Clinical Application Of Multiplex Molecular Diagnosis Methods For Respiratory Tract Pathogens,and Association Of Adenovirus Serotype With Severe Pneumonia In Children

BackgroundAcute respiratory tract infection(ARTI)is a common disease in children,especially in children under 5 years old.The symptom of ARTI can varies from mild amygdalitis to severe pneumonia or even die from complications,which cause high hospitalization rate and mortality,and bring huge burden to society.Most of ARTIs are caused by respiratory viruses.The co-infection is also common,of which detection rate varies from 30 % to 50 % in children with ARTI.Thus,fast and accurate method for pathogen detetion contributes to better diagnosis and clinical care.The realtime quantitative PCR(qPCR)method has been widely used in routine clinical diagnosis for many years.However,the turn around time is long due to manual operation of nucleic acid extraction.It’s hard to detect more than 10 pathogens in a run.With the development of multiplex PCR,several commercialized multiplex PCR methods are available in recent years.For example,the Filmarray based on multiplex nested qPCR can be independent of PCR laboratory,and finish the procedures of extraction,reverse transcription and qPCR in a blind reaction strip within 70 minutes.It can detect 18 types of respiratory pathogen in a run.Although the cost is expensive,Filmarray is suitable for patients with emergent and severe disease because of the short turn around time and wide spectrum of detection.For another example,the Health Technology System is a method based on the multiplex PCR and capillary electrophoresis.The extraction of nucleic acid relies on manual operation and PCR laboratory.It can detect 180 samples for 14 types of respiratory pathogen in a run,which has high throughput and lower cost.These multiplex molecular diagnosis methods help better diagnosis of respiratory pathogens substantially.However,these methods have not been approved by CFDA,which needs evaluation of clinical application as soon as possible.Adenovirus(ADV)is one of the respiratory viruses which are frequently detected in children with ARTI,especially in children aged under 5 years.The detection rate of ADV varies from 5 % to 25 %.Some previous studies figured out that ADV serotype 7(ADV-7)was frequently isolated in children with severe pneumonia,and indicated that ADV-7 might had association with severe pneumonia in children.However,the inadequate data of few studies are not yet enough to declare that ADV-7 is more likely to cause severe pneumonia compared to other serotypes.The pathogenic mechanism of ADV-7 in severe pneumonia still remains unclear.Objective1.The multiplex molecular diagnosis methods for respiratory pathogen should be evaluated,in order to provide reference on developing suitable platform for clinical laboratory.2.Through investigating the pathogen infection rate and co-infection rate in hospitalized children diagnosed as ARTI,and the association of co-infecion with clinical features,this study aims to promote the understanding on etiology of ARTI.It also aims to explore the correlation of pathogens in between upper and lower respiratory tract,for evaluating the method of specimen collection.3.For further mechanism study,this study was designed to investigate the distribution of different serotypes of ADV in the hospitalized children infected with ADV,and to explore the association of serotype with clinical features.Some analysis was also designed to figure out the genomic feature of ADV-7.Methods1.qPCR methods for detection for 18 types of respiratory pathogen,including Influenza A virus(IFA),Influenza B virus(IFB),Respiratory syncytial virus(RSV),Human adenovirus(ADV),Rhinovirus(RV),Enterovirus(EV),Human metapneumovirus(HMPV),Coronavirus OC43(CoV OC43),CoV NL63,CoV 229 E,CoV HKU1,Mycoplasma pneumoniae(MP),Chlamydia pneumoniae(CP),and Bordetella pertussis(BP),were developed by laboratory.The laboratory developed qPCR method,together with the multiplex nested qPCR based method Filmarray,and the multiplex PCR based method Health Technology were used to detect respiratory pathogens in 55 nasopharyngeal swab(NPS)and 30 bronchoalveolar lavage fluid(BALF)collected from 55 hospitalized children who were diagnosed as ARTI.The detection results provided by these three methods were compared.The detection rates of each pathogen and co-infection were calculated.The clinical features between co-infections and single-infections,and the pathogens in NPS and BALF from the same individual were analyzed.2.During 2014 June to 2016 August,the hosipitalized children at the First Affiliated Hospital of Guangzhou Medical University and Guangzhou Children and Women Hospital,and infected by ADV were included in this study.The nucleic acid of respiratory tract specimens from these children were collected and performed sero-typing by qPCR or Sanger sequencing.The detection rates of of each serotype were calculated.The data of clinical features of all children were collected,and performed further analysis of the association of serotype with clinical features.3.The metagenomic analysis using high-throughtput sequencing,mutation analysis,and phylogenetic analysis were performed on the whole genome and main fuctional genes of the ADV-7 strain isolated from a case developing severe pneumonia.Results1.The results obtained from the Filmarray showed that 74.5 % of children(41/55)diagnosed as ARTI were found to be infected by at least one pathogen in their NPS,and the co-infection rate was 40 %(22/55).The highest detection rate was observed in RV,followed by MP and RSV.The most common pattern of co-infection was RV co-infected with MP.No significant difference was observed between the association of single-infection and co-infection with clinical features.The pathogens in NPS from 76.7 % of cases included the pathogens detected in the BALF from the same individual,and nearly 53.3 % of cases had exactly the same pathogens in their NPS and BALF.The results mentioned above were totally consistent with the results provided by the Health Technology and the laboratory developed qPCR method.The Filmarray and the Health Technology showed concordant rate at 84.7 % and 75.4 % with laboratory developed qPCR method respectively.2.Adenovirus type 3 was the dominant serotype during 2014 to 2016 in all ADV positive samples included in this study,with a detection rate at 77.5 %,followed by ADV-7(20.4 %).The proportion of children aged under 2 years in ADV-7 infected children was higher than those in ADV-3 infected children.Furthermore,several clinical features such as the incidence of pneumonia,severe pneumonia,complications,pleural effusion,and assisted mechanical ventilation of children infected by ADV-7 were higher than those of children infected by ADV-3,with statistically significant differences(P <0.01)。3.A whole genome from one children infected by ADV-7 and developing severe pneumonia was successfully obtained using high-throughput sequencing.No large scale of insertion or deletion was observed in the genome compared to ADV-7 genomes in the database of GeneBank.Phylogenetic analysis showed that the strain obtained in this study was highly homologous with the strain isolated from a fatal case in 2011 in Guangzhou.The whole genome,Hexon gene,Fiber gene,and the E3 region of ADV-7 also showed high homology and identity with other ADV-7 strains isolated in China in these years.Compared to the ADV-7 genome of the fatal case in Guangzhou in 2011,only one nucleotide substitution of C3218 A located at the transformation-associated protein 55 kDa exon1 was found,resulting in synonymous mutation.ConclusionsThe three methods for detection of respiratory pathogen showed good concordance,which are all applicable for clinical laboratory to apply to patients with different infection states.Co-infection is common in hospitalized children diagnosed with ARTI.However,the clinical significance of co-infection remains unclear and warrants further analysis.The spectrum of pathogens is mostly concordant in the upper and lower respiratory tract.Collecting NPS for detection can be a non-invasive and more convenient method compared to collecting BALF.The main serotype of ADV is ADV-3 during 2014 to 2016.However,ADV-7 has stronge association with the severe pneumonia in children.The whole genome and several main functional genes of ADV-7 are highly conserved.Thus,the ability of ADV-7 to cause severe pneumonia in children is not due to mutation.The pathogenic mechanism of ADV-7 worths further study on tropism,viral replication,and the host immune response against ADV-7.