Objective:To prepare seven standard strains of 2019 novel coronavirus (2019-nCoV), including wild type (WT) strain, Beta variant, Delta variant, Omicron variants (BA.2, BA.5, BQ.1, XBB.1 branches), which could be used to apply for national standard strains.Methods:According to cytopathic effect (CPE), virus titer, whole-virus-genome-sequencing and detection of mycoplasma, the basic biological characteristics of clonal isolation were determined through plaque purification technology.Results:The CPE was mainly characterized by cell shrinkage and exfoliation in Vero cells after infection with seven 2019-nCoV clonal isolations (WT, Beta, Delta, Omicron, BA.2, BA.5, BQ.1 and XBB.1 branches). The result of mycoplasma detection were negative and titers of the clonal isolation from 2nd to 5th generations were stable at 10 5-10 8 TCID 50/ml; the electron microscope showed that the virions were all round or elliptical, with a diameter between 60 nm and 140 nm. The subtypes of 7 strains were identified by whole-virus-genome-sequencing and phylogenetic analysis, with genomic stability after the fifth successive generations of clonal isolation. Conclusions:The series clonal isolation of 2019-nCoV with typical CPE of coronavirus, clear morphological structure, good viral activity and stable genetic characteristics were prepared, and they could be used to apply for national standard strains.

Objective:To understand the serotype distribution, drug resistance and molecular characterization of invasive non-typhoid Salmonella (iNTS) in Guangdong Province from 2018 to 2022 and provide scientific evidence for the prevention and treatment of blood flow infection caused by Salmonella. Methods:Serological identification, antimicrobial susceptibility testing, multilocus sequence typing (MLST), and whole genome sequencing were performed on Salmonella isolated from blood and stool samples in Guangdong from 2018 to 2022. Simultaneously, annotated the sequencing results for drug resistance genes and virulence factors by a microbial gene annotation system. Results:The 136 iNTS strains were divided into 25 serotypes, and Salmonella enteritidis accounted for 38.24% (52/136). The OR of other iNTS serotypes were calculated with Salmonella typhimurium as the control. The OR values of Oreninburg, Rysson, and Pomona serotypes were the highest, which were 423.50, 352.92, and 211.75, respectively. The drug resistance rate of iNTS was 0.74%-66.91%, which was lower than that of non-iNTS (3.90%-77.21%). The main iNTS of drug resistance were ampicillin and tetracycline, with resistance rates of 66.91% (91/136) and 50.00% (68/136), respectively, while the resistance rates to ciprofloxacin (5.88%,8/136), ceftazidime (5.88%,8/136), gentamicin (5.13%,7/136) and cefoxitin (0.74%, 1/136) were relatively low. iNTS carried a variety of drug-resistance genes and virulence factors, but no standard virulence factor distribution has been found. MLST cluster analysis showed that iNTS was divided into 26 sequence types, and ST11 accounted for 38.24% (52/136). Conclusions:The iNTS strains in Guangdong were dominated by Salmonella enteritidis, of which three serotypes, Oreninburg, Rison, and Pomona, may be associated with a higher risk of invasive infection during 2018 to 2022 . iNTS was sensitive to clinical first-line therapeutic drugs (cephalosporins and fluoroquinolones), with highly diverse sequences and clear phylogenetic branches. ST11 was the local dominant clone group.

Objective:To analyze the clinical characteristics and pathogenic distribution of severe pneumonia in adults in order to provide basis for clinical diagnosis and treatment.Methods:From June 2021 to April 2022, 145 patients with pneumonia admitted to the Department of Respiratory and Critical Care Medicine of the Second People's Hospital of Guangdong Province. According to whether they meet the diagnostic criteria for severe pneumonia, they were divided into severe ( n=63) and mild ( n=82) groups, and the clinical features between the two groups were compared. At the same time, the role of FilmArray detection in severe pneumonia was discussed. The measurement data were tested using independent sample t test or Mann-Whitney U test, and the counting data were tested using Chi-square test or Fisher exact probability method. Results:The age of the patients in the severe group was (72.67±1.71) years, male patients accounted for 84.1%, and the median hospitalization time was 16 days. Nine patients died in hospital; most of them had fever, shortness of breath, and change of consciousness, accompanied by hypertension, diabetes, cerebrovascular disease, chronic kidney disease, and tumor history. Compared with the mild group, the total number of leukocytes, neutrophil ratio, procalcitonin, and C-reactive protein were higher in the severe group, but the CD3 +, CD4 +, and CD8 + cell counts were lower ( P<0.05). The positive rate of FilmArray detection in the severe group was 81%, and the mixed infection of multiple bacteria accounted for 50%, which was higher than that of traditional culture ( P<0.05). The top four pathogens in severe group were Pseudomonas aeruginosa, Acinetobacter baumannii complex, Klebsiella pneumoniae, and Staphylococcus aureus, which were significantly higher than that in the mild group ( P<0.05). Resistance genes were detected in patients with severe disease, which was significantly higher than that in patients with mild disease (70.7% vs. 17.5%, P<0.05). Conclusions:Severe pneumonia is more common in elderly men, with more basic diseases and poor immunity. FilmArray has a high positive rate and can detect multiple pathogens, which may have a role in the rapid diagnosis of severe pneumonia.

Objective:To detect blood samples from clinically confirmed cases infected with the 2019 novel coronavirus (2019-nCoV) by fluorescence immunochromatography, colloidal gold immunoassay and micro neutralization test and compare differences in result and provide useful approaches to clinical and epidemiological investigation.Methods:The 2019-nCoV IgG/IgM antibody kit (Fluorescent immuno-chromatography) and the 2019-nCoV antibody test kit (Colloidal gold immunoassay) from Guangzhou Wanfu biotechnology Limited by Share Ltd, and the micro neutralization test established by a 2019-nCoV strain isolated by the laboratory in Guangdong Provincial Center for Diseases Control and Prevention were used to detect serum samples of clinically confirmed patients, in the Guangdong Province Second People′s Hospital, China.Results:A total of 113 serum samples from clinically confirmed cases infected with the 2019-nCoV were collected in Guangdong 2 nd People′s Hospital. The median age of the patients was 47.50 (32.00, 57.00) years and the gender ratio was 2.77∶1; The highest neutralizing antibody titer of micro neutralization test was 1∶1 024; Taking the result of micro neutralization test as gold standard, the sensitivity for colloidal gold immunoassay was greater than that of fluorescence immunochromatography (94.74% vs 82.46%), and the Kappa value for colloidal gold immunoassay and fluorescence immunochromatography was 85.84% and 75.24% respectively; at the same time, the negative predictive value and the positive predictive value for them were 94.44%, 91.53% and 83.87%, 92.16% respectively. Conclusions:In the serological method for the detection of the 2019-nCoV infection, the sensitivity and Kappa value for colloidal gold immunoassay were higher than those of fluorescent immunochromatography when the result of micro neutralization test was taken as the gold standard, which was more suitable for rapid detection of cases with the 2019-nCoV infection.

Objective:To establish quality control products for 2019-nCoV detection, and provide reliable control materials for the quality evaluations of the 2019-nCoV detection kit based on fluorescence PCR.Methods:Virus strain was diluted to concentrations of 10 6, 10 5, 10 4, 10 3, 10 2 copies/ml which were used as positive control products. The mean value, standard deviation, coefficient of variability (CV) and tendency of the Ct values were calculated. The homogeneity and stability of the quality control products were tested. Results:Within the concentration gradients, Ct value and concentration of the control products were linearly related. In homogeneity test, the CVs of the quality control products with concentrations of 10 6, 10 5, 10 4, 10 3, 10 2 and 0 copies/ml were 4.60%, 2.67%, 2.22%, 2.04% and 2.50% respectively. In stability test, there was no significant linear trend with extended test time at 4 ℃. Conclusion:Evaluations of homogeneity and stability indicated that the quality control products were established successfully. And the products can be used for evaluation of 2019-nCoV detection kit based on fluorescence PCR.

Objective:To understand the pathogenic spectrum and epidemiological characteristics of febrile respiratory syndrome in Guangdong province, and to provide reference for disease prevention, clinical diagnosis and treatment.Methods:The pathogens of 1 891 nasopharyngeal swabs collected from 2011 to 2018 were detected by real-time fluorescent polymerase chain reaction. The different rates were compared by chi-square test or Bonferronni method of comparison.Results:Among 1 891 samples, 810 samples were positive for viral nucleic acid, with a total detection rate of 42.78%. The main infectious agents were Parainfluenza virus (PIV), Rhinovirus (RhV) and Coronavirus(CoV). The pathogen detection rate was higher in <5 years group and 5~ years group. Pathogen detection in patients with different symptoms showed that the detection rate of Metapneumovirus (MPV) in pneumonia patients was relatively high. Influenza virus A (FluA), PIV, CoV and RhV had higher detection rate in patients without pneumonia. FLuA and Adenovirus (ADV)had a relatively high detection rate in the high and ultra-high fever groups. The detection rate of CoV and Mycoplasma pneumonia(MP) in moderate fever group was relatively high. The detection rate of PIV in cough patients was relatively high. The prevalence of several viruses has strong seasonality, and the detection rate was generally higher in winter and spring. Different virus detection rates also have strong regularity in years.Conclusions:PIV, RhV and CoV are the main pathogens of febrile respiratory syndrome in Guangdong in recent years.

Objective: In this study, phage display technology was used to construct the human anti-Zika virus(ZIKV), phage antibody library and to obtain and express the monoclonal antibody. The aim was to master the preparation and expression of human phage antibody library screening method for highly specific antibodies. Methods: The whole blood samples of Zika patients were collected and the lymphocytes were isolated. The RT-PCR method was used to amplify the antibody light chain and heavy chain Fab gene from lymphocyte Ig mRNA. The pComb3H system was used to construct the gene with genetic diversity Preparation of human anti-ZIKV phage antibody library. The purified antibody library was screened by using the purified ZIKV and the obtained ZIKV E protein antigen. Results: The monoclonal antibody Fab fragment gene was successfully obtained for the ZIKV E protein antigen. The gene can be efficiently expressed in Escherichia coli. Conclusions According to the sequence analysis, this study showed that the monoclonal antibody was a new human genetically engineered antibody against ZIKV, which laid the foundation for the early diagnosis of ZIKV, and obtain a specific monoclonal antibody to ZIKV for human treatment of ZIKV infection.

Objective To investigate the infection status, serotype distribution, drug sensitivity and molecular characteristics of diarrheagenic Escherichia coli (DEC) in patients with diarrhea in Guangdong Province. Methods Fecal samples were collected, cultured and isolated by traditional methods. Suspected Escherichia coli isolates were confirmed by multiplex PCR used for detecting specific virulence genes and bio-chemical methods. Positive strains were serotyped, characterized for drug sensitivity and analyzed by pulsed-field gel electrophoresis ( PFGE). Results The total positive rate of DEC in patients with diarrhea was 6.26%. The positive rates of enteropathogenic Escherichia coli (EPEC), enterotoxigenic Escherichia coli (ETEC), enterohemorrhagic Escherichia coli (EHEC), enteroadherent Escherichia coli (EAEC) and en-teroinvasive Escherichia coli (EIEC) were 2. 47% , 1. 54% , 1. 32% , 0. 62% and 0. 09% , respectively, with infections primarily in children aged 0-<7 years. The total seropositive rate was 52. 54% , with EHEC accounting for 15. 00% . DEC showed high sensitivity to imipenem, ciprofloxacin, ceftazidime and cefo-taxime. The multidrug resistance rate of DEC was 58. 45% , with EPEC being the most serious for multidrug resistance. PFGE results showed that ETEC, EHEC, EPEC and EAEC had a high degree of polymorphism. Conclusion EPEC is the predominant type of DEC circulating in Guangdong Province. Third-generation cephalosporins are the first drugs of choice for treating infections in children. Ciprofloxacin can be used to treat adults. The problem of multiple drug resistance of DEC is severe and efforts to monitor DEC infections and drug resistance should be strengthened.

Objective: To develop a micro-neutralization test for determination of neutralizing antibody against ZIKA virus (ZIKV) in human sera and to verify the acute and convalescent serum samples of 10 ZIKA virus-infected cases diagnosed by nucleic acid detection and/or virus isolation. Methods: ZIKV isolated from ZIKA cases was used to determine micro-neutralization antibody. The virus solution was prepared by infecting BHK21, VERO and VERO-E6 cell lines and viral titer was tested; 100 TCID₅₀ viral solution and 4 times diluted sera which were inactivated at 56 ℃ for 30 min were neutralized, then added the cell suspension and incubated in 5% CO₂ incubator at 37 ℃ for 7 d. The CPE was observed every day. Results: The sensitivity of BHK21, VERO and VERO-E6 was different after infection with ZIKA virus. VERO cell line was the most sensitive and showed typical CPE. VERO cell line was used to establish a micro-neutralization test for determination of neutralizing antibody against ZIKA virus in sera. Conclusions The neutralizing antibody test for zika virus in sera is a special and usefulmethod to diagnose human infection of ZIKV and to conduct population based epidemiological investigation.

Objective: To analyze the etiological of herpangina(HA) in Guangzhou City in 2015, and to provide laboratory data for the epidemic control. Methods: Two hundred and eleven herpangina samples (stool and throat swab) were collected.Real-time (RT)-PCR and semi-nested (Sn)-PCR assays were performed to detect human enteroviruses (HEVs)-positive samples. The human rhabdomyosarcoma (RDa) cell lines were used to inoculate virus from HEVs-positive samples. The entire sequences of viral genes encoding VP1 of CVA6 positive samples or strains were amplified and sequenced. The phylogenetic analysis was performed to analyze the full-length gene sequences encoding VP1 of CVA6 by using DNAStar6.0 and MEGA5.2 software packages. Results: According to the laboratory test results, 115 cases were HEVs-positive and positive rate was 93.50%, eight serotypes of EV including CVA6, CVA10, CVA2, EV71, CVA16, CVB2, Echo14 and Echo30 were detected.The CVA6 positive rate was the highest with a percentage of 60.98%, followed by CVA10 with a percentage of 13.01%. The enterovirus positive rate of stool samples (χ²=29.88, P<0.01) and viruses isolated positive rate (χ²=8.67, P<0.01) were higher than that in throat swab samples. Phylogenetic analysis showed that all CVA6 strains detected in this study belonged to D3 subgenotype, and shared 96.9%-99.9% homologies in nucleotide and 99.0%-100.0% in amino acid. Conclusions CVA6 of the enterovirus A group accounted for the main pathogen of herpangina in Guangzhou City in Guangdong province in 2015, which belonged to D3 subgenotype.