The use of newborn screening dried blood spots for research: the parental perspective.

OBJECTIVE: To investigate the attitudes of Chinese parents regarding the storage of dried blood spots collected for newborn screening (NBS) and their use in research. METHODS: We conducted a hospital-based survey of parents and examined parental attitudes regarding (a) allowing NBS sample storage, (b) permitting use of children's NBS samples for research with parental permission, and (c) permitting use of children's NBS samples for research without parental permission. RESULTS: The response rate was 52 percent. Of parents surveyed, 68 percent would permit their infant's NBS sample to be stored for at least some length of time. If permission is obtained, 69 percent of parents "strongly agreed" or "agreed" to permit use of the NBS sample for research. If permission is not obtained, only 14 percent of parents "strongly agreed" or "agreed." There was no significant association between permitting use of NBS samples for research and parental gender, education, household income, number of children, or site of residence. CONCLUSIONS: This is the first survey of Chinese parents regarding the use of NBS samples for different types of research, with results indicating that most parents would permit their infant's sample to be stored and would support the use of NBS dried blood spots for research purposes.

Newborn screening for inborn errors of metabolism in mainland china: 30 years of experience.

The history of the Newborn Screening Program in Mainland China begins in 1981, when a pilot plan was developed that demonstrated the feasibility of its implementation. It has so far focused on the detection of congenital hypothyroidism (CH) and phenylketonuria (PKU) to prevent or reduce mental and physical developmental retardation in children. Throughout this period, a total of 35,795,550 dried blood samples (DBS) of newborns (NB) have been analyzed for PKU, and 35,715,988 for CH. During this period, 3,082 cases with PKU have been diagnosed, resulting in an incidence of 1 case per 11,614 (95% confidence interval 11,218-12,039) live births. In relation to CH, 17,556 cases have been confirmed, arriving at an incidence of 1 case per 2,034(95% confidence interval 2,005-2,065) live births. The biggest challenge for universal newborn screening is still to increase coverage to mid-western area. In Mainland China, MS/MS newborn screening started in 2004. In a pilot study, 371,942 neonates were screened, and 98 cases were detected with one of the metabolic disorders, and the collective estimated prevalence amounted to 1 in 3795 (95% confidence interval 3,168-4,732) live births, with a sensitivity of 98.99%, a specificity of 99.83%, and a positive predictive value of 13.57%. The most important is to get the government's policy and financial support for expanded screening.

[Study on the pathological and molecular characteristics of AHC epidemic in Zhejiang Province in 2010].

To identify and trace the pathogen of acute hemorrhagic conjunctivitis (AHC) epidemic in Zhejiang Province in 2010. Viral nucleic acid of Enterovirus (EV) and Coxsackievirus A24 variant (CA24v) were directly detected by real-time RT-PCR from the conjunctival swab collected from suspected patients. The virus was isolated from the swab samples using Hep-2 cell. The viral RNAs were extracted from the isolated viruses and followed by RT-PCR to amplify VP1 gene and 3C protease region(3C). The amplified fragments were sequenced and phylogenetic trees were also constructed. Eight out of 13 swab samples from suspected patients were both positive for EV and CA24v RNA (61.5%), 6 CA24v strains were isolated (46.2%). The complete VP1 genes of CA24v in 4 sequenced virus strains were 915 nt in length and the complete 3C genes were 549 nt in length. All VP1 and 3C genes were confirmed without any insertion or deletion. The identity of nucleotide and amino acid in 3C between the 2010 isolated strains and the prototype strain EH24/70 were 85.2%-85.8% and 96.2%-96.7%, and that between the 2010 Zhejiang strains and the Zhejiang,Yunnan and Guangdong CA24v strains isolated between 2007-2008 were 93.4%-93.8% and 96.7%-97.3%, respectively. The phylogenetic tree of 3C indicated that the isolated CA24v viruses of Zhejiang in 2010 located in the CA24v IV genotype cluster 4 (GIV-C4) and all the VP1 genes located in the human Enterovirus C (EV-C) CA24v. These findings indicated that AHC epidemic in Zhejiang Province in 2010 was caused by CA24v GIV-C4 viruses and they most likely evolved from CA24v viruses circulating locally in external environment from 2002.

[Study on the etiological and molecular characteristics of aseptic meningitis epidemic in Zhejiang Province in 2002-2004].

In order to confirm the cause of the outbreak of aseptic meningitis in Zhejiang Province in 2002-2004, trace the pathogen and analyze the molecular characteristics, 271 cerebrospinal fluid (CSF) and faeces specimens were collected from suspected patients. The virus strains from the specimens were isolated with RD and Hep-2 cell lines. The VP1 and VP4/VP2 genes of the isolated viruses were sequenced, and their phylogenetic and homology trees were also constructed. Of the total 271 samples, 78 Echovirus type 30 (E30) strains were isolated. All of the complete VP1 genes in 31 sequenced virus isolates of E30 were composed of 876 nt without any insertion or deletion, encoding 292 amino acids (aa). The identity of nucleotide and amino acid in VP1 gene were 84.7%-86.3% and 92.1%-94.2% between the 31 Zhejiang strains and the prototype strain Bastianni of E30, and 87.1%-99.4% and 96.2%-100% among the 31 Zhejiang strains, respectively. The Zhejiang strains of E30 in the phylogenetic tree of the VP1 gene were attributed into two branches of the G and H genotype, respectively. In G genotype, the Shangdong and Jiangsu E30 strains in 2003 among domestic strains and Ukraine E30 strain in 1999 among overseas strains had maximum similarity with the Zhejiang strains, while H genotype had maximum similarity with the Korea E30 strains in 2008. The phylogenetic tree of the VP4/VP2 genes was similar to that of VP1 gene. The results indicated that the outbreak of aseptic meningitis in Zhejiang Provinec in 2002-2004 was caused by the G and H genotypes of E30 strains existing simultaneously. The H genotype was a new variant strain, which was first isolated in Zhejiang Province in 2002.

[Molecular characteristics of noroviruses causing outbreaks of acute gastroenteritis in Huzhou].

To study the molecular characteristics of Noroviruses causing outbreaks of acute gastroenteritis in Huzhou. During April 2008 and February 2009, fecal specimens of patients collected from 2 outbreaks of acute gastroenteritis were tested for Norovirus by real-time RT-PCR. Partial sequence of RNA dependent RNA polymerase(RdRp) of the positive samples were amplified by RT-PCR, the PCR products were then purified, sequenced and phylogenetic analysis was conducted. Both genogroup II (GII) and genogroup I (GI) noroviruses were detected in 2 outbreaks. Phylogenetic analysis revealed that two of the GI norovirus strains isolated from 2008 belonged to genotype GI/2 and one of the GI Norovirus strain isolated from 2009 belonged to genotype GI/3. The other GIIú norovirus strains isolated from 2009 had high nucleotide identity with GIIb genotype that had been reported frequently in European countries during 2000 and 2001 and in Asian countries recently. These results suggested that the epidemic strains of norovirus isolated in Huzhou had a high degree of genetic diversity and prevalent genotypes at different times were also different. To our knowledge this is the first report of detecting GIIb variant in outbreaks of acute gastroenteritis in China.

[Molecular characteristics of norovirus in 3 outbreak-episodes of gastroenteritis in Zhejiang from 2008 to 2009].

OBJECTIVE: To study the molecular characteristic of norovirus in 3 outbreaks of gastroenteritis in Zhejiang province. METHODS: During January 2008 and December 2009, fecal specimens of patients were collected from 3 outbreaks of acute viral gastroenteritis. Noroviruses were detected by Real-time RT-PCR. Part of the positive samples were randomly selected and detected by RT-PCR. PCR products were sequenced. Sequence analysis was undertaken based on partial sequence of RNA dependent RNA polymerase (RdRp) and capsid protein gene. Some positive samples were amplified by 3'RACE (rapid amplification of cDNA 3'ends), 3200 bp in length. The exact whole ORF2, ORF3 and 3'untranslation regions (UTR) gene of norovirus were identified. RESULTS: There were in total 3 outbreaks of viral gastroenteritis caused by norovirus being reported. A total of 62 stools were obtained from cases with acute gastroenteritis. Noroviruses were detected in 41 cases including 27 strains of genogroup I norovirus and 9 strains of genogroup II norovirus, 5 strains of genogroup I + II norovirus. Four genotypes including GI.8, GII.b, GI.2/GI.6 recombination together with co-infection of GI.8 and GII.b were detected. CONCLUSION: Norovirus was confirmed as the major cause of outbreaks of viral gastroenteritis in Zhejiang province and multiple genotype of norovirus were identified from the outbreaks. It was the first time to have found a recombinant of GI.6 capsid and GI.2 polymerase norovirus as well as the co-infection of GI.8 and GII.b norovirus in the same sample.

[Study on the pathological and molecular characteristics of viral meningitis outbreaks in Linhai county, Zhejiang province, 2004].

OBJECTIVE: In order to confirm the causes of viral meningitis outbreaks in Linhai county, Zhejiang province in 2004, and to analyze the relationship between hereditary variation and evolution of the pathogen. METHODS: 60 cerebrospinal fluid (CSF) specimens were collected from the suspected patients. Virus strains from the specimens were isolated with RD and Hep-2 cell lines, and identified through neutralization test. VP1 and VP4/VP2 genes of the isolated viruses were sequenced. Both phylogenetic and homological trees were also constructed. RESULTS: 19 Echovirus type 30 (E30) strains were isolated from 60 CSFs, in which E30 accounted for 31.7%. All of the complete VP1 genes in 4 sequenced virus isolates of E30 were composed of 876 nt, encoding 292 amino acids (aa). The identity of nucleotide and amino acid in VP1 gene were 82.4% - 84.1% and 93.5% - 94.2% between the 4 Linhai strains and the prototype strain Bastianni of E30, were 87.1% - 99.9% and 97.9% - 100.0% among the 4 virus strains of E30 from Linhai, respectively. The 4 Linhai strains could be classified into two classes. The diversity of nt and aa was minimal in the same class but obvious between the two classes, with the range of diversities as 12.9% and 2.1%, respectively. The Linhai E30 strains had maximum similarity with the Zhejiang E30 strains in 2002 - 2003. The 4 Linhai strains of E30 in the phylogenetic tree of the VP1 gene were attributed into two branches of the G and H genotype, respectively. The G branch also included the E30 strains from Zhejiang, Jiangsu and Shangdong in 2003, while the H branch including E30 strains from Zhuji, Zhejiang in 2002. The phylogenetic tree of VP4/VP2 genes was similar to that of VP1 gene. CONCLUSION: The outbreak of viral meningitis in Linhai county in 2004 was caused by the two classes of E30 strains with G and H genotype existed simultaneously. The Linhai E30 strains had maximum genetic relations to the Zhejiang, Jiangsu and Shangdong strains of E30. The H genotype was inferred to be a new variant strain, which was first isolated in Zhejiang province in 2002.

[Analysis on VP1 gene variation of Coxsackie virus B5 for aseptic meningitis isolated from Zhejiang Province in 2008].

OBJECTIVE: Study meningoencephalitis virus isolated from the Coxsackie B5 virus(CVB5 virus) VP1 gene characteristics of Zhejiang Province in 2008 and compare with other countries CVB5 prototype isolates, and to explore the relationship of variation of the Virus VP1 areas and epidemic viral meningoencephalitis METHODS: Hep-2 and RD cells in cerebrospinal fluid and stool specimens for virus isolation, positive isolates combination with intestinal type of serum. On the separation of virus extracted RNA, and then RT-PCR amplified VP, gene CVB5 virus fragments, and purification of sequencing products, using DNAMAN and Bioedit analytical processing software. RESULTS: The VP1 gene of CVB5 isolated from Zhejiang province meningoencephalitis viral in 2008 was 735bp. There were no missing and insertion of nucleotide . Strains isolated from ZJ/12/02 with the nucleotide and amino acid sequence of the highest phylogenetic tree showed that they were not the same branch. CONCLUSION: The mutation caused by viral meningoencephalitis virus CVB5 Zhejiang province in 2008 was smaller. Viral meningoencephalitis distributed in some areas in the province had no significant prevalence.

Ortho effects on the change in electronic absorption spectrum of pyridinium salts of saturated bromohydrocarbon.

The quaterisation process of 1,2-dibromoethane and pyridine is in situ traced by electronic absorption spectrum. Two absorption peaks, induced by mono- and bis-pyridinium salt of 1,2-dibromoethane, appear at 429 nm and 313 nm, respectively. To explain the phenomena, several kinds of alkyl bromides with special structures were selected and compared by experimental measurement and theoretical calculation. The results indicate that for mono-pyridinium salt of 1,2-dibromoethane, the electron donor property of ortho-bromine group increases the electron cloud density of the carbon atom associated with pyridinium cation, which induces red-shift of absorption wavelength.

[Molecular epidemiology of norovirus in outbreaks of gastroenteritis in Zhejiang from 2006 to 2007].

OBJECTIVE: To study the molecular epidemiological characteristics of Norovirus gastroenteritis outbreaks in Zhejiang. METHODS: During January 2006 and December 2007, fecal specimens of patients collected from outbreaks of acute viral gastroenteritis were tested for Norovirus. Epidemiological data were also collected. Noroviruses were detected by a reverse transcription polymerase chain reaction (RT-PCR) and Real-time RT-PCR. Some positive samples were randomly selected and RT-PCR products were sequenced. Comparing to the nucleotide sequences of norovirus genotype I, II reference strains from GenBank, sequence analysis was undertaken based on partial sequence of RNA dependent RNA polymerase (RdRp) and capsid protein (VP1) gene. RESULTS: 5 outbreaks of viral gastroenteritis caused by Norovirus were reported. A total of 63 stools were obtained from cases with acute gastroenteritis. Noroviruses alone were detected in 45 cases and the illness appeared in autumn. Phylogenetic analysis revealed that Norovirus belonged to G II/G II 4 type. The strains isolated from Zhejiang were almost identical on G II/4 variants that causing epidemics in Beijing and in The Netherlands with the homology of 99.7% and 98.5%-99.0% respectively. Phylogenetic analysis revealed that the isolates were located at the same branch as the norovirus G II/4 variants found in Beijing and Netherlands. CONCLUSION: Norovirus is a major cause of outbreaks of viral gastroenteritis in Zhejiang province Genogroup II/4 variants viruses were the prevalent strains.

[An immunofluorescence assay for the detection of SARS associated coronavirus antibody based on recombinant nucleocapsid antigen and its application].

OBJECTIVE: To establish a new technique for SARS-CoV antibody test to detect infection of severer acute respiratory syndrome (SARS). METHODS: Nucleocapsid gene was obtained by reverse transcription and polymerase chain reaction from a SARS patient and inserted into the vector pFastBacHTa expressing baculovirus. Insect Sf9 cells were transfected with the recombinant baculovirus expressing SARS nucleocapsid antigen and then cultured, fixed by acetone so as to make SARS-specific antigen. Immunofluorescence assay (IFA) technique and plaque reduction neutralization test (PRNT) were used to detect 7 samples of sera of 4 newly diagnosed SARS patients collected in different days, 48 samples of convalescent sera of SARS patients, 24 serum samples of healthy person undergoing physical examination, and 40 serum samples from non-SARS patients with fever by double blind test. RESULTS: The recombinant SARS-specific antigen reacted only with SARS positive sera but not with normal sera. Double blind test showed that 45 of the 46 PRNT positive sera were IFA positive with an accordance rate of 97.8%. 7 samples of sera from 4 SARS patients in acute progressive stage in Guangdong province were all IFA positive. SARS antibody could be detected since the sixth day after onset, and the titer increased from 1:40 to 1:600 on the ninth day. CONCLUSION: Immunofluorescence assay is highly specific and sensitive in detection of SARS. This reagent is safe and easy to prepare.

[Isolation and sequencing of VP1 region of enterovirus 71 strains in Zhejiang, China].

OBJECTIVE: To study the gene characterization of enterovirus 71 (EV71) virus strains isolated from clinical specimens of children with hand-foot-and-mouth disease (HFMD) in Zhejiang province. METHODS: Virus were isolated from clinical samples including stool, throat swab and vesicle from patients with HFMD. The EV71 isolates were identified by microneutralization assay and reverse transcriptase PCR (RT-PCR) with specific primer pair for VP1 genes of EV71. Complete VP1 gene sequences (891 nucleotides) for recent 6 EV71 isolates were determined and compared with that of A, B, C genotype reference EV71 strains and 11 EV71 China isolates available from GeneBank by homogeneity and phylogenetic tree analyses. RESULTS: 9 strains of EV were isolated from 14 clinical specimens. Data from microneutralization and RT-PCR results indicated that all the strains belong to EV71. The nucleotide and amino acid homogeneity of these 6 Zhejiang strains with the representative isolates of A and B genotypes were 82.9%-85.5% and 94.9%-98.0% respectively; with the representative isolates of C were 89.2%-94.1% and 97.0%-99.0% respectively. There were 91.0%-92.2%, 90.2%-90.3%, 89.2%-89.5%, 96.7%-96.9% nucleotide, homology with representative strains of C1, C2, C3,C4 subgenotypes of EV71. The nucleotide homogeneity of these 6 EV71 isolated strains with 9 previously isolated Chinese strains appeared to be 93.8%-97.1%. These 6 EV71 isolated strains were within genotype C subgenogroup C4 in the phylogenetic tree. CONCLUSION: The recently identified EV71 isolates in Zhejiang province belonged to subgenogroup C4.