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>

Screening for phenylketonuria in 726,998 neonates in Zhejiang Province / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To analyze the results of screening for neonatal phenylketonuria (PKU) in Zhejiang Province.</p><p><b>METHODS</b>The screening for neonatal PKU was conducted among 726,998 newborns in Zhejiang Province. Heel prick blood specimens were collected around 72 h after birth with 6 intakes of high protein milk and the specimens were dried on S and S903 filter papers. Phenylalanine (Phe) levels were determined quantitatively with Perkin Elmer Neonatal Fluorometric PKU kits.</p><p><b>RESULTS</b>Among 726,998 newborns, elevated blood Phe levels were found in 152 infants. They were all recalled for serum amino acid analysis and 32 were confirmed to have PKU with 19 males and 13 females. The earliest time of confirmation was 16 d and latest was 105 d with the median of 32 d.</p><p><b>CONCLUSION</b>The data shows that the detection rate of screening for neonatal phenylketonuria in Zhejiang Province was 1/22,718.</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>

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>

Molecular diagnosis of the specific DNA patterns of 16S-23S rRNA gene of bacteria / 中华儿科杂志

<p><b>OBJECTIVE</b>To establish the specific 16S-23S rRNA gene spacer regions pattern in different bacteria using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), DNA cloning and sequences analysis.</p><p><b>METHODS</b>A pair of primers were selected from highly conserved sequences adjacent to the 16S-23S rRNA spacer region. Bacterial DNA of sixty-one strains of standard bacteria and corresponding clinical isolates representative of 20 genera and 27 species was amplified by PCR, and further studied by RFLP, DNA cloning and sequences analysis. Meanwhile, all specimens were examined by bacterial culturing and PCR-RFLP analysis.</p><p><b>RESULTS</b>The 27 different standard strains showed one, two, three or more than three bands. The sensitivity of PCR reached 2.5 colony-forming unit (CFU), and there was no cross reaction to the human, fungal or viral genomic DNAs. Fifteen species could be distinguished immediately by PCR, while another 10 species were further identified by Hinf I or Alu I digestion. Klebsiella pneumoniae (Kp) and Enterococcus durans (Ed) could not be differentiated from each other by Alu I or Hinf I digestion. The spacer sequences of the Kp and Ed were 908 bp and 909 bp, respectively, and they differed only at the site of the 779th nucleotide. The former was G, and the latter was A. The 760 - 790 bp sequence of Kp was as follows: CGACTGCACCGCCTCCTAC / GGCCGCGTATTC. The 760 - 790 bp sequence of Ed was as follows: CGACTGCAC CGCCTCCTAC / AGCCGCGTATTC. Only one enzyme XmaIII, could discriminate the two. The cleaving site of XmaIII is C downward arrow GGCCG. Kp DNA was cleaved into 778 bp and 130 bp fragments, while E. durans was not. Of 42 specimens with suspected septicemia, 15 were positive (35.7%) on blood culture, and 27 on PCR (64.29%). The positive rate of PCR was significantly higher than that of blood culture (P < 0.01). Of the six CSF specimens, one was positive for Staphylococcus epidermidis (Se) on culture as well as by PCR, while two specimens which were negative on cultures were positive by PCR and were diagnosed as Se according to its DNA pattern. One specimen was culture-positive for Cryptococcus neoformans (Cn) but was negative by PCR. The other two specimens were negative by both PCR and culture. Fifteen blood samples from healthy children were negative by both blood culture and PCR.</p><p><b>CONCLUSIONS</b>The method of detecting bacterial 16S-23S rRNA spacer regions using PCR-RFLP techniques was specific, sensitive, rapid and accurate in detecting pathogens in clinical bacterial infections.</p>

Analysis of TSH screening results of 42,979 newborns in Zhejiang Province / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>Document the prevalence of congenital hypothyroidism in Zhejiang Province, by neonatal screening of TSH.</p><p><b>METHODS</b>DELFIA neonatal TSH kit was applied for the quantitative determination of thyrotropin in blood specimens dried on filter paper.</p><p><b>RESULT</b>Among the 42 979 newborns, 112 had elevated hTSH concentration in blood. All had T3, T4 and TSH concentrations measured in serum and 29 were diagnosed as congenital hypothyroidism. The diagnosis time was 13 to 59 days and mean time was (27.9+/-9.2) days.</p><p><b>CONCLUSION</b>The incidence of congenital hypothyroidism in Zhejiang Province is not low. The TSH screening in newborns is significant in eugenics and the improvement of population quality.</p>