Application of nested PCR and sequencing technique to detect point mutations of the 23S rRNA gene of Mycoplasma pneumoniae / 中华儿科杂志

<p><b>OBJECTIVE</b>To establish a quick method to detect drug resistance of Mycoplasma pneumoniae (MP) and study the condition of drug resistance in MP infection.</p><p><b>METHODS</b>MP 23S rRNA target gene in throat swab specimens from 200 patients with suspected MP infection was detected by using nested PCR and DNA sequencing. The result of 23S rRNA gene detection was confirmed by MP isolation and drug susceptibility test in vitro for reliability.</p><p><b>RESULTS</b>Of the 200 clinical specimens, 64 were proved to be positive for MP through MP-IgM antibody, MP specific 16S rRNA nested PCR and MP isolation . The 23S rRNA gene was amplified and the gene sequence was compared with MP reference strain in Genbank, 26 were identical to the reference strain, 38 had a point mutation in 23S rRNA. Among them, 35 had A to G mutation at position 2063, 1 had A to C mutation at position 2063 and 2 had A to G mutation at position 2064, the percentage of drug resistance was 59.4%. The sensitivity of the gene detection method was 10(2) ccu/ml and it was confirmed to be reliable by MP isolation and drug susceptibility test.</p><p><b>CONCLUSIONS</b>The gene detection method could detect MP drug resistant gene directly from clinical specimen, which has the advantages of high specificity, high sensitivity and quickness. It is of great significance for diagnosis of MP infection because MP isolation is difficult and time-consuming.</p>

In vitro inhibitory mechanism of respiratory syncytial virus with solution prescription of zhidanhuayu / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the effective part of solution prescription of Zhidanhuayu (ZDHY) against respiratory syncytial virus (RSV) in vitro.</p><p><b>METHODS</b>Observe the pathology of RSV to Hep-2 under the condition of different concentrations and each effective part of ZDHY.</p><p><b>RESULTS</b>The concentration limit causing celluar toxicity of ZDHY is 5.5 mg/ml. The ZDHY failed to block the absorption of RSV to Hep-2 within this concentration, and consequently the cell fell into the full pathological changes. During the concentration of 2.75-5.50 mg/ml, the ZDHY directly destroyed virus array,meanwhile, the infected cells that treated by the medicine kept healthy also.</p><p><b>CONCLUSION</b>ZDHY could not defend the infection of RSV, but is able to destroy the RSV directly and inhibit the RSV inhabiting in the cell.</p>

Difference of T Lymphocyte Subsets Expression in Blood of Children with Bronchiolitis or Asthma / 实用儿科临床杂志

Objective To understand the difference of helpful T lymphocyte subsets in patients diagnosed with bronchiolitis or asthma.Methods There were 34 patients in bronchiolitic group including 20 males and 14 females,the age of the patients was 1-15(6.65?4.22) months.They were confirmed by indirect immunofluorescence of nasopharyngeal secretion and (or) virus IgM antibody detected in plasma.All of the patients were infected with virus,23,4,4,and 3 patients were infected with respiratory syncytial virus,parainfluenza virus,influenza virus and adenovirus,respectively.All patients showed wheeze and small to medium blistering sound to different extend in both lungs,and all patients got fever at various degrees (20 patients blow 38 ℃,12 patients at 38-39 ℃ and 2 patients above 39 ℃).The patients′ capillary white cell counts were relatively normal when they were admitted to the hospital.There were 21 patients in asthmatic group including 12 male and 9 female,the age of the patients was 3-13 (8.05?2.85) years.Blood cells of 34 patients with bronchiolitis and 21 patients with asthma were collected by flow cytometry,and their peripheral T lymphocyte subsets were analyzed.The percentages of CD4+ cells secreting IFN-? or IL-4 were counted.They represented the percentage of helpful T lymphocyte Th1 and Th2,respectively.Results The percentage of CD4 positive cells and the ratio of CD4/CD8 were significantly higher in the bronchiolitic group than that in the asthmatic group (Pa=0.002).There was no significant difference of the percentage of Th1 cells between 2 groups.The percentage of Th2 cells in the bronchiolitic group,however,was significantly lower than that of the asthmatic group (P=0).Thus the Th1/Th2 ratio was significantly higher in the bronchiolitic group (P=0.031).Conclusions Compared with asthmatic patients,the patients with bronchiolitis show relative Th1 predominant response.Therefore,the treatment strategy should be different for these 2 diseases.

Changes of Serum Myocardial Zymogram in Children with Mycoplasma Pneumoniae Pneumonia and Its Clinical Significance / 实用儿科临床杂志

180 U/L.CK-MB raised in only 5 cases(15.6%),and the highest was 28 U/L.Of the 218 MPP patients,only the 5 patients(2.3%) with raised CK-MB levels could be diagnosed as myocardial damage complicated with MPP.Conclusions The abnormal ECG is more important than myocardial zymogram in the diagnosis of myocardial damage complicated with MPP.Only higher level of certain myocardial zymogram without abnormal ECG can not be diagnosed as myocardial damage if lack of clinic and other objective evidences for myocardial damage.In watching out for myocardial damage,excessive examinations and treatments shall be avoided if possible.

Apoptosis of peripheral blood cells of children with viral pneumonia / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To study apoptosis of peripheral blood cells of children with viral pneumonia, explore immunopathogenesis and the possibility of immunotherapy of patients with viral pneumonia.</p><p><b>METHODS</b>Fresh peripheral blood samples were collected from 28 patients with viral pneumonia and 24 healthy children were treated and run through the flow cytometry. The data were acquired using Cell Quest software and the percentage of live cells, viable apoptotic cells, non-viable apoptotic cells and dead cells of neutrophils and lymphocytes were counted. The patients with viral pneumonia were hospitalized at our hospital. The average age of patients was 1.3 years; 24 healthy children were served as control group (age 1.8 years, on average). T-test and variance analysis by SPSS FOR WINDOWS 10.0 software was used for statistical analysis.</p><p><b>RESULTS</b>The percentage of live neutrophils and lymphocytes in the acute stage and recovery stage in patients were significantly lower than that in control group (P < 0.01). The percentage of viable apoptotic neutrophils and lymphocytes in two stages in patients were significantly higher than that in control group (P < 0.05). Except for the percentage of live cells, non-viable apoptotic cells and dead lymphocytes, others had no difference between the patients and control groups.</p><p><b>CONCLUSION</b>Apoptosis of neutrophils and lymphocytes of peripheral blood cells of children with viral pneumonia increased. Whereas the percentage of live cells decreased. Drugs that can accelerate apoptosis may be helpful in treatment of viral pneumonia.</p>

Th1/Th2 Balance in Children with Bronchiolitis / 实用儿科临床杂志

Objective To study the lymphocytes subsets including Th1 and Th2 cells in PBMC of children with bronchiolitis in acute period.Methods The present study enrolled 34 patients with bronchiolitis,23 of them were respiratory syncytial virus(RSV)and 11 non-RSV,and 22 age-matched normal infants.Fresh peripheral blood samples were treated and run through the flow cytometry.The percentage of lymphocytes subsets were acquired using simultest IMK-lymphcyte software.Results The results showed the percentage of CD3+ and CD4+ cells in bronchiolitis group was higher than that in control group(P

Epidemiological study of rotavirus diarrhea in Beijing, China - a hospital-based surveillance from 1998 - 2001 / 中华流行病学杂志

<p><b>OBJECTIVE</b>To provide information on epidemiology of rotavirus infection in Beijing, China.</p><p><b>METHODS</b>An ongoing hospital-based surveillance was conducted among children < 5yr old with acute diarrhea according to WHO generic protocol (CID-98). During a 3-year study (Apr. 1998 to Mar. 2001), a total of 484 stool samples were collected from 1 457 patients, including 275 samples from 1 048 outpatients and 209 samples from 409 inpatients.</p><p><b>RESULTS</b>The overall detection rate of rotavirus infection was 25.4%. Rotavirus was responsible for 27.3% of diarrhea inpatients on a yearly base, and 46.2% during rotavirus season. Two peaks of diarrhea were observed each year, one in the summer (June-Sep.) due to bacterial dysentery (16.7%) and another in fall winter (Oct.-Dec.) due to rotavirus infection (23.0%). The detection rate on rotavirus was the highest in age group of 6 - 11 months (38.2%), followed by 1 - 2 years old (28.5%). Ninety six point eight percentage of children were infected under 3 years of age. The number of deaths, possibly caused by rotavirus diarrhea were accounted for 40% of all diarrhea deaths and 11.1% of the total deaths. Serotyping of 123 rotavirus isolates showed that serotype G1 (55.3%) was predominant, followed by G2 (26.8%), G3 (9.8%), G4 (0.8%), and 10 isolates (8.1%) remained non-typeable. Mixed infections (0.8%) seemed to be rare.</p><p><b>CONCLUSION</b>Rotavirus diarrhea was an important infectious disease among children in Beijing. Safe and effective rotavirus vaccines for the prevention of severe diarrheas and the reduction of treatment costs are of significant importance to China.</p>

Effect of zhidan huayu oral liquid on mycoplasmal pneumonia in mice / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study the effect of Chinese herbal medicine for activating blood circulation to remove stasis in treating mycoplasmal pneumonia (MP) in mice.</p><p><b>METHODS</b>One hundered and thirty-five BALB/C mice were randomly divided into the control group, the MP model groups IF1 and IF2, the Rexithromycin treated groups LH1 and LH2, and the Rexithromycin plus Zhidan Huayu oral liquid treated groups LZ1 and LZ2. The changes of pathologic scoring, graphic analysis and thrombus counting of lung were observed.</p><p><b>RESULTS</b>In the 3rd day of treatment, the pathologic scores in LH1 and LZ1 were significantly lower and their values of graphic analysis were significantly higher than those in IF1 (P < 0.01 and P < 0.05 respectively), but with inflammation of lung significantly milder than that in IF1. The difference of therapeutic effect between LH1 and LZ1 was insignificant. In the 4th day of treatment, pathologic scores in LZ2 was significantly lower and value of graphic analysis higher than those in IF2 respectively (P < 0.01), with the improvement better than that of LH2 (P < 0.05). In 3rd and 4th day of treatment, the difference of thrombus counting between the Rexithromycin treated groups and the model groups was insignificant (P > 0.05), but it was significantly lower in the combined treated groups than that in the model groups (P < 0.05).</p><p><b>CONCLUSION</b>Zhidan Huayu oral liquid could assist Rexithromycin to alleviate the condition of mice with MP, its mechanism may be related with the effect of reducing thrombosis and improving microcirculation.</p>

T(H1) and T(H2) cells in children with mycoplasma pneumonia / 中华儿科杂志

<p><b>OBJECTIVE</b>To Study T lymphocyte subsets, including T(H1) and T(H2) cells in peripheral blood mononuclear cells (PBMC) of children with mycoplasma pneumonia, understand immunopathogenesis and explore the possibility of immunotherapy of patients with mycoplasma pneumonia.</p><p><b>METHODS</b>Fresh peripheral blood samples of patients from two groups, group 1, mycoplasma pneumonia (MP) group (35 cases, 15 males and 20 females, age range 3 - 13 years, mean 9 years), and control group consisted of 28 healthy children (14 males and 14 females, age range 3 - 12 years, mean 7 years) were treated and run through the flow cytometry. The data were obtained by using Simultest IMK-Lymphocyte software and the percentage of CD(3)(+), CD(3)(+)CD(4)(+), CD(3)(+)CD(8)(+), CD(3)(-)CD(19)(+) and CD(3)(-)CD(16 + 56)(+) cells were counted. The percentage of T(H1) and T(H2) cells were gained through determination of intracellular cytokines IFN-gamma or IL-4 in CD(4)(+) cells by flow cytometry. The 35 patients with MP were hospitalized at our hospital. In addition to fever and cough, all the patents had abnormal X-ray findings and/or moist rale on auscultation of the lungs. The IgM antibody to Mycoplasma pneumoniae was positive in each patient. Immunoglobulins were measured, and PPD skin tests were performed in 30 out of the 35 patients with MP. T test and rank sum test by SPSS FOR WINDOWS 10.0 was used for statistical analysis.</p><p><b>RESULTS</b>The percentage of CD(3)(+) and CD(4)(+) T lymphocyte was 68.00 +/- 6.66 and 37.86 +/- 5.84, respectively, in MP group, and 63.71 +/- 7.92 and 34.54 +/- 6.23 in control group (P < 0.05). The percentage of T(H1) cells was 14.13 +/- 8.46 in patients and 20.77 +/- 6.89 in normal control group (P = 0.001). The percentage of NK cells was 15.57 +/- 12.16 and 20.39 +/- 9.64 in MP and control group (P < 0.01). The ratio of T(H1)/T(H2) in MP group was lower than that in control group (P < 0.05). However the percentage of CD(8), T(H2), B cells and CD(4)/CD(8) had no difference between the MP and control groups. The levels of IgG, IgA, and IgM in serum were normal in most of patients except for a few patients who had elevated IgA and IgM levels. The PPD skin tests were negative in 30 out of 35 patients.</p><p><b>CONCLUSION</b>In this study a higher percentage of CD(3)(+), CD(4)(+) T lymphocyte and lower percentage of T(H1), NK cells in PBMC of patients with mycoplasma pneumonia were found. The ratio of T(H1) and T(H2) cells in patients was also lower. None of thirty patients had positive PPD skin tests. Unbalanced cell-mediated immunity with a tendency toward T(H2) existed in patients with MP. Therefore, immunomodulators may be useful in treatment of mycoplasma pneumonia.</p>

In vitro inhibition of respiratory syncytial virus with combined ribavirin and an oral preparation of traditional Chinese medicine Hu Fei / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the in vitro antiviral effect of ribavirin combined with an oral preparation of traditional Chinese medicine "Hu Fei" (protecting the lung) on respiratory syncytial virus (RSV).</p><p><b>METHODS</b>Cytopathic effects (CEP) of RSV on Hep2 cells were observed after adding different concentrations of ribavirin, Hu Fei and combination of both into the culture medium.</p><p><b>RESULTS</b>The minimum concentration of ribavirin and Hu Fei for complete inhibition of CPE caused by RSV was 7.80 microg/ml and 5.00 mg/ml, respectively. When the combination of ribavirin and Hu Fei was applied, their minimum concentrations needed for complete inhibition were decreased to 0.98 ?g/ml and 0.63 mg/ml.</p><p><b>CONCLUSIONS</b>Both ribavirin and Hu Fei showed in vitro anti-RSV effect, but the inhibitory effect of combined ribavirin and Hu Fei was more potent than either of the preparation alone.</p>

Cellular and Humoral Immunity in Children with Recurrent Respiratory Tract Infection / 实用儿科临床杂志

Objective To explore the cellular and humoral immunity in children with recurrent respiratory tract infection(RRI).Methods Fresh peripheral blood samples were treated and run through the flow cytometry,the percentage of lymphocytes subsets were acquired using Simultest software simulaneously. Immunoglobulin IgA,IgG,IgM and complement C3 and C4 were measured.Results The levels of IgG and IgA in the RRI group were lower than the normal reference value in only 1 patient respectively.The level of IgM decreased in 7cases.Complement C3 and C4 decreased in 3 and 1 patients, respectively.The percentage of CD8 in the RRI group was higher than that in healthy control group(P=0),and the CD4/CD8 was obviously lower than that in healthy control group(P=0.002).Conclusions The disorder of cellular and humoral immunity are existing and cell-mediated immune disorder is more obviously in RRI.The immune-modulation agents are necessary in the prevention and treatment for RRI.

Immune Response to Respiratory Syncytial Virus Infection in Ovalbumin Sensitized Mice / 实用儿科临床杂志

Objective To observe the lung inflammation and the expression of T - helper cell related cytokines in respiratory syncytial virus(RSV) infected Balb/c mice pre - sensitized with ovalbumin(OVA). Methods Mice were randomly divided into two series: nonsen-sitized and sensitized animal model with nebulized OVA once a day for 10 days. Each of the series was divided again into two groups: control group,RSV infected group with nasal draping plus nebulizing. All the mice were sacrificed 5 days later of infection. Viral isolation of lung organization was performed in each group. Pulmonary pathological detection and the levels of interleukin - 4(IL-4), interferon-? (IFN- ?) mRNA were evaluated by RT- PCR. Results The RSV were found only in RSV infected groups. The Balb/c mice developed typical interstitial pneumonia after RSV infection. When the mice pre- sensitized were infected with RSV, the pulmonary inflammation, lymphocyte and eosinophils infiltration and cell - collar peribronchiles were more severe in lung organization and bronchiole than those in the single RSV infection group. In the lung organization of control group, there were no mRNA expression of IL - 4 and IFN - ?. The mRNA of IFN -? was expressed in the lung organization of RSV infected groups while had not showed mRNA of IL- 4. In the OVA+ RSV group the mRNA expression of IL - 4 was obvious, while the expresses of IFN -? scarcely. Conclusions Pulmonary inflammation is more severe in the infection after OVA sensitization. RSV infection alone results in a Th1 - like cytokine response, while the infection after OVA sensitization results in Th2 - like response.

Study on Antimicrobial Resistance of Moraxella Catarrhalis in the Nasopharyngeal Secretions of Children with Respiratory Infection / 实用儿科临床杂志

Objective To explore the antimicrobial resistance of Moraxella Catarrhalis in children with respiratory infection.Methods Eleven strains of Moraxella Catarrhalis were isolated from nasopharyngeal secretion in 145 children with community-acquired respiratory tract infection in Beijing Friendship Hospital from 2004 to 2006.Segregated stocks were isolated from upper respiratory infection in 5 cases,bronchitis in 3 cases and pneumonia in 3 cases.Agar dilution method was used to determine minimal inhibition concentrations (MIC),including 8 kinds of antibiotics,and ?-lactamase was detected.WHONET 5 and SPSS 11.5 software were used to analyze data.Results Ten of the 11 strains were ?-lactamase positive.The rates of resistance to ampicillin,cefuroxime and erythromycin were 81.8%,63.6% and 18.2% respectively,however,all the strains were susceptible to ceftriaxone.MIC90 of penicillin and cefradine was 32.0 mg/L and 8.0 mg/L respectively.MIC90 of roxithromycin and azithromycin was 2.0 mg/L and 0.25 mg/L respectively.Conclusions Moraxella Catarrhalis is an opportunistic pathogen.The ?-lactamase positive rate of Moraxella Catarrhalis from children is high,and there is also a high resistance percen-tages of Moraxella Catarrhalis to penicillin,ampicillin,first and second generation cephalosporins.Moraxella Catarrhalis is susceptible to Cefuroxime.