Abstract: Objective To develop an ELISA kit to detect IgG antibodies of Chikungunya virus (CHIKV), providing a new method for epidemiological investigation and detection in the field for CHIKV infection. Methods Using the CHIKV-specific recombinant protein pMal-chik23 as diagnostic antigen, HRP-labeled anti-IgG antibody as color-developing antibody, and the working concentration of diagnostic antigen, serum to be tested and second antibody were optimized using orthogonal. The reaction conditions of ELISA reaction, such as coating, blocking, incubation, and color-developing were systematically optimized. The cut-off value for ELISA detection was established based on the assessment of a large clinical sample set. On this basis, the specificity, sensitivity, and stability of the ELISA response were evaluated to develop and assemble a rapid ELISA kit for the detection of Chikungunya fever IgG antibodies. Results On the basis of systematic conditions optimization, an indirect ELISA kit for the detection of IgG antibodies against CHIKV was developed and assembled. The optimal reaction conditions were identified as 1.0 μg/mL antigen was coated using carbonate buffer at 4 ℃ for 24 hours. Then the microplate was blocked using HBV blocking solution at 37 ℃ for 4 hours. 100 μL/well samples to be tested were diluted at 1∶101, reacted at 37 ℃ for 40 minutes, and washed 4 times with PBST. Thus, HRP-labeled rabbit anti-human IgG was diluted at 1∶20 000, HRP-labeled sheep anti-mouse IgG was diluted at 1∶10 000, reaction at 37 ℃ for 30 minutes, and washed 5 times with PBST. Finally, 100 μL/well TMB solution was added and incubated at 37 ℃ for 10 minutes. Then terminate the reaction with 50 μL of 20% H2SO4 and measure the A450 value at dual wavelengths of 450/630 nm (A450) . The evaluation results showed that ELISA A450 of Chikungunya fever-positive samples were more than 0.43, while the ELISA A450 of negative samples was less than 0.04, and the S/N ratio > 10. Specificity test showed that the developed kit had no cross-reaction with 9 other similar arbovirus species such as Sindbis, Geta, Ross River, and Dengue virus. The stability evaluation of the reagent kit indicated that it had high stability, with a coefficient of variation (CV) within the microplate ranging from 0.76% to 2.12%, the coefficient of variation between the microplate ranged from 0.64% to 1.85%, and the coefficient of variation between batches ranged from 0.83% to 2.31%, all of which were less than 3%. The sensitivity of the kit did not decrease significantly after being stored at 4°C for 1 year. Conclusions A rapid indirect ELISA kit for the detection of Chikungunya fever IgG antibodies was successfully developed, exhibiting good sensitivity, specificity, and stability.

Objective: To analyze the molecular typing characteristics by pulsed field gel electrophoresis (PFGE), drug resistance and virulence genes of Salmonella typhimurium in Longyan city in order to provide reference for the prevention and control. Methods: A total of 79 Salmonella typhimurium strains were isolated from sporadic cases of diarrhea and food poisoning and raw poultry meat samples during 2010 to 2017. PFGE was performed to measure the minimum inhibitory concentrations (MIC) of 15 commonly used drugs against them. PCR was used to detect nine virulence genes (sopB, invA, sifA, sscA, sseE, spvB, pefA, spvR, spvC) in 55 strains. Results: The 79 Salmonella typhimurium strains belonged to 61 PFGE types. There were 10, three and four strains of P1, P3 and P21 types, respectively. Seven P1 type strains were isolate from one food poisoning event. According to the 85% classification standard, 79 Salmonella typhimurium strains could be divided into five predominant gene clusters (G1-G5). Drug susceptibility test showed that the 79 strains had the highest resistance rate to ampicillin (88.61%), followed by that to tetracycline (87.34%) and streptomycin (73.41%). Multidrug resistant bacteria resistant to three or more antibacterial drugs accounted for 84.81% (67/79). All of the 55 strains carried invA, sopB, sseE and sscA genes. The other five genes, sifA, spvC, spvB, spvR and pefA, were detected in 54, 31. 10, 11 and 12 strains, respectively. There were 76.4% (42/55) of the strains carrying five or six virulence genes and all were positive for invA, sopB, sseE, sscA and sifA, and negative for spvB, spvR and pefA. The strains carrying all of the nine virulence genes accounted for 18.2% (10/55). Conclusions Salmonella typhimurium strains isolated in Longyan city had a diverse PFGE type. P1, P3 and P21 types were the three predominant PFGE types. In the food poisoning event, PFGE molecular typing could quickly alert the outbreak and traceability of Salmonella typhimurium. Attention should be paid to the multidrug resistance in Salmonella typhimurium. Monitoring of multidrug-resistant strains and supervision on antibacterial drug usage should be strengthened. Salmonella typhimurium had high virulence as it carried many virulence genes.

Objective To observe the thyroid hormone (TH) changes in patients with acute cerebral infarction in acute phase and convalescence,and to explore its clinical value.Methods Fifty cases of acute cerebral infarction patients from March 2015 to May 2016 in Heilongjiang Provincial Hospital were selected as observation group,at the same time 30 cases of healthy check-up people as control group.Serum levels of triiodothyronine (T3),thyroxine (T4),free triiodothyronine (FT3),free thyroxine (FT4) and thyroid stimulating hormone (TSH) were measured in 50 cases of acute cerebral infarction patients on the 2nd day (acute phase) and the 14th day (recovery) after onset of the disease,by using chemiluminescence method,and 30 cases of people underwent healthy physical check-up were treated the same way.Neurological injury and recovery of patients with cerebral infarction were evaluated using NIHSS.According to the recovery level of FT3,patients with cerebral infarction were divided into low FT3 group (FT3 ＜ 3.10 pmol/L) and normal FT3 group (FT3 ≥ 3.10 pmol/L).Prognosis of the patients was judged according to the NIHSS scores 90 days after discharged from the hospital,and NIHSS score improving acuity of 2 was judged as good prognosis.Results The T3 and FT3 levels in patients with acute cerebral infarction were significantly lower than those of people underwent healthy physical examination,the differences were statistically significant [(0.68 ± 0.22) vs(1.82 ± 0.31) nmol/L,(2.08 ± 0.31) vs (4.19 ± 0.75) pmol/L,all P ＜ 0.05].The T4,FT4 and TSH levels in patients with acute cerebral infarction were increased significantly,the differences were statistically significant [(142.56 ± 20.78) vs (109.89 ± 12.37) nmol/L,(12.88 ± 1.15) vs (9.77 ± 0.96) pmol/L,(5.15 ± 1.16) vs (2.95 ± 1.31) mU/L,all P ＜ 0.05].Compared with the acute phase,convalescence of cerebral infarction patients' serum T3 and FT3 levels [(1.75 ± 0.19) nmol/L,(3.97 ± 0.61) pmol/L] increased significantly,the differences were statistically significant (all P ＜ 0.05),and T4,FT4 and TSH [(115.64 ± 14.38) nmol/L,(10.05 ± 1.02) pmol/L,(3.16 ± 0.98) mU/L] obviously decreased,the differences were statistically significant (all P ＜ 0.05).Compared with control group,convalescence of cerebral infarction patients' serum T3,T4,FT3,FT4 and TSH were not statistically different (all P ＞ 0.05).There was a negative correlation between FT3 levels and NIHSS scores on admission (r =-0.586,P ＜ 0.05).Ninety days after discharge,7 cases (38.89％) of the patients had a good prognosis in low FT3 group,and normal FT3 group had 22 cases (68.75％) of patients with good prognosis,and the difference was statistically significant (x2 =7.186,P ＜ 0.05).Conclusions The thyroid hormone has a protective stress reaction in patients with acute cerebral infarction.The thyroid hormone level has changed significantly in the acute phase,and returned to normal level with improvement of the patients.As a biochemical indicator thyroid hormone detection can be used to estimate the prognosis of patients with acute cerebral infarction.

Objective To analyze the pulse-field gel electrophoresis (PFGE) patterns and antibiotic resistance of Salmonella enteritidisstrains isolated in Longyan city in order to provide reference for effective control, clinical diagnosis and treatment of outbreaks of food-borne diseases.Methods Seventy-seven Salmonella enteritidis strains isolated from food poisoning cases, livestock and poultry meat samples collected for food safety risk monitoring and sporadic cases in 2014 and 2016 were analyzed by PFGE.Antibiotic susceptibility testing was carried out to determine the minimum inhibitory concentrations (MIC) of 16 common antibiotics to 57 strains.Results Seventeen PFGE types were identified among the 77 Salmonella strains including three predominant types (P4, P6 and P8) and were grouped into three major pulsotypes (Cluster1-3) at 90% similarity level.The predominant types causing outbreaks of Salmonella enteritidis infection were P8, P6, P4 and P14, which were responsible for five cases of food poisoning.These isolated strains were highly resistant to nalidixic acid (93%), followed by resistance towards ampicillin (74%) and streptomycin (54%).Forty-one strains were resistant to three or more antibiotics (71.93%).Conclusion The PFGE types of Salmonella enteritidis strains isolated in Longyan city exhibit polymorphism and are clustered into three major pulsotypes.PFGE molecular typing can provide early-warning of sporadic outbreaks and help to identify the sources of infections in Salmonella enteritidis-related food poisoning.Attentions should be paid to multidrug resistant Salmonella enteritidis.Surveillance for multidrug resistant strains and usage of antibiotics should be strengthened.

Objective To explore value of early detection of cardiac troponin I(cTnI) and lactate in evaluating prognosis of emergency elderly patients with severe sepsis and septic shock.Methods A total of 98 elderly patients with severe sepsis oRseptic shock were selected.The patients were divided into survival group and death group according to the prognosis with follow-up of 28 d.The levels of cTnI,lactate,APACHE Ⅱ score and SOFA score were compared between different groups.The correlation analysis was performed between cTnI,lactate and APACHE Ⅱ score,SOFA score.Results The level of lactate and cTnI,APACHE Ⅱ score and SOFA score in septic shock group were significantly higheRthan those in severe sepsis patients,and the level of lactate,cTnI,APACHE Ⅱ score and SOFA score in death group were significantly greateRthan those in survival group (P<0.05).The level of lactate and cTnI showed a certain correlation with APACHE Ⅱ score and SOFA score (P<0.05).The areas undeRthe ROC curve of cTnI and arterial blood lactate were 0.846 and 0.809 respectively.Conclusion The cTnI and arterial blood lactate can evaluate condition and prognosisof emergency elderly patients with severe sepsis and sepsis shock.

Objective To study the value of England National Early Warning Score(NEWS) for the prognosis of elderly patients with severe sepsis and septic shock in emergency department.Methods A total of 116 elderly patients with severe sepsis and septic shock in Xuanwu Hospital of Capital Medical University were selected.Routine physiological and biochemical indicators were collected and NEWS score, APACHEⅡscores and SOFA scores were given to them.The follow-up was lasted for 28 d, and afterwards the patients were divided into survival group and death group according to the prognosis.The NEWS scores, APACHEⅡ scores and SOFA scores of two groups were compared.And above indicators were also compared between the severe sepsis and septic shock patients.Meanwhile, the correlation between NEWS scores, and APACHEⅡ scores, SOFA scores were analyzed.The value of NEWS scores for the prognosis of elderly patients with severe sepsis and septic shock was predicted by the area under the ROC curve(AUC).Results The NEWS scores, and APACHEⅡ scores, and SOFA scores in septic shock group were higher than that in severe sepsis group, and the death group was higher than survival group(P<0.05).NEWS scores showed a correlation with APACHEII scores and SOFA scores(P<0.05).The areas under the ROC curve of NEWS scores, APACHEII scores and SOFA scores were 0.870, 0.880, 0.865, respectively.Conclusion NEWS scores could evaluate the condition and prognosis of elderly patients with severe sepsis and sepsis shock, and the consistently higher scores of NEWS is associated with poorer prognosis.

Objective To explore value of early detection of cardiac troponin I(cTnI) and lactate in evaluating prognosis of emergency elderly patients with severe sepsis and septic shock.Methods A total of 98 elderly patients with severe sepsis oRseptic shock were selected.The patients were divided into survival group and death group according to the prognosis with follow-up of 28 d.The levels of cTnI,lactate,APACHE Ⅱ score and SOFA score were compared between different groups.The correlation analysis was performed between cTnI,lactate and APACHE Ⅱ score,SOFA score.Results The level of lactate and cTnI,APACHE Ⅱ score and SOFA score in septic shock group were significantly higheRthan those in severe sepsis patients,and the level of lactate,cTnI,APACHE Ⅱ score and SOFA score in death group were significantly greateRthan those in survival group (P<0.05).The level of lactate and cTnI showed a certain correlation with APACHE Ⅱ score and SOFA score (P<0.05).The areas undeRthe ROC curve of cTnI and arterial blood lactate were 0.846 and 0.809 respectively.Conclusion The cTnI and arterial blood lactate can evaluate condition and prognosisof emergency elderly patients with severe sepsis and sepsis shock.

Objective To study the value of England National Early Warning Score(NEWS) for the prognosis of elderly patients with severe sepsis and septic shock in emergency department.Methods A total of 116 elderly patients with severe sepsis and septic shock in Xuanwu Hospital of Capital Medical University were selected.Routine physiological and biochemical indicators were collected and NEWS score, APACHEⅡscores and SOFA scores were given to them.The follow-up was lasted for 28 d, and afterwards the patients were divided into survival group and death group according to the prognosis.The NEWS scores, APACHEⅡ scores and SOFA scores of two groups were compared.And above indicators were also compared between the severe sepsis and septic shock patients.Meanwhile, the correlation between NEWS scores, and APACHEⅡ scores, SOFA scores were analyzed.The value of NEWS scores for the prognosis of elderly patients with severe sepsis and septic shock was predicted by the area under the ROC curve(AUC).Results The NEWS scores, and APACHEⅡ scores, and SOFA scores in septic shock group were higher than that in severe sepsis group, and the death group was higher than survival group(P<0.05).NEWS scores showed a correlation with APACHEII scores and SOFA scores(P<0.05).The areas under the ROC curve of NEWS scores, APACHEII scores and SOFA scores were 0.870, 0.880, 0.865, respectively.Conclusion NEWS scores could evaluate the condition and prognosis of elderly patients with severe sepsis and sepsis shock, and the consistently higher scores of NEWS is associated with poorer prognosis.

Objective To analyze the genetic characteristics of echovirus type 25( Echo25) strains isolated from patients with viral encephalitis in Longyan city. Methods Cerebrospinal fluid(CSF) specimens were collected from hospitalized patients with viral encephalitis or central nervous system infection in Longyan,2012. Enteroviruses(EV)were isolated from the specimens and then identified. Four strains of Echo25 were screened out by using serum neutralization test. Coding sequence of the VP1 region of the 4 Echo25 strains were amplified by RT-PCR and then sequenced. A phylogenetic tree was constructed to ana-lyze the nucleotide sequence homology between those sequences and the sequences of reference Echo25 strains available in the GenBank database. Results The VP1 nucleotide sequences of Echo25 strains isola-ted in Longyan were 498 bp in length,encoding 166 amino acid residues. The homology analysis showed that the VP1 nucleotide sequences of 3 strains were identical,sharing 97% homology in nucleotide with the rest strain. The Echo25 strains isolated in Longyan were highly similar to KJ957190( Beijing,2010)and HM031189(Henan,2008)strains. Phylogenetic analysis revealed that the Echo25 strains isolated in Longyan belonged to genotype B1. Conclusion Echo25 was one of the pathogens causing viral encephalitis in Longyan in 2012 and different transmission chains of Echo25 had emerged. This study indicates that it is necessary to strengthen the surveillance for EV and understand the genetic variation of the Echo25 for provi-ding better supportive evidences for the prevention and control of related diseases.