Analysis of Cell Immunity for Children Infected with SARS-CoV-2 and Those Vaccinated against SARS-CoV-2 Using T-SPOT®.COVID.

Cellular immunity is critical for the regulation of viral diseases, including coronavirus disease 2019 (COVID-19), and is generally considered immature in childhood. However, the details of cellular immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection among children are unclear. We assessed cellular immunity in eight children post-vaccination against SARS-CoV-2 and 11 children after SARS-CoV-2 infection using the T-SPOT®.COVID assay for the spike (S) and nucleocapsid (N) proteins. In the vaccinated group, the T-SPOT®.COVID assay for the S protein yielded positive results in seven children. In the post-infection group, the assay for the N protein was positive for 5 of 11 children, with 3 of these 5 children requiring hospitalization, including 2 who needed mechanical ventilation. The T-SPOT®.COVID assay is thus valuable for assessing cellular immunity against SARS-CoV-2, and most children infected with SARS-CoV-2 may not develop such immunity unless the disease severity is significant.

Recent acute reduction in macrolide-resistant Mycoplasma pneumoniae infections among Japanese children.

INTRODUCTION: Mycoplasma pneumoniae contributes to numerous pneumonia cases among children and young adults. Therefore, this study aimed to investigate the prevalence of M. pneumoniae infections among Japanese children, occurring since 2008. METHODS: Nasopharyngeal swab specimens were obtained from all cases, following which real-time PCR was performed to identify M. pneumoniae. Further, the p1 genotypes of isolates were determined using the PCR restriction fragment length polymorphism typing method. RESULTS: The annual rate of macrolide-resistant M. pneumoniae (MRMP) infections peaked at 81.8% in 2012 and decreased annually until 2015. Although the infection rate increased to 65.3% in 2016, it decreased again to 14.3% in 2018. Although >90% of isolates harbored the type 1 genotype until 2012, this rate decreased, and approximately 80% harbored p1 genotypes other than type 1 in 2018. Furthermore, the occurrence rate of MRMP among the type 1 isolates was very high (82.4%), whereas that among p1 genotypes other than type 1 was very low (6.5%). CONCLUSIONS: MRMP occurrence potentially decreased owing to changes in not only antibiotic usage but also in the distribution of p1 genotype among isolates.

Low prevalence of Chlamydia pneumoniae infections during the Mycoplasma pneumoniae epidemic season: Results of nationwide surveillance in Japan.

OBJECTIVE: Chlamydia pneumoniae and Mycoplasma pneumoniae are both common causes of atypical pneumonia. We conducted an annual national survey of Japanese children to screen them for C. pneumoniae infections during the M. pneumoniae epidemic season. METHODS: Nasopharyngeal swab specimens were collected from children aged 0-15 years with suspected acute lower respiratory tract infection due to atypical pathogens, at 85 medical facilities in Japan from June 2008 to March 2018. Specimens were tested for infection using real-time polymerase chain reaction assays. RESULTS: Of 5002 specimens tested, 1822 (36.5%) were positive for M. pneumoniae alone, 42 (0.8%) were positive for C. pneumoniae alone, and 20 (0.4%) were positive for both organisms. In children with C. pneumoniae infection, the median C. pneumoniae DNA copy number was higher in those with single infections than in those with M. pneumoniae coinfection (p = 0.08); however it did not differ significantly according to whether the children had received antibiotics prior to sample collection (p = 0.34). CONCLUSIONS: The prevalence of C. pneumoniae infection was substantially lower than that of M. pneumoniae infection during the study period. The change in prevalence of C. pneumoniae was not influenced by that of M. pneumoniae. Children with single C. pneumoniae infection are likely to have had C. pneumoniae infection, while those with coinfection are likely to have been C. pneumoniae carriers.

Comparing Antimicrobial Susceptibilities among Mycoplasma pneumoniae Isolates from Pediatric Patients in Japan between Two Recent Epidemic Periods.

We compared the antimicrobial susceptibility of Mycoplasma pneumoniae isolates from pediatric patients in Japan in 2011-2012 and 2015-2016, when epidemics occurred. The antimicrobial activity of macrolides and tetracyclines against M. pneumoniae infection tended to be restored in 2015-2016. There was no change in the antimicrobial activity of quinolones against M. pneumoniae infection.

Macrolide-Resistant Mycoplasma pneumoniae Infection, Japan, 2008-2015.

We evaluated isolates obtained from children with Mycoplasma pneumoniae infection throughout Japan during 2008-2015. The highest prevalence of macrolide-resistant M. pneumoniae was 81.6% in 2012, followed by 59.3% in 2014 and 43.6% in 2015. The prevalence of macrolide-resistant M. pneumoniae among children in Japan has decreased.

Cellular and humoral immunity after vaccination or natural mumps infection.

BACKGROUND: This study measured cell-mediated immunity (CMI) and serum antibody to clarify the basis of breakthrough after vaccination and reinfection after mumps. METHODS: From a pool of 54 college students, 17 seronegative subjects and 14 subjects with intermediate level of antibodies against mumps were vaccinated with a monovalent mumps vaccine, and CMI was assessed using interferon-γ release assay. RESULTS: CMI positivity according to pre-existing antibody level, defined as titer <2.0 index units, negative; 2.0-3.9 index units, intermediate; and ≥4.0 index units, positive, was 8/17 (47.1%), 9/14 (64.3%) and 19/23 (82.6%) before vaccination, respectively. Of the 17 seronegative subjects, seven (41.2%) had a history of vaccination and/or natural infection, four (57.1%) of whom were CMI positive or intermediate. Ten (71%) of 14 subjects with intermediate antibody level had a history of vaccination or natural infection, eight (80%) of whom were CMI positive or intermediate. After vaccination the interferon (IFN)-γ and antibody titers increased significantly, but seven (41.2%) of the 17 seronegative subjects and 13 (92.9%) of the 14 intermediate-level subjects tested positive for both antibody and CMI. In a comparison of the natural infection group (confirmed as IgG seropositive and/or CMI positive without vaccination) versus the vaccination group, IgG antibody titer (mean ± SD) was 14.4 ± 8.0 versus 3.6 ± 2.4 index units (P < 0.01) and IFN-γ was 122.7 ± 90.0 pg/mL versus 59.5 ± 37.8 pg/mL (P > 0.05), respectively. CONCLUSION: Vaccination or even natural mumps infection did not always induce both cellular and humoral immunity.

Rubella specific cell-mediated and humoral immunity following vaccination in college students with low antibody titers.

OBJECTIVE: This study measured cell-mediated immunity (CMI) and antibodies to clarify the basis of rubella reinfection after vaccination. METHODS: In a pool of 65 college students, 39 who exhibited hemagglutination-inhibition (HI) antibody titers against rubella of ≤ 1:16 were vaccinated with a rubella vaccine. The CMI was assessed with interferon-gamma release assay. RESULTS: There was low correlation (r = 0.24) between the antibody titers and interferon-gamma levels at pre-vaccination status. Preexisting interferon-gamma levels were low in some subjects with low HI antibody titers of 1:8 and 1:16. Fifty-seven percent (4/7) of the subjects who were antibody-negative with past history of rubella vaccination at entry onto the study exhibited CMI. And 57% (4/7) of the subjects remained antibody-negative following a second vaccination, despite exhibiting CMI. HI antibody titers increased significantly after vaccination, whereas post-vaccination interferon-gamma levels did not exhibit significant increases. When subjects were divided (based on their past history of vaccination and antibody values) into natural infection and vaccination groups, HI antibody titers (mean ± SD) increased to 1:2(4.4 ± 1.4) from 1: 2(3.2 ± 0.4) (p = 0.065) in the natural infection group and to 1:2(4.4 ± 1.0) from 1:2(3.0 ± 0.8) (p < 0.00001) in the vaccination group following vaccination. The same classification revealed that interferon-gamma values did not increase significantly in either group following vaccination, but the interferon-gamma values at pre- and post-vaccination in the natural infection group were significantly higher than those at pre- and post-vaccination in the vaccination group (p < 0.05 and p < 0.05, respectively). CONCLUSION: Pre-vaccination interferon-gamma levels in each HI antibody titer group were similar. And there were some subjects with antibody-positive exhibited CMI-negative. These data may explain why rubella reinfection can occur in vaccinated seropositive individuals.

Varicella-zoster virus-specific, cell-mediated immunity with interferon-gamma release assay after vaccination of college students with no or intermediate IgG antibody response.

This study measured Varicella-zoster virus (VZV) specific cell-mediated immunity (CMI) and antibodies to clarify immune response after vaccination in 68 college students with negative or intermediate IgG antibody status. The enrolled numbers of negative, intermediate, and positive VZV-IgG antibody were 27, 41, and 28 students, respectively. The positive rates of CMI were 3.7% (1/27), 41.5% (17/41), and 96.4% (27/28) before vaccination, respectively. After vaccination, the IgG antibody titers became significantly higher in the intermediate IgG group compared to those in the negative IgG group (P < 0.01), but CMI did not differ significantly between the two groups. Ninety-three percent (38/41) of the intermediate IgG antibody group and 41% (11/27) of the negative IgG antibody group became positive for the IgG antibody after vaccination (P < 0.0001). When subjects were divided into negative, intermediate, and positive CMI by interferon-gamma values before vaccination, the IgG antibody and interferon-gamma values increased significantly in the positive CMI group compared to the negative CMI group after vaccination (P < 0.01 and P < 0.01, respectively). All (17/17) of positive CMI group and 61% (27/44) of negative CMI group became positive for the IgG antibody after vaccination (P < 0.01). Ninety-four percent (16/17) of positive CMI group and 59% (28/44) of negative CMI group became positive for CMI after vaccination (P < 0.05). Ninety-six percent (22/23) of the subjects with a history of vaccination became IgG seropositive after a second dose of vaccination, but 22% (5/23) of them remained negative for CMI. CMI is valuable information to identify potential non-responders to vaccination and to predict risk of clinical VZV infection.

Uniparental disomy of chromosome 8 leading to homozygosity of a CYP11B1 mutation in a patient with congenital adrenal hyperplasia: implication for a rare etiology of an autosomal recessive disorder.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder that usually results from paternally and maternally transmitted mutations in genes for steroidogenic enzymes. Recent studies on steroid 21-hydroxylase deficiency, the most common form of CAH, have revealed that a small percentage of patients have a non-carrier parent; uniparental disomy (UPD) and de novo mutations were reported as disease-causing mechanisms in these patients. However, it remains unknown whether UPD and de novo mutations underlie other forms of CAH. Here, we report a male patient with steroid 11ß-hydroxylase deficiency (11OHD) born to a non-carrier mother. The patient was identified by an elevated 17-hydroxyprogesterone level at a neonatal mass-screening test. His clinical features were comparable to those of previously reported patients with 11OHD. Direct sequencing of CYP11B1 identified a homozygous IVS7+1G>A mutation in the patient, which was not shared by his mother. Comparative genomic hybridization of the patient detected UPD of chromosome 8 [UPD(8)]. Microsatellite analysis indicated non-maternal origin of the UPD(8) and confirmed parentage of other chromosomes. This study shows for the first time that 11OHD can be caused by UPD in the presence of a non-carrier parent. Awareness of such rare cases should improve the accuracy of genetic counseling for families with CAH. Our data support the importance of UPD as an underlying mechanism of autosomal recessive disorders.

[Long effect of a booster on rubella antibodies from each original HI titer following vaccination].

This study was performed to clarify which titers of a pre-existing antibody could be efficiently boosted by vaccination and to assess the persistence of the antibodies. Two hundred healthy volunteer students with HI antibody titers of < or = 1:32 were enrolled. There were 6-16% of subjects with the negative HI antibody who had B-cell memory against rubella, because the EIA-IgM antibody remained negative and/or the avidity of the EIA-IgG antibody was high after vaccination. Furthermore most of them had already been vaccinated just once before. The ratio of those in whom the antibody levels increased significantly at one month after vaccination were 98%, 87%, 67% and 32% in subjects with an HI antibody titer of <1:8, < or =1: 8, < or =1:16 and < or =1:32 at pre-vaccination, respectively. The titers decreased significantly at two years after vaccination, however the ratio of decrease under each original level being 4%, 21.9%, 42.6% and 73.5% in each group of <1:8, < or =1: 8, < or =1:16 and < or = 1: 32, respectively. In comparison with the numbers of the subjects with <1: 8, the ones with < or = 1: 8, < or = 1:16 and < or = 1:32 increased 1.5-, 2.5- and 4.7-fold, respectively. Therefore, the recommendation of an HI antibody titer < or = 1:16 for vaccination in Japan is thought to be loose, although this is to decrease the risk of congenital rubella syndrome. We think that a new assay for cellular immunity for rubella should be developed in the future in order to ascertain whether congenital rubella syndrome will be prevented or not.

Management of refractory Mycoplasma pneumoniae pneumonia: utility of measuring serum lactate dehydrogenase level.

It has been suggested that cytokines are associated with refractory Mycoplasma pneumoniae pneumonia, and steroid administration is reported to be effective in this situation. In order to elucidate the characteristics of refractory M. pneumoniae pneumonia, we analyzed five pediatric patients with refractory M. pneumoniae pneumonia, which was defined as showing prolonged fever and deterioration of clinical and radiological findings despite administration of appropriate antibiotics, compared with 15 pediatric patients with M. pneumoniae pneumonia who responded to treatment promptly (control group). Serum lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and interleukin (IL)-18 levels were significantly higher in the refractory group than in the control group at the initiation of corticosteroid use (LDH: 571 vs 292 IU/L, p = 0.0129; ALT: 25 vs 11 IU/L, p = 0.0143; AST: 41 vs 26 IU/L, p = 0.0404; IL-18: 579 vs 365 pg/mL, p = 0.0402). Significant correlation was found between serum values of IL-18 and LDH (r(2) = 0.504, p = 0.0433). The administration of corticosteroids to patients in the refractory group resulted in the rapid improvement of symptoms and decrease in serum LDH levels in all patients. A serum LDH level of ≥410 IU/L, which was calculated from receiver operating characteristic curve analysis, seemed to be an appropriate criterion for the initiation of steroid therapy. In conclusion, serum IL-18 and LDH levels can be used as parameters to determine which patients are candidates for corticosteroid therapy. In addition, serum LDH levels seem to be a useful marker for the evaluation of therapeutic efficacy in refractory M. pneumoniae pneumonia.

Nationwide surveillance of macrolide-resistant Mycoplasma pneumoniae infection in pediatric patients.

We conducted nationwide surveillance to investigate regional differences in macrolide-resistant (MR) Mycoplasma pneumoniae strains in Japan. The prevalence of MR M. pneumoniae in pediatric patients gradually increased between 2008 and 2012. Although regional differences were observed, high levels of MR genes were detected in all seven surveillance areas throughout Japan and ranged in prevalence from 50% to 93%. These regional differences were closely related to the previous administration of macrolides.

Therapeutic efficacy of macrolides, minocycline, and tosufloxacin against macrolide-resistant Mycoplasma pneumoniae pneumonia in pediatric patients.

The importance of macrolide-resistant (MR) Mycoplasma pneumoniae has become much more apparent in the past decade. We investigated differences in the therapeutic efficacies of macrolides, minocycline, and tosufloxacin against MR M. pneumoniae. A total of 188 children with M. pneumoniae pneumonia confirmed by culture and PCR were analyzed. Of these, 150 patients had a strain with an MR gene and 134 had one with an A-to-G mutation at position 2063 of M. pneumoniae 23S rRNA domain V. Azithromycin (n = 27), clarithromycin (n = 23), tosufloxacin (n = 62), or minocycline (n = 38) was used for definitive treatment of patients with MR M. pneumoniae. Defervescence within 48 h after the initiation of antibiotic therapy was observed in 41% of the patients in the azithromycin group, 48% of those in the clarithromycin group, 69% of those in the tosufloxacin group, and 87% of those in the minocycline group. The average number of days of fever after the administration of antibiotic treatment was lower in the minocycline and tosufloxacin groups than in the macrolide groups. The decrease in the M. pneumoniae burden, as estimated by the number of DNA copies, after 48 to 96 h of treatment was more rapid in patients receiving minocycline (P = 0.016) than in those receiving tosufloxacin (P = 0.049), azithromycin (P = 0.273), or clarithromycin (P = 0.107). We found that the clinical and bacteriological efficacies of macrolides against MR M. pneumoniae pneumonia was low. Our results indicated that minocycline rather than tosufloxacin can be considered the first-choice drug for the treatment of M. pneumoniae pneumonia in children aged ≥ 8 years.

In vitro activities of 11 antimicrobial agents against macrolide-resistant Mycoplasma pneumoniae isolates from pediatric patients: results from a multicenter surveillance study.

Macrolide-resistant Mycoplasma pneumoniae is emerging in several countries, and it is mainly observed in children. To our knowledge, we conducted the first multicenter prospective epidemiological study of macrolide-resistant M. pneumoniae in order to investigate regional differences in the susceptibility of macrolide-resistant M. pneumoniae to antibacterial agents. The in vitro activities of 11 antimicrobial agents against macrolide-resistant M. pneumoniae isolates from 5 different areas of Japan were investigated. Among 190 M. pneumoniae isolates from pediatric patients, 124 (65.2%) isolates showed macrolide resistance and possessed an A2063G transition in domain V of the 23S rRNA. These isolates showed high resistance to erythromycin, clarithromycin, and azithromycin with minimum inhibitory concentrations (MICs) ≥ 16 µg/ml. Conversely, quinolones such as garenoxacin, moxifloxacin, tosufloxacin, and levofloxacin exhibited potent antimycoplasmal activity. No regional differences were observed with respect to the MICs among the 5 areas in Japan.

Clinical efficacy of macrolide antibiotics against genetically determined macrolide-resistant Mycoplasma pneumoniae pneumonia in paediatric patients.

BACKGROUND AND OBJECTIVE: Since 2000, the prevalence of macrolide-resistant (MR) Mycoplasma pneumoniae has increased among paediatric patients in Japan. To determine the efficacy of macrolides against MR M. pneumoniae pneumonia, microbiological and clinical efficacies were compared during the antibiotic treatment. METHODS: Samples from a total of 30 children with M. pneumoniae pneumonia, as confirmed by PCR and serology, were analyzed. Primers for domain V of 23S rRNA were used, and DNA sequences of the PCR products were compared with the sequence of an M. pneumoniae reference strain. RESULTS: Isolates from 21 patients demonstrated point mutations, and these patients were defined as MR. The remaining nine patients, whose isolates showed no point mutations, were categorized as control (macrolide-sensitive) patients. The number of M. pneumoniae in nasopharyngeal samples from the control group decreased rapidly 48 h after initiation of macrolide treatment and showed a close relationship with clinical outcome. In contrast, the number of M. pneumoniae 48 h after initiation of macrolide treatment were significantly higher in samples from MR patients than in samples from macrolide-sensitive patients. In 15 of 21 MR patients, fever persisted for more than 48 h after the initiation of macrolide treatment. When treatment was changed to minocycline, fever disappeared within 48 h in all these MR patients. There were no differences between MR patients who demonstrated a reduction in fever and those in whom fever persisted after 48 h of macrolide treatment. CONCLUSIONS: The microbiological and clinical efficacies of macrolides for treating patients with MR M. pneumoniae pneumonia were low. These results show that macrolides are clearly less effective in patients with MR M. pneumoniae pneumonia.

[What EIA assay levels correspond to rubella antibody HI assay titer?].

In 2004, a Japanese-government-supported research group recommended that women without rubella antibody or with low titers < or = 1:16 of HI antibody should be vaccinated to decrease the risk of congenital rubella syndrome. We compared HI antibody titer with EIA-IgG levels in 520 college students with < or = 1:64, HI antibody measuring rubella antibodies in the same specimen using HI and EIA assay kits from Denka Seiken Co. (Japan) and Dade Behring Co. (Germany). The positive predictive value of the EIA assay using the kit from Denka Seiken Co. was 99.8% and the negative predictive value 91.3%, respectively, compared to 97.9% (positive > or = 15 IU/mL), 98.1% (positive > or = 10 IU/mL), and 93.4%, using the kit from Dade Behring Co. Between HI titers and EIA-IgG measured with the Denka kit, the coefficient index was 0.715 (p < 0.0001). Between those measured with the Dade kit, the coefficient index was 0.610 (p < 0.0001). Between EIA-IgG levels measured using the two kits, the coefficient index was 0.753 (p < 0.0001). The HI value of 1:16 corresponds approximately to 9.0 with the kit (positive > or = 4.0) from Denka, and to 30 IU/mL with the kit from Dade. EIA-IgG levels > or = 10 IU/mL are considered globally as protective antibody titers, meaning that the Japanese recommendation of < or = 1:16 for vaccination is too loose. Japanese EIA kit values for the rubella antibody should also be expressed in IU/mL using the global standard.

Characteristics of facial nerve palsy during childhood in Japan: frequency of varicella-zoster virus association.

BACKGROUND: The aim of this study was to clarify the clinical characteristics of facial nerve palsy and the frequency of varicella-zoster virus association in Japanese children, retrospectively. METHODS: The subjects were 30 facial nerve palsy patients less than 15 years old, treated in the Department of Pediatrics, Kawasaki Medical School Hospital, Okayama, Japan, during the last 10 years. RESULTS: The male/female and right/left ratios were 16/14 and 16/13, respectively. The patients included 21 cases (70%) of Bell's palsy, four cases (13%) due to otitis media, three cases (10%) of Ramsay Hunt syndrome and two cases (7%) due to birth trauma. There were six cases of zoster sine herpete among the Bell's palsy cases. CONCLUSION: Varicella-zoster virus-associated facial palsy was found in nine (36%) of the 25 patients examined. Zoster sine herpete was more frequently encountered in children than adults. Ramsay Hunt syndrome was found in school-age children and zoster sine herpete was often found in preschool children. The period of recovery was fast for facial nerve palsy due to acute otitis media, which occurred within 23 months of age, after myringotomy and administration of antibiotics.

Elevated serum C-reactive protein in mumps orchitis.

A case of mumps orchitis with a high concentration of C-reactive protein (CRP) prompted us to evaluate the inflammatory response in mumps complications. We compared the CRP titers in mumps patients with orchitis and meningitis. The serum CRP titers were significantly higher in the patients with orchitis than in those with meningitis.