Modulation of host immune defenses by cytomegalovirus: advanced insights from evolutionary game theory / 病毒学报

Human cytomegalovirus (HCMV) is an ubiquitous pathogen that infects a majority of the world's population. The virus can establish lifelong infection once the human body is infected by HCMV and virus can be reactivated from a latent state in immune suppressed individuals. HCMV has developed several strategies to evade host immune surveillance after millions of years of co-evolution with mankind. One of the classical tricks is encoding homologous to human immune factors or stealing host cellular genes that have significant functions in immune system. Virus encoded immune modulators which participate in regulating the major histocompatibility complex, cellular immunity, humoral immunity, cytokines and chemokines are supposed to play a significant role in the pathogenesis of HCMV. Evaluation of "mutually assured survival" relationship between virus and host provides important insights into viral immunopathogenesis and study of viral immunomodulatory proteins might help us to uncover new human genes that control immunity.

Determination of ascitic bacterial 16S rRNA by quantitative PCR-microarray in the diagnosis of spontaneous bacterial peritonitis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To evaluate the significance of determining ascitic bacterial 16S rRNA by quantitative PCR combined with microarray (PCR-microarray) in the diagnosis of spontaneous bacterial peritonitis (SBP).</p><p><b>METHODS</b>Ascitic bacterial 16SrRNA was determined by real time fluorescent quantitative PCR-microarray in 76 cases of suspected SBP and 6 cases of non-infectious ascites with chronic liver diseases. The results were compared with ascitic bacterial culture simultaneously.</p><p><b>RESULTS</b>Of 76 ascitic samples, 17 were detected bacteria positive by PCR-microarray, including 8 Grams positive(G+) and 9 Grams negative(G-), which was higher than that by bacterial culture which had only 6 ascitic samples detected positive (all G-); the positive rates were 22.4% vs 7.9%, respectively (P < 0.01). The bacterial strains detected by both methods in 6 cases had a consistency with each other. No bacteria were detected in another 6 cases of non-infectious ascites with chronic liver diseases.</p><p><b>CONCLUSIONS</b>Determination of ascitic bacteria 16S rRNA by PCR-microarray has a higher specificity and sensitivity in the diagnosis of SBP as compared with the bacteria culture. Application of this novel method can not only accelerate SBP diagnosis but also stratify the different pathogens.</p>

Analysis of the epidemic characteristics of the etiological agents in children with hand, foot and mouth disease and its clinical significance / 中华儿科杂志

<p><b>OBJECTIVE</b>To investigate the epidemic characteristics of etiological agents in children with hand, foot and mouth disease (HFMD) and analyze the differences between the severe and mild cases with HFMD seen from 2008 to 2009 in the Children's Hospital.</p><p><b>METHODS</b>A total of 154 patients with HFMD were enrolled from May 2008 to September 2008 and from May 2009 to September 2009, including 28 severe HFMD patients. Data from 80 cases with suspected herpangina were collected as control. Enterovirus universal type, enterovirus type 71 (EV71) and coxsackie virus group A 16 (CA16) were detected by real-time RT-PCR respectively.</p><p><b>RESULTS</b>The positive rate of enterovirus universal type in the 154 patients with HFMD was 81.82%(126/154). EV71 positive rate in these 126 patients with enterovirus universal type infection was 57.14%(72/126). The positive rate of enterovirus universal type in the 80 cases with suspected herpangina was 68.75%(55/80). There was no EV71 infection in these 80 cases with suspected herpangina. EV71 infection was mainly popular in 2008. Both EV71 and CA16 were prevalent in 2009. The epidemic characteristics of enterovirus infection with HFMD between 2008 and 2009 had significant differences (χ(2) = 23.50, P = 0.000) (P < 0.01). The epidemic characteristics of enterovirus infection between severe and mild HFMD patients also had significant differences (χ(2) = 29.85, P < 0.01). There were 28 cases with severe HFMD, in whom the EV71 positive rate was 92.86% (26/28). EV71 positive rate in the mild HFMD was 36.51% (46/126) (χ(2) = 29.22, P < 0.01). There was no significant difference in the gender (χ(2) = 0.135, P = 0.714) and virus load (t = 0.141, P = 0.889) between the mild and severe HFMD cases. But the age of mild and severe HFMD showed a significant difference (t = 2.926, P = 0.009). Patients who were less than 2 years of age had a proportion of 88.89% (8/9) with severe HFMD. The mean age of mild HFMD patients was 3.19 years.</p><p><b>CONCLUSION</b>HFMD showed different epidemic characteristics at different times of enterovirus infection. There was no significant difference in the gender and virus load between the mild and severe cases with HFMD. Children under 3 years of age with EV71 infection were at high risk for severe HFMD.</p>

Establishment and clinical application of a new real time PCR assay for simultaneous detection of human herpesvirus-6A and human herpesvirus-6B / 中华儿科杂志

<p><b>OBJECTIVE</b>Human herpesvirus 6 (HHV-6) isolates are classified into two variants, HHV-6A and HHV-6B, based on distinct genetic, antigenic and biological characteristics. HHV-6 has been associated with encephalitis in children recently. This study aimed to establish a real time PCR assay for simultaneous detection of the two subtypes of HHV-6, and apply this new assay to children with suspected encephalitis, then analyze the relationship between the infection with HHV-6 and encephalitis in children.</p><p><b>METHOD</b>The universal primers and variant-specific TaqMan probes were designed based on the highly conserved sequences of the DNA polymerase gene (U38) of HHV-6. The 5' end of the probes for HHV-6A and HHV-6B was labeled with the fluorescein reporter tetrachloro-6-carboxyfluorescein and 6-carboxyfluorescein (6-FAM), separately, while the 3' end were quenched with 6-carboxy-tetramethylrhodamine. The real time PCR assay for simultaneous detection of HHV-6A and HHV-6B was established. Then, the plasmids of HHV-6A and -6B which were diluted by a 10-fold series from 10(9) to 10(0) copies/microl, together with controls were used for testing both sensitivity and specificity of the real time PCR assay. The cerebrospinal fluid (CSF) specimens from 445 cases of suspected encephalitis were tested with this real time PCR and positive samples were then sequenced.</p><p><b>RESULT</b>Both HHV-6A (strain ZJ-159) and HHV-6B (strain GS) were positive on the real time PCR assay. There were no cross-reaction with herpes simplex virus type 1, type 2 (HSV-1, HSV-2), varicella-zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), hepatitis B virus, Staphylococcus aureus, Mycoplasma pneumoniae and human DNA. A linear regression curve was obtained when plotting Ct values against the log10 of the viral DNA input for both subtypes of HHV-6. The sensitivity threshold was 10 copies/microl for the real time PCR. HHV-6 positive rate by the real time PCR assay was 4.72% (21/445), including 4 cases with HHV-6A infection, 16 cases of HHV-6B infection and 1 case with mixed HHV-6A and HHV-6B infection. The new PCR assay usually took 2 to 3 hours to provide results.</p><p><b>CONCLUSION</b>This new real time PCR assay can simultaneously detect both subtypes of HHV-6, and have high specificity and sensitivity. It will provide an early and sensitive diagnosis of HHV-6 encephalitis in children.</p>

Relationship of nonsyndromic cleft lip and/or palate and poliovirus receptor-related 1 exon 3 polymorphisms in Han people of Jiangzhe area / 中华整形外科杂志

<p><b>OBJECTIVE</b>To study the relationship of nonsyndromic cleft lip and/or palate (NSCL/P) and poliovirus receptor-related 1 exon3 (PVRL1exon3) polymorphisms in Han People of Jiangzhe area.</p><p><b>METHODS</b>PVRL1exon3 was examined by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique in the 50 patients with NSCL/P and 85 healthy parents.</p><p><b>RESULTS</b>No W185X mutation was found in the PVRL1exon 3.</p><p><b>CONCLUSION</b>It indicates that there is no relationship between NSCL/P and PVRL1exon3 in Han People in Jiangzhe area.</p>

Establishment of quantifying and typing analysis of 16S rRNA gene by real-time PCR / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To explore a new method of rapid and reliable diagnosis of bacterial infectious diseases such as purulent meningitis and septicemia.</p><p><b>METHODS</b>A pair of universal primers and a set of probes (including universal fluorescence probe, Gram-positive probe and Gram-negative probe) were designed based on the bacterial highly conserved region of 16S rRNA gene. By using the FQ-PCR method, 12 standard strains, 23 clinical cultural isolations and the controls such as HBV, Cryptococcus histolyticus, Blastomyces albicans and human DNA were detected with the three kinds of probes. The correlation among the results of the three kinds of probes detection was analyzed.</p><p><b>RESULTS</b>The determination of 16S rRNA gene with FQ-PCR was a highly specific and sensitive method and not cross-reactive with human DNA, virus or fungi. The least amount of 10 copies of 16S rRNA gene which corresponded to 2 bacteria could be detected with FQ-PCR. Twelve standard strains and 23 clinical cultural isolations were detected by FQ-PCR with the three kinds of probes mentioned above. All samples presented positive results using the universal probe. The results of 16S rRNA gene detected by the Gramjpositive probe were positive to the 18 G+ strains. The results of 16S rRNA gene detected by the Gram-probe were positive to the 17 G- strains.</p><p><b>CONCLUSIONS</b>The FQ-PCR technique was established for bacteria quantifying and typing using the universal primer and the double type probes. This method was convenient and rapid in detecting, quantifying and typing bacteria, with a high specificity and sensitivity.</p>

A broad-range 16S rRNA gene real-time PCR assay for the diagnosis of neonatal septicemia / 中华儿科杂志

<p><b>OBJECTIVE</b>To evaluate the usefulness of a broad-range real-time PCR assay aimed at the 16S rRNA gene of bacteria in a clinical setting in rapid and reliable diagnosis of neonatal septicemia for improving the speed and accuracy of bacterial detection.</p><p><b>METHODS</b>The universal primer and TaqMan probe were designed based on the highly conserved sequences of the bacterial 16S rRNA gene. The chosen primers and probe did not show any likely cross hybridization with human, viral or fungal genome sequences. The TaqMan assay used the fluorescent signal on the probe, such as 6-carboxyfluorescin (6-FAM), and quenched by the standard 6-carboxytetramethylrhodamine (TAMRA) probes. The broad-range 16S rRNA gene real-time PCR array was established. Then, three common pathogenic microorganisms including Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli, which were prepared by a 10-fold dilution series respectively from 10(8) colony forming unit (CFU)/ml to 10(3) CFU/ml, as well as controls, were used for testing of both sensitivity and specificity of the real-time PCR assay. The blood samples from 830 cases of suspected septicemia, who were hospitalized in our neonatal ward and the neonatal intensive care unit (NICU) and developed clinical signs suggestive of infection, were tested with routine culture and bacterial 16S rRNA genes real-time PCR separately. In addition, 30 neonates without infection were enrolled as the negative control group.</p><p><b>RESULTS</b>All the three common pathogenic bacterial species were positive on the 16S rRNA genes real-time PCR assay. There were no cross-reaction with cytomegalovirus (CMV), Epstein-Barr virus (EBV), hepatitis B virus (HBV), fungi, human DNA and blank control, and the technique showed high specificity and sensitivity. The detection limit of the TaqMan assay was tested by amplifying serial dilutions of the three common pathogenic bacterial DNA. The minimal detection limit of the TaqMan system was equivalent to 3 CFU of bacteria, the threshold cycle (CT), which is inversely proportional to the log of the amount of target DNA initially present, was 37.90 by calculation. The real-time PCR assay was evaluated on 830 blood specimens for suspected neonatal septicemia, as compared to the results obtained from the routine bacterial cultures. The positive rate by the real-time PCR assay was 5.18% (43/830) in 830 samples, and was significantly higher than that of blood culture [2.41% (20/830) (P < 0.01)]. The real-time PCR was positive in all the 20 positive blood culture samples. Thirty non-infectious blood samples were negative by both the PCR assay and blood cultures. When blood culture was used as control, the sensitivity of the real-time PCR assay was 100%, the specificity was 97.16%, and the index of accurate diagnosis was 0.972. Moreover, three of the PCR positive amplicons were confirmed by sequencing to confirm the accuracy of the real-time PCR assay in testing clinical specimens. The sequencing showed that except for one sequence, all the others were demonstrated to be Staphylococcus aureus and Escherichia coli respectively, which was in accord with the results of the blood cultures.</p><p><b>CONCLUSIONS</b>The bacterial 16S rRNA genes real-time PCR had been established to diagnose the neonatal septicemia. The sensitivity and specificity the real-time PCR assay were higher than those of blood culture. This technique can provide a rapid way for the etiological diagnosis of neonatal septicemia, and was a convenient and accurate method in etiologic diagnosis of neonatal septicemia.</p>

Pathogenic bacteria of childhood lower respiratory tract infection / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the pathogenic bacteria of lower respiratory tract infection (LRTI), and age and gender distribution and drug resistance of the pathogenic bacteria in children.</p><p><b>METHODS</b>Sputum specimens for bacterial cultures were collected in sterile tubes from all of the children with LRTI who had been admitted to the Children's Hospital of Zhejiang University between August 2001 and July 2002. Antibiotic susceptibility tests were performed using the Vitek system, the Kirby-Bauer diffuse method and the Etest method after bacteria were identified.</p><p><b>RESULTS</b>Among the 4,238 patients with LRTI during the study period, 1,181 patients were bacteria-positive, with a positive rate of 27.9%. Streptococcus pneumoniae (S. pneumoniae) was the most common (222 strains), followed by Haemophilus influenzae (H. influenzae) (216 strains), Klebsiella pneumoniae (K. pneumoniae) (216 strains), Escherichia coil (E. coli) (169 strains) and Staphylococcus aureus (S. aureus) (89 strains). The isolation rate of S. pneumoniae in females was significantly higher than in males (6.2% vs 4.7%; P < 0.05). However, the isolation rates of K. pneumoniae and S. aureus in males were higher than in females (5.1% vs 4.1% and 2.5% vs 1.5%, respectively; P < 0.05). A higher incidence of LRTI due to S. pneumoniae and H. influenzae was found in the 1-3 years group, while the incidence of LRTI due to K. pneumoniae, E. coli, S. aureus and E. cloacae was higher in patients under 1 year of age. Antibiotic susceptibility tests showed that rates of penicillin non-susceptible S. pneumoniae, ampicillin resistant H. influenzae, oxacillin-resistant S. aureus and ESBL-positive K. pneumoniae and E. coli were 55.0%, 16.5%, 41.2%, 42.6% and 4.5%, respectively.</p><p><b>CONCLUSIONS</b>S. pneumoniae, H. influenzae, K. pneumoniae, E. coli and S. aureus were common pathogens of LRTI in children. The infection rate varied with age and gender. Antibiotics for treating LRTI should be selected based on the drug susceptibility test.</p>

Drug-resistance of staphylococcus aureus and detection of mecA gene in all strains isolated from children in Hangzhou / 中华儿科杂志

<p><b>OBJECTIVE</b>To study the resistance of staphylococcus aureus (S. aureus) isolated from children in Hangzhou to antibiotics and analyze the clinical value of mecA-PCR in determining oxacillin-resistant isolates.</p><p><b>METHODS</b>S. aureus isolates were screened by using latex agglutination test and identified with GPI card of Vitek system. Antibiotics sensitivity tests were performed using disk diffusion methods and tests for sensitivity to oxacillin and vancomycin were performed with a further E-test method. The mecA gene was detected with polymerase-chain reaction (PCR).</p><p><b>RESULTS</b>Of all 259 S. aureus strains, 185 from clinical specimens in inpatients and 74 from pharyngeal swabs in healthy children, 247 strains (95.8%) were beta-lactamase-positive and resistant to penicillin, while 91.1% of all strains were sensitive to oxacillin. All the strains were sensitive to vacomycin and 91.9% of all the strains were susceptible to cefotaxime and ceftriaxone. Resistance to erythromycin, tetracycline, clindamycin, trimethoprim-sulfamethoxazole, chloramphenicol, ofloxacin and rifampin were 48.3%, 30.9%, 21.6%, 11.2%, 10.0%, 2.3% and 1.5%, respectively. The resistance rate to oxacillin, cefotaxime, and ceftriaxone in clinical strains were significantly higher than that in carried strains (P < 0.05), while erythromycin-resistance rate was significantly higher in carried strains than that in clinical isolates (P < 0.05). The mecA-PCR showed that the control strain ATCC25923 and all oxacillin-sensitive S. aureus were mecA-negative, while all oxacillin-resistant strains were mecA-positive instead. Only one strain was mecA-positive in 7 oxacillin-intermediate S. aureus strains.</p><p><b>CONCLUSION</b>Oxacillin-resistance in S. aureus isolates was low, and mecA-PCR method is a good choice for rapid examination oxacillin-resistant strains.</p>

Inhibitive effects of anti-oxidative vitamins on mannitol-induced apoptosis of vascular endothelial cells / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVE</b>Study blood vessel injury and gene expression indicating vascular endothelial cell apoptosis induced by mannitol with and without administration of anti-oxidative vitamins.</p><p><b>METHODS</b>Healthy rabbits were randomly divided into four groups. Mannitol was injected into the vein of the rabbit ear in each animal. Pre-treatment prior to mannitol injection was performed with normal saline (group B), vitamin C (group C) and vitamin E (group D). Blood vessel injury was assessed under electron and light microscopy. In a second experiment, cell culture specimen of human umbilical vein endothelial cells were treated with mannitol. Pre-treatment was done with normal saline (sample B), vitamin C (sample C) and vitamin E (sample D). Total RNA was extracted with the original single step procedure, followed by hybridisation and analysis of gene expression.</p><p><b>RESULTS</b>In the animal experiment, serious blood vessel injury was seen in group A and group B. Group D showed light injury only, and normal tissue without pathological changes was seen in group C. Of all 330 apoptosis-related genes analysed in human cell culture specimen, no significant difference was seen after pre-treatment with normal saline, compared with the gene chip without pre-treatment. On the gene chip pre-treated with vitamin C, 45 apoptosis genes were down-regulated and 34 anti-apoptosis genes were up-regulated. Pre-treatment with vitamin E resulted in the down-regulation of 3 apoptosis genes.</p><p><b>CONCLUSION</b>Vitamin C can protect vascular endothelial cells from mannitol-induced injury.</p>

Diagnosis of Cytomegalovirus Infection in Children / 实用儿科临床杂志

Cytomegalovirus(CMV) is an important pathogen of congenital and postnatal infections in children,which causes a series of acute and chronic infectious diseases and nervous system sequelaes.Early and accurate diagnosis of pediatric CMV infection is an effective way to improve health in children.This paper will introduce the types,laboratory techniques and diagnostic strategies of CMV infection based on the diagnostic standards at home and abroad,and also focus on current progress in diagnosis of pediatric CMV infection.

Determination of six major human herpes viruses in cerebrospinal fluid and blood of children with consensus primers / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To identify 6 major human herpesviruses with consensus primers and to explore its clinical application.</p><p><b>METHODS</b>Based on the highly-homogeneous regions of DNA polymerase gene in human herpesviruses,Two pairs of primer were synthesized. One pair was designed to amplify herpes simplex virus type 1, type 2, Epstein-Barr virus and cytomegalovirus; and another was used to amplify varicella-zoster virus or human herpesvirus 6. Virus species identification was performed by restriction enzyme digestion with BamH I and BstU I. Thirty-eight CSF specimens of clinically diagnosed viral encephalitis,and 49 blood specimens from 27 confirmed cases and 22 clinically diagnosed ones were tested for herpes virus DNA using the PCR-RFLP assay with these primers.</p><p><b>RESULTS</b>Thirteen out of 38 CSF specimens (34.2%) were herpes virus positive. All blood specimens from 27 confirmed cases showed positive results, while for 22 clinically diagnosed cases 16 (72.7%) were positive. The types of herpes virus were determined using restriction enzyme digestion with BamH I and BstU I. Two CSF specimens from the patients, who were treated with aciclovir for 2 - 3 days, were still positive for herpes virus DNA by this method. None of the control blood or CSF controls were positive for herpesvirus by PCR.</p><p><b>CONCLUSION</b>The PCR-RFLP method used in this study is a specific, sensitive and practicable one for diagnosis of herpes virus infection.</p>

The binding of MBL to common bacteria in infectious diseases of children / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVE</b>To purify Mannan-binding lectin (MBL) from human serum and detect its binding ability to several kinds of bacteria common in infectious diseases of children.</p><p><b>METHODS</b>MBL was purified from human serum by affinity chromatography on mannan-Sepharose 4B column. Its binding ability to eight species, 97 strains of bacteria was detected by enzyme-linked lectin assay (ELLA).</p><p><b>RESULTS</b>MBL has different binding ability to bacteria and shows strong binding ability to Klebsiella ornithinolytica and Escherichia coli, but shows relatively lower binding ability to Staphylococcus haemolyticus, Enterobacter cloacae and Staphylococcus epidermidis. To different isolates of Klebsiella pneumoniae, Haemophilus influenzae and Staphylococcus aureus, MBL shows quite different binding ability.</p><p><b>CONCLUSIONS</b>MBL has different binding ability to different bacteria, and has relatively stronger binding ability to Gram-negative bacteria. Its binding ability to different isolates of certain kinds of bacteria is quite different.</p>

Analysis of VP7 serotype of rotavirus in infantile diarrhea in Hangzhou area from 2001 to 2003 / 中华儿科杂志

<p><b>OBJECTIVE</b>To investigate molecular epidemiologic features of rotavirus (RV) infection in infantile diarrhea in Hangzhou area.</p><p><b>METHODS</b>Stool specimens of 683 infants with suspected acute viral enteritis in the autumn and winter of 2001 - 2003 were collected. RV (group A) was detected by using latex agglutination test (LAT). VP7 serotype (G) positive specimens were detected by using enzyme linked immunosorbent assay (ELISA) and then the RNA of the virus was determined with reverse transcription polymerase chain reaction (RT-PCR). cDNA of VP7 gene fragment was sequenced by automatic gene analyzor (ABI3730) and compared with the RV VP7 gene sequences stored in Genebank.</p><p><b>RESULTS</b>RV was detected in 297 of 683 (43.5%) specimens by LAT. The highest frequency of RV (group A) detected was 52.9% (228/431) in patients aged 7 - 18 months. The prevalent serotypes were G1 (36.7%, 109/297) and G3 (30.9%, 92/297), followed by mixed type (11.8%, 35/297), untyped (9.4%, 28/297), G4 (7.1%, 21/297) and G2 (4.0%, 12/297). The prevalent serotypes seen each year were different. G1 (54.9%, 45/82) was the major serotype in 2001 followed by G3 (14.6%, 12/82). In 2003, the major serotype was G3 (43.0%, 63/146) and followed by G1 (29.5%, 43/146). The reliability of ELISA was confirmed by RT-PCR, gene sequencing and homology analysis.</p><p><b>CONCLUSION</b>The main prevalent serotypes of VP7 of rotavirus were G1 and G3. The dominant serotypes of rotavirus varied in Hangzhou area from 2001 to 2003.</p>

Long-term therapeutic effect of triple therapy consisted of omeperazole, clarithromycin and amoxycillin in children with Helicobacter pylori infection and approach to re-treatment after failure of the treatment / 中华儿科杂志

<p><b>OBJECTIVE</b>Helicobacter pylori (Hp) infection presents high prevalence in the world, but there are few pediatric assays evaluating antimicrobial treatment using a short regimen of triple therapy. To evaluate the eradication rate and long term therapeutic effect of a triple therapy consisted of omeperazole, clarithromycin (CLA) and amoxycillin (AMO) on Hp infection, the authors explored the alternative therapeutic programs and their effects after first therapeutic failure.</p><p><b>METHODS</b>A total of 192 children with Hp infection were divided into two groups: 157 children were given the triple therapy for one week (CLA group); 35 children were given another triple therapy composed of omeperazole, metronidazole (MET) and AMO for two weeks (MET group). All of the children were followed up for 1 - 36 months after the therapies ended. Twenty-two children in whom Hp was eradicated with CLA triple therapy were followed up for 3 years. The children of the two groups who had therapeutic failure were given re-treatment as follows. CLA triple therapy was given for one week to the children who had failure after MET triple therapy; increased doses of CLA with longer treatment course was given to the children who had failure after CLA triple therapy. A tetra therapy consisted of omeperazole, colloidal bismuth subcitrate (CBS), furazolidone (FUR) and AMO was given to the children in whom the re-treatment failed.</p><p><b>RESULTS</b>The Hp eradication and ulcer recovery rate of CLA group was 90.4% (142/157) and 96.9% (32/33), respectively; the Hp eradication rate of MET group was 77% (27/35). There was significant difference between eradication rates of the two groups (chi(2) = 4.69, P < 0.05). The recurrence rate of 22 Hp eradicated children treated with CLA triple therapy was 4.5% (1/22) during the 3-year follow-up. The eradication rate of the three re-treatment programs for 29 children was 75% (6/8), 77% (11/15) and 100% (6/6), respectively.</p><p><b>CONCLUSION</b>(1) Omeperazole, CLA and AMO triple therapy for one week was the best to eradicate Hp infection with high eradication rate, few side effects, short period of treatment, good compliance and low recurrence rate. (2) Proper increase of CLA dose and longer therapeutic course may increase the eradication rate. Omeperazole, CBA, FUR and AMO tetra therapeutic program may be used as an alternative treatment in patients who develop resistance to CLA triple therapy.</p>

Rapid diagnosis of neonatal sepsis by 16SrRNA genes PCR amplification and genechip hybridization / 中华儿科杂志

<p><b>OBJECTIVE</b>To explore a method for rapid diagnosis of sepsis in newborn infants.</p><p><b>METHODS</b>(1) The primers and oligonucleotide probes were designed and synthesized based on the sequences of bacterial 16SrRNA gene. The gene chip was prepared through the probes printed onto special glass slides. The gene chip included 18 special probes: universal probe 1, universal probe 2, Gram positive bacterial probe, Gram negative bacterial probe 1, Gram negative bacterial probe 2, Staphylococcus aureus, coagulase negative staphylococcus (CoNS) 1, CoNS 2, Escherichia coli, Hemophilus influenzae, Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus agalactiae, Bacteroides fragilis, Bacillus, Meningococcus, Corynebacterium, Propionibacterium; (2) Blood specimens from 285 cases of suspected septicemia were cultured and bacterial 16S rRNA gene was detected separately; DNA isolated from blood specimens and cerebrospinal fluid was amplified by PCR, and PCR products were hybridized with the probes on the gene chips. Hybridization results were scanned and read by laser-scanner.</p><p><b>RESULTS</b>(1) Of the 285 cases, 17 were positive by PCR and the positive rate (5.96%) was significantly higher than that of blood culture (2.81%) (P < 0.01). When blood culture was taken as control, the sensitivity of PCR was 100% and Specificity was 96.75%, the index of accurate diagnosis was 0.968. (2) The 17 specimens which showed positive results by PCR were further hybridized on the gene chip. All were positive by universal probes. Among all of them, 5 were positive by E. coli probe; 4 were positive by Staphylococcus epidermidis; two were positive by Bacillus and Propionibacterium probes, separately; 4 were positive by CoNS. The 8 specimens which showed positive results by both PCR and blood culture, the result of gene chip hybridization coincided with the result of blood culture.</p><p><b>CONCLUSION</b>Detection of the bacterial 16SrRNA genes in clinical specimens by gene chip hybridization technology can diagnose neonatal septicemia rapidly. This method has higher sensitivity and specificity than blood culture or other methods and can provide a rapid way for the etiological diagnosis of neonatal septicemia. Therefore the genechip method may be valuable and practical in early diagnosis of neonatal septicemia.</p>

Antibiotics-resistance pattern and genetic type of Streptococcus pneumoniae isolated from children in Hangzhou / 中华儿科杂志

<p><b>OBJECTIVE</b>To investigate the antibiotics-resistance type and molecular epidemiology of Streptococcus pneumoniae isolated from children in Hangzhou.</p><p><b>METHODS</b>The sensitivities of 323 strains of Streptococcus pneumoniae to 9 antibiotics were determined in vitro by Kirby-Bauer diffuse methods, and MICs of penicillin and cefotaxime were determined by E-test methods.</p><p><b>RESULTS</b>Among all 323 strains isolated from children during the period from August 2001 to July 2002, 136 strains (42.1%) were sensitive to penicillin, while 57 strains (17.7%) were penicillin-resistant. Penicillin MICs ranged from 0.012 microg/ml to 4.0 microg/ml. All the strains were sensitive to cefotaxime and its MICs ranged from 0.012 microg/ml to 4.0 microg/ml. The most resistant antibiotic was erythromycin and it's resistant-rate was as high as 90.7%, followed by tetracycline (87.6%), trimethoprim-sulfamethoxazole (48.6%) and chloromycetin (14.9%). Totally 197 strains (61.0%) were multi-drug-resistant pneumococci and most of them were resistant to trimethoprim-sulfamethoxazole, erythromycin and tetracycline at the same time. Two strains (0.6%) were resistant to rifampin and none was resistant to vancomycin and ofloxacin. BOX PCR typing was carried out and no overwhelming fingerprinting pattern was found among penicillin resistant Streptococcus pneumoniae strains which were isolated from patients, while the banding patterns were always similar or identical among the strains isolated from the same specimen or from the same patient at different time, respectively.</p><p><b>CONCLUSION</b>The antibiotics-resistant rate of pneumococci was high in Hangzhou, but the third-generation cephalosporins were still the best antibiotics against Streptococcus pneumoniae. One child could be infected or colonized by more than one pneumococci clone at the same or different time.</p>

Detection of four human herpesviruses DNA and virus-specific IgM antibody in blood specimens of infants / 中华儿科杂志

<p><b>OBJECTIVE</b>To establish a restriction endonuclease pattern which could detect and differentiate four major human herpesviruses by polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), DNA cloning and sequence analysis.</p><p><b>METHODS</b>A pair of primer, which was designed according to sequences in well-conserved regions of the DNA polymerase gene in human herpesviruses, was designed to amplify herpes simplex virus type 1 and 2 (HSVI/II), Epstein-Barr virus (EBV) and cytomegalovirus (CMV). Sequences of the primers are as follows: P(1) (5'-CGACTTTGCCAGCCTGACC-3') and P(2) (5'-AGTCCGTGTCCCCGTAGATG-3'). DNA of four strains of standard herpesviruses were amplified by PCR, and further studied by DNA cloning, sequence analysis and RFLP. At last, the authors established the PCR-RFLP technique to differentiate the four different herpesviruses. Meanwhile, 75 clinical blood specimens from infants with suspected viral infection and 38 blood specimens from healthy children were evaluated for herpesviruses DNA or virus-specific IgM antibody by PCR-RFLP or by ELISA.</p><p><b>RESULTS</b>The PCR amplified products of four human herpesviruses were from 510 bp to 592 bp in length and were analyzed for herpesvirus types with restriction endonuclease technique. The specificity and sensitivity of this PCR-RFLP were examined. There was no cross-reaction with Escherichia coli, Staphylococcus aureus, hepatitis B virus (HBV), Clostridium neoformans and human-genomic DNA and the lowest detection level was 0.1 fg DNA. Among 75 specimens, 23 were positive by PCR and the positive rate was 30.7%, including 13 for CMV, four for EBV, five for HSVII and one for HSVI after restriction enzyme digestion with BamHI and BstUI, while 10 were positive by ELISA and positive rate was 13.3%. All ELISA-positive specimens were likewise positive by PCR. Thirteen of 65 specimens that were ELISA-negative were tested positive by PCR. An infant with CMV infection was determined with viral DNA and virus-specific IgM antibody in blood at 3, 4 and 6 months after birth, respectively. The result showed that she was still CMV DNA-positive in blood whereas IgM antibody was positive only at month 3 after birth. None of the 38 control blood specimens was positive for herpesvirus by this PCR-RFLP or by ELISA.</p><p><b>CONCLUSIONS</b>This PCR-RFLP technique was specific, sensitive, rapid and accurate in diagnosing herpesviruses infection in infants, and it could detect herpesviruses DNA in specimens which were negative for IgM antibody by ELISA.</p>

Serotypes and antibiotics-resistance patterns of 247 strains of Haemophilus Influenzae isolated from children in Hangzhou / 中华儿科杂志

<p><b>OBJECTIVE</b>To investigate the serotypes and antibiotics-resistance patterns of Haemophilus influenzae isolated from children in Hangzhou.</p><p><b>METHODS</b>Isolates were identified with api-NH card. Serotypes were determined with slide agglutination method. The sensitivities of 13 antibiotics against 247 strains of Haemophilus influenzae were determined in vitro with Kirby-Bauer diffusion methods and MICs of ampicillin were determined with E-test. Nitrocefin test was used to detect beta-lactamase.</p><p><b>RESULTS</b>Of the 247 strains isolated from children during the period from August 2001 to July 2002, 153 strains were non-typable, while 94 strains (38.1%) were typable and 90.4% and 1.1% of them belonged to type d and type b, respectively. Higher incidence of typable Haemophilus influenzae was found in male than in female children and the difference was significant (chi(2) = 5.30, P < 0.05), while between upper and lower respiratory tract infected children the difference was not statistically significant (chi(2) = 3.60, P > 0.05). Forty-one isolates (16.6%) were beta-lactamase-positive and 14 strains could not grow on medium in antibiotics sensitivity test. Of all 233 isolates tested successfully, 85.4% were susceptible to ampicillin, and the sensitivity rate to cefaclor, ceftriaxone, cefotaxime, imipenem, rifampin, clarithromycin, and chloramphenicol were as high as 98.7%, 99.6%, 99.6%, 99.6%, 98.7%, 91.0%, and 90.6%, respectively. All strains were sensitive to amoxicillin/clavulanic acid, ampicillin/sulbactan and ofloxacin, while 107 strains (45.9%) were resistant to trimethoprim-sulfamethoxazole, followed by that of tetracycline (14.6%). Resistance to ampicillin and trimethoprim-sulfamethoxazole in typable isolates was statistically significantly higher than in non-typable strains. Twenty-six strains (10.5%) were multi-resistant isolates and the multi-resistance rate in beta-lactamase-positive strains were significantly higher than that in beta-lactamase-negative strains (chi(c)(2) = 146.8, P < 0.001).</p><p><b>CONCLUSION</b>Non-typable Haemophilus influenzae was the most common type in clinical strains isolated from children in Hangzhou, while type d was the overwhelming type and type b was uncommon in typable isolates. Incidence of typable isolates was higher in male than in female children, and it was apt to intergrow with other species of pathogenic bacteria. The proportion of beta-lactamase-positive strains was not high and ampicillin or other beta-lactam actibiotics were still the treatment of choice for infections with Haemophilus influenzae.</p>

Expression of brain-derived neurotrophic factor at acute inflammatory injury of the brain / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the expression of brain-derived neurotrophic factor (BDNF) mRNA and immunoreactivity in experimental acute inflammatory brain injury.</p><p><b>METHODS</b>Ten rats were inoculated with pneumococcus to establish the model of bacterial inflammatory brain injury and other 6 rats were used as normal controls. At 24 h after inoculating, the expression of BDNF mRNA and BDNF protein in brain tissue was detected by in situ hybridization and immunohistochemical methods, respectively.</p><p><b>RESULT</b>The necrosis of neuron in cerebral cortex and hippocampus was observed after infection. The increase of BDNF mRNA expression in the cerebral cortex and hippocampus of experimental animals was demonstrated at 24 h after inoculation: (0.1194 +/- 0.02941 compared with 0.0662 +/- 0.01176)A and (0.1608 +/-0.01854 compared with 0.0680 +/- 0.00946)A (P<0.01), respectively. Compared with controls the expression of BDNF protein in the cerebral cortex and hippocampus was enhanced at 24 h of inoculation:(177.04+/-43.66 compared with 79.79+/-7.23)mm(2) (P<0.01) and (81.78 +/-37.47 compared with 42.98 +/-20.44)mm(2) (P<0.01), respectively. Strong positive hybridization and immunoreactivity were observed in the infiltrated inflammatory cell in leptomeninges, subarachnoid cavity, ventricles and brain parenchyma in the brain from the experimental rats.</p><p><b>CONCLUSION</b>The expression of BDNF mRNA and BDNF protein increases following brain inflammatory injury, which supports the hypothesis that BDNF may constitute intrinsic neuroprotective mechanism as a part of the inflammatory response.</p>