PURPOSE: Early identification of causative agents in lower respiratory infection of pediatric patients can reduce morbidity and prevent an overuse of antimicrobials. Two kinds of multiplex polymerase chain reaction (PCR) and a commercial shell vial viral culture were performed to identify causative agents in pediatric patients. MATERIALS AND METHODS: Nasopharyngeal aspirates of 220 children diagnosed with viral pneumonia were obtained. Two kinds of multiplex PCR (Seeplextrade mark RV detection kit, and Labopasstrade mark RV detection kit), and a shell vial culture by R-Mix were performed. RESULTS: Positive samples from 220 total samples by two multiplex PCRs were 52.7% and 46.4%, respectively. We also cultured 103 samples that showed positive results of the adenovirus, influenza virus, parainfluenza virus, and respiratory syncytial virus (RSV) by two multiplex PCR. The RSV was most frequently detected in 53.0% (Seeplex) and 51.7% (Labopass) of patients. The detection rate of adenovirus (AdV) was 10.3% and 12.1%, influenza virus (IFV) A and B was 12.5% and 3.4%, and parainfluenza virus (PIFV) 1, 2, and 3 were 2.9% and 2.6%. Shell vial cultures showed concordant results with each multiplex PCR by 96.1% and 77.7%, respectively. Sequencing results were 90% consistent with multiplex PCR. CONCLUSION: Multiplex PCR showed more positivity than the shell vial culture and it can be an effective primary test. Other complementary efforts such as viral cultures and sequencing analysis could be considered, according to clinical and laboratory conditions.
Adenoviridae/genetics/*isolation & purification ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Orthomyxoviridae/genetics/*isolation & purification ; Pneumonia, Viral/*virology ; Polymerase Chain Reaction/*methods ; Respiratory Syncytial Viruses/genetics/*isolation & purification ; Respirovirus/genetics/*isolation & purification

OBJECTIVETo establish a convenient and high-throughput respiratory virus detection method to facilitate epidemiological viral monitoring.

METHODSWe used high-throughput microsphere-based flexible multi-analyte profiling technology (xMAP) coupled with signal amplification molecules to simultaneously detect RNAs of 8 viruses including influenza viruses A and B, parainfluenza viruses type 1, 2 and 3, respiratory syncytial viruses A and B, and metapneumovirus in a 96-well plate format. The sensitivity and specificity of the method for the synthetic viral RNAs were evaluated.

RESULTSThere was no cross-reactivity among the 8 respiratory viral target RNAs. The detection limits for the 8 viral in intro-transcribed RNAs ranged from 1204 to 4695 RNA copies.

CONCLUSIONWe establish a sensitive, specific, convenient, and high-throughput multiplex detection method suitable for detecting multiple respiratory viral RNAs for epidemiological viral monitoring.

High-Throughput Screening Assays ; methods ; Influenza A virus ; genetics ; isolation & purification ; Influenza B virus ; genetics ; isolation & purification ; Limit of Detection ; Metapneumovirus ; genetics ; isolation & purification ; Nucleic Acid Amplification Techniques ; methods ; RNA, Viral ; analysis ; Respiratory Syncytial Viruses ; genetics ; isolation & purification ; Respiratory System ; virology ; Respirovirus ; genetics ; isolation & purification ; Reverse Transcription ; Sensitivity and Specificity

To investigate the genetic characterization of Human parainfluenza virus-3 (HPIV-3) circulating in Gansu and Shaanxi Provinces of China, 719 throat swabs were collected from pediatric patients with acute respiratory infections from 2009-2011. Multiplex RT-PCR was used to screen common respiratory viral pathogens. For HPIV-3-positive specimens, nested RT-PCR was used to amplify the HN gene of HPIV-3. The nucleotides of Hemagglutinin-neuraminidase(HN)gene of 13 HPIV-3 positive strains identified in Gansu and Shaanxi Provinces were successfully sequenced and compared with those downloaded from GenBank. The phylogenetic analysis based on the nucleotides sequence of HN gene showed that 13 HPIV-3 strains belonged to sub-cluster C3 with little sequence variation (overall nucleotide divergence of 0.2%-2.3% and amino acid divergence at 0-1.1%). Compared with the complete gene of HPIV-3 strains from U.S.A., Canada, and Australia, the biggest divergence of the nucleotide and amino acid lovels was 6.0% and 3.4%, respectively. The nucleotide divergence between shaanxi09-2 and shaanxi10-H0091 was 0.9%, while the nucleotide divergence between shaanxi10-H005 and gansull-62110372 was 0.5%, between shaanxi09-2 and BJ/291/09 was 0.6%. However, there was no amino acid divergence among them. It is likely that HPIV-3 virus had been transmitting in Gansu and Shaanxi Provinces for several years. Human parainfluenza virus-3 (HPIV-3) circulated in Gansu and Shaanxi Provinces from 2009 to 2011 belonged to sub-cluster C3.
Adolescent ; Adult ; Child ; China ; epidemiology ; Female ; Genetic Variation ; HN Protein ; genetics ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; Parainfluenza Virus 3, Human ; classification ; genetics ; isolation & purification ; Phylogeny ; Respirovirus Infections ; epidemiology ; virology ; Seasons ; Young Adult

OBJECTIVETo understand the impact of human parainfluenza virus (HPIV) on acute respiratory infections in infants and young children in Beijing.

METHODSMultiplex reverse transcription-PCR was used to amplify the hemagglutinin (HA) gene fragment of HPIV from clinical specimens. Primer pairs derived from a conserved region of the HA genes of HPIV were used to develop the multiplex RT-PCR for detecting and typing HPIV. The sensitivity and specificity of the method were determined by using various RNA and DNA viruses as controls. Specimens collected from 3519 children with acute respiratory infections from Aug. 2003 to Apr. 2006 were analyzed for HPIV by the multiplex RT-PCR as well as for other respiratory viruses by virus isolation and/or indirect immunofluorescent assay (IFA). Ten amplicons with expected molecular weight matching different types of HPIV were randomly selected for sequence analysis.

RESULTSOnly the cDNA from the isolated strains of HPIV 1 and 3 was positive by the multiplex RT-PCR. Phylogenetic analysis for those 10 amplicons' sequences which belong to HPIV 1 - 4 types respectively as determined by multiplex-PCR indicated that these specimens were truly HPIV positive. These 10 HPIV positive specimens included two specimens of type 4 which was further subtyped as HPIV4A and 4B by sequence analysis. With the multiplex RT-PCR, HPIV were detected in 349 out of 3519 specimens with the positive rate of 9.9% (349/3519), which is higher than 4.8% by the methods of virus isolation and/or IFA. And the HPIV positive rates were high in patients with not only acute upper but also lower respiratory tract infection. No regular seasonality distribution of HPIV infection was found. HPIV 1 and 3 were more common than HPIV 2 and 4.

CONCLUSIONWith higher sensitivity and specificity than virus isolation and IFA, multiplex RT-PCR is beneficial for the etiologic and epidemiologic studies on HPIV, as well as for HPIV typing. The data from this study indicate that HPIV is one of the important etiological viruses of acute respiratory tract infections in infants and young children in Beijing.

Child, Preschool ; China ; epidemiology ; Genes, Viral ; HN Protein ; genetics ; Humans ; Infant ; Paramyxoviridae Infections ; epidemiology ; virology ; Phylogeny ; Prevalence ; Respiratory Tract Infections ; epidemiology ; virology ; Respirovirus ; genetics ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; Sensitivity and Specificity