Objective:To analyze the clinical characteristics of dengue fever patients, summarize the course and characteristics of the disease, and analyze the risk factors that affect the condition.Methods:Retrospective collection of general information, clinical symptoms, medical history, laboratory tests, prognosis and other clinical data of dengue fever patients that admitted to Jinghong First People's Hospital and severe dengue fever patients at People's Hospital of Xishuangbanna Dai Autonomous Prefecture from June to December 2023 was conducted using a case report form (CRF). According to the diagnostic criteria of the World Health Organization (WHO), patients were divided into dengue fever group, dengue fever with warning signs group, and severe dengue fever group. The differences in clinical data between different groups of patients were analyzed and compared. Binary multiple factor Logistic regression analysis was used to explore the risk factors affecting the severity of dengue fever in patients. Receiver operator characteristic curve (ROC curve) was drawn to analyze the predictive value of prediction models constructed for various risk factors for severe dengue fever. Subgroup analysis was performed on the prognosis of severe dengue fever patients, and the differences in clinical data between two groups of patients with different prognoses were compared. Binary multivariate Logistic regression analysis was used to explore the risk factors affecting the prognosis of severe dengue fever patients. ROC curve was drawn to analyze the predictive value of prediction models constructed for various risk factors on the prognosis of severe dengue fever patients.Results:A total of 2 264 patients were included, including 499 cases in the dengue fever group, 1 379 cases in the dengue fever with warning signs group, and 386 in the severe dengue fever group (43 deaths and 343 survivors). The most common symptom of dengue fever patients was fever (94.70%), followed by muscle soreness (70.54%), headache (63.12%), fatigue (58.92%), and chills (46.02%). Compared with the dengue fever group and the dengue fever with warning signs group, the ratio of thalassemia and the levels of cardiac troponin (cTnI, cTnT), MB isoenzyme of creatine kinase (CK-MB), and myoglobin were significantly increased in patients with severe dengue fever group, albumin (Alb) was significantly decreased in patients with severe dengue fever group. The levels of cTnT and myoglobin in patients with dengue fever with warning signs group were significantly higher than those in the dengue fever group, and the level of Alb in patients with dengue fever with warning signs group was significantly lower than that in the dengue fever group, the differences were statistically significant (all P < 0.05). Binary multivariate Logistic regression analysis showed that thalassemia [odds ratio ( OR) = 6.214, 95% confidence interval (95% CI) was 2.337-16.524, P < 0.001], Alb ≤ 36 g/L ( OR = 6.297, 95% CI was 4.270-9.286, P < 0.001), and cTnT levels ( OR = 1.008, 95% CI was 1.002-1.015, P = 0.016) were risk factors for severe dengue fever. ROC curve analysis showed that the area under the ROC curve (AUC) for predicting severe dengue fever based on the prediction models constructed for the above risk factors was 0.856, with the best predictive value of 0.067, sensitivity of 67.1%, and specificity of 99.4%. In the subgroup analysis of patients with severe dengue fever, compared with the survival group, the levels of hematocrit (HCT), cTnT, and CK-MB in the death group patients were significantly increased, while the level of Alb was significantly decreased, and the differences were statistically significant. Binary multivariate Logistic regression analysis showed that Alb ( OR = 0.839, 95% CI was 0.755-0.932, P = 0.001), HCT ( OR = 1.086, 95% CI was 1.010-1.168, P = 0.025), elevated troponin level ( OR = 10.119, 95% CI was 2.596-39.440, P < 0.001), and CK-MB ( OR = 1.081, 95% CI was 1.032-1.133, P < 0.001) were risk factors for mortality in patients with severe dengue fever. ROC curve analysis showed that the AUC for predicting death in severe dengue fever patients based on the prediction models constructed for the above risk factors was 0.881, with the best predictive value of 0.113, sensitivity of 75.0%, and specificity of 88.9%. Conclusion:Thalassemia, Alb≤36 g/L, and cTnT level are risk factors for severe dengue fever, while HCT level, Alb level, CK-MB level, and elevated troponin level are risk factors for death in patients with severe dengue fever.

Objective:To analyze the biological characteristics, phylogeny and the taxonomic status of strain 7712 (=CGMCC 1.17031=NBRC 113783=KCTC 15766) isolated from a clinical blood sample.Methods:Strain 7712 was identified by the cultural properties, cellular and colonial morphology, physiological and biochemical reactions, matrix-assisted laser desorption ionization time-of-flight mass spectrometry system, and genome correlation index analysis. The genomic phylogenetic tree was construct to analyze the taxonomic position. The virulence factors and resistance genes of strain 7712 and related strains were then compared by the online virulence factor database and online comprehensive antibiotic research database respectively.Results:Strain 7712 was urease negative, gram-negative nonfermenters, which was identified as Ochrobactrum anthropi by VITEK GN card. The 16S rRNA gene analysis showed that the strain was closely related to the members of genera Ochrobactrum and Brucella. The phylogenetic tree showed that strain 7712 was clustered together with Ochrobactrum teleogrylli LCB8 T and Ochrobactrum haematophilum CCUG 38531 T, along with genus Brucella and other Ochrobactrum species. The genome relatedness indexes analysis showed that the average nucleotide identity between strain 7712 and Ochrobactrum teleogrylli LCB8 T was 98.16%, which was higher than the threshold for prokaryotic species. Genetic prediction showed that strain 7712 carried several virulence-related genes and resistance-related genes, of which the existence of OCH gene might be responsible to the resistance to cephalosporin. Conclusions:A case of human infection caused by Ochrobactrum teleogrylli is identified, which would help promote the understanding of biodiversity of genus Ochrobactrum.

Objective:To construct an evaluation system for clinical thinking ability of general practitioners in the treatment of multimorbidity.Methods:This was a cross-sectional study. The draft of evaluation indexes for clinical thinking ability of general practitioners in treatment of multimorbidity was preliminary developed through literature review, collation, analysis and discussion. Nineteen clinical and teaching experts of general practice were selected for consultation via anonymous convenient sampling. From January to June 2022, 2 rounds of expert consultation were conducted using the Delphi method. During the first round of consultation, according to the survey feedback, we modified and improved the evaluation system of general practitioners′ clinical thinking ability for multi-disease co-treatment. During the second round, experts were asked to assess the importance of each index, and to calculate the weight of each index accordingly. Questionnaires were sent to experts via letters. The content of the questionnaires encompasses the basic information of experts, evaluation for various indexes and relevant opinions. The mean value of importance assignment ≥3.5, coefficient of variation ≤0.25 and the full score frequency ≥30% were taken as the criteria. Indexes unsatisfying the criteria were removed, so that the final index system could be constructed.Results:The average age of 19 experts was 50.2 years old, 9 of them were male. A total of 2 rounds of expert consultation were conducted, 19 questionnaires were issued in each round, and 19 effective questionnaires were received afterwards. In the first round of consultation, 10 experts put forward revised opinions, and some indexes were adjusted according to the definition criteria and the discussion of the research group. In the second round of consultation, 3 experts put forward suggestions for modification. According to the definition criteria, no need to delete the indexes. After discussion by the research group, some indexes were adjusted, and finally an evaluation system of clinical thinking ability for multi-disease co-treatment of general practitioners was established, including 4 first-level indexes and 30 second-level indexes. The weights of the 4 first-level indexes in descending order were "overall thinking ability" (38.01%), "diagnostic thinking ability" (33.96%), "evidence-based thinking ability" (14.75%), and "critical thinking ability" (13.28%). Among the 30 secondary indexes, the top 5 were "ability to identify and handle priority emergency incidents" (5.04%), "risk assessment and critical illness identification ability" (4.63%), "emergency referral ability" (4.61%), "communication and expression ability" (4.57%), and "standardized diagnosis and treatment ability" (4.23%).Conclusion:This study successfully constructed an evaluation system for clinical thinking ability of general practitioners in the treatment of multimorbidity.

Objective:To identify and characterize one Spiroplasma strain (designated as DGKH1) isolated from the blood of a patient with sepsis. Methods:The traditional bacterial culture, staining, morphological observation, physiological and biochemical identification, 16S rRNA gene sequencing, phylogenetic analysis, genome sequencing, and the genome-related index analysis were performed to accurately determine the taxonomic status of the strain DGKH1. Antibiotic susceptibility testing was performed using a specific kit for culturing and testing Ureaplasma urealyticum/ Metamycoplasma hominis. Results:The strain DGKH1 could weakly grow on Columbia blood agar, chocolate agar, and Haemophilus chocolate 2 agar. However, it did not grow in liquid culture medium containing tetracycline (4 μg/ml), doxycycline (1 μg/ml), minocycline (1 μg/ml), josamycin (2 μg/ml), roxithromycin (1 μg/ml), clarithromycin (1 μg/ml), or telithromycin (1 μg/ml). DGKH1 resembling Metamycoplasma hominis formed "fried egg-like colonies" on Mycoplasma solid culture medium. DGKH1 could not be stained by Gram staining. When observed under transmission electron microscopy (TEM) using phosphate buffer as the matrix, the bacteria were spiral-shaped. Results of 16S rRNA gene sequence alignment showed that DGKH1 was highly similar (99.85%) to Spiroplasma eriocheiris CCTCC M 207170 T. However, the urea decomposition test was positive, which was different from all of the known Spiroplasma species. The phylogenetic analysis based on whole genome showed that DGKH1 was clustered in a small branch along with Spiroplasma eriocheiris CCTCC M 207170 T. However, the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the two strains were 94.14% and 56.00%, respectively, both below the threshold for prokaryotic species identification. Conclusions:DGKH1 represented a potential new species of genus Spiroplasma, closely related to Spiroplasma eriocheiris. Some microbiological characteristics of DGKH1 were similar to Mycoplasmas. However, the natural host and epidemiological data of DGKH1 need to be further studied.

Objective:To analyze the molecular epidemiological characteristics of Campylobacter fetus subsp. testudinum ( Cft). Methods:Fifteen strains of Cft collected in our laboratory from 2010 to 2022 were subjected to whole-genome sequencing. Their epidemiological characteristics were analyzed based on the global genome data of Cft on GenBank database. MLST-GrapeTree software was used to obtain the genetic structure of Cft strains. A phylogenetic tree was constructed using core-genome single nucleotide polymorphism (cgSNP) analysis, and the sequence clusters were identified using rhierBAPS. Virulence genes and drug resistance genes of Cft strains were annotated using CARD, ResFinder and VFDB database. Their susceptibility to antibiotics was tested using E-test method and the results were analyzed using the CLSI-M45 sensitivity standard for Campylobacter jejuni/ Campylobacter coli. Results:Based on average nucleotide identity (ANI) analysis, the genome data of 41 Cft strains including 24 isolated from human, 13 from animals and four of unknown sources were collected from GenBank database. Among the 24 human-derived strains, 20 were linked to Asian descent and only one was linked to Caucasian descent (spouse of Asian descent), showing statistically significant differences in human ethnicity. All of the 13 animal-derived strains were originated from reptilian sources, including six from turtles, four from snakes and three from lizards. MLST revealed that ST46 was the predominant ST in China, while ST15 was the major sequence type in the United States. Grapetree analysis also demonstrated that the genetic diversity in China was greater than that in the United States. The phylogenetic tree constructed based on cgSNP and BAPS identified six distinct sequence clusters. The Chinese isolates were scattered in diverse sequence clusters and closely related to animal-derived strains, while the American isolates mainly belonged to ST15. The genes encoding virulence factors such as flagella, glycosylation systems and adhesins were carried by all of the 41 Cft strains (100.00%). The invasion-related virulence genes, such as the genes encoding the IV type secretion system ( virB4, virB9, virD4) and the resistance-related tetO efflux pump gene were specifically identified in the emerging ST74 clones. In vitro drug susceptibility testing of 15 Chinese isolates revealed 46.67% of the Cft strains were resistant to ciprofloxacin and 100.00% were sensitive to erythromycin. Conclusions:The global sequence clusters of Cft isolates showed a great genetic diversity. Most of the people with Cft infection had basic immune diseases and might have eaten or had contact with reptiles. Notably, the Chinese domestic infection of ST46 and the emerging ST74 should arouse our more attention.

Objective:To study the molecular phylogeny and virulence gene profile of Francisella salimarina. Methods:Phylogenetic analysis of Francisella salimarina was performed based on the global genome data of related Francisella species on GenBank database. The consistency in phylogenetic analysis based on single marker genes (such as 16S rRNA gene, rpoB gene and mdh gene) and the core genome as compared. Virulence genes and antibiotic resistance genes were annotated using the virulence factor database (VFDB) and the Comprehensive Antibiotic Resistance Database (CARD), respectively. The virulence of Francisella salimarina was analyzed with a Galleria mellonella (greater wax moth) infection model using Francisella philomiragia ATCC 25015 T as reference strain. Results:The phylogenetic analysis revealed that Francisella salimarina was closely related to Francisella philomiragia. The phylogenetic tree based on mdh gene was highly similar to that based on the core genome. Francisella salimarina could be differentiated from other related species by 16S rRNA gene or mdh gene, with the latter being more accurate. Eight Francisella salimarina strains carried multiple virulence genes, mainly involved in secretion, adhesion, immune regulation, motility and stress survival. Moreover, beta-lactam resistance gene blaFPH was identified in all eight strains. Francisella salimarina showed high lethality in the Galleria mellonella infection model, which was similar to Francisella philomiragia ATCC 25015 T. Conclusions:Francisella salimarina was a rare pathogen with similar pathogenicity to Francisella philomiragia. The mdh gene could be used as a molecular target for rapid identification of Francisella salimarina.

Objective: To investigate the epidemiological characteristics of clusters of hand, foot and mouth disease (HFMD) in kindergartens and schools in Jinshan District, Shanghai Municipality from 2016 to 2021, so as to provide insights into improving the prevention and control measurements of HFMD in Jinshan District. Methods: Data of HFMD cases in Jinshan District from 2016 to 2021 were collected through Chinese Disease Prevention and Control Information System, and data pertaining to HFMD clusters in kindergartens and schools were also collected. The scale, temporal distribution, regional distribution and distribution of cluster places were descriptively analyzed. Results: Totally 338 HFMD clusters involving 974 cases were identified in kindergartens and schools in Jinshan District from 2016 to 2021, with an average attack rate of 9.89%. The number of cases in each cluster ranged from 2 to 12 cases, with a median number of 2 (interquartile range, 1) cases, and there were 223 clusters involving 2 cases, accounting for 65.98%. The duration of clusters ranged from 1 to 16 days, with a median duration of 4 (interquartile range, 3) days. HFMD peaked from April to June (136 clusters, 40.24%) and from September to December (176 clusters, 52.07%). All the 11 streets and towns (high-tech zones) were reported HFMD clusters, and the three largest number of clusters were reported in Zhujing Town (72 clusters, 21.30%), Shanyang Town (63 clusters, 18.64%) and Tinglin Town (40 clusters, 11.83%). There were 268 HFMD clusters in kindergartens (79.29%) and 70 in schools (20.71%), and the prevalence of HFMD clusters was higher in kindergartens than in schools (35.51% vs. 17.03%; χ2=31.507, P<0.001). Conclusions HFMD clusters in kindergartens and schools showed seasonal characteristics from 2016 to 2021 in Jinshan District, which predominantly occurred in Zhujing Town, Shanyang Town and Tinglin Town, and kindergartens were the main places.

Objective:To identify a pathogenic strain JM-1 isolated from the pus of a patient stabbed by a sea shrimp and to analyze its antibiotic susceptibility and virulence genes, aiming to provide reference for screening clinically related infections caused by Cysteiniphilum litorale as a rare pathogen and improving prognosis. Methods:Biochemical phenotype identification, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), 16S rRNA gene sequencing, analysis of average nucleotide identity (ANI) and average amino acid identity (AAI) based on the whole genome and phylogenetic analysis of the 16S rRNA gene and the whole genome were performed to accurately determine the taxonomic status of the strain JM-1. E-test was used to detect antibiotic susceptibility, and the results were interpreted according to the interpretation standards of Francisella tularensis in CLSI M45-A3. The virulence factor database (VFDB) was used for genome-wide annotation and analysis of virulence genes. Results:After culturing the strain JM-1 on the Columbia blood plate for 3 d, some grey-white, medium-sized, smooth, round and convex hemolytic colonies were observed. Gram staining result showed lightly colored Gram-negative Coccobacillus. API NH identification results suggested that the isolate JM-1 was Moraxella catarrhalis (biochemical code: 3010), while there was no identification result in Vitek2 system NH card (biochemical code: 0211002121). The EXS3000 mass spectrometer self-built database identified the isolate JM-1 as Cysteiniphilum litorale. The phylogenetic analysis based on the 16S rRNA gene and the whole genome showed that the isolate JM-1 and Cysteiniphilum litorale DSM 101832 T clustered into the same branch, and the ANI and AAI values between the two strains were 95.07% and 95.65%, respectively. The biochemical phenotype identification indicated the isolate JM-1 producing β-lactamase and penicillinase. Antibiotic susceptibility test results showed the strain was resistant to penicillin and sensitive to gentamicin, streptomycin, doxycycline, tetracycline, ciprofloxacin, levofloxacin, and chloramphenicol. Genome annotation suggested the virulence genes of the isolate JM-1 were similar to those of Francisella, including Francisella pathogenicity island (FPI), type Ⅳ fimbriae, capsule and lipopolysaccharide. Conclusions:Cysteiniphilum litorale was a rare pathogen with virulence genes similar to those of Francisella, and its antibiotic susceptibility was also similar to that of Francisella. This study confirmed a case of clinical infection caused by Cysteiniphilum litorale. The self-built MALDI-TOF MS system could be used for its rapid identification.

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), which causes great economic losses. At the moment, no effective neutralizing antibody is available for scientific research and treatment. Therefore, developing a method for screening the neutralizing monoclonal antibodies is of great significance for the prevention and treatment of PRRSV and the screening of antigen sites. Monoclonal antibodies have been widely used in the treatment and diagnosis of many human and animal diseases. Therefore, screening effective neutralizing antibodies for different pathogens is an urgent task. Among the methods for monoclonal antibody screening, B cell immortalization is an effective method to obtain neutralizing monoclonal antibody. Specifically, in this study, the bcl-6 and bcl-xl genes were connected by f2a and then the yielded product was ligated to a vector for retrovirus packaging. The swine lymphocytes immunized with PRRSV were infected the yielded mature viruses and cultured in the complete medium containing CD40L and IL21 cytokines. Then, CD21 was used as the marker to screen B cells with the magnetic bead method. Finally, monoclonal B cells were obtained and the secretion of antibodies was tested. The results showed that the plasmid, either being transfected alone or with the packaged plasmids, could be expressed, and that the packaged retrovirus could infect the cells. Moreover, the infected lymphocytes secreted antibodies, so did the screened B cells. Therefore, the method for screening monoclonal antibody against PRRSV was successfully established.
Animals ; Antibodies, Monoclonal ; Antibodies, Neutralizing ; Antibodies, Viral ; Humans ; Porcine Reproductive and Respiratory Syndrome/prevention & control* ; Porcine respiratory and reproductive syndrome virus/genetics* ; Swine

Antigenic purity is important for quality control of the foot-and-mouth (FMD) whole virus inactivated vaccine. The recommended method for evaluation the antigenic purity of FMD vaccine is to check the serum conversion to non-structural protein (NSP) 3AB antibody after 2 to 3 times inoculation of animals with inactivated vaccine. In this study, we developed a quantitative ELISA to detect the amount of residual 3AB in vaccine antigen, to provide a reference to evaluate the antigenic purity of FMD vaccine. Monoclonal antibody (Mab) of NSP 3A and HRP-conjugated Mab of NSP 3B were used to establish a sandwich ELISA to quantify the NSP 3AB in vaccine antigen of FMD. Purified NSP 3AB expressed in Escherichia coli was serially diluted and detected to draw the standard curve. The detectable limit was determined to be the lowest concentration of standard where the ratio of its OD value to OD blank well was not less than 2.0. Results: The OD value was linearly corelated with the concentration of 3AB protein within the range between 4.7 and 600 ng/mL. The correlation coefficient R² is greater than 0.99, and the lowest detectable limit is 4.7 ng/mL. The amount of 3AB protein in non-purified inactivated virus antigen was detected between 9.3 and 200 ng/mL depending on the 12 different virus strains, whereas the amount of 3AB in purified virus antigen was below the lowest detectable limit. The amount of 3AB in 9 batches of commercial FMD vaccine antigens was between 9.0 and 74 ng/mL, whereas it was below the detectable limit in other 24 batches of commercial vaccine antigens. Conclusion: the sandwich ELISA established in this study is specific and sensitive to detect the content of 3AB protein in vaccine antigen of FMD, which will be a useful method for evaluation of the antigenic purity and quality control of FMD inactivated vaccine.
Animals ; Antibodies, Viral ; Enzyme-Linked Immunosorbent Assay ; Foot-and-Mouth Disease/prevention & control* ; Foot-and-Mouth Disease Virus ; Viral Nonstructural Proteins/genetics* ; Viral Vaccines