Objective To analyze the molecular characteristics of Salmonella paratyphi A ( S. pa-ratyphi A) strains prevailing in Hangzhou area in recent years. Methods Pulse field gel electrophoresis ( PFGE) and multiple-locus variable-number tandem repeat analysis ( MLVA) were performed for molecular typing and epidemiological analysis of 72 S. paratyphi A strains isolated in Hangzhou area during 2002 to 2013. Results The 72 S. paratyphi A strains were divided into 11 PFGE ( by using restriction enzymes of Xba Ⅰ and Bln Ⅰ) and 6 MLVA types. Among the selected 34 variable number tandem repeat ( VNTR) sites, 4 sites (1188K, 2075K, 2201K and 4346K) showed high polymorphism, in which PFGE displayed a higher resolution than MLVA. Except for the 5 PFGE types of X4B5, X7B7, X8B8, X9B9 and X10B10, the other 6 PFGE types belonged to a same clone sharing a similarity of greater than 95%, and the S. Paratyphi A strains in that clone accounted for 93. 1% of the total strains isolated in Hangzhou. Conclu-sion The occurrence of paratyphoid A in Hangzhou area from 2002 to 2013 was mainly caused by S. para-typhi A strains belonging to the same clone. Combination of PFGE with MLVA was conducive to epidemiolog-ical investigation of paratyphoid A.

Objective To determine the molecular characteristics of predominant Salmonella typhi and Salmonella paratyphi A strains prevalent in Hangzhou area from 2002 to 2008.Methods Pulse field gel electrophoresis (PFGE),multi-locus variable-number tandem repeat analysis (MLVA) and multi-locus sequence typing (MLST) were applied for typing as well as analysis of the molecular characteristics of 31 S.typhi isolates and 404 S.paratyphi A isolates from Hangzhou area during 2002 to 2008.Results The 404 S.paratyphi A isolates could be divided into six PFGE types (P1-P6).99.0％ of the S.paratyphi A isolates (400/404) belonged to the same one clone family (P1 and P2 types),in which P1 strains occupied 93.3％ (373/400) of the isolates.The 31 S.typhi isolates displayed a high diversity,which could be classified into 14 PFGE types,28 MLVA types with 90.3％ resolving power and 3 MLST types.The S.typhi strains prevalent in Hangzhou area were similar to those in Southeast Asia but different from those in Europe.The variable number tandem repeat (VNTR) sites with high polymorphism,TR1,TR2 and Sal02,could be used to the markers for diagnosis of S.typhi isolates in the area.The MLST types of 31 S.typhi isolates included all the three types currently found in the world but the ST2 type of S.typhi strains was predominant (23/31,74.2％).Conclusion The paratyphoid A prevalence in Hangzhou area in the recent years is caused by infection of the same clone family of S.paratyphi A whereas the S.typhi strains prevalent in the area display a high diversity.

Objective To investigate the epidemiological characters of human metapneumovirus (hMPV) infection in children with influenza-like illness (ILI).Methods A total of 1 164 throat swabs were collected from children with ILI symptoms in Children's Hospital,Zhejiang University School of Medicine from January 2011 to December 2012.hMPV was detected by using nucleic acid assay,the fusion (F) protein gene of hMPV was amplified by RT-PCR,gene sequencing was performed and the sequences were compared with those in GenBank.Positive rates of hMPV in different age groups were compared with Chi-square test.Results Among 1 164 samples,hMPV was positive in 73 (6.27％) samples.hMPV infection was the most popular in ＞2-4 y age group (33/220,15.0％),and the positive rates of hMPV in different age groups were of statistical significance (x2 =40.69,P ＜ 0.05).hMPV infection occurred throughout the year,but it was most common in winter and spring.The highest incidence of hMPV infection was observed in December 2012 (12/51,25.53％).Among 24 samples of hMPV,14 were with genotype B1,2 were with genotype B2,and 8 were with genotype A2.The most common genotype was B1 in 2011 (10/12),and A2 in 2012 (8/12).Homology between nucleotide sequences of the 24 samples of hMPV were 81.6％ to 100.0％.Conclusions hMPV infection exists in children with ILI in Hangzhou,and the epidemic seasons are winter and spring.hMPV infection is more likely to be found in children aged 2 to 4 years old,and different genotypes may predominated alternately.

Objective To develop a microsphere-based suspension array for simultaneous detec-tion and identification of Salmonella H antigens by using Luminex xTAG technology and to evaluate its capa-bility in serotyping Salmonella strains. Methods The fliC and fljB genes, encoding the H antigen of Salmo-nella, were selected as the target genes. Universal upstream primers were designed based on the highly con-served regions of fliC and fljB genes, and the corresponding specific reverse primers were designed based on the variable regions. While synthesizing, the 5′end of each upstream primer was labeled with biotin and the 5′end of each specific reverse primer was modified with its certain TAG sequence. After amplified and la-beled with biotin and TAG sequence, the PCR products of specimens were hybridized with the mixture of va-rious MagPlex-xTAGTM microspheres. Each set of microspheres contained its unique anti-TAG sequences. The results of hybridization were analyzed by using Luminex MagPix reader system and the median fluores-cence intensity ( MFI) was reported. The H antigens of 145 Salmonella strains were identified with this de-veloped xTAG suspension array, and the results were compared with those obtained by using traditional ser-um agglutination test. Results The PCR products of different H antigens ranged from 94 bp to 245 bp and could be identified by hybridizing with MagPlex-xTAGTM microspheres. There was no cross-reaction between different H antigens or with DNAs derived from Escherichia coli, Vibrio cholerae, Vibrio parahaemolyticus and Shigella flexneri. Compared with the traditional serum agglutination test, the sensitivity and specificity of the xTAG suspension array in the identification of H antigens of 145 Salmonella strains were 95. 1% and 100%, respectively. Conclusion The developed xTAG suspension array was a specific, accurate and effective method for simultaneous detection and identification of 31 H antigens of common Salmonella serovars strains. It could be used for determining the H antigens of more than 90 Salmonella strains within 5 hours.

Objective To understand the genomic epidemiology of Salmonella paratyphi A strains circulating in Hangzhou area in recent years. Methods Next generation sequencing(NGS) technology was used to obtain genomes of 60 Salmonella paratyphi A strains isolated in Hangzhou area from 2002 to 2013. Genomes of 391 Salmonella paratyphi A strains were downloaded from the Sequence Read Archive (SRA) and Assembly database. After removing recombinations, the phylogenetic tree of all 451 genomes based on single nucleotide polymorphisms(SNP) was constructed using ATCC9150 as the reference. SRST2 and mul-tilocus sequence typing (MLST) were used to analyze sequence types(ST). The Salmonella In Silico Typ-ing Resource (SISTR) was used for core genome multilocus sequence typing (cgMLST). Resistant genes were screened out with SRST2 and BLASTN. Seven kinds of antibiotics were selected to conduct drug sus-ceptibility test in the 60 strains isolated in Hangzhou. Results A total of 19 258 SNP loci were found in 451 genomes. The average distance in the phylogenetic tree between strains was 0.007 0 and the distance less than 0.05 accounted for 96.73%, indicating a little difference in the 451 Salmonella paratyphi A ge-nomes. Fifty-eight Hangzhou strains of ST85 were highly similar in genomic sequences,which suggested that the clonal spread of ST85 strains caused the epidemic of paratyphoid A in Hangzhou area during 2002-2013. Salmonella paratyphi A strains isolated in Hangzhou were distantly related to five domestic strains (average distance:0.057),but close to 15 Yunnan strains(average distance:0.003 2) and closest to strains isola-ted in Cambodia (average distance: 0.001 8), suggesting the possibility of transnational spread of ST85 strains. Among two Hangzhou strains of ST129,HZ333 was closely related to two strains isolated in Jiangsu Province(average distance:0.009 7),suggesting the possibility of domestic transmission of ST129 strains. Except for two untyped strains,the other 58 strains were divided into nine cgMLST types. Except for 57 un-typed strains,the other 334 strains obtained from public databases were classified into 165 cgMLST types. Fifty-six out of the 60 Hangzhou strains carried the resistant gene aac6-Iy and the other four carried aac6-Iaa gene. Thirteen out of the 391 strains obtained from public databases didn′t carry resistant genes, while the other 378 strains carried the resistant gene aac6-Iy. Among the 60 Hangzhou strains,56 were sensitive to all of the seven kinds of antibiotics;three were resistant to cotrimoxazole and one was resistant to ampicillin and tetracycline. Conclusion The epidemic of paratyphoid A fever in Hangzhou from 2002 to 2013 was mainly caused by the clonal spread of ST85 strains that had the possibility of transnational spread. Some Hangzhou strains of ST129 had the possibility of domestic transmission. SNPs analysis had an advantage over pulse field gel electrophoresis(PFGE) technology in resolution and could be used to accurately trace the cause of paratyphoid A fever. Resistant gene aac6-Iy was generally carried. NGS technology had a promising prospect in the control and prevention of infectious diseases.

Objective:To study the pathogenic spectrum of enterovirus (EV) in the samples of child influenza-like(ILI)cases in Hangzhou city .Methods:In 2019, 1 060 throat swab specimens of ILI cases were collected for serotyping of influenza virus and EVs by real-time RT-PCR. The positive rates of influenza virus and EV in spring, summer, autumn and winter were compared by chi-square test with SPSS16.0 software. Specific primers were synthesized and used to amplify the VP1 fragments of EV. PCR products were sequenced and the results were compared with the reference sequences by using Basic Local Alignment Search Tool (BLAST) to identify the serotypes of isolated EV. The clinical diagnoses of EV positive cases were classified and analyzed.Results:A total of 1 060 specimens were collected and 283(26.70%) were positive for influenza virus, 75(7.08%) were positive for EV, 3(0.28%)were positive for influenza virus and EV. The comparison of positive detection rate of spring, summer, autumn and winter showed that influenza virus were prevalent in winter and spring. EV were mostly popular in the summer months. VP1 sequences of 51 EV were successfully amplified and BLAST analysis revealed that these strains belonged to 10 serotypes, including five serotypes of EV-A species, four serotypes of EV-B species and one serotype of EV-D. The ten serotypes of EV, including coxsackievirus (CV)A2, A4, A5, A6, A9, A10, and echovirus (ECHO)7, ECHO11, ECHO18, and EV-D68 were obtained and the percentages of positive were 16.00%, 16.00%, 5.33%, 12.00%, 5.33%, 1.33%, 1.33%, 5.33%, 4.00% and 1.33%, respectively. The phylogram of EV VP1 sequence showed that 51 EV strains in Hangzhou had different degrees of variation compared with the reference strains. Acute upper respiratory tract infection was the main clinical diagnosis in EV positive children, with 44 cases (58.67%). Acute tonsillitis was followed by 14 cases (18.67%). Followed by herpetic pharyngitis, acute bronchitis, asthmatic bronchitis, pneumonia, accounting for 12.00%, 8.00%, 1.33%, 1.33%, respectively.Conclusions:EV causing influenza-like illness in children in Hangzhou in 2019 belonged to 10 serotypes, CVA2 and CVA4 were the predominant serotypes, and the positive rate of EV detection was higher in summer.

Objective: To determine the epidemic characteristics of respiratory viruses, mycoplasma pneumonia(MP) and chlamydia pneumoniae(CP) in outpatients and hospitalized children with acute respiratory tract infections(ARI), to lay a foundation for the prevention and control of ARI. Methods: From 2011 to 2013, children with ARI, including outpatients and inpatients, were involved in this study. One nasopharyngeal aspirate or throat swab specimen was collected from each patient.Real time PCRs were performed to detect common respiratory tract viruses, MP and CP. Results: At least one pathogen was identified in each of 610 out of 908 patients and the overall positive rate was 67.2%. The positive rate in inpatient(76.7%)was higher than that in outpatient(43.0%) (χ²=94.79, P<0.001). Simultaneous detection of two or more viruses was found in 206 cases.Co-infection was more frequent in inpatients than in outpatients(29.0% VS 6.6%, P<0.001). Significant differences of the detection rate were observed in RSV, PIV, HRV, Flu, human bocavirus (hBoV), adenovirus (AdV), saffold virus(SAFV), MP and CP between the inpatient and outpatient group. Respiratory syncytial virus(RSV)(34.5%) was the most prevalent virus detected among hospitalized children, followed by MP(15.0%)and human rhinovirus(HRV)(14.6%). Whereas adenovirus(AdV) (15.2%)was the most frequently identified virus in the outpatient group, followed by influenza virus(Flu)(11.7%))and PIV(7.8%). Except for RSV and Flu, co-infection of the other pathogens was more frequent than its mono-infection in inpatients. Significant differences of the AdV co-infection rate were observed between the inpatient and outpatient group(χ²=18.90, P<0.001). Compared with mono-infection, co-infection has no significant effect on the clinical presentation. Conclusions The detection rate of respiratory pathogens was higher in inpatients than in outpatients with ARI, and co-infections were more popular in children hospitalized, it may show that co-infection had some correlation with disease severity.

OBJECTIVETo understand the molecular epidemiologic features of human metapnenmovirus (hMPV) in children with respiratory tract infection in Hangzhou.

METHODS2 593 throat swabs were collected from patients with respiratory tract infections who visited the hospitals with sentinel surveillance programs from January 2011 to December 2013, including 1 676 outpatients and 917 inpatients. Total nucleic acid was extracted from the specimens and the fusion (F) protein gene of hMPV was amplified by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR), with positive samples picked to compare with the sequence of hMPV in GenBank, after the sequence of amplification products were determined. Other two types of common respiratory virus were tested using RT-PCR.

RESULTSThe overall positive rate in this study was 6.51% (169/2 593), with 6.62% (111/1 676) in outpatients and 6.32% (58/917) in inpatients, but no statistically significant difference was found (χ(2) = 0.086, P = 0.769). The rates was 7.01% in males and 5.72% in females, with no statistically significant difference in different sex (χ(2) = 1.676, P = 0.195). The positive rate was 14.14% (28/198)in the 2-year-olds, 14.01% (22/158)in 3-year olds. The rate in 2-year olds was higher than in other groups, with statistically significant differences between the groups (χ(2) = 38.654, P = 0.000). Of the 169 positive cases, 153 (90.53%) in the younger than 5 years olds. The rates of infection with hMPV in winter and spring were statistically higher than in summer and autumn (χ(2) = 67.032, P = 0.000). The rate of co-infection was 19.52% (33/169). 88 amplified productions were selected for gene sequence analysis, and the F gene homology were 81.6%-100.0% with reference strains in GenBank. Data showed that all the 4 viral subtypes: A2 (52.27% , 46/88), B1 (37.51%, 33/88), B2 (9.09%, 8/88) and A1 (1.13%, 1/88) co-circulated during the study. However, different subtypes appeared predominant in different years:hMPV subtype B1 was in 2011 and 2012, subtype A2 in the end of 2012 and in 2013. Of the 88 specimens, gene sequences were determinate, with A genotype accounted for 67.56% (25/37), B genotype for 32.43% (12/37)in children younger than 1-year olds, and A genotype accounted for 43.13% (22/51), B genotype for 56.86% (29/51)in children above 1-year olds. Significant differences between the two groups (χ(2) = 5.143, P = 0.023) were noticed.

CONCLUSIONIt was confirmed that hMPV was one of the substantial pathogens causing the respiratory tract infections. Data from our study suggested that the peak time of hMPV infection predominated during winter and spring in Hangzhou. Both hMPV subtype B1 and subtype A2 were found popular in this study, with hMPV genotype A dominating in children younger than 1-year olds.

Child ; Child, Preschool ; China ; epidemiology ; Coinfection ; Female ; Genotype ; Humans ; Infant ; Male ; Metapneumovirus ; genetics ; pathogenicity ; Molecular Epidemiology ; Paramyxoviridae Infections ; epidemiology ; Real-Time Polymerase Chain Reaction ; Respiratory Tract Infections ; epidemiology ; Seasons ; Sentinel Surveillance