Acute Disease ; Humans ; Respiratory Tract Infections ; diagnosis ; etiology ; microbiology ; virology

Gastroenteritis ; etiology ; virology ; Human bocavirus ; isolation & purification ; Humans ; Parvoviridae Infections ; diagnosis ; Respiratory Tract Diseases ; etiology ; virology

A GeXP based multiplex PCR assay was developed to simultaneously detect six different chicken respiratory viruses including H5, H7, H9 subtypes of avian influenza virus(AIV), new castle disease virus (NDV), infectious bronchitis virus(IBV) and infectious laryngotracheitis virus(ILTV). According to the conserved sequences of genes of each pathogen, seven pairs of specific primers were designed, and the reaction conditions were optimized. The specificity and accuracy of GeXP were examined using samples of single and mixed infections of virus. The sensitivity was evaluated by performing the assay on serial 10-fold dilutions of cloned plasmids. To further evaluate the reliability, thirty-four clinical samples were detected by GeXP. The corresponding specific fragments of genes were amplified. The detection limit of GeXP was 10(2) copies/microL when all of 7 pre-mixed plasmids containing target genes of six chicken respiratory viruses were present. In the detection of thirty-four clinical samples, the results of GeXP were accorded with the viral isolation completely. In conclusion, this GeXP assay is a rapid, specific, sensitive and high-throughput method for the detection of chicken respiratory virus infections. It can be applied in rapid differential diagnosis for clinical samples, and also provide an effective tool to prevent and control chicken respiratory diseases with similar clinical symptoms.
Animals ; Chickens ; Influenza A virus ; classification ; genetics ; isolation & purification ; physiology ; Influenza in Birds ; diagnosis ; virology ; Multiplex Polymerase Chain Reaction ; methods ; Poultry Diseases ; diagnosis ; virology ; Respiratory Tract Infections ; diagnosis ; veterinary ; virology

Acute respiratory tract infections (ARTIs) are widely distributed among the population, mainly caused by respiratory viruses. ARTIs are responsible for significant morbidity and mortality among the elderly and infants or young children, causing a serious economic burden. The rapid and accurate identifi cation of a pathogen will provide a guideline for the clinical diagnosis and therapy. Multiplex polymerase chain reaction (PCR) technologies combine the rapidness and high sensitivity of PCR with high through put, thus achieving the capability of detecting multiple pathogens simultaneously. The commercial kits based on these multiplex PCR methods allow to detect more than twelve respiratory viruses simultaneous ly, reaching the comparable sensitivities and specificities to those of real-time PCR. The recent progress of novel multiplex PCR assays and their principles as well as applications in respiratory virus diagnosis were reviewed in this paper.
Animals ; Humans ; Multiplex Polymerase Chain Reaction ; methods ; Respiratory Tract Infections ; diagnosis ; virology ; Virus Diseases ; diagnosis ; virology ; Viruses ; classification ; genetics ; isolation & purification

OBJECTIVEThe impact of human bocavirus (HBoV), a newly identified human parvovirus, on childhood persistent wheezing has not been identified. In this study, the clinical features of infantile persistent wheezing induced by HBoV was analyzed.

METHODSTracheal aspirates were collected by bronchofibroscope or nasopharyngeal (NP) aspirates from April, 2006 to January, 2007. HBoV DNA in the tracheal aspirates of 33 children with persistent wheezing and in NP aspirates of 6 children with persistent wheezing, who had at least or more than four weeks wheezing. RSV was identified by virus isolation in Hep-2 cells and antigen detetion by direct immunofluorescence assay (DIFA) which was also used for diagnosis of adenovirus, influenza A and B, parainfluenza 1, 2, 3 infection.

RESULTSOf the 39 children with persistent wheezing, 12 cases (31%) were positive for HBoV DNA. Age of HBoV-positive patients ranged from 2 month to 1 year. The results of sequencing of PCR products proved that sequences of HBoV DNA from these 12 samples were exactly identical to the those of HBoV stored in GeneBank (accession numbers DQ000495 and DQ000496). Two cases with HBoV infection were found to be co-infected with RSV. Ten of the 12 HBoV-positive samples were collected during the period from winter to spring (1 in November, 4 in December, 2 in January and 3 in April), the other two HBoV-positive samples were collected during the period from summer to autumn (1 in May and the other in July). Seven of the 12 HBoV DNA-positive patients had fever, 5 of them had high fever. Significantly more patients with HBoV infection had fever as compared to patients with RSV infection. All the HBoV positive patients showed abnormal findings on chest X ray such as interstitial infiltrates, lung infiltration and hyperinflation. Abnormal findings on chest X ray were found in higher proportion of HBoV positive patients as compared with RSV positive patients. And other manifestations such as wheezing, cough and respiratory distress had no significant difference between HBoV and RSV infected patients.

CONCLUSIONSThis study further demonstrated that HBoV probably is a common pathogen of lower respiratory infection in children and might particularly be associated with persistent wheezing.

Child, Preschool ; Cough ; etiology ; Female ; Fever ; etiology ; Human bocavirus ; pathogenicity ; Humans ; Infant ; Male ; Nasopharynx ; pathology ; Paramyxoviridae Infections ; physiopathology ; Parvoviridae Infections ; physiopathology ; Respiratory Sounds ; etiology ; Respiratory Syncytial Virus Infections ; classification ; physiopathology ; Respiratory Tract Infections ; diagnosis ; physiopathology ; virology

BACKGROUND: Human bocavirus (HBoV) is a newly identified viral pathogen, and its clinical epidemiology and significance in respiratory infections have not yet been completely elucidated. We investigated the prevalence of HBoV infection and the association between viral (HBoV) load and clinical features of the infection in patients of all age-groups. METHODS: Nasopharyngeal aspirates from patients with symptoms of respiratory infection were tested for presence of HBoV by using real-time polymerase chain reaction. HBoV-positive patients were categorized into low- and high-viral-load groups using 1.0x10(6) copies/mL as the threshold value of viral load. RESULTS: Detection rate of HBoV was 4.8% (N=93) in a total of 1,926 samples with peak incidence of infection being observed in patients aged 6-12 months. HBoV infection was more frequently observed in young children, especially, in children aged less than 5 yr, and the HBoV load decreased with increase in age. HBoV was codetected with other respiratory viruses in 17 (18.3%) of the 93 HBoV-positive patients and 15 patients (88.2%) belonged to the low-viral-load group. Patients infected with HBoV alone showed a higher viral load than those patients in whom HBoV was codetected with other respiratory viruses (median load, 3.78x10(5) copies/mL vs. 1.94x10(4) copies/mL, P=0.014). Higher pulse rate (P=0.007) and respiratory rate (P=0.021) were observed in patients with a high-viral-load. CONCLUSIONS: Our results suggest that HBoV may be the causative agent of respiratory infection in the high-viral-load group.
Adolescent ; Adult ; Aged ; Child ; Child, Preschool ; DNA, Viral/analysis ; Female ; Human bocavirus/*isolation & purification ; Humans ; Infant ; Male ; Middle Aged ; Nasopharynx/virology ; Parvoviridae Infections/diagnosis/*epidemiology/virology ; Polymerase Chain Reaction ; Prevalence ; Respiratory Tract Infections/diagnosis/*epidemiology/virology ; Viral Load

BACKGROUND: Human bocavirus (HBoV) is a newly identified viral pathogen, and its clinical epidemiology and significance in respiratory infections have not yet been completely elucidated. We investigated the prevalence of HBoV infection and the association between viral (HBoV) load and clinical features of the infection in patients of all age-groups. METHODS: Nasopharyngeal aspirates from patients with symptoms of respiratory infection were tested for presence of HBoV by using real-time polymerase chain reaction. HBoV-positive patients were categorized into low- and high-viral-load groups using 1.0x10(6) copies/mL as the threshold value of viral load. RESULTS: Detection rate of HBoV was 4.8% (N=93) in a total of 1,926 samples with peak incidence of infection being observed in patients aged 6-12 months. HBoV infection was more frequently observed in young children, especially, in children aged less than 5 yr, and the HBoV load decreased with increase in age. HBoV was codetected with other respiratory viruses in 17 (18.3%) of the 93 HBoV-positive patients and 15 patients (88.2%) belonged to the low-viral-load group. Patients infected with HBoV alone showed a higher viral load than those patients in whom HBoV was codetected with other respiratory viruses (median load, 3.78x10(5) copies/mL vs. 1.94x10(4) copies/mL, P=0.014). Higher pulse rate (P=0.007) and respiratory rate (P=0.021) were observed in patients with a high-viral-load. CONCLUSIONS: Our results suggest that HBoV may be the causative agent of respiratory infection in the high-viral-load group.
Adolescent ; Adult ; Aged ; Child ; Child, Preschool ; DNA, Viral/analysis ; Female ; Human bocavirus/*isolation & purification ; Humans ; Infant ; Male ; Middle Aged ; Nasopharynx/virology ; Parvoviridae Infections/diagnosis/*epidemiology/virology ; Polymerase Chain Reaction ; Prevalence ; Respiratory Tract Infections/diagnosis/*epidemiology/virology ; Viral Load

OBJECTIVETo characterize the prevalence and occurrence of subgroups of human respiratory syncytial virus (RSV) in infants and young children with acute respiratory infections (ARI) in Beijing area.

METHODSRSVs were identified from nasopharyngeal aspirates and throat swabs collected from infants and children with ARI who visited the Children's Hospital Affiliated to Capital Institute of Pediatrics during the period of November 2000 to March 2006, by virus isolation in Hep-2 cells and antigen detection by indirect immunofluorescence assay (IFA). RT-PCR was used to differentiate subgroups A and B of RSV from part of the positive specimens.

RESULTSOut of 10 048 specimens including 7176 nasopharyngeal aspirates from inpatients and 2872 throat swabs from outpatients, 2286 (22.8%) were RSV positive. The positive rate for RSV identification were 30.0% (2153/7176) in specimens from the hospitalized patients, which was higher than that from outpatients (4.6%, 133/2872). The youngest of the RSV positive patients was 1 day after birth and the oldest was 15 years of age, with 73.0% younger than 1 year. Among those RSV positives, only 1.6% were older than 5 years. The ratio of male to female who were RSV positive was 2.4:1 (1598:674). The clinical diagnosis for 91.2% (1991) of those RSV positive patients was severe lower respiratory infections including bronchiolitis and pneumonia, whereas in only 8.8% (192) the diagnosis was upper respiratory infections. The data revealed that RSV started to be detected in October each year during the survey period and November to next April was the RSV season. The detection rate declined in May and almost no RSV could be found in summer. Positive rates for RSV detection were 42.3%, 41.0% and 40.5% in the seasons of 2001 - 2002, 2003 - 2004, 2005 - 2006, which were higher than those in seasons of 2000 - 2001 (14.0%), 2002 - 2003 (18.2%), 2004 - 2005 (20.4%). Subtyping of A and B during the surveillance period showed that 73.7% (691/938) were subgroup A and 26.3% (247/938) were subgroup B. Subgroup B was predominant in the 2000 - 2001 and 2004 - 2005 seasons, whereas subgroup A predominated in the 2001 - 2002, 2002 - 2003 and 2003 - 2004 seasons. Almost equal proportions of subgroup A and B appeared in 2005 - 2006 seasons.

CONCLUSIONThe data indicate that RSV is an important etiological agent for lower respiratory infections in infants and young children in winter and spring during the survey period. The pattern of RSV circulation varied alternately with higher rate every other year. The predominant subgroup changed between A and B, and co-circulated in equal proportion in some years.

Adolescent ; Cell Line, Tumor ; Child ; Child, Preschool ; China ; epidemiology ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Population Surveillance ; Prevalence ; Respiratory Syncytial Virus Infections ; diagnosis ; epidemiology ; Respiratory Syncytial Virus, Human ; genetics ; isolation & purification ; Respiratory Tract Infections ; diagnosis ; epidemiology ; virology ; Seasons

Antibodies, Viral ; analysis ; Cerebrospinal Fluid ; virology ; Child ; Child, Preschool ; Feces ; virology ; Genotype ; Humans ; Infant ; Meningoencephalitis ; diagnosis ; virology ; Parechovirus ; classification ; genetics ; isolation & purification ; Picornaviridae Infections ; diagnosis ; epidemiology ; virology ; RNA, Viral ; genetics ; Respiratory Tract Infections ; diagnosis ; virology ; Sepsis ; diagnosis ; virology ; Sequence Analysis, DNA

Resequencing Pathogen Microarray (RPM) is a new pathogen detection and identification technology based on DNA microarray. In order to apply RPM in the detection of unexplained infection and as a result, to improve the emergency response capacity, a new RPM-based respiratory pathogens detection assay was developed to simultaneously detect 19 common respiratory viruses, 9 influenza A viruses (Flu A),11 human rhinoviruses(HRV), 28 enteroviruses and 18 rare respiratory viruses. The specificity of multiplex system was examined by confirmed positive specimens for 16 common respiratory virus. The sensi-tivity was evaluated by serial ten-fold dilutions of plasmids or in vitro-transcribed RNA. RPM could detect and differentiate 16 virus types/subtypes at 10 - 1 000 copies/reaction level. Nucleic acids of 8 throat swabs with unexplained respiratory tract infections were pooled and detected by the new assay. The RPM result was verified by common PCR followed by sequencing as well as PLEX-ID (Abbott). Except for a false-positive of PIV1, no difference among the three assays was found. These results indicate the assay based on the new RPM is a highly sensitive, high throughput test for the detection of respiratory virus infections, which is significant for the management of emergent and epidemic infectious disease.
Child ; Child, Preschool ; Female ; Humans ; Male ; Oligonucleotide Array Sequence Analysis ; methods ; Respiratory Tract Infections ; diagnosis ; virology ; Sensitivity and Specificity ; Viruses ; classification ; genetics ; isolation & purification