Macro Virology Of MP Infection And Epidemiology Of HMPV In Hospitalized Children With ARTI In Beijing

Objective:Acute respiratory tract infections（ARTI）is one of the most common infectious diseases in human beings and an important cause of death for children in the world,which not only seriously threatens children’s health,but also brings a huge burden to the world economy.Mycoplasma pneumonia（MP）and human metapneumovirus（HMPV）are common causes of ARTI,and they are often co-infected with other respiratory viruses.Since the end of 2019,SARS-Co V-2 was discovered in Wuhan,China,and then widely spread around the world,various countries have formulated corresponding epidemic prevention policies,under the implementation of epidemic prevention measures,the epidemic of some respiratory pathogens has changed,such as RSV,IFV,etc.Metagenomic next generation sequencing（m NGS）detects all pathogens in a single sample,so all pathogens in clinical samples can be detected.In this study,m NGS technology was used to describe the pathogenic spectrum of nasopharyngeal aspirate samples with MP infection in ARTI-hospitalized children with MP infection and the co-infection with common respiratory viruses,analyze the impact of SARS-Co V-2epidemic on the pathogenic spectrum of MP infection,and elucidate the molecular epidemiological characteristics of HMPV infection in ARTI-hospitalized children in Beijing and the impact of the new crown epidemic on HMPV infection.Methods:1.To establish a real-time fluorescence quantitative Polymerase chain reaction（TaqMan qPCR）detection method using MP probe method:Download the sequences of all MP CARDS genes（68）from NCBI,use Bio Edit software for sequence comparison,and select a conserved region.Then primers and probes are designed on primer3 software and sent to gene companies for synthesis.The nucleic acid of known MP positive samples of the primer was used for PCR amplification,and the PCR products were identified with 1%agarose gel,and the target bands were correctly identified and recovered.The recovered product was then connected to p MD-18T-Vector,which was transformed,cloned and shaken.The bacterial solution was amplified by PCR and identified by agarose gel.The correct bacterial solution was identified and sent to the sanger sequencing.The plasmid was extracted from the bacterial solution as the standard substance and its copy number was calculated.real time PCR was performed after gradient dilution.The standard curve,amplification efficiency and R²values were obtained.The sensitivity,specificity and repeatability of the primers and probes were verified.2.Pathogen spectrum analysis of nasopharyngeal suction samples of MP hospitalized children:（1）TaqMan qPCR detection was performed on all samples using established primers and probes,and CT value<37 was positive.（2）Second-generation sequencing was performed on MP positive samples to build the database.The process of building the database included:two-step centrifugation（removing large particle impurities and organelles first）,nuclease treatment,nucleic acid extraction,reverse transcription,and Multiple displacement amplification（MDA）.The qualified amplified products detected by protein nucleic acid concentration tester were sent to Wuhan Huada Gene Technology Co.,Ltd.for second-generation sequencing.（3）Among the 104 MP-positive samples collected from April 2018 to March 2019,42 cases（40.38%）were co-infected,of which34 cases（32.69%）were co-infected with one respiratory virus,6 cases（5.77%）were co-infected with two respiratory viruses,and 2 cases（1.92%）were co-infected with three respiratory viruses.The number of HRV and HCo V-NL63 co-infection cases was higher,with 15（33.33%）and 10（22.22%）cases,respectively.Among MP-positive samples collected during the SARS-Co V-2 epidemic period（September 2020 to August 2022）,there were 4 cases（18.2%）of mixed virus infections.There was no significant difference in clinical symptoms between MP infection combined with other viruses and MP single infection.3.To explore the epidemic characteristics of HMPV:HMPV TaqMan q RT-PCR was performed for nucleic acid detection of all samples.The virus typing steps for nucleic acids of positive samples include reverse transcription,nested PCR reaction,agarose gel electrophoresis,generation sequencing of positive products,construction of evolutionary tree,and finally epidemiological analysis,as well as comparison of differences in HMPV epidemic characteristics before and after the COVID-19 epidemic.Results:1.Establishment and application of MP TaqMan qPCR detection method:The detection method of MP qPCR showed good specificity of primers and probes,the detection limit was 10copies/μL,the sensitivity was good,the linear range of standard curve was10-10¹⁰copies/μL,R²=0.9954,the linear relationship was good,the coefficient of variation was less than 5%,and the repeatability and stability were good.This method was used to detect 50 nasopharyngeal suction samples of hospitalized children,and the detection results were consistent with the results of a literature,with two positive cases.2.Pathogen spectrum analysis of nasopharyngeal aspiration samples of MP hospitalized children:（1）A total of 1572 nasopharyngeal aspiration samples were collected from pediatric ARTI inpatients in Beijing Friendship Hospital from April 2018to March 2019.A total of 104 cases of MP infection were detected by self-priming probe,with a detection rate of 6.6%（104/1572）.A total of 622 NPAs samples were collected from September 2020 to August 2022 during the novel coronavirus epidemic,and the MP detection rate was 3.5%（22/622）.Statistical analysis showed that the detection rate of MP was lower than that before the epidemic under the novel coronavirus prevention measures（P<0.05）.（2）A variety of common virus-related sequences were detected in 99MP-positive respiratory samples before the epidemic（April 2019 to March 2018）,with a total number of 40,941,878 reads and an average number of 413,554.These virus sequences were from 11 virus families.Among them,Coronaviridae,Pneumoviridae and Papillomaviridae contain 33661003（82.22%）,2878396（7.03%）and 2391603（5.84%）,respectively.During the COVID-19 epidemic（September 2020 to August 2021）,NGS of MP-positive respiratory samples detected six virus families,with a total of 5,265,393reads and an average of 239,336 reads.In conclusion,the number of viral species and reads decreased in the pathogen spectrum of MP-positive samples detected by NGS during the COVID-19 epidemic.（3）Among the 104 MP-positive samples collected from April 2018 to March 2019,there were 42 cases（40.38%）of mixed infections,including34 cases（32.69%）of single virus infection,6 cases（5.77%）of double virus infection and2 cases（1.92%）of triple virus infection.There were 15（33.33%）and 10（22.22%）cases of HRV and HCo V-NL63 mixed infection,respectively.Among the MP-positive samples collected after the novel coronavirus epidemic（September 2020-August 2022）,there were 3 cases（13.6%）of mixed virus infection,and all were single virus infection（HRV）.There was no significant difference in clinical symptoms between MP combined with other viruses and MP single infection（P>0.05）.3.Analysis of HMPV epidemic characteristics From April 2018 to March 2019,the detection rate of HMPV was 7.9%（124/1572）,with the majority of children under 5years old（78.2%,92/124）;Spring（11.7%,43/360）and winter（11%,47/426）were significantly higher than summer and autumn（P<0.05）.HMPV was often accompanied by other respiratory virus co-infection（37.1%,46/124）,the most common co-infection was HPIV3,and there was no significant difference in viral load between single HMPV infection and co-infection.The clinical diagnosis was mostly pneumonia（92.7%,115/124）,and the main symptoms were high fever and cough.A2b subtype（82.1%,64/78）was the main endemic strain.During the epidemic period of the novel coronavirus（September 2020 to August 2022）,12 cases were positive for HMPV,the detection rate was 1.9%（12/622）,and the B1 subtype（87.5%,7/8）was the main circulating strain.Conclusion:1.The TaqMan qPCR method for detection of Mycoplasma pneumoniae was established,which showed good sensitivity,specificity and repeatability.2.Under the strict national prevention and control measures of SARS-Co V-2,the detection rate of MP and HMPV in hospitalized children with ARTI decreased significantly compared with that before the epidemic,and the epidemic strain of HMPV in Beijing changed from subtype A2b to subtype B1.3.NGS data analysis showed that the types of respiratory viruses in MP-positive respiratory samples decreased compared with that before the epidemic of novel coronavirus,and the infection rate of mixing with common respiratory viruses decreased.