| Objective:Establish a relible and stable diagnostic method of TaqMan-MGB real-time reverse transcription-polymerase chain reaction (RT-PCR) to detect human metapneumovirus (hMPV) in the clinical respiratory samples. The aim of this study was to understand the clinical and molecular epidemiology of hMPV and other common respiratory pathogens among children with acute low respiratory tract infection (ALRTI) from Oct2006to Feb2008in Shanghai. In the mean time, we tested other seven common respiratory virus by direct immunofluorescence assays (DFA). We also analyzed the detection results of respiratory bacteria, mycoplasma pneumonia and Chlamydia among the targeted samples and enrolled patients to display the spectrum of respiratory pathogens and the changing pattern of pathogens with the time.Subjects and Methods:1.1347nasopharyngeal secretion samples were collected from children clinically disgnosed with ALRTI from Oct2006to Feb2008. One sample was taken from each patient within48hours of admission.2. All specimens were routinely detected for seven common respiratory viruses, bacteria, mycoplasma pneumonia and Chlamydia. Seven common respiratory viruses including respiratory syncytial virus (RSV), influenza virus (IFV) A and B, parainfluenza virus (PIV) type1-3and adenovirus (ADV) were detected by direct immunofluorescence assay. Mycoplasma pneumonia and Chlamydia were detected by real-time PCR. Bacteria were identified by culture.3. We selected622samples collected on every Tuesday and Thursday to detect N gene of hMPV by real-time RT-PCR. A forward primer with a sequence of5'-CATCAGGTAATATCCCACAAAATCAG-3'and a reverse primer with a sequence of5'-GTGAATATTAAGGCACCTACACATAATAA-3'and a probe with a sequence of5'-TCAGCACCAGACACAC-3'were used for real-time RT-PCR. The probe was labeled with6-carboxyfluorescein (FAM) at the5'end and with a quencher (NFQMGB) at the3'end. Traditional PCR was performed with another primer set (forward:5'-AACCGTGTACTAAGTGATGCACTC-3', reverse:5'-CATTGTTTGACCGGCCCCATAA-3') and PCR products were sequenced directly.4. The clinical characteristic of hMPV-positive cases and the prevalence of hMPV were analyzed. The nucleotide fragment of PCR products of hMPV target gene were confirmed in the Genbank by blast. Nucleotide sequences alignment and phylogenetic analysis of hMPV were performed with DNAMAN software.5. The clinical picture of seven respiratory viruses were analyzed and compared.6. The detection rates of mycoplasma pneumonia, chlamydia and bacteria were evaluated.Results:1. Of622samples, hMPV was detected to be positive in24(3.86%) samples.5hMPV-positive PCR products were sequenced. Nucleotide sequences of5hMPV gene were aligned with the submitted sequences of hMPV in Genbank, which revealed similarity at nucleotides level varied from84%to100%, which suggested2distinct genetypes. Phylogenetic tree analysis further demonstrated2different hMPV lineages circulating during the study period. By comparision of nucleotide sequences, point mutation of nucleotide was observed.2. The detection rates of hMPV were0.94%(2/213),3.95%(3/76),9.52%(6/63),6.98%(9/129) and3.23%(1/31) respectively among children aged<3months old,3months to6months,>6months to<1year,1year to<2years,2years to <5years and>5years. Among24hMPV-positive cases,14(58.33%) were less than2years of age and23(95.83%) were younger than5years old. hMPV infection occurred commonly from Nov to Jan of next year. The prevalence of hMPV in the winter of2006to2007was6.60%(14/212) and the prevalence of hMPV in the winter of2007to2008was1.11%(2/180). Of323boys,10(3.10%) were hMPV-positive; of237girls.14(5.91%) were hMPV-positive. There was no significant difference in detection rate between boys and girls (X2=2.63, P>0.05)。3. Of1347respiratory samples, seven common respiratory viruses were detected in452(33.56%) samples. Bacteria were isolated in329(24.64%) of1335samples. Mycoplasma pneumonia and Chlamydia were detected in103(7.85%) and210(15.59%) of1312samples, respectively. Co-infection rate was15.59%in1347samples. No etiological agents was identified in44.77%of samples。4. Of1347samples, RSV was identified in382(28.36%) which was the most common pathogen. The detection rates of RSV were31.16%(124/398),38.59%(120/311),21.05%(36/171),8.64%(7/81) and31.40%(92/293) respectively in the winter of2006, the spring, the summer and the autumn of2007and the winter of2007. Among382RSV-infected cases, children younger than2years old accounted for91.88%(351/382) and children under5years of age accounted for98.69%(377/382), which indicates children younger than2years old were the most susceptible population for RSV. RSV-infected children usually presented with wheezing.5. Of1347samples, the detection rates of PIV, ADV, IFV were2.82%(38/1347),1.86%(25/1347) and1.19%(16/1347), respectively. Virus co-infection was rarely found.6. The detection rates of Mycolasma pneumonia were7.85%(103/1312) and Chlamydia were2.97%(39/1312), respectively. Mycolasma pneumonia was identiifed most commoly in children>5years old with a detection rate of34.5%. Chlamydia was mainly detected in infants<3months of age with a detection rate of6.8%.Conclusions:1. TaqMan-MGB real-time RT-PCR is a rapid and sensitive assay for detecting hMPV, therefore, it can be used to screen hMPV in the clinical specimens. The N gene of field hMPV strains existed slight variation of nucleotide.2. The detection rate of hMPV was3.86%amongst children with ALRTI from Oct2006to Feb2008in Shanghai. However, the prevalence of hMPV in the winter season of2006to2007was higher than that in the winter season of2007to2008. The majority of children infected with hMPV were younger than5years old. 3. Viruses are the important respiratory pathogens among Shanghainese children with ALRTI. Most of virus infection occurred in children<2years old, bacteria infection mainly occurred in children<2years old, mycoplasma pneumonia infection occurred in children>5years old most frequently. Chlamydia infection commonly occurred in infants<3months. |
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