniotic fluids are 100 to 5,000 times higher than in those of maternal sera, and corresponded to the matching fetal serum. Amniotic fluids may substitute for the fetal sera in terms of quantitative estimation of fetal B19V infection at an early stage of pregnancy.

Large-scale multiplex polymerase chain reaction assay for diagnosis of viral reactivations after allogeneic hematopoietic stem cell transplantation.

Viral reactivations following hematopoietic stem cell transplantation are thought to result from the breakdown of both cell-mediated and humoral immunity. As a result, many viruses could be reactivated individually or simultaneously. Using a multiplex polymerase chain reaction (PCR), we prospectively examined many kinds of viral DNAs at a time in 105 patients who underwent allogeneic hematopoietic stem cell transplantation. In total, 591 whole blood samples were collected weekly from pre- to 42 days post-transplantation and the following 13 viruses were tested; herpes simplex virus 1 (HSV-1), HSV-2, varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpes virus 6 (HHV-6), HHV-7, HHV-8, adenovirus, BK virus (BKV), JC virus (JCV), parvovirus B19, and hepatitis B virus (HBV). Several viral DNAs were detected in 12 patients before hematopoietic stem cell transplantation. The detection rate gradually increased after transplantation and peaked at 21 days. The most frequently detected virus was HHV-6 (n = 63; 60.0%), followed by EBV (n = 11; 10.5%), CMV (n = 11; 10.5%), and HHV-7 (n = 9; 8.6%). Adenovirus and HBV were each detected in one patient (1.0%). Detection of HHV-6 DNA was significantly more common among patients undergoing cord blood transplantation or with steroid treatment. EBV DNA tended to be more common in patients treated with anti-thymocyte globulin. Multiplex PCR was useful for detecting many viral reactivations after hematopoietic stem cell transplantation, simultaneously. Cord blood transplantation, steroid treatment, or anti-thymocyte globulin use was confirmed to be risk factors after transplantation.

Pneumococcal vaccination reduced the risk of acute otitis media: Cohort study.

BACKGROUND: Seven-valent pneumococcal conjugate vaccine (PCV7) was introduced to Japan in 2009, and after that invasive pneumococcal disease has gradually decreased. There are few data, however, on the effectiveness of PCV7 against acute otitis media (AOM) in Japan. METHODS: From 10 daycare centers in Sapporo, Japan, 614 parents participated in the survey. Each parent reported whether their child subject had received one or more doses of PCV7, and, if so, the exact dates of receiving PCV7 were verified by reviewing their maternal and child health handbooks marked by a pediatrician. AOM was diagnosed by otorhinolaryngologist or pediatrician. Cox's proportional hazard model was used for calculating the hazard ratio (HR) of AOM incidence reduced by PCV7 inoculation. RESULTS: Inoculation of PCV7 significantly reduced the risk of AOM (crude HR, 0.63; 95%CI: 0.50-0.79). Adjusting for potentially confounding variables reduced the risk further (adjusted HR, 0.32; 95%CI: 0.23-0.44). On stratification by subject age on 30 April 2012, PCV7 was significantly associated with a reduced risk of AOM in both infants < 3 years old, and in children ≥ 3 years. CONCLUSION: PCV7 is effectiveness in reducing the risk of AOM both in infants < 3 years old, and in young children ≥ 3 years in Japan.

Quantitation of human parvovirus B19 DNA in erythema infectiosum and aplastic crisis.

Several publications concerning the methods of real-time PCR for human parvovirus B19 (B19V) have appeared and some case reports mention B19V DNA loads. However, no large-scale study quantitating levels of B19V DNA in common or representative B19V manifestations such as erythema infectiosum and aplastic crisis has been performed. Consequently, using the TaqMan PCR assay, the B19V load in a large sample of subjects with erythema infectiosum or aplastic crisis was quantitated. Sixty-five subjects in the acute phase of erythema infectiosum were involved, and in addition 22 serum samples from seven subjects with B19V-associated aplastic crisis complicating chronic hemolytic anemia were also analyzed. In the acute phase of erythema infectiosum the median B19V DNA load in the serum samples from the acute phase of erythema infectiosum was 7.63 × 10(5) genomes/ml, (range from 4.48 × 10(3) to 8.31 × 10(6) genomes/ml). The serum B19V DNA load during the acute phase of aplastic crisis complicating chronic hemolytic anemia was extremely high, that is 10(10) -10(13) genomes/ml, and decreased gradually to around 10(5) genomes/ml over 1-2 months. Although all subjects followed an almost uniform and typical clinical course of erythema infectiosum, there was a large individual variation of B19V DNA loads, that is differences of over 1,000 times. Extremely high B19V loads were observed in subjects with aplastic crisis. This study is the first large scale report of studies of the B19V DNA loads in subjects with erythema infectiosum and aplastic crisis, the most common and significant clinical manifestations by B19V infections.

Characterization of the NSP4 gene of group A human rotavirus G1P[8] strains circulating in Sapporo, Japan from 1987 to 2000.

The genetic diversity of the NSP4 gene of rotavirus G1P[8] strains obtained in Sapporo was analyzed, Japan from 1987 to 2000. Sixty-four strains, which were distributed across the whole study period, were included. All G1P[8] NSP4 genes detected in this study belonged to genotype E1, which divided into at least three lineages. The Sapporo rotavirus G1P[8] isolates were found in each lineage. The mean estimated substitution rate was 1.40 × 10(-3) nucleotide substitutions per site per year, which was comparable to that of the G1P[8] VP7 gene. Comparison of the deduced NSP4 amino acid sequences showed genetic diversity at the center of antigenic site II, but not in the enterotoxic domain. This report represents the first investigation of the genetic diversity and evolution of group A rotavirus NSP4 genes in Asia.

Longitudinal analysis of VP7 gene of group A human rotavirus G2P[4] strains circulating in the pre-vaccine era in Sapporo, Japan from 1991 to 2011.

Sequence analysis of the VP7 gene in 23 group A human rotavirus G2P[4] strains obtained during 1991-2011, that is, the pre-vaccine era, in Sapporo, Japan showed considerable genetic diversity, mainly in variable regions. Recent G2P[4] epidemic strains were located in sublineage IVa with a distinctive substitution of D96N. This study provides background data on the genetic variability of G2P[4] rotavirus-VP7 gene prior to the widespread use of rotavirus vaccines in Japan.

Effectiveness of influenza vaccine in children in day-care centers of Sapporo.

BACKGROUND: We conducted a retrospective cohort study for evaluating the effectiveness of the trivalent inactivated influenza vaccine (TIV) among children aged 0-6 years in the 2011-2012 season in Sapporo City, Japan, because of scarce evidence. METHODS: From 10 day-care centers in Sapporo City, Japan, 629 parents participated in the study. Each parent of the subjects described whether a subject received TIV once or twice in the 2011-2012 season, as well as the exact dates of receiving TIV from records in a maternal and child health handbook marked by a pediatrician. The incidence of influenza was defined as being affected with influenza as diagnosed by a pediatrician. Cox's proportional model was used for calculating a hazard ratio (HR) and its 95% confidence interval (95%CI) of TIV on an influenza incidence. RESULTS: After adjusting potential confounding variables, such as the day-care center, presence of comorbidity, size of household, number of siblings, and number of smokers in the home in addition to the age and sex of the child, HR was significantly reduced in the subjects aged 1 year (HR = 0.22, 95%CI 0.09-0.54) as well as in the total subjects (HR = 0.72, 95%CI 0.52-0.99). Consequently, the effectiveness of TIV was calculated as 78% for the subjects aged 1 year and 28% for the total subjects. CONCLUSION: Our study suggests that TIV is effective, especially in subjects aged 1 year. Further studies are necessary in different seasons, places, and populations to clarify the effectiveness of the influenza vaccine in children.

Risk factors for poor outcome in congenital cytomegalovirus infection and neonatal herpes on the basis of a nationwide survey in Japan.

BACKGROUND: Congenital cytomegalovirus (CMV) infection and neonatal herpes are major mother-to-child infections, and analyses of the important clinical issues, including risk factors for prognosis, are essential. METHODS: A secondary survey of congenital CMV infection and neonatal herpes was performed using questionnaires for cases reported in the primary survey between 2006 and 2008. RESULTS: Univariate analysis of 71 cases of congenital CMV infection showed that intrauterine growth restriction (IUGR) or other specific findings on fetal ultrasonography (US), microcephaly, intracranial calcification, disseminated intravascular coagulation, abnormal findings on computed tomography, and the use of i.v. gammaglobulin were all significantly correlated with poor outcome (death or severe sequelae). Multivariate analysis showed that only IUGR was significantly associated with poor outcome. Hearing impairment is one of the major abnormalities associated with congenital CMV infection. Automatic auditory brainstem response (automatic ABR) appeared to be useful for detection of hearing impairment in comparison with conventional ABR. Moreover, univariate analysis showed that specific fetal US or abnormal magnetic resonance imaging findings were correlated with sensorineural hearing loss. In 24 cases of neonatal herpes, fever and seizure were correlated with poor outcome on univariate analysis. All patients received acyclovir treatment, although substantial numbers of patients in severe clinical categories (disseminated or central nervous system diseases) received a low dose of acyclovir (<60 mg/kg per day). CONCLUSIONS: This secondary survey has identified the risk factors associated with outcome and important issues in diagnosis and treatment of two mother-to-child infections: congenital CMV and neonatal herpes, in Japan.