Objective To detect twenty types/subtypes of respiratory viruses with the GeXP (genomelab genetic analysis system) based mRT-PCR (multiplex reverse transcription-polymerase chain reaction) assay,and discuss its value in pediatric respiratory viral infection.Methods A retrospective study was conducted on a total of 1 305 inpatient children (0-6 years old) admitted to the Department of Respiratory in Children's Hospital of Hebei Province from May 2014 to November 2014.The sputum samples were collected and the viral DNA or RNA was extracted.After examining the specificity and sensitivity,the GeXP-based mRT-PCR assay was performed to test 20 types/subtypes of respiratory viruses.The obtained results were evaluated in comparison with those of fluorescence real-time PCR and direct sequencing.Chisquare test was conducted by SPSS16.0.Results Among 1 305 hospitalized children,the positive rate of respiratory virus was 58.31％ (761/1 305).The positive rate of respiratory syncytial virus (RSV,17.32％,226/1 305) was the highest followed by the parainfluenza virus 3 (PIV-3,16.02％,209/ 1 305),human rhinovirus (HRV,11.95％,156/1 305),adenovirus (ADV,5.06％,66/1 305),human bocavirus (HBoV,3.14％,41/1 305) and the parainfluenza virus 1 (PIV-1,2.07％,27/1 305).Multiple virus infection was found in 92 children (7.05％).The highest positive rate of respiratory virus was observed in 1-2 years old children (83.28％,249/299);the lowest rate was found in 5-6 years old children (37.68％,26/69).Conclusions The proposed GeXP-based mRT-PCR assay possessed the advantage of high throughput,sensitivity,specificity and rapidity in the detection of twenty types/subtypes of respiratory viruses.It provided a thorough investigation of viral pathogens involved in acute respiratory infection thus could satisfy the clinic requirement and provide epidemic data on the disease prevention.

Objective:To analyze the changes in T lymphocyte subsets, B lymphocytes and NK cells in children with active tuberculosis (TB) and their clinical significance.Methods:T lymphocyte subsets, B lymphocytes and NK cells in peripheral blood samples of 106 patients with acute TB (TB group) and 106 healthy children (healthy control group) were detected by flow cytometry and compared between different groups.Results:The percentages of CD3 + T, CD4 + T and NK cells as well as the CD4 +/CD8 + T cell ratio were significantly lower in the TB group than in the healthy control group ( Z=-3.783, P=0.000; Z=-5.401, P=0.000; Z=-3.434, P=0.001; Z=-2.014, P=0.044). The percentages of double negative T (DNT) and B cells in the TB group were significantly higher than those in the healthy control group ( Z=2.765, P=0.006; Z=6.880, P=0.000). No significant difference in the percentage of CD8 + T or double positive T (DPT) cells was observed between the two groups ( P>0.05). The expression of peripheral lymphocyte subsets varied in TB children of different age groups (0-<3, 3-<6, 6-<10 and 10-<16 years old). There were significant differences in CD3 + T, DNT and B cells among the four age groups ( H=10.081, P=0.018; H=14.583, P=0.002; H=8.498, P=0.037). The percentage of CD4 + T cells was significantly lower in children with extrapulmonary TB than in those with pulmonary TB ( Z=-3.068, P=0.002). No statistically significant difference in other lymphocyte subsets was found between children with extrapulmonary and pulmonary TB ( P>0.05). Conclusions:Tuberculosis could lead to immune dysfunction in children. Dynamic monitoring of the changes in peripheral lymphocyte subsets in children with TB could be conducive to better assessment of immune status and providing personalized treatment.

Objective To develop a tetra-primer amplification refractory mutation system PCR(T-ARMS-PCR)assay for detecting four single nucleotide polymorphisms(SNPs): rs1801133,rs1801131, rs1805087 and rs1801394 associated with folate metabolism in a single reaction tube.Methods Methodology was developed.Then, it applied the established method to analyze 150 physical examination children′s anticoagulant blood samples admitted to the Department of Clinical Laboratory, Children′s Hospital of Hebei Province from January 2017 to April 2017.Four sets of chimeric primers consisting of a universal Tag sequence and targeting rs 1801133,rs1801131,rs1805087 and rs1801394 fused to the specific sequence were designed according to the T-ARMS-PCR principle and the multiplex chimeric primers strategy.A single tube PCR was then conducted after optimizing the primer concentrations and the reaction conditions.The amplified products were analyzed by QIAxcel capillary electrophoresis, and the corresponding SNP genotypes of 150 samples were identified.Furthermore,all samples were verified by direct sequencing.And the Hardy-Weinberg Equilibrium(HWE)testing of four SNPs from 150 samples were conducted by SPSS17.0 Chi-square test.Results The improved T-ARMS-PCR combined with capillary electrophoresis can accurately verify eight different alleles of the four SNPs associated with folate metabolism at one time in 3 hours.Four SNPs of 150 whole blood samples were accurately classified and the results were completely consistent with direct sequencing.All the genotype frequencies of these four SNPs were in HWE (χ2rs1801133=0.69, Prs1801133=0.40; χ2rs1801131=0.21, Prs1801131=0.64; χ2rs1805087=3.32, Prs1805087=0.07;χ2rs1801394=1.91, Prs1801394=0.17).Conclusions The proposed improved T-ARMS-PCR combined with capillary electrophoresis in this study can accurately verify four SNPs associated with folate metabolism in a single reaction tube.This method might be a valuable tool to specifically guide the folate supplement in general population.

Objective To analyze the molecular epidemiology of adenovirus ( ADV) causing acute respiratory diseases and to investigate the mixed infection of ADV and other respiratory pathogens in Hebei Province. Methods Sputum samples were collected from inpatient children with acute respiratory diseases at Children′s Hospital of Hebei Province between June 2017 and May 2018. Multiplex reverse transcription PCR assay was used to detecte 13 kinds of respiratory pathogens. Nested PCR was performed to amplify ADV hexon gene and the amplified products were then sequenced. Results A total of 353 ADV-positive speci-mens were detected in 8839 specimens with a positive rate of 3. 99％. Significant difference in the positive rate of ADV was not observed between male and female patients (χ2=0. 0003, P=0. 99), but found among different age groups (χ2=115. 69, P<0. 001). All isolated ADV strains belonged to 11 serotypes, which were type 1 (16. 15％, 57/353), type 2 (35. 98％, 127/353), type 3 (21. 25％, 75/353), type 4 (1. 13％, 4/353), type 5 (11. 33％, 40/353), type 6 (3. 97％, 14/353), type 7 (8. 22％, 29/353), type 31 (0. 28％, 1/353), type 41 (0. 28％, 1/353), type 55 (0. 28％, 1/353) and type 57 (1. 13％, 4/353). Among the 353 ADV-positive specimens, 259 were mixed infections mainly caused by ADV and human rhinovirus (35. 52％). ADV and respiratory syncytial virus co-infections accounted for 12. 74％ and 33. 20％ of the mixed infections involved three or more pathogens. ADV could be detected throughout the year, especially in September and April to May. The predominant serotypes were types 1, 2 and 3. The av-erage ages of the two groups of ADV infection alone and ADV mixed infection were (27. 56±24. 67) months and (21. 33 ±20. 28) months, respectively, and the difference between them was statistically significant (P=0. 037). The incidence of ADV 2 infection alone was 25. 77％ (25/97), which was lower than that of ADV 2-involved mixed infection [39. 84％ (102/256),χ2=6. 05, P=0. 014]. However, the rate of ADV 7 infection alone was significantly higher than that of ADV 7-involved mixed infection [16. 49％ 16/97) vs 5. 08％ (13/256),χ2=6. 05, P<0. 001]. Conclusion ADV 1, ADV 2 and ADV 3 were the predominant serotypes circulating in Hebei Province from June 2017 to May 2018, especially in September and April to May. The younger the patients were, the higher the incidence would be. ADV 2 was prone to cause mixed infections with other respiratory pathogens, while ADV 7 was less common in mixed infections. Younger pa-tients were more susceptible to mixed infections. The most common co-infection was caused by ADV and hu-man rhinovirus.

Objective:To analyze the clinical characteristics of pertussis cases diagnosed by two pathological detection methods: bacterial culture and real-time polymerase chain reaction (RT-PCR), and to explore the applicable value of two pathological detection methods in the diagnosis of pertussis.Methods:Bilateral nasopharyngeal swabs and clinical information of 165 children suspected of pertussis were collected by Hebei Children′s Hospital from April 2019 to January 2020. The bacterial culture and RT-PCR for nasopharyngeal swab specimens were performed in all cases. Chi-square test was used to analyze the cases of pertussis diagnosed by the above two methods.Results:Based on clinical diagnosis, the sensitivity of bacterial culture and RT-PCR for the diagnosis of pertussis was 61.70% (58/94) and 86.17% (81/94), and the specificity was 92.96% (66/71) and 71.83% (51/71), respectively. The positive rate of RT-PCR in children of all ages, seasons and cough courses is higher than that of bacterial culture. Children with pertussis diagnosed by bacterial culture and RT-PCR were basically similar in age, season, and cough course distribution, with the most common cases ≤3 months old, a high incidence trend in summer and autumn, and the course of coughing in children was mostly within 15-21days. The positive rate of bacterial culture in the diagnosis of pertussis in children is affected by the age of the children, and there are statistical differences between children in different age groups (χ2= 11.929, P=0.036). The positive rate of bacterial culture was the highest in children with >3 years old (51.85% [14/27]), followed by children with ≤3 months old (48.72% [19/39]), and the lowest in children with >6-12 months old (15.00% [3/20]). Moreover, the positive rate of bacterial culture in the diagnosis of pertussis in children is also affected by the cough course of the children, and there are statistical differences between children in different cough course groups (χ2=9.841, P=0.020). The positive rate of bacterial culture was the highest in children with cough course 15-21 days (49.23% [32/65]), followed by 43.59% (17/39) in children with cough course 8-14 days, and the lowest in children with cough course of less than 7 days (22.86% [8/35]). Conclusions:Compared with RT-PCR, bacterial culture has lower sensitivity and higher specificity in the detection of pertussis. These two detection methods have their own advantages and limitations. Medical institutions at all levels should comprehensively analyze different laboratory detection methods. Only by combining the two methods can the diagnostic value and level be effectively improved.