Clinical And Molecular Epidemiologic Characteristics Of Human Metapneumovirus Infection In Children Of Chonghua City

Background and Purpose Respiratory tract infection (RTI) is one of the leading cause of morbidity and mortality of young children. Respiratory syncytial virus (RSV), influenza viruses (Flu), parainfluenza viruses (PIV) are recognized as etiologic agents of viral RTI. Nevertheless, despite the availability of sensitive diagnostic methods, infectious agents cannot be identified in up to one-third of the cases, suggesting the presence of unknown or unrecognized pathogens. In 2001, a new respiratory virus, the human metapneumovirus (hMPV), was isolated from children with RTI in Netherlands. Virologic data, sequence homology and gene organization revealed that this new pathogen was the first nonavian virus of the Metapneumovirus genus in the Pneumovirinae subfamily of the Paramyxoviridae family. The ubiquitous and worldwide distribution of hMPV was subsequently demonstrated by detection of the virus in patients from Europe, North America, South America, Australia, Asia ,Africa and so on. Numerous clinical and epidemiologic data related to hMPV infection have been documented from these previous reports. This virus seems to have a seasonal distribution given that most infections occur during the winter and spring months. Although hMPV has been detected in patients from all ages, young children seem to be particularly susceptible to hMPV infection, and the clinical diagnoses in children range from limited upper respiratory tract disease to severe bronchiolitis and pneumonia. There was no report about the clinical and molecular epidemiologic characteristics of hMPV infection in children of Chonghua City at present. It is significant to investigate the clinical and molecular epidemiologic characteristics of hMPV infection, and helpful to prevention and cure in Chonghua City. The purposes: (1) To explain the clinical and molecular epidemiologic characteristics of human metapneumovirus (hMPV) infection in children of Chonghua City. (2) To explore the relationshipn between hMPV and lower respiratory tract infection. such as bronchopneumonia, bronchiolitis, bronchitis and bronchial asthma. (3) To explore the homology and genotyping discrepancy of hMPV infection with other countries in chlidren through gene sequencing. (4) To understand the clinical and molecular epidemiologic characteristics of other respiratory virus infection.Material and methods Two hunderd and sixty specimens were collected from children younger than 6 years old from Dem. 2008 to Oct. 2009 in Chonghua central Hospital of Chonghua City. (1) Nested PCR was applyed to detect hMPV infection. Two rounds of PCR(hMPV-1,hMPV-2) products were isolated by 2% agarose gel electrophoresis and imaged. (2) The conventional respiratory virus including RSV-A, RSV-B, Flu-A, Flu-B, PIV-1,PIV-2 and PIV-3 were detected by multiplex PCR. The PCR products were isolated by by 2% agarose gel electrophoresis and imaged. (3) Selected seven hMPV-1 positive PCR products randomly and were sequenced by Invitrogen Commercal Corporation. (4) The sequence of 7 hMPV-1 positive PCR products were compared with those in GenBank. Clustalx software was adopted to do multiple sequence comparison. Used DNAStar software to drew cladogram.Results (1) hMPV-1 positive PCR products were isolated by 2% agarose gel electrophoresis and imaged. The positive strap appeared between 450bp and 600bp. It was consistent with expectant strap. hMPV-2 positive strap appeared between 400bp and 500bp. It was also consistent with expectant strap. (2) Among hMPV-1 positive specimens, there were 17 male and 4 female, the positive incidence were 9.83% and 4.60% respectively. There was no statistical significance between them (χ2=2.132,P>0.05). The male and femal positive specimens of hMPV-2 were 36 and 12 and the positive incidence were 20.81% and 13.79%, no statistical significance between them.The male and female positive incidence between hMPV-2 and hMPV-1 had significant difference (χ2=8.04, P<0.05;χ2=4.41, P<0.05). Among the 21 hMPV positive specimens, there were 13 specimens' age younger than 6 months ,the positive incidence was 7.51%; 8 specimens between 6 months and 1 year , the positive incidence was 13.11%. No statistical discrepancy between them (χ2=1.731,P>0.05). About hMPV-2, there were 24 specimens' age younger than 6 months ,the positive incidence was 13.87%; 19 specimens between 6 months and 1 year , the positive incidence was 31.15%; 5 specimens between 1 year and 3 years and the positive incidence was 20.83%. There was no difference among them. The positive incidence of the second age group about hMPV-2 and hMPV-1 had significant difference (χ2=5.76, P<0.05). The epidemic seasons of hMPV infection were spring and winter and the morbility was higher than other months. The morbility crest-time of hMV infection was overlaped with RSV infection. (4) The positive specimens of hMPV-1 were 21 and the positive incidence was 8.08% (21/260). hMPV-2 positive specomens were 48 and the positive incidence was 18.46% (48/260). The positive incidence of two rounds had no statistical significance (P>0.05). The major clinical characteristics of hMPV infection were cough and pursiness. There were statistical significance in pursiness (χ2=4.219,P<0.05) and neutrophil percentage (NEUT%) (t=-2.424,P<0.05) between hMPV-1 and PIV-3. Significant difference was seen in NEUT% between hMPV-2 and PIV-3. All of hMPV-1 positive children showed LRTI, 12 children were diagnosed with bronchiolitis (57.14%), 3 children were bronchopneumonia (14.29%) and 6 children were bronchitis (28.57%). hMPV-2 positive children were diagnosed with URTI and LRTI. 1 (2.08%) kid was laryngitis, 25 (52.08%) were bronchiolitis, 14 (29.17%) were bronchitis, 6 (12.5%) were bronchopneumonia and 2 (4.17%) were bronchial asthma. (5) The positive specimens of other conventional respiratory virus infection were 126 and the positive incidence was 48.46%. RSV-A positive specimens were 18 , the positive incidence was 6.92%. RSV-B positive specimens were 27 and the positive incidence was 10.83%. RSV positive specimens were 45 and the total positive incidence was 17.31%. The major clinical symptom were cough , pursiness, fever and breath holding and all of RSV infected children were diagnosesed with LRTI. The epidemic seasons of RSV infection were spring and winter. Flu-A positive specimens were 4 and the positive incidence was 1.54%. Flu-B positive specimens were 10 and the positive incidence was 3.85%. Flu positive specimens were 14 and the total positive incidence was 5.38%. The major clinical symptom were cough , pursiness, fever. The major diagnoses were LRTI, 1 chaildren was laryngitis and 2 were bronchial asthma. The epidemic seasons of Flu infection were spring and winter. 67 specimens were PIV-3 infected and positive incidence was 25.77%. Undeteced PIV-1 and PIV-2 infection. The major clinical symptom were cough , pursiness, fever, breath holding and expectoration. The major diagnoses were LRTI, 2 chaildren was laryngitis. The epidemic seasons of PIV-3 infection were spring and winter. The coinfected specimens of hMPV-1 were 19 and the incidence was 15.08%. 1 specimen was RSV-B/Flu-B-coinfected, 14 specimens were RSV-B/PIV-3-coinfected, 1 specimen was Flu-A/PIV-3-coinfected and 1 specimen was Flu-B/PIV-3-coinfected. The coinfected specimens of hMPV-1 were 17 and the incidence was 13.49%. 1 specimen was RSV-B/Flu-B-coinfected, 12 specimen was RSV-B/PIV-3-coinfected, 1 specimen was Flu-A/PIV-3-coinfected, 3 specimen was RSV-B/PIV-3-coinfected. The coinfected incidence between them had no significant difference. (6) There were 7 specimens coinfected with other respiratory virus of hMPV-1 positive specimens, the coinfected incidence was 33.33%. 1 specimen was hMPV/RSV-B -coinfected, 3 specimens were hMPV/Flu-B-coinfected and 3 specimens were hMPV /PIV-3-coninfected. The coinfected specimens and incidence of hMPV were 4 and 35.42%. 4 specimens were hMPV/RSV-A coinfected, 1 specimen was hMPV/RSV-B–coinfected, 3 specimens were hMPV/Flu-B-coinfected and 7 specimens were hMPV /PIV-3-coninfected. 2 specimens were hMPV/RSV-B/PIV-3-coinfected. The coinfected incidence had no significant difference. All of hMPV infected children were diagnosesed with LRTI and the major clinical symptom were cough and pursiness. (7) The sequences of hMPV-1 positive PCR products were compared with those in GenBank and the homology with HMPVgz01 (GenBamk code: GQ153651.1) and BJ (GenBank acession: DQ843659.1) was 98～100%. Germline analyze revealed the genotype of 7 sequences belonged to subgroup A2 of group A.Conclusions (1) The hMPV positive specimens were 48 and the incidence was 18.46%, no difference in gender and age group. The epidemic season of hMPV infection was spring, the positive incidence was higher between Feb. and Apr.. The major clinical symptom were cough and pursiness. All of hMPV positive chlidren were diagnosed with LRTI. hMPV was a major pathogen of viral respiratory tract infection. (2) Among 48 hMPV positive specimens, there were 17 specimens coinfected with other conventional virus and the coinfected incidence was 35.42%. The clinical symptom and epidemic features were similar to hMPV infection. (3) The identities of 7 sequences with HMPVgz01 (GenBamk code: GQ153651.1) and BJ (GenBank acession: DQ843659.1) was 98～100%. They were homology. Germline analyze revealed the genotype of 7 sequences belonged to subgroup A2 of group A. (4) Other conventional respiratory virus positive specimens were 126 and the positive incidence was 48.46%. The peak infection rate was PIV-3, RSV was secondary. The clinical symptom and objective were similar and the epidemic season was crossing. It was hard to discriminate and diagnose.