Investigation On Characteristics Of 500 Cases Of Human Metapneumovirus Infection In Guiyang

Part I Development of a new Real-time PCR Assay for Detecting Human Metapneumovirus in Deep Nasopharyngeal Aspirate ofObjective: To compare the specificity and sensitivity of real-time quantitative PCR( Real-time PCR) and Conventional reverse transcription-polymerase chain reaction(RT-PCR)in detecting human metapneumovirus, and to develop an accurate quantitative molecular biological method to detect h MPV from deep nasopharyngeal aspirate of children. Methods: DNAstar was used to analyze and locate the conservative gene locus of h MPV prototype and subtypes. Primers and probes were designed to correspond to conserved regions of h MPV gene segment. A new Real-time PCR method for h MPV F gene detection was then developed. Primers for RT-PCR were also designed according to the principles of primer design. Next, the specificity, sensitivity and reproducibility of the two methods as well as their applicability in detection of h MPV in deep nasopharyngeal aspirate of children were assessed. Results: Real-time PCR ensured not only specific detection but also quantitative analysis of h MPV in deep nasopharyngeal aspirate of children. The sensitivity of quantitation was up to 102 copies. h MPV was detected in 16 % cases by Real-time PCR assay and 9.8% by conventional RT-PCR assay. Compared to Real-time PCR assay, the sensitivity and specificity of RT-PCR were 31.3 % and 94.3% respectively. Conculsion: The sensitivity of Real-time PCR was significantly higher than conventional RT-PCR assay in detecting h MPV. It may serve as an effective test method for quantitation of h MPV in deep nasopharyngeal aspirate of children in clinic and can be used in the epidemiological investigation in Guiyang area.Part â…¡ Investigation on Characteristics of 500 Cases of HumanObjective: To investigate the epidemiological characteristic of human metapneumovirus(h MPV) infection in children with acute lower respiratory infection(ALRTI)in Guiyang. Methods: Five hundred nasopharyngeal aspirates(NPAs) specimens and venous blood samples were collected from hospitalized children with ALRTI between Januaryr 2014 to December 2014. NPAs were used for bacteria culture and Real-time PCR assay. F gene of h MPV was amplified via reverse transcription polymerase chain reaction(RT-PCR). The positive PCR products were assessed via direct sequencing. Blood samples were screened for seven common viruses relevant to respiratory diseases by indirect immunofluorescence. Clinical data were recorded and analyzed. Results: Specimens from 500 ALRTI patients were collected, 80(16%) of which were positive in Real-time PCR. The sequencing results of the positive cases showed 91%-99% homology with the published sequence of HMPV in Gene Bank. All of the 80 positive cases were under 6 years old. The highest infection rate was seen in infancy. Children 6 or older complicated with chronic diseases were also susceptive to HMPV infection. h MPV infection occur in all months, although a spring and winter seasonality and a summer peak prevalence has been observed. Wheezing was common in h MPV infected cases. Children with mixed infections of h MPV and other pathogens could also be found, some of which presented as severe pneumonia. Conculsion: The h MPV is an important pathogen of Children with acute lower respiratory tract infections in Guiyang.