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Objective:To investigate the distribution of pathogen species isolated from cerebrospinal fluid culture (CSF) in children and analyze the antibiotic-resistance of the main isolates in vitro, which provides reference for interpreting the pathogens and choosing antibiotics in empiric therapy for pediatric patients. Methods:The results of cerebrospinal fluid culture were collected by checking laboratory information system of the Children′s Hospital of Zhejiang University and the clinical characteristics of these children were analyzed retrospectively by checking electronic medical record system.Results:A total of 1 312 isolates were detected, including 1 294 isolates of bacteria and 18 isolates of fungi. A total of 497 (37.9%) isolates were pathogenic microorganisms, of which 288 (57.9%) isolates were gram-positive, 200 (40.3%) isolates were gram-negative, and 9 (1.8%) isolates were fungi. The top 5 pathogens were Escherichia coli (102 isolates, 20.5%), Streptococcus pneumoniae (64 isolates, 12.9%), Streptococcus agalactiae (52 isolates, 10.5%), Enterococcus faecium (33 isolates, 6.6%) and Staphylococcus aureus (28 isolates, 5.6%). Most of the Streptococcus pneumoniae strains were isolated from children more than 1 year old (76.6%, 49/64), while the other top 4 bacteria were mainly isolated from infants less than 1 year old, with the rate of 95.1%(97/102) for Escherichia coli, 98.1%(51/52) for Streptococcus agalactiae, 81.8%(27/33) for Enterococcus faecium and 71.4% (20/28) for Staphylococcus aureus. A total of 815 (62.1%) isolates were considered to be contaminated pathogens according to the analysis on clinical manifestations and other laboratory findings in CSF, and coagulase-negative Staphylococcus (680 isolates), Micrococcus (50 isolates), Corynebacterium (28 isolates) and Enterococcus faecium (23 isolates), which accounted for 41.1% (23/56) of the total detected Enterococcus faecium, were the top 4 contaminated bacteria. During the study period, the isolation rate of the pathogenic microorganisms increased year by year (χ2=34.84, P<0.001), while the isolation rate of the contaminated pathogens, which detected mainly in summer and autumn, decreased year by year (χ2=13.26, P<0.001). Conclusions:The predominant bacteria causing pediatric purulent meningitis were Escherichia coli, Streptococcus pneumoniae, Streptococcus agalactiae, Enterococcus faecium and Staphylococcus aureus. Coagulase-negative Staphylococcus, Micrococcus, Corynebacterium and Enterococcus faecium were common contaminated bacteria in CSF culture, therefore clinicians should interpret the results of CSF culture cautiously according to the bacterial species and clinical manifestations.
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Objective:To study the drug resistance patterns of Bordetella pertussis in vitro, and to know the clinical characteristics of pediatric pertussis and evaluation the treatment outcomes, which may provide references for experiential diagnosis and treatment of this disease. Methods:Nasopharyngeal swabs of the hospitalized children with suspected pertussis in Children′s Hospital, Zhejiang University School of Medicine in 2017 were collected for culture. And the clinical data of the children were collected. The strains were identified by pertussis-specific antiserum agglutination and finally confirmed by mass spectrometry. The drug sensitivity test was performed using the E-test method. The efficacy of therapy with antibiotic was evaluated after two weeks of treatment. Statistical analysis was performed with Mann-Whitney U test and chi-square test. Results:Of 1 029 children, 211 (20.5%) nasopharyngeal swabs were positive for Bordetella pertussis culture, and the isolation rate of the specimens was highest (31.2%, 45/144) in July. Of the 211 pertussis patients, 105 (49.8%) were male and the age were 3.8 (2.2, 6.9) months, 114 (54.0%) were not vaccinated with pertussis diphtheria tetanus mixed vaccine and 192 (91.0%) were prescribed with previous antibiotics. There were 142 (67.3%) children from families with two or more than two children, and 136 (95.8%) of which were the youngest siblings. One hundred and fifty-nine (75.4%) patients had paroxysmal cough and 61 (28.9%) had whooping. The white blood cell counts were higher than 20×10 9/L in 94 (44.5%) patients, and the lymphocyte counts were higher than 10×10 9/L in 97 (46.0%) of patients. The drug susceptibility results showed that 138 (65.4%) strains were against erythromycin, azithromycin and clindamycin with minimum inhibitory concentration (MIC)>256.000 mg/L. The MIC 90 of the isolates to ampicillin, ceftriaxone, cefoperazone/sulbactam, meropenem and trimethoprim/sulfamethoxazole were 0.190 mg/L, 0.190 mg/L, 0.094 mg/L, 0.094 mg/L and 0.750 mg/L, respectively. All strains had a MIC of <0.016 mg/L for piperacillin/tazobactam. After treatment, symptoms were improved in 195(92.4%) patients when they were discharged from hospital. Seventy-six (57.1%) children whose symptoms did not improve after seven-day treatment with macrolides, were prescribed with other antibiotics or other antibiotic with macrolides in combination. Compared with the patients treated with macrolides, more patients treated with cefoperazone/sulbactam or piperacillin/tazobactam had negative nasopharyngeal culture results after two weeks of therapy (46/48(95.8%) vs 46/57(80.7%)), or on day seven (45/46(97.8%) vs 39/47(83.0%)) and on day 14 (45/45(100.0%) vs 41/47(87.2%)) since discharged. The differences were all statistically significant ( χ2=5.50, 5.86 and 6.15, respectively, P=0.019, 0.015 and 0.013, respectively). Conclusions:The majority of children with pertussis do not have whooping, and the resistant rate of Bordetella pertussis to macrolides is high. Further study is needed to evaluate the feasibility and reasonability of cefoperazone/sulbactam and piperacillin/tazobactam in treating pediatric pertussis caused by macrolides-resistant Bordetella pertussis.
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Objective To construct the engineering bacteria expressing the recombinant human Kunitz protease inhibitor domain of amyloid protein precursor variant (rhKD/APPvar)in Pichia pastoris,and to establish the methods suitable for large-scale fermentation and purification of rhKD/APPvar.Methods The rhKD/APPvar expression vector was constructed based on the rhKD/APPvar-pPICZαexpression vector. Two restriction enzyme loci (ApaⅠ and SacⅡ)were added to two flanks of KD/APP and human KD/APP activity center RAM was replaced by the active site of BPTI KAR.After the rhKD/APPvar-pPICZαexpression vector was transformed into Pichiapastoris,optimized expression and purification of rhKD/APPvar was performed.The rhKD/APPvar was purified with cation exchange chromatography and desalting.Results The results of digestion identification and DNA sequencing analysis demonstrated that the recombinant plasmid rhKD/APPvar-pPICZα was successfully constructed and transfected into pastoris X-33. The SDS-PAGE analysis results indicated that rhKD/APPvar expressed after the induction of methanol and the relative molecular weight was 6 700.After a series of experiments the optimal expression conditions of rhKD/APPvar were obtained as follows:the optimal pH was 6.0 and the optimal induction time point was about the 5 th day for the strain.After purified the purity of rhKD/APPvar was about 95%.Conclusion KD/APPvar-pPICZ is successfully constructed;after expression in Pichia pastoris and purification,the rhKD/APPvar protein is obtained.
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Objective To evaluate the efficacy of intravenous immunoglobulin (IVIG) in the treatment of severe EV71 infection in children.Methods The clinical data of children with severe EV71 infection,who admitted in our hospital from January 2010 to November 2012 were retrospectively analyzed.According to the time of admission and treatment principles,the patients were divided into the 2010 group and the 2012 group.The patients of the 2010 group routinely received IVIG treatment,but patients in the 2012 group received IVIG treatment in serious condition.The obvious effective rate,effective rate,ineffective rate,the incidence rate of critically ill and mortality of the two groups were compared.Results There were no significant differences in the obvious effective rate (86.0％ and 85.2％),effective rate (9.6％ and 11.8％),ineffective rate (4.4％ and 3.0％),the incidence rate of critically ill (4.0％ and 2.2％) between the two groups (x2 =0.011,1.269,1.657,3.304,all P ＞ 0.05).The mortality of the 2010 group (2.8％) was higher than that of the 2012 group (0.6％)(x2 =8.213,P ＜0.05).Conclusion IVIG has no effect on patients with severe EV71 infection,and is not recommended.
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Objective To investigate the clinical feature, treatment and prognosis of severe hand-footmouth disease without typical skin lesions. Methods Clinical data from 24 patients with severe hand-footmouth disease without typical skin lesions collected from January 2010 to May 2010 were retrospectively analyzed.Results There were 17 males and 7 females among the 24 patients. Of them, 3 patients with positive EV71 showed no skin lesions at the first visit, 2 presented with only herpes of mouth, 3 with only skin rashes in gluteal regions, 5 with only skin rashes on the palms, 2 with only skin rashes of the knees, 9 with skin rashes in hands, feet and gluteal regions simultaneously. The skin rashes were sparse with an atypical distribution.Fever occurred in all the 24 patients and lasted 6 - 7 days. Nervous system was involved in all the patients,and pulmonary hemorrhage occurred in 4 patients. RT-PCR and real-time PCR showed that the pharyngeal swab was positive for EV71 in 13 patients, for CoxA16 in 2, for other enterovirus in 3, and feces samples were positive for EV71 in 9 patients, for CoxA16 in 1, for other enterovirus in 2. Five patients were positive for EV71 in both pharyngeal swab and feces samples. Four patients died of multiple organ failure, the other 20 patients were cured and discharged from hospital. Conclusions The major pathogen causing severe hand-footmouth disease is EV71 in the 24 patients; children under 2 years are liable to this disease; high fever is common with various rashes. Early recognition of atypical skin lesions and timely management are most important for the control of severe hand-foot-mouth disease and improvement in overall survival of patients with this entity.
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Objective To investigate the clinical and laboratory features of severe cases of hand,foot and mouth disease (HFMD) in Wenzhou, Zhejiang Province. Methods Clinical data of 107 children with HFMD, including 97 severe and 10 critical cases treated in Children' s Hospital Affiliated to Wenzhou Medical College during January and May 2010 were retrospectively analyzed. One hundred and fifty children with mild HFMD were also selected as the controls. Clinical features and laboratory results were compared between the two groups. Results Fever, rash and infection in central nervous system were observed in all patients with severe HFMD, and symptoms on respiratory, digestive and cardiovascular systems were more serious than those of mild HFMD cases. White blood cell counts (WBC) were higher in severe group than those in controls (t = 12.72, P ＜0.01). Hyperglycemia (9. 2 mmol/L) and abnormal troponin (0. 3 -9. 0 ng/mL) were presented in all the critical patients. Cerebrospinal fluid WBC counts were raised in 97 severe HFMD patients (98.5 × 106/L for average) with predominance of lymphocytes. Among 107 severe patients, EV71 was positive in 70, including all 10 critical cases. Conclusion Involvement of nervous,respiratory and digestive symptoms is common in severe cases of HFMD, and EV71 is the predominant pathogen.
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Objective To investigate the contents of IL-1β and IL-1ra in cerebrospinal fluid of children with Japanese encephalitis, and their clinical significance. Methods Enzyme linked immunosorbent assay (ELISA) was employed to detect IL-lβ and IL-lra contents in 50 children with Japanese encephalitis and 20 children without nervous system disease (controls). Results IL-1β contents in climax stage, convalescent stage and the controls were (49. 43±14. 59) , (24. 73±14. 50) and (8. 98± 1.26)μg/L (F = 79.88, P<0.01); IL-lra contents in climax stage, convalescent stage and the controls were (177. 39±60. 19), (78. 24±44. 63) and (21. 09±3. 10) μg/L (F = 91. 53, P <0. 01). There were significant differences on IL-lβ and IL-lra contents among children with mild, moderate and severe encephalitis (climax: F = 82.36 and 66.50, P<0.01; convalescence; F = 55. 17 and 79.50, P<0.01). IL-1β content was positively correlated with IL-lra in both climax and convalescent stages (climax; r = 0. 815, P < 0.01; convalescent; r= 0.728, P < 0.01). Conclusions IL-lβ and IL-lra contents in cerebrospinal fluid are significantly increased in children with Japanese encephalitis in climax stage, which are closely correlated with the disease severity. The two indicators may participate in the pathological process of brain damages with Japaness encephalitis.
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Objective To investigate Smac/DIABLO and cytochrome c(cyt-c)mRNA levels in liver tissue of rats with acute hepatic failure treated by microencapsulated hepatocyte.Methods Acute hepatic failure were induced by intraperitoneal injection of D-galactosamine in rats.and the rats were treated with microencapsulated hepatocytes,free hepatocytes and physical saline(contr01),respectively.Smac/DIABLO and cyt-c mRNA in liver tissue was detected by RT-PCR and the mRNA expression levels among three groups were compared.Results Smac/DIABLO and cyt-c mRNA levels in liver tissues of rats with acute hepatic failure were higher than those of normal rata(F=4.345,14.821,47.565,42.178 and 62.961,P<0.05).The peak values of Smac/DIABLO and cyt-C mRNA expressions in free hepatocytes and control groups were at 48 h.while that in microencapsulated hepatoeytes group was at 24 h.Conclusion Smac/DIABLO and cyt-c mRNA expression is an indicator of apoptosis of hepatocytes.