Expert consensus on nucleic acid amplification test of respiratory pathogens in children / 中华实用儿科临床杂志

Acute respiratory tract infection is the most common infectious disease in children, which seriously threatens children′s health.Rapid and accurate etiological diagnosis is of great significance for the clinical treatment and control of these diseases.Pathogen nucleic acid test was applied and became the main method of respiratory tract infection diagnosis for its high sensitivity and specificity.To regulate the application of pathogen nucleic acid amplification test in respiratory tract infection in children, improve the diagnosis level, expert consensus on nucleic acid amplification test of respiratory pathogens in children was prepared to guide the application and promote pathogens diagnosis ability.

An accurate analysis of the viral pathogen test results in children with acute respiratory tract infection / 中华实用儿科临床杂志

Acute viral respiratory tract infections among children have caused a substantial health burden worldwide.Early, rapid, and accurate diagnosis of viral etiologies is of great significance to optimal treatment, prevention of viral outbreaks and unnecessary use of antibiotics, and a favorable prognosis.At present, there are numerous laboratory diagnostic methods.It is urgent to correctly analyze the viral pathogen test results in children with acute respiratory tract infection using optimal methods.This review summarizes the diagnostic methods currently in use or soon to be applied in clinical practice and their interpretations of clinical reports, aiming to improve the understanding of the pathogenic results of respiratory viruses.

Correlation between human bocavirus 2 infection in children and acute diarrhea in Beijing / 中华儿科杂志

Objective@#To investigate the relationship between human bocavirus 2 (HBoV2) infection and acute diarrhea in children younger than 5 years of age in a case-control study.@*Methods@#This was a prospective case-control study. During May 2016 to December 2016, fecal specimens were collected from children ≤5 years of age with acute diarrhea who visited the Affiliated Children's Hospital of Capital Institute of Pediatrics (case group), or from children ≤5 years of age without diarrhea from Longtan Community Medical Service Center, Beijing (control group). The case group (n=240) and the control group (n=240) were divided into 8 age subgroups: ≤1 month old, >1-3 months old, >3-6 months old, >6-12 months old,>1-2 years old,>2-3 years old,>3-4 years old and >4-5 years old, and there were 30 cases in each age subgroup. The specimens were tested for 7 types of diarrhea-associated viruses, especially for HBoV2 by real-time PCR method. The HBoV2 viral load was predicted according to the cycle threshold (Ct). Finally, t-test was used to compare the differences between groups.@*Results@#In the case group (n=240), the positive rate of norovirus was 16.7% (40 cases); rotavirus, 10.8% (26 cases); HBoV2, 7.5% (18 cases); adenovirus, 7.1% (17 cases); astrovirus, 6.3% (15 cases); parachovirus, 3.8% (9 cases); and Aich virus, 0.4% (1 case). The positive rates of HBoV2 in case group (7.5%, 18 cases) and control group (5.0%, 12 cases) showed no significant difference (χ2=1.280, P=0.258), as well as in different age groups (all P>0.05) . However, the mean viral load of the HBoV2 in the case group (1×109copies/L with cycle threshold (Ct) 25.8) was higher than that of control group (1×105copies/L with Ct 33.8), showing a significant difference (t=0.597, P=0.000).@*Conclusions@#Norovirus and rotavirus are still the important viral pathogens in children with acute diarrhea. A higher load of HBoV2 may indicate a higher risk of acute diarrhea in children ≤5 years of age in Beijing.

Epidemiological features of prevalent influenza A viruses in children with influenza-like illness during the 2004-2017 season in Beijing / 中华儿科杂志

Objective@#To analyze and compare the epidemiological features of prevalent influenza A viruses in children in Beijing during 13 consecutive surveillance seasons from 2004 to 2017.@*Methods@#This was a repeated cross section study. Throat swabs were collected weekly from children with influenza-like illnesses (ILI) who presented to the outpatient/emergency department of Children's Hospital, Capital Institute of Pediatrics during the period from September, 2004 to August, 2017. All of the specimens were inoculated into Madin Darby canine kidney (MDCK) cells to isolate influenza viruses followed by identifying different types of influenza viruses with reference antisera by hemagglutination-inhibition assay. Descriptive statistics, t test and chi-square test were used to analyze the characteristics of prevalent influenza and characteristics of children infected with different types of influenza viruses.@*Results@#Out of 10 984 specimens from ILI tested for influenza viruses, 1 052 (9.6%) were positive for influenza A viruses, and the positive rate was higher than that of influenza B viruses (6.7%, 741/10 984). Out of 1 052 cases positive for influenza A viruses, 70 cases of seasonal H1N1, 302 cases of 2 009 pandemic H1N1 and 680 cases of H3N2 were identified. The mean age of children with influenza A was (4.2±2.9) years, in whom 55.5% (584/1 052) were male. The mean age of children infected with seasonal H1N1, 2009 pandemic H1N1 and H3N2 was (4.6±2.1) , (4.3±3.1) and (4.2±2.9) years, respectively. There was no significant difference in the mean age among children infected with different subtypes of influenza A viruses (seasonal H1N1 vs. H3N2: t=1.139, P=0.255; 2009 pandemic H1N1 vs. H3N2: t=0.631, P=0.528; seasonal H1N1 vs. 2009 pandemic H1N1: t=0.720, P=0.472), while the mean age of children with influenza B was higher than that of the patients with influenza A ((5.2±2.7) vs. (4.2±2.9) years, t=7.120, P=0.000). The infection rate of influenza A in children with each age group was significantly different from that of influenza B. The infection rate of 2009 pandemic H1N1 and H3N2 increased with age, except for the patients of 0-6 months. Meanwhile, the infection rate of H3N2 in children aged 6 months to 12 years was higher than that of seasonal H1N1 and 2009 pandemic H1N1 (all P<0.05). The influenza A epidemic peaked earlier than that of influenza B when the positive rate of influenza A was higher than that of influenza B, and vice versa. After 2009, circulating strain was substituted by 2009 pandemic H1N1 virus with higher positive rate, while previous seasonal H1N1 had not been detected. The 2009 pandemic H1N1 circulated at high level in two consecutive seasons, which was followed by low level in next season. H3N2 epidemic peaked mostly in winter and spring each year, however, the epidemic wave of H3N2 with high virulence occurred so early in the summer in the year of 2009 H1N1 pandemic.@*Conclusions@#The characteristics of prevalent influenza A viruses in children were different among 13 surveillance seasons from 2004 to 2017 in Beijing. The 2009 pandemic H1N1 and H3N2 became the predominant strains of influenza A virus.

Association of human rhinovirus infection with asthma in children / 中华实验和临床病毒学杂志

Asthma is a serious global health problem affecting all age groups. It has been recognized as a complex disease resulting from interactions between multiple genetic and environmental factors. Human rhinovirus (HRV) infections in early life constitute a major environmental risk factor for the development of childhood asthma and the most common cause of asthma exacerbation. HRV-C, the novel HRV, using cadherin-related family member 3 (CDHR3) as receptors, plays a key role in the development of childhood asthma. This review discusses the role and the potential underlying mechanisms of HRV infections in the development and exacerbation of childhood asthma based on recent clinical and experimental evidence.

Clinical analysis of children in hospital with adenovirus pneumonia in Beijing from 2015 to 2016 / 中华实验和临床病毒学杂志

Objective@#To study the clinical characteristics of children with adenovirus pneumonia and provide evidence for clinical diagnosis and treatment timely.@*Method@#This retrospective study included 89 children who were confirmed to have adenovirus pneumonia in hospital from January 2015 to December 2016. All the immunofluorescence test result of the 89 children showed that the exfoliated nasopharyngeal cells from the 89 children were all adenovirus antigen positive. All the severe type children reached the diagnostic criteria of severe pneumonia by the respiratory group in the society of pediatrics, Chinese Medical Association. The children were divided into 2 groups (severe type group and common type group). Different factors such as epidemiologic feature, clinical manifestation, laboratory examination and imaging data were analyzed.@*Results@#Among the 89 pediatric patients, the male to female ratio was 1.5∶1. The ages ranged from 1 month to 14 years. Children under 5 years of age accounted for 96.6%(86/89). The incidence was 37.1%(33/89)in winter and 30.3%(27/89)in spring. The lengths of hospital stay were 3-48 days and the median length of stay was 8.25±4.75 days. All of these 89 cases had fever and cough. The proportion of severe adenovirus pneumonia was high among male, under 2 years of age, those with dyspnea, hepatosplenomegaly, tachycardia, leukocytosis, elevated C-reactive protein (CRP), PCT, myocardial enzymes, electrocardiogram abnormality and cluster shadow in chest CT. Differences were statistically significant (P<0.05).@*Conclusions@#Special attention should be paid to the male children under the 2 years of age with abnormal related indicators to consider severe adenovirus pneumonia. Early detection and treatment are the key to improve treatment and reduce death.

Clinical analysis of human bocavirus infection in children with severe lower respiratory tract infection / 中华实用儿科临床杂志

Objective To explore the clinical characteristics of human bocavirus (HBoV) infection in the children with severe lower respiratory tract infection.Methods The clinical data of the hospitalized children with sputum HBoV-positive were analyzed retrospectively,who were hospitalized at the Pediatric Intensive Care Unit of Children's Hospital Affiliated to Capital Institute of Pediatrics from September 1,2016 to March 31,2017.Results A total of 17 children were included in the study.The ratio of male to female was 15 ∶ 2.The diagnostic age ranged from 4 months to 4 years and 10 months old.82.4％ (14/17 cases) of the patients were infants less than 2 years old.Autumn and winter were high-occurrence seasons.Pediatric Critical Illness Scores(PCIS) were 68-88 scores(median 82 scores).PCIS was less than 70 scores in 1 case,and between 70 scores and 80 scores in 7 cases,and more than 80 scores in 9 cases.The main clinical manifestations were respiratory system involvement,including dyspnea in 17 cases (100％),fever in 14 cases (82.4％),cough in 16 cases (94.1％),wheezing in 13 cases (76.5％),and moist rales in 13 cases (76.5％).The main abnormal chest radiological findings showed patchy shadows in 7 cases (41.2％) and consolidation in 6 cases (35.3％).The oxygenation index was 73.9-296.0 mmHg(1 mmHg =0.133 kPa),and median was 176 mmHg.The oxygenation index was between 100-200 mmHg in 7 cases(41.2％),and less than 100 mmHg in 2 cases(11.7％).Type Ⅰ respiratory failure occurred in 11 cases (64.7％),while type Ⅱ respiratory failure occurred in 6 cases (35.3 ％).All of the patients need respiratory support with mechanical ventilation.Among them,6 patients (35.3％) were treated by non-invasive ventilation and their ventilation time were 25-128 h(median 65 h),while 11 patients (64.7％) were treated by invasive ventilation and their ventilation time was 42-178 h(median 70 h).Other organ or system dysfunction is mild.The length of hospital stay ranged from 3 days to 13 days.The cure rate was 100％.Conclusions HBoV infection in the children with severe lower respiratory tract infection is common in infants under 2 years old,with acute onset and rapid recovery.The obvious clinical symptoms are fever,cough,wheezing and dyspnea.Although respiratory failure is common,other organ or system dysfunction is mild,which may bring about a good prognosis.

Molecular biological and clinical characteristics of respiratory syncytial virus in children with bronchiolitis / 中华儿科杂志

Objective@#To investigate the clinical characteristics of respiratory syncytial virus(RSV)bronchiolitis and molecular biological characteristics of RSV in children in Beijing.@*Method@#In a systematic retrospective study, 2 296 nasopharyngeal aspirates (NPA) were collected from children diagnosed with bronchiolitis from July 2006 to June 2016 for respiratory virus screening using direct immunofluorescence assay (DFA). For specimens positive for RSV, subgroup A or B was confirmed by real time RT-PCR and genotype of RSV was determined by amplifying the full G glycoprotein gene and sequencing. Clinical data were evaluated by the modified Tal score to compare the severity between RSV subtypes, as well as genotypes. Statistical analyses were performed using t test, Mann-Whitney U test and χ2 test.@*Result@#In 2 296 bronchiolitis cases, 961(41.9%) were RSV positive, including 719(74.8%) RSV A and 236 (24.6%) RSV B. The dominant RSV subtype changed from year to year: A-A-B-B-A-A-B-AB-A-AB and more bronchiolitis cases were identified in RSV A dominant years. Six genotypes of RSV A (NA1, NA2, NA3, NA4, GA5 and ON1) and 5 genotypes of RSV B (BA3, BA7, BA9, BA10 and CB1) were prevalent in Beijing. The dominant genotypes of RSV A were NA1 (55.9%) with high rates (50.0%-100%) before 2014 and ON1 (39.1%), mainly detected after 2014, while BA9 (90.6%) was the absolute dominant RSV B genotype. No significant difference in the severity of bronchiolitis was shown between cases of RSV A and B. Children positive for NA1 were more likely to stay longer in hospital (Median time: 8 days) compared to the group positive for ON1(Median time: 6 days ) (U=1.035, P=0.005) and had higher proportion of moderate to severe degree symptoms (Moderate: 41.0%, Severe: 10.0%) compared with ON1 group (Moderate: 22.9%, Severe: 4.3%) (U=9.785, P=0.008). In the group positive for ON1, more children had fever (ON1: 38.6%, NA1: 15.0%) (χ2=11.064, P=0.001) and more were younger than 3 months(ON1: 54.3%, NA1: 33.0%) (χ2=77.408, P<0.001).@*Conclusion@#The dominant RSV subgroup changed from year to year with a shifting pattern. The correlation between RSV genotypes and the severity of disease was documented in the study.

Clinical value of a rapid respiratory syncytial virus antigen detection in point-of-care testing / 中华儿科杂志

Objective@#To evaluate the clinical value of a rapid respiratory syncytial virus (RSV) antigen detection in point-of-care testing (POCT).@*Method@#A total of 209 specimens, including 78 throat swabs (TS) and 131 nasopharyngeal aspirates (NPAs), were collected from inpatients who visited the Children′s Hospital Affiliated to the Capital Institute of Pediatrics and were diagnosed as acute respiratory infection from 5 January to 7 February, 2015. These specimens were tested for RSV by a rapid antigen detection kit which was compared with reverse transcription polymerase chain reaction (RT-PCR) and direct immunofluorescence assay (DFA) for RSV detection.@*Result@#Compared with DFA for NPAs, the sensitivity and specificity of rapid antigen detection were 83.9% and 97.3%, respectively, with Kappa value of 0.86; Compared with RT-PCR, the sensitivity (NPAs, 74.2%; TS, 77.8%) and specificity (NPAs, 100.0%; TS, 92.0%) of rapid antigen detection were high, too, with Kappa value of 0.74 in NPAs and 0.62 in TS. However, the RSV positive rate of rapid antigen detection in TS (21.7%) from pediatric patients with acute lower respiratory tract infection was lower than that in NPAs (78.3%), as well as that of RT-PCR (7.3% in TS verse 78% in NPAs). The RSV rapid antigen detection kit can be finished in about 10 minutes.@*Conclusion@#With characteristics of high specificity, high sensitivity, being rapid, efficient and easy to operate in comparison with DFA and RT-PCR, RSV rapid antigen detection in this study is suitable for POCT. For pediatric patients with acute respiratory tract infection, NPA was better than TS for RSV detection.

Clinical features of human metapneumovirus and human bocavirus infections among children with acute respiratory ;tract infections in Taiyuan / 临床儿科杂志

Objective To investigate the status and clinical and epidemiological characteristics of human metapneumovirus (hMPV) and human bocavirus (HBoV) infections in children with acute respiratory tract infections (ARTIs) in Taiyuan. Methods A total of 549 children with ARTIs from November 2012 to May 2013 and November 2013 to May 2014 were recruited. The pharyngeal swab specimens were collected. The hMPV and HBoV were detected by using real-time PCR. Results In 549 children, 56 children (10.2%) were hMPV positive on swab specimens, 15 children (2.7%) were HBoV positive on swab specimens. The detection rates of hMPV and HBoV in November 2012 to May 2013 were 12.3%and 2.0%, respectively, and in November 2013 to May 2014 were 6.5%and 4.0%, respectively. The detection rate of hMPV was signiifcantly different between two periods (P<0.05), while the detection rate of HBoV has no signiifcant difference between two periods. In different months, the detection rate of hMPV and HBoV showed no signiifcant difference. The highest detection rates of hMPV and HBoV were all in children younger than two years old. The highest detection rate of hMPV was in children with asthmatic bronchitis or bronchiolitis. Conclusion In Taiyuan, during the monitoring periods, the ARITS are associated with childhood hMPV and HBoV infection especially in infants and toddlers. hMPV is one of the most important pathogens in infants and toddlers with wheezing.

Analysis on the change of genotype of enteroviruses associated hand, foot and mouth disease in Beijing during 2013 to 2014 / 中华儿科杂志

<p><b>OBJECTIVE</b>To analyze the genotype, epidemic pattern and the characteristics of the disease of enteroviruses during the epidemic season of hand, foot and mouth disease (HMFD) in children from 2013 to 2014 in Beijing to provide the scientific evidence for prevention and treatment of HFMD.</p><p><b>METHOD</b>During April to September in 2013 and March to October in 2014, a total of 977 throat swabs were collected from children who visited the Children's Hospital Affiliated to Capital Institute of Pediatrics, including 147 from patients with HFMD in 2013, 343 with HFMD, 201 with atypical HFMD, 83 with herpangina, 25 with fever with convulsions, 64 fever with rash and 114 with rash in 2014. Enteroviruses universal type (EV), Enteroviruses type 71 (EV71) and Coxsackievirus group A 16 (CA16) were detected by real-time RT-PCR respectively. The nucleic acid of specimens which were identified with non-EV71, non-CA16 was tested by nested PCR and analyzed by VP1 sequencing. The detection rate and epidemic pattern of different genotypes of enterovirus were analyzed among different age groups and between 2013 and 2014.</p><p><b>RESULT</b>Of 977 throat swabs, 80. 1% samples were detected positive for enteroviruses. The positive rates of CA16, EV71, CA6, CA10, CA4 and other EVs were 25. 6% (250/977), 18. 9% (185/977), 20. 0% (195/977), 5. 0% (49/977), 1.5% (15/977) and 9.1% (89/977), respectively. Twenty six of the 89 other EVs included CA2, CA5, CA8, CA9, CA12, CA14, CB2, CB5, E6, E9 and E25, each genotype of which was no more than 3. The nucleotide homologies shared among CA6, CA10 and CA4 strains between 2013 and 2014 were 94. 3% - 100%, 93. 8% - 99. 1% and 92.7% - 99. 8%, respectively. The positive rates of ≤1 year group were 71. 1% (106/149), which was lower than that of other age groups (all P <0. 05), but similar to that of >5 year group (χ2 =1. 181,P = 0. 277). In 2013, the positive rate of EV was 85. 7% (126/147) and the predominant genotype was CA6 54. 8% (69/126), followed by CA16 20. 6% (26/126) and EV71 11. 9% (15/126). In 2014, the positive rate of EV was 85. 4% (293/343) in the 343 children with HFMD, the predominant genotypes were CA16 with the positive rate of 42. 7% (125/293), EV71 with 38. 2% (112/293) and CA6 with only 11. 3% (33/293). In 2014, the positive rates of EV in 201 atypical HFMD, 83 herpangina, 25 fever with convulsions, 64 fever with rash and 114 rash were 83. 6% (168/201), 80. 7% (67/83), 76. 0% (19/25), 64. 1% (41/64) and 60. 5% (69/114), respectively. All genotypes of enteroviruses peaked mainly during May to August every year, but there were no obvious epidemiological pattern about each genotype.</p><p><b>CONCLUSION</b>CA6 became the main causative agent of HFMD in 2013, however, CA16 and EV71 predominated again in 2014 in Beijing. The clinical manifestations caused by CA6, CA10, CA4 and other genotype of enteroviruses differed from EV71 and CA16. Besides EV71 and CA16, more attention should be paid to CA6, CA10, CA4 and other type of enteroviruses.</p>

Human parechovirus associated sepsis and central nervous system infections in hospitalized children / 中华儿科杂志

<p><b>OBJECTIVE</b>Human parechovirus (HPeV) is a single-stranded, positive sense RNA virus in the Parechovirus genus within the large family of Picornaviridae. As a possible new pathogen of neonatal sepsis, meningoencephalitis and other infections in young children, HPeV gets more and more attention. This study aimed to better understand the association of HPeV with central nervous system (CNS) infectious diseases and sepsis among hospitalized children in Beijing.</p><p><b>METHOD</b>A total of 577 cerebrospinal fluid (CSF) samples were retrospectively collected from 557 children suspected of CNS infections in 2012. Three hundred and fifty-one of them were male and 206 were female. HPeV was screened by reverse transcription-nested PCR (RT-nPCR) with the universal primers which target the highly conserved 5'UTR. The positive samples were genotyped by amplifying and sequencing for the VP3/VP1 junction region. The sequences were compared with the HPeV sequences from GenBank and performed phylogenetic analysis.Some samples other than CSF from HPeV positive children, including serum, nasopharyngeal aspirate and stool, were collected and carried out screening for HPeV.</p><p><b>RESULT</b>With the RT-nPCR by universal primers, HPeVs were detected in 18 out of 577 CSF samples obtained from 18 children with a positive rate of 3.1%. The ratio of male and female was 2: 1. There were no statistically significant differences on infection rate between boys (12/351, 3.4%) and girls (6/206, 2.9%). All of 18 positive CSF samples were negative for enterovirus, Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), and herpes simplex virus 1 and 2 (HSV).HPeVs from 10 positive CSF samples were genotyped successfully, consisting of 7 HPeV3 and 3 HPeV1. In addition, 2 of 8 serum samples were positive for HPeV3 and 1 of 2 stool samples were positive for HPeV 1. HPeVs were identified in CSF from children aged from 15 days to 14 years, in which 7 cases were infants younger than 3 months and 5 cases were infants from 3 months to one year. Three children older than the age of 9 years (9, 13 and 14 years) were positive for HPeV. Most of the children (6/8) infected with HPeV3 were younger than 3 months and were diagnosed as sepsis, while the rest of HPeV3 positive children were diagnosed as meningitis and bronchopneumonia. HPeV3 infection clustered in August, while HPeV1 in January.</p><p><b>CONCLUSION</b>HPeVs were associated with CNS infections and sepsis in hospitalized children in Beijing, especially in children younger than one year.HPeV3 was the predominant type identified in CSF.</p>

Effect of human rhinovirus infection in pediatric patients with influenza-like illness on the 2009 pandemic influenza A(H1N1) virus / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Some research groups have hypothesized that human rhinoviruses (HRVs) delayed the circulation of the 2009 pandemic influenza A(H1N1) virus (A(H1N1)pdm09) at the beginning of Autumn 2009 in France. This study aimed to evaluate the relationship between HRV and A(H1N1)pdm09 in pediatric patients with influenza-like illness in Beijing, China.</p><p><b>METHODS</b>A systematic analysis to detect A(H1N1)pdm09 and seasonal influenza A virus (FLU A) was performed on 4 349 clinical samples from pediatric patients with influenza-like illness during the period June 1, 2009 to February 28, 2010, while a one-step real-time RT-PCR (rRT-PCR) assay was used to detect HRV in 1 146 clinical specimens selected from those 4 349 specimens.</p><p><b>RESULTS</b>During the survey period, only one wave of A(H1N1)pdm09 was observed. The percentage of positive cases for A(H1N1)pdm09 increased sharply in September with a peak in November 2009 and then declined in February 2010. Data on the monthly distribution of HRVs indicated that more HRV-positive samples were detected in September (2.2%) and October (3.3%), revealing that the peak of HRV infection in 2009 was similar to that of other years. Among the 1 146 specimens examined for HRVs, 21 (1.8%) were HRV-positive, which was significantly lower than that reported previously in Beijing (15.4% to 19.2%) (P < 0.01). Overall, 6 samples were positive for both A(H1N1)pdm09 and HRV, which represented a positive relative frequency of 1.60% and 2.08% HRV, considering the A(H1N1)pdm09-positive and -negative specimens, respectively. The odds ratio was 0.87 (95% CI 0.32; 2.44, P = 0.80).</p><p><b>CONCLUSIONS</b>HRVs and A (H1N1)pdm09 co-circulated in this Chinese population during September and October 2009, and the HRV epidemic in 2009 did not affect A(H1N1)pdm09 infection rates in Beijing, China as suggested by other studies. However, the presence of A(H1N1)pdm09 might explain the unexpected reduction in the percentage of HRV positive cases during the period studied.</p>

Virus profile in children with acute respiratory infections with various severities in Beijing, China / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Acute respiratory infection (ARI) is one of the most common infectious diseases in infants and young children globally. This study aimed to determine the virus profile in children with ARI presenting with different severities.</p><p><b>METHODS</b>Clinical specimens collected from children with ARI in Beijing from September 2010 to March 2011 were investigated for 18 respiratory viruses using an xTAG Respiratory Viral Panel Fast (RVP Fast) assay. The Pearson chi-square analysis was used to identify statistical significance.</p><p><b>RESULTS</b>Of 270 cases from three groups of ARI patients, including Out-patients, In-patients and patients in the intensive care unit (ICU), viruses were detected in 176 (65.2%) specimens with the RVP Fast assay. The viral detection rate from the Out-patients group (50.0%) was significantly lower than that from the In-patients (71.1%) and ICU-patients (74.4%) groups. The virus distribution was different between the Out-patients group and the other hospitalized groups, while the virus detection rate and distribution characteristics were similar between the In-patients and ICU-patients groups. The co-infection rates of the Out-patients group, the In-patients group, and the ICU-patients group were 15.6%, 50.0% and 35.8%, respectively. In addition to respiratory syncytial virus (RSV) and adenovirus (ADV), human rhinovirus (HRV) was frequently detected from children with serious illnesses, followed by human metapneumovirus (hMPV), human bocavirus (HBoV) and coronaviruses. Parainfluenza virus 3 (PIV3) was detected in children with lower respiratory illness, but rarely from those with serious illnesses in the ICU-patient group.</p><p><b>CONCLUSION</b>In addition to so-called common respiratory viruses, virus detection in children with ARI should include those thought to be uncommon respiratory viruses, especially when there are severe ARI-related clinical illnesses.</p>

A duplex RT-PCR for identifying genotypes of human metapneumovirus from clinical samples / 中华检验医学杂志

Objective To develop a convenient reverse transcription PCR(RT-PCR)method for identifying genotypes of human metapneumovirus(hMPV)from clinical samples.Methods According to the gene sequences of hMPV G with different genotypes,the A and B genotype specific primers were designed.A diplex RT-PCR was applied to identify different genotypes according to the molecular weight of PCR products in agarose gel.37 clinical samples were detected through this method.Results It was convenient to distinguish different genotypes of hMPV(383 bp for A and 284 bp for B)by the diplex RTPCR,and there was no non-specific amplification for common respiratory viruses.so it meant that the specificity of primers was good.The results of genotyping 37 clinical samples showed that 20 samples were identified as genotype A by both sequence analysis of M gene and diplex RT-PCR,whereas 17 samples were identified as genotype B by sequence analysis of M gene.but in these 17 samples 14 samples were identified as genotype B by the diplex RT-PCR and remaining 3 samples could not be genotyped because there was no PCR product after amplification.The consistency rate for these two methods Was 91.9%[(20+14)/37].Conclusion The method of diplex RT-PCR Was developed successfully and can be used for identify genotypes of hMPV.

Sequence analysis for the 3' end of genomes from enterovirus 71 isolated in Beijing during 2008-2009 / 中华微生物学和免疫学杂志

Objective To investigate the sequences of the 3' end of genomes from enterovirus 71 isolated from pediatric patients with different symptoms in Beijing during 2008-2009.Methods Clinical specimens were collected from pediatric patients suffering from hand,foot and mouth disease(HFMD)and/or with neurological complications who visited the affiliated Children's Hospital during the epidemic seasons of 2008 and 2009 in Beijing.The samples were inoculated into the Vero cell line,and the virus isolates were further identified by nested reverse transcription-nested PCR(RT-nPCR)assay using universal enterovirus primers,type specific primers for EV71 and CA16.The 3' end of genomes(including 3D and 3' UTR regions)from 10 EV71 strains derived from various clinical presentations were amplified and sequenced.Results Analysis of the nucleotide sequences of the amplified fragments showed that the 3' end of genomes of 10 EV71 isolates include the 3D region of 1386 nucleotides(nt)encoding the 3D polymerase(3Dpol,462 amino acids),the terminator codon TGA and 3' UTR of 81 nt.Nucleotide sequence identities among 3D regions from these 10 EV71 isolates were in the range of 95.8%-99.6%,while the nucleotide sequence identities for 3' UTR were 96.3%-100%.The majority of nucleotide changes were located at the third codon positions which caused silent mutations,thus the amino acid sequence changes of 3Dpol among those 10 EV71isolates were scanty.The residues 140 and 263 which were R and 1 were substituted by K and V,respectively in 3 of 4 neurovirulent strains,whereas only 1 of 6 strains from mild cases had these 2 amino acid changes.The sequences of the 3D and 3' UTR regions of 10 EV71 isolates were compared to the representative strains of known genotypes from the GenBank.The nucleotide and amino acid sequences of 10 EV71 isolates in the 3D region exhibited highest homology to the subgenotype C4 of EV71(92.7%-94.2%and 96.8%-97.6%.respectively).However,3' UTR of 10 EV71 isolates shared the highest nucleotide identity with CA16/G10(88.9%-91.4%).The phylogenetic analysis based on the 3D regions demonstrated that 10 EV71 isolates had the closest genetic relationship with the representative isolate of CA sub-genotype of EV71 and shared more homology with CA16/G10 than other known genotypes of EV71.Conclusion Genetic analysis of the 3' end of genomes from 10 EV71 strains indicated that the 3' end of genome may play a role in the evolution of EV71.

Surveillance of influenza viruses attacking children in Beijing during 2009 pandemic influenza A(H1N1) / 中华微生物学和免疫学杂志

Objective To investigate the prevalence of influenza virus infections in infants and young children during the pandemic period of 2009 influenza A(H1N1)in Beijing.Methods Throat swabs were collected from children visited the affiliated Children's Hospital to Capital Institute of Pediatrics for influenza-like illness from June 1,2009 to February 28,2010.The specific gene segments of 2009 pandemic influenza H1N1 and seasonal influenza viruses were amplified from samples by real-time RT-PCR recommended by WHO and National Influenza Reference Center of China.Results Out of 4363 clinical samples tested by real-time RT-PCR,the total positive rate of influenza A viruses was 29.3%,including 623(14.3%)identified as 2009 pandemic influenza A(H1N1)and 657(15.1%)influenza A viruses without subtype identity.Among those pandemic influenza H1N1 positive,23 were severe cases with 5 deaths.The ages for 618 pandemic influenza H1N1 infected children with completed information were from 14 days to 16 years.The ratio of male to female wag 1.3:1.Among them,25.2% were patients in age group of 1 to 3 years old and distribution of children in age groups of 3 to 6 years old and 6 to 12 years old were similar(about 30.0%).During the survey period,it appeared only one prevalence wave of pandemic influenza H1N1.The positive rate of pandemic H1N1 increased in September and the peak(36.5%of positive rate)was in November and then declined to 2.7%in February 2010.The data from routine influenza virus surveillance from 20-30 clinical samples collected each week indicated an alternative prevalence of seasonal H3N2,pandemic H1N1 and influenza B during this study period.Respiratory syncytial virus(RSV)became predominant in children after the circulating of pandemic H1N1.Conclusion There was an epidemic of pandemic influenza H1N1 in children in Beijing from June 2009 to February 2010,especially in those of preschool and school aged children.Seasonal influenza viruses and pandemic influenza H1N1 were contributed alternatively.

Etiology study on hand, foot and mouth disease in children in Beijing during 2007 to 2008 / 中华检验医学杂志

Objective To investigate the etiological agents of hand, foot and mouth disease (HFMD) in children in spring and summer from 2007 to 2008 in Beijing and the characteristics of the disease by virus isolation and to provide the scientific evidence for prevention and treatment for HFMD. Methods During April to August, 2007 and May to September, 2008, 356 clinical specimens including 255 throat swabs and 101 vesicle fluids were collected from 256 patients with HFMD who visited the Children's Hospital Affiliated to Capital Institute of Pediatrics and children with severe HFMD with neural system complications from Ditan Hospital and Youan Hospital All of the specimens were inoculated into Vero cells for virus isolation. After the cell pathogenic effects (CPE) appeared, the isolates were identified by RT-PCR with the universal primers within 5'untranslated region of enterovirus and typed by specific primers for VP1 gene of EV71 and CA16, respectively. The throat swabs from all of 10 severe HFMD were tested for enterovirus by RT-PCR addition to virus isolation. Results Out of 256 patients, 188 were positive for enterovirus by virus isolation, with the overall positive rate of 73.4%. Among the 356 clinical specimens collected from these 256 patients, 239 enterovirus strains were isolated with the overall positive rate of 67.1%. The positive rate for virus isolation from vesicle fluid samples was 75.2% which was higher than the positive rate of isolation from throat swabs (63.9%), but the time for CPE appearing in cell culture showed no significant difference. The positive rate of virus isolation from throat swabs from children with severe HFMD was 50% (5/10) which was lower than overall positive rate (73.4%) from regular HFMD. The RT-PCR typing for virus isolates revealed that among 45 enterevirus strains isolated from the specimens collected in 2007 by the universal primer pairs, 43 were CAI6 (95.6%, 43/45) and 2 were EV71 (4.4%, 2/45), whereas for the specimens collected in 2008, out of 143 enterovirus isolates by PCR with universal primers, 117 were EV71 (82.4%, 117/142) and 24 were CA16 (16.8%, 24/142). All of 10 severe cases were positive for EV71 by RT-PCR directly from clinical specimens. Conclusion CA16 and EVT1 were the etiological pathogens of HFMD in Beijing during 2007 to 2008 HFMD seasons. The dominant type of enterovirus was different between 2007 and 2008. Enterovirus type CA16 was predominant in 2007, whereas EV71 was predominant in 2008. All of severe cases of HFMD in children in this study were caused by EV71.